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MANAGEMENT OF SCLEROTINIA WHITE ROT OF BEANS WITH ANTAGONISTIC MICROORGANISMS.

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 infection could 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 fungi and 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 bio-fungicides 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.

Low tunnel investigations:

Effect of soil application with antagonistic microorganisms on disease incidence and disease severity of white rot disease in beans

Disease incidence: Data in Table 5 clarify that there were significant differences between all treatments and control in suppressing the DI caused by S. sclerotiorum. C. minitans Campbell and T. hamatum (Bonorden) Bainier were the most effective in decreasing the DI of white rot disease by 94.6 and 94.0% living plants, respectively while untreated control gave 61.4% and topsin M-70 that giving 93.4%. These were followed by Contans(r) and Mycostop(r) which produced 90.6 and 89.4% living plants, respectively.

Disease severity: Coniothyrium minitans Campbell was the best antagonistic fungi of S. sclerotiorum that reduced the DS with 13.0% when compared with untreated controls (61.0%) and chemicals control (11.2% in case of Topsin M-70). Alternatively, T. hamatum and Contans(r) produced 14 and 16.2% DS, respectively (Table 5). Conversely, all treatment with antagonistic bacteria gave a moderate effective in the DS ranging from 21% (Mycostop(r)) to 23.2% (B. subtilis).

Effect of antagonistic microorganisms on growth characteristics

Number of branches: Data presented in Table 6 showed that T. hamatum (Bonorden) Bainier, C. minitans Campbell and P. fluorescens were the best treatments for affecting in number of branches that giving 17.33, 17 and 17 branch/plant, respectively when compared to untreated controls. There were followed by B. subtilis and Mycostop (r) which produced 16 and 15.62 branch/plant, respectively.

Table 1. Effect of antagonistic fungi on radial growth of Sclerotinia sclerotiorum.

###After 3 days###After 15 days

Fungi

###R.G.###Inh.%###R.G.###Inh.%

Trichoderma harzianum###43.8c###51.3###7.5cd###91.7

Trichoderma viride###44.0c###51.1###7.3cd###91.9

Trichoderma hamatum###35.8d###60.0###6.3d###93.0

Clonostachys rosea###54.8b###39.1###45.3b###49.7

Coniothyrium minitans###23.0e###74.4###8.0c###91.1

Control###90.0a###0.0###90.0a###0.0

LSD###5.5###1.96

Plant height: Results in Table 6 illustrate that the best treatments which affected the plant height were P. fluorescens and B. subtilis that provided 91.28 and 90 centimeters, respectively when compared with untreated control (55.17 cm.) and chemicals control (86.68 cm. in case of topsin M-70). While, C. minitans Campbell and T. hamatum (Bonorden) Bainier produced 89.66 and 87cm, respectively.

Yield per hectare: Trichoderma hamatum, C. minitans and Mycostop(r) were the best antagonistic microorganisms affecting total yield per hectare that produced 10.485, 10.325 and 10.080ton/hectare., respectively when compared to untreated control (8.537 ton/ha.) and Topsin M-70 (10.494 ton/ha.). Also, Contans(r) produced 9.877 ton/ha. Furthermore, the least effective treatment in the total yield was Clonostachys rosea provided 8.863 ton/hectare (Table 6).

Exportable yield per hectare: Data in Table (6) explained that there were significant differences between all treatments and the untreated control. The best treatments affecting exportable yield were C. minitans Campbell and T. hamatum that increased the productivity by 9.729 and 9.449 ton/ha., respectively when compared to untreated control (6.233 ton/ha.) and chemicals control (9.734 ton/ha. in case of Topsin M-70). While, T. viride, Mycostop (r) and Contans (r) produced 9.103, 9.133 and 9.160 ton/ha., respectively.

Table 2. Effect of antagonistic bacteria on radial growth of Sclerotinia sclerotiorum.

###After 3 days###After 15 days

Fungi

###R.G.###Inh. %###R.G.###Inh. %

Pseudomonas fluorescens###53.8c###40.2###34.3c###61.9

Bacillus subtilis###55.5bc###38.3###41.0b###54.4

Mycostop(r) (S. griseoviridis)###58.8b###34.7###36.3c###59.7

Control###86.8a###0.0###90.00a###0.0

L.S.D###4.09###4.02

Table 3. Viability of Sclerotinia sclerotiorum-sclerotia inoculated with antagonistic fungi.

###Percentage of Sclerotia according to the number of emerging

###viability

Treatments###hyphae

###index

###0###1-5###6-10###11-25###>25

Trichoderma harzianum###86###13###1###0###0###3.8

Trichoderma viride###100###0###0###0###0###0.0

Trichoderma hamatum###93###3###4###0###0###2.8

Clonostachys rosea###50###31###10###7###2###20.0

Coniothyrium minitans Campbell###100###0###0###0###0###0.0

Control###3###6###4###19###68###85.8

Table 4. Viability of Sclerotinia sclerotiorum-sclerotia inoculated with antagonistic bacteria.

###Percentage of sclerotia according to the number of emerging

###Viability

Treatments###hyphae

###index

###0###1-5###6-10###11-25###>25

Pseudomonas fluorescensns WCS365###50###30###15###2###3###19.5

Bacillus subtilis###57###22###10###7###4###19.8

Mycostop(r) (S. griseoviridis)###52###29###11###1###7###20.5

Control###3###6###4###19###68###85.8

Table 5. Effect of soil application with biocontrol agents on disease incidence and disease severity of white rot disease in beans.

###After 15 days###After 45 days###After60

Treatments###DS%

###No.###Mo.%###Sur.%###No.###Mo.%###Sur.%###No.###Mo.%###Sur.%

Non-treated###33.0i###34.0###66.0###31.7h###2.6###63.4###30.7j###2.0###61.4###61.0

T. harzianum###40.7hg###18.6###81.4###40.0fg###1.4###80.0###40.0hi###0.0###80.0###20.2

C. minitans Campbell###47.3a###5.4###94.6###47.3a###0.0###94.6###47.3ab###0.0###94.6###13.0

Contans(r)###46.0ab###8.0###92.0###45.3abc###1.4###90.6###45.3abc###0.0###90.6###16.2

Trifender(r)###43.7bcde###12.6###87.4###43.0cde###1.4###86.0###43.0def###0.0###86.0###21.7

T. viride###44.7bcd###10.6###89.4###44.3bcd###0.8###88.6###44.3cde###0.0###88.6###21.9

T. hamatum###47.7a###4.6###95.4###47.0a###1.4###94.0###47.0ab###0.0###94.0###14.0

C. rosea###39.3h###21.4###78.6###38.7g###1.2###77.4###38.7i###0.0###77.4###31.7

Mycostop(r)###45.7abc###8.6###91.4###45.0abc###1.4###90.0###44.7bcd###0.6###89.4###21.0

P. fluoroscens###41.7efg###16.6###83.4###40.7efg###2.0###81.4###40.7fghi###0.0###81.4###21.3

B. subtilis###43.3cdef###13.4###86.6###43.0cde###0.6###86.0###43.0defg###0.0###86.0###23.2

Captan 50-WP###41.0fgh###18.0###82.0###40.3fg###1.4###80.6###40.3ghi###0.0###80.6###33.7

RizolexTM###42.3defg###15.4###84.6###42.0def###0.6###84.0###42.0efgh###0.0###84.0###25.5

Topsin M 70WP###47.3a###5.4###94.6###46.7ab###1.2###93.4###46.7abc###0.0###93.4###11.2

LSD###2.4###2.6###2.5

Table 6. Effect of soil application with biocontrol agents on beans growth and yield.

###No. of###Plant###Total Yield###Exportable yield

Treatments

###branches###Height (cm)###(Ton/hectare)###(Ton/hectare)

Non-treated###10.00f###55.17e###8.537e###6.233e

T. harzianum###14.55cd###70.83cd###9.083de###7.533d

C. minitans###17.00ab###89.66a###10.325a###9.729a

Contans(r)###15.00bcd###86.00ab###9.877abc###9.160abc

Trifender(r)###14.87cd###79.47abcd###9.548bcd###8.579bc

T. viride###15.58abcd###85.78abc###9.806abc###9.103abc

T. hamatum###17.33a###87.00ab###10.485a###9.449ab

C. rosea###12.00ef###69.33de###8.863de###7.493d

Mycostop(r)###15.62abcd###72.93bcd###10.082ab###9.133abc

P. fluorescens###17.00ab###91.28a###9.258cd###8.272cd

B. subtilis###16.00bc###90.00a###9.380bcd###8.614bc

Captan 50-WP###11.53f###71.64cd###9.490bcd###8.844abc

RizolexTM###14.00de###81.60abcd###9.929abc###8.878abc

Topsin M 70 WP###16.68abc###86.68ab###10.494a###9.734a

LSD###2.25###15.02###0.52###0.67

DISCUSSION

Beans white rot disease is a serious disease under the favored agro-climatic conditions in Ismailia Governorate, Egypt. One of the restrictions to fulfilling biological control against plant diseases is the lack of knowledge on the use of in vitro tests for selection of BCAs. Furthermore, the disease is usually overlooked by beans growers because it appears by the end of the growing season at the lower parts of the stem.

In the present study, it was noticed that Trichoderma species were highly efficient against S. sclerotiorum in the sequence T. hamatum, T. harzianum and T. viride. This high antifungal activity of Trichoderma spp. mostly dependent on some lytic enzymes, which act as fungal cell-well-degrading agents such as N-acetyl-ss-D-glucosedeaminidase, chitinase, ss-1, 3 gluconase, chitobiosidase and protease (Harsukh et al., 2013). T. harzianum was found to restrain enzymes of foliar pathogens, the activities of exo-endopolygalacturonase, pectin methyl esterase, pectatelyase, chitinase and cutinase, which are thought to be involved in mycoparasitism process in leaves infested with fungi (Sharma et al., 2012). Furthermore, Trichoderma spp. could produce cynamidehydratase, rhodanese and ss-cyanoalnine synthases, which known to play an important function in reducing the growth of plant pathogenic fungi (Wilmari, 2010).

Under low tunnels studies, it was observed that T. hamatum was the greatest antagonistic fungi against S. sclerotiorum that produced 94% living plants and 14% DS when compared with controls. The results can be understood by synergistic involvement of a number of mechanisms, which may include activation of plant defense system (Manoj et al., 2011). The synthesis of pathogenesis-linked proteins is one of the most ordinary defense mechanisms triggered in plants following infection with inducing agents (Markus and Susanne, 2010). Induced resistance is recognized as an important mode of biocontrol in vegetative tissue (Aidemark et al., 2010). Induced systemic resistance caused by Trichoderma spp. and various microorganisms can protect plants against soil or foliar pathogens (Chowdappa et al., 2013). Salicylic acid produced by Trichoderma spp. induced resistance to B. cinerea in bean (Martinez-Medina et al., 2013).

Besides, root colonization with Trichoderma induced increased peroxidase and chitinase activities in many plants (de Santiago et al., 2011).

In the current study, we observed that C. minitans one of the best antagonistic fungi against S. sclerotiorum. This high antifungal activity of C. minitans Campbell may be attributed to causing destruction of hyphae and sclerotia of S. sclerotiorum (Whipps et al., 2008). The extra-cellular enzyme ss-1, 3-glucanase (EC 3.2.1.39) appears to be an important enzyme involved in the mycoparasitism of S. sclerotiorum by C. minitans Campbell, as the expression of the gene cmg1 encoding ss-1,3-glucanase increases during infection of sclerotia of S. sclerotiorum by C. minitans.

Under low tunnel studies, C. minitans significantly reduced disease levels with 94.6% living plants and 13.0% DS when compared with untreated controls and establishment of the BCA in the rhizosphere protects the bean roots from infection with the mycelium of S. sclerotiorum growing into this region (Khokhar et al., 2012).

From all tested antagonistic bacteria, it was observed that P. fluorescens gave a high influence on the mycelial growth and the VI of S. sclerotiorum-sclerotia with 61.9% and 19.5%, respectively. Also, it had highly reduced the DI and the DS caused by S. sclerotiorum with 81.4% living plants and 21.3% DS. This high efficiency of P. fluorescens against S. sclerotiorum is probably related to the degradation of chitin in hyphal and sclerotia cell by several hydrolyzing enzymes (Sebastian et al., 2010) such as endochitinase or, exochitinase and chitobiosidase. In addition, P. fluorescens produces three antibiotics which are possibly involved in its biocontrol activity: 2, 4-diacetylphloroglucinol, pyrrolnitrin and pyoluteerin (Sebastian et al., 2010). P. fluorescens has been reported to succeed in decreasing plant diseases and promoting plant growth by inducing systemic resistance (Phrueksa, 2010).

These mechanisms include competition for nutrients and place, and producing chitinase, lytic enzyme, siderophores besides many antibiotics viz, 2,4-diacetylphloroglucinol, pyoluteorin, pyrrolnitrin, pyocyanin and oomycin A (Vanitha and Umesha, 2011). Biocontrol bacteria may stimulate plants to secrete more chitinase when colonizing the rhizosphere, and the plant chitinase may play a more effective role in plant defense (Fgaier and Eberl, 2011). Hui et al. (2011) confirmed that P. fluorescens produced volatile hydrogen cyanide, which stopped the growth of S. sclerotiorum for a long period. In addition, Sharma et al. (2011) have discovered that P. fluorescens P13 could excrete siderophores that might inhibit S. sclerotiorum.

In thus study, it was indicated that Mycostop(r) was effective in controlling the fungus S. sclerotiorum. The effect of Mycostop(r) was similar to the previous reports above. It seems obvious that S. griseoviridis produces extracellular enzymes with lytic characteristics. S. griseoviridis is well known as a root colonizer and stimulates root growth during rhizosphere colonization (Doumbou et al., 2001). In some cases, stimulation of plant growth could explain the enhanced yield results when the antagonist was combined with soil solarization. The effectiveness of Mycostop(r) against Fusarium wilt of tomato was satisfactory when applied to artificially infested soil. On the other hand, higher yields of healthy crops after Mycostop(r) application indicate that a growth promoting factor may also be involved.

Another hypothesis for vigorous growth and yield increases is the control of minor pathogens. Sutthinan et al. (2010) had shown that S. griseoviridis produced indole-3-acetic acid which induces several effects in plant, among which the stimulation of growth is.

Conclusion: Finding harmless and ecological alternatives to chemical control is an urgent need to face increasing demand for safe, sustainable and effective management plan to white rot of beans counting on biocontrol agents, and other best disease management practices. This study demonstrated that the antagonistic fungi could be considered better biocontrol agents against S. sclerotiorum than the antagonistic bacteria in vitro and under the field conditions. The current study suggests that all tested biocontrol agents could be used in an integrated control program against S. sclerotiorum. Fungal biocontrol agents such Trichoderma spp. and C. minitans Campbell and bacterial biocontrol agents such P. fluorescens seemed to be efficient control elements if it is used in integration with other management practices, including cultural practices, using resistant varieties, and reduced chemical control.

Acknowledgments: This project was supported by King Saud University, Deanship of Scientific Research, College of Science Research Center. I would like to express my sincere gratitude and deep gratefulness to all our colleagues of the Department of Botany and Microbiology, King Saud University for their valuable criticism, advice and the authors have declared no conflict of interest.

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