The Rhizosphere Microecological Mechanisms Of Protozoa Reducing Soil Obstacle Of Ralstonia Solanacearum

The biological obstacle caused by soil-borne pathogens seriously restrict the sustainable development of agricultural production.A large number of beneficial microbial resources such as bacteria,fungi,phages and protozoa are colonized in the rhizosphere of crops.They form the first biological barrier to restrict soil-borne pathogens from invading plants through antagonistic competition,parasitism or predation.Interactions of complex beneficial microbes in the rhizosphere are unclear in relation to pathogen invasion,which is the main bottleneck for the development and application of soil-borne disease rhizosphere ecological control strategies.The researches on rhizosphere resistance with beneficial microorganisms mostly focus on bacteria or fungi,ignoring protozoa and the role of multi-trophic synergistic interactions on the rhizosphere invasion of soil-borne pathogens.In this study,we used tomato bacterial wilt as disease model which caused by Ralstonia solanacearum,to reveal the effect of rhizosphere protist community composition on the health of tomato through in situ experiments,and the mechanism of the rhizosphere protist community affecting the health of tomato by using metagenomic sequencing,screen protozoa from the rhizosphere of healthy tomato plants,the mechanism of protozoa affecting the invasion of R.solanacearum and the effect of synergistic control of bacterial wilt by combing protozoa and Pseudomonas.Through these studies,we try to reveal the biological mechanism of rhizosphere protozoa affecting the occurrence of bacterial wilt in tomato.Our main results show as followed:1.We conducted a in situ field experiment and collected rhizospheric soil by nondestructive sampling method,combined with high-throughput sequencing technology,to study the interpretability of the rhizosphere protist community on the occurrence of bacterial wilt in tomato.We found that in the sequencing results of the two primers targeting 18 s r RNA V4 region and V9 region,the community composition of eukaryotes and protists was different.The primer sequencing results of V4 region covered more protists,and the tendency of protist community with tomato growth was basically the same in both results.In addition,we compared the shannon index of bacteria,fungi and protist communities in the rhizosphere of healthy and diseased tomato and found that only the protist community was different during tomato growth,and the shannon index of bacteria and protist communities in healthy plants increased significantly with tomato growth.To link the different factors with plant health,we found that the diversity and structure of bacterial communities could significantly predict the abundance of R.solanacearum in health plants,the structure of the protist community in diseased plants could significantly predict the abundance of R.solanacearum.Combining the sequencing results of all plants,we found that the protist community structure was the key indicator for predicting the abundance of R.solanacearum.2.We categorized protist communities by phylogeny and function,and link different functional groups with plant health.We found that the initial protozoa community composition significantly affected plant health,and the protist species that could indicate tomato health were more in healthy plants than diseased plants.The relative abundance of protozoa in diseased plants decreased significantly with tomato growth,but the relative abundance of protozoa in healthy plants was relatively stable.We analyzed the correlation between the relative abundance of protozoa and the number of R.solanacearum and found that they were significantly negatively correlated in the diseased plants.Eight microbial metabolic genes were analyzed using metagenomic sequencing methods and found that [Q] genes involved in the biosynthesis,transport,and catabolism of secondary metabolites were significantly associated with the number of R.solanacearum.The co-occurrence network of rhizosphere protists,bacteria and [Q] genes in healthy plants was more complex than that in diseased plants.Using structural equation model analysis,it was found that the protist community in healthy plants affects plant health by driving the bacterial community to promote the expression of functional genes.3.We isolated three protozoa from healthy tomato rhizosphere and identified as Vahlkampfia inornata,Vahlkampfia avara and Naegleria sp.These three protozoa could prey on R.solanacearum and could significantly inhibit its growth.QL92 was selected for greenhouse experiments to explore its effect on the indigenous bacterial community in the rhizosphere.The results showed that QL92 inoculation could significantly change the structure of the indigenous bacterial community at the seedling stage,flowering stage and fruiting stage of tomato,and make the bacterial community network in the tomato rhizosphere more complex.Inoculation with protozoa significantly affected the composition of indigenous bacterial communities,and the abundance of Proteobacteria and Actinobacteria increased significantly at the seedling stage;at the genus level,the genus Pseudomonas abundant was increased.The relative abundance of Pseudomonas was negatively correlated with R.solanacearum.4.Pseudomonas fluorescens P1 that isolated from the rhizosphere of tomato can significantly inhibit the growth of R.solanacearum.It was found that P1 can resist the predation of QL92 and the predation of protozoa QL92 prompt P1 to produce more secondary metabolites to inhibit the growth of R.solanacearum.Compared with the inhibition rate of the fermentation when P1 was cultured alone,the inhibition rate of the fermentation of the co-cultures with P1 and QL92 increased by about 24%.The results of the greenhouse experiment showed that the incidence of bacterial wilt disease can be significantly reduced by inoculation of QL92 or P1,but the incidence of bacterial wilt disease can be significantly reduced by inoculation of the two,which can reduce the incidence of disease to about 25%.At the same time,the density of R.solanacearum in the rhizosphere also decreased significantly.In summary,the rhizosphere protozoa community is significantly related to the occurrence of bacterial wilt in tomato.Protozoa can not only prey on pathogenic bacteria,but also increase the abundance of antagonistic bacteria in the community by driving the bacterial community,thereby promoting the expression of functional genes and affect plant health.