MANAGEMENT OF SCLEROTINIA WHITE ROT OF BEANS WITH
ANTAGONISTIC MICROORGANISMS
Journal of Animal and Plant Sciences. 27.2 (Apr. 30, 2017): p542. From General
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Byline: Basheer A. Alsum, Mohamed Elsheshtawi, Maged T. Elkahky, Abdallah M.
Elgorban, Marwah M. Bakri and Manal M. Alkhulafi
ABSTRACT
In the present study, antagonistic activity of locally isolated bio agents including
five fungi and three bacteria was evaluated against Sclerotinia sclerotiorum (Lib.) de
Bary casual of white rot of snap beans. All tested biocontrol agents were able to
inhibit radial growth and sclerotia viability in dual culture assay. Trichoderma
hamatum (Bonorden) Bainier was the most effective agent in suppressing the mycelial
growth of S. sclerotiorum by 93% compared to control. Whereas, tested isolates of
Trichoderma viride Pers and Coniothyrium minitans Campbell were able to
completely deactivating all treated sclerotia. In field trial, same isolates were tested in
comparison of other commercial bio products. Naturally infested soil with S.
sclerotiorum treated with local isolated bio agents as well as some commercial bio
agents. Local isolate of C. minitans was the most effective in reducing disease
incidence and the disease severity by 94.6% living plants (5.4 % mortality) and 13.0,
respectively.
Trichoderma hamatum and Contans(r) (commercial product C. minitans) also
minimized disease severity by 14 and 16.2%, respectively when compared to
untreated control. Among tested bacterial bicontrol agents, Pseudomonas fluoroscens
was the best in reducing disease severity by 21.3% compared to controls. Yield data
showed that Trichoderma hamatum increased total yield (10.485 ton/ha) Conversely,
C. minitans was the best in increasing quality of yield in terms of exportable yield that
giving 9.729 ton/ha.
Keywords: Coniothyrium minitans; white rot; low tunnel; beans.
INTRODUCTION
Sclerotinia sclerotiorum (Lib.) de Bary, considers one of the most destructive soil
borne pathogens. It has been reported that this pathogen affects a wide range of wild
and cultivated cops. It can infect over 408 species and 42 subspecies of plants at all
stages of growth in field, moreover the infectioncould be developed during transit and
storage of the product during postharvest stages (Barari et al., 2010). The resulted
disease is commonly known as white mold, sclerotinia wilt or stalk rot. White rot
considers one of the most important limitation factor in producing green beans in
Egypt. S. sclerotiorum has been isolated from soil samples obtained from greenhouses
and protected agricultural areas. These areas - where bean plants are grown-usually
known to be very moist and cool. Such conditions seemed to be subsidizing factors to
incidence of white rot disease.
One of the major problems in controlling this disease, that the pathogen produces
large numbers of sclerotia which could stay viable for a long time in the soil. During
the growing season, depending on various diverse environmental factors, sclerotia
start to germinate and produce either mycelium or ascospores by developing an
apothecium (Elgorban et al., 2013). Ascospores are the primary inoculum for
epidemics in many crops. They can move for a long distance to neighboring fields and
infect plants in adhering fields. The fungus is capable of infecting flowers, leaves,
fruits or stems. Wide host range of S. sclerotiorum make the control process more
difficult. According to FAO STAT database Egypt exported about 37597 thousand
ton of green beans in 2013. Most of this yield were exported to European Union fresh
market.
Beside known problems of using chemical control of white rot in beans such human
health concerns, environmental pollution, and development of resistant isolates, most
exporting regulation restrict using chemical pesticides for controlling white rot of
snap beans. Biological control as a disease management strategy in protected
agricultural areas could be economical and durable. It helps in reducing potential
inoculum, which will lead to decreasing amount of disease produced by the
pathogenic fungus. Several biocontrol agents have been screened for the control of S.
sclerotiorum. For instance, Ulocladium atrum found to be a successful bio agent to
management of S. sclerotiorum (Fernando et al., 2007). Furthermore, Trichoderma
and Bacillus species seemed to be effective bio agents against S. sclerotiorum (Zhang
et al., 2004; Fernando et al., 2007;).
The aim of this study was to evaluate the effectiveness of some local isolates of
antagonistic fungiand bacteria in controlling white rot of snap beans and compare
their controlling level to other available commercial bio and chemical pesticides
taking in consideration the impact of that control level on the quantity and quality of
green beans yield.
MATERIALS AND METHODS
Pathogenic fungus: Sclerotinia sclerotiorum used in this study was retrieved from
sclerotia collected from diseased bean plants (Phaseolus vulgaris L.). Infected plants
samples showing typical symptoms of white rot were collected from Ismailia
governorate, Egypt. Collected sclerotia were surface sterilized with NaOCl solution
then plated on PDA and incubated at 25 degC for 7 days. The purified fungal isolates
were identified by Department of Plant Pathology, College of Agriculture, Mansoura
University according to Kora et al., 2005. PDA slants from isolated fungus were kept
at 4 oC for further studies.
Isolation, purification and identification of antagonistic fungi: Soils from 20 different
fields of Ismailia, Egypt were collected in sterile polyethylene bags. Standard serial
dilution method was used for isolation of antagonistic soil fungi. The soil suspensions
were done by suspending 1 g of each soil sample in 9 mL of 0.1 % peptone solution.
Serial dilution has been done by transferring 1mL of the previous suspension to 9 mL
of 0.1 % peptone to be diluted to 1/10. 0.1 mL from each dilution was plated onto
PDA supplemented with 300 mg/L of chloramphenicol. Petri plates were incubated at
25+-2oC for 7 days until sporulation was observed. Individual colonies with typical
Trichoderma characters such as green, velvety mycelia were transferred separately
onto new PDA plates, incubated at 25+-2 oC for 3-5 days.
The isolates that grew rapidly and formed greenish to white concentric circles were
transferred to the Trichoderma-selective medium Rose Bengal agar (Williams, et al.,
2003) (Magnesium sulphate heptahydrate 0.2 gm/L; Dipotassium hydrogen phosphate
0.9 gm/L; Ammonium nitrate 1.0 gm/L; Potassium chloride 0.15 gm/L; Glucose 3.0
gm/L; Rose Bengal 0.15 gm/L; and Agar 20 gm/L).
The isolates were confirmed as having the same morphotype as on PDA and then
stored as purified isolates in 50 % (v/v) glycerol at -80 oC. Trichoderma spp. and
Clonostachys rose were identified by microscopic observations according to
identification keys of Bissett (Bissett, 1991a; Bissett, 1991b; Rifai, 1969).
Conothyrium minitans Campbell, Isolate, the commercials product Trifender(r)
(Trichoderma asperellum Samuels, Lieckf. and Nirenberg) and Contans(r) (C.
minitans Campbell) were obtained from Plant Pathology Department, Plant Protection
Research Institute, Budapest, Hungary. Spore suspensions of antagonistic fungi were
prepared by subculture each fungus on PDA then incubated for 15 days at 25+-2 oC
in the dark then adding 5 ml of sterilized distilled water and 2 drops of tween 20 to
each plate and scrap the surface with sterilized spatula to harvest the spores.
The resulted suspension has been transferred to sterilized baker through two layers of
sterilized cheese cloth to get rid of mycelial fragments. Concentration of spore
suspension was adjusted to 1x106 using hemocytometer slide to count spores and
sterilized distilled water to dilute the suspension Isolation and Identification of
antagonistic bacteria: Pseudomonas fluorescens was isolated from the rhizosphere of
healthy green beans obtained from farmland in Ismailia, Egypt, using King's B
medium. The identification of P. fluorescens was based on morphology, Gram
staining, physiological and biochemical tests (Krieg and Holt, 1984).
Locally isolated Bacillus subtilis was obtained from Center Laboratory Organic
Agriculture, Agricultural Research Center, Egypt, where the commercial product
Mycostop(r) (lyophilized spores of Streptomyces griseoviridis) obtained as a kind gift
from the Kemira OY(r) of Finland. B. subtilis and P. fluorescens were grown on
Nutrient Agar medium (NA), while Streptomyces griseoviridis was used as spore
suspension from the commercial product Mycostop(r).
Effect of antagonistic fungi on mycelial growth of Sclerotinia sclerotiorum in vitro:
One mycelial discs 5 mm in diameter from 7 days old culture of
antagonistic fungi (Trichoderma harzianum, Trichoderma viride, Trichoderma
hamatum, Clonostachys rosea, and Coniothyrium minitans) were placed in facing 5
mm disc of S. sclerotiorum on in 90 mm Petri dish containing PDA, with four
replicates. Control treatments conducted same as in treatments but without
antagonistic fungi disc. All plates were incubated at 25+-2 oC for 15 days. Percentage
of mycelial growth inhibition was calculated after 3 and 15 days by comparing the
radial growth in treatments plates to control.
Effect of antagonistic bacteria on Sclerotinia sclerotiorum (Lib.) de Bary: One disc, 5
mm in diameter of mycelial growth of S. sclerotiorum was placed in side of Petri dish
and antagonistic bacteria were spot inoculated at 3 cm distance from pathogen's disc
on a PDA. The inhibition zone was observed after 3 and 15 days of incubation at 25+2 oC.
Viability of Sclerotinia sclerotiorum-sclerotia treated with antagonistic
microorganisms: Sclerotia were collected from 21 day old culture of S. sclerotiorum
grown on PDA and incubated at 25+-2 oC then dipped into a spore suspension 1x106
cfu/ml in case of antagonistic fungi or bacterial cell suspension 1x103 cfu/ml in case
of antagonistic bacteria for 5 min. After that, all sclerotia were dried on sterilized
filter paper in an air current for two hours under laminar flow hood. Untreated
sclerotia used as the control were dipped in sterilized distilled water. The sclerotia
were placed on the bottom of Petri plates, incubated at 20+-2 oC in a sterile humidity
chamber (100% Rh). After 30 days, the viability of S. sclerotiorum-sclerotia was
estimated by placing them on WA for 48 h at 25degC and counting the number of
emerging hyphae with phase contrast microscopy (100x).
The sclerotia viability was assessed on a scale from 0 to 4, and sclerotia viability
index (VI) was calculated according to Jager and Velvis, 1988. One hundred sclerotia
were used for each treatment.
Low tunnels experiments:
Soil preparation: The experiment was performed on loamy sand soil (pH 7.16) at the
protected agricultural area of exportable green bean var. Paulista under low tunnel
conditions. This experiment was established at Gamal Ahmed Farm, Faid city,
Ismailia governorate, where fields were naturally infested with S. sclerotiorum.
Fertilizers were applied according to the recommendation of Agricultural extension
department in that area, in amount per hectare were as follows, 168 kg agriculture
sulfur, 480 kg calcium phosphate Ca(H2PO4)2, 240kg Ammonium sulfate
(NH4)2SO4, and 120kg K2SO4, while the organic amendments were 24m3 chicken
manure and 12 m3 livestock manure. All fertilizers were applied at the beginning of
the first season. The experiment had randomized complete block design. The plots
were 0.75x5.0 meter, 10 cm distance between plants, with three replicates per
treatment.
Effect of antagonistic microorganisms on disease incidence and disease severity of
white rot in beans: This test was done to evaluating antagonistic ability of five fungal
antagonists and two bacterial antagonists in addition to three commercial biofungicides products; Contans(r) (C. minitns Campbell, 1x109 cfu/mL) and
Trifender(r) (T. asperllium 1x106 cfu/mL) and Mycostop(r) (S. griseoviridis, 1x106
cfu/gm) in controlling whit rot of green beans under natural infestation conditions.
Antagonistic fungi were applied as spore suspensions (1x 106 cfu/ml) through
drenching soil at 5 days after sowing (DAS) at rate 50 ml spore suspension per plant.
The control treatments were naturally infested soil without any treatment. Three
chemical pesticides were used as chemical check at recommended dose. Chemical
pesticides were also applied with same method (soil drenching 50 ml/ plant). Used
pesticides were Topsin M-70(r) (2 gm/L), Rizolex(r) and Captan(r) (3 gm/L).
The disease incidence calculated as average of dead plants numbers. The number of
surviving plants after 15, 45 and 60 DAS were recorded. Disease severity (DS) was
assessed on 0 to 4 scale. At the end of the season, plants were removed and washed to
be free of soil then roots were visually assessed for percentage of affected root area
that was due to Sclerotinia rot and each plant was assigned a root disease score 0-4 as
follows:-0=lesions and necrosis absent from roots; 1= 1-25 % of total root area
necrotic; 2 = 26-50 % of total root area necrotic; 3 = 51-75 % of total root area
necrotic; 4 = 76-100 % of total root area necrotic. The DS was calculated with the
following formula:
Disease sevarity = Sum of all rating / Total number of plants x Maximun score x 100
In addition, number of branches, plant height, total yield and exportable yield were
recorded as indicators for non-direct impact on controlling the pathogen.
Statistical analysis: Collected data were statistically analyzed using the Statistic
Analysis System Package (SAS institute, Cary, NC, USA). Differences between
treatments were studied using Fisher's Least Significant Difference (LSD) test and
Duncan's Multiple Range lest (Duncan, 1955). All analysis was performed at P: 5 %
level.
RESULTS
Effect of the antagonistic fungi on Sclerotinia sclerotiorum: After 3 days, C. minitans
Campbell was the most effective against S. sclerotiorum with 74.4% reduction in the
mycelial growth. This was followed by T. hamatum (Bonorden) Bainier that cause
60.0% inhibition in the mycelial growth (Table 1). Conversely, T. hamatum
(Bonorden) Bainier was the most effective against the radial growth of S.
sclerotiorum after 15 days, with 93.0%, followed by T. viride Pers, T. harzianum
Rifai and C. minitans Campbell that causing 91.9, 91.7 and 91.1% reduction in the
mycelia growth, respectively.
Effect of the antagonistic bacteria on Sclerotinia sclerotiorum: Data in Table 2 show
the tested antagonistic bacterial strains significantly reduced pathogens growth in
comparison to the control. P. fluoroscens was the most effective against S.
sclerotiorum that giving 40.2 and 61.9% inhibition at 3 and 15 days, respectively.
Viability of Sclerotinia sclerotiorum-sclerotia inoculated with antagonistic fungi:
Germination of S. sclerotiorum-sclerotia was extremely reduced by
antagonistic fungi (Table 3). S. sclerotiorum-sclerotia inoculated with T. viride Pers
and C. minitans Campbell were completely deactivated, while non-inoculated
sclerotia showed 85.8% viability index.
Viability of Sclerotinia sclerotiorum sclerotia inoculated with antagonistic bacteria:
The effect of antagonistic bacteria on sclerotia viability is presented in Table 4.
Treatment of sclerotia by antagonistic bacteria caused comparable results in VI of the
sclerotia ranging from 19.5% VI in case of P. fluorescens to 20.5% in case of
Mycostop(r), while the control was 85.8% VI.
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