Res. on Crops 15 (4) : 884-892 (2014) Printed in India Biological control of Sclerotinia sclerotiorum in beans with antagonistic microorganisms under greenhouse conditions ALI H. BAHKALI, MOHAMED ELSHESHTAWI1, RAMADAN A. MOUSA2, ABDALLAH M. ELGORBAN*3 AND AREEJ ABDULLAH ALZARQAA4 Department of Botany and Microbiology College of Science, King Saud University, P. O. Box 2455, Riyadh 11451, Saudi Arabia *(e-mail : aelgorban@ksu.edu.sa) (Received : June 2014/Accepted : July 2014) ABSTRACT The effects of the mycoparasites Coniothyrium minitans and Trichoderma harzianum, Trichoderma viride, Trichoderma hamatum, Gliocladium virens, Gliocladium roseum, Pseudomonas fluorescens, Streptomyces griseoviridis and Bacillus subtilis on the suppression of bean white rot caused by Sclerotinia sclerotiorum were evaluated in vivo during 2013. Results showed that soil drenching with C. minitans, T. viride and T. hamatum significantly suppressed the white rot disease incidence with 90% survival plants. In case of mixture, antagonistic fungi and antagonistic bacteria, the mixtures of T. hamatum+S. griseoviridis and C. minitans+S. griseoviridis completely inhibited the disease incidence that produced 100% survival plants when compared to controls. Key words : Antagonistic, Bacillus subtilus, beans, biocontrol, biological control, Coniothyrium minitans, Sclerotinia sclerotiorum INTRODUCTION Sclerotinia sclerotiorum (Lib.) de Bary is a destructive pathogen with worldwide distribution known to infect 408 species and 278 genera of plants nearly (Boland and Hall, 1994). Important crops affected include oilseed rape, sunflower, tobacco and a range of vegetables such as bean, lettuce, cauliflower, cabbage, carrot and potato as well as a number of flower crops. Control using crop rotations is unrealistic due to the persistence of survival structures as sclerotia in the soil for long periods and because Sclerotinia has such a wide host range (Nelson, 1998; Elgorban et al., 2013). These factors require the use of fungicides, which have been known to have adverse effects on non-target organisms (Gilmour, 2001). Also, the rising costs of soil fumigation, lack of suitable replacement for toxic fumigants and public concerns over repeated fungicide use for disease control have 1 meant that alternative control methods, such as biological control are gaining interest with growers. The use of microbial agents to control plant pathogens can be an eco-friendly and economical component of an integrated pest management programme (Mao et al., 1997). Several microorganisms have been reported effective as potential biocontrol agents for management S. sclerotiorum of beans (Li et al., 2006; Huang and Erickson, 2007). Coniothyrium minitans and Sporidesmium sclerotivorum, two specialized mycoparasites of Sclerotinia spp., have been reported to control disease in both glasshouse and field trials (Rabeendran et al., 2006; Wenting et al., 2012). Trichoderma spp., namely, Trichoderma harzianum and T. virens have also been shown to attack both sclerotia and mycelium of Sclerotinia spp. (Chitrampalam et al., 2008; de Figueirêdo et al., 2010) and have been reported to give some disease control in field trials against sclerotia (Ristaino et al., 1994; Wenting Plant Pathology Department, College of Agriculture, Mansoura University, Mansoura 35516, Egypt. Weed Research Central Laboratory, Agricultural Research Center, Giza, Egypt. 3 Plant Pathology Institute, Agricultural Research Center, Giza, Egypt. 4 Department of Biology, Umuluj College, Tabuk University, Saudi Arabia. 2 Control of Sclerotinia sclerotiorum in beans with antagonistic microorganisms et al., 2012). Bacterial biocontrol agents against S. sclerotiorum are rarely studied (Boyetchko, 1999). Some bacterial strains have shown antifungal activity to S. sclerotiorum, such as Erwinia herbicola (Godoy et al., 1990), Bacillus spp. and Pseudomonas species (Fernando et al., 2007). The objective of this study was to (1) evaluate the antagonistic activity of bioagents against S. sclerotiorum and (2) investigate the combination of antagonistic fungi and bacteria against S. sclerotiorum. MATERIALS AND METHODS Fungal and Bacterial Isolates Pathogenic fungus : Sclerotinia sclerotiorum (Lib.) de Bary used in this study was derived from sclerotia on diseased bean plants (Phaseolus vulgaris L.) from Ismailia governorate, Egypt during 2013. The purified fungal isolates were identified by Department of Plant Pathology, College of Agriculture, Mansoura University (Kora et al., 2005). PDA slants from each isolated fungus were kept in 4ºC for further studies. Antagonistic fungi : Soil samples were collected from 40 different places comprising different agricultural protected area and greenhouses in five governorates, Egypt. For isolation of Trichoderma strains, a serial dilution technique was followed and a 10 3 dilution of each sample was prepared. One milliliter of each solution was pipetted onto a Rose Bengal Agar plate and incubated at 20±2°C for one week. The plates were examined daily and each colony that appeared was considered to be one colony forming unit (CFU). After enumeration of CFU, individual colonies were isolated from the same plates and each uncommon colony was re-isolated onto a fresh potato dextrose agar (PDA) plate. Distinct morphological characteristics were observed for identification (Rifai, 1969, Bissett, 1991a and b; Barnett and Hunter, 1998). In case of Glicoladium roseum, after 10-12 days, the discs were checked under a stereoscopic microscope for G. roseum colonies with typical G. roseum conidiophores were transferred to PDA amended with chloramphenicol (50 mg/l). Identification was carried out based on microculture colony morphology (Barnett and Hunter, 1998; Schroers et al., 1999). Isolates 885 were preserved in PDA discs kept in sterile distilled water and in colonized wheat kernels with silica gel, both stored at 4°C. Antagonistic bacteria : Pseudomonas fluorescens was isolated from the rhizosphere of green bean in long term rotation farmland in Ismailia, Egypt, by using King’s B medium. Identification of P. fluorescens P13 was based on morphology, Gram staining, physiological and biochemical tests according to Bergey’s Manual of Systematic Bacteriology (Krieg and Holt, 1984). B. subtilis was obtained from Central Laboratory of Organic Agriculture, Agriculture Research Center, Giza, Egypt, where the commercial product Mycostop ® (Streptomyces griseoviridis) was obtained as a gift from the company Kemira OY of Finland. All pure cultures of B. subtilis and P. fluorescens were grown on nutrient agar medium (NA), while S. griseoviridis was used as spore suspension from the commercial product Mycostop®. Effect of antagonistic fungi on the disease incidence caused by Sclerotinia sclerotiorum : In this experiment, pots (30 × 25 × 30 cm) containing sterile soil (sand : loamy sand : compost, 1 : 2 : 1) were used. Before seven days from sowing, the fungal bioagents were applied to pots. For bioagents preparation, aliquots (100 µ) of a conidial suspension (1 × 107 conidia/ml) were pipetted onto PDA in Petri plates (9 cm diameter), spread on PDA using a sterilized glass rod, and incubated at 20±2°C in dark for four weeks. Conidia were harvested by adding 10 ml of sterile distilled water to each dish and the surface of the colony of bioagents gently rubbed using a sterilized glass rod to dislodge conidia. Conidial suspensions from several dishes were pooled and the mixture was filtered through four layers of sterilized cheesecloth to remove mycelial fragments. The concentrations of bioagents conidia in suspensions were determined using a haemocytometer under a compound light microscope. Antagonistic fungi suspension was amended to pots at the rate of 1 × 106 conidia/ ml (20 ml/pot). Seeds of bean (Phaseolus vulgaris L.) were surface sterilized in 0.1% sodium hypochlorite for 2 min and then washed three times with distilled water. S. scleroitiorum was grown on autoclaved wheat bran (20 g of wheat bran in 30 ml of water) for 10 to 14 days 886 Bahkali, Elsheshtawi, Mousa, Elgorban and Alzarqaa before use. The fungus was grown on the wheat bran mixture with bean seeds in petri dishes. When bean seeds were completely colonized, the petri dish lids were removed and the cultures were dried in a sterile airstream provided by laminar flow transfer hood. Bean seeds were sown in pots (5 seeds/pot). The number of survival plants after 15, 30, 45 and 60 days were recorded. Effect of antagonistic bacteria on the disease incidence caused by Sclerotinia sclerotiorum : For greenhouse assays in which antagonistic bacteria were applied as a soil drench, bacterial cell suspensions were prepared first by streaking each antagonistic bacteria taken from ultra-cold storage onto Luria-Bertani (LB) agar plates, then incubating the plates 20±2°C for 24 h to check for purity, and finally by transferring single colonies to fresh LB agar plates for two days. Bacteria were washed off the plates with 10-15 ml of sterilized distilled water. For use in our experiments, the bacterial suspensions were adjusted to 1×108 CFU/ml with sterilized distilled water. Spore suspensions used in experiments were adjusted to appropriate concentrations in sterilized distilled water with the help of a hemacytometer and a compound microscope. The number of survival plants after 15, 30, 45 and 60 days was recorded. Effect of combination of antagonistic fungi and bacteria on the disease incidence caused by Sclerotinia sclerotiorum : The effect of mixture of antagonistic fungi and antagonistic bacteria was studied. Antagonistic fungi were amended to pots 24 h prior to the inoculation of antagonistic bacteria. Antagonistic fungi were applied at the rate of 1 × 106 conidia/ml and bacterial antagonists were amended at the rate of 1 × 108 cfu/ml. The number of survival plants after 15, 30, 45 and 60 days was recorded. Statistical analysis : Data collected 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 analyses were performed at P 5% level. RESULTS AND DISCUSSION Effect of Antagonistic Fungi on the Disease Incidence Caused by Sclerotinia sclerotiorum Trichoderma viride, T. hamatum and C. minitans significantly reduced the white rot disease incidence on beans caused by S. sclerotiorum that produced 90% survival plants when compared to controls (Table 1). While T. harzianum and G. virens produced 85 and 80% survival plants, respectively. Effect of Antagonistic Bacteria on the Disease Incidence Caused by Sclerotinia sclerotiorum Results in Table 2 show that the Mycostop® was highly effective on reducing the disease incidence by 95% survival plants when compared to controls. These were followed by B. subtilis and P. fluorescens which produced 85% survival plants. Effect of Combination of Antagonistic Fungi and Antagonistic Bacteria on the Disease Incidence Caused by Sclerotinia sclerotiorum The highest inhibition for disease incidence came from the combinations of T. hamatum+S. griseoviridis and C. minitans+S. griseoviridis which produced 100% survival plants when compared to controls. These were followed by the combinations T. harzianum+ S. griseoviridis, T. hamatum+B. subtilis, G. virens+S. griseoviridis and C. minitans+B. subtilis which gave the same result by 95% survival plants when compared to controls (Table 3). Conversely, the lowest effect in reducing disease incidence came from the combination of G. roseum+P. fluorescens that giving 75% survival plants when compared to controls. In this study, we noticed that C. minitans had high effect on S. sclerotiorum. This high influence of C. minitans may be due to destroyed sclerotia (Whipps et al., 2008) and the hyphae of S. sclerotiorum (Li et al., 2005). Furthermore, the enzyme ß-1,3-glucanase seemed to be an important enzyme involved in the mycoparasitism of S. sclerotiorum by C. Table 1. Effect of antagonistic fungi on disease incidence of bean caused by S. sclerotiorum After 15 days Non-infested Infested T. harzianum T. viride T. hamatum G. virens G. roseum C. minitans LSD (P=0.05) After 30 days After 45 days No. Mortality (%) Survival (%) No. Mortality (%) Survival (%) No. 5.00a 3.25 b 4.50a 4.75a 4.75a 4.50a 4.25a a 4.75 0.93 0.0 35.0 10.0 5.0 5.0 10.0 15.0 5.0 100.0 65.0 90.0 95.0 95.0 90.0 85.0 95.0 5.00a 3.00 b 4.50a 4.75a 4.75a 4.50a 4.25a a 4.75 0.89 0.0 5.0 0.0 0.0 0.0 0.0 0.0 0.0 100.0 60.0 90.0 95.0 95.0 90.0 85.0 95.0 5.00 a 3.00c 4.25 ab 4.75 ab 4.75 ab 4.25 ab 4.00 ab ab 4.75 0.93 After 60 days Mortality (%) Survival (%) 0.0 0.0 5.0 0.0 0.0 5.0 5.0 0.0 100.0 60.0 85.0 95.0 95.0 85.0 80.0 95.0 No. 5.00 a 2.75c 4.25 ab 4.50 ab 4.50 ab 4.00 ab bc 3.75 ab 4.500 1.36 Mortality (%) Survival (%) 0.0 5.0 0.0 5.0 5.0 5.0 5.0 5.0 100.0 55.0 85.0 90.0 90.0 80.0 75.0 90.0 Values within a column followed by the same superscript are not significantly different according to Duncan’s multiple range test (P=0.05). Table 2. Effect of antagonistic bacteria on disease incidence of bean caused by S. sclerotiorum Treatment After 15 days Non-infested Infested P. fluoroscens B. subtilis Mycostop ® LSD (P=0.05) After 30 days After 45 days No. Mortality (%) Survival (%) No. Mortality (%) Survival (%) No. 5.00a 3.25b 4.75a 5.00a 5.00a 1.36 0.0 35.0 5.0 0.0 0.0 100.0 65.0 95.0 100.0 100.0 5.00 a 3.00 b 4.75 a 4.75 a 5.00 a 1.14 0.0 5.0 0.0 5.0 0.0 100.0 60.0 95.0 95.0 100.0 5.00 a 3.00 b 4.50 a 4.50 a 5.00 a 1.33 After 60 days Mortality (%) Survival (%) 0.0 0.0 5.0 5.0 0.0 100.0 60.0 90.0 90.0 100.0 No. 5.00 a 2.75 a 4.25 a 4.25 a 4.75 a 1.18 Mortality (%) Survival (%) 0.0 5.0 5.0 5.0 5.0 Values within a column followed by the same superscript are not significantly different according to Duncan’s multiple range test (P=0.05). 100.0 55.0 85.0 85.0 95.0 Control of Sclerotinia sclerotiorum in beans with antagonistic microorganisms Treatment 887 888 Treatment After 15 days No. Non-infested Infested T. harzianum+B. subtilis T. harzianum+S. griseoviridis T. harzianum+P. fluorescens T. viride+B. subtilis T. viride+S. griseoviridis T. viride+P. fluorescens T. hamatum+B. subtilis T. hamatum+S. griseoviridis T. hamatum+P. fluorescens G. virens+B. subtilis G. virens+S. griseoviridis G. virens+P. fluorescens G. roseum+B. subtilis G. roseum+S. griseoviridis G. roseum+P. fluorescens C. minitans+B. subtilis C. minitans+S. griseoviridis C. minitans+P. fluorescens LSD (P=0.05) Mortality (%) a 5.00 3.25c 4.75ab 5.00a 4.50ab 4.75ab 5.00a 4.50ab 4.75ab 5.00a 4.75ab 4.75ab 5.00a 4.50ab 4.25b 4.25b 4.25b 5.00a 5.00a 4.75ab 0.74 0.0 35.0 5.0 0.0 10.0 5.0 0.0 10.0 5.0 0.0 5.0 5.0 0.0 10.0 15.0 15.0 15.0 0.0 0.0 5.0 After 30 days Survival (%) 100 65 95 100 90 95 100 90 95 100 95 95 100 90 85 85 85 100 100 95 No. a 5.00 3.00c 4.75ab 5.00a 4.50ab 4.75ab 5.00a 4.50ab 4.75ab 5.00a 4.75ab 4.75ab 5.00a 4.50ab 4.25b 4.25b 4.25b 5.00a 5.00a 4.75ab 0.72 Mortality (%) 0.0 5.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 After 45 days Survival (%) 100 60 95 100 90 95 100 90 95 100 95 95 100 90 85 85 85 100 100 95 No. Mortality (%) a 5.00 3.00c 4.75a 5.00a 4.25ab 4.50ab 4.50ab 4.25ab 4.75a 5.00a 4.50ab 4.50ab 4.75a 4.25ab 4.25ab 4.25ab 3.75b 5.00a 5.00a 4.75a 0.81 0.0 0.0 0.0 0.0 5.0 5.0 10.0 5.0 0.0 0.0 5.0 5.0 5.0 5.0 0.0 0.0 10.0 0.0 0.0 0.0 After 60 days Survival (%) 100 60 95 100 85 90 90 85 95 100 90 90 95 85 85 85 75 100 100 95 No. Mortality (%) Survival (%) a 5.00 2.75c 4.50abc 4.75ab 4.00bc 4.50ab 4.50abc 4.25abc 4.75ab 5.00a 4.50abc 4.25abc 4.75ab 4.25ab 4.00bc 4.25abc 3.75bc 4.75ab 5.00a 4.50abc 0.90 0.0 5.0 5.0 5.0 5.0 0.0 0.0 0.0 0.0 0.0 0.0 5.0 0.0 0.0 5.0 0.0 0.0 5.0 0.0 5.0 Values within a column followed by the same superscript are not significantly different according to Duncan’s multiple range test (P=0.05). 100 55 90 95 80 90 90 85 95 100 90 85 95 85 80 85 75 95 100 90 Bahkali, Elsheshtawi, Mousa, Elgorban and Alzarqaa Table 3. Effect of the combination of antagonistic fungi and antagonistic bacteria on disease incidence of bean caused by S. sclerotiorum Control of Sclerotinia sclerotiorum in beans with antagonistic microorganisms minitans, as the expression of the gene cmg1 encoding ß-1,3-glucanase increased during infection of sclerotia of S. sclerotiorum by C. minitans (Giczey et al., 2001). The soil application of C. minitans is an effective strategy for management of Sclerotinia white rot of bean caused by S. sclerotiorum. The findings confirm previous reports on biocontrol of white mold of bean (Huang et al., 2000). Coniothyrium minitans survived in the soil throughout the glasshouse trial, its recovery being consistent with the amount applied and ability to survive in soil may be important for long term disease control (McQuilken and Whipps, 1995). Coniothyrium minitans infection of sclerotia recovered from the control plots was recorded. All the tested isolates of Trichoderma spp. inhibited mycelial growth of the pathogen, and formed an inhibition zone. The mechanism of inhibition may be competition for food or space (Dubey et al., 2007; Elgorban et al., 2014; El-Sheshtawi et al., 2014) or inhibitory extracellular metabolites produced by antagonists. One of the mechanisms involved in the antagonistic activity of Trichoderma spp. against a range of economically important pathogens is the mycoparasitism (Dennis and Webster, 1971), where the production of fungal cell wall-degrading enzymes by Trichoderma is believed to play a role. It was shown that extracellular lytic enzymes (1, 3-glucanase and chitinase) excreted by T. harzianum were involved in cell wall degradation of R. solani, S. sclerotiorum and Sclerotium rolfsii, and therefore in the biological control of these pathogens by T. harzianum (Elad et al., 1982; Ana and Alicia, 1998). The results of pot trials revealed that soil application of tested Trichoderma species was an effective strategy to decrease disease incidence of bean white rot. Several Trichoderma spp. including T. aureoviride, T. hamatum, T. harzianum, T. koningii, T. pseudokoningii, T. viride and T. virens had already shown good effectiveness against S. sclerotiorum, and acting as mycoparasites, they were reported for their ability to destroy sclerotia directly (Clarkson and Whipps, 2002). Trichoderma spp. are also known to provide plants with useful molecules such as glucose oxidase and growth stimulating compounds that can increase their vigour and as a result resistance to pathogens (Gravel et al., 2006). Moreover, these fungi produce antibiotics such 889 as gliotoxin, viridin and cell wall degrading enzymes and also biologically active heatstable metabolites such as ethyl acetate. These substances are known to be involved in disease incidents suppression (Mujeebur et al., 2004). Antagonistic Bacteria In this study, we observed that S. griseoviridis plant growth promoting as well as bioagent capabilities led to increased yields (Hamdali et al., 2008). In soil, antibiotic metabolites producing (Hyang et al., 2005) and antimicrobial compounds (Berdy, 2005) make actinomycetes able to restrict the attack by pathogenic organisms in the habitat (ElTarabily et al., 2000). There are many reports on the agricultural implications of these organisms in biological control of plant pathogens (Ghorbani et al., 2007) and causing of signal transduction in host plants to initiate defense responses to cope with the stresses at cell, tissue and organ level following inoculation of these organisms (Hasegawa et al., 2006). S. griseoviridis is a root colonizer and stimulates root growth during rhizosphere colonization (Kortemaa et al., 1994). In some cases, stimulated plant growth could explain the enhanced yield results (Minuto et al., 2006) when the antagonist was combined with soil solarization. The efficiency of Mycostop® against Fusarium wilt of tomato was satisfactory (Minuto et al., 2006) when applied to artificially infested soil. Also, Mycostop® had exhibited partial efficacy against F. oxysporum f. sp. basilici (Minuto et al., 1997). In the present study, it was observed that the maximum of survival plants in beans were observed in the T. hamatum+S. griseoviridis and C. minitans+S. griseoviridis which produced 100% survival plants. These results are in agreement with previous reports such as the combinations of fungi mixtures bacteria, worked better than a single isolate (Rini and Sulochana, 2007). Jetiyanon and Kloepper (2002) suggested a combinational use of diverse biocontrol agents for enhanced and stable biocontrol agents to manage a complex of diseases. 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