Deciphering The Rhizosphere Microbiome Associated With Healthy And Verticillium Wilt Diseased Cotton Plant And Screening Beneficial Microbes

Verticillum wilt,as an important soilborne fungal disease in cotton production,has caused serious economic losses around the world.Rhizosphere microorganisms have a great impact on plant health through multiple complex interactions with plants and pathogens.on the present study,the adjacent healthy and diseased plants were taken as the research object from two cultivars(Zhongzhimian 2 and Lumianyan 28)at the peak period of Verticillium wilt in two fields.The density of Verticillium dahliae microsclerotia and total rhizosphere microorganisms abundances in the soil were detected by wet-sieving method and real-time quantitative PCR in order to compare the difference of the density of pathogen and rhizosphere microbes in the healthy and Verticillium wilt diseased cotton plants.High-throughput sequencing technology was used to analyze the differences of rhizosphere microbial community between healthy and diseased strains and between two cultivars.Isolate culturable bacteria from the rhizosphere of healthy cotton by spread-plate method,combine strains with better indoor biocontrol effect,and examine the control effect on cotton Verticillium wilt by greenhouse experiments.The main results are as follows:1.The severity of cotton Verticillium wilt is not only significantly increased by the density of microsclerotia in the soil,but also by the rhizosphere microorganisms.The occurrence of cotton Verticillium wilt always accompanied by a substantial increase in the number of rhizosphere fungi.Furthermore,wilt severity increased highly significantly(P < 0.001)with increasing the ration between fungal and bacterial abundance,particularly from healthy to symptoms.2.Community composition analysis showed that the dominant rhizosphere bacteria mainly included Proteobacteria and Firmicutes at the phylum level,and the dominant species at the genus level were Lactobacillus and Serratia.The dominant rhizosphere fungi mainly include Ascomycota and Mucoromycota,and the dominant species on the genus level are Fusarium and Chaetomium.3.Rhizosphere soil samples from healthy plants had much larger(P < 0.001)values than from diseased plants both cultivars.However,fungal alpha diversity indices showed a different pattern from the bacterial one(Fig.3).LM28 showed the same pattern as for bacteria,namely alpha diversity indices were greater for healthy plants than for diseased plants.The opposite was true for ZHM2.Such a difference in the response between the two cultivars,i.e.the interaction between cultivar and plant disease status,was highly significant(P < 0.001).4.The variability among samples from cultivar LM28 was less than those from cultivar ZHM2.Second,the variability among diseased samples was far less than the healthy samples,particularly for the bacterial community in the rhizosphere cultivar LM28 plants.The cultivar and plant diseased status as well as their interaction on the beta diversity indices that plant diseased status,rather the cultivar,explained the major variations in the bacterial and fungal community structure.The cultivar effect appeared to be more pronounced for the fungal than for the bacterial community.5.Through the antibacterial test,89 strains with inhibitory effect on cotton Verticillium wilt were selected from the rhizosphere bacteria,followed by further screening by the plate dual culture method and the Oxford Cup method bacteriostasis test,12 strains with better bacteriostatic effect Strains were obtained,of which the antibacterial XJ6-96 can also increase the germination rate and promote the growth of cotton buds.In greenhouse experiments,the antagonistic strains XJ5-6 and XJ6-96 were selected for single inoculation and mixed inoculation.It was found that only mixed inoculation had a good prevention effect on cotton Verticillium wilt,with a control effect of 32.9%(After inoculation 28 d).The 16 S rRNA sequence was used to generate the phylogenetic tree,it was found that XJ5-6 and XJ6-96 belong to the genus Klebsiella.The above research results clarified the relationship between the number of microsclerotia in the soil,the abundance of rhizosphere fungi and bacteria,and the existence of community structure and the occurrence of cotton Verticillium wilt.In addition,the antagonistic antibacterial combination obtained from the cotton rhizosphere separation was later applied to cotton,which was beneficial to the prevention and treatment of Verticillium wilt.In the next step,according to the different mechanism of antagonistic antibacterial action,the combination of them under field conditions was used to verify the control effect of beneficial bacteria on cotton Verticillium wilt,in order to provide a theoretical basis for the green sustainable control of cotton verticillium wilt.