Rhizobacteria Isolation And Community Construct For Controlling Seed-Borne Fusarium In Maize

There are a lot of microorganisms in soil.The microbes can have close interaction with each other or with the plants.Many plant assosiated microorganisms are plant benifical and can promote plant growth and development.More attentions have been paid to the interactions between these microorganisms and plants.In this study,the compositional and functional traits of microbial communities in the rhizosphere and endosphere of maize were studied by traditional cultivation and high-throughput sequencing technology.The succession of maize rhizosphere and endophytic fungal communities under natural soil and sterilized soil were analyzed by high-throughput sequencing technology;the endophytic fungi were isolated and identified by traditional cultivation technology;the appropriate dilution degree for suppressing endophytic fungal pathogen in maize was found by inoculating gradient diluted soil suspension into sterilized soils,and the rhizosphere soil which could suppress endophytic fungal pathogen was selected for isolating and screening of biocontrol bacteria;the biocontrol bacterial community was constructed by artificial compounding strains,and the suppresseffect of biocontrol bacterial community on Fusarium spp.was evaluated through the re-inoculation of biocontrol bacterial community.The main research results are as follows:1.The fungal diversity and eveness are highest in soil,while the fungal diversity in rhizosphere and root are lower than in the soil and decreases with planting days.The dominant fungal group is Ascomycota.In natural soil,the compositions of fungal community in rhizosphere are similar to thaose in soil,hence we speculate that these fungal groups in rhizosphere are generally from soil,and the community assembly is mainly induced by horizontal transfering.However,in sterilized soils,the fungal community assembly in root and rhizosphere is mainly induced by vertical transfering due tothe community compositions are significantly different from those in natural soil.The Fusarium spp from seed is mainly present in endosperm.In addition,we found the abundance of Fusarium spp in rhizosphere from natural soil is significantly lower than in sterilized soil.2.Eight maize varieties are collected from five major planting areas（Northeast plain,North China plain,Southwest mountain,Northwest inland,middle and lower Yangtze river plain）,which are labeled as DHA,LCH,FT,LP,JKE,ZDN,ZDA and YYU,respectively.Eight Fusarium spp.strains were isolated from each variety by culturing method,and the phylogenetic tree is constructed.Among them,two of them are Fusarium oxysporum,five of them are Fusarium verticillioides and one of them is Fusarium avenaceum.The sequencing results of pot experiment by planting these seeds in natural soil and sterilized soil showed that Fusarium spp.is dominant in sterilized soil while suppressed in natural soil.The relative abundance of Fusarium spp.in rhizosphere and root of maize in natural soil was significantly lower than in sterilized soil,indicating that there should be some microorganisms with biocontrol function may suppress the seed-borne Fusarium spp.3.The gradient soil suspension dilution method is used to discoverthe potential functional microbes that can suppress the seed-borne Fusarium.The results show that the outbreak rate of Fusarium is 60.00%under the dilution degrees of 10-1,the outbreak rates of Fusarium are 64.00%and 66.67%under the dilution degrees of 10-2 and 10-3,respectively,and the outbreak rates of of Fusarium are 74.67%and 89.33%under the dilution degrees of 10^-4 and 10^-5,respectively.The outbreak rate increases with the increasing dilution level.The results of qPCR shouwed that the abundance of Fusarium are 5.89 × 10⁸ gene copies·g^-1 and 1.12 × 10⁹ gene copies·g^-1 in rhizosphereunder the dilution level of 10^-4 and 10^-5 respectively,while there are 4.57 × 10⁷ gene copies·g^-1 and 1.00 × 10⁸ gene copies·g^-1 in the control samples.Moreover,in the root samples,the abundance of Fusarium are 2.04 × 10⁷ gene copies·g^-1 and 3.72 × 10⁶ gene copies·g^-1 respectively while there are 1.51 × 10⁶ gene copies·g^-1 and 4.47 × 10⁶ gene copies·g^-1 in the control samples.The abundance of Fusarium in the root and rhizosphere are lower than those of the suppressive treatments but with no significant differences.The rhizosphere soil that can suppress Fusarium were collected for isolation of the biocontrol strains and to generate a potential functional bacterial seed bank of maize.A total of 350 bacterial strains were isolated.The main groups of culturable bacteria in the rhizosphere are Proteobacteria,Actinomycetes,Firmicutes and Bacteroidetes,most of them are Proteobacteria,accounting for 59.6%,followed by Actinomycetes（29.5%）,Bacteroidetes（7.0%）,and Firmicutes（3.9%）.4.Artificially compounded functional bacterial community with 12 strains was reinoculatedto sterilized soil,and we found that the overall suppression rate is 54%,indicating that the artificially generated biocontrol rhizosphere community can suppress Fusarium.High-throughput pyro-sequencing demonstrated that eight strains,including Chryseobacterium,Lysobacter,Acinetobacter,Pseudomonasstutzeri,Pseudoxanthomonas,Burkholderia,Stenotrophomonas,Bacillus,were successfully colonized in the rhizosphere in the suppression treatments,,in which the Acinetobacter and Bacillus were absent in the non-suppression treatments.In this study,we found that the Fusarium groups become dominant froups in the rhizosphere and root of maize in sterilized soil,whereas it can be suppressed in natural soil.Therefore,we speculate that the outbreak of seed-borne Fusarium in sterialized sol is generally existed.It is a potential menace for maize production.So by culturing the maize rhizosphere beneficial bacterial groups to controlthe seed-borne Fusarium is a new pathway for preventing the potential seed-borne disease.This practice will provide a good foundation for manipulating the seed microbiome and sustaining the global food security.