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Res. on Crops 15 (4) : 884-892 (2014) Printed in India Biological control of Sclerotinia sclerotiorum in beans withantagonistic microorganisms under greenhouse conditions ALI H. BAHKALI, MOHAMED ELSHESHTAWI 1 , RAMADAN A. MOUSA 2 ,ABDALLAH M. ELGORBAN* 3 AND AREEJ ABDULLAH ALZARQAA 4 Department of Botany and Microbiology College of Science, King Saud University, P. O. Box 2455, Riyadh 11451, Saudi Arabia *(e-mail :
[email protected]) (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 thesuppression 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 caseof mixture, antagonistic fungi and antagonistic bacteria, the mixtures of T. hamatum+S.griseoviridis and C. minitans+S. griseoviridis completely inhibited the disease incidencethat 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 isa destructive pathogen with worldwidedistribution known to infect 408 species and278 genera of plants nearly (Boland and Hall,1994). Important crops affected include oilseedrape, sunflower, tobacco and a range of vegetables such as bean, lettuce, cauliflower,cabbage, carrot and potato as well as a numberof flower crops.Control using crop rotations isunrealistic due to the persistence of survivalstructures as sclerotia in the soil for longperiods and because Sclerotinia has such awide host range (Nelson, 1998; Elgorban et al. ,2013). These factors require the use of fungicides, which have been known to haveadverse effects on non-target organisms(Gilmour, 2001). Also, the rising costs of soilfumigation, lack of suitable replacement fortoxic fumigants and public concerns overrepeated fungicide use for disease control havemeant that alternative control methods, suchas biological control are gaining interest withgrowers. The use of microbial agents to controlplant pathogens can be an eco-friendly andeconomical component of an integrated pestmanagement programme (Mao et al ., 1997).Several microorganisms have been reportedeffective as potential biocontrol agents formanagement S. sclerotiorum of beans (Li et al .,2006; Huang and Erickson, 2007). Coniothyrium minitans and Sporidesmium sclerotivorum , twospecialized mycoparasites of Sclerotinia spp.,have been reported to control disease in bothglasshouse and field trials (Rabeendran et al .,2006; Wenting et al ., 2012). Trichoderma spp., namely, Trichoderma harzianum and T. virens have also been shownto attack both sclerotia and mycelium of Sclerotinia spp. (Chitrampalam et al ., 2008; deFigueirêdo et al ., 2010) and have been reportedto give some disease control in field trialsagainst sclerotia (Ristaino et al ., 1994; Wenting 1 Plant Pathology Department, College of Agriculture, Mansoura University, Mansoura 35516, Egypt. 2 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. et al ., 2012). Bacterial biocontrol agents against S. sclerotiorum are rarely studied (Boyetchko,1999). Some bacterial strains have shownantifungal 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 bioagentsagainst S. sclerotiorum and (2) investigate thecombination of antagonistic fungi and bacteriaagainst S. sclerotiorum . MATERIALS AND METHODSFungal and Bacterial IsolatesPathogenic fungus : Sclerotinia sclerotiorum (Lib.) de Bary used in this study was derived from sclerotia on diseased beanplants ( Phaseolus vulgaris L.) from Ismailiagovernorate, Egypt during 2013. The purifiedfungal isolates were identified by Departmentof Plant Pathology, College of Agriculture,Mansoura University (Kora et al ., 2005). PDAslants from each isolated fungus were kept in4ºC for further studies. Antagonistic fungi : Soil samples werecollected from 40 different places comprisingdifferent agricultural protected area andgreenhouses in five governorates, Egypt. Forisolation of Trichoderma strains, a serialdilution technique was followed and a 10 3 dilution of each sample was prepared. Onemilliliter of each solution was pipetted onto aRose Bengal Agar plate and incubated at20±2°C for one week. The plates were examineddaily and each colony that appeared wasconsidered to be one colony forming unit (CFU).After enumeration of CFU, individual colonieswere isolated from the same plates and eachuncommon colony was re-isolated onto a freshpotato dextrose agar (PDA) plate. Distinctmorphological characteristics were observed foridentification (Rifai, 1969, Bissett, 1991a andb; Barnett and Hunter, 1998). In case of Glicoladium roseum , after 10-12 days, the discswere checked under a stereoscopic microscopefor G. roseum colonies with typical G. roseum conidiophores were transferred to PDAamended with chloramphenicol (50 mg/l).Identification was carried out based on micro-culture colony morphology (Barnett andHunter, 1998; Schroers et al ., 1999). Isolateswere preserved in PDA discs kept in steriledistilled water and in colonized wheat kernelswith silica gel, both stored at 4°C. Antagonistic bacteria : Pseudomonas fluorescens was isolated from the rhizosphereof green bean in long term rotation farmlandin Ismailia, Egypt, by using King’s B medium.Identification of P. fluorescens P13 was basedon morphology, Gram staining, physiologicaland biochemical tests according to Bergey’sManual of Systematic Bacteriology (Krieg andHolt, 1984). B. subtilis was obtained fromCentral Laboratory of Organic Agriculture,Agriculture Research Center, Giza, Egypt,where the commercial product Mycostop ® ( Streptomyces griseoviridis ) was obtained as agift 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 sporesuspension from the commercial productMycostop ® . Effect of antagonistic fungi on thedisease 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. Beforeseven days from sowing, the fungal bioagentswere applied to pots. For bioagents preparation,aliquots (100 µ) of a conidial suspension (1 ×10 7 conidia/ml) were pipetted onto PDA in Petriplates (9 cm diameter), spread on PDA using asterilized glass rod, and incubated at 20±2°Cin dark for four weeks. Conidia were harvestedby adding 10 ml of sterile distilled water to eachdish and the surface of the colony of bioagentsgently rubbed using a sterilized glass rod todislodge conidia. Conidial suspensions fromseveral dishes were pooled and the mixture wasfiltered through four layers of sterilizedcheesecloth to remove mycelial fragments. Theconcentrations of bioagents conidia insuspensions were determined using ahaemocytometer under a compound lightmicroscope. Antagonistic fungi suspension wasamended to pots at the rate of 1 × 10 6 conidia/ml (20 ml/pot). Seeds of bean ( Phaseolus vulgaris L.) were surface sterilized in 0.1%sodium hypochlorite for 2 min and then washedthree 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 Control of Sclerotinia sclerotiorum in beans with antagonistic microorganisms 885 before use. The fungus was grown on the wheatbran mixture with bean seeds in petri dishes.When bean seeds were completely colonized,the petri dish lids were removed and thecultures were dried in a sterile airstreamprovided by laminar flow transfer hood. Beanseeds were sown in pots (5 seeds/pot). Thenumber of survival plants after 15, 30, 45 and60 days were recorded. Effect of antagonistic bacteria on thedisease incidence caused by Sclerotinia sclerotiorum : For greenhouse assays in whichantagonistic bacteria were applied as a soildrench, bacterial cell suspensions wereprepared first by streaking each antagonisticbacteria taken from ultra-cold storage ontoLuria-Bertani (LB) agar plates, then incubatingthe plates 20±2°C for 24 h to check for purity,and finally by transferring single colonies tofresh LB agar plates for two days. Bacteria werewashed off the plates with 10-15 ml of sterilizeddistilled water. For use in our experiments, thebacterial suspensions were adjusted to 1×10 8 CFU/ml with sterilized distilled water. Sporesuspensions used in experiments were adjustedto appropriate concentrations in sterilizeddistilled water with the help of a hemacytometerand a compound microscope. The number of survival plants after 15, 30, 45 and 60 dayswas recorded. Effect of combination of antagonisticfungi and bacteria on the disease incidencecaused by Sclerotinia sclerotiorum : Theeffect of mixture of antagonistic fungi andantagonistic bacteria was studied. Antagonisticfungi were amended to pots 24 h prior to theinoculation of antagonistic bacteria.Antagonistic fungi were applied at the rate of 1× 10 6 conidia/ml and bacterial antagonists wereamended at the rate of 1 × 10 8 cfu/ml. Thenumber of survival plants after 15, 30, 45 and60 days was recorded. Statistical analysis : Data collectedwere statistically analyzed using the StatisticAnalysis System Package (SAS Institute, Cary,NC, USA). Differences between treatments werestudied using Fisher’s Least SignificantDifference (LSD) test and Duncan’s MultipleRange Lest (Duncan, 1955). All analyses wereperformed at P 5% level. RESULTS AND DISCUSSIONEffect of Antagonistic Fungi on the DiseaseIncidence Caused by Sclerotinia sclerotiorum Trichoderma viride, T. hamatum and C.minitans significantly reduced the white rotdisease incidence on beans caused by S.sclerotiorum that produced 90% survival plantswhen compared to controls (Table 1). While T.harzianum and G. virens produced 85 and 80%survival plants, respectively. Effect of Antagonistic Bacteria on theDisease Incidence Caused by Sclerotinia sclerotiorum Results in Table 2 show that theMycostop ® was highly effective on reducing thedisease incidence by 95% survival plants whencompared to controls. These were followed by B. subtilis and P. fluorescens which produced85% survival plants. Effect of Combination of Antagonistic Fungiand Antagonistic Bacteria on the DiseaseIncidence Caused by Sclerotinia sclerotiorum The highest inhibition for diseaseincidence came from the combinations of T.hamatum + S. griseoviridis and C . minitans + S.griseoviridis which produced 100% survivalplants when compared to controls. These werefollowed 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 inreducing disease incidence came from thecombination of G . roseum + P . fluorescens thatgiving 75% survival plants when compared tocontrols.In this study, we noticed that C.minitans had high effect on S. sclerotiorum . Thishigh influence of C. minitans may be due todestroyed sclerotia (Whipps et al ., 2008) andthe hyphae of S. sclerotiorum (Li et al ., 2005).Furthermore, the enzyme ß-1,3-glucanaseseemed to be an important enzyme involved inthe mycoparasitism of S. sclerotiorum by C. 886 Bahkali, Elsheshtawi, Mousa, Elgorban and Alzarqaa Table 1. Effect of antagonistic fungi on disease incidence of bean caused by S. sclerotiorum TreatmentAfter 15 daysAfter 30 daysAfter 45 daysAfter 60 daysNo.Mortality (%)Survival (%)No.Mortality (%)Survival (%)No.Mortality (%)Survival (%)No.Mortality (%)Survival (%)Non-infested5.00 a 0.0100.05.00 a 0.0100.05.00 a 0.0100.05.00 a 0.0100.0Infested3.25 b 35.065.03.00 b 5.060.03.00 c 0.060.02.75 c 5.055.0 T. harzianum 4.50 a 10.090.04.50 a 0.090.04.25 ab 5.085.04.25 ab 0.085.0 T. viride 4.75 a 5.095.04.75 a 0.095.04.75 ab 0.095.04.50 ab 5.090.0 T. hamatum 4.75 a 5.095.04.75 a 0.095.04.75 ab 0.095.04.50 ab 5.090.0 G. virens 4.50 a 10.090.04.50 a 0.090.04.25 ab 5.085.04.00 ab 5.080.0 G. roseum 4.25 a 15.085.04.25 a 0.085.04.00 ab 5.080.03.75 bc 5.075.0 C. minitans 4.75 a 5.095.04.75 a 0.095.04.75 ab 0.095.04.500 ab 5.090.0LSD (P=0.05)0.930.890.931.36Values 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 TreatmentAfter 15 daysAfter 30 daysAfter 45 daysAfter 60 daysNo.Mortality (%)Survival (%)No.Mortality (%)Survival (%)No.Mortality (%)Survival (%)No.Mortality (%)Survival (%)Non-infested5.00 a 0.0100.05.00 a 0.0100.05.00 a 0.0100.05.00 a 0.0100.0Infested3.25 b 35.065.03.00 b 5.060.03.00 b 0.060.02.75 a 5.055.0 P. fluoroscens 4.75 a 5.095.04.75 a 0.095.04.50 a 5.090.04.25 a 5.085.0 B. subtilis 5.00 a 0.0100.04.75 a 5.095.04.50 a 5.090.04.25 a 5.085.0Mycostop ® 5.00 a 0.0100.05.00 a 0.0100.05.00 a 0.0100.04.75 a 5.095.0LSD (P=0.05)1.361.141.331.18Values within a column followed by the same superscript are not significantly different according to Duncan’s multiple range test (P=0.05). C o n t r o l o f S c l er o t i n i a s c l er o t i or u m i n b e a n s w i t h a n t a g o n i s t i c m i cr o or g a n i s m s 8 8 7 Table 3. Effect of the combination of antagonistic fungi and antagonistic bacteria on disease incidence of bean caused by S. sclerotiorum TreatmentAfter 15 daysAfter 30 daysAfter 45 daysAfter 60 daysNo.Mortality (%)Survival (%)No.Mortality (%)Survival (%)No.Mortality (%)Survival (%)No.Mortality (%)Survival (%)Non-infested5.00 a 0.01005.00 a 0.01005.00 a 0.01005.00 a 0.0100Infested3.25 c 35.0653.00 c 5.0603.00 c 0.0602.75 c 5.055 T. harzianum+B. subtilis 4.75 ab 5.0954.75 ab 0.0954.75 a 0.0954.50 abc 5.090 T. harzianum+S. griseoviridis 5.00 a 0.01005.00 a 0.01005.00 a 0.01004.75 ab 5.095 T. harzianum+P. fluorescens 4.50 ab 10.0904.50 ab 0.0904.25 ab 5.0854.00 bc 5.080 T. viride+B. subtilis 4.75 ab 5.0954.75 ab 0.0954.50 ab 5.0904.50 ab 0.090 T. viride+S. griseoviridis 5.00 a 0.01005.00 a 0.01004.50 ab 10.0904.50 abc 0.090 T. viride+P. fluorescens 4.50 ab 10.0904.50 ab 0.0904.25 ab 5.0854.25 abc 0.085 T. hamatum+B. subtilis 4.75 ab 5.0954.75 ab 0.0954.75 a 0.0954.75 ab 0.095 T. hamatum+S. griseoviridis 5.00 a 0.01005.00 a 0.01005.00 a 0.01005.00 a 0.0100 T. hamatum+P. fluorescens 4.75 ab 5.0954.75 ab 0.0954.50 ab 5.0904.50 abc 0.090 G. virens+B. subtilis 4.75 ab 5.0954.75 ab 0.0954.50 ab 5.0904.25 abc 5.085 G. virens+S. griseoviridis 5.00 a 0.01005.00 a 0.01004.75 a 5.0954.75 ab 0.095 G. virens+P. fluorescens 4.50 ab 10.0904.50 ab 0.0904.25 ab 5.0854.25 ab 0.085 G. roseum+B. subtilis 4.25 b 15.0854.25 b 0.0854.25 ab 0.0854.00 bc 5.080 G. roseum+S. griseoviridis 4.25 b 15.0854.25 b 0.0854.25 ab 0.0854.25 abc 0.085 G. roseum+P. fluorescens 4.25 b 15.0854.25 b 0.0853.75 b 10.0753.75 bc 0.075 C. minitans+B. subtilis 5.00 a 0.01005.00 a 0.01005.00 a 0.01004.75 ab 5.095 C. minitans+S. griseoviridis 5.00 a 0.01005.00 a 0.01005.00 a 0.01005.00 a 0.0100 C. minitans+P. fluorescens 4.75 ab 5.0954.75 ab 0.0954.75 a 0.0954.50 abc 5.090LSD (P=0.05)0.740.720.810.90Values within a column followed by the same superscript are not significantly different according to Duncan’s multiple range test (P=0.05). 8 8 8 B a h k a l i ,E l s h e s h t a w i ,M o u s a ,E l g or b a n a n d A l z ar q a a
