Screening Of Biocontrol Isolates Against Verticillium Wilt Of Cotton And Finding Of A New Pathogen

Verticillium wilt of cotton is a soilborne disease,caused by Verticillium dahliae in China.The disease occurs in almost all cotton producing areas,caused great economic losses and already become an obstacle in the cotton industry development.V.dahliae survives in soil in form of microsclerotia for many years,resulting in the disease to be difficult to control in production.There are no high resistance varieties to cotton verticillium wilt in production and disease control dependends on chemical fungicides for long-term.With people understanding the harm of chemical pesticide residues to environment and human health,attention is being devoted to biological control against the diseases.Therefore,in this study,biocontrol isolates against V.dahliae were screened and identified by morphology and molecular data,and their biocontrol effects and biological characteristics were determined,laying the groundwork for application of biocontrol isolates.In addition,in the process of isolating V.dahliae isolates,a Scopulariopsis sp.was often isolated.They didn't belong to any known species of Scopulariopsis and list of endophytic fungi from cotton plants.So the fungal isolates were identified by morphological characteristics and molecular data and their pathogenicity was determined for illustrating their synergistic action with V.dahliae.1.Screening of biocontrol isolates against cotton verticillium wilt1)A total of five microorganisms showing significant zones of inhibition were screened by the method of double layer technique,and a best isolate SX-3 was obtained by disc diffusion assay.The isolate SX-3 was identified as Paenibacillus polymyxa by morphological characteristics,molecular data and fatty acid analysis.2)The antagonistic activity of P.polymyxa SX-3 against other plant pathogens was assessed using the disc diffusion assay.P.polymyxa SX-3 had a broad antagonistic activity against several plant pathogens and percentage inhibition of radial growth(PIRG)values showed that its antagonistic active against different pathogens was different.The highest PIRG values(75.0%)were observed against Colletotrichum truncatum and the lowest recorded(59.52%)were observed against Sclerotinia sclerotiorum,respectively.The PIRG values ranged from 73.02,72.22,70.63 to 69.84%against Botrytis cinerea,Phytophthora capsici,Rhizoctonia solani and Fusarium oxysporum f.sp.niveum,respectively,but there was no significant differences among them.3)The results of biocontrol assay,P.polymyxa SX-3 was able to delay foliage symptoms for about 7 days,significantly reduce disease incidence 76.9%and disease index 74.9%.Furthermore,P.polymyxa SX-3 significantly increased the biomass of cotton including dry weight,fresh weight and height,especially root system.Compared with the control,the root dry weight and fresh weight increased 38.5%and 47.8%,respectively.4)The optimum cell growth temperature,pH as well as the optimum endospore-producing temperature,medium and culture time were determined for the growth of SX-3 strain.The results showed that the optimum cell growth temperature was 37?,pH 7.0.While the optimum endospore-producing temperature was 30?,the optimal culture time and medium were 30 hours and NBS,respectively.When the temperature exceeds 37?,there are no endospore formation.2.Finding of a new pathogen1)A total of 291 isolates obtained from stems was consist of 209 from the diseased plants and 82 from the healthy plants.Among 209 isolates,90 isolates were identified as Verticillium dahlia,30 as Scopulariopsis sp.,and 89 for other unknown species.Among 82 isolates from the healthy plants,all isolates were recognized as other unknown species.Obviously,fungus of Scopulariopsis sp.found was only present in samples of the diseased plants but not in the healthy plants,not including in list of the endophyte for cotton plants.2)Scopulariopsis sp.isolates was identified as Scopulariopsis gossypii sp.nov.by using the three-locus combined TEF1,D1/D2 and TUB dataset with maximum likelihood(ML)and bayesian inference(BI)as well as morphological characteristics.3)The pathogenicity testing confirmed that in plants coinoculated with V.dahliae and S.gossypii,the wilting symptoms occurred earlier.Meantime,coinoculation with S.gossypii and V.dahliae significantly increased the disease severity,promoted defoliation,However,S.gossypii did not infect host plants with undamaged roots.The cut tissues of stems of cotton plants showed that vascular browning presented among different treatments but browning level was different.In plants coinoculated with V.dahliae and S.gossypii,the entire vascular tissues were dark brown,showing more severe symptoms comparing with brown vascular tissues inoculated with V.dahliae and partially brownish vascular tissues inoculated with S.gossypii.There was no vascular browning in control plants.