Isolation,Identification Of Antagonistic Bacteria And Its Mode Of Action Against Gray Mold Disease Of Tomato Caused By Botrytis Cinerea

Plant pathogens are the main threat for profitable agricultural productivity.Currently.chemical based pesticides are thought to be an effective and reliable agricultural management measure for controlling pests.Chemical pesticides are highly effective and convenient to use but they are a potential threat for the environment and all kinds of life on earth.Therefore,the use of biological control agents for the management of plant pathogens is considered as a safer and sustainable strategy for safe and profitable agricultural productivity.Bacillus-based biocontrol agents play a fundamental role in the field of biopesticides.Many Bacillus species have proved to be effective against a broad range of plant pathogens.They have been reported as plant growth promoter,systemic resistance inducer,and used for production of a broad range of antimicrobial compounds(lipopeptides,antibiotics and enzymes)and competitors for growth factors(space and nutrients)with other pathogenic microorganisms through colonization.So keeping in view the problems and constrains of synthetic fungicides the present study was conducted for isolation,screening and identification of potential biological control agent against Botrytis cinerea with special focus on its antagonizing mechanism,including plant growth promotion,trigging systemic induced resistance and alteration in different plant defense determinants.Furthermore,extraction and profiling of antimicrobial metabolites produced by biocontrol agent and their effects on morphology,physiology and ultrastructure of Botrytis cinerea were also investigated.The phylloplane bacteria were isolated from leaves of tomato plant collected from different cities of Liaoning Province,P.R.China.A total of 215 bacterial isolates were selected based on differences in colony morphology and pigment production by visual observations.Out of 215 isolates screened,only one isolate(JS-786)showed strong antifungal activity and was selected for further experiments in this study.The isolate JS-786 was identified as Bacillus aerius based on physiological,biochemical and 16S rRNA sequence comparison with the sequences deposited in the NCBI database.The phylogenetic tree revealed that isolate JS-786 belonged to the genus Bacilus and it has highest similarity(99%)with B.aerius.This is in agreement with the physiological and biochemical characteristics of isolate JS-786.The 16S rRNA sequence was submitted to NCBI Genebank under the accession number KX926557 and bacteria was deposited in China General Microbial Culture Collection Center with the accession number CGMCC 1.15932.The optimization of fermentation conditions is necessary for field application of biological control agents.The fermentation conditions for the B.aerius strain,JS-786 in terms of cell dry mass and its antifungal activity against B.cinerea with response surface methodology were optimized.After the success of preliminary antifungal activity tests,response surface methodology was used to optimize the medium pH,gelatin percentage,incubation period,rotatory speed and incubation temperature,to maximize the cell dry mass and antifungal activity against B.cinerea.A 25 factorial central composite design was employed and multiple response optimization was used to determine the desirability of the operation.The results of regression analysis showed that at the individual level,all of the experimental parameters were significant for cell dry mass;significant results were shown for antifungal activity by pH,incubation period,rotatory speed and incubation temperature.The interactive effect of the incubation period,rotatory speed and incubation temperature was significant.Maximum cell dry mass(8.7 g/L)and inhibition zone(30.4 mm)were obtained at pH 6.4,gelatin 3.2%,incubation period 36.92 h,rotatory speed 163 rpm,and temperature 33.5℃.This part of study should help to formulate a more rational and cost-effective biological product both in terms of bacterial growth and antifungal activity.Further study was conducted to appraise the extraction,profiling and antifungal activity of antimicrobial metabolites of strain JS-786.The antimicrobial metabolites were extracted with ethyl acetate and fractioned by silica gel chromatography.The fractions with strong antifungal activity against Botrytis cinerea were tested for their effect on spore germination,mycelial growth,morphology and physiology of Botrytis cinerea.Furthermore,scanning and transmission electron microscopes were used to visualize the alteration in ultrastructure and intracellular organelles of B.cinerea.After silica chromatography,three fraction(fraction number 7,8 and 13)showed antifungal activity.All the active friction and crude metabolic extract were tested at the concentration of 31.25μg/mL.Carbendazo(31.25μg/mL)and DMSO(1%)were used as positive and negative control,respectively.The results showed that,all the fractions and crude extract caused significant reduction in spore germination,mycelial growth and ergosterol contents in B.cinerea,while they enhanced the alkaline phosphatase activity and electrical conductivity of supernatants of treated hyphae compared to negative control.Similarly,the contents of saturated fatty acids were increased while unsaturated fatty acids were decreased.The fraction 7,8,13 and crude extract showed no effect on SOD and PAL activity while they significantly reduced the activity of POD.Scanning and transmission electron microscopy reveled markedly alteration in morphology and ultrastructure B.cinerea.Treated hyphae were severely raptured.shriveled,and collapsed with indistinct organelles and many of them were unidentifiable vesicular structures.The GC-MS profiling of active fractions revealed that the major compounds were Pyrrolo[1,2-a]pyrazine-1,4-dione,hexahydro-;4-Hydroxy-6-methyl-3-nitro-2(1H)-pyridinone;p-Hydroxybiphenyl;3,6-Diisobutyl-2,5-piperazinedione;and 2,6-Diamino-4-oxo-5-formamidopyrimidine.B.aerius strain JS-786 was further investigated for its role in plant defense activation and growth promotion in tomato under greenhouse conditions.Wettable powder formulation of strain JS-786 was prepared and tested as foliar spray and soil drenching at four different concentrations(i.e.2.5,5,7.5,and 10 g per litter of water as foliar spray or per port as soil drenching).Disease severity,plant growth promotion(root,shoot and photosynthetic parameters),alterations in reactive oxygen species,ROS;(H2O2,OH,O2-)and other plant defense determinants like,malondialdehyde,catalase,superoxide dismutase,peroxidase,polyphenol oxidase,ascorbate peroxidase,phenylalnine ammonialyase,glutathione reductase,chitinase,flavonoids and total phenols,were recorded.The results showed that the strain JS-786 was significant for reducing grey mold severity,growth promotion and plant defense activation.Minimum disease severity was recorded at 10 g/L as foliar spray while maximum plant growth was observed at 10 g/pot as soil drenching of JS-786.Botrytis cinerea triggered production the ROS in control plants but not in plant where strain JS-786 was applied at the concentration oflOg/L or pot.More plant treated with lOg concentration of strain JS-786 showed higher and timely elevated activities of antioxidants,which reveals its positive role in plant defense activation and plant hemostasis hence,suitable for the management of B.cinerea and development of biopesticide and fertilizer.