Scientia Horticulturae 171 (2014) 5157

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Scientia Horticulturae
journal homepage: www.elsevier.com/locate/scihorti

Antifungal activity of asparagus extracts against phytopathogenic

Fusarium oxysporum
C. Rosado-lvarez a , L. Molinero-Ruiz b, , R. Rodrguez-Arcos c , M.J. Basallote-Ureba a
a
IFAPA Centro Las Torres-Tomejil, CAPMA (Junta de Andaluca), Apdo. ocial, Alcal del Ro, 41200 Sevilla, Spain
b
Institute for Sustainable Agriculture (CSIC), Alameda del Obispo s/n, 14080 Crdoba, Spain
c
Instituto de la Grasa (CSIC), Avenida Padre Garca Tejero 4, 41012 Sevilla, Spain

a r t i c l e i n f o a b s t r a c t

Article history: Asparagus, carnation and strawberry are major horticultural crops in southern Spain, where diseases
Received 29 January 2014 caused by soilborne pathogens are frequent. Among them, Fusarium spp. are the fungi more frequently
Received in revised form 22 March 2014 associated to the infection of underground plant organs. Flavonoids are phenolic compounds which
Accepted 24 March 2014
possess a wide range of biological activities, including antifungal activity. In this work we evaluated the
Available online 18 April 2014
in vitro effect of an extract enriched in avonoids obtained from triguero asparagus by-products against
Fusarium oxysporum pathogenic to asparagus, carnation and strawberry, as well as its safety in aspara-
Keywords:
gus plants. A signicant inhibition of mycelial growth and sporulation of F. oxysporum f. sp. asparagi,
Asparagus ofcinalis L.
By-products
F. oxysporum f. sp. dianthi and F. oxysporum pathogenic to strawberry was obtained by the addition of
Flavonoid compounds the asparagus extract enriched in avonoids in three independent experiments and using the amended
Fusarium oxysporum f. sp. asparagi plate technique. Besides, in vitro tests were conducted aiming at assessing the effect of the extract in
Fusarium oxysporum f. sp. dianthi asparagus plants. Seed germination and growth of asparagus in medium amended with the asparagus
Fusarium oxysporum from strawberry extract enriched in avonoids were similar to those in the not amended medium. Our results constitute
the rst report of the antifungal properties of bioactive compounds, mainly avonoids, from asparagus
by-products, and show these extracts as a promising alternative within horticultural crop protection
strategies.
2014 Elsevier B.V. All rights reserved.

1. Introduction and strawberry are caused by F. oxysporum as well. Fusarium oxys-

Asparagus (Asparagus ofcinalis L.) is one of the main horticul-
tural crops worldwide. Spain is the fth country in importance of
asparagus exports (FAOSTAT, 2013). Carnation (Dianthus caryophy-
lus L.) and strawberry (Fragaria x ananassa Duch.) are other
horticultural crops of major importance in this same area. In fact,
Spain is the main producer of fresh strawberries and the second
of carnation within the European Union (MARM, 2012; FAOSTAT,
2013). Crown and root rot of asparagus are severe constraints to
production worldwide (Elmer, 2001) which have been reported
in Spain as associated to infections by Fusarium oxysporum f.
sp. asparagi S.I. Cohen (Foa) among other Fusarium spp. (Corpas-
Hervias et al., 2006; Wong and Jeffries, 2006). Diseases of carnation
Abbreviations: AEEF, asparagus extract enriched in avonoids.
Corresponding author at: Institute for Sustainable Agriculture, CSIC, Apdo. 4084,
14080 Crdoba, Spain. Tel.: +34 957 499272; fax: +34 957 499252.
E-mail address: lmolinero@ias.csic.es (L. Molinero-Ruiz).
porum f. sp. dianthi (Fod) is the causal agent of vascular wilt of
carnation, the most devastating disease of the crop worldwide
(Garibaldi and Gullino, 1987) and also in Spain (Prados-Ligero et al.,
2007). Fusarium oxysporum has been reported as associated to wilt
and crown and root rot of strawberry (Fo-S) in Australia (Fang et al.,
2011) and in Spain (Arroyo et al., 2009).
The most successful strategy for controlling Fusarium diseases
in many horticultural crops is the development of resistant culti-
vars (Mace et al., 1981). This approach has not been developed in
the asparagus/Foa pathosystem because of the difculty of nding
resistance to this pathogen. Neither Fusarium-resistant strawberry
cultivars are still available for growers, although important efforts
are devoted to it (Prez-Gimnez et al., 2012). Concerning genetic
resistance of carnation to Fusarium, resistant cultivars are infre-
quent due to pathogenic and genetic diversity of Fod (Gmez-Lama
et al., 2012).
The low level of Fusarium diseases control achieved with the
use of synthetic fungicides, the detrimental effects of these fungi-
cides against soil biota and the risk of damage to humans together

http://dx.doi.org/10.1016/j.scienta.2014.03.037
0304-4238/ 2014 Elsevier B.V. All rights reserved.
52 C. Rosado-lvarez et al. / Scientia Horticulturae 171 (2014) 5157
with delay of exports at frontiers due to residues in commercial-
ized products, foster the need for alternative methods of efcient
control of F. oxysporum.
Phenolic compounds which accumulate in plant tissues have
been proposed for some time to serve as useful alternatives for
the control of pathogens of agricultural crops (Langcake et al.,
1981). Among them, avonoids occur widely in plants and are
a biologically major and chemically diverse group of secondary
metabolites. Flavonoids possess a wide range of biological activ-
ities. Many avonoids are active principles of medicinal plants
and exhibit pharmacological effects (Kong et al., 2003; Marles
et al., 2003; Yilmaz and Toledo, 2004; Agrawal, 2011; Sandhar
et al., 2011; Kumar and Pandey, 2013). They are also benecial
for the plant itself as physiological active compounds, as stress
protecting agents, as attractants or as feeding deterrents, and, in
general, by their signicant role in plant resistance. Defence-related
avonoids are innate compounds that are synthesised during the
normal development of plant tissue or can be synthesised by
plants in response to physical injury, infection, or stress (Treutter,
2006). Flavonoids act as phytoalexins, phytoanticipins and biocides
against phytophagous insects and soil-borne pathogens (Akhtar
and Malik, 2000; Lattanzio et al., 2006). Grayer and Harborne
(1994) listed many avonoids from higher plants with antifun-
gal activity. Flavonoids (proanthocyanidins and small amounts of
dihydroquercetin) are involved in several defence mechanisms of
barley against Fusarium species (Skadhauge et al., 1997). In resis-
tance against vascular pathogens causing wilt diseases, such as
F. oxysporum and Verticillium albo-atrum, avonoids are stored in
specialised cells from where they can be infused into attacked tis-
sue, such as xylem vessels. Such leaching is probably involved in
hypersensitive response and programmed cell death as common
mechanisms of plant defence (Beckman, 2000). Steinkellner and
Mammerler (2007) found that avonoids are no specic incentive
for microconidia germination of the tomato pathogen Fusarium
oxysporum f. sp. lycopersici (Fol). Out of 12 avonoid compounds
only myricetin and luteolin exhibited a low stimulating activity
on microconidia germination of the fungus, whereas the other
avonoids tested were inactive when applied at ve different
concentrations. When healthy onion bulbs were inoculated with
F. oxysporum, four avonoids (two quercetin and two isorham-
netin derivatives) underwent concentration changes typical for the
defence materials against pathogens (Lee et al., 2012). Ardila et al.
(2013) have recently showed that the constitutive total phenolic
and avonoid contents and the antioxidant activity levels of nine
distinct commercial cultivars of carnation are related to their differ-
ential response to Fod. In roots, resistant cultivars exhibited higher
levels of avonoids and antioxidant activity than those found in
susceptible cultivars, which, as suggested by the authors, might be
associated with processes in the environment of the rhizosphere,
such as nutrient uptake, growth and interaction with soil microor-
ganisms.
From a functional point of view, asparagus and its by-products
are highly interesting resources due to their contents in several
phytochemicals, such as phenolics, mainly avonoids, saponins,
sterols and fructans (Salvatore et al., 2005; Fuentes-Alventosa
et al., 2013). Previous research about asparagus spear phenolic
characterization revealed that, whereas white spears mainly
contained hydroxycinnamic acid derivatives, avonoids were the
major phenolics in green asparagus (Guilln et al., 2008). Aspara-
gus known as triguero are tetraploid subspecies which proceed
from wild asparagus and are autochthonous to the Hutor-Tjar
area (Granada, southern Spain) area. Triguero asparagus have a
avonoid content that is similar to the one reported for green
asparagus from commercial varieties (Fuentes-Alventosa et al.,
2007). Although total contents of avonoids from both commer-
cial hybrids and triguero asparagus are comparable, there are
signicant differences related to their avonoid compositions
(Fuentes-Alventosa et al., 2008). Whereas the avonoid prole
from green asparagus commercial varieties is characterized by
containing rutin (quercetin-3-O-rutinoside) as main avonoid
and representing about 90% of total avonoid content, in triguero
asparagus as much as eight different avonoid glycosides derived
from three distinct aglycones (quercetin, kaempferol and isorham-
netin) have been identied, being the percent of rutin 50% of total
avonoid content (Fuentes-Alventosa et al., 2008). This greater
variety of avonoids found in triguero asparagus could be related
to its major antioxidant and antifungal activities, among others.
Within sustainability and respect to the environment required
to alternatives for crop protection in Europe, the use of plant
compounds that show biological activity against crop pathogens
appears as a potentially useful tool (Hussin et al., 2009; Abu-Taleb
et al., 2011; Chvez-Quintal et al., 2011; Ponts et al., 2011; Khan
et al., 2012; Curifuta et al., 2012; Karimi et al., 2013). Therefore, the
objectives of this work were to: (a) molecularly identify two iso-
lates of F. oxysporum from affected asparagus and carnation plants,
(b) evaluate the in vitro effect of an asparagus extract enriched in
avonoids obtained from triguero asparagus by-products against
Foa, Fod and Fo-S, and (c) evaluate the effect of the extract from
triguero by-products in seed germination and growth of asparagus.
2. Materials and methods
Three isolates of F. oxysporum from infected plants of aspara-
gus, carnation and strawberry sampled in Southern Spain were
included in this work. The pathogenicities of F. oxysporum from
asparagus (Foa) and from carnation (Fod) were previously con-
rmed (Corpas-Hervias et al., 2006). The isolate from strawberry
(Fo-S) was pathogenic and molecularly identied as F. oxysporum
by Arroyo et al. (2009).
2.1. Molecular identication of F. oxysporum from affected
asparagus and carnation plants
Mycelia of monoconidial isolates of (Foa) from asparagus and
carnation (Fod) were produced on PDA plates incubated at 25 C
for 7 days. One hundred mg mycelium of each isolate were
scraped with a scalpel, powdered in liquid nitrogen and the
DNA was puried using the Dneasy Plant Mini Kit (Qiagen,
Hilden, Germany) according to the manufacturers instructions.
Quality and concentration of DNA samples were determined
using a NanoDrop 1000 spectrophotometer (Thermo Fisher Sci-
entic Wilmington DE, USA). Fungal isolates were molecularly
analyzed using the sequence of the translation elongation fac-
tor 1 (EF1) gene. Primers used for DNA amplication and
sequencing were EF1: TGGGTAAGGAA/GGACAAGAC and EF2:
GGAA/GGTACCAGTC/GATCATGTT) (ODonnell et al., 1998).
The reaction mixture (50 l) consisted of: 20 ng of fungal DNA,
400 nM of each primer, 250 nM of each dNTP, 2 mM MgCl2, 5 l
10 reaction buffer and 1 U DNA polymerase (VelocityR, Bioline
Ltd, London, UK). The PCR cycling protocols were: denaturation
at 94 C for 5 min followed by 35 cycles of 1 min at 94 C, 1 min
at 58 C, 1 min at 72 C, and a nal step of 6 min at 72 C. The
amplication products (5 l) were separated by electrophore-
sis on 1% agarose gels in 1 TAE buffer, stained with ethidium
bromide and visualized under UV light. The 100 pb ladder gTP-
bio size marker was used for electrophoresis (C Viral, Seville,
Spain). The amplicon was excised from the gel and puried using
the Favor/Prep gel/PCR purication mini kit (Favorgen, Vienna,
Austria). Amplication products were sequenced at Stab Vida
(Costa da Caparica, Portugal). Sequences were edited with Mega
5.0 (http://www.megasoftware.net/) and a search for sequence
 C. Rosado-lvarez et al. / Scientia Horticulturae 171 (2014) 5157 53

similarities was performed with the BLAST program of GenBank Table 1

(http://www.ncbi.nlm.nih.gov/).
2.2. Obtention of extracts from asparagus by-products
Treatments applied as amends to potato dextrose agar (PDA) growing media onto
which Fusarium oxysporum f. sp. asparagi (Foa) (experiment 1), F. oxysporum f. sp.
dianthi (Fod) (experiment 2) and F. oxysporum pathogenic to strawberry (Fo-S)
(experiment 3) were cultured.

Treatmentsa Growth media

The extract was obtained from asparagus by-products, which
consist on the basal portions of the spears that are discarded prior Control Not amended PDA
AEEF-10B PDA + 10% AEEF added before autoclaving
to its industrial canning. Spears of varieties of triguero asparagus
AEEF-10A PDA + 10% AEEF added after autoclaving
were used for the extraction. AEEF-20B PDA + 20% AEEF added before autoclaving
The functional extract with potential antifungal activity was AEEF-20A PDA + 20% AEEF added after autoclaving
achieved as reported by Fuentes-Alventosa et al. (2013). The extrac- EtOH-10B PDA + 10% EtOH added before autoclaving
EtOH-10A PDA + 10% EtOH added after autoclaving
tion method consisted on the treatment of asparagus by-products
EtOH-20B PDA + 20% EtOH added before autoclaving
with water, as the extraction solvent at 121 C, for 2 h. The treat- EtOH-20A PDA + 20% EtOH added after autoclaving
ments were carried out by placing 10 kg asparagus by-products and a
AEEF: asparagus extract containing 6.33% avonoids was dissolved in 80% EtOH
20 l water in a closed container that was placed in an industrial
prior to amendment of the growing media.
autoclave (Steriow, Madinox, Barcelona, Spain). Two fractions
were separated after the hydrothermal treatment, consisting on an
aqueous functional extract containing most of the soluble bioactive added to a glass tube containing 5 ml of sterile distilled water and
compounds from the asparagus by-products and a brous residue ultrasonicated (90 U) for 10 min. Serial dilutions were prepared and
that constitutes the asparagus bioactive ber. conidia were counted using a hemacytometer.
The aqueous extract was fractionated and partially puried in The biological variables analysed for each treatment and repli-
order to obtain functional extracts enriched in a specic class of cation were: inhibition of mycelial growth (IMG) expressed as
phytochemicals: avonoids. Fractionation of the global extract was the percentage ratio of the diameter of mycelial growth in each
performed by adsorption chromatography in a column lled with treated plate relative to the diameter of growth on the control
Amberlite XAD polymeric adsorption resin (Rohm and Hass Espana treatment, standardized area under the fungal growth progress
SA, Barcelona, Spain) and the avonoid enriched fraction was eluted curve (SAUGPC) calculated by the trapezoidal integration method
with 40% ethanol aqueous solution. The asparagus extract enriched standardized by the duration fungal growth in days (Campbell and
in avonoids (AEEF) was used as powder after being freeze-dried. Madden, 1990) and fungal sporulation (conidia/mm2 ). Analysis of
The richness in avonoids of this partially puried fraction was variance (ANOVA) was performed to SAUGPC, angle-transformed
not high (6.33%), however it is remarkable that it was ten times percentage IMG, and transformed [log (conidia/mm2 + 1)] sporu-
greater than that quantied in the global asparagus extract. Major lation. Means were compared using Fishers protected least
components of the AEEF were proteins (29.54%) and phenolic com- signicant difference tests (P = 0.05).
pounds, mainly avonoids (6.33%); and these were accompanied
by low quantities of neutral sugars (3.14), pectins (3.49) and ash 2.4. In vitro effect of AEEF on asparagus seed germination and
(4.37%) (unpublished results). growth of the plants

2.3. In vitro effect of AEEF against Foa, Fod and Fo-S Two experiments (4 and 5) were conducted for studying the
effect of AEEF on asparagus seed germination. Cultivar Dariana
The toxicity of AEEF against Foa, Fod and Fo-S was assayed using was used in these experiments because it is commercially used for
the amended plate technique. The ability of the AEEF to inhibit the growing both green and white asparagus. Most importantly, Dar-
hyphal growth and sporulation of the fungi was evaluated. Three iana it is moderately tolerant to Fusarium, what makes it a good
independent experiments, 1 to 3, were individually conducted with option for controlling asparagus crown and root rot using inte-
Foa, Fod and Fo-S, respectively. The experiments were conducted at grated strategies. In experiment 4, AEEF was added to the medium
least twice. in which asparagus seedlings were grown (0.6% water agar, WA)
Fungal cultures were maintained in sterile soil tubes at 18 C at the rate of 20%, before or after autoclaving (when the tempera-
until used. Extracts from triguero asparagus spears of the Hutor- ture of the medium reached about 50 C) and the growth medium
Tjar population variety were obtained as above described. Prior was amended with AEEF which was previously diluted in EtOH, WA
to each experiment, the AEEF was eluted with ethanol (EtOH) and plates amended with EtOH at 20% (0.127 mg avonoids/ml) were
used to amend the PDA at 10 and 20% (nal concentrations of 0.063 also included. In experiment 5 the AEEF was added to the growth
and 0.127 mg avonoids/ml, respectively). Unamended PDA plates medium previously diluted (AEEF-20A) or without prior dilution
served as controls and treatments consisting on the amendment in EtOH (AEEFw-20A) and only after autoclaving. Un-amended WA
with EtOH at concentrations equal to those used for the dilutions plates served as controls in both experiments (Table 2).
of AEEF were also included. Additionally, AEEF was added before
(AEEF-B) or after autoclaving and when the temperature of the Table 2
Treatments, applied as amendments to water agar (WA) growing media, onto which
medium reached about 50 C (AEEF-A) (Table 1).
the effect of asparagus extract enriched in avonoids (AEEF) on asparagus seed
For hyphal growth inhibition experiments, 5-mm-diameter agar germination and growth was assessed (experiments 4 and 5).
disks were cut from the edge of actively growing colonies of the
Treatmentsa Growth media Experiment
corresponding F. oxysporum isolate and then transferred onto the
centre of the plates. Cultures were incubated at 25 C in the dark Control Not amended WA 4,5
for two days, and then with a 12-h photoperiod. Colony radial AEEF-20B WA + 20% AEEF added before autoclaving 4
AEEF-20A WA + 20% AEEF added after autoclaving 4
growth was measured, as the average of longest and shortest diam-
EtOH-20B WA + 20% EtOH added before autoclaving 4
eters, after incubation for 2, 5, 7, 9 and 12 days. Five Petri plates EtOH-20A WA + 20% EtOH added after autoclaving 4
(replications) were established for each treatment in a completely AEEF-20A WA + 20% AEEF added after autoclaving 5
randomised design. At the end of the experiments (12 days) fun- AEEFw-20A WA + 20% AEEFw added after autoclaving 5
gal sporulation in each plate was determined. Four 5-mm-diameter a
AEEF: asparagus extract containing 6.33% avonoids was dissolved in 80% EtOH
agar disks were cut at 2 cm from the center to the edge of the colony, prior to amendment of the growing media; AEEFw: without prior dilution in EtOH.
54 C. Rosado-lvarez et al. / Scientia Horticulturae 171 (2014) 5157

Table 3 Table 4
In vitro effect of asparagus extracts enriched in avonoids (AEEF) on the inhibi- In vitro effect of asparagus extracts enriched in avonoids (AEEF) on the standardized
tion of mycelial growth (IMG), standardized area under the growing progress curve area under the growing progress curve (SAUGPC), inhibition of mycelial growth
(SAUGPC), and sporulation of Fusarium oxysporum f. sp. asparagi (experiment 1)a . (IMG) and sporulation of Fusarium oxysporum f. sp. dianthi (experiment 2)a .

Treatments IMG (%) SAUGPC Sporulation (conidia/mm2 ) Treatments IMG (%) SAUGPC Sporulation (conidia/mm2 )

Control 0.0 0.0a 2.55 0.03a 18,651 9,570a Control 0.0 0.0a 2.98 0.25a 293,318 27,715a
EtOH-10B 2.4 2.3a 2.24 0.03a 5,595 2,152ab EtOH-10B 4.2 3.4ab 2.25 0.10c 120,610 15,535ab
EtOH-10A 26.0 1.1b 1.70 0.10b 1,616 313ab EtOH-10A 8.7 3.0b 2.08 0.09cd 151,446 32,013a
EtOH-20B 26.6 8.0b 1.63 0.19b 1,119 551bc EtOH-20B 39.5 3.2cd 1.37 0.04e 19,770 5,245b
EtOH-20A 63.3 14.3c 0.83 0.27c 373 373cd EtOH-20A 53.4 4.9d 1.11 0.12e 52,720 10,541ab
AEEF-10B 0.0 0.0a 2.40 0.05a 15,791 3,923a AEEF-10B 0.0 0.0a 2.62 0.07b 54,336 17,726ab
AEEF-10A 63.2 2.7c 1.01 0.04c 746 321bc AEEF-10A 30.6 7.8c 1.79 0.10d 122,350 15,754ab
AEEF-20B 50.4 4.0c 1.06 0.05c 0 0d AEEF-20B 45.3 6.3d 1.44 0.11e 84,794 16,026ab
AEEF-20A 85.5 1.6d 0.35 0.03d 124 124d AEEF-20A 84.6 2.1e 0.40 0.04f 2,238 1,193
a a
Percentage data of inhibition of mycelial growth were angle-transformed prior Percentage data of inhibition of mycelial growth were angle-transformed prior
to statistical analyses. Data of sporulation (conidia/mm2 ) were transformed accord- to statistical analyses. Data of sporulation (conidia/mm2 ) were transformed accord-
ing to [log (conidia/mm2 + 1)] prior to statistical analyses. Data followed by different ing to [log (conidia/mm2 + 1)] prior to statistical analyses. Data followed by different
letters in each column are signicantly different (P 0.05) according to Fishers letters in each column are signicantly different (P 0.05) according to Fishers
protected least signicant difference tests. protected least signicant difference tests.

Following the methodology by Corpas-Hervias et al. (2006),
asparagus seeds were surface-disinfested by immersion for 2 min
in 20% household bleach solution (50 g of active chlorine per liter);
then air-dried and transferred (ve per plate) to 20 ml of the hard-
ened amended growing media. Five or six plates (replications), in
experiments 4 and 5, respectively, were established for each treat-
ment in a completely randomised design. After incubation in the
dark at 28 C for 814 days, the percentage of seed germination
was recorded for each experimental unit (plate).
The effect of the AEEF on the growth of asparagus plants was
studied after seed germination. Seven germinated seeds from each
of the treatments control, AEEF-20B and EtOH-20B of experiments
4, and 36 germinated seeds of each of the experiment 5 treatments
were individually transferred under sterile conditions to test tubes
containing 20 ml of Hoagland medium (Tuite, 1969). Tubes were
kept at 25 C and with a 12-h photoperiod for 3 weeks. At the end
of the experiment lengths of stem and roots of the plants were
recorded.
Percentage germination was angle-transformed, and root and
stem lengths were transformed according to sqrt (length + 1) prior
to ANOVA, and means were compared using Fishers protected least
signicant difference tests (P = 0.01).
3. Results
3.1. Molecular identication of F. oxysporum from affected
asparagus and carnation plants
Amplications of the TEF1 gene resulted in 650 bp fragments.
Sequencing and comparison of sequence data to the GenBank
BLAST database showed that F. oxysporum isolated from aspara-
gus and carnation had 99 and 100% homology with F. oxysporum
f.sp. asparagi (Acc. No. DQ837691.1) and F. oxysporum f. sp. dianthi
(Acc. No. GU199329.1), respectively.
treatments with AEEF and with EtOH at the same dose were
observed. These differences were generally observed when the
amendments were not autoclaved (-10A and -20A treatments) and
they varied, at low and high doses, between 37 and 22%, 22 and 31%,
and 23 and 32%, for Foa, Fod and Fo-S, respectively (Tables 35).
Similarly, signicant effects of the treatments were also found for
the growth of the fungi along the experiments (SAUGPC, P < 0.0001
for all of them). In Foa, signicant differences of SAUGPC as com-
pared to that of the control were generally highest with AEEF
at the high dose and irrespective of the moment of the amend-
ment. In both, AEEF-20B and AEEF-20A treatments, the SAUGPC
was reduced two and seven-fold, respectively, as compared to those
of the control. Similarly, two and three-fold reduction of SAUGPC
were observed with EtOH-20B and -20A treatments (Table 3). Con-
cerning the experiments with Fod and Fo-S, the SAUGPC was also
signicantly reduced in all the treatments as compared to the
controls (P < 0.0001 both). Additionally, three-fold lower values of
SAUGPC were obtained in the AEEF-20A treatments as compared
to those with EtOH-20A, for both Fod and Fo-S (Tables 4 and 5).
Finally, a clear effect of the AEEF on the sporulation of the
three fungi was observed (P < 0.0001 in the three experiments). In
the case of Foa, the sporulation for the AEEF-20 treatments was
lower as compared to the EtOH treatments (average 60 and 750
conidia/mm2 , respectively) and highly reduced as compared to
the control (18,651 conidia/mm2 ) (Table 3). Similar effects of the
amendments were obtained concerning the sporulation of Fod
and Fo-S. In AEEF-20A treatments, conidia/mm2 was decreased
to minimum values of 2,240 and 1,120 for Fod and Fo-S, respec-
tively, as compared to 293,320 and 168,480 conidia/mm2 in the
Table 5
In vitro effect of asparagus extracts enriched in avonoids (AEEF) on the standardized
area under the growing progress curve (SAUGPC), inhibition of mycelial growth
(IMG) and sporulation of Fusarium oxysporum from strawberry (experiment 3)a .
Treatments IMG (%) SAUGPC Sporulation (conidia/mm2 )

3.2. In vitro effect of AEEF against Foa, Fod and Fo-S Control 0.0 0.0a 2.78 0.03a 168,480 33,338a
EtOH-10B 0.0 0.0a 2.23 0.10bc 115,512 15,964a
EtOH-10A 12.2 3.6b 2.00 0.13c 17,283 5,214b
When the effect of AEEF against the F. oxysporum isolates was EtOH-20B 46.1 7.6de 1.13 0.13e 7,212 2,817b
evaluated in vitro, signicant differences of all the biological vari- EtOH-20A 53.9 7.7e 1.05 0.16e 11,688 3,908b
ables IMG, SAUGPC and sporulation as compared to not amended AEEF-10B 0.0 0.0a 2.50 0.05ab 87,660 25,327a
controls were obtained in all the experiments. AEEF-10A 34.8 4.2dc 1.70 0.05d 16,288 4,849b
AEEF-20B 31.2 7.5c 1.50 0.30d 8,952 2,030b
Similar trends were observed in the mycelial growth of the three
AEEF-20A 85.7 1.1f 0.38 0.03f 1,119 373c
isolates as a response to the treatments with AEEF. A signicant
a
Percentage data of inhibition of mycelial growth were angle-transformed prior
(P < 0.0001) IMG occurred in all the AEEF treatments, and it was
to statistical analyses. Data of sporulation (conidia/mm2 ) were transformed accord-
highest when the AEEF was added at the highest dose and after ing to [log (conidia/mm2 + 1)] prior to statistical analyses. Data followed by different
autoclaving (86, 85 and 86% IMG for Foa, Fod and Fo-S, respec- letters in each column are signicantly different (P 0.05) according to Fishers
tively) (Tables 35). Differences of IMG of the three fungi between protected least signicant difference tests.
 C. Rosado-lvarez et al. / Scientia Horticulturae 171 (2014) 5157 55

Seed germination Length of roots Length of stem

100 120
A B

Germination of seeds (%)

80 100

Length (mm)
80
60
60
40
40

20
20

0 0

Control EtOH-B EtOH-A AEEF-B AEEF-A Control AEEFw-A AEEF-A

Fig. 1. In vitro effect of different amendments with asparagus extracts enriched in avonoids (AEEF) on seed germination and growth of asparagus plants in experiments
4 (A) and 5 (B). Asparagus seeds of Dariana were incubated on ve or six plates (ve seed per plate) in experiments 4 and 5, respectively. Vertical upper bars represent
standard errors of the mean. Percentage germination was angle-transformed, and root and stem lengths were transformed according to sqrt (length + 1) prior to ANOVA.
Means were compared using Fishers protected least signicant difference tests (P = 0.01).

corresponding controls (Tables 4 and 5). Additionally, values of The asparagus extracts used in this work contained avonoids
sporulation in AEEF-20A treatments were statistically lower than as main bioactive compounds. The avonoid prole consisted on
in EtOH-20A treatments (52,720 and 11,688 conidia/mm2 of Fod quercetin kaempferol and isorhamnetin glycosides being the
and Fo-S, respectively (Tables 4 and 5). quercetin derivatives the most abundant (Fuentes-Alventosa et al.,
2007). It has been reported that avonoids, such as proanthocian-
iodins and dihidroquercitins are involved in defence mechanisms
3.3. In vitro effect of avonoids on asparagus seed germination
of barley against Fusarium species (Skadhauge et al., 1997). On
and growth of the plants
the other hand, kaempferol along with other two avonoid glyco-
sides from carnation exhibited antifungal activity against different
When the effect of AEEF on asparagus seed germination was
F. oxysporum f. sp. dianthi pathotypes (Galeotti et al., 2008).
studied, signicances of the treatments were obtained (P < 0.0001
It is well established that minor differences within avonoid
in both experiments). In experiment 4, amendments with EtOH and
structure, such as sugar type and number are able to signicantly
with AEEF dissolved in EtOH before sterilization (-B), resulted in a
modify phenolic activity (Seyoum et al., 2006). Flavonoid pro-
lower germination of seeds (60 and 48%, respectively) as compared
le from triguero asparagus contain mono-, di- and tri-glycosides
to the control (88%) (Fig. 1A). No germination of seeds occurred
of three distinct aglycones that have distinct biological activi-
in the EtOH-A and in the AEEF-A treatments (Fig. 1A). When the
ties related to plant defence mechanism (Fuentes-Alventosa et al.,
AEEF was added to the medium without prior dilution in EtOH
2008). This composition may confer great antioxidant and antifun-
nor autoclaving (AEEFw-A, experiment 5), the percentage of ger-
gal activities to triguero asparagus extract derived not only from
minated seed was similar to the one in the control treatment (97.9
their molecular structure but also related to synergistic effects
and 96.1%, respectively) (Fig. 1B). The lowest germination percent-
among avonoid glycosides and/or other bioactive compounds
age in experiment 5 was obtained in the medium that was amended
present in the extracts.
with AE previously dissolved in EtOH (AEEF-A treatment, 16.7%)
The AEEF had a high activity against the three F. oxysporum
(Fig. 1B).
isolates, inhibiting their mycelial growth and decreasing their pro-
Concerning the effect of the AEEF on the growth of the plants,
gression in time (SAUGPC) in averages of 85% and 86%, respectively,
signicant differences of stem and root lengths were obtained in
when used at the highest dose. This agrees with the appreciable
experiment 4 (P < 0.0001 and P = 0.0184, respectively) and in exper-
inhibition of the mycelial growth of F. oxysporum f. sp. dianthi by
iment 5 as well (P < 0.0001 for both variables). In experiment 4
carnation avonoids (approximately 55%) obtained in biological
only the effects of EtOH and AEEF when added before autoclaving
tests by Galeotti et al. (2008). Other authors have also referred the
(-B) were analysed, because seeds of the no autoclaved amends (-
in vitro effect of avonoids against plant pathogens such as Pyricu-
A) did not germinate (Fig. 1A). Both treatments resulted in lower
laria oryzae, Xanthomonas oryzae pv. oryzae and Neurospora crassa
root and stem lengths (13 mm and 23 mm averaged across treat-
(Treutter, 2006). Besides, a very notorious reduction of the three
ments, respectively) than those of the control (42 and 115 mm,
fungi sporulation by the AEEF was observed in our work. Flavonoids
respectively) (Fig. 1A). Similarly to experiment 4, plants from the
seem to be the main responsible of the antifungal activity of AEEF
treatment with AEEF-A did not develop (0 mm of root and stem
according to several authors that have reported the fungicide effec-
lengths). A very interesting result was obtained for plants grown
tiveness of avonoid compounds similar to those present in AEEF
after the AEEFw-A treatment, since their roots and stems were as
(Skadhauge et al., 1997; Galeotti et al., 2008). On the contrary,
long as those of the control plants (66 and 109 mm as compared to
Ruan et al. (1995) found that various avonoids which occur in
60 and 106 mm, respectively) (Fig. 1B).
root exudates of legumes exhibit a strong stimulatory effect on the
macroconidia germination on the pea and bean pathogen Fusarium
4. Discussion solani (Mart.) Sacc.
Because EtOH is a disinfectant, the ethanolic amendment also
Our experiments show that extracts obtained from triguero had a reducer effect upon mycelial growth and sporulation of the
asparagus Morado de Huetor contain components of great impor- fungi. However, and for the three F. oxysporum isolates, both vari-
tance in the inhibition of mycelial growth and sporulation of F. ables were signicantly reduced by the AEEF as compared to the
oxysporum pathogenic to asparagus, carnation or strawberry. ethanolic amendment. Concerning the effectiveness of the AEEF,
56 C. Rosado-lvarez et al. / Scientia Horticulturae 171 (2014) 5157
and since the total content of avonoids is signicantly decreased
after heating and stirring (Fuentes-Alventosa et al., 2009), the AEEF
was more effective when added after autoclaving.
Regarding asparagus seed germination and growth of seedlings,
they were high and similar to those of the control treatment
when the growth medium was amended only with AEEF. In
contrast, zero or very low germination was obtained in the
EtOH amendment as well as when the AEEF was previously dis-
solved in EtOH, suggesting the toxicity of EtOH to asparagus
seeds.
To our knowledge, this is the rst report about the antifun-
gal properties of bioactive compounds, mainly avonoids, from
asparagus by-products. Future studies should consider the isola-
tion of these avonoid compounds in order to identify which of
them is/are responsible for the fungicide effect. Future research
might also explore on the in vivo effect of the AEEF, i.e. its
antifungal activity when the pathogens are infecting asparagus,
carnation or strawberry plants. Triguero asparagus are an impor-
tant source of avonoids ((Fuentes-Alventosa et al., 2007, 2008;
Guilln et al., 2008). Therefore, the use of these bioactive by-
products appears as a highly interesting and promising alternative
within crop protection strategies of a modern sustainable agri-
culture. Further investigations are required in order to determine
the specic activities of both individual compounds and mixtures
of avonoids from different green asparagus genotypes. It is also
of interest to investigate the possible synergistic or antagonistic
interactions among the different avonoids and/or other bioactive
compounds present in the asparagus extracts. These studies will
allow establishing the most adequate composition and effective
doses of these potential biofungicides based on natural extracts
from asparagus by-products, which is crucial for getting success on
the application of natural extracts as control agents on horticultural
crops.
5. Conclusions
The asparagus extract used in this work was obtained from by-
products consisting on the basal portions of the spears that are
discarded prior to its industrial canning, and it was characterized
as an asparagus extract enriched in avonoids (AEEF). The potential
antimicrobial activity of the AEEF was evaluated in this work, being
this, to our knowledge, the rst report about the antifungal prop-
erties of bioactive compounds, mainly avonoids, from asparagus
by-products.
The results from independent and replicated in vitro experi-
ments show that AEEF contain components of great importance
in the inhibition of F. oxysporum f. sp. asparagi, F. oxysporum f.
sp. dianthi and F. oxysporum pathogenic to strawberry. The anti-
fungal activity of AEEF was expressed as: inhibition of fungal
mycelial growth, decrease of its progression in time (SAUGPC)
when used at the highest dose, and a very notorious reduction
of the sporulation of the three fungi. Regarding asparagus seed
germination and growth of seedlings, our results show the harm-
lessness of the biocompound on the development of asparagus
plants.
Acknowledgments
This work was funded by CICE, Junta de Andalucia, Spain
(grant number P07-AGR02364) and by a IFAPA scholarship to C.
Rosado-lvarez. We are grateful to Asuncin Snchez-Contreras
for technical assistance. The critical reading of the manuscript and
valuable suggestions prior to submission of Dr. Guilln-Bejarano
are gratefully acknowledged.
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