The Construction And Internal Interaction Mechanism Of A Synthetic Rhizosphere Microbiota In Inhibiting The Seed-borne Fusarium Of Maize

Seed-borne pathogenic Fusarium is prevalent in seeds.Traditional methods always with low efficiency and may cause serious environmental pollution.Therefore,there is an urgent need to develop an enironment friendly and stable methods to control these seed-borne Fusarium.Rhizosphere microorganisms plays important roles in plant growth promotion and disease suppression.Taking full advantages and making full use of the function of rhizosphere microorganisms to prevent Fusarium is very important for crop production.This study collected the different maize cultivars from the main maize planting areas in China as the research object.Firstly,we studied the characteristics of maize rhizosphere bacterial communities in“Fusarium-suppressed”and“Fusarium-infected”states,and clearified 12 keystone species in the rhizosphere of Fusarium-suppressed maize seedlings.Then,12bacterial isolates were selected to constructe a systhetic microbiota.These 12 strains were optimized into a simplified rhizosphere systhetic microbiota of 8 strains by strain antagonistic relationship analysis and microbiota stability analysis.Finally,the mechanism of bacteria interaction in the simplified rhizosphere systhetic microbiota was studied by q-PCR technology.The main results are as follows:1.Maize was planted in natural soil and sterilized soil to study the effects of seed-borne Fusarium infected on maize growth and microbial community assembly characteristics of rhizosphere and endosphere.The results showed that the Fusarium infected could significantly inhibit the growth of maize,resulting in a significant decrease in plant height,fresh weight and SPAD value,which were38.0%,41.9%and 22.9%,respectively.At the same time,the activities of SOD,POD and CAT in Fusarium-infected maize leaves were significantly increased,which were 97.0%,134%and 5.4%,respectively.According to the analysis of the characteristics of bacterial community in maize rhizosphere and enzosphere during cultivation period,it was resulted that the structure of bacterial community in the rhizosphere of Fusarium-infected and Fusarium-suppressed maize seedlings was significantly different.Bacillus,Pseudomonas,Ochrobactrum and Agrobacterium were significantly enriched in the rhizosphere of Fusarium-suppressed maize seedlings.It can speculated that it is the key species to inhibit the outbreak of seed-borne Fusarium.2.The root exudates of Fusarium-suppressed and Fusarium-infected maize seedlings in different cultivation periods were collected,and the soil cultivation and microbial chemotaxis analysis were used the specific differed substances of root exudates,in order to study the bacteria which can be significantly enriched by the specific differed substances of root exudates of Fusarium-suppressed maize seedlings.The results showed that the significant increase in root exudates of Fusarium-suppressed maize seedlings was mainly concentrated in sugars,while the significant increase in root exudates was mainly concentrated in organic acids and alcohols.The specific differed substances of root exudates of Fusarium-suppressed and Fusarium-infected maize seedlings were compounded into solutions for soil cultivation.It was found that the root exudates of Fusarium-suppressed maize seedlings could enrich Pseudomonas,Pseudoxanthomonasand and Pantoea,while the root exudates of Fusarium-infected maize seedlings could enrich Chitinophage.The chemotaxis of soil microorganisms to specific differed substances of root exudates showed that Bacillus,Pseudomonas and Enterobacter could have chemotactic effect to the specific differed substances of root exudates of Fusarium-suppressed maize seedlings,while Lysinibacillus had chemotactic effect to the specific differed substances of root exudates of Fusarium-infected maize seedlings.Therefore,Pseudomonas,Pseudoxanthomonasand,Pantoea,Bacillus and Enterobacter may be the key species to inhibit the outbreak of seed-borne Fusarium.3.The soil microbial suspension were progressive diluted and then inoculated to sterilized soil in order to study the effects of loss of rare species of soil microorganisms on the control seed-borne Fusarium outbreak by rhizosphere and endosphere of maize seedlings microorganisms.The results showed that the inhibition rate of natural soil on the outbreak of seed-borne Fusarium was decreaseing with the increase of dilution.PCo A analysis showed that the rhizosphere was the key area for bacterial to control seed-borne Fusarium.9 key OTUs were confirmed by Randomforest and PLS-DA analysis,which were significantly enriched in the rhizosphere of Fusarium-suppressed maize seedlings.They are Pseudomonas,Bacillus,Lysobacter,Burkholderia,Enterobacter,Pseudoxanthomonasand,Stenotrophomonas,Acinetobacte and Chryseobacterium.The above strains may be the key species to inhibit the outbreak of seed-borne Fusarium.4.The potential key species which identified by the above three strategies were combined to 12non-redundant key species.Then 12 rhizosphere bacterias which were Enterobacter ludwigii（Ent）,Lysobacter soli（Lys）,Acinetobacter baumannii（Aci）,Pseudomonas stutzeri（Pse）,Pseudoxanthomonas japonensis（Pth）,Burkholderia cenocepacia（Bur）,Stenotrophomonas maltophilia（Ste）,Bacillus amyloliquefaciens（Bac）,Ochrobactrum anthropi（Och）,Agrobacterium tumefaciens（Agr）and Pantoea eucrina（Pan）respectively choosed from to construct a systhetic microbiota.Colonization experiment showed that 3 of the 12 strains could not colonized in the rhizosphere and endosphere of maize,which were identified as Och,Agr and Pan,respectively.Antagonistic experiment showed that the Bac in the systhetic microbiota was the core strain to against the seed-borne Fusarium.Promoting experiment showed that Chr could inhibit the growth of Bac,so the systhetic microbiota of 12 strains were optimized simplified systhetic microbiota of 8 strains.Furthermore,it was found that Ent is the key strain to maintain stability in the systhetic microbiota.The expriement of simplified systhetic microbiota controled seed-borne Fusarium which in 8 different maize breed otubreak resulted that the outbreak rate of seed-borne Fusarium decreased significantly after inoculated simplified systhetic microbiota,Yuyu22（42.85%）decreased most obviously showded cpoies of Fusarium in the rhizosphere of Fusarium-infected maize seedlings was 1.14×10⁶ copies.g^-1,and the copies of Fusarium in the rhizosphere of Fusarium-suppressed maize seedlings was 1.95×10⁴copies.g^-1.Compared with the Fusarium-infected maize seedlings,the maize seedlings which inoculated simplified systhetic microbiota showded plant height and fresh weight all significantly increased,Jingke 968 increased most obviously by 102.1%and 142.4%,respectively.Except Longping 206,others showed a significantly increase in SPAD value.The maize which inoculated simplified systhetic microbiota showed leaves enzyme activity of CAT,SOD and POD decreased to the same level as maize planted in natural soil.The most obvious decrease in enzyme activity among the three breeds was Jingke 968（48.1%）,Yuyu 22（34.1%）and Longping 206（80.17%）.Therefore,a simplified and stable systhetic microbiota of 8 strains was constructed in this study,which can effectively control seed-borne Fusarium outbreak in different maize breeds,Bac is the core strain to antagonistic seed-borne Fusarium and Ent is the key strain to maintain stability in the simplified systhetic microbiota.5.The interaction mechanism of simplified systhetic microbiota against seed-borne Fusarium was studied by q PCR technology.The results showed that,in the biological control genes,Pth and Ste could promote the expression of dfn,bac,fen and dhb genes in Bac;Lys and Aci could promote the expression of bac,srf and dhb genes in Bac;in the promoting plants growth genes,Aci and Pth could promote the expression of als S,ycl O,Aco A and phy genes in Bac,Ste could promote the expression of als S,aco A and phy genes in Bac,Pse could promote the expression of phy and Aco A genes in Bac,Lys and Ent could promote the expression of aco A genes in Bac,Bur could promote the expression of trp A and aco A genes in Bac.The specific differed substances of root exudates of Fusarium-infected maize seedlings could promote the expression of biological control genes in Bac,while the specific differed substances of root exudates of Fusarium-suppressed maize seedlings could promote the expression of promoting plants growth genes in Bac.In the Fumonisin produce genes,Pth,Aci,Pse,Bur and Ste could inhibit the expression of fum1 gene in seed-borne Fusarium,Pse could inhibit the expression of fum14 gene in seed-borne Fusarium.In the infected plants genes,Aci,Bac,Ent,Lys,Pse could inhibit the expression of abp1 gene in seed-borne Fusarium,Aci could inhibit the expression of fow2 gene in seed-borne Fusarium,Bur and Pse could inhibit the expression of xln2 gene in seed-borne Fusarium.The above results showed that Bac was the core strain to antagonistic Fusarium and promote plant growth.Other strains could enhance the effects,and also could reduce the Fumonisin produce and infection of seed-borne Fusarium.The simplified systhetic microbiota showed a new cooperative mode of"Main effect of core strains and others enhance the effects of core strains".In this study,the keystone species of the rhizosphere bacterial communityof the Fusarium-suppressed maize seedlings were identified through a three-pronged innovative approach.A stable and simplified systhetic microbiota of 8 strains was constructed,which had a strong effect on the prevention and control of the seed-borne Fusarium and on the growth promotgroin of maize.This study provides a new innovative approach to construct a simplified systhetic rhizosphere microbiota,and puts forward a new cooperative pattern of the systhetic microbiota,which is"Main effect of core strains and others enhance the effects of core strains".It can provide theoretical basis and practical proof for efficient prevention of seed-borne Fusarium and ensuring the security of global maize production.