Characterization And Management Of Microbial Community In Banana Suppressive Soils To Soil-borne Fusarium Wilt Disease

China,where 90%of the banana industry was comprised by Cavendish cultivar,is the second banana producer after India in the world.However,Cavendish cultivar is severly hampered by banana Fusarium wilt disease,which is caused by Fusarium oxysporum f.sp.cubense race 4(Foc 4).Because of high economic returns,almost all banana orchards in China are monocultured,which destroyed the banance of soil microbial community.Moreover,the Fusarium wilt disease was not managed efficiently,resulting in the soil-borne disease more worse and spread quickly,which is the biggest threaten to banana production in China.Soil health is one of the most vital requirements for crop production in agricultural systems while soil microorganisms play a major role in the development and maintenance of soil health.And the composition and diversity of soil microbial community responses for the soil-borne disease suppression.Therefore,characterization of microbial community in naturally and managed suppressive soils to induce the soil suppressiveness to banana Fusarium wilt disease is the important basis for overcoming the biological obstacles of banana continuous cropping.We used the plate counting method,real time PCR,denaturing gradient gel electrophoresis(DGGE)and high-throughput sequencing method to explore 1)soil microbial community in naturally banana suppressive soils to Fusarium wilt disease in Hainan banana main production area,2)the soil microbial community under different banana continuous cropping spans located at "Wan Zhong" farm in Ledong,Hainan province,China,and 3)soil microbial community after bio-organic fertilizer(BIO)application to banana orchard with high Fuarium wilt disease incidence.This study aims to reveal the potential microbial ecological mechanism of how banana Fusarium wilt disease happened under monoculture and decipher the microbial community in naturally formed and BIO application induced soils.The following results are obtained:1.Naturally suppressive soils to banana Fusarium wilt disease harbor unique microbial communities After compared and analyzed naturally suppressive soils to banana Fusarium wilt disease and co-located conducive soils for 6 experimental sites in Hainan banana main production area,we found that mean values of soil pH,total nitrogen,total carbon,available phosphorus and available potassium were significant higher in suppressive soils than conducive soils.The mean number of soil culturable actinomycete population was significant higher while the mean number of Fusarium population was significant lower than conducive soils.Interestingly,the number of isolated antagonistic Bacillus strains to Foc 4 was higher in disease suppressive soils than in conducive soils.Moreover,the dominated antagonistic Bacillus strains in disease conducive soils were affiliated to B.amyloliquefaciens,B.methylotrophicus and B.subtillis,which were different to those isolated from conducive soils.Next-generation sequencing of the 16S rRNA genes and ITS sequences using the MiSeq platform showed microbial community from different field sites were obviously different as revealed by principal coordinate analysis.Interestingly,bacterial community from disease suppressive soils separated from disease conducive soils within the same field site.The mean relative abundance of Proteobacteria,Gp4,Trichoderma and Pseudomonas in disease suppressive soils were significantly higher while Fusarium was significantly lower than those in disease conducive soils.Compared to microbial network constructed from disease conducive soils,microbial network constructed from disease suppressive soils based on random matrix theory was a better organized or a better operated community.Furthermore,microbial network constructed from disease suppressive soils contained more meta-modules,generalists and supergeneralists.Differed from network constructed from disease conducive soils,the composition of generalists and supergeneralists in the network constructed from disease suppressive soils was made up of Gemmatimonas,Flavobacterium,Sphingobacterium,Terrimonas,Gp3 and so on.2.Dynamic microbial community of banana orchard soils under different continuous cropping spans After compared and analyzed the banana orchards of newly planted for 1 year,monocultured for 2,6,9,10 and 11 years,we found that the banana Fusarium wilt disease incidence increased gradually while the total yield decreased gradually with the monoculture time.Next-generation sequencing of the 16S rRNA genes and internal transcribed spacer sequences(ITS)using the MiSeq platform showed that bacterial and fungal communities altered obviously with monoculture time,especially for the fungal communities.The relative abundance of Fusarium in banana soils from orchard newly planted for 1 year and monocultured for 10 years was significantly lower compared to soils from other treatments.Notably,the relative abundance of Fusarium was not significantly correlated to monoculture time,however,was significantly and positively correlated to banana Fusarium wilt diseae incidence while significantly and negatively correlated to banana total yield.Moreover,the relative abundance of Fusarium showed a significant and negative correlation to soil pH value and organic matter content.Soil bacterial richness index showed a significantly negative correlation while fungal richness index showed a significantly positive correlation to banana Fusarium wilt disease incidence while showed a significantly negative correlation to total yield.The common potential biocontrol taxa,such as Flavobacterium and Gp4,were not significantly correlated to monoculture time,but significantly correlated to banana Fusarium wilt disease incidence and yield.Microbial molecular ecological networks based on random matrix theory(RMT)were constructed to compare the organization,modularity and stability in soils under short and long term monoculture,respectively.The results illustrated that monoculture makes microbial community network to be a worse organized or a worse operated community with a low modularity,stability and limited co-assciation in the network.3.Effects of bio-organic fertilizer sustainable application on soil microbial community under greenhouse condition A 4-year pot experiment was performed to evaluate the effects of sustainable BIO application,which integrated the biocontrol agent Bacillus amyloliquefaciens NJN-6 with mature composts,at low and high levels on banana Fusarium wilt disease suppression,soil chemical properties and microbial communities.The results showed sustainable BIO application effectively controlled the disease,especially at a high level.Next-generation sequencing of the 16S rRNA genes and ITS sequences using the MiSeq platform showed that the soil bacterial and fungal communities in the treatment amended with a high level of biofertilizer(HBIO)were significantly different from a low level biofertilizer treatment(LBIO)or chemical fertilizer control(CF).Moreover,the abundance of Firmicutes and Bacillus was significantly increased,while the abundance of Acidobacteria,Bacteroidetes and Ascomycota was significantly decreased in the HBIO treatment compared with the CF control.Furthermore,the abundance of Fusarium was significantly reduced in the HBIO treatment compared to CF control and was slightly reduced(not significant)compared with the LBIO treatment.Spearman correlation and redundancy analysis showed that Bacillus,Spartobacteria_genera and TM7_genera dominated in the HBIO treatment and they were positively correlated with the soil pH and the contents of total nitrogen and carbon and available phosphorus,which were negatively correlated with disease incidence.4.Effects of bio-organic fertilizer application on banana Fusarium wilt disease and soil microbial community in monocultured banana orchard under field condition Compared to general control(GCK),the bio-organic fertilizer(BIO)applied to monocultured banana orchard with high Fusarium wilt disease incidence showed a 47.1%decrease in disease incidence for the new re-planted season.The BIO application also showed a decrease in titratable acidity(TA)and an increase in total soluble sugars(TSS)and the ratio of total soluble sugars to titratable acidity(TSS/TA)compared to GCK.The highest total soil bacteria population,culturable actinobacteria population and lowest fungi and Fusarium population were observed from the BIO treatment.The ratio of bacteria to fungi(B/F)and actinomycetes to fungi(A/F)values from the plate count method and the B/F value from the real-time PCR analysis were also highest in the soils collected from the BIO application.Based on PCR-DGGE results,the soil bacteria structure was significantly altered in the soils collected from BIO treatment compared to the other treatments.Compared to the other soil samples,BIO-treated soil revealed higher abundances of Gemmatimonadetes and Acidobacteria,while lower abundance of Bacteroidetes.Meanwhile,on genus level,higher abundances compared to other treatments were observed for Gemmatimonas and Gp4.Correlation and redundancy analysis showed that the abundance of Gemmatimonas and Sphingomonas and the soil total nitrogen and ammonium nitrogen content were higher after BIO application,and they were all negatively correlated with disease incidence.Compared to chemical fertilizer application control(CF),the BIO application showed a 40.6%decrease in disease incidence and an increase of 22.4 t/ha in fruit yield in the second sucker cropping year.The highest culturable bacteria,actinobacteria and Bacillus populations,culturable bacteria to fungi(B/F)value,antagonistic Bacillus ratio and lowest Fusarium were observed in the BIO treatment.Based on culture-dependent denaturing gradient gel electrophoresis(CD DGGE)results,the BIO application significantly altered the rhizopshere soil bacterial structure and showed highest richness and diversity.The phylogenetic analysis of selected bands showed that the most abundant phyla were Proteobacteria and Bacteroidetes and BIO application enriched the genera Comamonas,Chitinophaga,the species Bacillus flexus and uncultured Bacillus.Compared with the treatment of pig manure compost(PM)and CF control,the alpha diversity of bacteria significantly increased,whereas the value for of the fungi was significantly reduced following 2 consecutive years of biofertilizer application.Moreover,the abundance of Acidobacteria(Gpl and Gp3),Firmicutes,Leptosphaeria and Phaeosphaeriopsis was significantly increased,while the abundance of Proteobacteria and Ascomycota was significantly decreased in the BIO treatment.Furthermore,the abundance of Fusarium,a causal pathogen for Fusarium wilt disease,was significantly reduced in the BIO treatment compared with the CF control.Interestingly,the disease incidence was negatively correlated with the enriched taxa of Acidobacteria(Gp1 and Gp3)and Firmicutes,Leptosphaeria and Phaeosphaeriopsis but positively correlated with abundance of Proteobacteria,Ascomycota,Fusarium,Cylindrocarpon,Gymnascella,Monographella,Pochonia and Sakaguchia taxa in the rhizopshere soil.In conclusion,long term monoculture unbalanced the soil microbial community,and aggravated the banana soil-borne Fusarium wilt disease.A better operated community for microbial ecological network,a lower relative abundance of Fusarium,and higher relative abundances of beneficial taxa,such as Pseudomonas,Gp4 and Trichoderma,were found in disease suppressive soils.Both under greenhouse and filed condition,the bio-organic fertilizer application could manipulate the microbial community in monocultured soil to increase disease suppression ability,thus controlled the banana soil-borne Fusarium wilt disease.