Manipulation Of Microbial Community In Banana Rhizospherer To Suppress Fusarium Wilt Of Banana

Continuous monocropping system of cash crops often increases soilborn pathogens,but beneficial species decrease,leading to a shift of microbial community from bacteria dominant type to fungi dominant one.Pathogens can easily invade plant roots resulting in severe plant disease such as Fusarium wilt of banana.Thus the study of soil microbial community is of great importance to soil-borne disease control in agricultural production.In this study,firstly we estimated how different fungal marker genes and databases influence soil microbial community study and screened the proper fungal marker gene and database for further study.Secondly,we compared the microbial communities between healthy and diseased banana rhizosphere soils and found out the indicator microbial community in healthy banana rhizosphere.We further isolated the bio-control agent that can strongly suppress the growth of the pathogen of Fusarium wilt of banana.The whole genome of the isolate was sequenced and annotated to estimate its biocontrol potential.Specific fragment of this isolate was screened through genome comarison and proper primers for quantitative PCR of this isolate were designed and estimated.The colonization ability of this isolate in soil was evaluated as well.In the end,banana Fusarium wilt specific bioorganic fertilizer(BIO)was produced by combining biological control agent with amino-acid compost.BIO was applied to control Fusarium wilt of banana by manipulating soil microbial community in banana rhizosphere.The main results obtained were summarized as follows.1.Both ITS fungal marker gene coupled with WARCUP and UNITE classification databases and 28S rRNA fungal marker gene coupled with LSU classification database revealed the fungal community in corn rhizosphere was most different among corn,switchgrass,and Miscanthus.Crop type was the key driving factor to the fungal community variation in this study.These results indicate the choice of fungal marker genes and classification databases had little influence on comparisons of community structure between treatments.However,it resulted in a marked influence on fungal community composition results due to differences in taxonomic conflicts and varying coverage of high abundance fungal groups.The combination of 28S rRNA marker gene and LSU database failed to detect Fusarium,Phoma,and Verticillum,while these fungal groups were in high abudance in the results based on ITS marker gene coupled with WARCUP and UNITE databases.Orbicula was not detected using ITS marker gene.Eupenicillium was not detected using ITS marker gene coupled with UNITE database,while it was in low abudance in ITS plus WARCUP database dataset,but it was the forth most abundant fungal group in LSU dataset.The use of WARCUP database resulted in better classification for ITS reads than UNITE.The pathogen for Fusarium wilt of banana was Fusarium oxysporum f.sp.cubense(FOC),so ITS fungal marker gene coupled with WARCUP classification database was used for fungal community profiling in our downstream study.2.Plant health status was correlated to soil microbial community composition.Clone library data showed Bacillus was the most abundant bacterial group in healthy banana rhizosphere soils.Pyrosequencing results showed Bacillus was the top contributing bacterial group to the bacterial community difference between healthy and diseased banana samples.Bacillus was more abundant in healthy banana rhizosphere than in diseased samples.Fusarium was more abundant in diseased banana rhizosphere soils compared to healthy samples,and it was also the top contributing fungal group to the overall fungal community dissimilarity between healthy and diseased banana rhizosphere soil samples.Bacillus and Fusarium were negatively correlated in abundance,thus the increase of Bacillus but the decrease of Fusarium in banana rhizosphere might be the key to succsessfully control Fusarium wilt of banana,and the ratio of Bacillus and Fusarium might be appled in banana Fusarium wilt early warning.3.By in vitro culture against Fusarium oxysporum f.sp.cubens(FOC),strain NJN-6 which can strongly suppress the growth of FOC was isolated from healthy banana rhizosphere soil.This strain was further identified as Bacillus amyloliquefaciens based on whole genome sequencing and 16S rRNA phylogenetic tree.The genome anotation results showed the genome of strain NJN-6 encoded 11 genes associated with penicillin systhesis,43 genes associated with lipopeptide systhesis,and 91 genes associated with spore formation and germination,indicating strain NJN-6 was a potential bio-control agent.Unique fragments on the genome of strain NJN-6 was screened by genome comparison with B.amyloliquefaciens FZB42.Specific primers to quantify strain NJN-6 was designed and screened based on the sequences of these unique fragments.This specific primer set was further applied to quantify the population of strain NJN-6 in soil after inoculation.To estimate the survive ability of strain NJN-6 in soil,two treatments were included:1)Strain NJN-6 was inoculated to the soil directly(IS);2)the fermentation of strain NJN-6 was pre-mixed with compost and then the mixture was inoculated into the soil(BIO).After inoculation,in the first 7 days,strain NJN-6 could survive in soil in high abundance.After 15 days,it decreased dramatically.Thirty days later,the population of strain NJN-6 in soil is stable.Combination of compost with strain NJN-6 before inoculation could increase the population of strain NJN-6 in soil and slow down the decrease of strain NJN-6,which might be benifit for biological control process conducted by this strain.4.Field results demonstrated that the practical application of the BIO is a promising strategy for disease control and growth promotion in banana production.Application of BIO significantly decreased Fusarium wilt disease incidence with the biological control efficacy of 68%resulting in doubled yield.Pyrosequencing results again confirmed Bacillus was the most abundant rhizo-bacterial group of healthy banana and it was also the indicator bacterial group in healthy banana rhizosphere.Fusarium was more abundant in diseased banana rhizosphere than the healthy samples,and it was negatively correlated to banana yield but positively correlated to disease incidence.Bacillus and Fuasrium were negatively correlated in abundance.Soil microbial community ws correlated to plant health status.The community in healthy banana rhizosphere was significantly different from the diseased samples.However,MRT analyses showed BIO application overwhelmed the effected caused by plant health status on rhizo-bacterial community resulting in the leading shaping factor for rhizo-bacterial community in this study.BIO application altered the rhizo-bacterial community by establishing beneficial strains(Bacillus spp.)dominated microbial community and decreasing pathogen colonization in the banana rhizosphere,which plays an important role in the management of Fusarium wilt disease in banana plantations.We described the roadmap to develop Panama disease specific bioorganic fertilizer to control this disease and promote banana growth including comparisons of microbial communities between healthy and diseased banana rhizosphere soils,screening of biocontrol agents,and nursery pot applications of the bio-product to suppress Fusarium wilt of banana.Plant health status was correlated to soil microbial community composition indicating the potential to control soil-borne disease by manipulation of rhizo-microbial community.BIO application altered the rhizo-bacterial community by establishing beneficial strains(Bacillus spp.)dominated microbial community and decreasing pathogen colonization in the banana rhizosphere resulting in plant growth promotion and biological control of Fusarium wilt of banana in the field.