International Research Journal of Microbiology (IRJM) (ISSN: 2141-5463) Vol. 2(6) pp.

192-199, July 2011 Available online http://www.interesjournals.org/IRJM Copyright 2011 International Research Journals

Full Length Research Paper

Fusarium pallidoroseum A new biofertilizer responsible for enhancing plant growth in different crops
Rashmi Srivastava, C.M. Mehta and A.K. Sharma*
Department of Biological Sciences, College of Basic Sciences and Humanities, G.B. Pant University of Agriculture and Technology, Pantnagar, Uttarakhand, India *Associate Director, Department of Biological Sciences, College of Basic Sciences and Humanities, G.B. Pant University of Agriculture and Technology, Pantnagar-263 145, Udham Singh Nagar, Uttarakhand, India.
Accepted 15 June, 2011

Arbuscular mycorrhizal fungi (AMF) are of potential significance in plant growth and development. Though AMFs are considered as unique biofertilizer yet they are difficult to mass multiply as they are biotrophs and their propagation under axenic condition has not been possible. An alternate bioagent i.e. Fusarium pallidoroseum, a saprophytic fungus, has been discovered by us that plays a significant positive role in plant growth promotion for which Indian Patent (#237 946) has been obtained. Inoculation of tomato seeds with F. pallidoroseum enhanced proline content; acid and alkaline phosphomonoesterase activity; and peroxidase activity by 89.92, 8.57, 31.71 and 167.22%, respectively. The fungus enhanced shoot dry weight and shoot length of wheat, maize, marigold, okra, moongbean and brinjal over control. Keywords: Glomus intraradices, Fusarium pallidoroseum, Piriformospora indica, tomato INTRODUCTION Introduction of chemical fertilizers and continuing search for high yielding varieties have significantly contributed to the enhancement of plant yield thus resulting self sufficiency in food grain production globally. However, in achieving this goal for increased population, the quality of food grains and soil has deteriorated considerably (Mahajan and Gupta, 2009). Serious efforts are, therefore, required to adopt alternate technologies for sustenance of food grain production wherein biofertilizers can play a significant role. Towards this end, a number of biofertilizers like N2 fixers: Azospirillium sp., Azotobacter sp., rhizobia; P solubilizers/mobilizers: Pseudomonas fluorescens, Aspergillus sp., AMF are being used at a commercial scale. Amongst these, AMF have gained most attention because of their varied characteristics: non-specificity towards crops except in members of the
*Corresponding author E-mail: anilksharma_99@yahoo.com; Tel or Fax: +91 5944 233309

family Brassicaceae and Chenopodiaceae where they are not responsive; uptake of the limiting nutrient in soils e.g., phosphorus; enhanced disease resistance in crops; capacity to withstand drought conditions and similar other functions (Sharma et al., 1992; Ferrol, 2002). Though, AMF are widespread (Giovannetti 2006), their biotrophic nature has proved the biggest bottleneck in application in agriculture. Piriformospora indica has also been reported to play a role in enhancing the yield of plants (Waller et al., 2005). This fungus associates with roots of various plant species: rice, wheat, and barley as well as many dicotyledonous plants, including Arabidopsis (Varma et al., 1999; Peskan-Berghofer et al., 2004). However, interaction of this endophytic fungus with Arabidopsis roots is accompanied by a considerable requisition of nitrogen from the environment (Peskan-Berghofer et al., 2004). The present investigation was carried out to search for an alternate saprophytic fungus, which could be used as efficient plant growth promoter.

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MATERIALS AND METHODS Isolation Rhizospheric soil of healthy tomato (Solanum esculentum L.) and chilli (Capsicum capscii L.) plants was collected from farmers fields in the District Bareilly (Altitude 550ft AMSL, 280 22 N and 790 27 E). The fungus isolation was done on Armstrong medium (Armstrong & Armstrong, 1965) at 27+20C. Roots were sterilized in NaClO solution (0.75%Cl) and washed six times with sterile water, teased and kept on the medium placing teased side towards the medium. The isolation was also carried out -3 -4 from soil employing soil dilution (10 and 10 ). The 0 0 plates were kept at 27 C 2 C in a BOD incubator. A total of 400 samples (100 root samples from different plants and 300 soil samples) were screened. After three days, two hundred fungal colonies were isolated, subcultured and maintained at 40C on Potato Dextrose Agar (PDA). Growth Promotion Test Seeds of 15 different varieties of tomato and chilli were sown separately in steam sterilized soil filled in plastic trays (21x10x3 LxWxH). Seedlings were removed from the soil after 21 days and washed under running tap water followed by washing with sterilized water twice. Roots were pruned to approximately half the length and seedlings were inoculated by dipping the roots in a conidial suspension (1.5 x 106 conidia mL-1) of 200 isolated fungi individually for 2 min (Burger et al. 2003). Inoculum was prepared by inoculating potato dextrose broth (PDB) by employing 2 discs of 5 mm size taken from fully-grown plate of tested fungi on potato dextrose agar (PDA). The 250ml Erlenmeyer flasks containing 100ml PDB, inoculated with fungal discs were placed at 0 120 rpm at 27+2 C in an incubator shaker for 4 days; roots dipped in PDB only served as control. Treated and non-treated plants were transferred to plastic pots containing 200g steam sterilized soil that contained sandy loam mixture, pH 6.81, Electrical Conductivity - 0.3 -1 -1 dS m , Organic matter - 0.81%, Olsens P 12kg ha , -1 Total N 95 kg ha , Zn 0.545 ppm, Mn 23.1 ppm, Fe 16.81 ppm, Cu 1.01 ppm, and S 9.5 ppm. Pots were placed in a glasshouse with day and night temperature of 25-270C and 15-170C, respectively; plants were exposed to fluorescent lights for 12h day length. Each treatment was replicated four times. From 200 fungal isolates, two fungal isolates from chilli roots (isolate number 2 and 10) increased plant growth of both the crops. They were identified as Fusarium pallidoroseum by CABI Bioscience, UK and were used for further studies. This experiment was repeated six times (Data not shown).

Optimization of growth condition of F. pallidoroseum Three media: Armstrong, Potato dextrose and Czapek were used to standardize growth of F. pallidoroseum in shake flask culture. Flasks were kept at 120 rpm and 27+20C. Armstrongs medium was further modified using glucose or sucrose as a carbon source @ 20.0gL-1and calcium nitrate was replaced by ammonium chloride or ammonium nitrate or ammonium sulphate or urea as a -1 nitrogen source @ 5.9gL . Flasks (250ml cap.) containing 50mL modified Armstrong medium were inoculated with 2 discs of 5mm size from a full-grown plate of F. pallidoroseum on PDA and were incubated at 120 rpm and 27+20C for 4 days. Formulation and dose standardization Cell biomass with fresh mycelia and conidia (0.20g mycelium on air dry wt. basis in 50 ml and 1.5 x 106 conidia mL-1) was filtered through sterilized whatman filter paper 1 using vaccum pump under aseptic conditions. A total of four different concentrations: 20 (T1), 15 (T2), 10 (T3), and 5 (T4) mL of fungal culture broth were taken and mixed in 100g of talcum powder. To maintain the uniformity amongst the treatments, the volume of broth of each treatment was adjusted to 20mL by adding filtered medium (without mycelium and conidia) to maintain the level of nutrients and secondary metabolites under identical condition. There were three controls: 20mL filtered broth with talc (T5); 20mL sterilized uninoculated broth with talc (T6); absolute control (without talc and broth) (T7). Pre-germinated tomato seeds (Variety Holland louis) were sterilized in NaClO solution (0.75%Cl) and washed six times with sterile water. Sterilized tomato seeds were coated with this inoculum. The experiment was conducted in steam-sterilized soil mentioned in section 2.2. Comparison of F. pallidoroseum with AMF and Piriformospora indica The experiment was conducted with formulation of F. pallidoroseum; arbuscular mycorrhizal fungus, Glomus intraradices (100 IP) produced through monosporal culturing on maize by repeating three cycles of 60 days each; and P. indica formulation supplied by Dr. Vikram Sahai, Department of Biochemical Engineering, IIT, New Delhi, India. The talcum powder based formulation of both F. pallidoroseum and P. indica was used to coat the seeds of tomato. The experiment was conducted in steam-sterilized soil (section 2.2) filled in 500mL pots with five replications each. Control seeds were coated with talcum powder only. Mycorrhizal inoculation was done by making holes in the soil and providing infectious propag-

194 Int. Res. J. Microbiol.

ules in these holes before placing the seeds. The experiment was laid out under glasshouse condition mentioned under section 2.2. Watering with sterilized water was done as and when required. Plants were harvested after 45 days of seed germination. Dry shoot 0 weight was measured by placing the shoots at 60 C for 48h in a hot air oven. Dry shoot was digested in tri-acid mixture (HNO3:H2SO4:HClO4; 10:1:4) and the phosphorus content was estimated (Murphy and Riley, 1962); zinc content was measured using an atomic absorption spectrophotometer (GBC 902, Switzerland). Proline (Bates et al., 1973), acid and alkaline phosphomonoesterase (Gianinazzi-Pearson and Gianinazzi, 1976) and peroxidase activity (Bestwick, et al. 1998) were also measured. Root pieces from all the treatments were stained (Sharma et al. 1988) and infection rating was recorded employing formula: % colonization = No. of root segments colonized x 100/Total number of segments examined. Re-isolation of the inoculated F. pallidoroseum inevitable Plants were uprooted from the pots sown with seeds coated with F. pallidoroseum and surface sterilized; longitudinally teased out root pieces were placed on PDA at 27+20C for three days. Fungal hyphae emanating from the root pieces were subcultured. Conidial structures of re-isolated fungus were compared morphologically with original culture of F. pallidoroseum and were found to be similar. DNA extraction

280 nm. Polymerase chain reaction ITS region of both inoculated and re-isolated fungus was amplified using primers ITS1 (5'-TCC GTT GGT GAA CCA GCG G-3') (Waalwijk et al. 1996) and ITS4 (5'-TCC TCC GCT TAT TGA TAT GC-3') (White et al., 1990). Each PCR reaction mixture contained 5-10 ng of genomic DNA, 1M each of the primers ITS1 and ITS4, 10X PCR Buffer (100mM Tris, pH 9.0; 500mM KCl and 0.1% Gelatin), 3 mM MgCl2, 200M each of dNTP and 2.0 U of Taq DNA polymerase in a total volume of 50 l. An initial denaturation step for 2 min at 950C was followed by 30 cycles of denaturation for 1 min. at 940C, annealing for 30 sec at 540C and extension for 1 min. at 0 0 72 C before a final extension step for 5 min at 72 C. The PCR product was run on 1% agarose gels, stained with ethidium bromide (EtBr) and visualized under an UV transilluminator. The amplified product was sent to M/s Ocimum Biosolutions, Hyderabad, India for sequencing. Restriction Fragment Length Polymorphism (RFLP) Analysis Amplified products were digested for 3 h at appropriate temperature with the following 3 restriction enzymes, MspI, RsaI and TaqI (Fermentas). Digested DNA fragments were separated on 2.5% agarose gels. Gels were stained with ethidium bromide and photographed under a UV transilluminator. Plant growth responses

Genomic DNA of coated and reisolated F. pallidoroseum was extracted using a modified method of Kim et al. (1992). Fungal mycelial mat (approximately 0.5 g) was suspended in CTAB extraction buffer (0.7 M NaCl, 50 mM Tris-HCl (pH 8.0), 10 mM EDTA, 1% 2mercaptoethanol and 1% CTAB) and extracted with phenol/chloroform/isoamylalcohol [25:24:1] solution and chloroform/ isoamyl alcohol [24:1] solution. RNA was degraded by treatment with RNase (50 mg mL-1) for 30 min. at 370C. DNA was then precipitated by adding 2.5 volumes of absolute ethanol and pelleted by centrifugation for 15 min at 12,000 rpm. The pellet was washed with 70 % ethanol, air-dried and resuspended in 1 mM TE buffer (10 mM Tris-HCl, 1 mM EDTA (pH 8.0). DNA concentration and purity were measured using a spectrophotometer (Rayleigh UV-2601) at 260 nm and

Seeds of six plants viz. wheat (Triticum aestivum L.) variety UP 2338, maize (Zea mays L.) variety Naveen, marigold (Tagetus erecta L.) variety Pusa Narangi Gainda, okra (Abelmoschus esculentus L.) variety Parbhani Kranti, moongbean (Vigna radiata L.) variety Pant M-1 and brinjal (Solanum melongena L.) variety Pant Rituraj were treated with formulation of F. pallidoroseum as mentioned in section 2.4 and were sown in 500 ml plastic pots containing soil as per section 2.2. Control plants were treated with talcum powder only. Each treatment was replicated five times and each replicate contained two plants. The plants were harvested after 45 days. Glasshouse conditions were maintained as mentioned in section 2.2. The experiment was repeated twice.

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0 .1 0 00 0 .0 9 00 0 .0 8 00 Shoot dry weight (g) 0 .0 7 00 0 .0 6 00 0 .0 5 00 0 .0 4 00 0 .0 3 00 0 .0 2 00 0 .0 1 00 0 .0 0 00 T1 T2 T3 T4 T r e a tm e n t T5

L S D P = 0.0 5 = 0 .0 0 6

T6

T7

Figure 1: Influence of different inoculum doses of F. pallidoroseum on shoot dry weight of tomato Lines of bars represents standard deviation. Fungal culture broth (ml) + Filtered media (without mycelium and conidia) (ml) T1= 20 0 T2= 15 5 T3= 10 10 T4= 5 15 T5= 0 20 T6= 20ml Uninoculated fresh broth with talc T7= Absolute control (without talc and broth)

Data analysis Data was analyzed using one-way ANOVA. RESULTS Two hundred fungal isolates were recovered from tomato and/or chilli roots or their rhizospheric soil on Armstrong medium. All of these isolates were screened for their plant growth promoting (PGP) properties on tomato. Two isolates (2 and 10) were found to have strong PGP activity. Both isolates exhibited similar conidial structures. However, on the basis of higher PGP activity, isolate no. 10 was used for further studies. The fungus was identified as Fusarium pallidoroseum by CABI Bioscience, UK (IMI No. 393490) and was described as a common soil and plant debris inhabiting fungus found in tropical and sub- tropical regions. The sequence of ITS region of the fungus has been submitted to Gene Bank with Accession No. GU372974. Growth studies of F. pallidoroseum were conducted with the three different media: Potato dextrose agar, Armstrong, and Czapek medium. On the basis of dry cell biomass, this fungus showed significantly higher growth -50mL on Armstrong medium (0.186g ) compared to -50mL Czapeks medium (0.118g ). By modifying carbon and nitrogen sources as sucrose and ammonium nitrate, respectively in Armstrong medium, the dry cell biomass -50mL could be increased to 0.65g .

Comparing inoculum doses, a significantly higher shoot dry weight in tomato was observed in T1. Lowest dose of inoculum showed lower shoot dry weight (Figure 1). Concentration of inoculum in T1 was further used in all the growth related experiments. Comparing the growth performance of F. pallidoroseum with either Glomus intraradices (AMF) or P. indica, F. pallidoroseum significantly increased shoot and root dry matter of tomato when compared to control (Figure 2a and 2b). High amount of phosphorus and zinc was also observed in shoot tissue inoculated with F. pallidoroseum followed by plants inoculated with either G. intraradices or P. indica (Figure 2c). No significant difference in dry matter and nutrient concentration was observed between plants inoculated with either G. intraradices or P. indica. Root infection by G. intraradices, F. pallidoroseum and P. indica was 65, 56 and 57%, respectively. A significantly enhanced level of proline in plants infected by F. pallidoroseum, G. intraradices and P. indica showed their role in drought resistance (Figure 3a). The enzymes responsible for induced systemic resistance: peroxidase (Figure 3b) and for phosphorus uptake: acidic and alkaline phosphomonoesterases were significantly enhanced by F. pallidoroseum compared to control (Figure 3c) and were comparable with mycorrhizal plants and plants inoculated with P. indica. Re-isolation from the inoculated plant roots showed the endophytic nature of the fungus. Further, amplicons obtained by amplification with primers ITS1 and ITS4 were subjected to RFLP analysis. The pattern of

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0 .7 0 .6 Shoot dry weight (g) 0 .5 0 .4 0 .3 0 .2 0 .1 0 c o n tr o l P. in d my c Fp 1 0 L S D ( P= 0 .0 5 ) 0 . 0 6 1

Figure 2a

0 .3 5 0 .3 0 Root dry wt (g) 0 .2 5 0 .2 0 0 .1 5 0 .1 0 0 .0 5 0 .0 0 c on tr o l P. in d my c Fp 1 0

LSD (P=0.05) 0.038

Figure 2b

3 .0 0 2 .5 0 Concentration 2 .0 0 1 .5 0 1 .0 0 0 .5 0 0 .0 0

L S D ( P= 0 . 0 5 ) f o r P = 0 .0 8 0 a n d Z n = 0 . 2 2

c o n tr o l

P. in d ic a

my c

Fp 1 0

P (% )

Z n (x 1 0 p p m )

Figure 2c Figure 2: Effect of bioagents on (a) shoot dry matter, (b) root dry matter, and (c) P and Zn concentration in shoot. Line on the bars represents standard deviation. P. ind =P. indica, Myc = Glomus intraradices, Fp10 = Fusarium pallidoroseum isolate number 10

250 Proline concentration (ug/g fr. wt.) 200 150 100 50 0

LSD (P=0.05) 0.864

control

P. ind

myc

Fp10

Figure 3 a

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Phosphomonoesterases (n moles of pnitrophenol released/h/g fr. wt.)

LSD (P=0.05) Ac. Phos = 0.561, Alk. Phos = 0.153

14 12 10 8 6 4 2 0 control P. ind myc Fp10

Acid phosphomonoesterase

Alkaline phosphomonoesterase

Figure 3 b

Peroxidase content ( A470 nm/min/g fr. wt.)

0.35 0.3 0.25 0.2 0.1 5 0.1 0.05 0

LSD (P=0.05) 0.0351

control

P. ind

myc

Fp10

Figure 3c Figure 3: Effect of bioagents on (a) proline content; (b) acid and alkaline phosphomonoesterase activity, and (c) peroxidase activity in leaves of tomato. Lines on bars represents standard deviation. P. ind = Piriformospora indica, Myc = Glomus intraradices, Fp10 = Fusarium pallidoroseum isolate number 10

restricted ITS region of re-isolated fungus from the infected plant roots and its comparison with the inoculated F. pallidoroseum matched by 100% with all the restriction enzymes, thus, proving the similarity between the two of them (Figure 4 a and b). The fungus enhanced the shoot dry weight and length by 18.6 and 14% in wheat; 35.6 and 10.9% in maize; 25.2 and 62.14% in marigold; 19.1 and 27.6% in okra; 133.6 and 25.5% in moongbean; 11.0 and 15.2% in brinjal, respectively, over control. The enhancement of growth of different plants clearly showed the non-specific nature of F. pallidoroseum.

DISCUSSION An enhanced growth and nutrient uptake in plants inoculated with AM fungi (Ferrol, 2002) and P. indica (Waller et al., 2005) has been reported. Enhanced levels of phosphomonoesterases and peroxidases in mycorrhizal and P. indica infected plants have been reported by Gianinazzi-Pearson and Gianinazzi (1976) and Waller et al. (2005), respectively. Their contribution in phosphorus uptake and disease resistance has also been reported (Sharma et al., 1992). The endophytic nature of F. pallidoroseum reflects on similar mechanism

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Figure 4 a Figure 4 a: Confirmation of presence of F. pallidoroseum as an endophyte by comparing the restriction profile of inoculated and re-isolated fungus 1Kb = Molecular weight marker 1 Kb DNA ladder Lane1 = Inoculated F. pallidoroseum (Restricted with MspI) Lane2 = Re-isolated F. pallidoroseum (Restricted with MspI) Lane3 = Inoculated F. pallidoroseum (Restricted with RsaI) Lane4 = Re-isolated F. pallidoroseum (Restricted with RsaI) Lane5 = Inoculated F. pallidoroseum (Restricted with AluI) Lane6 = Re-isolated F. pallidoroseum (Restricted with AluI) Lane7 = Inoculated F. pallidoroseum (Restricted with HaeIII) Lane8 = Re-isolated F. pallidoroseum (Restricted with HaeIII) Lane9 = Inoculated F. pallidoroseum (Restricted with TaqI) Lane10 = Re-isolated F. pallidoroseum (Restricted with TaqI) 100bp = Molecular weight marker 100 bp DNA ladder

Figure 4b Figure 4 b: Dendrogram obtained from restriction profile of Inoculated and re-isolated F. pallidoroseum 1 = Inoculated F. pallidoroseum (Restricted with MspI) 2 = Re-isolated F. pallidoroseum (Restricted with MspI) 3 = Inoculated F. pallidoroseum (Restricted with RsaI) 4 = Re-isolated F. pallidoroseum (Restricted with RsaI) 5 = Inoculated F. pallidoroseum (Restricted with AluI) 6 = Re-isolated F. pallidoroseum (Restricted with AluI) 7 = Inoculated F. pallidoroseum (Restricted with HaeIII) 8 = Re-isolated F. pallidoroseum (Restricted with HaeIII) 9 = Inoculated F. pallidoroseum (Restricted with TaqI) 10 = Re-isolated F. pallidoroseum (Restricted with TaqI)

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of plant growth promotion as in case of AMF and P. indica. The higher level of alkaline and acidic phosphomonoesterases shows the role of F. pallidoroseum in phosphorous uptake mechanism. Enhanced level of peroxidases in the F. pallidoroseum infected plants indicates greater resistance mechanism of fungus treated plants.

CONCLUSION The results of plant growth promotion in different crops and enhanced enzymatic activity in tomato taken as model crop using F. pallidoroseum indicates the need of further exploitation of this saprophytic and endophytic fungus for its commercial use. The present investigation is the first report of use of F. pallidoroseum as plant growth promoter. ACKNOWLEDGEMENT Authors are grateful to Prof. Michel Aragno, Laboratory of Microbiology, Institute of Biology, University of Neuchatel, Neuchatel, Switzerland and Dr. Adrian Leuchtmann, ETH, Zurich, Institut fr Integrative Biologie (IBZ), kologische Pflanzengenetik, Universittstrasse 16, Switzerland for critically reading and suggesting the corrections in the manuscript. Authors are also thankful to Dr. V.S. Bisaria, Dr. Vikram Sahai, and Mr. Vinod of Indian Institute of Technology, New Delhi, India for providing the formulation of Piriformospora indica.
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