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ANALELE TIINIFICE

ALE

UNIVERSITII Alexandru Ioan Cuza


DIN IAI (SERIE NOU)

SECIUNEA II a. BIOLOGIE VEGETAL

TOMUL LIV, FASCICULA 1

2008

Editura Universitii Alexandru Ioan Cuza Iai

Editura Universitii Alexandru Ioan Cuza din Iai


ANALELE TIIN IFICE ALE UNIVERSIT II Alexandru Ioan Cuza DIN IA I (SERIE NOU ), SEC IUNEA II a. BIOLOGIE VEGETAL Comitetul de redac ie:
Dr. Constantin TOMA Universitatea Alexandru Ioan Cuza din Ia i Dr. Toader CHIFU Universitatea Alexandru Ioan Cuza din Ia i Dr. Mihai MITITIUC Universitatea Alexandru Ioan Cuza din Ia i Dr. Maria Magdalena ZAMFIRACHE - Universitatea Alexandru Ioan Cuza din Ia i Conferen iar Dr. C t lin T NASE Universitatea Alexandru Ioan Cuza din Ia i Conferen iar Dr. L cr mioara IV NESCU - Universitatea Alexandru Ioan Cuza din Ia i Profesor Profesor Profesor Profesor

Comisia de referen i tiin ifici:


Academician Nicolae BO CAIU Academia Romn , Filiala Cluj-Napoca Academician Valeriu COTEA Academia Romn , Filiala Ia i Profesor Dr. Constantin TOMA Universitatea Alexandru Ioan Cuza din Ia i, membru corespondent al Academiei Romne, Filiala Ia i Profesor Dr. Leontin tefan PTERFI Universitatea Babe -Bolyai din Cluj-Napoca, membru corespondent al Academiei Romne, Filiala Cluj-Napoca Profesor Dr. Jean Pierre AUQUIRE Universitatea Catolic din Louvain la Neuve, Belgia Profesor Dr. Maria COULADIS Universitatea din Atena, Grecia Profesor Dr. Cvetomir DENCHEV Academia de tiine din Bulgaria Profesor Dr. Franco PEDROTTI Universitatea din Camerino, Italia Profesor Dr. Andrei MARIN Universitatea din Bucure ti Profesor Dr. Ioan BURZO Universitatea Agronomic i de Medicin Veterinar din Bucure ti Profesor Dr. Toader CHIFU Universitatea Alexandru Ioan Cuza din Ia i Profesor Dr. Mihai MITITIUC Universitatea Alexandru Ioan Cuza din Ia i Profesor Dr. Ursula ST NESCU Universitatea de Medicin i Farmacie Gr. T. Popa din Iai Conferen iar Dr. C t lin T NASE Universitatea Alexandru Ioan Cuza din Ia i Redactor responsabil: Profesor Dr. Constantin TOMA , membru corespondent al Academiei Romne Secretar de redac ie: ef lucr ri Dr. Naela COSTIC Tehnoredactare computerizat: Dr. Ramona Crina GALE

CONTENTS
IRINA STNESCU, C. TOMA The leaf structure of some Nepenthes Danser species 5

IRINA BERCIU, C. TOMA Histo-anatomical aspects reffering to the vegetative organs of two subspecies of Thymus pannonicus All. 16 LUMINIA HUANU-BASHTAWI, C. TOMA Contributions to the histoanatomical study of the Calendula officinalis L. leaves treated with thiophanate methyl (topsin M) 22 IOANA MARCELA PDURE, LILIANA BDULESCU, TEODORA DEDIU, I. BURZO Morpho-anatomical and phytochemical researches regarding Pseudotsuga menziesii (Mirbel) Franco (Pinaceae) 33 ALEXANDRINA MURARIU, CORINA GRDINARIU, ANIOARA STRATU The ecophysiological reaction of some varieties of apple tree, pear tree and quince tree to the pathogenic agents attack 40 ELENA CRISTINA ROU, MARIA MAGDALENA ZAMFIRACHE, I. I. BRA - Physiological effects induced by purinic substances at Capsicum annuum L. 46 M. RCA, L. FRTI, ANA LEAHU The influence on the Mn2+ ions effects on the wheat (Triticum aestivum L. ) seed germination 50 NICOLETA IANOVICI, I. E. JUHSZ, P. RADISIC, M. JUHSZ, B. SIKOPARIJA - Plantago atmospheric pollinic season in the Danube-KrisMures-Tisza euroregion (2000-2004) 54 L. POP, DORINA CACHI Araucaria excelsa L. vitrocultures initiation 64 ADRIANA PETRU VANCEA, C. F. BLIDAR, ANCA BACIU African violet (Saintpaulia ionantha L.) exvitroplantlets acclimatization, in different types of substratum 71 TATIANA EUGENIA ESAN, J. KHL, WILMA M. L. MOLHOECK Ulocladium atrum preuss - biological control agent of grey mould (Botrytis cinerea Pers.) of cropped plants 78 M. COSTIC, NAELA COSTIC - A new site in Romania for Spirulina 92 (Arthrospira) platensis

T. CHIFU, C. MNZU, OANA ZAMFIRESCU Contribution to the study of grassy vegetation in the Ceahlu Mountain 94 J. HANGANU, M. DOROFTEI, N. TEFAN - Assesment of ecological status of Danube Delta lakes using indicator macrophytes species 103 OANA ZAMFIRESCU The plant communities with Phragmites australis from The hayfields of Valea lui David natural reservation (Iasi county) 109 IRINA BLAJ-IRIMIA Associations of the Molinio-Arrhenatheretea R. Tx. 1937 class in Vaslui river basin 113 LOREDANA ASOLTANI Contributions to the study of paludal vegetation from Neagra arului rivers basin (Suceava county) 121 CARMEN AONCIOAIE Protected taxa from the Bistria river basin between Piatra Neam and Bacu 129 MIHAELA AURELIA DANU, T. CHIFU Contribution to the study of the class Molinio-Arrhenatheretea R. Tx. 1937 in the upper basin of river Dorna (Suceava county) (I) 136 I. M. CIUMAU, NAELA COSTIC - Environmental education: education for transition to sustainable development 146 C.TOMA, MARIA MAGDALENA ZAMFIRACHE - Review C. DRGULESCU - Review 153 154

Analele tiinifice ale Universitii Al. I. Cuza Iai Tomul LIV, fasc. 1, s. II a. Biologie vegetal, 2008

THE LEAF STRUCTURE OF SOME NEPENTHES DANSER SPECIES IRINA STNESCU , C. TOMA
Abstract: The authors analyze a few aspects referring to the modified leaf of four Nepenthes species, at different levels, stress being laid on the structure of the vascular bundles, digestive and nectariferous glands. Key words: Nepenthes, digestive glands, nectariferous glands, hydathodes.

Introduction The Nepenthes genus consists of more than 80 tropical species [8], spread around SE Africa, Sri-Lanka and Madagascar. Etienne de Flancourt described it in 1658 for the first time [3] and in 1753 Linn called it Nepenthes . The plant is a climbing, weakly branched liana. It presents a basal rosette of leaves with short internodes when young; on sexual maturity, the internodes become elongated and the plant starts being climbing or prostrate, according to the species. The plant creates a special impression by its bizarre leaves, consisting of a basal assimilatory part, a tendril which rolls up around different supports and a trap [2]. This trap is like a pitcher with a lid, which covers the trap, avoiding the dilution of the liquid from inside the trap by the rainwater. Some authors believe that the assimilatory part, the tendril and the pitcher belong to the petiole of an archaic leaf, while the lid represents the limb. Others consider that the pitcher and the lid form the limb, and the assimilatory part and the tendril form the petiole. Darwin stated that a carnivorous plant attracts, captures and digests the prey; the supplementary nutritive elements brought by the prey are necessary in developing and blooming. The capturing system in Nepenthes is passive; the plant does not need to move to capture the prey, unlike those which have active traps, such as Dionaea muscipula. The plants bear flowers with shiny colours and abundant nectar to attract the pollinating insects; on the other hand, the plants use different traps with different attraction elements: shiny colours or the reflection of the UV radiation, attracting odours or nectar secreted by the nectariferous extrafloral glands; all these characteristics belong to the leaf. Some authors [5, 7] evidenced the structure of the digestive glands; others [1, 6] considered that the digestive glands are closely associated with the vascular bundles. Some histoanatomical aspects were evidenced in a previous work [9] devoted to Nepenthes maxima.

Botanical Gardens of Iasi, Dumbrava Roie Street, no. 7-9, Romania

Al. I. Cuza University, Faculty of Biology, Carol I. Bd., no. 20A, 700506, Iai, Romania

Materials and methods The material under study, coming from the collection of the Alexandru Borza Botanical Gardens of Cluj-Napoca, belongs to four taxa: N. x coccinea Mast, N. distillatoria L., N. maxima Reinw. ex Nees and N. northiana Hook f. The material subjected to analysis (the modified leaves of the plants) has been fixed and preserved in 70% ethylic alcohol. The sections (from the assimilatory part, tendril and pitcher) were cut with a microtome, then coloured with iodine green and alaun-carmine, mounted in gel and analyzed on a Novex (Holland) light microscope. The light micrographs were performed by means of Novex (Holland) microscope, using a Canon A95 camera. Results and discussions As already mentioned, the leaf of Nepenthes consists of three parts: a basal, assimilatory one, a tendril and a pitcher which represents the trap of the plant. In front side view, the upper epidermis of the assimilatory part appears as formed of polygonal cells (Fig. 1); here and there, a few hydathodes are present. The lower epidermis consists of small cells, bearing weakly waved walls (Fig. 2). Here and there, stomata of the anomocytic type and hydathodes are present. A hydathode bears a short pedicel formed of a few cells and a stellate part, formed of 4-10 cells. Another author [4] suggests that the hydathodes do not only secrete water, but even absorb it from time to time. In cross section, the upper epidermis evidences small cells covered by a thick cuticle. Just beneath the epidermis, a few isodiametric-celled layers are present, forming an acviferous tissue (Fig. 3); some authors [5] call it an acviferous hypodermis. Then, a 2-3 layered palisade tissue, with short cells, in which chloroplasts can be observed, is present. The lacunary tissue is multi-layered, with small aeriferous spaces between the component cells. A lot of isolated mechanical cells (idioblasts) with spiral thickenings can be observed in the mesophyll; these were evidenced by other authors [5], too. The lower epidermis consists of small, isodiametric cells, covered by a cuticle thinner than the one covering the upper epidermis. Numerous stomata are present, as well as numerous calcium oxalate crystals in the mesophyll. The midvein is very prominent at the lower side of the assimilatory part (Fig. 4). A large number of vascular bundles is present (8 big bundles, one of its being situated in the centre or 6 bundles and a central one at N. northiana); most of them are implanted in a thick sclerenchyma ring, formed of sclerenchymatic fibres with thickened and lignified walls (or unlignified at N. coccinea). The vascular bundles have different orientation in the sclerenchymatic ring. A vascular bundle (Fig. 5) consists of a phloem (sieved tubes and companion cells) and a xylem (xylem vessels separated by celulosic parenchyma). Sometimes, the sclerenchyma sheath bears very small vascular bundles, consisting of a few phloem elements or of phloem and 1-2 xylem vessels. In the fundamental, external parenchyma, idioblasts and small, isolated vascular bundles are present, often consisting of a few phloem elements, surrounded by a thin sclerenchymatic sheath.

The tendril In cross section, the tendril shows a circular shape, with 7-8 ribs at N. maxima (Fig. 6). Small cells, covered by a thick cuticle, form the epidermis. Here an there, hydathodes, short, sometimes branched tector hairs and stomata prominig above the epidermis are present. The cortical parenchyma is formed of 5-6 layers of large cells. Most of the vascular bundles are implanted in a strong sclerenchyma ring. There is a high variability regarding the number of bundles and on their position (N. coccinea and N. maxima present a lot of vascular bundles of different size in the sclerenchyma ring and a central one in the fundamental parenchyma; N. distillatoria presents 2 big vascular bundles in the center of the parenchyma and a smaller one, close to them, while N. northiana shows the largest number of vascular bundles, implanted in the sclerenchyma ring, and also two smaller ones in the fundamental parenchyma, but close to the sclerenchyma. The tendril has an homogenous parenchyma, formed of big, turgescent cells and a few idioblasts. Near the pitcher, the cross section of the tendril is quite circular. The vascular bundles form 2-3 rings (the internal bundles are bigger than the external ones, in the fundamental parenchyma). A sclerenchymatic sheath surrounds each vascular bundle. Numerous calcium oxalates are present in the fundamental parenchyma. At the inferior level of the pitcher, a typical limb structure is present. In front side view, the internal epidermis presents polygonal elongated cells, with thick walls. Here and there, a lot of multicellular digestive glands are present (Fig. 7); a small epidermal prolongation can be observed near each gland, yet without touching it. The external epidermis (Fig. 8) consists of small polygonal cells with thin walls, anomocytic stomata, hydathodes and nectariferous glands, which appear like multicellular, massive structures, communicating with the exterior through a short channel. In cross section, the wall of the pitcher is quite thick. The internal epidermis shows elongated cells, covered by a thick cuticle. Numerous big digestive glands are present in small epidermal cavities (Fig. 9); the epidermal cells form a small fold, without touching the gland. A digestive gland shows 2-3 layers of oblate cells, 1-2 layers of isodiametrical cells and an external layer of columnar-shaped cells. Each digestive gland is associated with small vascular bundles (tracheids with ringed and spiral thickenings). In longitudinal section, the small fold can be better observed (Fig. 10). The external epidermis consists of small cells, covered by a thin cuticle. The nectariferous glands are formed of three layers of cells delimiting a cavity which opens towards the exterior through a short channel (Fig. 11). The nectariferous glands attract the insects (the prey) to the trap and make them climb the wall of the pitcher to reach the slippery peristome. The assimilatory parenchyma is thick, homogenous, formed of small cells outside and bigger inside. A lot of calcium oxalates are present all over the parenchyma. The vascular bundles have different sizes, the biggest ones occupying the external part of the parenchyma, while the smallest ones occupy the centre of it. Each vascular bundle consists in phloem facing the exterior part of the pitcher and a xylem facing the internal one, so that the internal epidermis represents the old upper epidermis of an archaic leaf and the external epidermis represents the old lower epidermis. All the studied species show mechanical sheaths surrounding the vascular bundles, consisting of fibres with

moderately thickened and lignified walls. The assimilatory parenchyma also presents a few idioblasts. The middle level of the pitcher presents a quite similar structure to that of the anterior level. In front side view, the internal epidermis consists of polygonal cells with thick walls and secretory glands, smaller than those occurring at the inferior level; the integumentary fold do not touch the gland (Fig. 12). The external epidermis is formed of polygonal cells, anomocytic stomata, multicellular tector hairs, nectariferous glands and hydathodes (Fig. 13). In cross section, the pitcher shows a thinner wall. The internal epidermis presents digestive glands which communicate with the tracheids (Fig. 14). In longitudinal section, the fold can be better observed (Fig. 15). The external epidermis consists of small cells covered by a thin cuticle. Anomocytic stomata, hydathodes, nectariferous glands (Fig. 16) and multicellular tector hairs, often branched, are present. In the assimilatory parenchyma, numerous calcium oxalates can be observed. Each vascular is bounded by a sclerenchymatic sheath (Fig. 17). There are vascular bundles consisting only of a few phloem elements. The assimilatory parenchyma presents idioblasts, too. The superior level of the pitcher shows the same structure as the other levels. In front side view, the internal epidermis presents large polygonal cells and digestive glands (Fig. 18) smaller than those occurring at the other levels. The epidermal fold covers more than half of the digestive glands. The external epidermis (Fig. 19) consists of small polygonal cells, with waved lateral walls, tector hairs, hydathodes, nectariferous glands and anomocytic stomata. Cross section through the superior level of the pitcher shows the digestive glands in their incipient stage of development (Fig. 20), consisting of a small number of cells. Almost half of the gland is covered by the integumentary fold (Fig. 21); the developing stages of the glands during ontogenesis were previously presented [9]. The external epidermis shows similar structures to those of the anterior levels (Figs. 22 and 23). The assimilatory parenchyma is thinner; the vascular bundles are bounded by a very thin mechanical sheath, consisting of fibres with thickened walls, but weakly lignified; some of the bundles present only a few phloem elements; calcium oxalates are not present. The lid In front side view, both the upper and the lower epidermis present polygonal cells, with waved lateral walls, anomocytic stomata, secretory glands surrounded by an integumentary fold (Fig. 24) and hydathodes (Fig. 25); tector hairs, sometimes branched, are present only in the lower epidermis. The cross section of the lid is similar to that occurring in the pitchers wall. Numerous digestive glands (Fig. 26) are present in both epidermis. The fundamental parenchyma presents vascular bundles of different sizes (Fig. 27), the largest occupying the centre of the parenchyma. All vascular bundles are surrounded by a thin sclerenchyma sheath formed of thin-walled and weakly lignified fibres. The peristome (Fig. 28) is a common characteristic of the pitcher plants. All four investigated Nepenthes species have a ridged peristome. The epidermal cells are small, covered by a very thick cuticle. Stomata and hydathodes are present in the lower epidermis.

The peristome has an homogenous parenchyma, with large cells, a few idioblasts and small vascular bundles, most of them consisting of phloem elements surrounded by sclerenchyma fibres, with cellulosed unthickened walls. Conclusions In spite of the high variability of the pitcher size recorded from one species to another, their histo-anatomy is quite similar. There have been observed mostly quantitative differences (number of the vascular bundles, size of the digestive glands at different levels, thickness of the sclerenchymatic sheath, length of the integumentary fold which covers each digestive gland) and not qualitative differences. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.
ANDERSON A. N., 1994 - Secretion and absorbtion in glands of the carnivorous plant Nepenthes alata. B. A. honors thesis, Connecticut College, New London, C. T. DALTON M. JOS., 1859 - Note sur l origin et le dveloppement des urnes dans les plantes du genre Nepenthes. Ann. des Sci. Nat.; sr.Bot., 12: 125-129 LLOYD F. E., 1942 - The Carnivorous Plants. Chronica Botanica, 9. Ronald Press, New York MACFARLANE J. M., 1889 - Observations on pitchered insectivorous plants. Part I. Ann. of Bot., 3: 253265 METCALFE C. R., CHALK L., 1972 - Nepenthaceae in Anatomy of the Dicotyledons. 1: 1105-1111, Clarendon Press, Oxford STERN K., 1917 - Contribution to the knowledge of Nepenthes. Flora, 109: 213 283 PARKES D. M., 1980 - Adaptive mechanisms of surfaces and glands in some carnivorous plants. MSc Thesis, Monash University, Clayton, Victoria, Australia STAROSTA P., LABAT J.-J., 1993. - Lunivers des plantes carnivores. d. Du May, Paris TOMA I., TOMA C., STNESCU I., 2002 - Histo-anatomical aspects of the Nepenthes maxima Reinw. ex Nees metamorphosed leaf. Rev. Roum. Biol., sr. Biol. vgt., 47, 1-2: 3-7

Acknowledgments The authors gratefully thank to Felician Micle PhD, Ex-Director of the Botanical Garden of Cluj-Napoca and to Elena Rnba for supplying the material for the present investigation.

Explanation of plates: Plate I 1. The upper epidermis of the assimilatory part of N. maxima, in front side view 2. The lower epidermis of the assimilatory part of N. distillatoria, in front side view 3. The mesophyll of the assimilatory part of N. distillatoria 4. Cross section through the midvein of the assimilatory part of N. maxima 5. The biggest vascular bundle of the midvein belonging to the assimilatory part of N. maxima 6. Cross section through the tendril of N. maxima Plate II 7. The internal epidermis of the inferior level of N. distillatoria pitcher, in front side view 8. The external epidermis of the inferior level of N. distillatoria pitcher, in front side view 9. Cross section through the inferior level of N. distillatoria pitcher 10. Longitudinal section of the inferior level of N. maxima pitcher 11. Cross section through the inferior level of N. distillatoria pitcher 12. The internal epidermis of the middle level of N. northiana pitcher, in front side view Plate III 13. The internal epidermis of the middle level of N. maxima pitcher, in front side view 14. Cross section through the middle level of N. northiana pitcher 15. Longitudinal section through the middle level of N. maxima pitcher 16. Cross section through the middle level of N. distillatoria pitcher 17. Cross section through the middle level of N. maxima pitcher 18. The internal epidermis of the superior level of N. coccinea pitcher, in front side view Plate IV 19. The internal epidermis of the superior level of N. distillatoria pitcher, in front side view 20. Cross section through the superior level of N. distillatoria pitcher 21. Longitudinal section through the superior level of N. maxima pitcher 22. Cross section through the superior level of N. distillatoria pitcher 23. Cross section through the superior level of N. northiana pitcher 24. The upper epidermis of the lid belonging to N. northiana pitcher, in front side view Plate V 25. The lower epidermis of the lid belonging to N. northiana pitcher, in front side view 26. Cross section through the lid of N. coccinea pitcher 27. Cross section through the lid of N. northiana pitcher 28. Cross section through the peristome of N. northiana pitcher

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IRINA STNESCU, C.TOMA

PLATE I

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IRINA STNESCU, C.TOMA

PLATE II

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PLATE III

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PLATE IV

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PLATE V

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Analele tiinifice ale Universitii Al. I. Cuza Iai Tomul LIV, fasc. 1, s. II a. Biologie vegetal, 2008

HISTO-ANATOMICAL ASPECTS REFFERING TO THE VEGETATIVE ORGANS OF TWO SUBSPECIES OF THYMUS PANNONICUS ALL. IRINA BERCIU*, C. TOMA*
Abstract: The authors analyze comparatively two Thymus pannonicus subspecies (Thymus pannonicus ssp. auctus and Thymus pannonicus ssp. pannonicus). The vegetative organs (root, stem and leaf) of the two studied taxa were histo-anatomically investigated, evidencing the structure, localization and frequency of the secretory structures of essential oils. The secretory structures of essential oils are always multicellular, consisting of a basal cell, a unicellular pedicel and a gland which bears 1, 2 or 8 cells. Key words: Thymus, anatomy, vegetative organs, trichomes.

Introduction 1 Thymus pannonicus is wide spread in our country, living in meadows, bushes, forest glades, sands, heated rocks, from the steppe region to the holm level [2]. The plant presents vigorous, ascending at the basis or until the inflorescence, branched stems, the entire surface being covered by trichomes measuring the same length as does the diameter of the stem. The plant presents a capitulum inflorescence, the calyx of the flowers reaching 3-4 mm; the corolla is red to mauve, reaching 6-7 mm [2]. In our country, T. pannonicus presents two subspecies which are focused in the present paper: - T. pannonicus ssp. pannonicus - a villous-hairy plant, which presents a lot of trichomes on both sides of the leaves; - T. pannonicus ssp. auctus (Lyka) So - a plant which bears glabrous leaves. Most of the Lamiaceae species have been investigated from anatomical point of view; their general structure characteristics were presented in the fundamental tractates regarding the anatomy of the dicotyledons [3] or of the angiosperms (Napp-Zinn, 1973, 1974). Regarding the Thymus genera, in our country, Toma and Rugin have investigated only the anatomy of T. vulgaris. We have publicized until now a paper referring to the seedling structure of Thymus vulgaris. Material and methods The material under study is represented by two subspecies: T. pannonicus ssp. pannonicus, collected on 12.06.2007 from Potoci (Neam district) and T. Pannonicus ssp. auctus, collected on 16.07.2007 from Flticeni (Suceava district).

Al. I.Cuza University, Faculty of Biology, Carol I Bd., no. 20A, 700506, Iasi, Romania

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The vegetal material has been fixed and preserved in 70% ethylic alcohol. The sections were cut with a microtome and a botanical razor. The vegetative organs, subterranean and overhead, were cross sectioned, on different levels, from the top to the basis. First of all, the sections were submitted to a discoloration process, using sodium hypochlorite (20-25), then coloured (with iodine green and with alaun-carmine) and mounted in gel. The drawings were performed by means of a Romanian (Projektionszeichenspiegel) microscope and the micrographs were performed by means of a Novex (Holland) microscope, using a Canon A95 camera. Results and discussions The root (Plate I. 1, 2). There have not been observed major differences at the two subspecies; both of them present a root with an early secondary structure generated by the activity of both lateral meristems: the cambium and the phellogen. The phellogen is generated by a profound cortical layer. The cambium forms a thin phloem ring and 2-3 secondary xylem rings (the vessels are spread in the fundamentally massive of libriform). All the rings present a lot of early xylem vessels of large diameter and less lately xylem vessels of smaller diameter, resulting very easy to establish the limit between different rings. The stem (Plate I. 3, 4, 5, 6; Plate II. 1, 2). The superior level of the stem belonging to T. pannonicus ssp. pannonicus has a quadratic shape in cross section, with less prominent ribs where sheaths of angular collenchyma are present. The epidermis consists in isodiametric cells, having a bellied external wall thicker than the others and covered by a thick cuticle. T. pannonicus ssp. auctus presents tangentially elongated epidermal cells. Here and there both species present stomata (promining above the external part of the epidermis) and trichomes of two types: tector trichomes (almost all the trichomes are bi- or three-celled, with obtuse point) and short, multicellular secretory trichomes. T. pannonicus ssp. pannonicus presents a thicker cortex (5-6 layers) than T. pannonicus ssp. auctus (3-4 layers). The cortex does not present a casparian endodermis, opposite to other Lamiaceae species. Both analyzed species show a central cylinder consisting in annular vascular tissues: an external, thin phloem ring and an internal, thick xylem ring (formed by xylem vessels and cells belonging to the xylem cellulosed parenchyma); a large meristematic region is present between them; the tracheogenesis is in process. The middle level of the stem shows a quadratic shape in cross section, too, with very prominent ribs. T. pannonicus ssp. pannonicus presents longer tector trichomes and more numerous on unit area; the epidermal cells are covered by a thick cuticle (resulting a ridged profile); the internal layer of the cortex may be considered as playing the role of a casparian endodermis. T. pannonicus ssp. auctus does not present a typical endodermis yet. The central cylinder shows a typical secondary structure at both taxa: a continuous, thin phloem ring and a thick xylem ring where the libriform is prevalent; the libriform fibers present thick and intense lignified walls.

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The meristematic region (which is present between the phloem and the xylem) is very thick. The tracheogenesis is still in process. Some of the cells belonging to the central part of the pith start the disarrangement process. At the inferior level, the stem still shows a quadratic shape, but presenting less prominent ribs at both species. The epidermal cells are covered by a thicker cuticle; the cortex is thinner that the one belonging to the others levels because the most internal layers consist in strong oblate cells (with hardly noticeable lumina), the external ones are moderately collenchymatized and the collenchyma bands are present in the ribs. Only at this level, a casparian endodermis can be distinguished at T. pannonicus ssp. auctus. The structure of the central cylinder is quite the same, but the xylem ring is thicker and stronger lignified. Most of the pith is disorganized, appearing a large aeriferous cavity, of irregular shape at T. pannonicus ssp. pannonicus and more narrowed at T. pannonicus ssp. auctus. The foliar limb (Plate II. 3) In front side view, the epidermis consists in elongated cells, with uncurved lateral walls, at both analyzed subspecies. A lot of tector trichomes, of different size, are present at the edges of the limb belonging to T. pannonicus ssp. pannonicus; their structure varies from unicellular to multicellular, having an obtuse or narrow point; some of them are inflected toward the epidermis, bearing a large basal cell and a thick wall; they are frequently present in the lower epidermis. The leaves are glabrous at T. pannonicus ssp. auctus; only the edges of the limb present some short aculeiform trichomes which are not mentioned in the literature. The vascular bundles are big and present a very thick sclerenchymatic sheath at the phloemic pole; the sclerenchymatic fibers have a thick and lignified wall, at both analyzed subspecies. The mesophyll is formed by palisade tissue and spongy tissue; the former is bilayered, but the cells belonging to the hypodermic layer are higher than the others. Both taxa shows multicellular secretory trichomes, bearing a basal cell, smaller than the adjacent epidermic cells, a short unicellular pedicel and a gland formed by 1, 2 or 8 secretory cells, all of them being covered by a common bellied cuticle. The stem presents shorter secretory trichomes, their frequency rises from the basis to the apex of the organ. In front side view, the epidermic cells of the limb, which surrounds the secretory trichomes, are radialy elongated towards the cuticle and bear uncurved walls.

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Conclusions The superior level of the stem does not present a casparian endodermis at both subspecies; it appears at the middle level of the stem belonging to T. pannonicus ssp. pannonicus or at the inferior level of the stem belonging to T. pannonicus ssp. auctus. The xylem vessels are spread all over the libriform at T. pannonicus ssp. auctus or only in its internal part at T. pannonicus ssp. pannonicus. The aeriferous cavity appears earlier at T. pannonicus ssp. pannonicus than at T. pannonicus ssp. auctus. T. pannonicus ssp. pannonicus presents the largest number of secondary xylem vessels on unit area. The frequency of the tector trichomes decreases toward the basis of the stem at both subspecies. REFERENCES
1. 2. 3. 4. 5. CIOCRLAN V., 2000 - Flora ilustrat a Romniei. Pteridophyta et Spermatophyta. Ed. Ceres, Bucureti: 670-675 GUULEAC M., 1961 - Thymus, In Flora Republicii Populare Romne, VIII, Ed. Acad. RPR, Bucureti: 301-334 METCALFE C.R., CHALK L., 1950 - Anatomy of Dicotyledons, vol II, Clarendon Press, Oxford: 10411053 TOMA C., BERCIU I., 2007 - Morphological peculiaries of germination and structure of seedling in Thymus vulgaris L.; Rom. Biol. Sci., 5, 1-2: 136-137 TOMA C., RUGIN R., 1998 - Anatomia plantelor medicinale. Atlas. Ed.Acad. Rom., Bucureti: 169-172

Explanation of the plates: Plate I 1. Cross section through the root of T. pannonicus ssp. pannonicus (x400) 2. Cross section through the root of T. pannonicus ssp. auctus (x400) 3. Cross section through the inferior level of the stem of T. pannonicus ssp. pannonicus (x400) 4. Cross section through the inferior level of the stem of T. pannonicus ssp. auctus (x400) 5. Cross section through the middle level of the stem of T. pannonicus ssp. pannonicus (x400) 6. Cross section through the middle level of the stem of T. pannonicus ssp. auctus (x400) Plate II 1. Cross section through the superior level of the stem of T. pannonicus ssp. pannonicus (x 200) 2. Cross section through the superior level of the stem of T. pannonicus ssp. auctus (x 200) 3. Cross section through the limb of T. pannonicus ssp. pannonicus (x400) 4. Glandular trichome of T. pannonicus ssp. auctus (x400) 5. Tector trichome of T. pannonicus ssp. pannonicus (scale bars=50m) 6. Aculeiform trichome of T. pannonicus ssp. auctus (scale bars=50m) 7. Multicellular secretory trichome of T. pannonicus ssp. auctus (x400) 8. Tector and glandular trichome of T. pannonicus ssp. pannonicus (x400)

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IRINA BERCIU, C. TOMA

PLATE I

1.

20

IRINA BERCIU, C. TOMA

PLATE II

3 5 6

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Analele tiinifice ale Universitii Al. I. Cuza Iai Tomul LIV, fasc. 1, s. II a. Biologie vegetal, 2008

CONTRIBUTIONS TO THE HISTO-ANATOMICAL STUDY OF THE CALENDULA OFFICINALIS L. LEAVES TREATED WITH THIOPHANATE METHYL (TOPSIN M) LUMINIA HUANU-BASHTAWI , C. TOMA
Abstract. The study analyzes the histo-anatomical modifications of the Calendula officinalis leaf, caused by the treatment with thiophanate methyl, applied 3 times, in two different concentrations, of 0.1% and, respectively, 0.4%. The cross-sections made at the three levels of the leaf, as well as the surface ones, evidenced some quantitative differences between the two variants of treatment and the reference, while the differences of qualitative type are minimum, referring to the different distribution of the pallisadic tissue on the two sides of the foliar limb; consequently, the leaf structure is different: bifacial unequally equifacial in the reference and bifacial heterofacial, respectively, in the treated samples. The quantitative type modifications are related to the prominence extent of the median nervure, thickness of the meristematic area, size and number of the conducting fascicles and the xylem vessels (which are intensely stimulating parameters in the two treatments), the presence of secretory hairs, width and thickness of the foliar limb which, at a concentration of 0.4%, are slightly inhibited, in spite of the fact that the median nervure is much more prominent, even comparatively with the 0.1% concentration treatment. Keywords: Calendula, Topsin M, cytokinin hormone-type action, foliar limb, histo-anatomical modifications.

Introduction The influence of fungicides on plants productivity is usually attributed to the primary fungicide/fungistatic effect of such substances, although, quite frequently, they may show secondary physiological effects, which may be either toxic or, on the contrary, beneficial to the plants subjected to such treatments [3]. The thiophanate methyl, a systemic fungicide belonging to the benzimidazole class, is largely utilized, as due to its large spectrum of action, as a curative and protecting substance for the cultivation of alimentary, industrial as well as medicinal plants [9]. Involvement of benzimidazoles and of some of their derivatives in the regulation of certain physiological processes developed at plant level has been extensively studied, being usually defined as a cytokynin hormone-type action [6, 10]. In the case of both thiophanate methyl and carbendazime, the main metabolite and the fungicides active substance, respectively, cytokinin-like effects have been demonstrated in some culture plants, being manifested by the inhibition of leaves senescence, lower degradation of chlorophyll, proteins and AND; more than that, synthesis of the photosynthetic pigments is stimulated, so that the treated leaves maintain their green colour over a longer period of time [1, 4, 5, 6]. The (20 g ml-1) carbendazime solution applied on the leaves of wheat

Al. I. Cuza University, Faculty of Biology, 20A Carol I Bd., 700506, Iai , Romania

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prevents the loss of electrolytes and amino acids, as well as disorganization of the cellular organites, the main mechanism of the antisenescence activity exercised by carbendazime being its protecting effect upon the membranary system. At higher concentrations (100 g ml-1), the fungicide losses its cytokinin-like activity; more than that, it even stimulates this loss of electrolytes and amino acids at the level of the membranary system of the treated leaves [7]. It is expected that the action exercised by carbendazime on the treated plants will be possibly extrapolated to the thiophanate methyl, in spite of the fact that, according to some authors [6], the cytokinin hormone-type action might be partially caused by carbendazime, in certain cases fungicides being more active, in this context, then pure carbendazim; on the other hand, the results of the experiments performed with various commercial formulae of the benzimidazolic fungicides are quite controversial as to their secondary effects [8]. Considering all these observations, the present paper analyzes the histo-anatomical modifications induced by thiophanate methyl and/or its main metabolite (MBC) upon the Calendula officinalis leaf, along their correlation with the cytokinin hormone-type effect of the fungicide, anatomically evidenced on the Cynara scolymus leaves [2], comparatively with the non-treated reference sample. Material and methods The experimental material, cultivated in the Anastasie Ftu Botanical Gardens of Iasi, was obtained from seeds of the Petrana kind, provided by the Research Station for Medicinal and Aromatic Plants of Fundulea. Besides the treated plants (TM70 0.1%, a concentration, applied in agriculture and, respectively, a TM70 0.4%, a concentration value recommended for fungicides similar to thiophanate methyl), a sample batch, formed of nontreated plants, was prepared for comparative purposes. The administration of fungicide, as a moisty powder, was made three times (at intervals of 7 and 10 days), in the moment of branching or of the first anthodium formation, the plants possessing 30-35 nomophyles. The vegetal material, harvested 10 days after the last treatment, was fixed and conserved in 70% ethanol, then processed according to the methods commonly applied in studies of vegetal anatomy. Measurements were performed on a photonic microscope, by means of a micrometer (ocular and objective), while the light micrographs were performed on a Novex (Holland) microscope, using a Cannon A95 camera. In this paper we used the following abbreviations: Ca. of. M - Calendula officinalis, control (untreated plants); Ca. of. TM 0,1% - Calendula officinalis, treated with Topsin M 0,1%; Ca. of. TM 0,4% - Calendula officinalis, treated with Topsin M 0,4%. Results and discussions Cross-sections - the basal leaf, the middle third In the reference the median nervure is visibly prominent on the inner side of the limb, evidencing only one conducting fascicle (Fig. 1, 2). The mesophyl shows 2 layers of low pallisadic cells, with sinuous lateral walls on the upper side, and lacunary tissue (6-7

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layers of cells) on the inferior side, the structure of the limb being bifacial heterofacial (dorsi-ventrally) (Fig. 9, 10). In the TM 0.1% treated sample the median nervure strongly prominent on the inferior side of the limb, evidencing a semi-circular contour and including 3 large conducting fascicles which, in the case of Asteraceae, is an exception (Fig. 1, 2). The mesophyl includes 2 layers of law pallisadic cells, on the upper side, and 6 layers of lacunary tissue, so that the structure of the limb is bifacial heterofacial (dorsi-ventrally) (Fig. 9, 10). In the TM 0.4% treated sample the median nervure strongly prominent on the inferior side of the limb, with a semi-elliptical contour, forming 3 ribs: a large one in the middle and two, smaller, lateral ones (Fig. 1, 2). The secretory hairs are more numerous on the surface unit, similarly with the tectory ones, which form large bunches on the edge of the limb (Fig. 14 a). The mesophyl evidences 2 layers of low pallisadic cells on the upper side, and 5 layers of lacunary tissue, with isodiametric cells. The cells of the abaxial layer do not form a typical palisade, being rather square-shaped, their ratios being of 1/1.5, while the mesophyl is visibly thinner at this sample (Fig. 9, 10; Tab. IV). Cross-sections - the leaf from the mid strain, the middle third In the reference the median nervure is pronouncedly prominent on the inferior side of the limb and moderately prominent, respectively, on the upper side (Fig. 4). The mesophyl is of the pallisdic type under both epidermes, yet the cells from the adaxial side are taller; between the two pallisades, the cells of the assimilatory parenchyma are isodiametric, the structure of the leaf being therefore bifacial unequally equifacial (Fig. 11, 12). The median conducting fascicle is collaterally open, having a collenchyma girdle at each of the two poles (Fig. 5). All the other conducting fascicles are small, the latter ones possessing only phloem elements. In the TM 0.1% treated sample the median nervure, very thick and highly prominent on the inferior side of the limb, includes 3 conducting fascicles (of which, the median one is thicker), all of the open collateral type, each with a collenchyma girdle at both poles (Fig. 4, 5). The mesophyl from the adaxial side is of the pallisadic type, yet with lower cells, similar to those of the reference, while the one from the inferior side is mostly of lacunary type, which explains the different bifacial heterofacial structure of the limb, comparatively with the reference (Fig. 11, 12). The frequency of the secretory hairs is approximately equal to that recorded in the standard sample. In the TM 0.4% treated sample the median nervure is highly prominent on both sides of the limb, the hypodermic layer being of collenchymatic type (Fig. 4). The fundamental parenchyma of the median nervure evidences a single conducting fascicle of open collateral type (Fig. 4, 5); the lateral nervures of the first order show, too, relatively large conducting fascicles. The mesophyl is more lax, with the pallisadic tissue at its adaxial side (with visibly lower cells, as actually in the case of the untreated sample) and lacunary tissue on its inferior side, the limbs structure being bifacial heterofacial, as in the 0.1% treatment (Fig. 11, 12).

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Cross-sections - the upper leaf, the middle third In the reference the mesophyl is almost wholly of the pallisadic type, yet with visibly higher cells in the hypodermic cells; the cells in the middle of the mesophyl are not always perpendicularly elongated on the epidermis, yet oblique; the structure of the limb is, at this level, too, bifacial equifacial (Fig. 13). In the TM 0.1% treated sample the median nervure is prominent on both sides of the limb, evidencing isodiametric epidermal cells, with an extremely thick, almost wholly cutinized wall (Fig. 6). The fundamental parenchyma of the median nervure includes a single conducting fascicle, of open collateral type, its wooden vessels occurring in parallel radial rows (8-10) as well as a thin girdle of mechanical fibers, in the course of formation in periphloemic position (Fig. 7); the generating area between the xylem and the phloem has 4 cell layers, comparatively with the reference which evidences only 2 layers (Fig. 8). The mesophyl is homogeneous, formed of isodiametric cells with large aeriferous spaces among them; it is only the cells of the adaxial hypodermal layer that appear slightly higher, reminding of the pallisadic form, with sinuous lateral walls; here and there, up to two layers of pallisadic cells may be observed (Fig. 13). The secretory hairs are thicker than in the reference. In the TM 0.4% treated sample the secretory hairs are more frequent on the median nervure, yet fewer then in the untreated and 0.1% treated samples. The mesophyl is almost homogeneous, of lacunary type, only the cells of the adaxial hypodermal layer being slightly taller (Fig. 13). In front of the median nervure, the epidermis has isodiametric cells, with their internal and external wall thicker than the others, the external one being covered by a thin cuticle. Long tectory hairs are visible at the edges of the limb (Fig. 14 a); where, too, the mesophyl appears typically lacunary within the whole thickness of the limb. The fascicle of the median nervure evidences numerous (10-12) parallel rows of woody vessels (Fig. 7); the generating area between the xylem and the phloem is thicker (5-6 cell layer) (Fig. 8), while the phloemic pole has a girdle of sclerenchymatic fibers with moderately thickened and lignified walls. Epidermis in front side view (the upper leaf) In the reference the upper epidermis is formed of polygonal cells with straight lateral walls. Here and there, stomatae of anomocytic type and secretory hairs may be observed. In the TM 0.1% treated sample, more stomatae occur on the unit of the surface. Besides the secretory hairs, very long tectory hairs, with a very long filiform terminal cell, have been also noticed. In the TM 0.4% treated sample, the epidermal cells are more numerous on the unit of surface, therefore they are smaller, yet the hairs have the same frequency as in the previous sample (Fig. 15 a). In the reference the inferior epidermis evidences some epidermal cells with slightly waved lateral walls. The stomatae and the secretory hairs have the same frequency on both sides of the limb, the gland showing secretory cells arranged on 3-4 levels, the ones situated at superior levels having a convex wall. In the TM 0.1% treated sample, the inferior side shows some cells with slightly waved walls. The frequency of hairs is the same on both sides, yet more numerous than in the untreated sample (3-4 in a microscopic field). In the TM 0.4% treated sample, all epidermal cells show slightly waved lateral walls.

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The secretory hairs are more rare (1-2 in a microscopic field), yet the stomatae show the same frequency as in the TM 0.1% treatment (Fig.15 b). Conclusions The histo-anatomical modifications induced by the treatment with Topsin M depend on both the administered dose and the leaves development stage in the moment of fungicides application. The response reactions of the tissues are more frequently of quantitative order, although mention should be also made of certain structural aspects that might influence the physiology and biochemistry of the plant and, consequently, the active principles synthesized by Calendula officinalis. The first and most evident effect of thiophanate methyl involves an increase of the foliar surface, a process to be decompensated by a weaker development of the pallisadic tissue, along with a more lax texture of the lacunary one (especially in the 0.4% treatments). At higher concentrations, the fungicide visibly reduces the thickness of the foliar limb, by reducing the sizes of the pallisadic cells (Tab. IV, V, VI), the ratio of which become 1.5/1 on the upper side (mainly in the terminal leaves which, in the moment of spraying, appear in an incipient stage of development), and 1/1.5, respectively, on the inferior side (almost quadratic cells); consequently, the structure of the foliar limb gets modified, from bifacial equifacial in the reference, to bifacial heterofacial in the leaves collected from the middle of the stem, and to an almost wholly lacunary (isofacial) structure in those from the top of the Calendula officinalis stem (Fig. 9-14). However, the median nervure is much thicker, with a modified (either triangular or semi-circular) contour in cross-section, the growth being intensely stimulated by the fungicide action, through an increased number of conducting fascicles (Tab. I, II, III). In Asteraceae, the presence of more numerous conducting fascicles in the median nervure constitutes an exception, to be possibly explained by inclusion of the first order nervures, as a result of the stimulating action exercised by the thiophanate methyl (the cytokinin hormone-type action of this fungicide being acknowledged). The woody conducting tissue shows a visible reaction to the treatment, by increasing the number of its vessels and, equally, of their diameter (TM 0.1%) (Tab. I, II, III); between the xylem and the phloem, the generating zone is thicker in the treated samples, including several cell layers (TM 0.4%) (Fig. 3, 5, 8). The epidermal cells are more numerous and smaller, with more waved lateral walls than in the reference (Fig. 15), while the stomatae are more numerous on the unit surface, the stomatic index recording higher values in the treated materials (Tab. 7); the frequency of secretory hairs increases in inverse ratio to the concentration of Topsin M and with the development stage of leaves in the moment of fungicides application so that, in the terminal leaves, it records higher values in the TM 0.1% treatment (stimulating dose) and lower values, respectively, in the TM 0.4% one (inhibiting dose).

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REFERENCES
1. EL MASHAD A.A.A., 2002 - Effect of thiophanate methyl on the growth and some metabolic activities of soybean plant. Egyptian J. Physiol. Sci., 24, 1: 83-102 2. HUANU-BASTAWI LUMINIA, TOMA C., 2006 - Considerations on the histo-anatomical study of the leaves of Cynara scolymus L. treated with thiophanate methyl (Topsin M). 4th Conference on Medicinal and Aromatic Plants of South-East European Countries - Iai, Romnia, 28th 31st of May 2006 :126-132 3. PETRCZI, M.I., MATUZ, J., KTAI C., 2002 - Study of pesticide side-effects in winter wheat trials. Acta Biologica Szegediensis, 46, 3-4: 207-208 4. PRIESTELY, R.H., BAYLES, A. ROSEMARY, 1982 - Effect of fungicide treatment on yield of winter wheat and spring barley cultivars. Plant Pathology, 31, 1: 3137 5. STASKAWICZ B., KAUR-SAWHNEY R., SLAYBAUGH R., ADAMS W., GALSTON A.W., 1978 - The cytokinin-like action of methyl-2-benzimidazolecarbamate on oat leaves and protoplasts. Pesticide Biochemistry Physiology, 8, 1:106-110 6. THOMAS T.H., 1974 - Investigations into the cytokinin-like properties of benzimidazole-derived fungicides. Ann. Appl. Biol., 76: 237-241 7. TRIPATHI R.K., TANDON, K., SCHLSSER E., HESS W.M., 1981 - Effect of fungicides on the physiology of plants. Part IV: Protection of cellular organelles of senescent wheat leaves by carbendazim. Pesticide Science, 13, 4: 395 400 8. VAN IERSEL, M.W., BUGBEE, B., 1996 - Phytotoxic effects of benzimidazole fungicides on bedding plants. J. Am. Soc. Hort. Sci., 121, 6: 1095-1102 9. YEOUNG-SEUK, B., BYEONG-YONG P., TAE-JIN A., BYEONG-YEON Y., SUNG-WOO L., NAKSUL S., 2006 - Selection of potential fungicides for control of Ginseng seedling damping-off and research on fungicide application for disease control in farms. Treat. Crop Sci., 7: 679-698 10. YOSHIDA Y., 1970 - Effect of benzimidazole on the senescence of wheat chloroplasts and their boat shape transformation. Plant Cell Physiology, 11, 3: 435-444

Table I. Numerical values - basal leaves (median nervure, middle level) Diameter Thickness No. of vessels No. of Variant Length/width (m woody fascicles (m Oc.10 Oc.10 x Ob.4) vessels x Ob.40) Ca. of. M 1200/10751500/1400 1 78-89 15-22,5 Ca. of. TM 0.1 % 1850/23501925/2475 3 81-94 22,5-32,5 Ca. of. TM 0.4 % 2075/33252250/3500 3 111-115 22,5-30 Table II. Numerical values - middle leaves (median nervure, middle level) Diameter Thickness No. of vessels No. of Variant Length/width (m woody fascicles (m Oc.10 Oc.10 x Ob.4) vessels x Ob.40) Ca. of. M 1375/7501500/1000 1 80-88 22,5-27,5 Ca. of. TM 0.1 % 1700/22501800/2600 3 -5 95-109 25-32,5 Ca. of. TM 0.4 % 2000/19002200/2075 2-3 132-139 22,5-27,5

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Table III. Numerical values - superior leaves (median nervure, middle level) Diameter Thickness No. No. of of vessels Variant Length/width (m woody fascicles (m Oc.10 Oc.10 x Ob.4) vessels x Ob.40) Ca. of. M 950/975-1000/1000 1 50-56 20-22,5 Ca. of. TM 0.1 % 1075/10501250/975 1 61-72 25 Ca. of. TM 0.4 % 1375/12001600/1450 2 68-88 25-30 Table IV. Numerical values - basal leaves (limb, middle level) (Oc.10 x Ob.20) Thickness of the No. of layers in Thickness of the Variant limb (m) the limb pallisade (m) Ca. of. M 375-450 7-8 135-160 Ca. of. TM 0.1 % 350-435 6-7 125-150 Ca. of. TM 0.4 % 325-375 7-8 115-130 Table V. Numerical values - middle leaves (limb, middle level) (Oc.10 x Ob.20) Thickness of the No. of layers in Thickness of the Variant limb (m) the limb pallisade (m) Ca. of. M 350-400 6-7 150-175 Ca. of. TM 0.1 % 375-410 7-8 140-170 Ca. of. TM 0.4 % 300-350 6-7 110-120 Table VI. Numerical values - superior leaves (limb, middle level) (Oc.10 x Ob.20) Thickness of the No. of layers in Thickness of the Variant limb (m) the limb pallisade (m) Ca. of. M 335-375 6 125-145 Ca. of. TM 0.1 % 325-375 7-8 125-135 Ca. of. TM 0.4 % 275-300 7 100-120

Table VII. Numerical values epidermis of the upper leaves, middle level (Oc.10 x Ob.20) Upper epidermis Inferior epidermis No. of No. of Stomatic No. of No. of Stomatic Samples cells stomatae index cells stomatae index Ca. of. M 63 9 11,11 76 13 12,74 Ca. of. TM 0.1 % 71 12 12,63 78 15 13,88 Ca. of. TM 0.4 % 75 15 14,28 88 17 13,93

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Ca. of. M Ca. of. TM 0.1% Ca. of. TM 0.4% Fig. 1. Cross-sections median nervure of the limb, basal leaf, middle third

Ca. of. M Ca. of. TM 0.1% Ca. of. TM 0.4% Fig. 2. Cross-sections median conducting fascicle, basal leaf, middle level

Ca. of. M Ca. of. TM 0.1% Ca. of. TM 0.4% Fig. 3. Cross-sections median conducting fascicle, basal leaf, middle level

Ca. of. M Ca. of. TM 0.1% Ca. of. TM 0.4% Fig. 4. Cross-sections median nervure of the limb, middle leaf, middle level

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Ca. of. M Ca. of. TM 0.1% Ca. of. TM 0.4% Fig. 5. Cross-sections median conducting fascicle, middle leaf, middle level

Ca. of. M Ca. of. TM 0.1% Ca. of. TM 0.4% Fig. 6. Cross-sections median nervure of the limb, superior leaf, middle level

Ca. of. M Ca. of. TM 0.1% Ca. of. TM 0.4% Fig. 7. Cross-sections median conducting fascicle, superior leaf, middle level

Ca. of. M Ca. of. TM 0.1% Ca. of. TM 0.4% Fig. 8. Cross-sections median conducting fascicle, superior leaf, middle level

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Ca. of. M Ca. of. TM 0.1% Ca. of. TM 0.4% Fig. 9. Cross-sections limb, basal leaf, middle level

Ca. of. M Ca. of. TM 0.1% Ca. of. TM 0.4% Fig. 10. Cross-sections mesophyl, basal leaf, middle level

Ca. of. M Ca. of. TM 0.1% Ca. of. TM 0.4% Fig. 11. Cross-sections limb, middle leaf, middle level

Ca. of. M Ca. of. TM 0.1% Ca. of. TM 0.4% Fig. 12. Cross-sections mesophyl, middle leaf, middle level

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Ca. of. M Ca. of. TM 0.1% Ca. of. TM 0.4% Fig. 13. Cross-sections mesophyl, superior leaf, middle level

b
Fig. 14. Calendula officinalis: tectory hairs (a), uni- and biseriated secretory hairs (b)

b
Ca. of. M Ca. of. TM 0.1% Ca. of. TM 0.4% Fig. 15. Epidermis in front side view - upper (a) and inferior epidermis (b), superior leaf, middle level

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Analele tiinifice ale Universitii Al. I. Cuza Iai Tomul LIV, fasc. 1, s. II a. Biologie vegetal, 2008

MORPHO-ANATOMICAL AND PHYTOCHEMICAL RESEARCHES REGARDING PSEUDOTSUGA MENZIESII (MIRBEL) FRANCO (PINACEAE) IOANA MARCELA PDURE *, LILIANA BDULESCU, TEODORA DEDIU, I. BURZO
Abstract: The paper presents morpho-anatomic and phytochemical researches of needles and young shoots of Pseudotsuga menziesii (Mirbel) Franco in Romania. The composition of needle and young shoots essential oils in Douglas-fir needles from different population are studied for the first time. Essential oil extraction of P. menziesii was performed using a neo-Clevenger-type apparatus and the analysis procedures with a GC-MS system. The major constituents of the oils were found to be sabinene, terpinolene, terpinene 4-ol, -pinene, -terpinen, -phellandrene and -pinene in various chemical concentrations due to analyzed vegetative organs (needles / young shoots), ecological conditions (chemotypes), analytical methods or plant phenophases. Keywords: Pseudotsuga menziesii, Pinaceae, needles, chemotype, essential oil, GC-MS, chromatogram.

Introduction Pseudotsuga menziesii [syn. P. douglasii (Lindl.) Carrire] is an evergreen trees; bark initially smooth with transverse resin blisters, with age becoming reddish brown, thick and corky, deeply fissured into scaly ridges or flaking; branches often pendulous, irregularly whorled; short shoots absent; leaf scars transversely elliptic, slightly raised proximally but essentially flush with twig distally. Buds elongate, not or slightly resinous, apex acute. Leaves borne singly, persisting 6-8 years, alternate, short-stalked, linear, flat, green and grooved above, with 2 white stomatal bands containing 5-8 lines of stomata beneath; 2 marginal resin ducts and 1 vascular bundle. Seed cones maturing first season, terminal on short branchlets, consisting of numerous spirally arranged scales, each scale 2ovulated. Mature cones shed whole, deflexed or pendent from a 2-10 mm long peduncle, ellipsoid, ovoid, or cylindric, lacking apophysis and umbo; scales persistent, apex rounded; bracts exserted, apex 3-lobed with the middle lobe long and narrow. Seeds winged; cotyledons 2-12 [6]. It is in leaf all year, in flower from March to May, the seeds ripen from September to November. The scented flowers are monoecious and are pollinated by wind. The plant prefers light (sandy), medium (loamy) and heavy (clay), acid to neutral soils. It cannot grow in the shade. It requires moist or wet soil. The plant can tolerate strong winds but not maritime exposure. Very slow growing for its first few years, growth soon becomes extremely fast with new shoots of up to 1.2 metres a year. New growth takes place from May to July. The genus Pseudotsuga contains about 7 species native to North America, and eastern Asia. Douglas-fir is named for Henry Douglas (1798-1834), a Scottish botanist who traveled in North America. The word Pseudotsuga means false

University of Agronomic Sciences and Veterinary Medicine (U.S.A.M.V.), Faculty of Horticulture, Department of Botany and Plant Physiology, Mrti 59 Bd., 011464, Bucharest, Romania

33

hemlock", while menziesii is used in recognition of Archibald Menzies (1754-1842), a Scotch physician and naturalist, who discovered Douglas-fir in 1793 on Vancouver Island. Douglas fir was often employed medicinally by various native North American Indian tribes who used it to treat a variety of complaints: an antiseptic resin is obtained from the trunk; it is used as a poultice to treat cuts, burns, wounds and other skin ailments. The resin is used in the treatment of coughs and can be chewed as a treatment for sore throats. Infusions: the green bark has been used in the treatment of excessive menstruation, bleeding bowels and stomach problems; the leaves has been used as a wash and a sweat bath for rheumatic and paralyzed joints; the young sprouts has been used in the treatment of colds; the shoots has been used in the treatment of kidney and bladder problems. A decoction of the buds has been used in the treatment of venereal disease. Young shoots have been placed in the tips of shoes to keep the feet from perspiring. A mouthwash is made by soaking the shoots in cold water [5], [7]. Bogar et al. (1965), Spicer et al. (2000) and Apple et al. (2002) made anatomical investigations of seedling roots, needles and stem growth in P. menziesii [1]. The chemical composition of P. menziesii has been reported in several studies. Snajberk et al. (1974), Wagner et al. (1989), Gambliel & Cates (1995) determined that the essential oil of needles and shoots in Douglas-fir consists of terpenes (monoterpene, sesquiterpene, oxygenated monoterpene) [10] and oleoresins (linalool, methylsalicylate, bornyl acetate citronellol, geranyl acetate, methylthymol, citronellyl acetate, terpinen 4-ol, borneol, isopulegol, anethole, terpinen 4-ol acetate, camphor, geraniol, neryl acetate, and nerol) [8]. The oil of P. menziesii from different Bulgarian populations was constituted as main compounds the monoterpenes: -pinene, sabinene, -ocimene, -terpinolene, -terpineol, citronellyl acetate, -terpinene, limonene and -terpinene [4]. Material and methods The mature shoots with needles were collected from two different populations with P. menziesii from Bucharest: chemotype 1: I. Todor Botanic Garden and chemotype 2: Faculty of Biotechnology in U.S.A.M.V. in 2005-2006. The fresh vegetal organs were free hand sectioned. The material were cleared with chloral hydrate, stained in carmine red and green iodine and mounted in gelatinized glycerin. Numerical characteristics were undertaken at ML-4M IOR microscope. The prepared material was viewed and photographed with a Nikon camera; anatomical photos are presented in Fig. 1-3. Fresh needles of the collected plant were subjected to hydrodistillation for three hours using a neo-Clevenger-type apparatus to produce essential oils. Analysis conditions: FISIONS gas chromatograph with DB 5 column 25 m length and 0.25 mm internal diameter. Carrier gas has been helium; work temperatures by injector 250C, interface 280C and ionization source 200C; EI mode 70eV; mass range 41-550 amu. Chemical compounds identification has been performed by library search represented by NIST and ESO 85. Kovats indices were used as a confirmation for the chromatographic peak position. These determinations were performed in Research Center for the Study of Food Quality and Useful Plant Compounds from U.S.A.M.V. Bucharest.

34

Results and discussions The needle and young shoots anatomy in Pseudotsuga menziesii: The two adaxial resin ducts of Pseudotsuga needle are located in direct contact with epidermis. The needles present a central midvein with variable diameter (31,25-68,7 m) surrounded by a thin endodermis. Each canal is sometimes partial surrounded by sclerenchyma fibers with lignified walls (fig. 1, 2). The resin ducts of young shoots with different diameter are located in cortical zone of stem. The stem presents secondary structure with three annual rings in the moment of analysis. In the cortex, each canal with different diameters (25-137,5 m) is surrounded by secretory cells with thin walls (fig. 3). The anatomical characteristics of needle and young shoots are presented in Tab. I.
Table I Anatomical characteristics of needle and young shoot in Pseudotsuga menziesii
Anatomical characteristics Needle 1. Stomata length (40x) 2. Stomata wide (40x) 3. Number of stomata / mm2 4. Stomatiferous zone wide (10x) 5. Number of stomata strings / on leaf wide (10x) 6. Midvein height, including endodermis (10x) 7. Vascular bundle height (10x) 8. Vascular bundle wide (10x) 9. Resin ducts diameter (10x) 10. Mesophyllum height (10x) 11. Palisade tissue height (10x) 12. Number of palisade cell layers 13. Septate cells height 14. Number of septate cell layers 15. Xylem thickness (40x) 16. Sclerenchyma thickness (40x) 17. Endodermis thickness (40x) 18. Phloem thickness (40x) 19. Adaxial epidermis thick (40x) 20. Abaxial epidermis thick (40x) 21. Cuticle thickness (40x) Young shoot 22. Resin ducts number / leaf section 23. Secretory hair length (10x) 24. Xylem thickness (primary and secondary xylem) (10x) 25. Resin ducts diameter (10x) 26. Cortex length (10x) 27. Number of cell layers in cortex 28. Phloem thickness (primary and secondary phloem) (10x) 29. Annual ring 2004 thickness (10x) Minimum value [m] Ecotype 1 2 19,4 17,8 16,2 13 266 207 206,2 200 4 4 187,5 125 162,5 31,3 331,3 93,75 1 187,5 4 19,4 12,9 8,1 19,44 6,4 4,8 1,62 7 19 375 37,5 162,5 5 125 56,2 268,7 218,7 185,3 31,25 500 143,7 2 281,2 3 40,5 29,1 9,7 23,4 9,7 6,5 6,5 6 31,2 75 25 375 7 12,5 125 Maximum value [m] Ecotype 1 2 32,4 32,4 19,4 24,3 414 335 281,2 312,5 6 7 243,5 168,7 218,7 62,5 375 156,2 3 250 7 43,74 32,4 27,5 59,9 8,1 6,4 8,1 10 125 419 137,5 394 7 187,5 87,5 293,7 231,2 220 68,7 562,5 218,7 3 406,2 6 65 42,1 39 40,5 24,3 11,34 11,3 11 156 156 56,2 500 12 25 187,5 Average value [m] Ecotype 1 2 24,3 18,5 17,8 18,5 239 257,8 5 6 200 143,8 184,4 45 347,9 120,6 2 226,2 5 30,29 25,5 15,4 42,28 7,2 5,6 5,7 8 62,5 400 70,3 255 6 157 70 282,6 220,8 203,5 51,3 534,4 172,2 2 348,2 5 48,3 33,7 22 32,6 14 9 9 8 106,2 106,2 42,5 411 10 20 144,6

35

30. Annual ring 2003 thickness (10x) 31. Annual ring 2002 thickness (10x) 32. Primary xylem thickness (10x) 33. Pith diameter (10x) 34. Epidermis thickness (40x) 35. Subepidermic cell layers number 36. Bark thickness (40x) 37. Number of bark cell layers 38. Pheloderm thickness (40x) 39. Number of pheloderm cell layers

125 75 31,2 375 8,1 2 11,3 4 11,3 2

6,25 437,5 4,8 4,8 1 48,6 3

150 125 50 500 13 2 24,3 4 21 3

31,2 500 9,7 9,7 3 64,8 4

135,4 99 40,6 441,6 10 2 17,4 4 18,3 3

18,7 465,6 7,2 7,2 2 58 4

The needle and young shoots phytochemistry in P. menziesii: The yield and composition of the essential oils of P. mensiesii chemotypes can be seen in Table II, III. Twenty eight components representing between 91.08% and 99.56% of the total oil composition were identified for the needles, and twenty six for young shoots oil representing between 95.5 to 98.97% of the total oil composition. The remaining percentage consists of traces or remained unidentified by chemical searched library. The major components of essential oils in needles and young shoots of P. menziesii in different chemotypes and phenophases are represented by sabinene (14.34-31.73%), terpinolene (12.71-23.23%), terpinen-4ol (7.53-14.82%), -pinene (3.88-9.23 %) in needle oil and -pinene (3.88-9.23%), -pinene (12.6-15.22%), -terpinene (21.05-32.87%), terpinolene (13.7-15.4%) in young shoots. The most significant quantities of different compounds are noticed as follows: in needle oils- -pinene 9.23% (chemotype 1, April), sabinene 31.73% (chemotype 1, March), -pinene 30.28% (chemotype 2, April), -terpinene 5.64% (chemotype 1, October), terpinen-4ol 14.82% (chemotype 1, October) and terpinolene 23.23% (chemotype 2, April) (Tab. II). In the oils of young shoots the most important components are represented by pinene 10.6 % and sabinene 12.3% (chemotype 2, April), -terpinene 32.87% (chemotype 1, January), terpinolene 15.4% and terpinen-4ol 7.12% (chemotype 2, January) (Tab. III).
Table II. Chemical composition of essential oils in needle of Pseudotsuga menziesii
Chemical components October 2005 1.84 3.88 0.15 19.16 7.23 1.23 0.64 1.01 5.64 0.93 1.93 0.18 0.102 8.09 CHEMOTYPE 1 January 2006 1.95 7.31 0.32 24.36 21.22 1.64 0.3 0.69 3.07 0.33 2.12 0.24 0.07 4.79 March 2006 2.00 4.72 0.14 31.73 9.22 1.43 0.41 1.21 4.09 0.32 1.68 0.17 0.13 6.4 April 2006 1.82 3.9 0.15 29.99 7.22 1.37 0.42 1.17 4.62 0.5 1.7 0.25 0.14 7.17 CHEMOTYPE 2 January 2006 1.97 4.44 0.13 29.57 7.94 1.39 0.48 1.15 4.74 0.51 1.73 0.19 0.11 7.14 April 2006 1.81 9.23 0.48 14.34 30.28 1.85 0.37 0.64 3.31 0.29 2.49 0.29 0.09 5.11

-thujen -pinene Camphene Sabinene -pinene -myrcene -phellandrene p-cymene -terpinene Cymol Limonene Eucalyptol Cis -ocimene -phellandrene

36

Trans -ocimene Terpinolene Linalool Fenchol Citronellal Isoborneol Terpinen-4ol -terpineol Cis-piperitol L-terpinen 4-ol propenil-anisol Trans-piperitol Citronellol-acetate Selinenol Total

0.16 20.31 0.23 0.41 0.24 0.11 14.82 0.80 0.26 0.53 0.17 0.95 0 0.08 91.08 %

0.41 16.05 0.34 0.37 0.25 0.23 7.53 0.71 0.29 0.26 0.24 2.27 0.81 0.24 98.41 %

0.3 22.99 0.27 0.41 0.25 0.11 9.03 0.47 0.17 0.47 0.07 1.24 0 0.13 99.56 %

0.35 23.23 0.31 0.52 0.33 0.12 11.49 0.61 0.23 0.38 0.14 1.03 0.07 0.1 99.33 %

0.19 23.06 0.21 0.44 0.28 0.18 10.68 0.55 0.25 0.49 0.09 1.14 0 0.08 99.13 %

0.24 12.71 0.3 0.37 0.23 0.18 9.16 1.13 0.27 0.25 0.32 2.12 0.8 0.15 98.81 %

Table III Chemical composition of essential oils in young shoot of Pseudotsuga menziesii
Chemical components -thujen -pinene Camphen Sabinene -pinene -myrcene -phellandrene -terpinene P-cymene Cymol Limonene Cis -ocimene Trans -ocimene Terpinolene Linalool - terpineol terpinen-4ol -terpineol Propenil-anisol -cubebene Isoeugenol -cadinene Selinenol Cembrene Kauren Norkauren Total CHEMOTYPE 1 January 2006 1.16 7.89 0.25 9.46 12.6 2.52 0.39 21.05 2.39 0.68 3.15 0.29 3.98 15.4 0.84 0.08 7.12 1.07 0.61 1.32 1.05 0.22 0.42 0.27 0.34 1 95.55 % CHEMOTYPE 2 January 2006 April 2006 1.38 1.29 10.47 10.6 0 0 5.65 12.3 14.89 15.22 2.45 2.66 0.304 0.27 24.56 32.87 2.7 1.74 0.59 0.71 3.78 3.13 0.77 0.25 4.48 2.85 14.61 13.7 0 0.13 0.22 0.36 2.77 1.96 0.4 0.22 0.27 0.74 0.12 1.76 0 0.09 0 0.205 0.25 0.39 0 0.28 0 0.36 0 1.023 98.97 % 96.80 %

Conclusions The major components of essential oils in needles and young shoots of P. menziesii in different chemotypes and phenophases are represented by sabinene (14.34-31.73%), terpinolene (12.71-23.23%), terpinen-4ol (7.53-14.82%), -pinene (3.88-9.23 %) in needle

37

oil and -pinene (3.88-9.23%), -pinene (12.6-15.22%), -terpinene (21.05-32.87%), terpinolene (13.7-15.4%) in young shoots. The most significant quantities of different compounds are noticed as follows: in needle oils- sabinene 31.73%, -pinene 30.28% terpinen-4ol 14.82% and terpinolene 23.23%. In the oils of young shoots the most important components are represented by pinene 10.6 % and sabinene 12.3%, -terpinene 32.87% and terpinolene 15.4%. The each chemotype or populations are represented by several compounds varying from month to month in the investigated periods or type of analyzed organs. The chemical composition of essential oils in needle of P. menziesii are represented in Chemotype 1 by 27 compounds in October 2005 and March 2006, 28 compounds in January and April 2006; in Chemotype 2 there are 27 compounds in January 2006 and 28 compounds in April 2006. The chemical composition of essential oils in young shoots of P. menziesii are represented in Chemotype 1 by 26 compounds in January 2006 and Chemotype 2 by 19 compounds in January 2006, respectively 25 in April 2006. Several anatomical and morphological characters were found significant for an analysis of the relationship of Pseudotsuga populations such as resin ducts diameter in stem or needle or number of resin ducts in cortical zone of stem. This original study bring new information in the general knowledge on the morphology and anatomy of cultivated plants from Pinaceae family and fill the gaps regarding the phytochemical composition of oil in Pseudotsuga menziesii, studied for the first time in Romania. REFERENCES
APPLE M., TIEKOTTER K., SNOW M., YOUNG J., SOELDNER A., PHILLIPS D., TINGEY D. & BOND B.J., 2002 - Needle anatomy changes with increasing tree age in Douglas-fir. Tree Physiol., 22:129 136 2. BOGAR G.D. & SMITH F.H., 1965 - Anatomy of seedling roots of Pseudotsuga menziesii. Am. J. Bot. 52, 7: 720-729 3. GAMBLIEL H.A. & CATES R.G., 1995. Terpene changes due to maturation and canopy levels in Douglasfir (Pseudotsuga menziesii) flush needle oil. Bioch. Syst. Ecol. 23, 5: 469-476 4. JIROVETZ L., PUSCHMANN C., STOJANOVA A., METODIEV S. & BUCHBAUER G., 2000 - Analysis of the essential oil volatiles of Douglas fir (Pseudotsuga menziesii) from Bulgaria. Flavour Fragrance J. 15: 434-437 5. JOHNSTON W.H., KARCHESY J.J., CONSTANTINE G.H. & CRAIG A.M., 2001 - Antimicrobial activity of some Pacific Northwest Woods against Anaerobic Bacteria and Yeast. Phytotheraphy Res. 15: 586588. 6. LIPSCOMB B., 1993 - Flora of North America North of Mexico, vol II, University Press Oxford 7. MOERMAN D., 1998 - Pseudotsuga in Native American Ethnobotany. Timber Press, Oregon 8. SNAJBERK K., LEE C.J. & ZAVARIN E., 1974 - Chemical composition of volatiles from cortical oleoresin of Pseudotsuga menziesii. Phytochemistry 74, 13: 185-188 9. SPICER R., GARTNER B.L. & DARBYSHIRE R.L., 2000 - Sinuous stem growth in a Douglas-fir (Pseudotsuga menziesii) plantation: growth patterns and wood-quality effects. Canad. J. For. Res. 30, 5: 761768 10. WAGNER M.R., CLANCY K.M. & TINUS R.W., 1989 - Maturational variation in needle essential oils from Pseudotsuga menziesii, Abies concolor and Picea engelmannii. Phytochemistry 28, 3: 765-770 1.

38

A
epidermis endodermis midvein

secretory duct

Fig. 1 The anatomy of needle (A) and median vein (B) in P. menziesii [ob. 6x / 25x, oc. 12,5x, orig.]
A
phloem cambium xylem II cortex

B
medullary rays

xylem I pith

pith

xylem

xylem II (I II III: annual rings)

Fig. 2 The anatomy of shoots in P. menziesii: cortical cells (A) and central cylinder zone (B) [ob. 6x / oc. 12,5x, orig.]
epidermis epidermis bark secretory cells phelogen

resin ducts from stem

chlorenchyma

cortex

Fig. 3 Pseudotsuga mensiesii needle (A) and cortical resin ducts of stem (B) [ob. 40x, oc. 12,5x, orig.]

39

Analele tiinifice ale Universitii Al. I. Cuza Iai Tomul LIV, fasc. 1, s. II a. Biologie vegetal, 2008

THE ECOPHYSIOLOGICAL REACTION OF SOME VARIETIES OF APPLE TREE, PEAR TREE AND QUINCE TREE TO THE PATHOGENIC AGENTS ATTACK ALEXANDRINA MURARIU *, CORINA GRDINARIU *, ANIOARA STRATU *
Abstract: The research was carried out in 2004, on material from horticultural collections of the Didactical Station Vasile Adamachi within the University of Agricultural Sciences and Veterinarian Medicine of Iai. The investigations were performed on three fruit tree species: Cydonia oblonga Mill., Pyrus communis L., and Malus domestica Borkh. and consisted in revealing the morphological symptoms induced by the attack of the pathogen Erwinia amylovora (Burrill 1882) Winslow et al. The biochemical (contents of water, assimilatory pigments and total mineral) and physiological analyses (intensity of photosynthesis, respiration and transpiration) were carried out on healthy and with different infection degree material of two varieties for each species: Jonathan and Generos (apple tree), Williams and Argesis (pear tree), Mona and De Hui (quince tree). The results revealed the correlation between the physiological alterations and the severity of the pathogenic attack: in weakly attacked (5 8% per tree) Jonathan (apple), Argesis (pear) and De Hui (quince) samples we recorded high contents of water and minerals and an increase of the net photosynthesis intensity compared to the Williams (pear) variety and relatively strong bacterial attack (12%). Large differences between healthy and infected leaves were recorded in Mona (quince) at a bacterial attack of 38%, which induced a decrease in pigment content (20%), net photosynthesis (50%), water content (76%), transpiration (38%), and an increase in respiration (117%). Key words: Cydonia oblonga Mill., Pyrus communis L., and Malus domestica Borkh., Erwinia amylovora, physiological indicators

Introduction Fruit trees can be attacked by a large number of pathogens (viruses, mycoplasmas, bacteria, and fungi), nematodes and insects, which results in important economical losses in orchards and storehouses. Just for the apple tree, the scientific literature describes over 150 diseases and pests, 50 of which are presumably worldwide spread [ 1 ]. In Romania, there are cited three bacterial diseases in apple tree and pear tree and one in quince tree [ 8 ]. With regard to the attack of the bacteria Erwinia amylovora on species of the Family Rosaceae, the first investigation was completed by T. Svulescu (1938). In 1992, two contagion centers were identified in Brila and Mrcineni, and after 1993, the disease spread into almost all the Romanian fruit growing areas [ 4 ]. The most susceptible species belong to the following genera: Cydonia, Pyrus, Malus, followed by Cotoneaster, Pyracantha, and Sorbus.

Al. I. Cuza University, Faculty of Biology, Carol I Bd., no. 20A, 700506, Iai, Romania

40

The plant reaction to the pathogen attack represents the expression of some genetically induced characteristics, and hence, the understanding of the genomic interaction implies biochemical and physiological investigations. Material and methods The research was carried out in 2004, on material from horticultural collections of the Didactical Station Vasile Adamachi within the University of Agricultural Sciences and Veterinarian Medicine of Iai. The research material consisting of fresh leaves was sampled from healthy and infected trees of two varieties of each species: Jonathan and Generos (apple tree), Williams and Argesis (pear tree), Mona and De Hui (quince tree). The material was sampled during the second decade of June, when the trees were at the beginning of fruit growth phenophase, given that the pathogen optimum period comes after blooming. The fresh material was used to determine the water and assimilatory pigments contents, the intensity of photosynthesis, respiration and transpiration. After the inactivation of the enzymes (60C exposure) the material was dried with air and was used to determine the total mineral content, through the analysis of the ash. Additionally, a visual assessment of the Erwinia amylovora attack was made. The strength of the attack was expressed as percentage (Tab. I) and varied with the climatic conditions of the year (extreme heat and drought).

Table I. The strength of Erwinia amylovora attack in 2004 Species Malus domestica Pyrus communis Cydonia oblonga Variety Jonathan Generos Williams Argensis Mona De Hui Strength (%) 5.0 1.0 12.0 7.0 38.0 8.0 Resistance category* Susceptible Resistant Susceptible Very susceptible Very susceptible Susceptible

* after Cimpoe, 2001 and Grdinaru, 2002

41

Results and discussions Even though the primary infection occurs in flowers, it spreads out to leaves, braches and trunk, causing ulcerations [5]. Metabolic dysfunctions appear in leaves due to profound biochemical and physiological alterations such as imbalances of the water content, photosynthesis and respiration, and biosynthesis of complex organic substances. 1. Imbalance of water content The general effect of the bacterial attack was the reduction of the leaf water content in all the varieties (Tab. II). Leaf dehydration in infected varieties compared to the healthy leaves varied from 2% in Generos resistant variety (apple tree) to 76% in Mona very susceptible variety (quince tree). Total mineral content of mature leaves, at fructification, varies as a function of the strength of the bacterial attack. In apple tree and pear tree the bacterial attack induced a 10 45% increase, whereas in quince tree (Mona) the infected leaves contained 27% less mineral elements. The reduction of the leaf area because of necroses and irregularities in stomatal movements occurs concurrently with transpiration decrease in all analysed varieties. The bacterial attack favoured the transpiration reduction in susceptible varieties with 6% in apple tree (Jonathan), 45% in pear tree (Argesis), and 43% in quince tree (De Hui). 2. Imbalance of photosynthesis and respiration During the pathological process, the chloroplasts suffer alterations that lead to fragmentation and loss of chlorophyll due to increased quantities of chlorophyllase. Our research on the content of the assimilatory pigments (total and by forms chlorophylls and carotenoids) revealed the same correlation of the strength of bacterial attack and the quantitative fluctuations in infected and healthy leaves, comparatively. Only in De Hui variety (quince tree), the infected leaves were richer in pigments, which entails the activation of a certain defensive mechanism against the pathogen. The quantity of chlorophylls (a + b) in leaves stays high during fructification, varying between 1.78 3.85 mg in healthy leaves and between 1.43 2.14 mg in infected leaves. The carotenoids showed little differences in all the analysed varieties. Fruit presence represents a stimulating factor for the photosynthesis intensity in healthy leaves (1.24 5.14 mg CO2/g/h). In the infected leaves the simulative effect is weaker (1%) because of the resistance to the bacterial attack (Generos variety pear tree). The obvious water deficit of quince tree infected leaves (Mona variety) diminishes the photosynthesis with 50%. The bacterial attack induces an intensification of the respiration in the host plant because of the redox enzymes and toxins or represents a tendency to counteract the very intense respiration of the pathogenic agent. Our results showed that in all the cases an increase in respiration intensity with approximately 44% in apple tree and pear tree, and with 117% in quince tree (Mona variety).

42

Regarding the total organic substances content, the bacterial attack generally reduces the synthesis of complex organic substances, but intensifies their hydrolysis, which increases the content of soluble forms of sugars and proteins with approximately 121% (Mona variety). All the mentioned physiological modifications could be disease indicators, through their accumulation in cells, tissues and finally organs (Fig.1). Conclusions By correlating the obtained results with the strength of the bacterial attack, we revealed the existence of a powerful relation between the physiological and biochemical modifications and the gravity of the attack of the studied pathogen. In the varieties Jonathan (apple tree), Argesis (pear tree) and De Hui (quince tree) in which the pathogen attack was weak (5 8%) there is a higher content of water and minerals and an increase in the net photosynthesis, in comparison to the variety Williams (pear tree) and situations of relatively strong bacterial attack (12%). Large differences between healthy and infected leaves were recorded in Mona (quince) at a bacterial attack of 38%, which induced a decrease in pigment content (20%), net photosynthesis (50%), water content (76%), transpiration (38%), and an increase in respiration (117%). REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9. BRANITE N., ANDRIE N., 1990 - Soiuri rezistente la boli i duntori n pomicultur. Ed. Ceres. Bucureti CIMPOE GH., BACARCIUC V., CAIMACAN I., 2001 - Soiuri de mr. Ed. I. E.P. tiina, Chiinu. GRDINARU G., 2002. Pomicultura special. Ed. Ion Ionescu de la Brad, Iai MAC HARDY, W.E. 1996 - Apple scab - Biology, Epidemiology and Management, APS Press, Sant Paul, Minnesota MITITIUC M., 1994 - Fitopatologie. Ed. Universitii Al. I. Cuza, Iai OKTEM, Y.E.; BENLIOGLU, K. , 1988 - Studies on fireblight [Erwinia amylovora (Burr.) Winsl. et al.] of pome fruits. Journal of Turkish Phytopathology, 17, 106 SEVERIN V., 1996 - Focul bacterian al rozaceelor (Erwinia amylovora). Ed. Ceres, Bucureti VAN DER ZWET T., KEIL H. L., 1979 - Fire Blight: A Bacterial Disease of Rosaceous Plants. United States Department Agriculture Handbook, 510, Washington DC YOSHKAWA M., 1983 - Macromolecules, recognition and the traggerin of resistance. In :Biochemical Plant Pathology (edited by: Callow J. A.), Plant Physiology, 73: 497- 506

43

Table II. The ecophysiological reaction of some varieties of apple tree, pear tree and quince tree to Erwinia amylovora in 2004
Strength of Attack (%) Transpiration (mg/h) substance 33.14 35.90 33.41 32.85 39.06 43.50 34.20 34.62 36.03 79.93 34.97 34.95 Organic (%) Assimilatory Pigments (mg/g fresh substance) Chlorophyll b Chlorophyll a Carotenoids Photosynthesis (mg CO2/g/h) Dry substance (%) 38.67 42.01 39.84 40.71 43.56 48.72 38.47 41.02 45.36 86.76 41.77 43.37 Net Photosynthesis Gross Photosynthesis 6.22 4.176 2.87 3.681 3.432 1.984 4.992 1.560 1.992 2.240 2.616 3.432

Malus domestica

Jonathan Generos

Healthy 5% Healthy 1% Healthy 12% Healthy 7% Healthy 38% Healthy 8%

61.33 57.99 60.16 59.29 56.44 51.28 61.53 58.98 54.64 13.24 58.23 56.63

62 58 25 14 22 12 198 144 71 44 79 45

5.53 6.11 6.43 7.86 4.50 5.22 4.27 6.40 9.33 6.83 6.80 8.42

3.27 1.36 1..60 1.22 1.97 1.45 1.53 1.42 1.53 1.24 1.65 1.81

0.58 0.26 0.29 0.21 0.45 0.25 0.25 0.24 0.25 0.19 0.24 0.33

0.30 0.18 0.25 0.16 0.21 0.18 0.16 0.14 0.20 0.18 0.20 0.23

4.15 1.80 2.14 1.59 2.63 1.88 1.94 1.80 1.98 1.61 2.09 2.37

5.148 2.624 2.496 3.120 2.496 0.624 3.744 0.624 1.248 0.624 1.872 2.496

1.072 1.552 0.374 0.561 0.963 1.360 1.248 0.936 0.744 1.616 0.744 0.936

Pyrus communis

Williams Argensis

Cydonia oblonga

Mona

De Hui

44

Respiration

Water (%)

Minerals

Species

Variety

Total

90 80 70 60 50 40 30 20 10 0 Healthy Strength Healthy Strength Healthy Strength Healthy Strength Healthy Strength Healthy Strength of Attack of Attack of Attack of Attack of Attack of Attack 5% 1% 12 % 7% 38 % 8% Generos Williams Argensis Mo na De Hu i

Jonathan

Malus domestica Water content ( %)

Pyrus communis Total mineral elements content ( %)

Cydonia oblonga Organic substance content ( %)

Fig.1. The physiological modifications in leaves of studied species

45

Analele tiinifice ale Universitii Al. I. Cuza Iai Tomul LIV, fasc. 1, s. II a. Biologie vegetal, 2008

PHYSIOLOGICAL EFFECTS INDUCED BY PURINIC SUBSTANCES AT CAPSICUM ANNUUM L ELENA CRISTINA ROU , MARIA MAGDALENA ZAMFIRACHE, I. I. BRA
Abstract: The paper present the physiological effects after the treatment with two purinic substances, at Capsicum annuum L. plantles in seedling degree. For the treatment was using 1, 3, 7 trimetil-xanthine (coffeine, theine) and 1,3-dimethil-xanthine (theophyline). As physiological parameters was using quantity of assimilatory pygments, water percentage and dry substance percentage. The treatment has determinated the decrease of quantity assimilatory pygments and the dry substance percentage, comparatively with the control variant. Keywords: purinic compounds, coffeine, theophylline, assimilatory pigments.

Introduction Purinic compounds are substances with purinic nucleus, wich besides physicals characteristic and chemicals properties, may replaced the nitrate bases on DNA and causing different mutations. For these properties, the substances were used in plant amelioration and for the study of aberations in mitotic divisions. The Capsicum genre, is an important genre in Solanaceae family, including more than 245 species, initial in Central America. This plantes containes: capsaicinoids: (0.05 1.5 %), pungent phenolic amides including mostly capsaicin, dihydrocapsaicin and derivatives; carotenoids: carotene, capsanthin; volatile oil: trace; proteins, vitamins A, C, coumarins, steroidal alkaloids including solanidine, flavonoids. Medicamentary properties: stimulant to heart and circulation; peripheral of circulatory insufficiency, intermittent claudication. Low vitality, cold, weak, debility syndromes. Stimulant and tonic to the gastro-intestinal tract; warms digestion, poor appetite, atonic conditions; membranes pale, relaxed, or flabby; impaired secretion; chronic gastric catarrh in absence of inflammation; atonic dyspepsia; gastric flatulence; migraines and cluster headaches. Materials and methods The biological material was represented by leafs grow from plantles of Capsicum annuum L. three varietys: grossum, longum and tetragonum, in seedling degree. The treatment was used on seeds until germination, after that the seeds was hotbed planting. Methods: The treatment The seeds was treated with four concentrations, coffeine and theophylline solutions: V1 (1 variant) = 0.025%, V2=0.05%, V3=0.1% , V4 =0.25% and the control variant with distilled water.

Al. I. Cuza University, Faculty of Biology, Carol I Bd., no. 20A, 700506, Iai, Romania

46

Physiological determinations: The assimilatory pygments dosage according to spectrophotometrical method after acetone extraction; The determination of humidity and dry substance through gravimetrical method bringing to constant weight, at 1000C. Results and discussions The quantity of assimilatory pigments: The substances applied on germination seeds, had establish the modifications of physiological aspects trhough decrease of assimilatory pygments quantity comparatively with control variant. In theophylline treatment case, the 0,1% concentration has determinated the decrease of pygments quantity (exception tetragonum variety), obtaining lesser values comparatively even with maxim concentration used (0,25%). The 0,25% concentration had a different effect only in longum variety case, where it determinated the increasing of pygments quantity comparatively with those treatement variants, without outrun the control variant value (fig.1). The coffeine has determinated the decrease of pygments quantity, the minim value obtaned at 0,25% concentration, with exception of grossum variety, where in this case the pygment quantity breede outruning the control variant value (fig. 1). The quantity of carothenoids pygment, is generally decreased in all treatment variants inclusively at control variant. The treatment with coffeine has a strongly effect in tetragonum variety case, where the quantity of carothenoid pygments decreasee in proportions with the increasing of substance conentration (fig.2). The other varietys, inregistrated an variation of pygments quantity, with decreasing values at 0,025% and 0,1% concentrations, with the difference as whether longum variety case, the minim value is obtained at 0,25% concentration, and at grossum variety the same concentration determinated maxim value of pygments quantity, outruning even the control value . Comparatively, between this two substances applied, the theophylline has a strongly effect as decreasing of pigments quantity comparatively with coffeine effect, and relation proportion doze-effect, with litlle exceptions, the quantity of assimilatory pygments decrease in proportion with increasing the substance concentration applied in treatment. The percent of water and the drying substance: The treatment with both substance, has not determineted very large variation of water percentage, only at tetragonum variety was observed a decrease of water percent at control variant comparatively with the percentages obtained at treated variant. In case of theophylline using, we remarked a decreas of the drying substance percentage comparatively with control variant at longum and tetragonum varieties, unlike of grossum variety were with exception 0,25% concentration, obtained higer percentage comparatively with the control variant (fig. 3).

47

The report of chlorophyll a/b: The analysis of diagrams, showe, that at the treatment with theophylline, the report chlorophyll a/b, varies much comparatively with control variant in longum variety case, were decrease unexpectedly at a minim value at 0,025% concentration, following an easye increasing at 0,25% without outrun the control variant value. At grossum and tetragonum variety the minim values obtaned at V1 and V3 and maxim at V2 and V4 without outrun the control variant value. The coffeine has determinated at grossum and longum varietyes case an increasing of report in proportion with increasing the substance concentration, outrun the control variant. At tetragonum variety, obtained minim values at V2, the value increasing until 0,25% were the control variant value is equalised (fig.4).

Fig.1. The quantity of assimilatory pygments after de treatment with coffeine (left) and theoffylline (right) at Capsicum annuum L

Fig 2. The quantity of carothenoids pygment after the tretment with coffeine (left) and theoffylline (right) at Capsicum annuum L

48

Fig 3. The percent of water and the dry substance after the tretment with coffeine (left) and theoffylline (right) at Capsicum annuum L

Fig 4. The report of chlorophyll a/b after the tretment with coffeine (left) and theoffylline (right) at Capsicum annuum L Conclusions Comparatively, between those two substances used, the theophylline has un strongly effect by decreasing of pygments quantity up to coffeine and abaut the relation of dozeeffect report, with little exceptions, the pygments quantity decrease in proportion with increasing of substance concentration applied in treatment . The report of chlorophyll a/b, in the both substance case, had a general tendency by decreasing of the report at minim treatement variant, then (with exception longum variety), the report increased sometimes outgruning the control variant value. REFERENCES
1. 2. BRA I. I., CMPEANU MIRELA, 2003 - Genetica, Edit. Corson, Iai: 208-209 DIACONU P., 1971 - Ereditatea i factorii mutageni, Edit. Ceres, Bucureti

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Analele tiinifice ale Universitii Al. I. Cuza Iai Tomul LIV, fasc. 1, s. II a. Biologie vegetal, 2008

THE INFLUENCE OF THE Mn2+ IONS EFFECTS ON THE WHEAT (TRITICUM AESTIVUM L.) SEED GERMINATION I. M. RCA , L. FRTI, ANA LEAHU
Abstract: The testing of the Mn2+ ions on wheat seed were conducted in a growth chamber with controlled parameters and the results showed that germination rate and shoot length varies according to the ions concentration. Significant increases of the germination rate at high concentrations were observed as a probable consequence of the seeds enzymatic system activation. The probable biochemical action mechanisms are discussed. Keywords: manganese ions, wheat, germination rate

Introduction The microelements have a complex role in the living structures, with both negative and positive effects, depending inter alias on the nature of the elements, their acting form and also their concentration. The essential role of some elements like Fe, Mg, Mn, Zn, Cu, B or Mo in the plant kingdom or Co, Se, Fe and I in the animal one, is well-known [2]. The role of other microelements is not yet well known. The role of manganese in the unfolding of the oxidative processes at the cellular level and in the functioning of some enzymatic systems [2, 3, and 5] is also a matter of common knowledge; its action unfolds in direct connection with those of the iron [1]. Thus, as bivalent ion, the manganese is part of the superoxyd-dismutase (SOD) from the prokaryotes, an enzyme that annihilates at the mitochondrial level the super oxide anion which induces multiple negative biological effects, due to the formation of hydrogen peroxide, aggressive towards the cells [5]: 2O- 2 + 2H+ H2O2 + O2 Further, the hydrogen peroxide is removed by the metal enzyme: 2H2O2 2 H2O + O2 Among the biological effects of the superoxyd against animals and humans we can enumerate: destruction of the endothelial cells, increase of the micro vascular permeability, formation of some chemotactic factors like leukotriene B4, peroxidation and oxidation of lipids, deterioration of AND singlet chains and formation of peroxynitrite anion (ONOO-), a strong cytotoxic and pro-inflammation agent, according the reaction [5]: O- 2 + NO NOO-2 In the green plants, the photosystem II uses another manganoenzyme that is involved in the water dissociation and the production of molecular oxygen, of protons and neutrons. On the other side, manganese is cytotoxic in high concentrations, those effects were studied especially on the animal kingdom were it produces Parkinson-like effects (rhythmical

tefan cel Mare University, Universitii 13 Street, Suceava, Romania

50

trembling and muscular rigidity) but also effects at the psychical level like behavioural aggressiveness, probably due to the neurotoxic accumulations of manganese in globus pallidus. In fact it is well known that the professional exposure to manganese is a risk factor for the Parkinson disease. The paper studies first of all the manganese effects on the wheat seed germination but also the effects on the wheat growth after the germination. Material and methods Apparatus. The germination was accomplished in a CONVIRON MP4030 - G30 growth chamber with the parameters settled as follows: temperature 200C, humidity 90%, without illumination. Biological material. The wheat samples (Triticum aestivum) we used came from the Magistral variety, 37.5 g/1000 seeds, harvested in 2005 at S.C.D.A Suceava. We measured the germination (FG), according to the standards [4] and also the hypocotyls length (LH) of the germinated plants. Reagents. We used MnCl2, analytical reagent (Chimopar) and bidistilled water. Applied treatments. The wheat seeds were treated with MnCl2 solutions; 7 concentrations were used: 1M, 0,5 M, 0,1 M, 5 x 10-2 M, 10-2 M, 5 x 10-3 M, 10-3 M, 5 x 10-4 M and a blank with distilled water, 3 x 50 seeds for each concentration, the witness included, in Petri dishes on filter paper. Two treatment schemes were used: 1.The seeds were immersed for one hour in the treatment solutions, washed thereafter and placed in Petri dishes with distilled water; 2. The seeds were maintained throughout the germination period in the treatment solutions. After 7 days the number of germinated seeds and the hypocotyls length for the germinated plants were measured. The data obtained was statistically analysed with an application that makes a multiple variance analysis. Results The experiments were fulfilled in order to establish the biological answer of the wheat seed under the influence of mn2+ ions; the results are synthetically showed below (table I and figures 1 and 2). Table I. Germination and hypocotyl length values under the influence of the treatment with mncl2 solutions
concentration of 2+ Mn

witness

1m

0,5 m

0,1 m

0,05 m

0,01 m

0,005 m

0,001 m

measured parameter (average values) germination 1h (%) germination 7 d (%) hypocotyls length 1h (mm) hypocotyls length 7 d (mm) 88,00 88,00 60,29 90,67 100,00 8,78 78,00 100,00 20,35 91,33 98,00 51,72 90,67 88,00 50,02 94,67 92,66 51,02 93,33 91,33 53,09 96,67 94,66 62,50

60,29

1,00

1,00

8,69

26,71

56,82

65,27

64,60

51

120

100

80

60

7 days 1 hour

40

20

0 1M 0,5 M 0,1 M 0,05 M 0,01 M 0,005 M 0,001 M

Fig. 1: Germination analysis


70

60

50

40
7 days 1 hour

30

20

10

0 1M 0,5 M 0,1 M 0,05 M 0,01 M 0,005 M 0,001 M

Fig. 2: Hypocotyl length analysis

52

Conclusions and discussions A first finding is that the stimulation effects of the manganese becomes manifest at high dilution. Thus, beginning with the dilution of 10-1M we observed an approach to the witness of the value of the hypocotyls length, especially for the short-term treatment (fig. 2). The drastically inhibition of the hypocotyls, especially at high concentrations and longterm treatment, could be generated by the secondary toxicity of the manganese ions on the plantlets. On the other hand, analysing the influence of the manganese ions on the germination, an obvious positive reaction at high concentrations and long-term treatments (fig. 1) comes out, so that, for concentrations of 1M and 0.5 M, the germination is practically 100% and for the concentration of 0,1M 98%. At lower values of the concentration the effect of manganese ions on the germination is not so obvious; the obtained differences are not so significant (table I). The probable action mechanism is as follows: the high concentrations and the long action times of the manganese ions on the seed permits the diffusion of the ions through the seed tegument in a sufficient high concentration to permits the activation of the enzymatic systems controlled by the Mn2+ ions. At low concentrations and/or short action times this process does not take place any longer, according to the obtained results. In the case of the action of manganese ions on the hypocotyls, this mechanism is no longer valuable (the plantlet has not a protection teguments like the seed) so that the higher Mn2+ ions concentrations act aggressively and therefore the growth of the wheat embryos is inhibited (fig. 2). REFERENCES
1. 2. 3. 4. 5. CRICHTON, R., 2001 - Inorganic Biochemistry of Iron Metabolism. John Wiley & Sons, Ltd. Chichester New York Weinheim Brisbane Singapore Toronto DAVIDESCU D., DAVIDESCU VELICICA, LCTUU R., 1988 - Microelementele n agricultur. Edit. Acad. Rom., Bucureti, 280 p. Institutul Romn de Standardizare, 1999 - Semine pentru nsmnare. Determinarea germinaiei. SR1634: iunie 1999, Bucuresti KHAN A. A. EDITOR, 1980 - The physiology and biochemistry of seed dormancy and germination. New York, Oxford ROAT-MALONE, ROSETTE M., 2002 - Bioinorganic Chemistry A short course. John Wiley & Sons, Inc., Hoboken, New Jersey

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Analele tiinifice ale Universitii Al. I. Cuza Iai Tomul LIV, fasc. 1, s. II a. Biologie vegetal, 2008

PLANTAGO ATMOSPHERIC POLLINIC SEASON IN THE DANUBE-KRISMURES-TISZA EUROREGION (2000-2004) NICOLETA IANOVICI , I. E. JUHSZ, P. RADISIC, M. JUHSZ, B. SIKOPARIJA
Abstract: The aim of the present study is to compare the pollinic season of Plantago in four aerobiological stations within the Danube-Kris-Mures-Tisza euroregion. The Plantago pollen has generally been considered a minor cause of pollinosis. It is difficult to evaluate the role of Plantago airborne pollen in the pollinosis symptomatology because of the low rate of monosensitive patients. moreover, the allergy to Plantago airpollen is connected with the allergy to Poaceae pollen because of the simultaneous presence of the two types of pollen in the air. Since the onset of the allergy also depends on the airpollen concentration, in this study we intend to present the concentrations of the Plantago pollinic type within the Danube-Kris-Mures-Tisza euroregion. The analysis uses the data obtained during five years of monitoring with the help of four volumetric traps located in timioara (Romania), Szeged (Hungary), Novi Sad and Ruma (Serbia). The Plantago pollen is present in the air from may until august. The highest total annual concentration was recorded at novi sad in 2001 (1326 pg/m3). The highest daily airpollen concentrations seldom exceed 30 pg/m3. The highest daily concentration was 64 pg/m3 (recorded in Novi Sad, 2001). In Szeged and Novi Sad the annual concentrations are on the decrease, while in Timioara they are slightly on the increase. The presence of the pollen in the airplankton was considerably long in Timioara, Novi Sad and Ruma in 2004. Key words: airborne pollen, airplankton of cities, Plantago

Introduction Several authors have already reported that Plantago lanceolata (English plantain, ribwort) belong to the most important pollens and should therefore be included in the test spectrum for allergological examinations. Allergic sensitization to Plantago pollen is fairly common. It was first reported by Bernton (1925) and there is a relatively large bibliography available on the subject; numerous researchers have carried out studies on this pollen type, including Tuft & Blumstein (1937), Serafini (1957), Duchaine & Spapen (1961), Charpin et al. (1962), Izco et al. (1972), Lewis (1977), Saenz de Rivas (1978), Bousquet et al. (1984), Subiza Martin et al. (1986), Watson & Constable (1991). Few studies have tried to identify the allergens in Plantago pollen. In 1980, Baldo et al. detected at least six IgE-binding allergens in the molecular weight range of 10300 kDa. More recently, Dreborg et al. found at least 13 allergens by immunoblotting with one component of 15 kDa with a high degree of specific IgE binding. Only a 30 kDa plantain allergen cross-reactive with the grass group 5 allergens has been identified to date, yet this cross-reactivity shows little or no clinical relevance, as suggested by Asero et al. (2004).

Department of Biology, Faculty of Chemistry-Biology-Geography, West University of Timisoara, Romania Department of Botany, University of Szeged, Hungary Laboratory of Palynology, Institute of Biology, Faculty of Sciences, University of Novi Sad, Serbia

54

These authors also reported in the same article that IgE from monosensitized plantainallergic patients mainly reacted with 17, 19 and 40 kDa allergens. Calabozo et al. found that the 17 and 20 kDa allergens are the unglycosylated and glycosylated forms of the same protein (Pla l 1) respectively, and that the 3236 kDa protein is a dimeric form of the same Pla l 1 allergen. Moreover, the major complex N-glycan of Pla l 1 might be a potential source of cross-reactivity with other glycosylated pollen allergens that could be misleading in terms of false positive diagnoses of allergy to plantain when using natural extracts. Therefore the generation of a recombinant Pla l 1 without the complex N-glycan as part of its structure would be a very useful tool to diagnose patients specifically sensitized to plantain. The glycoprotein Pla l 1 is the major allergen from Plantago lanceolata pollen, which is a common cause of pollinosis in temperate areas.The major allergen of Olea europaea pollen has been found to share sequence similarity to Pla l 1[10]. Material and methods The aerobiological monitoring concerning the pollen content of the airplankton was carried out by using the volumetric method of collecting data. This is the method that most researchers use for qualitative and quantitative studies of airborne pollen and fungi. It implies the repeated succession of two phenomena: the absorption of a constant volume of air and the immediate trapping of the airborne particles as they impact a trapping surface. The traps used were of the Hirst type, model VPPS 2000, Lanzoni. The trap allows for an evaluation of the dynamics of the allergenic atmospheric pollen in the town/ city and its surroundings. In order to regularly collect the data and to get correct statistics, the traps are placed at locations higher than 20 meters, far from industrial areas and barriers which might prevent the circulation of the air currents. The bands inside the volumetric traps were changed weekly. Pollen identification was carried out according to morphological criteria [23, 46]. The pollen concentration is expressed in number of pollen grains per m of air. Our bulletins were published weekly, from February until October on the following websites: Euroregional Polleninformation Service Danube-Kris-Mures-Tisza Euroregion (www.pollinfo.ini.hu) and www.nspolen.com. The DKMT Euroregion includes the following counties: Bcs Kiskun, Csongrd, Jsz-Nagykun-Szolnok, Bks (Hungary), Arad, Timi, Hunedoara, Cara Severin (Romania), and Vojvodina (Serbia).

55

Fig.1. The map of The Danube-Kris-Mures-Tisza Euroregion

Results and discussions The main pollen types with a role in allergic sensitization come from anemophilous plants. The pollination duration matches the symptomatology of clinic manifestations. Spieksma (1991) included Plantago pollen into sporomorphs revealing high level of allergenicity, and these taxa were placed on the list comprising the most important plants which should be considered in pollen monitoring in European research centres. In 2000, the highest total annual concentration was recorded in Szeged (472 PG/m3). In Timioara the concentration reached only half of the result recorded in Szeged, representing 1.72% of the pollinic range of the year 2000. In 2001, the Novi Sad station reported a total concentration of 1326 PG/m3. In Timioara, the concentration of Plantago pollen (148 PG/m3) diminished as compared to the concentration recorded the previous year and represented a mere 0.94% of the annual pollinic range. In 2002, the airpollen concentrations recorded in Novi Sad and Timioara were relatively similar: 797 PG/m3 and 669 PG/m3. In Timioara, the concentration represented 3.28% of the total annual of 20068 PG/m3. In 2003, the Ruma monitoring station started recording data in Serbia, alongside the Novi Sad station. The total annual concentration was highest in Timioara, representing 2.67% of the total annual of 24557 PG/m3. In 2004, the highest concentration (486 PG/m3) was recorded in Timioara. As to the interannual variation (fig.2), the linear regression model shows the decreasing trend in the annual concentrations for Szeged and Novi Sad, while an increasing trend can be noticed for Timioara. We can state that in the DKMT Euroregion the Plantago airpollen is a constant presence in the pollinic range, yet its quantity is moderate. This situation is also present in other parts of Europe, where the annual concentrations do not exceed 10% [20]. In Europe, this pollinic type proved to be dominant in Montpellier, France [43], Athens [1], Bitlis, Turkey [12], London, Leiden, Brussels, Munich, Marseille [41], Madrid [44], Salamanca [22], Northwestern Spain [36], Estepona, Southern Spain [35], Belgium [42, 43].

56

The longest period (8 days) when the number of pollen grains exceeded 30 PG/m3 was reported in Novi Sad in 2001. Another two values over 30 PG/m3 were also registered in Novi Sad: one in 2000 and the second, in 2002. In Szeged and Ruma the threshold value was never exceeded. In Timioara, the sensitization threshold value was exceeded for three days in 2003 and one day in 2002. The stations in the Euroregion did not register excessive values in 2004. The highest daily concentration (fig.3), 64 PG/m3, was registered in Novi Sad in 2001. For Szeged the highest value (22 PG/m3) was registered in 2000; for Timioara the highest value (42 PG/m3) was registered in 2003. These concentrations are exceptions from the usual values throughout the pollinic season. Similar situations were recorded in Spain [20], Poland [50], Turkey [7, 21], Hungary [26], and Croatia [32]. The Plantago species pollinate from May until the end of August. By considering the data which refer to the number of days when the pollen was present in the airplankton, very wide variations were noted: 97 to 107 days in Szeged, 108 to 137 days in Novi Sad, 102 to 138 days in Ruma, and 76 to 136 days in Timioara. In 2004, the presence of the Plantago airpollen until the first decade of September did not correlate with an increase in the daily or annual concentrations. In this paper, we determined the APS (Atmospheric Pollen Season) in accordance with the criteria used by the following authors: Nilsson and Persson (corresponding to 90% of the total pollen catch -the 90% method), Andersen and Torben (corresponding to 95% of the total pollen catch- the 95% method) [25]. The longest pollinic season was that in Ruma and the shortest, in Szeged (tab.I). The duration of the pollinic season and the total annual airpollen concentration were relatively close in Timioara and Novi Sad. Similar variations were recorded in Poland: 95 to 105 days in Rzeszw, 69 to 92 days in Krasne [28], 62 to 98 days in Lublin [50]. In Ankara, Plantago was included in the pollinic group with a maximum pollinating period longer than 15 weeks [27].

Fig.2. Dynamics of annual concentrations of Plantago airpollen (2000-2004)

57

Tabel I. The duration of the atmospheric pollinic season in 2004


2004 First identification of the airpollen Last identification of the airpollen Duration of the atmospheric pollinic season (Nilsson & Persson, 1981); 90% Duration of the atmospheric pollinic season (Andersen, 1991; Torben, 1991); 95% Novi Sad 7V 21 IX 89 days 107 days Ruma 4V 19 IX 108 days 128 days Szeged 15 V 28 VIII 73 days 85 days Timisoara 1V 13 IX 95 days 109 days

Fig.3. Number of Plantago airpollen on the peak day

Several authors report that between 3%-36% of patients are allergic to Plantago, most being polysensitized and, therefore, also allergic to the pollen of other plants, mainly Poaceae. Most patients positive to the skin prick test (SPT) with plantain-pollen extracts are hypersensitive to Poaceae [6, 49; 38], a fact which suggests that at least one allergen in grass and plantain pollens cross-reacts. In a series of 242 consecutive grass-pollen-allergic patients, 71 (29%) were positive in the SPT with plantain-pollen extract. The results suggest the existence of common antigenic epitopes in melon and Plantago pollen, and in melon and grass pollen [18]. The pollen contains epitopes made up of major and minor determining (antigenic, allergenic) groups of amino acids. An individual may be sensitized either to several major and minor epitopes or to a single epitope, be it a minor one. Cross-reactivity phenomena may occur especially because of homology, but also because of the structural mimocrimy of some epitopes, both within the

58

same species and between different species. The cross-reactivity phenomenon may be produced not only by various pollen types, but also by food [34]. In other countries, a relationship between changes in crops and variation in pollen sensitization has been observed [38]. Subiza et al. (1995) reported an average airborne Plantago pollen count in Madrid of 3.6%, with positive skin tests of 53% to P. lagopus pollen, 32% to P. lanceolata and 55% to P. lagopus and/or P. lanceolata. Garcia Gonzales (1995) in Mlaga reported that 8% of patients were allergic to P. lancelata. Recently, several articles have yielded clinical results from tests carried out in several Spanish cities, with sensitization percentages varying between 15% for Mlaga [47]) and 78.24% for Toledo [30]. Regarding patient sensitization, sensitivity was detected in Thessaloniki - Greece to plantain in 194 patients (14.6%) [19]. In the period of plantain pollination, Bryant et al. reported from Sydney that the patients developing asthma symptoms were simultaneously allergic to the plantain allergens. The allergy to plantain allergens was noted in 84% patients with asthma [8]. Of the 629 patients, 459 gave positive SPT results to at least one pollen. No statistical differences were found with respect to gender, habitat (rural or urban) and age. Sensitizations to the different botanical families were as follows: 384 patients were sensitized to Poaceae family pollen, 348 patients to the Oleaceae family, 249 patients to the Plantaginaceae family, 211 patients to the Chenopodiaceae family, 153 patients to the Cupressaceae family, 94 patients to the Platanaceae family, 94 patients to the Compositae family, 80 patients to the Betulaceae family and 27 patients to Urticaceae pollen. Multiple Correspondence Analysis proved the existence of associations among pollen sensitizations, showing that they clustered into two groups: Group I which included Poaceae, Oleaceae, Cupressaceae, Chenopodiaceae and Plantaginaceae and Group II, which included Betulaceae, Platanaceae and Compositae. Pollens of the association Group I do not coincide with those collected in largest numbers in the Madrid atmosphere, since the total annual pollen grain count is highest for Poaceae, followed by Cupressaceae, Platanus, Olea and Plantago [44, 4]. Two distinct behaviors could be observed in Milan: a) a high propensity to develop new respiratory allergies characterized patients allergic to Poaceae (46%), Parietaria (35%), and Betula pollen (37%) whereas b) patients allergic to house dust mite (15%), Ambrosia (15%), Alternaria (11%), Artemisia (22%), and Plantago (20%) showed a much lower propensity to develop new allergies. The new allergens (Ambrosia and Betula) caused 228/256 (89%) new sensitizations detected in the whole study group, included patients allergic to Plantago [2]. It has been observed that is not a clear relationship between the amount of pollen collected in the air and the incidence in allergy people [38]. Percentage of patients displaying reactions to Plantago pollen type according to SPT was 13.33% in Cordoba and 21.42 % in Evora. Sum of daily pollen counts during the study period was 606 in Cordoba and 184 in Evora. Result of correlation between Plantago pollen counts and symptoms suggest a lower incidence of allergic diseases related to pollen in the city of Evora. This fact could be explained by two main causes. Firstly, 73.34 % of patients in the city of Cordoba were aged between 11 and 30 years old, whereas only 50% of patients were included in group. A study performed across

59

Spain reported that the 14-25 age-group was the most affected by pollinosis. Secondly, the population in Evora has maintained a rural lifestyle for a long time whereas in Cordoba people have changed to an urban lifestyle along the last decades. Some studies suggest that acquisition of certain infections or exposure to naturally occurring microbial exposures as encountered in the rural environment could confer protection from allergic diseases. The atmosphere in Evora is by far less contaminated than in Cordoba where the main source of solid material emissions into the air is road traffic, since the city lies on the route for goods transported from the southern to the central regions of the country. Crdoba has recently seen a reduction in the number of ecosystems where grasses are able to grow, due to the expansion of the city and to town-planning changes [38, 11]. Prevalence of allergic reactions to Plantago pollen type in each area was 9.24 in North, 10.15 in West, 11.21 in South, 6.05 in Centre, 12.52 in East. Percentage of patients displaying reactions to Plantago pollen type was: 29.16% (between 1984 -1990) and 33.87% (between 1999 -2001). A positive and significant correlation was observed between monthly pollen indices and antihistamine sales for Plantago [38]. In the sample of population of Sarajevo region during the 2002 year has been investigated on the pollen of weed plant species and Poaceae pollen. In the mixture of pollen weed plant species have been following plants: Plantago lanceolata, Chenopodium album, Solidago gigantea, Artemisia vulgaris and Urtica dioica. 589 have been tested patients by mixture of pollen mentioned plant species and found 115 as a sensitive on pollen alergy; 65 male and 50 females. Even 61 are children to 14 years, or 53% of total sick patients [39]. In Romania, hypersensitization to the mixture of pollen coming from grasses (Artemisia vulgaris, Plantago lanceolata, Rumex acetosella, Urtica dioica) was found in 13.13% of the cases [33] and 2.77% of the patients [16]. A study carried out in France points out that some of the children suffering from atopic dermatitis (9.8%) are also sensitive to Plantago aeroallergens [9]. The number of people allergic to plant aeroallergens has substantially increased in big cities and industrial areas [31]. Thus, monitoring of the pollen counts in the airplankton of cities is of relevant medical importance. The concentration of pollen grains in the air over a city is determined by the individual rhythm of plant pollination, meteorological conditions, composition of local flora, geographic location and kind of urban structure (loose or compact housing, areas with many gardens or with scarce vegetation, industrial areas, agricultural areas or forests) [51]. The higher temperatures in a town can cause a longer vegetative period. The microclimate of towns is characterized by reduced levels of relative air humidity, specific winds, an increased content of aerosols in the air, and a greater frequency of fogs. The generally accepted conclusion is that the participation of arboreal pollen in the pollen fall reflects regional conditions, while the content of pollen of herbaceous plants reflects local ones [20]. Results of this study demonstrate that Plantago seasons occurred at regular intervals between May and August each year; however, individual daily and seasonal Plantago counts were heterogeneous. The start of APS (Atmospheric Pollen Season) was relatively constant for Plantago, while the end of APS showed significant variations. Registered data

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confirm the fact that at least quantitatively Plantago pollen is not an important allergenic factor. Overall, the pollen shedding course of the Plantago in Danube-Kris-Mures-Tisza Euroregion corresponds to that already described during the pollen season in other European areas [23]. Conclusions In the DKMT Euroregion the Plantago airpollen is a constant presence in the pollinic range, but it is moderately represented from a quantitative point of view. The highest annual concentration (1326 PG/m3) was recorded in Novi Sad in 2001, while the lowest annual concentration (134 PG/m3) was recorded in Ruma in 2004. In Szeged and Novi Sad the annual concentrations are on the decrease, while in Timioara they are slightly on the increase. The highest daily concentration (64 PG/m3) was recorded in Novi Sad in 2001. The threshold value (30 PG/m3/day) was seldom exceeded. There is no correlation between the longer presence in the airplankton and an increase in the concentrations. REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. APOSTOLOU E. K., YANNITSAROS A. G., 1977 - Atmospheric pollen in the area of Athens, Acta Allergol, 32, 2:109-117 ASERO R., 2004 - Analysis of new respiratory allergies in patients monosensitized to airborne allergens in the area North of Milan, J Invest Allergol Clin Immunol; 14, 3: 208-213 BALDO B. A., CHENSEE Q. J., HOWDEN M. E. H., SHARPS P. J., 1982 - Allergens from plantain (Plantago lanceolata). Studies with pollens and plant extracts. Int Arch Allergy Appl Immunol; 68:295-304 BELVER M. T., CABALLERO M. T., CONTRERAS J., CABAAS R., SIERRA E., MADERO R., LPEZ SERRANO M. C., 2007 - Associations Among Pollen Sensitizations From Different Botanical Species in Patients Living in the Northern Area of Madrid, J Investig Allergol Clin Immunol, 17, 3: 157-159 BERNTON H. S., 1925. Plantain hay-fever and asthma. JAMA, 84: 944-946 BOUSQUET J., COUR P., GUERIN B., MICHEL F.B. 1984 - Allergy in the Mediterranean area. I. Pollen counts and pollinosis of Montpellier. Clin Allergy; 14: 249 258 BICAKCI A., TATLIDIL S., SAPAN N., MALYER H., CANITEZ Y., 2003 - Airborne pollen grains in Bursa, Turkey, 19992000. Ann Agric Environ Med, 10: 3136 BRYANT D.H., BURNS W.M., LAZARUS L., 1975 - The correlation between skin tests, bronchial provocation tests and the serum level of IgE specific for common allergens in patients with asthma. Clin Allergy, 5: 145-157 CABON N., DUCOMBS G., MORTUREUX P., PERROMAT M., TAIEB A., 1996 - Contact allergy to aeroallergens in children with atopic dermatitis: comparison with allergic contact dermatitis, Contact Dermatitis, 35, 1: 2732 CALABOZO B., DIAZ-PERALES A., SALCEDO G., BARBER D., POLO F., 2003 - Cloning and expression of biologically active Plantago lanceolata pollen allergen Pla l 1 in the yeast Pichia pastoris, Biochem. J., 372: 889896 CARIANOS P., PRIETO J. C., GALN C., DOMNGUEZ E., 2001 - Solid suspected particles affecting the quality of air in urban environments. Bull Environ Contam Toxicol, 67: 385- 391 CELENK S, BICAKCI A., 2005 - Aerobiological investigation in Bitlis, Turkey, Ann Agric Environ Med., 12(1), 87-93 CHARPIN J., AUBERT J., CHARPIN H., 1962 - La pollinose. Monographie sur lallergie. Expansion Scientific Franaise, Paris

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14. DREBORG S., SJGREN I., ERIKSSON N. E., EINARSSON R., 1987 - Selection of patients for biological standardization of mugwort, goosefoot and English plantain pollen allergens extracts/preparations. Allergy; 42: 485-495 15. DUCHAINE J., SPAPEN R., 1961 - Lallergie respiratoire au pollen des Composees en Europe, etudiee par les epreuves de provocation. Int. Arch. Allergy, 18:121-130 16. FAUR A., IANOVICI N., NECHIFOR C., 2003 - Airpalynology Research Implications in Allergic Diseases, Annals of West University, ser. Biol., 3-4: 7-14 17. GARCIA GONZALES J. J., 1995 - Calendario polnico de la ciudad de Mlaga. Prevalencia de test cutneos. Tesis Doctoral. Univ. de Mlaga 18. GARCIA ORTIZ J., COSMES C., MARTIN P., LOPEZ-ASUNSOLO A., 1995 - Melon sensitivity shares allergens with Plantago and grass pollens. Allergy, 50, 3: 269-273 19. GIOULEKAS D., PAPAKOSTA D., DAMIALIS A., SPIEKSMA F., GIOULEKA P., PATAKAS D., 2004 Allergenic pollen records (15 years) and sensitization in patients with respiratory allergy in Thessaloniki, Greece, Allergy. 59, 2:174-184 20. GUTIRREZ A.M., SAENZ C., CERVIGN P., ALCZAR P., DOPAZO A., RUIZ L., TRIGO M.M., VALENCIA R., VENDRELL M., 1999 - Comparative Study of the Presence of Airpollen from Plantago Sp. at Several Locations In Spain, Polen, 10:111-122 21. GUVENSEN A., OZTURK M., 2003 - Airborne pollen calendar of Izmir - Turkey. Ann Agric Environ Med, 10: 3744 22. HERNNDEZ PRIETO M., TOLEDANO L.F, CORTINA R.A,, GONZLEZ I., YGES L.E, BULLN C.A., 1998 - Pollen calendar of the city of Salamanca (Spain). Aeropalynological analysis for 1981-1982 and 1991- 1992., Allergol Immunopathol (Madr)., 26, 5: 209-22 23. IANOVICI N., 2007 - Atmospheric Pollen Season of Plantago in Timisoara, Studia Universitatis Babes Bolyai, Biologia, 52, 2: 63-68 24. IZCO J., LADERO M., SAENZ DE RIVAS C., 1972 - Flora alerggena de Espaa. Anal. Real Acad. Farmacia 38, 3: 521-570 25. JATO V., RODRIGUEZ-RAJO F.J., ALCAZAR P., DE NUNTIIS P., GALAN C., MANDRIOLI P., 2006 May the definition of pollen season influence aerobiological results?, Aerobiologia 22: 1325 26. KADOCSA E., JUHASZ M., 2002 - Study of airborne pollen composition and allergen spectrum of hay fever patients in South Hungary (19901999), Aerobiologia, 18: 203209 27. KAPLAN A., 2004 - Predominant aeroallergen pollen grains in the atmosphere of Ankara, Turkey, Allergy, 59: 670672 28. KASPRZYK I., 2006 - Comparative study of seasonal and intradiurnal variation of airborne herbaceous pollen in urban and rural areas, Aerobiologia, 22:185195 29. LEWIS W.H., IMBER W.E., 1975 - Allergy epidemiology in the St. Louis Missouri area. IV. Weeds. Ann. Allergy 35, 180187 30. MORAL DE GREGORIO A., SENENT SANCHEZ C., CABANES HIGUERO N., GARCIA VILLAMUZA Y., GOMEZ-SERRANILLOS R., 1998 - Plenes alergnicos y polinosis en Toledo durante 1995-96. Rev. Esp. Alergol. Inmunol. Cln. 13, 2: 126-134 31. NILSSON S., PERSSON S., 1981 - Tree pollen spectra in the Stockholm region (Sweden), 1973-1980, Grana, 20: 179-182 32. PETERNEL R., ULIG J., MITI B., VUKUI I., OSTAR Z., 2003 - Analysis of airborne pollen concentrations in Zagreb, Croatia, Ann Agric Environ Med, 10: 107112 33. POPESCU F.D., VIERU M., 2001 - Consideraii privind sensibilizarea la polenul de Artemisia vulgaris, Conferina Naional de Alergologie i Imunologie Clinic "Alergia - o problem de sntate public", Trgu- Mure, 2001, http://www.astmasan.ro/rezumate/22.html [03.08.2007 22:58:24] 34. RADU J.R., 1998. Alergiile reaginice. Imunoterapia specific cu vaccinuri alergenice. Ed. Medical Amaltea, Bucureti 35. RECIO M., DEL MAR TRIGO M., TORO F., DOCAMPO S., GARCIA-GONZALEZ J., CABEZUDO B., 2006 - A three-year aeropalynological study in Estepona (southern Spain). Ann Agric Environ Med., 13, 2:201-207 36. RODRIGUEZ-RAJO F.J., JATO V., AIRA M.J., 2003 - Pollen content in the atmosphere of Lugo (NW Spain) with reference to meteorological factors (19992001). Aerobiologia, 19: 213225 37. SAENZ DE RIVAS C., 1978 - Polen y Esporas. Introduccin a la Palinologa y Vocabulario Palinolgico. H. Blume ed., Madrid

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38. SNCHEZ MESA J.A., BRANDAO R., LOPES L., GALAN C., 2005 - Correlation between pollen counts and symptoms in Cordoba and Evora. J Invest Allergol Clin Immunol, 15, 2: 112-116 39. SARACEVIC E., REDZIC S., TELACEVIC A., 2005 - The frequency of pollen allergy at the population of Sarajevo region during the 2002 year. Med Arh; 59, 4:221-223 40. SERAFINI U., 1957 - Studies on hay fever (with special regard to pollinosis due to Parietaria officinalis). Acta Allergolog. 11:3-27 41. SPIEKSMA F. T. M., CHARPIN H., NOLARD, N., STIX E., 1980 - City spore concentration in the European Economic Community (EEC) in summer weed pollen (Rumex, Plantago, Chenopodiaceae, Artemisia) in 1976 and 1977. Clin. Allergy 10: 319329 42. SPIEKSMA F.T.M., 1990 - Pollinosis in Europe: New observations and developments. Rev Paleobot Palynol, 64: 35-40 43. SPIEKSMA F.T.M., NOLARD N., JAGER S., 1991 - Fluctuations and trends in airborne concentrations of some abundant pollen types, monitored at Vienna, Leiden and Brussels. Grana, 30: 309-312 44. SUBIZA J., JEREZ M., JIMNEZ J. A., NARGANES M.J., CABRERA M., VARELA S., SUBIZA E., 1995 - Clinical aspects of allergic disease. Allergenic pollen and pollinosis in Madrid. J. Allergy Clin. Immunol., 96: 1523 45. SUBIZA MARTIN E., SUBIZA GARRIDO-LESTACHE J., JEREZ LUNA M., 1986 - Arboles, hierbas y plantas de inters alergolgico en Espaa. In: A. Basomba et al. Tratado de Alergologa e Immunologa Clnica, Ed. Luzn, Madrid: 257-366 46. TARNAVSCHI I.T., ERBNESCU JITARIU G., MITROIU RDULESCU N., RDULESCU D., 1980 Monografia polenului florei din Romnia, vol. III, Edit. Acad. Romne 47. TORRECILLAS M., GARCIA GONZALES J.J., PALOMEQUE M.T., MUOZ C., BARCELO J.M., FUENTE J.L. DE, VEGA CHICOTE J.M., MIRANDA A., 1998 - Prevalencia de sensibilizaciones en pacientes con polinosis de la provincia de Mlaga. Rev. Esp. Alergol. Inmunol. Cln. 13, 2:122-125 48. TUFT L., BLUMSTEIN G. L., 1937 - Incidence and importance of tree pollen hayfever with particular reference to Philadelphia and vicinity. J. Allergy, 8: 464 49. WATSON H.K., CONSTABLE D.W., 1991 - Allergenic significance of Plantago pollen. In: D'Amato G, Spieksma FThM, Bonini S, editors. Allergenic pollen and pollinosis in Europe. Blackwell Scientific: 132134 50. WERYSZKO-CHMIELEWSKA E., PIOTROWSKA K., 2004 - Airborne pollen calendar of Lublin, Poland. Ann Agric Environ Med, 11: 9197 51. WERYSZKO-CHMIELEWSKA E., PUC M, RAPIEJKO P., 2001 - Comparative analysis of pollen counts of Corylus, Alnus and Betula in Szczecin, Warsaw and Lublin (20002001). Ann Agric Environ Med, 8: 235240

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Analele tiinifice ale Universitii Al. I. Cuza Iai Tomul LIV, fasc. 1, s. II a. Biologie vegetal, 2008

ARAUCARIA EXCELSA L. VITROCULTURES INITIATION L. POP ; DORINA CACHI*


Abstract: Araucaria excelsa L. is a well-known conifer, mostly used as an indoor ornamental plant. For the initiation of Araucaria excelsa L. vitrocultures, we have studied the reactions of explants, in the presence of different growth regulators, added in the aseptic nutritive media. We have prelevated apexes from a unique plant and used them as biological material. The explants were sterilized and inoculated on BM media, with and without growth regulators. This experiment, which lasted for 90 days, has brought forth the following conclusions: On V0 (control variant BM without growth regulators), the inoculs have presented a very week regenerative capacity; the best medium for elongation of Araucaria excelsa L. was V2 (BM with 2 mg/l BA + 2 mg/l NAA); the most ramifications and buds can be obtained if using V4 experimental variant (BM with 0.5 mg/l NAA + 0.5 mg/l KIN); at any experimental variant the rhysogenesis wasnt observed. Keywords: Araucaria excelsa L., vitroculture, growth regulators, conifer

Introduction Araucaria excelsa L. is a beautiful conifer from Canar and Mader Irelands, and commonly cultured to decorate different indoor and outdoor places. The multiplication of this tree is problematical [4]; therefore the in vitro micropropagation remains a good alternative to be studied. The purpose followed on this experiment was the Araucaria excelsa L. vitroculture initiation and its evolution during 90 days observation. Material and methods For this research we have collected 2 cm length apexes from a 2 m height adult Araucaria excelsa L. plant, which is founded in the glasshouse of University of Oradea. The apexes were prelevated from basal zone of crown and sterilized in 96 alcohol for 1 minute submersion, followed by a Natrium Hypochloride 0.8%, for 15 minutes resubmersion. After these were done, the biological material was washed, more times, in sterile water [1]. In aseptic environment, the resulted pieces were shorted at 1 cm length and so the inoculs were obtained. They were inoculated (fig.1) on 5 different variants of nutritive media. The control experimental variant have consisted in Araucaria excelsa L. apexes, placed on Murashige and Skoog (1962) nutritive standard medium [3], abbreviated here BM (basic medium). The other variants have contained, in addition, different growth regulators.

University of Oradea, Faculty of Science, Universitii Street, no.1, 410087, Oradea, Romania

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inoculation

Murashige and Skoog medium

Original plant 4 cm

1 cm Explant 1 cm

Fig. 1. Making minicuttings and the inoculation process The Murashige&Skoog (1962) [4] mineral medium, which have consisted in macroellements, FeEDTA, Heller microellements, vitamins (B6, B1 and PP), m-inositol, sucrose and agar, was used as basic medium (BM). In this mixture, growth regulators were added, as following: - V0 (control variant) BM without growth regulators; - V1 BM with 2 mg/l BA + 2.5 mg/l IBA; - V2 BM with 2 mg/l BA + 2 mg/l NAA; - V3 BM with 0.5 mg/l KIN + 2.5 mg/l IBA; - V4 BM with 0.5 mg/l NAA + 0.5 mg/l KIN; The growth media were sterilized at 121C, during 30 minutes [2]. After their cooling, in the sterile room, we proceeded to inoculate the minicuttings, one piece per culture recipient, and place them on shelves, at 20-22C, under fluorescent white light, at 1700 lux, with a 16h light/24h photoperiod. Results and discussions The Araucaria excelsa L. vitroplantlets evolution has been observed during 90 days, and the watched parameters were noted and compared. At 30 days after inoculation, the Araucaria excelsa L. vitroplantlets have presented stagnation, their height modification being mostly insignificant (fig.2, fig.4). The highest elongation was founded on V2 medium (BM with 2 mg/l BA + 2 mg/l NAA). No buds or ramifications were observed. The rhysogenesis was missing, too. We didnt find any infection on cultures, but some necroses have occurred in a few growth recipients, at all experimental variant, excepting V3 (BM with 0.5 mg/l KIN + 2.5 mg/l IBA), where all plantlets have survived. The lowest survival level (84.61%) was observed on V0 (control variant), where the growth regulators were missing (fig.3). The general survival percents were pretty good, the best being founded on V3 (BM with 0.5 mg/l KIN + 2.5 mg/l IBA) (fig.3)

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Fig. 2 Araucaria excelsa L. cultures after 30 days from inoculation (V0 (control variant) BM without growth regulators, V1 BM with 2 mg/l BA + 2.5 mg/l IBA, V2 BM with 2 mg/l BA + 2 mg/l NAA, V3 BM with 0.5 mg/l KIN + 2.5 mg/l IBA, V4 BM with 0.5 mg/l NAA + 0.5 mg/l KIN)

% 100
84,61

95,2 91,66

100 93,1

80

60 variants

V0

V1

V2

V3

V4

Fig. 3. The survival percent of Araucaria excelsa L. plantlets, at 30 days after inoculation

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1.2 1.18 1.16 1.14 1.12 1.1 1.08 1.06 1.04 V0 V1 V2 V3 V4

Stalk length

Fig. 4. The vitroplantlets elongation at 30 days after inoculation on aseptic media At 60 days after inoculation the best elongation was observed also on V2 medium (BM with 2 mg/l BA + 2 mg/l NAA) (fig.5, fig.6), but the ramifications were more on V4 medium (BM with 0.5 mg/l NAA + 0.5 mg/l KIN), (fig.5, fig.7).

Fig. 5. Araucaria excelsa L. cultures after 60 days from inoculation (V0 (control variant) BM without growth regulators, V1 BM with 2 mg/l BA + 2.5 mg/l IBA, V2 BM with 2 mg/l BA + 2 mg/l NAA, V3 BM with 0.5 mg/l KIN + 2.5 mg/l IBA, V4 BM with 0.5 mg/l NAA + 0.5 mg/l KIN)

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2,5 2 1,5 1 0,5 0 V0 V1 V2 V3 V4

Stalk le ngth

Fig. 6. The vitroplantlets elongation at 60 days after inoculation on aseptic media

6 5 4 3 2 1 0 V0 V1 V2 V3 V4

Ram ifications

Fig. 7. The vitroplantlets stalk ramification at 60 days from inoculation The 90th day of this experiment has revealed us that in the V2 medium (BM with 2 mg/l BA + 2 mg/l NAA) the vitroplantlets were 30% taller than those from the control variant (BM without growth regulators) (fig.8, fig.9). The most ramifications were found again at V4 experimental variant (BM with 0.5 mg/l NAA + 0.5 mg/l KIN) (fig8, fig.10) The control experimental variant V0 (BM without growth regulators) hasnt manifested any ramification (fig.8, fig.10). No one of experimental variants has manifested rhysogenesis.

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Fig. 8. Araucaria excelsa L. cultures after 90 days from inoculation (V0 (control variant) BM without growth regulators, V1 BM with 2 mg/l BA + 2.5 mg/l IBA, V2 BM with 2 mg/l BA + 2 mg/l NAA, V3 BM with 0.5 mg/l KIN + 2.5 mg/l IBA, V4 BM with 0.5 mg/l NAA + 0.5 mg/l KIN)

3 2,5 2 1,5 1 0,5 0 V0 V1 V2 V3 V4

Stalk le ngth

Fig. 9. The vitroplantlets elongation at 90 days after inoculation on aseptic media

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6 5 4 3 2 1 0 V0 V1 V2 V3 V4

Ramifications

Fig. 10. The vitroplantlets stalk ramification at 90 days from inoculation Conclusions According to this research, the initiation of Araucaria excelsa L. vitoculture is possible, and this is a useful tool for micropropagation of this ornamental conifer specie. On V0 (control variant BM without growth regulators), the inoculs have presented a very week regenerative capacity. No ramifications were observed on standard MS medium (V0) The best medium for elongation of Araucaria excelsa L. was V2 (BM with 2 mg/l BA + 2 mg/l NAA). The most ramifications and buds can be obtained if using V4 experimental variant (BM with 0.5 mg/l NAA + 0.5 mg/l KIN). 90 days seems to be a to short vitroculture period, for rhysogenesis occurrence. These positive results stimulate us to go forward with the experiments concerning Araucaria excelsa L. in vitro micropropagation. Abreviations: MS Murashige&Skoog (1962), BM basic medium, BA benzyladenine; IBA indolilbutilic acid; NAA -naftilacetic acid; KIN kinetin REFERENCES
1. 2. 3. 4. CACHI, C.D., 1987 - Metode in vitro la plantele de cultur. Bazele teoretice i practice, Ed. Ceres, Bucureti CACHI, C. D., SAND, C., 2000 - Biotehnologie vegetal. Baze teoretice i practice. Vol.1, Ed. Mira Design, Sibiu: 272-276 MURASHIGE, T., SKOOG, F., 1962 - A revised medium for rapid growth and bioassays with tobacco tissues cultures. Physiologia Plantarum, 15: 155-159 PREDA, M, 1979 - Floricultura (ed. a II-a), Edit. Ceres, Bucureti: 231-232

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Analele tiinifice ale Universitii Al. I. Cuza Iai Tomul LIV, fasc. 1, s. II a. Biologie vegetal, 2008

AFRICAN VIOLET (SAINTPAULIA IONANTHA L.) EXVITROPLANTLETS ACCLIMATIZATION, IN DIFFERENT TYPES OF SUBSTRATUM ADRIANA PETRU VANCEA , C. F. BLIDAR, ANCA BACIU
Abstract: In micropropagation, the success of cloning depends on the surviving process of the exvitroplantlets after the acclimatization and on the quality of the resulting planting material. For the planting material, coming from vitroculture, to be competitive with the one obtained through classic vegetative multiplying, especially for African violets, it has to be cheaper and better quality. So, the ex vitro planting of the African violet plantlets, directly into greenhouse conditions, is possible and favorable if the substratum in which the acclimatization is being made is efficient and low cost. From the seven types of substratum we tested for exvitroplantlets acclimatization, the most efficient, regarding the post acclimatization survival percent, was 100%, the Top soil substratum, a worm compost, the exvitroplantlets growth spores set on this type of substratum having a very good rooting, these data being meaningful statistically speaking. Key words: Saintpaulia ionantha L., ex vitro acclimatization, substratum

Introduction The proper characteristics for the ex vitro planting substratum for cultivating African violets are the following [4]: consistence 0,75g/cm3, porosity 75,10%, ventilation 19,53%, available water 26,03% and easy available water 23,71%. Analysing different types of substratum mixtures, that had the previously described characteristics, namely: common peat : eucalyptus saw : sand; earthworm soil: vermiculite : sand; pine saw : earthworm soil; peat : vermiculite or other commercial substratum (Eucatex, made of peat and Vida Verde, a commercial substratum especially produced for African violets), in equal proportion each, the authors [4] demonstrated that all variants proved themselves to be proper for cultivating African violets. African violet exvitroplantlets (after removing them from vitro) can be acclimatized successfully to a septic medium, in different types of unconventional substratum [5]. In this sense, it was recommended the use of poplar saw, but never the beech one and no additional thermic treatments [2]. Also, glassy wool may be an ex vitro acclimatization substratum for African violets, as a cheap and good alternative in placing the African violet exvitroplantlets, for future planting in a septic medium [5], and adding the biogel [6], and also the zeolits [7], in the exvitroculture substratum, especially mixed with Top soil, proved to be favorable to this species, the post-acclimatization survival percent being of 100%, and the growth in the adapting to a septic medium period was suitable. In this last type of substratum, the peroxides activity from the rootlets of the African violet exvitroplantlets as a marker of the rizogenesis process determined after 30 days from

University of Oradea, Faculty of Science, Universitii Street, no. 1, 410087, Oradea, Romania Potato Research & Development Station from Trgu Secuiesc, Romania

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their acclimatization to a septic medium, presented very significant positive differences statistically speaking, compared to the control (the rootlets of the plants grown in a natural medium, in a greenhouse) [3], which proves the presence of a intense process of rootlets forming. Material and methods The acclimatized vegetal material consisted by individual propaguls of African violets (Saintpaulia ionantha) witch were detached from vitrobushes, having 3 4 rootlets, 1 1,5 cm waist and 4 5 leaflets with 1 cm foliar limb diameter, kept 60 days in vitroculture, in basal MURASHIGE-SKOOG (1962) medium [1], containing vitamins (HCL thiamine, HCL pyridoxine and nicotinic acid, 1 mg/l each), myo-inositol 100 mg/l, sucrose 20 g/l and agar agar 7 g/l, without grown regulators; pH was adjusted to 5,7, prior autoclaving. The Saitpaulia propaguls were inoculated and cultivated in vitro in glass jar by 200 ml with 12 cm height and 7 cm diameter, in each recipient was shared 50 ml medium. The sterilization of recipients with culture medium was made by autoclaving at 121 C for 25 minutes. The inoculation was operated after medium cooling. After the inoculation, the jars were covered with colourless, transparent, polyethylene folia. The cultures were incubated at irradiance with white fluorescent light with 1700 lx intensity and 16/24 h light photoperiod, at 23 C 2C in the light period and 20C 2C in the darkness period. After 60 days of vitroculture, the agarized medium was removed, by washing with tap water at lab temperature and the vitroplantlets were transferred to the septic medium, in greenhouse. In acclimatization process, was tested the efficiency of seven mixture types, as ex vitro culture substratum, with some characteristics (tab.1), namely: V0 river sand, with fallow soil (3:1); V1 - white peat, with manure and river sand (1:1:1); V2 - perlite with white peat (1:1); V3 - river sand with white peat (1:1); V4 - manure with white peat (1:1); V5 perlite; V6 Top soil. Top soil is commercial name of a substratum made in a biobase (from Stei City, Bihor County), soil resulted from a vital activity of worm cultures growth on vermicompost. To ensure an optimum humidity in the atmosphere around plantlets and to avoid an excessive evapoperspiration, each plantlet was placed under a colorless plastic case, which was pierced in the upper part. This was made to ensure the evacuation of the excessive humidity from the interior vessel. During acclimatization period, all the substratum types were humidified with foul tap weather, and after 14 days from planting, those were weathered with Knop (1865) mineral solution, 100 ml/100 cm2; between 1100 1400 hours, exvitroplantlets were protected, by direct action of sunray, covering them with paper sheet. Both in the moment of the ex vitro vitroplantlets transfer and also after 30 days from the beginning of the acclimatization process, were operated biometrics of the growth indices and was calculated the post-acclimatization survival process, the data being statistically insured. We have calculated the arithmetic average on each sample, at 30 days after their transfer into ex vitro culture substratum was related to values registered at the start moment of acclimatization, these being considered 100%.

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Results and discussions The survival percent registered at 30 days from ex vitro transfer of African violet vitroplantlets, given to initiation moment of acclimatization has noted maximal values to the exvitrocultures that were made on perlite with white peat (1:1) (V2), river sand with white peat (1:1) (V3), perlite (V5) and Top soil (V6), but higher values, meaning 98%, were recorded at the exvitroplantlets lot placed on the white peat with manure and river sand (1:1:1) mixture (V1) or on manure with white peat (1:1) (V4). A little less exvitroplantlets, 95%, were survived on river sand, with fallow soil (3:1) (V0) (fig. 1), but the percent is still a reasonable one. During the acclimatization period, the rizogenesis was influenced by the exvitroculture substratum nature. So, the rootlets length increased given to the moment of planting the exvitroplantlets on soil, registered insignificant increased from statistically point of view (tab. 2), with 17% (fig. 1) percentage efficiency, in case of those placed on river sand with fallow soil (3:1) (V0), those size reaching only medium values of 1,35 cm (tab. 2), to the 130%. On the other hand, for the rootlets of exvitroplantlets cultivated on Top soil (V6), whose length was to the 2,95 cm, the efficiency was to the 130% and the difference against the control was statistically very significant. At exvitroplantlets placed on perlite with white peat (1:1) (V2) to the 30 exvitroculture days was reported a doubling of radicular system length, reaching values of 2,29 cm, related to rootlets size from the moment of vitroplantlets transfer on soil. At the end of acclimatization period, a little less rootlets, just in average of 4,5 samples was counted at the base of exvitroplantlets planted on river sand with fallow soil (3:1) (V0), the increased given to initiation moment being to 34%, and a lot of rootlets, 7,5 samples, was identified similar to the his length at exvitroplantlets planted on Top soil (V6), the efficiency given to control lot was 130%, all the values being statistically very significant (tab. 2). The perlite substratum were identified to be optimal, alone or mixed 1:1 with peat (V2), according to variant V5, when the benefits, in comparison to the control lot, were 66%, respective 106% (fig. 1), same like in the case of rootlets length. We can observe in the figure 1 histograms that the highest benefits of exvitroplantlet growth indicators, in dependence of exvitroculture substratum, were registered to the indexes that express the rizogenesis, these ones touching highest values, 130%, referred to the plantlet rootlets size growth, in Top soil. To those cultivated on mixture of river sand with fallow soil (3:1) (V0), white peat with manure and river sand (1:1:1) (V1), river sand with white peat (1:1)(V3) and manure with white peat (1:1)(V4), total leaflets number was represented by the summing of the leaflets with 0 0,4 cm diameter, those with 0,5 0,9 cm and 1,0 1,4 cm diameter, because exvitroplantlets did not disposed of higher diameter leaflets (tab. 2), those market medium values between 4,6 samples (V0), showing significant differences to the start moment of acclimatization and 6,05 samples (V4), witch represented statistically significant efficiency, percentage expressed by 12% values, respectively 41%, to the initiation of the acclimatization (fig. 1), though the leaflets number with reduced diameter (which is in class dimensions of the 0 - 0,4 cm and 0,5 0,9 cm), registered statistically significant minuses to 47% (V0), at the end of acclimatization period. Only those exvitroplantlets, cultivated on

73

the substratum containing perlite (V2 and V5) and those on the Top soil (V6) presented leaflets with an diameter between 1,5 1,9 cm (fig. 1), total leaflets number to these lots reaching values of 7,5 and 8,54 samples, to the first two lots, and to the final lot the efficiency was 59% and 92%, respectively 9,1 samples and the higher increase of 109%, the difference between those values, given to the acclimatization initiation, being very significantly, statistically point of view (tab. 2). The efficiency of the total leaflets number, registered to the end of the acclimatization period, given to the initiation moment, represent those leaflets which ex vitro new formed. Conclusions Analyzing the results of the present experiment, we can state that, in the case of the African violets, all tested substratum led to a good or even very good post acclimatization survival, especially remarked was the mixture of perlite with peat, in a report of 1:1 (V2), or perlita (V5) and Top soil, as independent substratums from rooting, in this last case the growth parameters being the highest. REFERENCES
1. MURASHIGE T., SKOOG F., 1962 - A revised medium for rapid growth bioassays with tobacco tissue cultures. Physiol. Plant., 15: 473 - 497 2. PETRU - VANCEA ADRIANA, 2005 - The acclimatization of African violet exvitroplantlets in substratum consisting in sawdust. Analele Univ. Oradea, fasc. Biol., t. XII: 119 123 3. PETRU VANCEA ADRIANA, CACHI C. DORINA, IPO MONICA, 2004 - Activitatea peroxidazic n rdciniele vitro- i exvitroplantulelor de crizanteme, violete africane i de Cymbidium. An. SNBC, t. IX, 1., cap. III Biologie celular vegetal: 392 395 4. SALVADOR E.D., MINAMI K., JADOSKI S.O., 2003 - Evaluation of different substrates on African violets (Saintpaulia ionantha Wendl.) growth. International Symposium on Soilless Culture and Hydroponics. Acta Horticulturae, 697, www.actahort.org/books/697 5. VANCEA ADRIANA, CACHI C. DORINA, 2002 - Aclimatizarea vitroplantulelor de Saintpaulia ionantha, prin plantarea acestora pe substraturi neconvenionale. n: Lucrrile celui de al X-lea Simpozion Naional de Culturi de esuturi i Celule Vegetale. (eds. Cachi C. Dorina, Rakosy T. Lenua, Ardelean A.). Edit. Risoprint, Cluj- Napoca: 310 315 6. VANCEA ADRIANA, CACHI C. DORINA, 2002 - Utilizarea biogelului n substratul destinat aclimatizrii vitroplantulelor de saintpaulia ionantha la mediul septic de via. n: Lucrrile Simpozionului tiinific 90 de ani de nvmnt agronomic universitar la Iai. CD, Agrosoft U.S.A.M.V., Iai 7. VANCEA ADRIANA, CACHI C. DORINA, FENCE DANIELA, 2003 - Utilizarea zeolitului ca substrat de aclimatizare a vitroplantulelor la mediul septic de via. n: Lucrrile celui de al XI-lea Simpozion Naional de culturi de esuturi i celule vegetale. (eds. Cachi C. Dorina, Ardelean A. Edit. Daya, Satu-Mare: 212 224

74

V0 250% 200% 150% 100% 50% 0% Survival percent

V1

V2

V3

V4

V5

V6

I zi

Rootlets length

Rootlets number

Propaguls number

Total Leaflets Leaflets number of number with number with leaflets 0-0,4 cm 0,5-0,9 cm diam. diam.

Fig. 1. The survival percent and growing of the African violet (Saintpaulia ionantha L.) exvitroplantlets, at 30 days from their ex vitro transfer and planting on different substratum types: V0 river sand with fallow soil (3:1); V1 - white peat with manure and river sand (1:1:1); V2 - perlite with white peat (1:1); V3 - river sand with white peat (1:1); V4 - manure with white peat (1:1); V5 perlite; V6 Top soil, expressed in percentage values against the parameters registered at the level of exvitroplantlets biometred in first days of acclimatization, which were considered 100%

Table I. Qualitative characteristics of some substratum type witch were used by us in African violet exvitroplantlets acclimatization, obtained from chemical analyses made at Chemical and Agrotechnical Research Center Oradea
Determinations Substratum type M eather H
2 2O5

mid. aO gO g/100

ineral residue %

river sand + fallow soil, 3:1 white peat + manure + river sand, 1:1:1 perlite + white peat, 1:1 river sand + white peat, 1:1 manure + white peat, 1:1 Top soil

0 ,1 ,4 ,3 ,2 ,1 0 2 6 9 6 2 5 - 30 0 1 3 1 - 12 7 8 0 - 60 3 3 1 8 5 2 2 5 0 - 50 - 15 0 8 4 1 9 5 0 ,13 0 ,14 0 ,15 0 ,18 0 ,25 0 ,14

,9 Normal values

Table II. Statistic processing of biometric measurements done at the level of African violet extrovitroplantlets (Saintpaulia ionantha L.), at 30 days of their ex vitro transfer and their plantation into different substratum type, namely: V0 river sand with fallow soil (3:1); V1 - white peat with manure and river sand (1:1:1); V2 - perlite with white peat (1:1); V3 - river sand with white peat (1:1); V4 - manure with white peat (1:1); V5 perlite; V6 Top soil Type V0 (control) Rizogenesis Caulogenesis
Biometrics Rootlets length Statistic count 1 2 Rootlets number Propaguls number Total number of leaflets Leaflets number with 0-0,4 cm diam. 6 Leaflets number with 0,5-0,9 cm diam. 7 Leaflets number with 1,0-1,4 cm diam. 8 Leaflets number with 1,5-1,9 cm diam. 9

76

Sx p

1,35 0,08 ns 2 1,59 0,06 *** 2,29 0,06 *** 1,95 0,03 *** 1,81 0,03 *** 2,46 0,07 *** 2,95 0,05 ***

4,50 0,11 *** 3 4,70 0,10 *** 5,65 0,11 *** 5,20 0,09 *** 5,10 0,12 *** 7,40 0,11 *** 7,50 0,11 ***

1,00 0,00 ns 4 1,00 0,00 ns 1,00 0,00 ns 1,00 0,00 ns 1,00 0,00 ns 1,00 0,00 ns 1,00 0,00 ns

4,60 0,15 * 5 5,20 0,09 *** 7,55 0,22 *** 5,80 0,09 *** 6,05 0,05 *** 8,45 0,11 *** 9,10 0,16 ***

0,95 0,05 *** 6 1,20 0,09 *** 1,90 0,07 ** 1,40 0,11 *** 1,75 0,10 ** 2,10 0,07 ns 2,10 0,07 ns

1,90 0,07 ** 7 2,05 0,09 ns 2,50 0,11 ns 2,25 0,12 ns 2,30 0,10 ns 2,65 0,11 * 2,85 0,08 ***

1,75 0,10 *** 8 1,95 0,09 *** 2,30 0,10 *** 2,15 0,13 *** 2,00 0,00 *** 2,55 0,11 *** 2,70 0,10 ***

0,00 0,00 ns 9 0,00 0,00 ns 0,95 0,05 *** 0,00 0,00 ns 0,00 0,00 ns 1,15 0,08 *** 1,45 0,11 ***

Type V1 1

Sx p

Type V2

Sx p

Type V3

Sx p

Type V4

Sx p

Type V5

Sx p

Type V6

Sx p

Note: x S x (average standard deviation of the average), s (standard deviation), S% (variability coefficient), p (significance limit of the difference against the control): ns insignificant, * - significant, ** - distinctively significant, *** very significant

77

Analele tiinifice ale Universitii Al. I. Cuza Iai Tomul LIV, fasc. 1, s. II a. Biologie vegetal, 2008

ULOCLADIUM ATRUM PREUSS - BIOLOGICAL CONTROL AGENT OF GREY MOULD (BOTRYTIS CINEREA PERS.) OF CROPPED PLANTS TATIANA EUGENIA ESAN , J. KHL , WILMA M. L. MOLHOECK
Abstract. Ulocladium atrum Preuss is a promising candidate as biological control agent against Botrytis cinerea Pers. from different plants: onion, lily, geranium, cyclamen, kiwifruit, strawberry, grapevine, etc. During year 2000 the authors have been worked together in the Plant Research International Wageningen, The Netherlands, for the EU project 1898 - BIOSPORSUPPRESS - The biological control of air-borne necrotrophic plant pathogens by suppression of spore production -, leaded by PhD J. Khl. The objectives of that research were: (1) Testing culture media for Ulocladium atrum (isolate 385 and isolates from soil organic fragments and necrotic leaf fragments) in order to select the most favourable one for detecting the fungus in soil organic material and necrotic leaf samples; (2) Detecting of U. atrum incidence in necrotic leaf and soil samples (soil organic fragments soil suspensions) collected from U. atrum treated strawberry fields plated on ARSA medium and (3) Reisolation and purification of U. atrum from colonies developing on organic material or soil dilution plates from all the experimental plots, obtaining of U. atrum isolates for their characterization in comparaison with the isolate 385 used in the experimental field plots as a biocontrol agent against the grey mould of strawberry. Key words: biological control (biocontrol) of Botrytis-diseases, Ulocladium atrum biological control agent, strawberry, culture media.

Introduction Ulocladium atrum Preuss (Mitosporic fungi/ Conidial Ascomycetes/ Ord. Hyphomycetales/ Fam. Dematiaceae) (fig. 1) is an attractive candidate for applications as a biocontrol agent in the field as well as in greenhouses for suppressing sporulation of Botrytis cinerea and other Botrytis spp. of different crops, like: onion, lily, geranium, cyclamen, kiwifruit, strawberry, grapevine, etc. [1], [2], [4], [5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [17], [18], [19], [20], [21], [22]. For application of the antagonistic fungus as a bioproduct it is necessary to be studied his action on the environment.

University of Bucharest, Faculty of Biology, Aleea Portocalilor, no. 1-3, 060101, Bucharest 35, Romania Plant Research International, Bornesesteeg 65, 6708 Wageningen, The Netherlands

78

10m Fig. 1 Ulocladium atrum Preuss: conidiofores and conidia (after David, 1995 [3])

The objectives of this research, in the frame of the international project 1898 BIOSPORSUPPRESS - The biological control of air-borne necrotrophic plant pathogens by suppression of spore production - were: 1. Choice of culture media for Ulocladium atrum (isolate 385 and isolates from soil organic fragments and necrotic leaf fragments) in order to select the most favourable one for detecting the fungus in soil organic material and necrotic leaf samples; 2. Incidence of U. atrum in necrotic leaf and soil samples (soil organic fragments soil suspensions) collected from U. atrum treated strawberry fields plated on ARSA medium. Material and methods Biological materials used for this investigation consisted of: strawberry necrotic leaves only collected from the overwintering trial (1999-2000) and soil organic fragments obtained from soil samples collected on 16 February 2000 from 8 field experiments performed between 1997 and 2000 (table 1) in the biocontrol group of Plant Research International Wageningen.

79

year 1997-1

Table I. Samples from strawberry trials 1997-2000 treatments plot numbers 1. 2. 3. 4. 1. 2. 3. 4. 1. 2. 3. 4. 5. 1. 2. 3. 4. 5. 1. 2. 3. 4. 5. 1. 2. 3. 4. 5. 1. 2. 3. 4. 5. Control Ua weekly during season fungicides Ua weekly during flowering Control Ua weekly during season fungicides Ua weekly during flowering Control Ua monthly sept.(IX)-april (IV) (a) removal senescing/dead leaves Ua planned during flowering; not performed Ua monthly sept.(IX)-june (VI) (b) Control Ua at planting; weekly from bud to flowering; twice weekly during flowering fungicides Ua twice weekly at flowering removal senescing/dead leaves Control Ua at planting; weekly from bud to flowering; twice weekly during flowering fungicides Ua twice weekly at flowering removal senescing/dead leaves Control (Tween water) fungicides Ua fortnightly from winter to flowering; twice weekly during flowering Ua twice weekly during flowering removal senescing/dead leaves Control Tween water every second day during flowering Ua 5x105 ml-1 every 4th day during flowering Ua 5x105 ml-1 every 2nd day during flowering fungicides 2,5,10,13,17 3,8,11,15,18 4,7,12,16,19 1,6,9,14,20 1,6,11,13,20 4,7,10,16,18 3,8,9,15,19 2,5,12,14,17 1,8,15,19 3,7,12,17 4,10,11,18 2,6,14,16 5,9,13,20 1,8,15,19 3,9,12,17 4,6,11,18 2,10,14,16 5,7,13,20 1,6,13,20 4,8,15,19 3,9,14,18 5,10,11,17 2,7,12,16 1,8,15,19 3,9,12,17 4,6,11,18 2,10,14,16 5,7,13,20 1,8,15,20 2,6,13,17 3,7,12,18 4,10,11,16 5,9,14,19

date of collecting1)
18.07.2000

1997-2

12.07.2000

1997/ 1998-a 19971998-b 1998-1

28.06.2000

27.06.2000

1998-2

26.06.2000

1998/ 1999

14.06.2000

1999

08.06.2000

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30.05.2000 1999/ 1. Control 3,10,14,16 2. Ua December 1999 (a) 2,6,12,18 2000-a 3. Ua January 2000 (b) 5,9,13,20 2000-b 4. Ua March 2000 (c) 1,7,15,19 2000-c 5. Ua May 2000 (d) 4,8,11,17 2000-d Soil samples plus vegetation cover, 5 sub-samples per plot, collected in trays (40 x 30 x 8 cm) - left in field - on 16 February 2000, except for trial 8 (1999-2000); 1) Date of collecting material for laboratory work from the trays with soil put aside since 16 February. Samples of the 1999-2000 experiment were taken directly from the field. Legend: Performed laboratory tests

1. Choice of culture media for growth of Ulocladium atrum. Isolate 385 of U. atrum has been tested on different culture media: ARSA (Alternaria radicina selective agar), WA (water agar), MA (malt agar) and PDA (potatodextrose-agar). ARSA medium was prepared in two parts: part A consisted of 16.0 g agar 1.0 g KH2PO4, 1.0 g KNO3, 0.5 g KCl, 0.5 g MgSO4, and 500 ml H20; and part B consisted of 5.0 g sodium polypectate (Sigma P-1879) and 500 ml of H20. Parts A and B were autoclaved separately, cooled to 50C, and combined. Subsequently 50 mg chlortetracycline HCl (Sigma C-4881), 50 mg streptomycin sulphate (Sigma S-6501), 4 mg dicloran (5 mg Botran 75PW), 100 mg triadimefon (200 mg Bayleton 50WP), 106 mg thiabendazole (0.25 g Mertect 340-F), and 10 mg 2,4-D (Sigma D-8407). The herbicide 2,4-D was added from a stock solution that consisted of 200 mg 2,4-D dissolved in 5 ml of hot ethanol and added slowly to 100 ml H20 [16]. WA (water-agar) medium was prepared from 15 g agar for 1 l of distilled water (Tuite, 1969, p. 75), 50 mg streptomycin sulphate (Sigma S-6501) was added to control bacteria. MA (malt extract) medium was prepared from 25 g malt extract and 20 g agar for 1 l distilled water [23], 50 mg streptomycin sulphate (Sigma S-6501) was added to control bacteria. PDA (potato-dextrose-agar) was prepared from 39 g Oxoid PDA powder suspended in 1 l distilled water. Observations on U. atrum 385 have been performed by daily measuring the colony diameter 3 to 17 days after inoculation (table 2), till the whole surface of the culture medium has been covered by the fungal colony.

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Soil organic material for plating has been obtained using the following method: COLLECTING SOIL SAMPLES FROM THE FIELD (obtaining a composite sample consisting of 7 cores of soil, 15 mm , 5 cm deep, collected from different points of each plot/tray) WEIGHING SOIL SAMPLES (70 - 100 g each) WASHING SOIL SAMPLES (with running with tap water on two nematological sieves with mesh 0.5 and 1.0 mm diameter) COLLECTING SOIL ORGANIC FRAGMENTS From the sieves in a tube containing 10 ml sterile water FIRST WASHING with STERILE WATER (10 ml sterile water per tube) SECOND WASHING with TWEEN 80 1% (9 ml sterile water + 1 ml Tween 80 per tube) COLLECTING SOIL ORGANIC FRAGMENTS PLATING SOIL ORGANIC FRAGMENTS ON CULTURE MEDIA (5 fragments/ plot/ 5 replications) Incidence of U. atrum on leaf and soil organic fragments (0.5 and 1.0 mm sieves) has been established after plating the biological material in replicates on discs of the three media: MA WA and ARSA (10 discs of 1.0 cm / Petri dish/ replicate) [24], [25]. Observations have been performed after 14 days under the stereomicroscope. 2. For checking/estimating incidence of U. atrum on necrotic leaf and soil organic fragments the following methods were used: Leaf fragments (about 1 mm diameter) were detached from necrotic leaves collected from the field of trial 8 (5 leaflets/plot). They have been plated directly on Petri plates with culture media, without washing or sterilizing.

82

Soil organic fragments have been plated in the same way (5 pieces / plot/ 5 replicates) (fig. 2).

Fig. 2 - Soil organic fragments plated in Petri plates with culture media Soil suspensions have been prepared from each soil sample. One ml of a suspension of 1 g soil in 9 ml sterile water has been plated on each of 3 Petri plates ( 9 cm ) containing culture media (ARSA) (fig. 3).

Fig. 3 Laboratory preparing of suspensions from soil samples collected from different strawberry experiments

83

3. Isolation/ reisolation and purification of Ulocladium atrum-like fungi from colonies developing on organic material or soil dilution plates has been attempted for all plots sampled (3-5 per plot). Isolates were randomly transferred to slants or Petri dishes (5 cm diameter) with PDA and incubated at 18oC. If necessary for purification a second or third transfer was done. Selection of obtained isolates has been performed by checking them under the stereomicroscope. Purified isolates are stored in a cold room at 4oC. Results and discussions 1. Growth of Ulocladium atrum (isolate 385 and isolates from soil organic fragments and necrotic leaf fragments) on different culture media. As presented in table 2 and in fig. 3a and 3b, the development of U. atrum 385 was different on the three tested culture media. Table II. Colony diameter (cm) of Ulocladium atrum 385 colony on different culture media after 3-17 days of inoculation
Culture media MA WA ARSA 3 1.500 1.150 0.225 4 1.925 1.500 0.225 5 2.420 2.800 0.350 6 2.675 3.625 0.575 Days after inoculation: 7 9 12 2.875 3.175 3.200 5.000 6.900 7.900 0.725 1.000 1.150 13 3.450 8.050 1.200 14 3.450 8.275 1.360 17 3.950 8.320 1.450

Fig. 3a - Diameter (cm) of Ulocladium atrum colony on different culture media

10 8 Colony diameter (cm) 6 4 2 0 0

MA WA ARSA

10 Incubation time (days)

15

20

84

The fastest growth of U. atrum mycelium has been noticed on WA medium the maximal value of the colony diameter (8.050-8.320 cm) being recorded after 13-14 days of incubation. Contrary on the same culture medium a poor sporulation has been recorded.

3b1

3b2

3b3

Fig. 3b - Growth of Ulocladium atrum on different culture media (after 70 days): 3b1. on ARSA medium; 3b2.on Malt Agar; 3b3. on Water agar media On MA culture medium the U. atrum isolate 385 had a moderate growth as well as a moderate sporulation. Colony diameter recorded after 14 days of incubation was 3.450 cm. The lowest value of U. atrum growth has been recorded on ARSA medium with a colony diameter of 1.360 cm after 14 days of incubation. On this medium U. atrum sporulation was abundant. Based on these first observations necrotic leaf and soil organic fragments have been plated on the three culture media under test in order to check incidence of U. atrum (tables 3-4). Table III. Incidence of Ulocladium atrum (%) in organic soil fragments (0.5 and 1.0 mm sieves) from strawberry plots treated with U. atrum 1 month before soil sampling Experiment 1999-2000 ( i ) 1999-2000 ( ii ) Culture media MA WA ARSA MA WA ARSA Incidence of U. atrum % 0.5 mm 1.0 mm 20.83a 17.50b a 19.17 5.83c a 33.33 31.67a a 10.0 6.67a a 6.67 1.33b a 8.00 8.00a

The data on the incidence of U. atrum on soil organic fragments collected from the 0.5 and 1.0 mm diameter sieves an effect of culture media.

85

For the soil organic particles from the 1.0 mm sieve the highest incidence of U. atrum was noticed on ARSA medium (31.67% in experiment i and 8.00% in experiment ii). The incidence on MA culture medium was second (17.50% in experiment i and 6.67% in experiment ii). The lowest incidence of U. atrum was on WA medium: 5.83% in experiment i and 1.33% in experiment ii. No significant differences were found with particles from the 0.5 mm sieve. On necrotic leaf fragments plated on the three culture media no difference was found (table 4). Table IV. Incidence of Ulocladium atrum (%) in leaf fragments from strawberry plots treated with U. atrum 1 month before soil sampling Experiment 1999-2000 ( i ) Culture media MA WA ARSA Incidence of U. atrum (%) 38.23a 29.32a 38.23a

U. atrum grew and sporulated well on ARSA medium. On the other media - MA and WA - saprophytic fungi and bacteria quickly developed. This did not permit the estimation of U. atrum incidence on necrotic leaf pieces, soil organic fragments and in soil suspensions. 2. Incidence of U. atrum on necrotic leaf and soil samples (soil organic fragments soil suspensions) collected from U. atrum treated strawberry fields and plated on ARSA medium. Incidence of U. atrum on organic soil fragments from strawberry plots treated with U. atrum 1-37 months before soil sampling was about twice as in the controls (table 5, fig. 4). The lowest incidence of U. atrum (30-45%) was recorded in the U. atrum treated plots from the experiment 1999-2000 and 1997-1. Higher incidence of U. atrum (67-91%) was recorded in the other experiments performed from 1997 until 1999. Table V. Incidence of Ulocladium atrum (%) in organic soil fragments from strawberry plots treated with U. atrum 1 37 months before soil sampling Experiment 1997-1 1997-2 1997-1998 1998-1 Time span between last U. atrum treatments and sampling (months) 35 37 22 24 23 Incidence of U. atrum (%) untreated 21 14a 38a 38a 74a
a1)

Ua treated 39a 44b 72b 87c 85b

a b

86

24 62a 91b a 13 55 86b a 11 41 67b a a 6 19 36a a b 4 19 34a a c 2 19 45a a d 1 19 30a 1) Figures for U. atrum-treatment in the same time with same latter are not different from the control 1998-2 1998-1999 1999 1999-2000 -

Fig. 4 Incidence of Ulocladium atrum (%) in organic soil fragments Incidence of U. atrum in soil suspensions from strawberry plots (cfu/ml) treated with U. atrum 1-37 months before soil sampling was higher in the treated plots in comparison with the controls (table 6, fig. 5). The incidence of U. atrum was lowest in the 1999-2000 trial (227-326 cfu/ml soil suspension); the maximum was 1033 cfu/ml soil suspension in the 1998-1 trial. The difference between U. atrum treatment and control was not significant at low levels of incidence.

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Table VI. Incidence of Ulocladium atrum (cfu/ml) in soil suspensions from strawberry plots treated with U. atrum 1 37 months before soil sampling Experiment Time span between last U. atrum treatments and sampling (months) 35 37 22 24 23 24 13 11 6 4 2 1 Incidence of U. atrum (cfu/ml soil suspension) untreated Ua treated 316.7a1) 346.7a 669.5b 669.5 742.5a 316.7a 341.7a 548.3a 168.3a 168.3a 168.3a 168.3a 418.7a 834.7b 765.2b 934.6c 1032.5b 725.0b 570.0a 810.0b 226.7a 262.5a 327.5a 276.5a

1997-1 1997-2 1997-1998 1998-1 1998-2 1998-1999 1999 1999-2000 -

a b

1)

a b c d

See table 5

Fig. 5 - Incidence of Ulocladium atrum (cfu/ml) in soil suspensions

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Incidence of U. atrum on necrotic leaf fragments from strawberry plots treated with U. atrum 1-6 months before sampling was twice to 3 times as high in the treated plots in comparison with the controls (table 7, fig. 6). Incidence tended to decrease with increasing time span between the last U. atrum treatments and sampling (1-6 months). Table VII. Incidence of Ulocladium atrum (%) on necrotic leaves from strawberry plots treated with U. atrum 1 - 6 months before soil sampling Experiment Time span between last U. atrum treatments and sampling (months) 6 4 2 1 Incidence of U. atrum (cfu/ml soil suspension) untreated Ua treated 27 a1) 27a 27a 27a 50ab 62bc 72bc 85c

1999-2000 -

a b c d 1) See table 5

Fig. 6 - Incidence of Ulocladium atrum (%) on necrotic leaves Incidence tended to decrease with increasing time span between the last U. atrum treatments and sampling (1-6 months).

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Conclusions 1. From the three culture media (MA, WA, ARSA) tested for use to detecting Ulocladium atrum in necrotic leaf and soil organic fragments as well as in soil suspensions from U. atrum treated strawberry plots, ARSA medium (Pryor et al., 1994) proved to be the proper one for development of the fungus. MA and WA media were covered by other saprophytic fungi and bacteria, that did not permit the estimation of U. atrum incidence on the biological material collected from the field. 2. Incidence of U. atrum on all material (soil organic fragments, soil suspensions, necrotic leaf pieces) and in all experiments (1997-2000) was higher in the treated plots in comparison with untreated ones. U. atrum incidence was lower for semples from the oldest and the most recent field experiment than for the experiment in the intermediate period (1997-1998 to 1999). 3. A number of 168 isolates of U. atrum was purified from the biological material analyzed (leaf necrotic and soil organic fragments, soil suspensions) plated on ARSA medium. REFERENCES
BOFF P., 2001 - Epidemiology and biological control of grey mould in annual strawberry crops. PhD Thesis, WAU 2. BOFF P., KHL J., JANSEN M., HORSTEN P.J.F.M., LOMBAERS-VAN DER PLAAS KARIN, GERLAGH M., 2002 - Biological control of grey mould with Ulocladium atrum in annual strawberry crops, Plant Disease 86, 3: 220-224 3. DAVID J.C. 1995. Ulocladium atrum, IMI Descriptions of Fungi and Bacteria, No. 1224 4. ELMER P. A. G. KHL J., 1998 - The survival and saprophytic competitive ability of the Botrytis spp. antagonist Ulocladium atrum in lily canopies. Eur. J. Plant Pathology, 104: 435-447 5. FRUIT LAETITIA, NICOT PH., 2007 - Use of Ulocladium atrum for biological control of Botrytis cinerea stem infections in greenhouse tomatoes, INRA, http://www.ubourgogne.fr/UVV/P56.pdf. 6. GERLAGH T., AMSING J.J., MOELHOEK WILMA M.L., BOSKER-VAN ZESSEN A.I., LOMBAERSVAN DER PLAAS KARIN, KHL J., 2001 - The effect of treatment with Ulocladium atrum on Botrytis cinerea attack of geranium (Pelargonium zonale) stock plants and cutting. Eur. J. Plant Pathology, 107: 377386 7. KESSEL G.J.T., KHL J., POWELL J.A., RABBINGE R., VAN DER WERF W., 2005 - Modeling spatialk characteristics in the biological control of fungi at the leaf scale: competiting substrate coloniyation by Botrytis cinerea and the saprophytic antagonists Ulocladium atrum. Phytopathology, 95: 439-448 8. KHL J., 1997-2000 - Contract FAIR-CT96-1898, BIOSPORSUPPRESS The biological control of airborne necrotrophic plant pathogens by suppression of spore production 9. KHL J. MOELHOEK WILMA M.L., VAN DER PLAAS KARIN, FOKKEMA N. J., 1995 - Effect of Ulocladium atrum and other antagonist on sporulation of Botrytis cinerea on dead lily leaves exposed to field conditions. Phytopathology 85, 4: 383-403 10. KHL J. FOKKEMA N. J. 1998. Strategies for biological control of necrotrophic fungal foliar pathogens, in BOLAND G. J. & KUYKENDALL L. D., 1998 - Plant-microbe interactions and biological control. Marcel Dekker Inc. New York-Basel-Hong Kong : 49-88 11. KHL J. GERLAGH M. DE HAAS B. H. KRIJGER M. C., 1998 - Biological control of Botrytis cinerea in cyclamen with Ulocladium atrum and Gliocladium roseum under commercial growing conditions. Phytopathology, 88: 568-575 12. KHL J. GERLAGH T., DE HAAS B.H., KRIEGER M.C., 1998 - Biological control of Botrytis cinerea in cyclamen with Ulocladium atrum and Gliocladium roseum under commercial growing conditions. Phytopathology, 88, 6: 568-575 1.

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13. KHL J. LOMBAERS-VAN DER PLAAS KARIN, MOELHOEK WILMA M.L., KESSEL G.J.T., GOOSEN-VAN DER GEIJN HELEN M., 1999 - Competitive ability of the antagonists Ulocladium atrum and Gliocladium roseum at temperatures favourable for Botrytis spp. Development. BioControl, 44: 329-346 14. KHL J. GERLAGH M. GRIT G., 2000 - Biocontrol of Botrytis cinerea by Ulocladium atrum in different production systems of cyclamen. Plant Disease, 84: 569-573 15. KHL J. KESSEL G.J.T., 2000 - Epidemiology of Botrytis spp. in different crops determines success of biocontrol by competitive substrate exclusion by Ulocladium atrum, p. 63, in Sixth Workshop of the IOBC/wprs Phytopathogens WG: Biocontrol agents modes of action and their interaction with other means of control, 30 Nov.-3 dec.200, Sevilla, Spania 16. PRYOR B. M., DAVIS R. M., GILBERTSON R. L., 1994 - Detection and eradication of Alternaria radicina on carrot seed, Plant Dis. 78 (5): 452-456 17. ROUDET J, DUBOS BERNADETTE., 2000 - Evaluation of a three year study of Ulocladium atrum (strain 385) as a biological control agent of vine grey rot in the Bordeaux region, 12th International Botrytis Symposium, Reims (France): 58 18. SCHOENE P. KHL J., 1999 - Biologische Bekmpfung von Botrytis cinerea mit Ulocladium atrum in Reben und Cyclamen. Gesunde Pflanzen, 51, 3: 81-85 19. SCHOENE P., 2002 - Ulocladium atrum as an antagonist of grey mold (Botrytis cinerea) in grapevine. PhD Thesis Rheinische Friederich Wilhelms University, Germany 20. ESAN T. E., 2003 - Sustainable management of gray mold (Botrytis cinerea) on grapevine, strawberry and ornamentals, in H.-C. Huang & Acharya Suryia, Advances in plant diseases management. Research Signpost, Kerala, India: 121-152 21. ESAN T. E., 2005 - Bibliografia romneasc n domeniul combaterii biologice a micozelor plantelor. Sntatea plantelor, ediie special: 15-22 22. ESAN T. E., 2006 - Integrated control of strawberry diseases, Phytopathologia Polonica, 39: 133-148 23. TUITE J., 1969 - Plant pathological methods. Fungi and bacteria, Burgess Publishing Company, Minneapolis 55415: 44, 52, 75 24. WHIPPS J. M., 1987 - Behaviour of fungi antagonistic to Sclerotinia sclerotiorum on plant tissue segments. Journal of General Microbiology, 133: 1495-1501 25. WHIPPS J. M., BUDGE S. P., 1990 - Screening for sclerotial mycoparasites of Sclerotinia sclerotiorum. Mycological Research, 94, 5: 607-612

Acknoledgments Acknoledgments for the Biocontrol Group in Plant Research International Wageningen, The Netherlands, leaded by PhD Jurgen Khl, for the opportunity to work together for six monthes in 2000 for the project 1898, BIOSPORSUPPRESS. Deep thanks to a special friend PhD Thijs Gerlagh from the same working group for his kindness, perenial help and cooperation in our research activity in the field of Botrytis and Sclerotinia biocontrol.

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Analele tiinifice ale Universitii Al. I. Cuza Iai Tomul LIV, fasc. 1, s. II a. Biologie vegetal, 2008

A NEW SITE IN ROMANIA FOR SPIRULINA (ARTHROSPIRA) PLATENSIS M. COSTIC *, NAELA COSTIC *
Abstract: Spirulina (Arthrospira) platensis Geitl was found in the perimeter of the Tomesti village (district Iasi) for the first time in Moldavia (Romania). Its coenotic ambience and distribution in Romania are given. Key words: Spirulina (Arthrospira ) platensis, a new site in Romania

Introduction In 1827, P.J.Turpin isolated Spirulina from a fresh water sample. In 1844, Wittrock and Nordstedt reported the presence of a green - blue microalgae named Spirulina jenneri f. platensis in the sample collected near the city of Motevideo. In 1852, Stizenberger gave the name Arthrospira for the septal form and multicellular structure, and Spirulina for without septal, but helical form. Geitler, in 1932 reunified the two genera under the designation Spirulina, considering only morphological similarity. [2]. The microalgae exploited as food belongs to the genus Arthrospira, but it has been called Spirulina for some time. The taxonomy of the genus Arthrospira is quite confused, and at least 12 binomials are currently recognized: A. funiformis, A. fusiformis, A. geitleri, A. gomontiana, A. indica, A. jenneri, A. khannae, A. massartii, A. maxima, A. miniata, A. platensis, and A. tenuis. However, different interpretations were given to their descriptions, and these species are difficult to distinguish [4]. Material and methods The samples were collected from small pools in the perimeter of the Tomesti village. and analysed at optical microscope. The morphological characterisation has been defined through measurement of microscopic details, such as: trichome wide, helix wide, coils of helix apart. The results were compared with the species description from speciality literature [5]. The identified species has been verified by phycologist, Prof.dr. alaru Vasile (University from .Kishinev , The Republic of Moldovia) Results and discussions The basin of the Bahlui river is situated on North - Eastern of Romania with strong continental influence (9.6 Celsius degree; 518 mm precipitation; there is a large variation of these values in the years with low precipitations). There are many microhabitats, in this

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basin, where we could find some interesting species. In the perimeter of the Tomesti village we found a pool where Spirulina (Arthrospira) platensis grows. Around this pool, there is the vegetal association (Cynodonto Atriplicetum tataricae Morariu 1943), where we identified the species: Aster tripolium, Spergularia marina, Juncus gerardi. Cenotic ambience The characteristics of the water at the sampling site, at a depth of 20 25 cm are: pH= 8.9; temperature = 24 Celsius degree (in August 2004). The Spirulina grows together with species: Chlorella vulgaris, C. minutissima, Oscillatoria brevis, Synecoccocus sp., Chlamydomonas sp., Carteria sp., Sphaerellopsis sp. Conclusions Chorology in Romania Spirulina platensis was identified in basin of the Cri River (at Petea, Homorog), of the Arge River (at Caldarusani, Chirnogi), of the Danube River (at Pardina and the Danube Delta) [1]. Site from Tometi is unique in the province of Moldovia.and the third quotation from Europe. A short history of Spirulina in human consumption The first writing about this alga belonged to Bernal Diaz del Castillo (a member of Hernan Cortez,s troops). He reported in 1521 that Spirulina was harvested from the Lake Texcocco, dried and consumed by people in a Tenochtitlan market (today Mexico City) [2]. In 1940 P. Dangeard (phycologist) mentioned that Spirulina has been consumed by people near the African Lake Chad (confirmed by Jean Leonard, 1966 cf. [2].). In 1970, Germany supported studies on human consumption of Spirulina in Peru, Thailand and India. Spirulina is marketed in Germany, Brasil, Chile, Spain, France, Canada, Ireland, Argentina, India etc. [3]. Spirulina is one of the most extensively used microalgae for animal and human nutrition; its main interest is centered in its high protein content, 60-65% on a dry weight basis. REFERENCES
1. 2. 3. 4. 5. CRU I., 2002 - The Algae of Romania. St. Cerc., Univ. Bacu, (Biol.), 7: 1-694 CIFERRI O., 1983 - Spirulina, the edible microorganism. Microbiol. Rev., 47: 551- 578 LUCIA HELENA PELIZER et al ., 2003 - Influence of inoculum age and concentration in Spirulina platensis cultivation. J. Food Engeneering, 56, 4: 371-375 KOMREK, J., LUND, J.W.G., 1990 - What is Spirulina platensis in fact ?. Algological Studies, 58: 1-13. PASCHER A., 1925 - Cyanophyceae In: Pascher A.(ed) Suswasser- Flora Deutschlands, Osterreichs und der Schweiz, Gustav Fischer, Jena.

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Analele tiinifice ale Universitii Al. I. Cuza Iai Tomul LIV, fasc. 1, s. II a. Biologie vegetal, 2008

CONTRIBUTION TO THE STUDY OF GRASSY VEGETATION IN THE CEAHLU MOUNTAIN T. CHIFU , C. MNZU*, OANA ZAMFIRESCU*
Abstract: This paper presents five types of grassland phytocoenoses, widely spread in the Ceahlu Mountain and classified by us, into five associations belonging to Cynosurion R. Tx. 1947 Alliance, Arrhenatheretalia R. Tx. 1931 Order, Molinio Arrhenatheretea R. Tx. 1937 Class: Pastinaco Arrhenatheretum elatioris Passarge 1964, Anthoxantho Agrostietum capillaris Sillinger 1933, Festuco rubrae Agrostietum capillaris Horvat 1951, Agrostio Festucetum rupicolae Csrs Kaptalan 1964 and Poo Trisetetum Knapp ex Oberd. 1957. For these associations, we present the distribution in the studied territory, the ecology, the use, as well as the synthetic table. Key words: Arrhenatheretalia R. Tx. 1931 Order, grassland vegetation, Ceahlu Mountain.

Introduction The phytocoenoses we analyzed are located at altitude between 530 m and 1270 m, corresponding to the mixed beech and coniferous trees forests altitudinal horizon [6]. The primary vegetation of this altitudinal horizon consists in phytocoenoses of the Pulmonario rubrae Fagetum (So 1969) Taber 1987, Symphyto cordati Fagetum Vida 1963, Leucanthemo waldsteinii Fagetum (So 1964) Taber 1987, Hieracio transsilvanico Fagetum (Vida 1963) Taber 1987, Geranio robertianae Fagetum (Burduja et al. 1974) Chifu et tefan 1994, Galio schultesii Fagetum (Burduja et al. 1973) Chifu et tefan 1994 associations. The main secondary vegetation is represented by the meadows of the Festuco rubrae Agrostietum capillaris Horvat 1951 association, togheter with Pastinaco Arrhenatheretum elatioris Passarge 1964, Anthoxantho Agrostietum capillaris Sillinger 1933, Agrostio Festucetum rupicolae Csrs Kaptalan 1964 and Poo Trisetetum Knapp ex Oberd. 1957 associations [6]. Material and methods For the study of vegetation in the Ceahlu Mountain, we used the classical method of the phytosociological surveys, elaborated by the Zurich-Montpellier school [2]. To identify each association we carried out an appropriate number of surveys (between 5 and 16) on 25 to100 m2 sample areas. The investigation lasted from June to August. For the identification of the species with special status in the analysed communities, we used The Red list of superior plants in Romania [13], and whereas the ecological demands of the species was set according to The Abstract of the spontaneous cormophytes in Romania [14].

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Results and discussions 1. The communities of the Association Pastinaco Arrhenatheretum elatioris Passarge 1964 grow on fresh, fertile soil, plane or less inclined slopes, generally southern orientated, at the base of the Ceahlu Mountain, between 600 m and 700 m of altitude. The characteristic species, Arrhenatherum elatius, dominates in the communities together with Trisetum flavescens, Dactylis glomerata, Festuca rubra, Agrostis capillaris sau Medicago falcata. Over 70% of the component species are characteristic to the sintaxa of the Class Molinio-Arrhenatheretea, and many of them are constant: Campanula patula, Centaurea phrygia, Trifolium pratense, T. repens, Leucanthemum vulgare, Briza media, Tragopogon pratensis ssp. orientalis, Medicago lupulina, Taraxacum officinale etc. (Table I, column 1). The plant communities display an obvious stratification: a superior layer, uniform and dense, consisted of Arrhenatherum elatius, Dactylis glomerata, Trisetum flavescens etc.; medium layer, consisted of Festuca rubra, Agrostis capillaris, Filipendula vulgaris, Achillea millefolium etc., and an inferior layer, consisted of Trifolium repens, Prunella vulgaris, Taraxacum officinale, Leontodon hispidus, L. autumnalis etc. The communities of Arrhenatherum elatius are among the most productive and with good quality meadows, making excellent hayfields. Their floristic composition includes the endemit Primula elatior ssp. leucophylla, and the rarities Leontodon hispidus ssp. hyoseroides and Ranunculus acris ssp. friesianus. 2. The Association Anthoxantho Agrostietum capillaris Sillinger 1933 is widely spread in hills and mountains, up to the limit of the common spruce woods. In the Ceahlu Mountain, the association occupies plane fields or gentle slopes, with sufficiently humid soil. The communities are rich in species. Many species are characteristic to the Alliance Cynosurion, Order Arrhenatheretalia and Class Molinio Arrhenatheretea, and some of them are highly constant: Anthoxanthum odoratum, Leucanthemum vulgare, Rhinanthus minor, Leontodon hispidus, Festuca rubra, Plantago lanceolata, Prunella vulgaris etc. (Table 1, column 2). The floristic composition of the communities reflects the habitat conditions, 90% of the species are mesophilous and mesohygrophilous. These meadows have mixed use: they are grazed in spring and autumn and mowed in summer. For this reason, the floristic composition is heterogeneous and the fields are often overgrazed. 3. The Association Festuco rubrae Agrostietum capillaris Horvat 1951 is widely spread throughout the Carpathians, in mesophilous locations, mainly at the decidous forest level, descending in the common oak sub-level, and ascending in the common spruce level. In Ceahlu, the association is widely spread at the altitude of 550 1000 m, in the fields generally with gentle or medium inclined slopes, with varied orientation, and more rarely on flat fields. The phytocoenoses are mainly formed of Festuca rubra and Agrostis capillaris, which are in different relations of co-dominance. In other phytocoenoses, they associate also with other species with significant dominance, such as Leucanthemum vulgare, Trifolium montanum, T. alpestre, Filipendula vulgaris, Carex montana. To these species, several highly constant ones can be added: Campanula patula, Holcus lanatus, Briza media, Lotus corniculatus, Galium verum, Plantago media etc.

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In the structure of the association we distinguish three layers: the superior one, 40 50 cm high, where predominates Festuca rubra and Agrostis capillaries; a median layer, 20 - 30 cm high, formed of several species such as Anthoxanthum odoratum, Cynosurus cristatus, Trifolium pratense, Lotus corniculatus, Leucanthemum vulgare etc., and the inferior layer, up to 10 - 15 cm, which includes Plantago lanceolata, P. media, Trifolium repens, Carlina acaulis, Euphrasia stricta etc. Two sub-associations represent the association: - typicum Coldea 1991, with a rich, more homogenous, floristic composition (Table I, column 3a); - nardetosum strictae (Csrs et Resm. 1960) Oroian 1998, which occupies fields, less rich in nutritive substances and more acid; the differential species are Nardus stricta, Antennaria dioica, Potentilla aurea and others characteristic to the Class Juncetea trifidi (Table 1, column 3b). These meadows have a mixed use, however, the overgrazing and degradation let a series of weeds to appear. The evolution towards nardetosum strictae is the effect of grazing. The need to protect these fields is also supported by the presence of some endemic (Silene nutans ssp. dubia, Primula elatior ssp. leucophylla etc.) and rare species (Dactylorhiza maculata, Ranunculus oreophilus, Leontodon hispidus ssp. hyoseroides etc.). 4. Association Agrostio Festucetum rupicolae Csrs Kaptalan 1964 (Table I, column 4). The phytocoenoses established by Festuca rupicola and Agrostis capillaris occurs on humid to dry soils in hill and mountain regions. The dominant of the two species is Festuca rupicola, which is accompanied in some phytocoenoses by Agrostis capillaris, Festuca rubra, Trifolium montanum, Thymus pulegioides, Leucanthemum vulgare etc. Apart from the mesophyllous species characteristic to the class, a large group of xero-mesophyllous species from Class Festuco Brometea is included in the floristic composition. These fields are used for grazing, and therefore they are highly degraded and invaded by weeds. 5. Association Poo Trisetetum Knapp ex Oberd. 1957 (Table I, column 5) The meadows of Trisetum flavescens are scarcely distributed in the Romanian Carpathians, in the inferior and medium mountain level, on flat or gently inclined terrain, with moderately humid and poor in nutrients soils. These meadows are distributed in patches, at altitudes between 600 m and 700 m, in the broad valleys of the Ceahlu Mountain. Trisetum flavescens dominate the communities. In some phytocoenoses, this species is associated with Trifolium pratense, Festuca rubra, Taraxacum officinale etc. In addition, we must remark the presence of some constant species, such as Trifolium repens, Campanula patula, Centaurea phrygia, Carum carvi, Leucanthemum vulgare, Achillea millefolium, Medicago lupulina, Dactylis glomerata, Cerastium holosteoides, Ranunculus acris, Prunella vulgaris, Tragopogon pratensis ssp. orientalis etc. The floristic composition is clearly dominated by the mesophilous species (approximately 85 %), in relation to the habitats of the communities belonging to this association.

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From the economic point of view, these meadows are valuable because they are used as hayfields. Table I. Associations from the Arrhenatheretalia R. Tx. 1931 Order
Association Altitude (m. o. s.) Exposure 1 620 - 690 S, SE 2 630 - 1230 NV, S 0 - 20 90 - 100 8 3a 600 - 1004 NV, NE, S, SE, V, E 0 15 75 - 100 16 IV V V I IV IV I I I III II I II III I II III I III V I I V I 3b 970 1270 SV, S, NV, V 0 10 95 100 8 IV V V III V III II II V II I V II 4 530 780 SE, S 0 - 25 75 - 100 6 III V V II I III III III II III IV IV 5 570 820 90 - 100 10 II III II III II IV IV I II V I II III IV II III V I V II -

Slope (degrees) 0-5 Vegetation coverage (%) 90 - 100 No. of surveys 5 Associations characteristic species Pastinaca sativa I Anthoxanthum odotarum Festuca rubra III Agrostis capillaris II Poa pratensis III Subassociations differential species Antennaria dioica Nardus stricta Potentilla aurea Arrhenatherion Arrhenatherum elatius V Campanula patula V Centaurea phrygia IV Daucus carota I Equisetum arvense Geranium pratense Pimpinella major Taraxacum officinale IV Cynosurion Bellis perennis Cynosurus cristatus III Leontodon autumnalis II Lolium perenne Phleum pratense I Plantago major Trifolium repens IV Phyteumo Trisetion Aegopodium podagraria Gladiolus imbricatus II Hypericum maculatum I Trisetum flavescens IV Trollius europaeus Veratrum album I Arrhenatheretalia Achillea millefolium III Ajuga reptans Avenula pubescens Briza media IV Bromus hordeaceus -

IV IV V I III III I III III II I IV I III I I II II -

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Campanula glomerata II III III III Carum carvi III III II III Crepis biennis I I III Dactylis glomerata IV II II Heracleum sphondylium I I Holcus lanatus I IV I Knautia arvensis I I III Leontodon hispidus ssp. IV III hispidus Leucanthemum vulgare V V V II Luzula campestris II V Medicago lupulina IV I I Rhinanthus minor III V III Stellaria graminea I Thymus pulegioides III III V V Tragopogon pratensis IV I III ssp. orientalis Trifolium campestre I Molinietalia (incl. Alopecurion, Filipendulion, Deschampsion, Molinion, Calthion) Agrostis canina I Agrostis stolonifera I Carex ovalis I I Carex pallescens III III III Carex tomentosa I Chaerophyllum hirsutum I Colchicum autumnale III I III Dactylorhiza maculata I Deschampsia caespitosa I I I IV Dianthus superbus I Festuca pratensis III III Filipendula ulmaria I Gymnadenia conopsea I I III Hypericum tetrapterum I Juncus articulatus I Laserpitium prutenicum I Linum catharticum III V Lychnis flos-cuculi I Lysimachia vulgaris I Lythrum salicaria Myosotis scorpioides I I Molinia caerulea II Platanthera bifolia I Polygonum bistorta I Serratula tinctoria I Stachys officinalis I V Succisa pratensis II III Symphytum officinale Valeriana officinalis I I Potentillion anserinae et Potentillo Polygonetalia Bromus commutatus II I Carex distans Elymus repens I -

II II I III V III III V III I II I II I I -

II IV II II III I I V IV III III IV II II I I I I I I -

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Inula britanica Ranunculus repens Molinio Arrhenatheretea Alchemilla vulgaris agg. II Centaurea jacea Cerastium holosteoides II Euphrasia officinalis ssp. I pratensis Euphrasia stricta Lathyrus pratensis Leontodon hispidus ssp. I hyoseroides Lotus corniculatus III Plantago lanceolata II Polygala vulgaris II Poa trivialis Primula veris Prunella vulgaris III Ranunculus acris II Rhinanthus angustifolius I Rumex acetosa I Stellaria graminea Trifolium alpestre Trifolium montanum III Trifolium pratense V Vicia cracca II Festuco Brometea s. l. Ajuga genevensis Anthemis tinctoria Anthericum ramosum Anthyllis vulneraria Arenaria serpyllifolia Asperula cynanchica Astragalus cicer Astragalus onobrychis Bupleurum falcatum Carex montana Carlina vulgaris Centaurea stoebe Cirsium pannonicum Dianthus carthusianorum Echium vulgare Elymus hispidus Eryngium campestre Euphorbia cyparissias Festuca rupicola II Filipendula vulgaris I Galium verum II Gentiana cruciata Helianthemum nummularium ssp. obscurum

II I I III I IV III IV II II III III II III I III V III I I III I I

I III I II I I III II V IV III I III IV II I II III III V V II I I I III I I I I III I I III II I I V IV I III

V II IV I V IV III IV III V -

I II I V III III II III III IV III II I I III II II I III I III I IV II IV I I I V IV II III

I II IV I III II II I I IV IV III I V II I I I I I -

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Hieracium bauhinii Hieracium pilosella Hypochoeris maculata Koeleria macrantha Linum austriacum Medicago falcata Onobrychis viciifolia Pimpinella saxifraga Plantago media Potentilla argentea Potentilla arenaria Prunella grandiflora Ranunculus bulbosus Salvia nemorosa Salvia pratensis Scabiosa ochroleuca Teucrium chamaedrys Trifolium aureum Trifolium ochroleucon Veronica orchidea Juncetea trifidi s. l. Botrychium lunaria Campanula abietina Campanula serrata Carlina acaulis Genista tinctoria Gentianella austriaca Hieracium aurantiacum Hieracium umbellatum Hypochoeris radicata Ligusticum mutellina Potentilla erecta Viola canina Festuca supina Variae syntaxa Achillea stricta Acinos arvensis Astragalus glycyphyllos Astrantia major Bunias orientalis Campanula persicifolia Campanula rapunculoides Campanula trachelium Carduus acanthoides Carex echinata Carex flava Cerastium arvense Cichorium intybus Cirsium arvense Cirsium decussatum Cirsium erisithales

III II II II II I III I -

I I I I I V I I I I III III I I I I I I I I I I I

II III I I III III V II I II I II I I I I I III I II I III I IV III I III I I I I I II

II II III I II I I II III II I I I -

II III I II II III IV III II III I II I III III I II II II II I II I I I I -

I III I II III I II I I I III I

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Cirsium oleraceum Cirsium vulgare Clinopodium vulgare Convolvulus arvensis Coronilla varia Cruciata glabra Cruciata laevipes Dianthus deltoides Dianthus tenuifolius Erigeron acris Festuca heterophylla Fragaria vesca Galium schultesii Gentiana asclepiadea Gentiana verna Gnaphalium sylvaticum Inula salicina Laserpitium latifolium Listera ovata Luzula luzuloides Luzula pilosa Matricaria recutita Melampyrum cristatum Myosotis alpestris Nepeta cataria Origanum vulgare Polygonatum verticillatum Primula elatior ssp. leucophylla Pteridium aquilinum Ranunculus acris ssp. friesianus Ranunculus oreophilus Ranunculus polyanthemos ssp. polyanthemoides Reseda lutea Rumex acetosella Sagina saginoides Salvia verticillata Senecio jacobea Senecio umbrosus Silene alba Silene nutans ssp. dubia Tanacetum corymbosum Telekia speciosa Thalictrum aquilegiifolium Thalictrum minus Trifolium medium Trifolium pannonicum

I III I I I I II I -

I I III I II I I I II I I I I I I II

I I I I I I I I I III I I I I II II III II II II I I I II II II V

II I I I I -

I I II I III IV II III I I I I II II I I III I III II -

I II I I I II I I I I -

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Veratrum album ssp. lobelianum Verbascum lychnytis I Veronica chamaedrys III III I Vicia sepium Vicia sylvatica I I 1. Pastinaco Arrhenatheretum elatioris Passarge 1964 2. Anthoxantho Agrostietum capillaris Sillinger 1933 3. Festuco rubrae Agrostietum capillaris Horvat 1951 a. typicum b. nardetosum strictae (Csrs et Resm. 1960) Oroian 1998 4. Agrostio Festucetum rupicolae Csrs Kaptalan 1964 5. Poo Trisetetum Knapp ex Oberd. 1957

III -

I -

REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. BELDIE AL., 1968 - Asociaiile vegetale din Carpaii Romniei. Com. de Bot., Soc. t. Biol., Bucuresti, 6: 133-238 BORZA AL., BOCAIU N., 1965 - Introducere n studiul covorului vegetal, Edit. Acad. R. S. R., Bucureti BURDUJA C.,1968 - Muntele Ceahlu flora i vegetaia, Ocrot. Nat., Bucureti, 6: 63 92 BURDUJA C., DOBRESCU C., GRNEANU A., RVRU M., CZCEANU I., BRC C., RACLARU P., TURENSCHI E., 1956 - Contribuii la cunoaterea pajitilor naturale din Moldova sub raport geobotanic i agroproductiv. St. cerc. t. Acad. R. P. R. Fil. Iai, Biol. t. Agric.7, 1: 1 37 CHIFU T., 1995 - Contribuii la sintaxonomia vegetaiei pajitilor din clasele Molinio-Arrhenatheretea Tx. 1937 i Agrostietea stoloniferae Oberd. in Oberd. et al. 1967 de pe teritoriul Moldovei. Bul Grd. Bot. Iai, 5: 125 132 CHIFU T., MNZU C., ZAMFIRESCU O., 2006 - Flora i vegetaia Moldovei (Romnia), vol. II, Edit. Univ. Al. I. Cuza Iai: 11 - 21, 211 303 CHIFU T., MITITELU D., DSCLESCU D., 1987 - Flora i vegetaia judeului Neam. Mem. Sec. t. Acad. Rom. , Seria IV, 10, 1: 281 302 COLDEA GH., 1991 - Prodrome des associations vgtales des Carpates du sud-est (Carpates Roumaines), Docum. Phytosoc., Camerino, 13: 458 470 COLDEA GH., NEGREAN G., SRBU I., SRBU A.(coord.), 2001 - Ghid pentru identificarea i inventarierea pajitilor seminaturale din Romnia. Edit. alo, Bucureti!: 17 -58 ELLMAUER T., MUCINA L., 1993 - Molinio Arrhenatheretea, in Mucina L., Grabherr G., Ellmauer Th. 1993. Die Pflanzengesellschaften sterreichs, I, Anthropogen Vegetation, Gustav Fischer Verlag JenaStuttgart-New York: 297 401 GRINTESCU I., 1924 - Considerations gobotaniques sur le Mont Ceahlu (Carpates Orientales), Bul. Soc. t. Cluj-Napoca, 2, 2: 104 - 112 NYRDY E., 1924 - Contribuiuni la cunoaterea vegetaiei i florei muntelui Ceahlu, Bul. Grd. Bot. Cluj-Napoca, 4: 2 3 OLTEAN M., NEGREAN G., POPESCU A., ROMAN N., DIHORU G., SANDA V., MIHILESCU SIMONA, 1994 - Lista roie a plantelor superioare din Romnia, Stud., Sint., Docum. de Ecol., Acad. Rom., Bucureti, I POPESCU A., SANDA V., 1998 - Conspectul florei cormofitelor spontane din Romnia, Acta Bot. Hort. Bucurestiensis POPESCU A., SANDA V., DOLTU M. I. 1983 - Conspectul vegetaiei ierboase din Romnia. St. i Com., Muz. t. Nat. Brukenthal, Sibiu, 25: 182 255 RESMERI I., 1977 - La classe des Molinio - Arrhenatheretea Tx. 1937, dans les Carpathes Roumaines, Docum. Phytosoc., Lille, vol. I: 241 267 ZANOSCHI V., 1971 - Flora i vegetaia masivului Ceahlu. Tez de doctorat, Univ. Babe- Bolyai, ClujNapoca

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ASSESMENT OF ECOLOGICAL STATUS OF DANUBE DELTA LAKES USING INDICATOR MACROPHYTES SPECIES J. HANGANU , M. DOROFTEI , N. TEFAN
Abstract: Implementation of European Water Framework Directive (WFD) legislation requires valuation of chemical and ecological status of surface water bodies. One of the biological indicators prescribes for such assessment is aquatic macrophytes taxonomic composition and abundance. In many member states the trophic status of the lakes is asses by calculating trophic index. This paper show the results of applying Schaumburg reference index for 39 water bodies in Danube Delta . Total P, secchi depth, connectivity and substrate type were the main environmental variables calculated versus index values. As lakes differs little in tot. P content, distribution of aquatic vegetation seems to be mainly determined by connectivity type, substrate and lake morphology. Key words: submerged macrophytes, lakes, classification, trophic index, hydromorphology.

Introduction Danube Delta refers to the area between 3 main branches of the Danube river (from north to south: Chilia, Sulina and Sfntu Gheorghe branch). This depresionary area is covered by over 200 km2 of reed beds and 140 km2 open water area. Total numbers of lakes is over 300 and lake size varies from 14 to 4530 ha. Lakes are supplied with fresh river water through vast networks of natural and artificial canals. Water level in lakes is variable and dependent of the river pulse. Higher water level is recorded in spring (May-June). With water depths of 1.5 - 4 meters and a chloride concentration below 60 mg/l, the Danube Delta lakes are characterized as shallow freshwater lakes and included in LCB2 GIG group. In the Danube River the median phosphorus concentration is 0.12 - 0.17 mg P/l. In the lakes the seasonal variation in phosphorus is more pronounced than in the river. In spring the concentration of P-total in the lakes is lower than in the river; in this season also P-ortho is lower in the lakes than in the river. Relative to spring the summer phosphorus concentration increases in all lakes to the level in the river; this increase occurs both in particulate and in dissolved phosphorus. Differences between lakes are small. Based on the classification scheme of Vollenweider [5] all lakes are eutrophic with respect to phosphorus. P-ortho concentrations in spring can be quite low, but are considerably higher in summer [3]. Some lakes in the Danube Delta are dominated by submerged vegetation, high water clarity and a high diversity of benthos and fish in contrast with other lakes dominated by phytoplankton, low water clarity and a low diversity of benthos and fish. The man-made network of canals in the Delta has intensified the water circulation and input of river water into the lakes. Also nutrient content in river water has increase in
Danube Delta National Institute for Research & Development Tulcea, 165 Babadag Street, 820112, Tulcea, Romania Al. I. Cuza University, Faculty of Biology, Carol I Bd., no. 20A, 700506, Iasi, Romania

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the last century few times. As a results lakes reported before 1960 to be dominated by Charecee at present the dominate vegetation is Ceratophyllids and Potamides type. Material and methods 691 vegetation surveys of the submerged vegetation from 39 water bodies in the Danube Delta made between 1993 and 2002 were used to test Schaumburg reference index. All vegetation surveys were made in June at fully development of vegetation. Each vegetation surveys had an approximate diameter of 5 m, and were made from canoe. Submerged plants were collected using a rake, and the abundance of each species in the vegetation was visually estimated using a 7-point scale corresponding with the BraunBlanquet scale. Only true aquatic macrophytes and characeae were included in the analyses. Additionally, measurements of water depth and transparency (secchi-depth: water depth) were made in each sampling point [2]. For each water body type dominant bottom substrate from the soil map was assigned. The substrate was classified in 4 classes according with the % of clay content; 1 = 5; 2 = 5 - 32, 3= 32-60, 4 = organic Type of connectivity for each lake has been use in the analyses. The connectivity type for each lake was calculated from modeling (SOBEK). Lake was classified in 3 connectivity classes: 1 = lakes with direct connectivity to the river and short residence time; 2= large lakes with long residence time; 3 = remote lakes, surrounded by reed beds. For calculation of Schaumburg trophic index in Danube delta lakes we use the REBECCA reference list of species, slightly modified (tab. I) The quantity of species was estimated from the original data and transformed in 5 degree scale [2]. Calculation (1) is the same as Reference Index in Schaumburg et al. (2004):

TI ( S ) =

Q Ai Q Bi
i =1 i =1 gi

nA

nB

Q
i =1

ng

*100

Where: TI(S) = trophy-index based on quantity (identical to Reference index in Schaumburg et al. 2004), QA = quantity of species i in group A (see table I), QB = quantity of species i in group B, QC = quantity of species i in all groups, nA = total number of species in group A, nB = total number of species in group B, nC = total number of species in all groups. Quantity = (semi quantitative score) Results Almost all TI values are negative (tab. II). The lower values are in lakes that are relatively deep (2 - 4 m) and large (> 200 ha), with sand-silt substrate and an intermediate inflow of river water with direct connectivity. Potamogeton trichoides, P. pectinatus, P. crispus are dominant species in those lakes and usually the cover is 100%.

104

Intermediate TI values are represented by lakes that are of medium size and water depth, loamy to clayey loamy top bottom substrate and are intensively flushed with river water and have a high seasonal dynamics in water level. Ceratophyllum demersum and Trapa natans may cover large part of the lakes. Higher TI values are related to relatively small and shallow lakes, with peat bottom, surrounded by extensive reedbeds, hydrologically isolated from the river and dominated by Characeae. Nymphaeids as Nuphar luteum, Nymphaea alba/candida can create large field at the border of large lakes or be dominant in small insulated lakes with peat bottom and surrounded by reed beds. We could not found for Danube Delta lakes good relationship between P-total and Schaumburg trophic index (fig. 1). It confirmed previously detailed studies [3] on linking trophic statutes of the Danube delta lakes with biological elements as chlorophyll -a, aquatic vegetation and fish species composition in the lakes. For the TP It was expected to be so as at present, the lakes differing little in nutrient concentrations. Transparency may be influence by other environmental factors and is quite variable during growing season and lakes types.

Total P vs. RI
100 50 RI 0 0 -50 -100 P-tot 100 200 300 R2 = 0.0011

Fig.1. Trophic Reference Index (Schaumburg) vs. P-total However, hydro morphological parameters as soil substrate and connectivity type we found to correlate better with above biological elements (fig. 2, 3). One explanation is that soil texture is dependent of lake hydrology (connectivity) and geomorphology as shown in [3]. Lakes with direct connectivity are supplied with fresh sediments during flooding and large lake is former lagoon with sandy substrate. In the insulated lakes water is filtered by suspended solids by reed beds and more clear and accumulation of organic matter from decay of aquatic plant debris or dieback of peaces of floating reed beds is the dominant process. The input of toxic substances (e.g. humic acids

105

or H2S) from the surrounding reed beds may give charophytes a competitive advantage over Potamogeton species in these lakes and low redox values in organic soils may favor
RI vs. Soil texture classes 100 80 60 40 20 0 -20 0 -40 -60 -80 -100

R2 = 0.1273

RI

soil texture 1 2 3 4

development of Nympaeides. Fig.2. Trophic Reference Index (Schaumburg) vs. Soil texture classes

Conectivity vs.RI
100 50 0 0 -50 -100 1 2 3 Conectivity R2 = 0.228

Fig.3. Trophic Reference Index (Schaumburg) vs. Connectivity classes

RI

106

Conclusions Connectivity and residence time seems to play an important roll in distribution of aquatic macrophytes in the Danube delta lakes. In lakes with low residence time of the water the algae may be flush away and have not sufficient time to fully develop and so the transparency is higher giving a chance to some specific submerged aquatic vegetation to grow [3]. The use of Schaumburg TI for the Danube Delta lakes revealed the eutrophication stage of the lakes and the biological quality gradient between lakes as is perceived in the field. In the future a possible reduction in nutrients correlated with hydrological works to reduce the direct inflow of river water in lakes may lead to a switch of tolerant dominant vegetation species to sensitive dominant species as Characeae group. REFERENCES
1. 2. 3. 4. 5. CARLSON B., 1995 - The Secchi Disk and the Volunteer Monitor. LakeLine, N. Am. Lake. Manage. Soc., 15, 1: 28-29, 35-37 KOHLER A., 1978 - Methoden der Kartierung von Flora und Vegetation von Swasserbiotopen. Landschaft + Stadt, 10: 23-85 OOSTERBERG W. et al., 2000 - Ecological gradients in the Danube Delta; present state and man-induced changes. RIZA The Netherlands, Danube Delta National Institute Romania and Danube Delta Biosphere Reserve Authority, RIZA raport nr. 2000.015 SCHAUMBURG J., SCHRANZ C., HOFMANN G., STELZER D., SCHNEIDER S., SCHMEDTJE U., 2004 - Macrophytes and phytobenthos as indicators of ecological status in German lakes a contribution to the implementation of the Water Framework Directive, Limnologica, 34: 302314 VOLLENWEIDER R.A., KEREKES J., 1982 - Eutrophication of waters. Monitoring, assessment and control. OECD Cooperative programme on monitoring of inland waters (Eutrophication control). Environment Directorate, OECD, Paris

Acknowledgments This study was funded by the European Commission under the 6th Framework Program, Contract No.:SSP1-CT-2003-502158 REBECCA.

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Table I. List of species groups with regard to eutrophication


A Sensitive species Chara globularis Elodea canadensis Nitella flexilis Nitellopsis obtusa Nitella mucronata Potamogeton gramineus Potamogeton nodosus Potamogeton mucronatus Tolypella glomerata B Tolerant species Hydrocharis morsus-ranae Nuphar luteum Nymphaea alba Nymphaea candida Nymphoides peltata Potamogeton lucens Potamogeton perfoliatus Potamogeton pusillus Potamogeton natans Ranunculus aquatilis Trapa natans Zannichellia palustris C Indifferent species Ceratophyllum demersum Elodea nuttallii Lemna gibba Lemna minor Lemna trisulca Myriophyllum spicatum Myriophyllum verticillatum Najas marina Potamogeton crispus Potamogeton berchtoldii Potamogeton compressus Potamogeton trichoides Potamogeton pectinatus Salvinia natans Spirodella polyrrhiza Stratiotes aloides Utricularia vulgaris TI -52 -73 -54 -58 -53 -35 -87 -24 -56 -6 -32 -48 -61 -16 -49 -93 -52 -62 -100 -6 -1 51 -41 -92 -76 -92 -87 -35 -60

Table II. TI values / lake


Lake name Baclaneti 1996 Cuibul cu Lebede 2002 Isac W 1998 Plin 1997 Raducu 1997 Rosu 1998 Rosulet 1998 Serbata 1997 Uzlina 1998 Iacub Miazazi Nebunu Oprio Raduculets 1997 L. Erenciuc 1993 L. Merheiu Mare 1995 Fortuna 1996 Cuibul cu Lebede 1998 Isac 2002 Uzlina 2002 Cn. Draghilea 1995 Cn. L.Ttaru 1995 Cn. Mila 35 1995 Cn. Potcoava 1995 Cn. Sireasa 1995 Cn. Sulimanca 1995 Grl. Somova 1994 Grl. ontea Veche 1995 J. Sulimanca 1995 J. Urechea 1994 TI -99 -64 -75 -81 -73 -100 -98 -79 -68 -20 -13 -15 -8 -11 -14 -39 -98 -93 -82 -78 -58 -63 -58 -68 -92 -37 -50 -65 -75 -39 Lake name Grl. Bratusca 1995 Grl. Sireasa Veche1995 L. Corciovata 1994 L. cu Cotete 1995 L. Gasca 1994 L. Lung 1995 L. Merheiu Mic 1995 L. Mesteru 1995 L. Parches 1994 L. Puiu 1993 L. Rdcinosu Mare 1995 L. Rdcinosu Mic 1995 L. Rotund 1994 L. Somova 1994 L. Telincea 1994 Lopatna 1996 M. Sacalin 1993 O. Babina 1995 Sf. Gheorghe 1995 Sf. Gheorghe 1993 Tataru 1998 Chiril 2002 Ghearsim 2002 Gorgova 2002 Gorgova 1998 HO02_1996 LacBabina 1997 Pojarnia 2002 L. Casla 1994

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THE PLANT COMMUNITIES WITH PHRAGMITES AUSTRALIS FROM THE HAYFIELDS OF VALEA LUI DAVID NATURAL RESERVATION (IASI COUNTY) OANA ZAMFIRESCU
Abstract: The association that comprises the arid reed communities was described based on an insufficient number of relevs. The plant communities with Phragmites australis from the arid and saline slopes of the natural reserve are significantly different from the typical reed beds that cover water banks. Such communities are phytosociologicaly classified in the Association Xero-Phragmitetum. These communities arise on the wide tolerance of reed to substrata water conditions. Keywords: reed beds, xerophylous vegetation, steppic meadow

Introduction The natural reserve The secular hayfields of Valea lui David, located at 13 km from Iai, comprises a patchy vegetation because of the rough relief and the large variation of humidity and salinity of the soil on relatively small areas. The dominant vegetation of the protected area is mostly xerophilous and generally belongs to the Class Festuco-Brometea. Among the six associations of this class, we discuss the Association Xero-phragmitetum erbnescu 1955, which has not been studied from this zone. Additionally, the relevs from the areas where it was identified are not sufficiently numerous to justify the coenotaxonomical classification. Material and methods We used the phytocoenological relev method created by the Zrich-Montpellier floristic-phytosociological school, and adapted by us to the local conditions. Therefore, we carried out many field observations, in optimal sampling periods, in order to obtain the relevs. For the vernal aspect, the observations were sampled in April, whereas for the aestival aspect, the relevs were sampled from May to July. The sample area varied between 10m2 and 25m2, depending on area of the investigated habitat. Due to the small areas occupied by these plant communities, we managed to draw just five relevs. Nevertheless, these relevs were very consistent with regard to the type of vegetation, and consequently they allowed the phytosociological classification.

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Results and discussions The communities formed by Phragmites australis grow on arid habitats, on flat areas or in small depressions where the water table riches the surface. The areas of these habitats are generally small, 10m2 to 20m2, with western orientation, and 100% vegetation covering. The analysis of the relevs demonstrated the phytosociological classification through the high number of species fitting in the Class Festucion valesiacae, Order Festucetalia valesiacae, and in the Class Festuco-Brometea.

Table I Ass. Xero-Phragmitetum Relev No. 1 2 3 4 Altitude (m) 103 105 123 98 Exposition NV V V V Slope (degree ) 20 10 15 5 Cover (%) 100 100 100 100 Area (m) 10 10 15 10 Caract. Sp. Phragmites australis + 1 + 2 Festucion valesiacae Festuca valesiaca 1 + 1 + Phlomis pungens + + + Adonis vernalis + + + + Agropyron cristatum 1 + 1 Linum austriacum + + + Salvia nemorosa + + + Euphorbia glareosa + + Festucetalia valesiacae Achillea setacea + + + Centaurea biebersteinii + Elymus hispidus 2 + + + Inula hirta + + Erysimum odoratum + + Bromus inermis + Festuco-Brometea Galium verum + + + Medicago falcata + + Dianthus carthusianorum + + + Muscari racemosum + + + + Thlaspi perfoliatum + + +

5 94 V 10 100 25 1 + + + + + + + + + + + + -

K V

V V IV V IV III IV III III II V III III I IV III III IV III

110

Relev No. Agrimonia eupatoria Thalictrum minus Salvia verticillata Melica ciliata Asperula cynanchica Molinio-Arrhenatheretea Dactylis glomerata Elymus repens Knautia arvensis Rhinanthus rumelicus Variae syntaxa Melampyrum arvense Convolvulus arvensis Asparagus officinalis Peucedanum latifolium Nepeta nuda Marrubium vulgare Centaurea orientalis Chenopodium album Conium maculatum Polygonum dumetorum Stachys annua Tribulus terestris

1 + + + + + + + + +

2 + + + + + + + + + + + -

3 + + + + + + + + + + +

4 + + + + + + -

5 + + -

K II III II I II IV II II III II I III IV II I II I I I I II

We compared our five relevs with other 4 from the literature, sampled from Frumoasa-Moara (Mititelu and Cojocaru, 1970) and Prut River Valley (Mititelu and Baraba, 1975), and we noted their strong resemblance, given that it is the same type of vegetation. On the other hand, our relevs differ very much from another type of community formed by Phragmites australis, i. e. the Association Phragmitetum vulgaris So 1927, both in floristic composition and in ecological characteristics. The latter occupies the banks of the lakes, ponds, and slow rivers, and its floristic composition is dominated by species characteristic of the Alliance Phragmition, Order Phragmitetalia, Class PragmitiMagnocaricetea, and other subordinated taxa of this class.

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Conclusions The plant communities with Phragmites australis from The hayfields of Valea lui David natural reserve differ significantly from those of the Association Phragmitetum vulgaris So 1927. Consequently, our observations support the classification of these types of communities in the Class Xero-Phragmitetum erbnescu 1955. The characteristic species Phragmites australis is highly tolerant to soil moisture, given that it grows, not only in wet habitats but also in arid ones. REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. BORKMAN J., MORAVEC J. & RAUSCHERT S., 1985 - Code de nomenclature phytosociologique Vegetatio. Haga, 67, 3: 177-187 BURDUJA, C., 1959 - O rezervaie tiinific care trebuie nfiinat "Fneele din Valea lui David" - Iai. Ocrot. nat., Bucureti, 4: 154 157 CHIFU T., MNZU C. & ZAMFIRESCU O., 2007 - Flora i vegetaia Moldovei. Edit. Univ "Al. I Cuza" Iasi MITITELU, D. & BARABA N., 1972 - Rspndirea unor asociaii ierboase n lunca Prutului. St. Com., Muz. t. Nat. Bacu, 5: 189 196 MITITELU D. & BARABA N., 1975 - Vegetaia din lunca Prutului. St. Com. Muz. t. Nat. Bacu, 8: 219 285 MITITELU D. & COJOCARU V., 1970 - Flora i vegetaia rezervaiei Frumoasa - Suceava. Ocrot. nat., Bucureti, 14, 1: 35 40 MITITELU D. & COJOCARU V., 1981 - O nou contribuie la flora rezervaiei botanice de la Frumoasa - Moara ( jud. Suceava ). St. Com. ocrot. nat. Suceava, 5: 394 395 MITITELU D., MOIU T., DSCLESCU D., TEU C. & VIALARIU C., 1969 - Flora i vegetaia rezervaiei "Valea lui David" - Iai. St. Com. Muz. t. Nat. Bacu, 2: 81 100 SANDA V., 2002 - Vademecum ceno - structural privind covorul vegetal din Romnia. Ed. Vergiliu, Bucureti SANDA V., POPESCU & A. DOLTU M. I., 1980 - Cenotaxonomia i corologia gruprilor vegetale din Romnia. St. Com. t. Nat. Muz. Brukenthal, Sibiu, 24

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ASSOCIATIONS OF THE MOLINIO ARRHENATHERETEA R. TX. 1937 CLASS IN VASLUI RIVER BASIN IRINA BLAJ - IRIMIA
Abstract: The paper presents 4 vegetal associations belonging to the Molinio Arrhenatheretea R. Tx. 1937 class, associations found on the territory of Vaslui river basin. Each association is accompanied by a phytosociological table and an analysis of the bioforms, floristic elements and ecological indices. Key words: phytosociology, bioforms, floristic elements, ecological indices.

Introduction The coenotaxonomic classification of the vegetal associations identified is the following: MOLINIO ARRHENATHERETEA R. Tx. 1937 Class MOLINIETALIA CAERULEA Koch 1926 Order CALTHION R. Tx. 1937 Alliance Scirpetum sylvatici Ralski 1931 Association POTENTILLO POLYGONETALIA R . Tx. 1947 Order POTENTILLION ANSERINAE R. Tx. 1947 Alliance Ranunculetum repentis Knapp ex Oberd. 1957 Association Junco inflexi Menthetum longifoliae Lohmeyer 1953 Association PLANTAGINETALIA MAJORIS R. Tx. et Preising in R. Tx. 1950 Order LOLIO-PLANTAGINION R. Tx. 1947 Alliance Schlerochloo Polygonetum avicularis So ex Korneck 1969 Association Material and methods For the identification of plant associations, we used phytosociological research methods according to the CentralEuropean school. The establishment of the bioforms and floristic elements was made on the basis of Flora ilustrat a Romniei Pteridophyta et Spermatophyta, by V. Ciocrlan (2000). The ecological indices were noted having in mind the works of H. Ellenberg [4]. Results and discussions Ass. Scirpetum sylvatici Ralski 1931 (Syn.: Scirpetum sylvatici Schwickerath 1944, Scirpetum sylvatici Maloch 1935)

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Chorology: Codeti, Dobrov Ecology: The coenoses represented by Scirpus sylvaticus are met on alluvial soils, gleic and pseudogleic, having a large altitude distribution. The association was identified on plane surfaces, on soils with excessive humidity almost all the year. The phytocoenological composition: Together with the characteristic and dominant species, Scirpus sylvaticus, we can find numerous hygrophilic species, Lysimachia nummularia, Lythrum salicaria, Carex vulpina, but also some mesophilic ones, fact denoting the belonging to the classes Phragmiti-Magnocaricetea and MolinioArrhenatheretea (Table I). After the analysis of the surveys undertaken, the following was noticed: - from the spectrum of bioforms it is noticed the net predominance of the hemicryptophytes (70.83%), followed by geophytes (16.66%), hydrohelophytes (4.17%), hydrophytes (4.17%) and chamephytes (4.17%); - from the phytogeographical spectrum we notice the dominance of the Euro Asian elements (37.5%) and circumpolar ones (37.5%), followed by the cosmopolite ones (16.66%) and European (8.34%); - from the spectrum of ecological indices we notice that the species bare weakly the shadow (47.82%), are amphitolerant to temperature (52.17%), with area of spreading in central Europe (34.78%), developing on humidwet soils (usually not aerated) (26.08%), amphitolerant to the reaction of the soil (47.83%) and the quantity of mineral nitrogen in the soil. Observations: This association is quoted for the first time in Vaslui river basin. Ass. Ranunculetum repentis Knapp ex Oberd. 1957 (Syn.: Ranunculetum repentis Knapp 1946, Ranunculetum repentis Knapp 1948, Agrostio-Ranunculetum repentis (Knapp ex Oberd. 1957) Oberd. et al. 1967) Chorology: Tcuta (Mititelu D.,1975), Vaslui, Vleni (Mititelu D. and collab., 1996), Soleti Ecology: The association vegetates on plane fields, with excess of humidity met in courtyards of houses. It stands well stagnant water for a period, after the withdrawal Ranunculus repens develops fast, covering by means of stolons, important surfaces. During the summer, it stands severe dryness of the soil at the surface. The phytocoenological composition: The floristic composition is not very rich because Ranunculus repens covers almost all the surface. Among the species frequently met in the association we mention: Potentilla reptans, Rumex crispus, Elymus repens etc. (Table II). After the analysis of the surveys undertaken, the following was noticed: - the spectrum of bioforms indicates the predominance of the hemicryptophytes (81.82%), followed by geophytes (18.18%); - the phytogeographical spectrum indicates the predominance of the EuroAsian elements (63.63%), followed by the circumpolar ones (27.28%) and the cosmopolite ones (9.09%);

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- the spectrum of ecological indices indicates the presence of the species standing weakly the shadow (45.45%), developing on humid to wet soils (5-27.27%, 6-27.27%) and with a moderate content of mineral nitrogen (45.46%). They are amphitolerant to temperature (54.54%) and the reaction of the soil (63.64%). Observations: The association was mentioned without floristic surveys by D. Mititelu (1975, 1996). Ass. Junco inflexi Menthetum longifoliae Lohmeyer 1953 (Syn: ass. Mentha longifolia-Juncus inflexus Passarge 1964) Chorology: Popeti, Satu Nou (Mititelu D., 1975), Vaslui (Mititelu D. and collab., 1996), Ciorteti, Codeti, Dobrov, Fereti Ecology: This association was frequently met in the river watersides, on soils with excess of humidity, where it develops isolated, in thick groupings. The phytocoenological characterization: The species characteristic and representative are Mentha longifolia and Juncus inflexus, together with which it also participate Agrostis stolonifera, Inula britannica, Potentilla reptans, Ranunculus repens etc. species characteristic to the class Molinio-Arrhenatheretea (Table III). The presence of the species in the classes Phragmiti-Magnocaricetea and Bidentetea is explained by the conditions of higher humidity characterizing this association. After the analysis of the surveys undertaken, the following was noticed: - the spectrum of bioforms illustrates the dominance of the hemicryptophytes (62.49%), followed by geophytes (17.86%), terrophytes (12.50%), hemiterrophytes (5.36%) and fanerophytes (1.79%); - the phytogeographical spectrum indicates a predominance of the Euro-Asian elements (55.36%). Apart from them, there are also circumpolar elements (16.08%), cosmopolite ones (14.28%), continental EuroAsian (5.35%), Mediterranean (3.56%), European (1.79%), AtlanticEuropean (1.79%) and ponticBalkan elements (1.79%); - the spectrum of ecological indices indicates us the fact that the species stand weakly the shadow (45.28%), they are mesothermal (30.20%), with the area of spreading in central Europe (28.30%), mesohygrophilic (15.09%), amphitolerant to the reaction of the soil (54.72%) and develop on soils with a moderate content of mineral nitrogen (24.52%). Observations: The association was mentioned in the Vaslui river basin by D. Mititelu (1975, 1996), but without presenting a table with floristic surveys. As. Schlerochloo Polygonetum avicularis So ex Korneck 1969 (Syn.: Polygonetum avicularis Gams 1927, Schlerochloo-Polygonetum avicularis So 1945) Chorology: Brnova (Mititelu D. and collab., 1995), Dneti, Tcuta, Vaslui, Vleni (Mititelu D. and collab., 1996), Deleni, Moara Grecilor, Popeti Ecology: Both species characteristic Schlerochloa dura and Polygonum aviculare are adapted to hardened fields, growing by the roads, paths, in courtyards, where the field is compact, but also with a content of nitrogen substances of organic nature.

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The phytocoenological composition: The association resists to human, animals or even light vehicles stepping over. The species Schlerochloa dura being an annual and vernal species, it develops in the first part of the season of vegetation, forming the aspect of spring (Sanda V. et al., 2001) (Tabel IV). After the analysis of the surveys undertaken, the following was noticed: - the spectrum of bioforms indicates the predominance of the terrophytes (58.62%) and hemicryptophytes (37.92%), followed by geophytes (3.46%); - the phytogeographical spectrum indicates a dominance of the EuroAsian elements (48.27%) and cosmopolite (31.05%), followed by the Mediterranean EuroAsian ones (6.88%), Mediterranean (3.45%), adventive (3.45%), continental EuroAsian (3.45%) and ponticMediterraneancentral European (3.45%); - the spectrum of ecological indices indicates the fact that the species stand weakly the shadow (33.33%), they are to temperature (41.66%), develop on dry to moderately humid soils (41.66%), amphitolerant to the reaction of the soil (75%), with a high content of mineral nitrogen (29.16%). Observations: The association was mentioned in Vaslui river basin by D. Mititelu (1996), but without presenting a table with floristic surveys. REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. CIOCRLAN V., 2000 - Flora ilustrat a Romniei Pteridophyta et Spermatophyta. Ed. Ceres, Bucureti CHIFU T., 1995 - Contribuii la sintaxonomia vegetaiei pajitilor din clasele Molinio Arrhenatheretea Tx.37 i Agrostietea stoloniferae Oberd. in Oberd. et al.67 de pe teritoriul Moldovei. Bul. Grd. Bot. Iai, 5: 125-132 CHIFU T., MNZU C., ZAMFIRESCU O., 2006 - Flora & vegetaia Moldovei (Romnia), vol. II, Ed. Univ. Al. I. Cuza Iai: 211-303 ELLENBERG H., 1974 - Indicator values of vascular plants in Central Europe. Scripta Geobotanica, vol. IX, Verlag Erich Goltze K.G., Gttingen: 1-97 ELLMAUER T., MUCINA L., 1993 - Molinio Arrhenatheretea In: MUCINA L., GRABHERR G., ELLMAUER T., 1993 Die pflanzengesellschaften sterreichs, Gustav Fischer Verlag Jena Stuttgart New York, vol. I: 297-401 MITITELU D., 1975 - Flora i vegetaia judeului Vaslui. St. i Com. Muz. t. Nat. Bacu, Biol. veget.: 67162 MITITELU D., CHIFU T., SCARLAT A., ANIEI L., 1995 - Flora i vegetaia judeului Iai. Bul. Grd. Bot. Iai, 5: 99-124 MITITELU D., HUANU M., 1996 - Noi contribuii la flora i vegetaia judeului Vaslui. St. i Cerc. Muz. t. Nat., Piatra-Neam, 8: 193-211 MUCINA L., 1997 - Conspectus of classes of European vegetation. Folia Geobot. Phytotax., Praha, 32, 2: 117-172 SANDA V., 2002 - Vademecum ceno-structural privind covorul vegetal din Romnia. Bucureti, Ed. Vergiliu SANDA V., POPESCU A., 1991 - Studiul fitocenozelor clasei Molinio-Arrhenatheretea Tx. 37 din Romnia. Acta Bot. Horti Bucurestiensis: 49-59 SANDA V., POPESCU A., BARABA N., 1997 - Cenotaxonomia i caracterizarea gruprilor vegetale din Romnia. St. i Comun. Muz. t. Nat. Bacu, Biol. veget., 14: 2-365 SANDA V., POPESCU A., STANCU D. I., 2001 - Structura cenotic i caracterizarea ecologic a fitocenozelor din Romnia. Ed. Conphis, Bucureti

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Table I. Ass. Scirpetum sylvatici Ralski 1931


Number of survey Altitude (m.s.m.) Cover of the vegetation (%) Surface of survey (m) Number of species Associations characteristics Scirpus sylvaticus Poa palustris Epilobium hirsutum Myosotis scorpioides Galium palustre Lysimachia nummularia Lythrum salicaria Juncus effusus Symphytum officinale Ranunculus repens Juncus inflexus Mentha aquatica Mentha longifolia Agrostis stolonifera Ajuga reptans Poa pratensis Veronica beccabunga Veronica anagallis-aquatica Phragmites australis Alisma plantago-aquatica Carex vulpina Juncus gerardii Equisetum arvense Lemna minor 1 210 100 10 13 2 121 95 25 14 3 121 90 25 11 4 210 100 10 10 5 + + + + + 1 + + + + 5 210 100 10 8 5 + + + + + + 1 -

K V IV III II IV IV III I I V III II II II I I III II II I I II I I

5 5 5 Calthion + + + + Molinietalia + + + + + + + + Potentillo-Polygonetalia 1 + + + + + + + + + Molinio-Arrhenatheretea + + Phragmiti-Magnocaricetea + + + + + + + Variae syntaxa + + + -

Place and date of the surveys: 1,4,5. Dobrov, 1.07.2004, 18.07.2004; 2,3. Codeti, 1.07.2004, 18.07.2004 Table II. Ass. Ranunculetum repentis Knapp ex. Oberd. 1957
Number of survey Altitude (m.s.m.) Cover of the vegetation (%) Surface of survey (m) Number of species Associations characteristics Ranunculus repens Potentilla reptans 1 120 90 20 5 2 120 95 20 4 3 120 100 20 6 5 + 4 120 85 20 6 4 2 5 120 100 20 5 5 +

K V V

5 5 Potentillion anserinae + 1

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Elymus repens Rumex crispus Agrostis stolonifera

+ + + Potentillo-Polygonetalia Rorippa sylvestris ssp. sylvestris + Plantago major Carex vulpina Molinio-Arrhenatheretea Taraxacum officinale Phragmiti-Magnocaricetea Phragmites australis + Poa palustris -

+ + + + -

+ + + + -

+ + +

III III II II II I I I I

Place and date of the surveys: 1-5. Soleti, 6.06.2004 Table III. Ass. Junco inflexi Menthetum longifoliae Lohmeyer 1953
Number of survey Altitude (m.s.m.) 1 170 2 210 3 94 4 110 5 110 N 85 25 9 3 2 2 + + 6 110 SV 60 15 7 3 1 + + 1 + 7 120 NV 70 25 10 4 + + + 8 110 V 100 20 17 4 2 + + + + 1 + + 9 110 NV 85 50 11 4 1 + + + + 10 210 loc plan 85 20 18 3 2 + 2 + + + + + + +

Exposition SV V SE N Slope () Cover of the 60 100 70 70 vegetation (%) Surface of survey (m) 25 20 20 25 Number of species 8 7 8 7 Associations characteristics Juncus inflexus 3 5 4 3 Potentillion anserinae et Potentillo-Polygonetalia Mentha longifolia 2 1 + Mentha pulegium 1 Agrostis stolonifera 2 Ranunculus repens Trifolium repens Inula britannica + Verbena officinalis Myosoton aquaticum Potentilla reptans Molinietalia Galium palustre Trifolium hybridum Molinio-Arrhenatheretea Daucus carota + Lotus corniculatus + Trifolium pratense + Cichorium intybus + Plantago major Medicago lupulina Centaurea jacea Ranunculus acris ssp. acris Festuca pratensis -

K V IV II II II II I I I I I I II II I I I I I I I

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Festuco-Brometea Prunella vulgaris Galium humifusum Achillea setacea Salvia nemorosa Plantago media Artemisietea vulgaris Artemisia absinthium + Tanacetum vulgare + Setaria viridis Arctium lappa Equisetum arvense Stellarietea mediae Cannabis sativa ssp. ruderalis Lathyrus tuberosus Chenopodium vulvaria Linaria vulgaris Sonchus arvensis Anagallis arvensis Phragmiti-Magnocaricetea Lythrum salicaria + Typha angustifolia + Lycopus europaeus Equisetum palustre Carex riparia Typha latifolia Phragmites australis Carex vulpina Bidentetea Bidens tripartita Polygonum hydropiper Rumex conglomeratus Rumex crispus Variae syntaxa Schoenoplectus tabernaemontani + Polygonum aviculare Salix alba Chaemerion angustifolium Trifolium fragiferum Juncus gerardi -

+ + + + -

+ 1 + -

+ + + 1 -

+ + 1 + -

+ -

+ + 1 + + + -

+ + + + + + + + + -

+ + 1 1 + -

+ + + + + + + +

I I I I I I I I I I I I I I I I II I I I I I I I III I I I I I I I I I

Place and date of the surveys: 1. Ciorteti, 27.07.2003; 2, 10. Dobrov, 23.08.2003, 1.07.2004; 3. Vaslui, 12.08.2003; 46,8,9. Codeti, 24.08.2003, 6.08.2003; 7. between Codeti and Dobrov, 24.08.2003

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Table IV. Ass. Schlerochloo-Polygonetum avicularis So ex Korneck 1969


Number of survey Altitude (m.s.m.) Exposition Slope () Cover of the vegetation (%) Surface of survey (m) Number of species Associations characteristics Schlerochloa dura Lolio-Plantaginion et Plantaginetalia Polygonum aviculare Poa annua Lolium perenne Lepidium ruderale Matricaria perforata Hordeum murinum Cynodon dactylon Malva pusilla Cichorium intybus Molinio-Arrhenatheretea Plantago major Trifolium repens Plantago lanceolata Verbena officinalis Stellarietea mediae Capsella bursa-pastoris Cardaria draba Malva neglecta Convolvulus arvensis Thlaspi arvensis Bromus arvensis Salvia nemorosa Chenopodium album Atriplex tatarica Amaranthus retroflexus Portulaca oleracea Artemisietea vulgaris Xanthium strumarium Taraxacum officinale Artemisia absinthium Artemisia annua 1 220 E 1-2 95 50 14 + 5 + + + + + + + + + + + + 2 220 E 1-2 100 25 14 5 1 + + + + + + + + + + + + 3 270 70 25 11 3 2 + + + 1 + + + + + 4 95 80 50 16 + 4 + + + 1 + + + + + + + + + + 5 190 NE 2-3 75 25 14 4 + + + + + + + 1 1 + + + + -

K II V V III III II II I I I IV III III II IV III III III II II II I I I I III III II I

Place and date of the surveys: 1,2. Micleti, 11.08.2004; 3. Deleni, 10.08.2004; 4. Moara Grecilor, 11.08.2004; 5. Popeti, 11.08.2004

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Analele tiinifice ale Universitii Al. I. Cuza Iai Tomul LIV, fasc. 1, s. II a. Biologie vegetal, 2008

CONTRIBUTIONS TO THE STUDY OF PALUDAL VEGETATION FROM THE NEAGRA ARULUI RIVERS BASIN (SUCEAVA COUNTY) LOREDANA ASOLTANI
Abstract: This paper presents three vegetal paludal associations identified in Neagra arului rivers basin: Epilobio Juncetum effusi Oberd. 1957, Scirpetum sylvatici Ralski 1931 i Filipendulo Geranietum palustris W. Koch 1926., described in a phytocoenological table and analysed from the point of view of bioforms, floristic elements and ecological indices. Key words: paludal vegetation, phytocoenology, Neagra arului river basin.

Introduction Neagra arului river basin is situated in the north-east side of central groupe from Oriental Carpathians, in the Dornelor Depression, ascending the higher peaks of the Climani Mountains in its south side. The Neagra arului river springs on the northern slope of Climani Mountains, at an absolute altitude of 1832 m, and flows into Bistria river downstream to Vatra Dornei town [9]. Regarding the paludal vegetation existing in the investigated area, we present in this paper three associations, included in the following phytocoeno-system [1, 4, 6, 7]: MOLINIO ARRHENATHERETEA R. Tx. 1937: MOLINIETALIA CAERULEAE Koch 1926 CALTHION PALUSTRIS R. Tx. 1937 Scirpetum sylvatici Ralski 1931 Epilobio Juncetum effusi Oberd. 1957 FILIPENDULION Segal 1966 Filipendulo Geranietum palustris W. Koch 1926 Ass. Scirpetum sylvatici Ralski 1931 (Syn.: Scirpetum sylvatici Schwickerath 1944, Scirpetum sylvatici Maloch 1935) Results and discussions The association has a large spreading in Neagra arului river basin, populating the rivers valleys on alluvial soils, at 824 1250 m altitude, on plane or easy sloping soils. Phytocoenosis of this association have been identified in Plaiul arului area, on Srioru Mic river valley, in Coverca area on Negru and Deluganu rivers valley, in Pltini area, in Gura Haitii area, on Tamu and Haita rivers valley.

Al. I. Cuza University, Faculty of Biology, Carol I Bd., no. 20A, 700506, Iasi, Romania

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As an observations, the association was first recorded in 1975, by M. Toma in his doctors theses, in aru Dornei with two releves, but we have to underline that this results didnt find again into a speciality publication. In 1989, it was also mentioned by D. Mititelu, without attaching a table of floristic releves. The floristic composition of the assocition is relatival richly in species, respective 45 species. Sirpus sylvaticus is the main species in this association, realizing coverings of 90-100%. In the analysed phytocoenosis were identified 5 species characteristic to Calthion alliance (11,11%), 8 species characteristic to Molinietalia caeruleae order (17,78%) and 10 species characteristic to Molinio Arrhenatheretea class (22,22%). The excess of humidiy favours the presence of some species characteristic to Filipendulion alliance and Phragmiti Magnocaricetea and Scheuchzerio Caricetea fuscae classes (tab. I). After the analysis of the releves, we notice the following: - the bioform spectrum shows the dominance of hemicryptophytes (75,56%) followed by geophyte (11,11%), hydro-helophytes (6,67%) and chamaephytes, phanerophytes and the therophytes in equal proportions (2,22% each); - the phytogeographic spectrum shows the predominance of Eurasian elements (42,22%), followed by circumpolar elements (28,89%), European (8,89%) and central element European (6,67%), cosmopolitan (11,11%) and alpine ones (2,22%); - within the spectrum of ecological indices, there is a predominance of species which have a low lever of tolerance of shade (51,11%), amphitolerant towards the temperature indice (44,45%), with a spreading area in oceanic climate (46,67%), adapted to excessive humidity (48,89%), amphitolerant to the soil reaction (48,49%) and to the content of mineral nitrogen in soil (20%). Ass. Epilobio Juncetum effusi Oberd. 1957 (Syn.: Ranunculus repens Juncus effusus Pauc 1941) The phytocoenosis enlightened by juncus effusus have been found on plane surfaces, with a humidity surplus of soil and a low content of nutritive substance, at an altitude of 900-1370 m, in Neagra arului, Srior, Coverca, Panaci and aru dornei area. The association was mentioned before in Neagra arului river basin by M. Toma, in 1975 (Neagra aruluI), presenting just a floristic list, and also by T. Seghedin in 1986 (Coverca) and D. Mititelu in 1989 (Panaci, Pltini), without attaching a table of floristic releves. the restrictive conditions from stations populated by this phytocoenosis are reflected by the presence of species characteristic Epilobium palustre and Juncus articulatus and dominant species Juncus effusus which is realizing coverings of average 95%, and also by the presence of species Deschampsia caespitosa in most of the analised phytocoenosis, sometimes realizing important covering. alongside of this, there are other species that are part of the Calthion alliance and Molinietalia order, but also of the Phragmiti Magnocaricetea and Scheuchzerio Caricetea fuscae classes, characteristics to the biotopes with a hight humidity of soil (tab. II). After the analysis of the releves, we notice the following:

122

- the bioform spectrum shows the dominance of hemicryptophytes (80%) followed by geophyte (12,72%), chamaephytes (4,25%), therophytes (2,13%) and hydrohelophytes (1,82%); - within the phytogeographic spectrum, one may notice the presence of a large number of Eurasian (40%) and circumpolar (36,37%) elements, followed by cosmopolitan and central European in equal proportions (7,27% each), European (5,45%) and alpine elements (3,64%); - within the spectrum of ecological indices, there is a predominance of species which have a low lever of tolerance of shade (50,91%), amphitolerant towards the temperature indice (52,73%), with a spreading area in oceanic climate (47,27%), adapted to excessive humidity (41,81%), amphitolerant to the soil reaction (49,09%) and in equal proportions, amphitolerant to the content of mineral nitrogen in soil and adapted to a low content of mineral nitrogen in soil (18,18% each). Ass. Filipendulo Geranietum palustris W. KOCH 1926 The association has been found on humid fields, with a high level of nitrates owing to a high antropo-zoological influence, at an altitude of 825-1200 m, in Plaiul aruluI, aru Dornei, aru Bucovinei, Srior, Coverca and Tarnia river valley. The association was not quoted before in the investigated area; it only was mentioned by D. Mititelu, in 1989, in Dornelor depression, but with no precise location. The main species in this association, Filipendula ulmaria, realize an average covering degree of 75-100%; in some of the analized phytocoenosis, the other dominant species, Geranium palustre, realize an important covering of 25% average. the hight humidity of soils populated by the investigated phytocoenosis, induces the presence of some species characteristic to the Filipendulion and Calthion alliances and Molinietalia order, also to the Phragmiti Magnocaricetea AND Scheuchzerio Caricetea fuscae classes (tab. III). After the analysis of the releves, we notice the following: - within the bioform spectrum, one may notice the net dominance of the hemicryptophytes (81,36%), followed by geophyte (8,48%), hydro-helophytes (5,08%), hemitherophytes (3,39%) and therophytes (1,69%); - within the phytogeographic spectrum, one may notice the presence of a large number of Eurasian elements (47,46%), followed by circumpolar (23,73%), cosmopolitan (11,87%), European (10,17%), central European (5,08%) and alpine elements (1,69%); - within the spectrum of ecological indices, there is a predominance of species which have a low lever of tolerance of shade (50,84%), amphitolerant towards the temperature indice (54,24%), with a spreading area in oceanic climate (44,07%), adapted to moderate to hight humidity (20,34%), amphitolerant to the soil reaction (55,93%) and to the content of mineral nitrogen in soil (23,73%).

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REFERENCES
1. CHIFU T., MNZU C., ZAMFIRESCU O., 2006 - Flora i vegetaia Moldovei (Romnia), vol. I, II, Edit. Univ. Al. I. Cuza Iai 2. CIOCRLAN V., 2000 - Flora ilustrat a Romniei Pteridophyta et Spermatophyta. Ed. Ceres, Bucureti 3. MITITELU D., CHIFU T., PASCAL P., 1989 - Flora i vegetaia judeului Suceava, Anuar. Muz. t. Nat. Suceava, t. Nat., 10: 93-120 4. MUCINA L., 1997 - Conspectus of classes of European vegetation. Folia Geobot. Phytotax (Praha), 32, 2: 117-172 5. POPOVICI D., CHIFU T., MITITELU D., CIUBOTARIU C., LUPACU GH., DAVIDESCU G., PASCAL P., 1996 - Pajitile din Bucovina, Edit. Helios 6. SANDA V., 2002 - Vademecum ceno-structural privind covorul vegetal din Romnia. Ed. Vergiliu, Bucureti 7. SANDA V., POPESCU A., BARABA N., 1997 - Cenotaxonomia i caracterizarea gruprilor vegetale din Romnia. St. i Comun. Muz. t. Nat. Bacu, Biol. veget., 14 8. SEGHEDIN T. G., 1986 - Flora i vegetaia munilor Bistriei, Rezumatul tezei de doctorat, Inst. Agr. Ion Ionescu de la Brad Iai 9. STOICA D. L., 2007 - Cercetri de geografie fizic pe versantul nordic al Masivului Climani. Rezumatul tezei de doctorat, Univ. Al. I. Cuza Iai 10. TOMA M., 1975 - Cercetri asupra florei i vegetaiei din Depresiunea Dornelor (jud. Suceava), Rezumatul tezei de doctorat, Univ. Babe - Bolyai Cluj-Napoca

Table I SCIRPETUM SYLVATICI RALSKI 1931


1 Number of relev 1209 Altitude (m.s.m.) 100 Covering (%) 50 Surface (m2) 15 No. of species Associations characteristics 5 Scirpus sylvaticus Calthion palustris + Caltha palustris + Chaerophyllum hirsutum Cirsium rivulare + Geum rivale + Myosotis scorpioides Deschampsion Carex ovalis Deschampsia caespitosa Juncus conglomeratus Filipendulion Equisetum arvense + Filipendula ulmaria Lysimachia vulgaris + Mentha longifolia Molinietalia caeruleae + Cirsium palustre + Equisetum palustre + Galium palustre + Juncus effusus Lychnis flos-cuculi Lythrum salicaria Symphytum officinale 2 920 100 100 15 5 + + + + + + + + + 3 910 100 100 17 5 + + + + + + + + + + 4 920 95 100 16 5 + + + + + + + + 5 1013 95 50 13 5 + + + + + + + + 6 1016 95 50 19 5 + + + + + + + + + + 7 1046 95 25 21 4 1 + + + + + + + + + + + + 8 1100 95 50 15 5 + + + + + + + + 9 1250 95 50 9 5 + + + + + + 10 1069 90 50 12 4 + + + 1 + + + + 11 831 90 100 11 5 + + + + 12 824 90 25 21 5 + + + + + + + + + + + V III II I III V III IV II II III II III IV III III V II I I K

124

ssp. officinale Trifolium hybridum + + + II ssp. hybridum Molinio Arrhenatheretea Agrostis stolonifera ssp. + + + + + III stolonifera + + + + II Alchemilla vulgaris + I Alnus incana juv. + + + + II Holchus lanatus + + + + + III Lysimachia nummularia + + I Phleum pratense + + I Prunella vulgaris + I Stellaria graminea Phragmiti - Magnocaricetea s. l. + + + + II Carex riparia + + I Epilobium palustre + I Glyceria notata + + I Lycopus europaeus + + + + + III Ranunculus repens + + + + + III Veronica beccabunga Scheuchzerio Caricetea fuscae s. l. + + + + II Carex flava + + + II Carex nigra ssp. nigra Variae syntaxa + I Epilobium montanum + I Festuca pratensis + I Luzula campestris + + + + II Potentilla erecta Rorripa sylvestris ssp. + I sylvestris + I Rumex crispus + + I Stellaria nemorum + + + II Valeriana tripteris Place and date of releves: 1. Tamu river valley (16.08.2007); 2, 3, 4. Srioru Mic river valley (14.07.2007); 5. Gura Haitii (20.08.2006); 6. Coverca Negru river valley (16.08.2006); 7. Coverca Deluganu river valley (16.08.2006); 8, 9. Neagra river valley (21.08.2006); 10. Pltini (13.07.2007); 11, 12. Plaiul arului (8.08.2007).

Table II EPILOBIO JUNCETUM EFFUSI OBERD. 1957


1 Number of relev 914 Altitude (m.s.m.) 95 Covering (%) 100 Surface (m2) 21 No. of species Associations characteristics Epilobium palustre + Juncus articulatus Calthion palustris Caltha palustris Chaerophyllum hirsutum Geum rivale + Myosotis scorpioides + Scirpus sylvaticus 2 920 95 50 19 + + + + 3 998 95 50 22 + + + + 4 1000 95 100 16 + + + + + 5 1124 95 50 25 + + + + + 6 1372 90 50 13 + + + 7 995 90 100 15 + + + + 8 993 90 50 17 + + + + 9 980 90 30 17 + + 10 906 90 100 18 + + III IV III I II V III K

125

Deschampsion + + Carex ovalis + + Deschampsia caespitosa + Juncus conglomerates Filipendulion Cirsium erysithales + + Filipendula ulmaria + + Mentha longifolia Molinietalia caeruleae + Cirsium palustre + Equisetum palustre + Galium palustre 5 5 Juncus effusus + + Succisa pratensis Trifolium hybridum ssp. hybridum Molinio Arrhenatheretea Agrostis capillaris Agrostis stolonifera ssp. stolonifera + Alchemilla vulgaris Anthoxanthum odoratum Briza media + Carex hirta + + Carex pallescens Cynosurus cristatus Dactylis glomerata Festuca rubra Holchus lanatus Luzula campestris + + Lysimachia nummularia Phleum pratense + + Prunella vulgaris Ranunculus acris + Rumex crispus Trifoiul repens ssp. repens Phragmiti Magnocaricetea s. l. Alisma plantago + aquatica Carex riparia Lycopus europaeus + Ranunculus repens Scheuchzerio Caricetea fuscae s. l. + + Carex flava Carex nigra ssp. nigra Eriophorum angustifolium Variae syntaxa Carduus personatus ssp. personatus + Cruciata glabra Epilobium montanum

+ + + + + 5 -

+ + + 5 -

+ + + + 5 + -

1 + 4 -

+ + 5 -

+ + + + 5 -

+ 2 + + + + 4 -

+ 1 + + + 4 +

III IV IV I II II II II IV V II I

+ + + + + + -

+ + + + + -

+ + + + + + -

+ 1 + + + -

+ + + + + + -

+ + + + + -

+ + + + -

1 + + + + +

I II IV II I I II I I I I II III I IV I I I

+ + + +

+ -

+ + + + -

+ + -

+ + +

+ + -

+ -

II I I III III II I

+ + -

+ -

I I II

126

+ I Equisetum sylvaticum + I Homogyne alpina + + I Hypericum maculatum + I Plantago media + + + + + + + + IV Potentilla erecta + + I Valeriana tripteris + + I Veratrum album Place and date of releves: 1. Neagra arului (14.08.2006); 2. Srioru Mic river valley (14.07.2007); 3. Coverca Climnel river valley (16.08.2006); 4. Coverca Negru river valley (16.08.2006); 5. Pltini (13.07.2007); 6. Panaci (18.082006); 7. Coverca Negru river valley (16.08.2006); 8. Coverca Bucini river valley (16.08.2006); 9. Panaci Rusului peak (18.08.2006); 10. on the outskirts Tinovului Mare aru Dornei (15.07.2007).

Table III Filipendulo Geranietum palustris W. Koch 1926


1 Number of relev 999 Altitude (m.s.m.) 100 Covering (%) 25 Surface (m2) 12 No. of species Associations characteristics 5 Filipendula ulmaria Filipendulion Chaerophyllum hirsutum Equisetum arvense Geranium palustre Lysimachia vulgaris Lythrum salicaria Mentha longifolia Calthion palustris Alchemilla vulgaris Briza media Caltha palustris Cirsium rivulare Geum rivale Myosotis + scorpioides Scirpus sylvaticus Deschampsion Carex ovalis Deschampsia caespitosa Juncus + conglomeratus Molinietalia caeruleae Cirsium oleraceum Cirsium palustre Equisetum palustre Galium palustre + Juncus effusus Lychnis flos-cuculi Succisa pratensis Molinio Arrhenatheretea Achillea millefolium 2 851 100 25 10 5 3 825 100 50 18 5 4 1037 95 50 10 5 5 1035 95 50 15 5 6 1016 90 25 18 5 7 1210 90 25 19 5 8 998 90 25 13 5 9 920 95 50 19 5 10 830 95 100 12 5 11 830 95 50 14 4 12 830 95 100 12 4 V K

+ + + + + -

+ + + + + + -

+ + + + -

+ + + + + + + +

+ + + + + + + -

+ + + + + + + + -

+ + + + + + + -

+ + + + + + + + +

1 + + + + +

2 + + + + + -

1 + + 1 + -

II I IV II III II III II I I II V III I IV II

+ + -

+ -

+ -

+ + + -

+ + + + +

+ + + -

+ + +

+ + -

+ + + -

+ + 1 + -

II II I I IV II II II

127

Agrostis stolonifera + + I ssp. stolonifera + I Ajuga reptans + + I Angelica sylvestris Campanula + + I glomerata + + I Centaurea jacea + + I Cynosurus cristatus + I Festuca pratensis + I Luzula campestris + + + II Phleum pretense + + + + II Prunella vulgaris + I Ranunculus acris + + I Rumex acetosa + I Rumex crispus Trifolium repens + + + + + III ssp. repens Phragmiti Magnocaricetea s. l. + + I Epilobium palustre + I Glyceria notata + + + + + III Ranunculus repens Veronica + I beccabunga Scheuchzerio Caricetea fuscae s. l. Carex nigra ssp. + I nigra + I Ligularia sibirica Variae syntaxa + I Agrimonia eupatoria Astragalus + I glycyphyllos Athyrium filix+ I femina + I Cirsium arvense Epilobium + I montanum Equisetum + I sylvaticum + + I Galeopsis tetrahit + I Holchus lanatus Hypericum + I perforatum Hypochoeris + I uniflora + I Mentha arvensis + + I Plantago media + + + II Potentilla erecta + + I Valeriana tripteris Place and date of releves: 1. Srior Sriorul Mare river valley (14.08.2006); 2. aru Dornei (14.08.2006); 3. Coverca Negru river valley (16.08.2006); 4. Coverca Deluganu river valley (16.08.2006); 5. Coverca Deluganu river valley (16.08.2006); 6. Coverca Bucini river valley (16.08.2006); 7. Tamu river valley (16.08.2007); 8. Plaiul arului (19.08.2006); 9. aru Bucovinei Srioru Mic river valley (14.07.2007); 10, 11, 12. Plaiul arului (8.08.2007).

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Analele tiinifice ale Universitii Al. I. Cuza Iai Tomul LIV, fasc. 1, s. II a. Biologie vegetal, 2008

PROTECTED TAXA FROM THE BISTRIA RIVER BASIN BETWEEN PIATRA NEAM AND BACU CARMEN AONCIOAIE
Abstract: The results presented in this article were obtained during a study made in 2005 and 2006 in the Bistria river inferior basin in the region between Piatra Neam and Bacu. As a result of this study were catalogued a number of 124 taxa of vascular plants in different categories of protection, using several specialty papers. Key words: Red List, Berne Convention, Bistria inferior basin, Bacu, Piatra Neam

Introduction The studied area belongs to the lower course of the Bistria river, being situated on two counties from Moldova region Neam and Bacu. Geomorphologicaly speaking, the studied region spreads on the following four natural units (1400 Km2): Oriental Carpathians (the Gomanu Mountains), the Moldavian sub-Carpathians with two subdivisions (Bistria sub-Carpathians and Cracu Bistria depression) and the Moldavian Plateau (a very small region between Racova and Bacu). The climate is temperate continental with variations, depending on the altitude of the relief and its particularities. The climate has excessive nuances in East and moderated nuances in West, presenting noteworthy variations with the altitude, with cold winters and hot, often dry, summers. The hydrographical network is represented by Bistria and its affluents. The most important affluent in his sector is Cracu, followed by a number of streams like Calu, Iapa, Nechit, Trebi, Negel and so on. Building hydro-electric power stations on the river leaded to the appearance of some artificial lakes like Bacu, Buhui, Grleni, Lilieci and Racova. Material and methods For analyzing the vascular flora of the region were used the classical methods and materials for this kind of research. The working stages begun with documentation and study of the bibliography, followed by a terrain research stage and then a herbarium stage, finally ended with a stage of data interpretation and a complete list of taxa. Among the identification guides used are: Flora Romniei (vol. I XIII) 1952 1976; Ciocrlan V. Flora ilustrat a Romniei. Pteridophyta et Spermatophyta., 2000; Srbu I. et al. Flora ilustrat a plantelor vasculare din estul Romniei (vol. I and II) 2001.

Al. I. Cuza University, Iai, Faculty of Biology, Carol I Bd., no. 20A, 700506, Iasi, Romania

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There were used two papers for including the taxa in different protection categories: Gh. Dihoru, Alexandrina Dihoru Plante rare, periclitate i endemice n flora Romniei Lista Roie, 1993 1994 and M. Oltean, G. Negrean, A. Popescu et al. Studii, sinteze, documentaii de ecologie, I/1994. Results and discussions At the moment, the floristic inventory (of the studied region) has a number of 1.436 taxa among witch there are species included in diverse categories of protection. These are presented below from the point of view of two well known scientific papers (from Romania) and also after the Berne Convention. The first column of the table presents the species registered in Studii, sinteze, documente de ecologie Lista Roie a plantelor superioare din Romnia by Oltean M., Negrean G., Popescu A., Roman N., Dihoru G., Sanda V., Mihilescu S. (1994) and the second column presents the analysis made after Plante rare, periclitate i endemice n flora Romniei Lista Roie by Dihoru Gh., Dihoru Alexandrina (1993 1994). Both columns of the table gather a number of 124 taxa. After the paper by Oltean M. et al. a number of 95 taxa are protected and after the paper by Dihoru Gh. only a number of 58. There is a difference of 37 taxa between the two papers and different visions on many species. Only 29 taxa are common in both papers and even about the protection category there are different opinions. This problem may occur because of the great variety of the Romanian territory and a list of protected species should be made for each region of the country, knowing that stationary factors are different and in certain places certain species may not be endangered (tab. I). Another analysis was made after the data presented by the Berne Convention (1979) to witch Romania adhered in 1993. The analysis showed that a number of 5 strictly protected species are present in the territory: Cypripedium calceolus L. Eleocharis carniolica Koch. Salvinia natans (L.) All. Typha minima Funk in Hoppe Typha schuttlewortii Koch et Sonder. Conclusions This article presents a number of 124 taxa of vascular plants from diverse categories of protection, representing 8,63 % out of the total number of taxa identified in the studied region.

130

REFERENCES
BELDIE AL., 1977 - Flora Romniei. Determinator ilustrat al plantelor vasculare. Vol. I, II, Ed. Acad. R.S.R., Bucureti 2. CIOCRLAN V., 2000 - Flora ilustrat a Romniei. Pteridophyta et Spermatophyta. Ed. Ceres, Bucureti 3. DIHORU GH., DIHORU A., 1993 1994 - Plante rare, periclitate i endemice n flora Romniei Lista Roie, Acta Botanica Horti Bucurestiensis, Bucureti, 1994: 173-179 4. DIHORU G., PRVU C., 1987 Plante endemice n Flora Romniei. Ed. Ceres, Bucureti 5. MITITELU DUM., BARABA N., TEFAN N. 1987. Contribuii la corologia unor plante rare n Moldova i Muntenia. An. t. Univ. Al. I. Cuza Iai , s. II, a., Biol. veget., t. XXXIII: 20 24 6. OLTEAN M. et al., 1994 - Lista roie a plantelor superioare din Romnia. Studii., Sinteze, Documentaii de Ecologie, Acad. Rom. 7. POPESCU A., SANDA V., 1966 - Consideraii corologice asupra plantelor endemice din Romnia. St. Cerc. Biol., ser. Bot., 18, 5;;437 466 8. RUGIN R., MITITIUC M., 2003 - Plante ocrotite n Romnia. Ed. Univ. Al. I. Cuza Iai 9. SRBU I., TEFAN N., IVNESCU LCRMIOARA, MNZU C., 2001 Flora ilustrat a plantelor vasculare din estul Romniei. Vol. I, II, Ed. Univ. Al. I. Cuza Iai 10. OPA E., 1979 - Ocrotirea naturii n Judeul Neam. Reflexii, istoric, realizri i sugestii. **Conventia de la Berna (1979) *** 1952 1976 Flora R. P. R-r. S. R., I XIII. Ed. Acad. R.S.R., Bucureti 1.

Table I: Protected taxa category E R R K R I R R R VR M. Oltean et al. specie Abies alba Miller Allium schoenoprasum L. ssp. sibiricum (L.) Hartman Anacamptys pyramidalis (L.) L.C.M. Richards Bromus racemosus L. Campanula carpatica Jacq. Carex brevicollis DC. Carex dioica L. Carex hallerana Asso Centaurea melanocalathia Borbs Centaurium littorale (D. Turner) Gh. Dihoru, Alexandrina Dihoru category specie R Adonis flammea Jacq. V Adonis vernalis L. R K K I (R) R R Alopecurus arudinaceus Poiret Bromus racemosus L. Bryonia dioica Jacq. Carex brevicollis DC. Carex dioica L. Centaurium littorale (D. Turner)

131

V V R V R R R VR V R VR R R R R R R VR R R R R R

Gilmour ssp. uliginossum (W et K) G. Beck Cephalanthera damassonium (Miller) Druce Cephalanthera longifolia (L.) Fritsch Cephalanthera rubra (L.) M.L. C. Richards Cirsium furiens Griseb. et Schenk Cirsium grecescui Griseb et Schenk Coeloglossum viride (L.) Hartman Colutea arborescens L. Corynephorus canescens (L.) P. Beauv. Crocus reticulatus Steven Cyperus serrotinus Rottb. Cypripedium calceolus L. Dactylorhizza incarnata (L.) So Dactylorhizza maculata (L.) So ssp. maculata Dactylorhizza maculata (L.) So ssp. schurii (Klinge) So Dactylorhizza sambucina (L.) So Dianthus collinus Waldst et Kit ssp. collinus Dianthus collinus Waldst et Kit ssp. glabriusculus (Kit) Thaisz Dictamnus albus L. Dryopteris cristata (L.) A.Gray Epipactis helleborine (L.) Crantz Epipactis palustris (L.) Crantz Epipactis purpurata Sm. Eryssimum witmannii Zawadzki ssp. transilvanica (Schur) P.W. Ball

K R E V I R V -

Gilmour ssp. uliginossum (W et K) G. Beck Cirsium furiens Griseb. et Schenk Cirsium grecescui Griseb et Schenk Corynephorus canescens (L.) P. Beauv. Cypripedium calceolus L. Dianthus collinus Waldst et Kit ssp. glabriusculus (Kit) Thaisz Dryopteris cristata (L.) A.Gray Eleocharis carniolica Koch -

132

K VR nt nt K nt V R R R R R VR R R R R R R R R R R R

Fragaria moschata Duchesne Fritillaria orientalis Adams Galanthus elwesii Hooker fil. Galanthus nivalis L. Galium sylvaticum L. Hepatica transsilvanica Fuss. Hippuris vulgaris L. Gymnadenia conopsea (L.) R. Br. Herniaria hirsuta L. Koeleria macrantha (Ledeb) Schultes ssp. transsilvanica (Schur) A. Nyr. Lactuca virosa L. Lapulla deflexa (Lehm.) Cesati Lepidium cartilagineum (J.Mayer) Thell ssp. crassifolium (W. et K.) Thell Listera ovata (L.) R. Br. Luzula pallescens Swartz. Melmpyrum saxosum Baumg. Monotropa hypopytis L. Myosotis stenophylla Knaf. Neottia nidus-avis (L.) L.C.M. Richards Oenanthe peucedanifolia Pollisch Orchis coriophora L. Orchis laxiflora Lam. ssp. elegans (Heuffel) So Orchis mascula (L.) L. ssp. signifera (Vest) So Orchis militaris L.

R E R R R nt R R R nt R V R R R R -

Evonymus latifolius (L.) Miller Galanthus elwesii Hooker fil. Galium tenuissimum Bieb. Gladiolus imbricatus L. Goodyera repens (L.) R. Br. Hepatica transsilvanica Fuss. Iris aphylla L. Iris sibirica L. Isolepis setacea (L.) R. Br. Koeleria macrantha (Ledeb) Schultes ssp. transsilvanica (Schur) A. Nyr. Lactuca virosa L. Lepidium cartilagineum (J.Mayer) Thell ssp. crassifolium (W. et K.) Thell Luzula pallescens Swartz. Melampyrum nemorosum L. Myosotis discolor Pers. Myosotis stenophylla Knaf. -

133

R R R R R R R R R R R R R R R R R R R R R R VR -

Orchis morio L. ssp. morio Orchis purpurea Hudson Orchis ustulata L. Orobanche lucorum A. Braun Pinus sylvestris L. Plantago schwarzenbergiana Schur Platanthera bifolia (L.) L.C.M. Richards Platanthera clorantha Pleurospermum austriacum (L.) Hoffm. Potamogeton trichoides Cham. et Schlecht. Primula elatior L. ssp. leucophylla (Pax.) H. Harrison ex W.W. Sm. et Fletcher Pulsatilla grandis Wenderoth Rhynchospora alba (L.) Vahl. Ribes spicatum Robson Rorippa islandica (Oeder) Borbs Rosa micrantha Sm. Salix aurita L. Salix daphnoides Vill. Salvinia natans (L.) All Scirpus radicans Schkuhr Sempervivum zeleborii Schott Serratula radiata (Welk) Bieb. Seseli hippomarathrum Jacq. -

V R R nt R R R R R R R R R R K E

Plantago cornuti Gouan. Pleurospermum austriacum (L.) Hoffm. Potamogeton trichoides Cham. et Schlecht. Primula elatior L. ssp. leucophylla (Pax.) H. Harrison ex W.W. Sm. et Fletcher Pyrola chlorantha Swartz Rhynchospora alba (L.) Vahl. Ribes spicatum Robson Rorippa prolifera (Heuffel) Weiche Rubus glandulosus Bellardi Rumex aquaticus L. Rumex longifolius DC. in Lam. et DC. Sedum caespitosum (Cav.) DC. Seseli tortuosum L. Seseli hippomarathrum Jacq. Silene italica Retz. ssp. italica Sisymbrium altissimum L.

134

R R R R VR K V R R R VR R K R R

Sorbus aria (L.) Crantz. Spirea crenata L. Stellaria palustris Retz. Symphytum tauricum Willd. Taxus baccata L. Thymus serpyllum L. Traunsteinera globosa (L.) Reichenb. Trisetum macrotrichum Hackel Trollius europaeus L. ssp. europaeus Typha minima Funk in Hoppe Typha shuttlewortii Koch et Sonder Vicia peregrina L. Vicia tenuifolia Roth. Viola jooi Janka Wolffia arrhiza (L.) Horkel ex Wimmer

E E E E R nt V V R R R -

Sisymbrium irio L. Sisymbrium loeselii L. Sisymbrium officinale (L.) Scop. Sisymbrium strictissimum L. Taxus baccata L. Thymus comosus Heuffel Thymus serpyllum L. Trollius europaeus L. ssp. europaeus Typha minima Funk in Hoppe Vicia peregrina L. Viola jooi Janka -

Abbreviations: I- indeterminate species and subspecies R- rare species and subspecies V- vulnerable species and subspecies Ex - extinct species and subspecies K less known species and subspecies nt - not endangered endemic species and subspecies P- endangered species and subspecies

135

Analele tiinifice ale Universitii Al. I. Cuza Iai Tomul LIV, fasc. 1, s. II a. Biologie vegetal, 2008

CONTRIBUTIONS TO THE STUDY OF THE CLASS MOLINIO-ARRHENATHERETEA R. TX. 1937 IN THE UPPER BASIN OF RIVER DORNA (SUCEAVA COUNTY) (I) MIHAELA AURELIA DANU , T. CHIFU
Abstract: This paper represents an analysis of 2 vegetal associations (Agrostideto Festucetum pratensis So 1949 and Festuco rubrae Agrostietum capillaris Horvat 1951) classified from the coenotaxonomical point of view in the class Molinio-Arrhenatheretea R. Tx. 1937. The phytocoenoses of these mesophilic association, identified on the territory of the upper basin of the river Dorna (district of Suceava), are described from both the phytocoenological point of view, as well as from the point of view of the bioforms, floristic elements and ecological indices. Key words: class Molinio-Arrhenatheretea, mesophilic phytocoenoses, upper basin of Dorna.

Introduction The upper basin of the river Dorna is located in the south-west part of the district of Suceava. Integrated in the central-northern part of the Oriental Carpathians, the basin is characterized by a temperate continental climate, the average annual temperatures being low (4.2C). The average value of precipitations is over 740 mm/an. In the area can be identified different types of soil belonging to 7 classes: non-evolved soils, truncated or cleaned, hydromorph soils, cambisoils, spodosoils, muddy luvisoils, shadowed soils, hysto soils. The vegetal associations analysed in this paper have not been noticed so far in the upper basin of the river Dorna. Material and methods For the study of the vegetation we used the method of the phytocoenological School in Zurich-Montpellier, perfected by J. Braun-Blanquet and J. Pavillard. On taking into consideration few phytosociological papers of classification [7], [8], [9], [10], the associations were framed in the following coenosystem: Cls. Molinio Arrhenatheretea R. Tx. 1937 Ord. Molinietalia caeruleae Koch 1926 Al. Alopecurion pratensis Passarge 1964 As. Agrostideto Festucetum pratensis So 1949 Ord. Arrhenatheretalia R. Tx. 1931 Al. Cynosurion R. Tx. 1947 As. Festuco rubrae-Agrostietum capillaris Horvat 1951

Al. I. Cuza University, Faculty of Biology, Carol I Bd., no. 20A, 700506, Iasi, Romania

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As. Agrostideto Festucetum pratensis So 1949 Corollogy: Piatra Fntnele Ecology. The phytocoenoses of Festuca pratensis and Agrostis stolonifera were identified in the upper basin of the river Dorna at altitudes between 1100 and 1130 m, on fields with slopes between 2 and 20, having a cover of vegetation between 80 and 100%. The phytocoenoses of this association are developed on humid soils, with high humidity especially in the vernal season. The floristic and phytocoenological characterization. The floristic composition of the association is rich (51 species), varied, with numerous mesophilic and meso-eutrophic species. The mesophilic character of the association is underlined also by the presence in big number of some species characteristic for the order Arrhenatheretalia: Achillea millefolium ssp. millefolium, Carum carvi, Dactylis glomerata, Leucanthemum vulgare ssp. vulgare, etc., species with a high constancy (tab. I). As for the spectre of bioforms, the basic found of these meadows is represented by hemicryptophytes (H.-70.59%) and, in proportion of over 11%, by terrophytes (T.-11.76%) (fig. 1 a). From the phytogeographical point of view, the important contribution in the flora composition is brought by the Euro-Asian and European elements (fig. 2 a). The spectre of ecological indices shows that, due to high humidity of the underlayer, in these phytocoenoses are present many (over 45%) mesohygrophilic species (Agrostis stolonifera, Festuca pratensis ssp. pratensis, Lychnis flos-cuculi, Rhinanthus angustifolius ssp. angustifolius, Alchemilla vulgaris). The analysis of the spectre of ecological indexes underlines also the fact that in the structure of these phytocoenoses are dominant the species of light, which prefer weakly the shadow (over 90%); as for the temperature, many species are amphitolerant, but over 30% are plants characteristic to the chilly areas. Concerning the content of mineral nitrogen, half of the species identified in these phytocoenoses prefer the soils with low content till moderate (fig. 2 b). As. Festuco rubrae Agrostietum capillaris Horvat 1951 The meadows with Festuca rubra and Agrostis capillaris have a wide spread in the Romanian Carpathians, on the coasts moderately sloped, with regime of moderate humidity, with brown rainy and brown acid soils, moderate-low acid and with a moderate content of nutritive substances. The species characteristic and representative, Agrostis capillaris and Festuca rubra, are in a proportion of co-dominance, according to the content of nutritive substances in the soil and the degree of aeration of the soil. Thus, the species Agrostis capillaris is dominant on the fields recently covered with herbal vegetation and fertilized, while Festuca rubra dominates on the fields more beaten and less rich in nutritive substances. Corollogy: Piatra Fntnele Ecology. The meadows with the phytocoenoses of this association were identified at altitudes between 1100 and 1150 m, on soils slightly sloped (1-20), having a cover of vegetation between 80% and 100%.

137

The floristic and phytocoenological characterization. The floristic composition is rich (62 species) and varied, being noticed the predominance of the species characteristic for the alliances Cynosurion (Bellis perennis, Cynosurus cristatus, Phleum pratense, Trifolium repens ssp. repens, Prunella vulgaris), Arrhenatherion (Campanula patula, Centaurea phrygia, Taraxacum officinale), order Arrhenatheretalia (Achillea millefolium ssp. millefolium, Briza media, Carum carvi) i clasei Molinio Arrhenatheretea (Anthoxanthum odoratum, Cerastium holosteoides, Euphrasia officinalis ssp. pratensis, Rhinanthus minor, Stellaria graminea, Trifolium pratense), in proportion of 62,9%, which denotes the character mainly mesophilic of this association. In the frame of the phytocoenoses of this association, there were also identified, in reduced proportions, species characteristic to the class Festuco Brometea (12.9%), Calluno Ulicetea (9.67%) and Juncetea trifidi (6.45%) (tab. II). The spectre of bioforms is dominated by hemicryptophytes (H.) situation underlined by the percentage of 74.19%. Follow the hemiterrophytes (Ht.) that realize 9.68%, and in lower quantities are represented the species terrophytes (T.) (9.68%), geophytes (G.) and camephytes (Ch.) being represented in equal proportion (each 4.84%) (fig. 1 b). Among the elements of flora, 50% belong to the Euro-Asian element (Euras.); follow the European element (Eur.) represented by 30.65% and the cosmopolite species (Cosm.) with 8.06%. The circumpolar species (Circ.) are represented equally as the alpine elements (each 3.23%); the pontic, Carpathian Balkan elements and endemic elements have one representative each in these phytocoenoses (fig. 3 a). The spectre of ecological indices indicates the following preferences of the species to the ecological factors: from the point of view of the preferences to light, the heliophylic species, which bear weakly the shadow, are dominant (over 90%). From the point of view of the temperature, dominate the species amphitolerant (62.9%), and almost 30% are mesothermal plants. The association has a mesophylic character (demonstrated by the presence of the species developed on soils moderately humid), 33.87% being mesophylic plants. We notice that 37.15% among the species are xerophylic, which can be explained by the fact that some phytocoenoses are located on sloped surfaces, with reduced capacity of retaining water. As for the preference of plants for the pH of the soil, over 53% are euriionic, and 24.19% are acidophilic and moderate-slight acidophilic plants. Most of the species belonging to the phytocoenoses develop on soils poor in mineral nitrogen (51.61%), 22.58% among the species prefer the soils with content of mineral nitrogen varying from moderate to excessive, and almost 21% of the species are amphitolerant from this point of view (fig. 3 b). REFERENCES
1. 2. BELDIE AL., 1977 - Flora Romniei Determinator ilustrat al plantelor vasculare, vol. I-II, Edit. Acad. R.S.R., Bucureti CHIFU T., 1995 - Contribuii la sintaxonomia vegetaiei pajitilor din clasele Molinio Arrhenatheretea Tx. 37 i Agrostietea stoloniferae Oberd. in Oberd. et al. 67 de pe teritoriul Moldovei. Bul. Grd. Bot. Iai, 5: 125-132

138

3.

CHIFU T., MNZU C., ZAMFIRESCU O., 2006 - Flora i vegetaia Moldovei, vol. II, Ed. Univ. Al. I. Cuza Iai 4. CHIRI V., 2003 - Depresiunea Dornelor: studiu fizico geografic. Edit. Univ., Suceava 5. CIOCRLAN V., 2000 - Flora ilustrat a Romniei Pteridophyta et Spermatophyta, Edit. Ceres, Bucureti 6. ELLENBERG H., 1974 - Indicator values of vascular plants in Central Europe, Scripta Geobotanica, vol. IX, Verlag Erich Goltze K.G., Gttingen: 1-97 7. SANDA V., 2002 - Vademecum ceno-structural privind covorul vegetal din Romnia, Edit. Vergiliu, Bucureti 8. SANDA V., POPESCU A., ARCU M., 1999 - Revizia critic a comunitilor de plante din Romnia, Edit. Tilia Press International1, Constana 9. SANDA V., POPESCU A., BARABA N., 1997 - Cenotaxonomia i caracterizarea gruprilor vegetale din Romnia. St. i Com. Muz. t. Nat. Bacu, Biol. veget., 14: 2-365 10. SANDA V., POPESCU A., STANCU D. I., 2001 - Structura cenotic i caracterizarea ecologic a fitocenozelor din Romnia, Edit. Conphis, Bucureti

Table I - As. Agrostideto Festucetum pratensis So 1949


Relev number 1 Altitude (m) 1130 Exposition E Slope () 5 Cover of the vegetation (%) 90 Surface of the relev (m) 100 Number of species 43 Caract. as. Agrostis stolonifera 3 Alopecurion pratensis Festuca pratensis ssp. pratensis 1 Phleum pratense + Molinion caeruleae et Molinietalia caeruleae Briza media + Gymnadenia conopsea ssp. conopsea + Lychnis flos-cuculi Arrhenatherion Campanula patula Centaurea phrygia 1 Cynosurion Cynosurus cristatus + Leontodon autumnalis ssp. + autumnalis Trifolium repens ssp. repens Arrhenatheretalia Achillea millefolium ssp. + millefolium Carlina acaulis ssp. acaulis + Carum carvi + Crepis biennis + Dactylis glomerata Heracleum sphondylium ssp. + sphondylium Knautia arvensis ssp. arvensis 2 1125 V 15 95 100 36 3 2 + + + + + 1 + + + + + + + 3 1125 NE 2 100 100 28 2 3 + + + + + + + + + 4 1100 E 20 90 100 34 3 2 + + + + + + + + 5 1120 NV 10 80 100 24 3 2 + + + + 1 + + 6 1120 V 10 85 100 27 3 1 + + + + + 1 + + + -

K V V IV IV II II IV IV V III II V V IV II III III II

139

Leucanthemum vulgare ssp. vulgare Tragopogon pratensis ssp. orientalis Thymus pulegioides Veronica chamaedrys ssp. chamaedrys Molinio Arrhenatheretea Anthoxanthum odoratum Campanula glomerata ssp. glomerata Cerastium holosteoides Euphrasia officinalis ssp. pratensis Lotus corniculatus Plantago lanceolata ssp. lanceolata Polygala vulgaris Ranunculus acris ssp. acris Rhinanthus angustifolius ssp. angustifolius Rumex acetosa Trifolium pratense Vicia cracca Potentillo Nardion Arnica montana Cruciata glabra Scorzonera rosea Juncetea trifidi Campanula serrata Dianthus deltoides Hypochaeris uniflora Nardus stricta Potentilla erecta Festuco Brometea Plantago media Trifolium alpestre Trifolium pannonicum Variae syntaxa Alchemilla vulgaris Galeopsis speciosa Hypericum maculatum ssp. maculatum Stellaria media Tanacetum corymbosum Viola tricolor

+ + + + + + + + + + 1 + + + + + + + + + + + + + 1 + + + + +

1 + + + 1 + + + + + + + + + + + + + + + + -

+ + + + 1 + + + + + + 1 + + + + + -

1 + 1 + + + + + + + + + + + + + + + + + + + +

+ + + + + + + + + + + + + + + -

+ + + + + + + + + + 1 + + + + + -

V IV V III V II IV V V IV III IV V III IV III II III II II II V V V III V II V II III II II II

Place and date of the releves: Piatra Fntnele 1 - 27.07.2006; 2, 3, 4 - 20.08.2006; 5, 6 - 1.09.2006

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Table II - As. Festuco rubrae Agrostietum capillaris Horvat 1951


Relev number Altitude (m) Exposition Slope () Cover of the vegetation (%) Surface of the relev (m) Number of species Caract. as. Agrostis capillaris Festuca rubra Cynosurion Bellis perennis Cynosurus cristatus Leontodon autumnalis ssp. autumnalis Phleum pratense Plantago major ssp. major Prunella vulgaris Trifolium repens ssp. repens Veronica serpyllifolia ssp. serpyllifolia Arrhenatherion Campanula patula Centaurea phrygia Taraxacum officinale 1 1130 V 5 95 100 30 1 3 + + + 2 1140 E 5 95 100 32 2 3 + + + + + + + + + + 3 1140 S 5 95 100 26 3 2 + + + + + 4 1100 NV 1 80 100 24 4 2 + + + + + + + + 5 1100 NV 3 90 100 37 + 4 + + + + 6 1125 NE 20 90 100 29 1 4 + + + + 7 1150 E 10 90 100 26 3 2 + + + + + + 8 1148 V 10 95 100 36 3 2 + + + + + + + + 9 1145 S 2 100 100 24 2 3 + + 1 + 10 1150 V 2 100 100 26 1 4 1 1 + + 11 1150 E 10 90 100 29 1 4 + + + + + + -

K V V II V II III II IV IV II IV III III

141

Phyteumo Trisetion Hypericum maculatum ssp. maculatum Luzula luzuloides ssp. luzuloides Arrhenatheretalia Achillea millefolium ssp. millefolium Briza media Campanula glomerata ssp. glomerata Carlina acaulis ssp. caulescens Carum carvi Leucanthemum vulgare ssp. vulgare Knautia arvensis Thymus pulegioides Tragopogon pratensis ssp. orientalis Molinietalia Gymnadenia conopsea ssp. conopsea Lychnis flos cuculi Molinio Arrhenatheretea Alchemilla vulgaris Anthoxanthum odoratum Centaurea jacea Cerastium holosteoides Euphrasia officinalis ssp. pratensis Lotus corniculatus Plantago lanceolata ssp. lanceolata

+ + + + + + + 1 + +

+ + + + + + + + + + + +

+ + + + + + + + -

+ + + + + +

+ + + + + 1 + + + + + + + +

+ + + + + + + + + +

+ + + + + 1 + + +

+ + + + + + + + 1 + + + +

+ + + 1 + + + + 1 + -

+ + + + + + + + + -

+ + + + + + + + + + + + + -

III II IV V I IV II V II V II II I V V III I III IV IV

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Polygala vulgaris Ranunculus acris ssp. acris Rhinanthus minor Rumex acetosa Stellaria graminea Trifolium pratense Festuco Brometea Anthyllis vulneraria ssp. vulneraria Echium vulgare Euphrasia stricta ssp. stricta Galium verum Hieracium pilosella Ranunculus polyanthemos ssp. polyanthemoides Trifolium alpestre Trifolium pannonicum Juncetea trifidi Campanula serrata Nardus stricta Potentilla ternata Scorzonera rosea Calluno Ulicetea Antennaria dioica Arnica montana Dianthus deltoides Genista tinctoria ssp. tinctoria Gentianella austriaca Potentilla erecta

+ 1 + + + + + + + + + + + -

1 + + + + + + + -

+ + 1 + + + + + + +

+ + + 1 + + -

+ + + + + + + + + + + + + + 1

+ + + + + + + + + + + +

+ + + + + + 1

+ + + + + + + + + + +

+ + + + + + +

+ + + + + + + + + +

+ + 1 + + + +

III II V II I III III I I II III II IV II III I III II I II II II II IV

143

Variae syntaxa Cirsium vulgare Cruciata glabra Pteridium aquilinum Rumex acetosella ssp. acetosella Tanacetum corymbosum ssp. corymbosum

+ +

+ -

+ + -

+ + -

+ -

+ +

+ + -

+ -

+ -

+ -

II II II II I

Place and date of the releves: 1 - Piatra Fntnele, 27.07.2006; 2 - 11 - Piatra Fntnele, 21, 22.08.2006

H - 74,19%
T - 11,76%

T - 6,45%

Ht - 9,68%
H - 70,59% Ht - 9,8%

Ch - 3,92% G - 3,92%

Ch - 4,84%

G - 4,84%

Fig. 1. The bioforms spectrum: a) as. Agrostideto-Festucetum pratensis So 1949; b) as. Festuco rubrae-Agrostietum capillaris Horvat 1951

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Circ. - 18,35% Centr. eur. 9,91%

Pont. - 1,47% Medit. -0,37% Atl. - 0,92%

Eur. - 12,11% Dacic - 0,18% Euras. 44,77%

% 80 70 60 50 40 30 20 10 0 1 2 3 4 5 6 7 b 8 9 10 11 x ?

L T Ct U R N

Fig. 2. a) The floristic elements spectrum; b) The ecological indices spectrum (L-light; T-temperature; Ct-continent; U-humidity; R-soil reaction; N-nitrogen) as. Agrostideto-Festucetum pratensis So 1949

Euras. - 50%

% 70 60 L T Ct U R N 1 2 3 4 5 6 b 7 8 9 x ? 50 40
Eur. - 30,65%

Cosm - 8,06%

30 20 10 0

End. - 1,61% Alp. - 3,23% Carp. balc. 1,61%

Circ. - 3,23% Pont. - 1,61%

Fig. 3. a) The floristic elements spectrum; b) The ecological indices spectrum (L-light; T-temperature; Ct-continent; U-humidity; R-soil reaction; N-nitrogen) as. Festuco rubrae-Agrostietum capillaris Horvat 1951

Analele tiinifice ale Universitii Al. I. Cuza Iai Tomul LIV, fasc. 1, s. II a. Biologie vegetal, 2008

ENVIRONMENTAL EDUCATION: EDUCATION FOR TRANSITION TO SUSTAINABLE DEVELOPMENT I. M. CIUMAU , NAELA COSTIC
Abstract: It is general accepted that the principles of sustainable development cant be reached without education, public awareness and training. In this regard, the present paper presents rationales and approaches in Environmental Education, as well as the importance of institutional and curricular aspects in implementing this type of education. Key words: Environmental Education, institutional and curricular aspects.

Rationales and educational approaches While civilization is being maintained through institutions, it is kept alive and growing through education. As the current unsustainability of humanity resides the inharmonious (conflicting) relationship between nature and human society, we need environmental education. Therefore we need a coherent program to train environmental educators. Chapter 36 of Agenda 21 calls each nation to bring together experts from various disciplines to prepare a national strategy for environmental education (EE) and training [33, 28, 17]. Lucas [16, 7] identified three meanings/facets of environmental education: 1. education about the environment (concerned with cognitive understanding of environmental issues); 2. education for the environment (concerned with environmental protection via particular purposes and aims); 3. education in the environment (concerned with environmental experience as educational mean outside the classroom). In EE institutions, the organisational strategy and the curricular strategy should be complementary. The first has a greater effect on values, attitudes and behaviour, whereas the second influence more the conceptual/knowledge understanding. EE is not effective if the organisational strategy contradicts the curricula [30]. For example, an Integrated

Al. I. Cuza University, Faculty of Biology, Carol I Bd., no.11, 700506, Iai, Romania

Associated researcher at: Al. I. Cuza University, Faculty of Biology, Centre of expertise on sustainable exploitation of ecosystems (CEXDUREC), B-dul Carol I no.11, 700506, Iai, Romania. and Technische Universitt Mnchen, Arcistrasse 21, 83000, Mnchen, Deutschland, Wissenschaftszentrum Weihenstephen fr Ernhrung, Landnutzung und Umwelt, Biowissenschaftlische Grundlagen, Alte Akademie 8, 85354, Freising, Deutschland, Lehrstuhl fr kologische Chemie und Umweltanalytik Weiehnstephaner Steig 23, 85350, FreisingWeihenstephan, Deutschland.

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Action Model [7] can be used to identify motivational profile of students (this also includes age categories). These can be used together with social and situational conditions to design adapted curricula for different categories of students (target groups). However, such precision must not be applied at the expense of community-based cooperation in EE, but in the same time with it (and integrating it). There are four possible target groups in Environmental Education, as related to their personality: 1. The Technical Group needs to know how to gauge environmental parameters; 2. The Subject Specialist Group needs to understand environmental systems; 3. The Management Group requires skills and abilities to resolve complex environmental problems; 4. The Lay Group needs to have attitudes, philosophies and values about the environment. The old-fashion one-direction model of teaching is already outdated (though it resists in many countries). The teacher must accept that, in the Internet era, he cannot hold control of the learning as in former times (when he was the one possessing information). On the contrary, the teacher must extend teaching beyond the walls of the classrooms, and capture students' attention in more subtle ways, on the basis of reciprocal respect of the other and of the common values. This is of course not to say that the teacher-student relationship should be loose. The do-ityourself learning (a lessez-faire approach) based on technology-in-the-classroom must be backed by reciprocal assuming of responsibility for the learning process. Otherwise, internet-chatting and other free-time activities will replace learning. Thus, internet in the classroom does not automatically bring educational progress [10]. An effective education approach is the two-ways, dialogical lesson. This dialogue requirement holds also when environmental teaching kits are being used, eventually in combination with web-based and other methods and materials. As such kits tend to be more employed at lower ages [4], they could be employed more heavily in kindergartens and progressively be replaced at older school ages with web-based lessons of appropriate difficulty. Given the ever changing and enlarging context of the teacher-student relationship, any curriculum must be rather elaborated for educating the teacher how to educate the student. Such "rehearsal curriculum" must be written in a way that motivates the teacher to learn and update and diversify its skills [11, 24]. Thus, in order to structure a problem-based EE, various types of questions can be identified and labeled [6]: - encyclopaedic, - meaning-oriented, - relational, - value-oriented, - solution-oriented

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For example, web-based systems for environmental management [21, 29] can (and should) be introduced in classrooms with internet availability through questions and dialogue. There exist even international web-based, hands-on EE programs, e.g. Global Learning and Observations to Benefit the Environment GLOBE is such a program which allows school children to be involved in a dynamic student-teacher-scientist partnership: they learn about the environment by taking carefully supervised scientific measurements of their natural surroundings (land cover, soil, hydrology, phenology, haze/aerosols, and atmosphere) and sharing information/data with scientists and other students in remote locations via Internet [34]. More, there have already been experimented internet-based, inter-institutional teaching systems, linking education institutions with with actors in the socio-economic environment (local authorties, companies, etc). In sucha case has a profoundly applied character, because it also includes the real world decision-taking processes [18]. The children's sense of participation and discovery is the best medium for EE and for nurturing responsible attitudes towards the environment. We will come back to this aspect when addressing "community-based learning". Institutional aspects We should no be left with the impression that there are no limits to this approach. For example, the idea of green schools looks shiny, but is dangerous. The big risk is that creating a new category of school out of the mainstream education will ascribe to EE a marginal importance in the collective perception of the citizens. In fact, EE is still largely without focus and side-lined [13, 27, 5]. I mean we must learn from the mistakes of politicization of the environmental issues, and not confound environmental education with political activism [8]. Thus the greens insist on the fact that only a green party can do the necessary change for environmental protection. While it is true that politic efforts must be focused, the focus should be on problems, not on political activism, and solutions must be found within mainstream politics: main parties, main governments. EE (and EE curricula) must be present at the core educational programs, not mere a specific but marginal one. Such marginalizing risks exist with green schools, despite being a great concept. Surely, sustainability and earth conservation is an emergency, and we want to achieve and see fast progresses toward sustainability. However, short-term fast progresses are all too often false progresses, and a pathway to profound deceit (they are a dead-end road). The idea that all schools will follow the example of the green schools and turn green themselves is rather wishful thinking. Undoubtedly, the fact that green schools exist is a working idea is good think, itself a sign of a healthy democratic, diverse society. The fact that green school can exist is a victory for sustainability. But if green curriculum can only be implemented as segregated from the regular school, in green school and the like, this is no victory, but a defeat.

148

Curricular aspects At present, EE is part of various disciplines/curricula. Our project is a way to synthesize and update EE methodologies and strategies in the partner institutions; hence it offers a model path for others. In addition, the project acts for the development and proposing of a common EE framework in the European Union. This work is therefore one of the international, real life efforts to implement sustainability. A common European Curriculum for training the trainers in Environmental Education cannot avoid overlapping some specialized programs and curricula. Our aim is not to propose some sort of imposition of a common European curriculum. Still, a European curriculum should exist, at least as an authoritative reference. The first obstacle to overcome is the fact that there is no unified theory or scientific body of knowledge regarding environmental knowledge. The same is true for the environmental education itself. In fact even local/institutional knowledge is both largely contingent and not monolithic [22]. But that's ok, this should be so. This variety of opinion may appear to hamper decision-making process. The idea is to create the necessary conditions and working framework that allow professional and democratic involvement, which is the scope of what is known as the science of governance (not governing; governing pertains to governmental decisions, while governance pertains to multiplayer decision governments, NGOs, scientists, and all stakeholders). For this, it is essential to establish the common grounds and the separate freedoms and responsibilities of each player. This is what we want to achieve with the current curriculum project. The workshop (kick-off meeting) held between May 30 June 01 in Iasi (Alexandru Ioan Cuza University), with the participation of all partners within the Leonardo da Vinci project RO/05/B/P/PP175010, allowed intensive discussions on the background of EE. The debates allowed reaching the common position that an environmental education (EE) curriculum must include the following character features: interdisciplinary and holistic [1, 31], value- and fairness driven, critical thinking and problem solving orientation, participatory, applicable in real-life and local contexts, favouring creativity, acceptance of change. The last point is particularly relevant because it is a condition for necessary reforms in Central Eastern European member countries of the European Union, but also in older members. A two-step education project at Purdue University in the US, a "dual-level professional development model for changing teacher practice" where Level I

149

participants were trained by University staff and trained at their turn their colleagues Level II participants) similar to our educating-educators project showed high effectiveness in changing classroom practice (83% in Level I participants and 68 % in Level II participants). Hands-on approaches were the most effective [26]. While these results suggests that peer-education can be employed as highly effective (68%), they also hint to a more effective education of educators through direct contact with University staff, probably because the later (besides being higher qualified; but being also research professionals) have a deeper hands-on experience. For instance, writing is an integral part of the Environmental Education research [15]. Therefore, innovative methods in the formation of EE teachers should include writing EE texts: conducting literature reviews, interviewing decision-makers and scientists, as well as synthesizing and documenting management problems (with related science and other issues that might constrain or drive the solution (legislation, social pressures, politics, personalities, etc) [3]. This should also include in every EE institution writing and periodically updating a document describing the organizational strategy of "greening" the EE institution. EE educators should acquire basic training and some working knowledge on what it means to green a Centre; plus related documents / working knowledge on environmental issues, environmental management, alternative systems, etc. This document should explain how the centres work (decision-making bodies, budgets, etc). It is recommended that "greening" mechanisms be professional, transparent and democratic (democratic does not signify lack of hierarchical responsibilities) [30]. A diversity of approaches is needed there is no single general valid method [25]. While EE methods are already very diverse, the EE outcomes in schools can be understood as both: 1. well-established evidence (EE outcomes: students' environmental knowledge, attitudes and behaviour) and 2. emerging evidence (EE processes: students' perceptions of nature, experience of learning and influence on adults). While later aspects have received less attention from researchers than the former, they disserve more dedication: as EE is not once and for all (but a life-long process), understanding EE processes insures better adaptability to new concerns and foci in time. Currently, there is a need to restore equilibrium in this sense [23]. Having in mind the importance of community involvement in governance and sustainable development, an interesting approach in EE is that of community-based schools, where parents and other community members are actively taking part in schoolbased EE curriculum and various indoor and outdoor EE activities [32]. Some authors even talk of a "school-family-community ecosystem" as approach in environmental education [2]. Local environmental knowledge is also influenced by active participation in land use practices and outdoor recreation [19]. This is valuable bottom-line experience. Learning about "places" associated with local cultures is a good way to do effective EE. This learning can be done via [20]: childhood experiences,

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learning from elders and family, action and observation, comparisons between places, via festivals and community events, external sources, seeing a place under different conditions (during summer, winter, conflict, drought, floods, etc, continuity in connection to a place. For example, in schools that are closed to significant water bodies, EE can be done through water quality analyses, e.g. in the sea [12] or across watersheds [9]. REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. Barrett, G.W., 2001 - Closing the ecological cycle: the emergence of integrative science. Ecosystem Health, 7, 2:79-84 Bennett, S.K., Bennett, D.B., 2004 - Lecture: regarding the ecology of science education: connections to environmental and science education. Journal of Science Education and Technology 13, 2:137-146 Bradley, M.P., Hanson, R., Walbeck, E.S., 2004 - Innovative environmental education contributes to improved management practices in the Mid-Atlantic region of the United States. Environmental Monitoring and Assessment, 94: 205-215 Chan, K., 2000 - Use of environmental teaching kits in Hong Kong. The Environmentalist, 20:113-121 Chapman, D., Sharma, K., 2001 - Environmental attitudes and behaviour of primary and secondary students in Asian cities: an overview strategy for implementing an eco-schools programme. The Environmentalist, 21:265-272 Dahlgren, M.A., berg, G., 2001 - Questioning to learn and learning to question: structure and function of problem-based learning scenarios in environmental science education. Higher Education, 41:263-282 Dempsey, R., Gresele, C., Bgeholtz, S., Martens, T., Mayer, J., Rode, H., Rost, J., 1997 - Empirical studies on environmental education in Germany: contributions by the Institute for Science Education. Research in Science Education, 28, 2: 259-279. Disinger, J.F., 1997 - Environmental education research news. The Environmentalist, 17:153-156 Donahue, T.P., Lewis, L.B., Price, L.F., Schmidt, D.C., 1998 - Bringing science to life through communitybased watershed education. Journal of Science Education and Technology, 7, 1:15-23 Elstad, E., 2006 - Understanding the nature of accountability failure in a technology-filled, laissez-faire classroom: disaffected students and teachers who give in. Journal of Curriculum Studies, 38, 4: 459-481 Fien, J., Poh, I. T.-C., Yencken, D., Sykes, H., Treagust, D., 2002 - Youth environmental attitudes in Australia and Brunei: implications for education. The Environmentalist, 22:205-216 Gough, G.A., Robottom, I., 1993 - Towards a socially critical environmental education: water quality studies in a coastal school. Journal of Curriculum Studies, 25, 4: 301-316 Harvey, T., 1995 - An education 21 programme: orienting environmental education towards sustainable development and capacity building for Rio. The Environmentalist ,15, 3: 202-210 John, P.D., 2006 - Lesson planning and the student teacher: re-thinking the dominant model. Journal of Curriculum Studies, 38, 4:483-498 Lotz-Sisitka, H. Burt, 2002 - Being brave: writing environmental education research texts. Canadian Journal of Environmental Education 7, 1: 9 http://www.uleth.ca/edu/research/ictrd/cjee/volume_7.1/9.pdf Lucas, A.M., 1980 - Science and environmental education: pious hopes, self praise and disciplinary chauvinism. Studies in Science Education, 7: 1-26 Lucie, S., 1999 - Environmental Education between modernity and postmodernity: searching for an integrating educational framework. Canadian Journal of Environmental Education, 4: 9-35 Manring, S. & Moore, S.B., 2006 - Creating and managing a virtual inter-organizational learning network for greener production: a conceptual model and case study. Journal of Cleaner Production, 14: 891-899

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19. McDaniel, J., Alley, K.D., 2005 - Connecting local environmental knowledge and land use practices: a human ecosystem approach to urbanization in West Virginia. Urban Ecosystems, 8: 23-38. 20. Measham, T.G., 2006 - Learning about environments: the significance of primal landscapes. Environmental Management, 38, 3: 426-434 21. Okada, M., Tarumi, H., Yoshimura, T., Moriya, K., Sakai, T., 2002 - Realization of a digital environmental education a future style of environmental education in dynamically changing virtual environment. Book chapter in: Digital cities II: second Kyoto Workshop on digital cities, Kyoto, Japan, October 18-20, 2001 22. Reid, A., Petocz, P., 2006 - University lecturers' understanding of sustainability. Higher Education , 51: 105123 23. Rickinson, M., 2001 - Learners and learning in environmental education: a critical review of the evidence. Environmental Education Research, 7, 3: 207-320. 24. Schwartz, M., 2006 - For whom do we write the curriculum? Journal of Curriculum Studies, 38, 4: 449-457. 25. Scott, D., 2004 - Transforming the "market-model University": environmental philosophy, citizenship and the recovery of the humanities. Worldviews 8, 2-3: 162-184 26. Shepardson, D.P., Harbor, J., 2004 - ENVISION: the effectiveness of a dual-level professional development model for changing teacher practice. Environmental Education Research, 10, 4: 471-492 27. Simmons, B., 2000 - Towards excellence in environmental education. A view from the United States. Water, Air and Soil Pollution, 123: 517-524 28. Smyth, J.C., 1996 - A national strategy for environmental education: an approach to a sustainable future? The Environmentalist, 16, 1: 27-35. 29. Sugumaran, R., Meyer, J.C., Davis, J., 2004 - A web-based environmental decision support system (WEDSS) for environmental planning and watershed management. Journal of Geographical Systems, 6: 307322 30. Sureda, J., Calvo, A.M., 2001 - Organization of school centres and environmental education: in search of action models for the greening of school organization. The Environmentalist, 21: 287-296 31. Stables, A., Scott, W., 2002 - The quest for holism in education for sustainable Development. Environmental Education Research, 8, 1: 53-60 32. Tal, R.T., 2004 - Community-based environmental education a case study of teacher-parent collaboration. Environmental Education Research , 10, 4: 523-543 33. UN Agenda 21. 2005 - United Nations Agenda 21. http://www.un.org/esa/sustdev/documents/agenda21/english/agenda21toc.htm 34. Wormstead, S.J., Becker, M.L., Congalton, R.G., 2002 - Tools for successful student-teacher-scientist partnerships. Journal of Science Education and Technology, 11, 3: 277-287

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REVIEW MARIA DUCA, 2006, Plant physiology, Stiinta Publishing House in Chisinau, 287 p. (ISBN: 978-9975-67-596-3) Plant physiology, book issued in 2006 at Science Publishing House in Chisinau, is considered to be a very important editorial event, which helps the increase of the knowledge level in plant physiology field. The author, Mrs. Maria DUCA, PhD Habilitated Professor, Corresponding Member of the Moldavian Academy of Sciences and Dean of The Faculty of Biology and Pedology from University of State from Moldavia, is an important figure for the Moldavian education and research activity, which, by this book, remarked once again in the field of expertise that she has been serving for the past two decades. As the professor stated, the volume does not only reflects the basic principles and directions of the plant physiology, using specialized literature filtered by own thinking, but also includes results of personal research, by synthesizing a large amount of experimental data, and offering concrete possibilities to direct application of obtained knowledge in practice.The theme is very ample - it is enough to prospect the content of each of the 11 chapters included in the volume so it will be a mistake to persist in the idea to make an exhaustive analysis of the book; even more, we would diminish the possibilities to know and to get thoroughly into the real novelties, exposed in a form inciting to meditation by running through the text. In the almost 300 pages, highly illustrated with suggestive color schemes and figures, the author approaches in a very systematic manner top contemporary plant physiology themes such as: auto regulation, signal perception and transduction, physiologic basis to perform the genetic program, biorhythms, compound elimination by the plants, as well as the resistance of these organisms to improper environmental factors. To ensure the possibility to evaluate the new acquired knowledge, each chapter ends with evaluation tests of different complexity degrees, a glossary for specific terms, as well as a short, but comprehensive and actualized bibliography, that comes to help the ones interested in further studies on the discussed phenomena. The data transmitted in this manner helps to form the biologists, by detailing the biochemical and molecular mechanisms of the vital processes and functions, firstly having an informative function, but also a pronounced formative character. The book tries to build a systematic and logical thinking on the plant organisms vital functions, as well as to form the readers competence in the field. The volume is correctly dimensioned, according to the contemporary biology requirements, and also offers to the biologists the necessary tools that allow the manipulation of plant organism, tools that are situated at the foundation of contemporary biotechnologies. The present volume, constructed in accord with the intrinsic changes in the Moldavian education system - that is trying to adhere to the Bologna process - by reviewing the information presentation mode and by offering landmarks for the self training of students, recommends itself to everyone and especially to the ones preoccupied with the study of plant physiology and interested in self education and up to date information in this scientific field. Prof. dr. C. TOMA, Prof. dr. MARIA MAGDALENA ZAMFIRACHE

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REVIEW TNASE C., ESAN TATIANA EUGENIA, 2006, Concepte actuale n taxonomia ciupercilor, Editura Universitii Alexandru Ioan Cuza, Iai: 510 pp. (ISBN 973-703144-X; 978-973-703-144-0) Rod al unei minuioase munci, Concepte actuale n taxonomia ciupercilor este cel mai complex i mai complet manual universitar tratnd att clasificarea ciupercilor ct i morfologia, citologia, ultrastructura, biologia, ecologia i reproducerea lor, cu informaii utile i asupra utilizrii multora dintre ele. Aceast carte se adreseaz n primul rnd specialitilor, dar i studenilor de la facultile de Biologie (cu secii de Biologie, Ecologie i Protecia Mediului, Biochimie, Biotehnologie), Silvicultur, Agricultur, Horticultur, Farmacie, Medicin veterinar i uman, Industrii alimentare, precum i celor interesai de cunoaterea i conservarea diversitii ciupercilor, de protecia patrimoniului documentar, de carte, a monumentelor istorice, a diverselor tipuri de materiale .a. Autorii cadre didactice din dou prestigioase universiti ale rii, Iai i Bucureti au proiectat aceast lucrare sub forma unui tratat pe baza sintezei informaiilor teoretice selectate din literatura de specialitate, a rezultatelor personale i a experienei acumulate n domeniul micologiei. Aceast experien, este concretizat n tratarea minuioas a unor aspecte originale care vizeaz att identificarea acestor organisme, ct i analiza critic a unor aspecte actuale referitoare la biologia, ecologia, corologia, biochimia i taxonomia ciupercilor. Tratatul este structurat n 5 capitole, care reunesc att aspecte teoretice ct i practice din domeniul micologiei. Ciupercile sunt prezentate ca un grup de organisme polifiletice reunite n 11 ncrengturi, care aparin la 3 regnuri: Chromista, Fungi i Protozoa. n aceste categorii taxonomice sunt descrise 1170 specii de ciuperci grupate n 115 familii. Lucrarea cuprinde 510 pagini, n care sunt inserate 366 de figuri, 120 fotografii color originale i 30 de tabele. Tratatul a fost realizat de cei doi autori, pe baza celor 580 de lucrri de specialitate consultate (marea majoritate aprute dup 1990), a rezultatelor personale i a experienei acumulate de-a lungul multor ani de activitate n domeniu. Prin analiza critic a cercetrilor din domeniul micologiei generale i aplicate, inserate n baze de date internaionale sau n reviste i volume ale unor manifestri tiinifice internaionale, autorii evalueaz reperele i realizrile obinute pn n prezent, precum i direciile de perspectiv. n acest context, evideniez experiena i rezultatele autorilor, racordate la realizrile internaionale, foarte multe comunicate i publicate n reviste de specialitate din ar i din strintate. Prof. dr. C. DRGULESCU

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INSTRUCTIONS TO AUTHORS The Journal Analele tiinifice ale Universitii Al. I. Cuza din Iai (serie nou), Seciunea II a. Biologie vegetal, includes original articles of cytology, morphoanatomy, physiology, taxonomy, phytosociology, mycology, phytopathology, along with book reviews and anniversary announcements. All the papers must be submitted to our redaction address (Dr. Naela COSTIC, Al. I. Cuza University, Faculty of Biology, Department of Biology, Bd. Carol I., no. 20A, 700506, Iasi) both as printed manuscripts and electronic format. For the graphic uniformity of the volume, please consider followings: PAGE FORMAT: 18 x 12,7 cm; margins settings: 5,8 cm top, 6 cm bottom, 4 cm left, 4 cm right. TEXT: the papers will be printed in one of the following languages: English, French, German the text must be typed (in a PC compatible text editor) with Times New Roman 10, single-spaced, on A4 paper; the Abstract and the Key words must be typed in English or French, using the font Times New Roman 8 points; the title must be typed in Times New Roman 10 bold capitals; authors' names and surnames must be typed in caps; male surnames must be abbreviated; the address of each author must be provided in footnote; the text will be partitioned as follows: Introduction, Material and methods, Results and discussions, Conclusions, References. All subtitles must be centred typed in Times New Roman, bold 10 ex.: Introduction the scientific names must be typed in italics; the text references of tables and figures (included in plates) must be typed between round brackets: ex: (fig. 2, Pl. I), (tab. II); the text references of the cited bibliography must be typed between square brackets: ex: [5]; the References subtitle must be centred and typed in bold 10 points caps:. ex.: REFERENCES bibliography references must be alphabetically ordered and typed in Times New Roman 8 point font, as follows: for books: 1. BELDIE Al., 1972 - Plantele din Munii Bucegi. Edit. Acad. Rom., Bucureti 2. for articles: DAUB E. M., 1981 - Cercosporin, a photosensitizing toxin from Cercospora species. Phytopathology, 72: 370 374

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REDZI S., TUKA M., PAJEVI A., 2006 - Research into microscopic structure and essential oils of endemic medicinal plant species Satureja subspicata Bartl. ex. Vis. (Lamiaceae). Bosn. J. Basic Med. Sci., 6, 2: 25-31 4. RUGIN R., TOMA C., IVNESCU L., 2007 Morphological and histo-anatomical aspects at some dicotyledonate seedlings related to the vascular transition. An. t. Univ. Al. I. Cuza Iai, s. II a., Biol. veget., 52: 133- 142 FIGURES (colour or black and white photos, drawings) all figures must be grouped in plates on separated pages; the number of the plate (ex. PLATE I) must appear in the top-right position and the names of the authors (Times New Roman 10, caps, bold) must appear in the top-left position of each plate; the Explanation of plates (including figures) must be typed on separated page (after References) the figures must be printed on tracing paper (photocopies are not acceptable, just original materials) and must not exceed 18 x 12,7 cm; all materials must be accompanied by graphical scale. TABLES the tables must be printed on separated pages and must be numbered with roman digits (Table I, Table II,). For Book Reviews: - mention the followings: author (name, surname, in caps), coma, year, title with italic bold characters, coma, place of publishment, number of pages, ISBN. Leave a blanc line and write the text of the reviews (paragraphs as few as possible) single spaced, on A4 paper with Times New Roman 10 font. Recommendations: The submitted paper must not exceed 10 pages (illustration included) and must have an even number of pages (including an even number of pages with colour plates). The papers will be further submitted to the reference comity and will be published for a charge in Analele tiinifice ale Universitii Al. I. Cuza din Iai, Sec. II a. Biologie vegetal. The editorial comity reserves the right to: reject certain papers (one paper as first author and another one in collaboration would be acceptable) reduce the number of figures, in case they are to many Only papers presented in the Plant Biology section of the scientific congress organised by our Faculty will be published. Responsibility upon the articles content belongs to the author (s). Papers that do not meat these rules will be returned to the author (s). Editorial comity

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