| Root-knot nematodes are one of the important diseases and insect pests that harm crops more seriously and limit crop continuous cropping.With the wide application of high-throughput sequencing technology,it has been found that the occurrence and control of plant diseases are closely related to the community structure and functional composition of closely related microorganisms.Therefore,exploring the mechanism of root-knot nematode infection and the formation of microorganisms in tomato roots and its correlation with root-knot nematodes is of great significance for the development of new biocontrol microresources and biological control strategies.Based on previous work,high-throughput sequencing technology and proteome sequencing technology were used to study the differences in the composition mechanism and bacterial structure of root endophytic bacterial communities during the development of healthy tomato plants and tomato plants infected with root-knot nematodes,revealing the composition mechanism,enrichment and functional differences of endogenous microorganisms in different time periods.Metagenomic analysis of the structure and function of the tomato root microbiome showed that as the tomato grows,the difference in microbial diversity becomes more pronounced.Among them,the samples infected by root knot nematodes were most affected by time(k = 0.00145).In addition,there was no significant difference in the distribution of NTI and βNTI among all treatment groups,and the value of βNTI was mostly between-2 and +2.The results show that the construction of microorganisms in the tomato growth process is dominated by a random process,but the probability of root microbes infected by root-knot nematodes to the deterministic process is greater than the healthy tomato root microbes.There are significant differences(p < 0.05)in structure,diversity and functional composition of bacteria in tomato roots infected by health and root-knot nematodes.Among them,the impact of the differences is mainly on the type and abundance of bacteria.There were also significant differences(p < 0.05)in the bacterial structure,diversity and functional composition of bacteria in the roots and rhizospheres of healthy and diseased tomatoes under different periods.Compared with healthy tomato root samples,Burkholderiales,Rhizobiales and Actinomycetales of tomato roots infected with root-knot nematodes were enriched.However,in healthy tomato roots,Actinomycetales,Pseudomonadales,Bacillales,Flavobacteriales,Xanthomonadales,Enterobacteriales Was enriched.Compared with healthy samples,metagenomic results showed that the actinomycetes in the root knot had higher functional abundance in the metabolism of aromatic compounds.Functional genes related to aromatic compound metabolism and nitrogen metabolism are enriched in the Burkholderia and Rhizobium enriched in root knots.potentially indicating that these bacteria may be involved in nitrogen metabolism in root knots and may be involved Nitrogen source in root-knot nematodes and providing part of the nitrogen supply for root-knot nematodes.What’s more,the functional protein changes of endophytic bacteria in tomato roots of health and infected by root nematode under different periods were analyzed by meta-proteomics.The results showed that in healthy tomato roots at different growth stages,the differential proteins were mainly involved in the growth and reproduction of microorganisms,including nucleic acid synthesis and repair,ATP synthesis,and cell membrane construction.It showed that the growth and reproduction of microorganisms in the healthy tomato root system were faster in different stages,and the microorganisms simply changed obviously,which further indicated that the diversity of healthy tomato root microorganisms at different times was significantly different.In tomato roots infected with root-knot nematodes,in the early the metabolic processes are still related to nucleic acid synthesis and repair,ATP synthesis,and microbial growth and reproduction.However,in the middle and late stages,differential proteins involve the secondary metabolic that respond to the surrounding environment,suggesting that microorganisms may be affected by the surrounding environment to respond.Nodule protein and nitrogenase produced by rhizobia were detected in the root knots of infected tomatoes,so it was mutually confirmed that the presence of rhizobiales was related to the infection of root-knot nematodes.By comparing the differential proteins in the roo nodes and non-root nodes infected by root-knot nematodes,it was found that the response to stimulus began to enrich at the beginning of the root node.Based on the analysis of the KEGG metabolic pathway,it was found that at different stages of healthy tomato growth,microorganisms differ greatly in nucleic acid repair,amino acid metabolism,photosynthesis,and carbon fixation.However,there are significant differences including amino acid metabolism and oxidative phosphorylation bacterial protein secretion on the metabolism of microorganisms in tomato infected by root-knot nematodes.Based on the analysis of the structure of tomato root endophyte bacteria,the tomato root endophyte with obvious inhibitory activity against root knot nematode infection are screened,and most of them are Bacillus bacteria.The effect of the introduction of different biocontrol strains in soil that changes the microbial community structure of tomato roots infected with root nematodes is further analyzed by 16 S amplification sequencing.The results show that different strains of infected tomato will cause differences in the classification of the Phylum level.Among them,the abundance of Firmicutes in sample Infected by Bacillus including CZ40,CZ45,CZ51,CZ52 and CZ53 is relatively higher than other samples,while the abundance of Proteobacteria in CZ6,CZ7 and CZ8 is higher than others.In the classification of the order level,the structural changes of the endophyte community structure of tomato roots infected by different strains are more obvious compared with the control group.Among them,the abundance of Enterobacterales in CZ6,CZ7,CZ8 and CZ29 is significantly higher than those of other treatment groups,while the abundance of Bacillales in CZ40,CZ51,CZ52 and CZ53 is significantly(p < 0.05)higher than that of other treatment groups.Similarly,the abundance of Pseudomonas in CZ57,CZ65,CZ66,CZ67,CZ69,CZ71 and CZ75 is also significantly(p < 0.05)higher than other samples.On the α index,CZ45,CZ51 and MR2 and the control group(Control)have significant differences in OTUs index and Chao1index(p < 0.05),the experimental group CZ53 and the control group(Control)also have significant differences in Shannon index and Simpson index(p < 0.05).The results of LEfSe analysis indicate that the differences between the endophytic bacteria in tomato roots of the experimental group and the control group are mainly focused on Enterobacterales,Bacillales,and Chloroflexi.Among them,the Bacillales and Enterobacterales are biomarker in the experimental group.Chloroflexi and Kiloniellales are significantly aggregated in the control group,while the LDA score of Firmicutes including Bacillus and Paenibacillus and Enterobacteriaceae is greater than 4.Therefore,the species that affect the growth and reproduction of root-knot nematodes in the experimental group are mainly concentrated on the Firmicutes and Enterobacterales.Based on the enrichment analysis of OTUs,the differential heatmap and the Manhattan map indicated that the enriched bacteria in the control group are mainly subordinate to the autotrophic microorganisms such as Rhizobiales and Chloroflexi.The OTUs enriched in the experimental group are all Bacillales,indicating that Bacillales may play an important role in inhibiting the growth and reproduction of root-knot nematodes.This is also consistent with the fact that most of the bacteria with obvious inhibitory activity against root-knot nematode are Bacillales obtained by our previous screening.Through the infection experiments of different strains,the theoretical basis is established for further cultivation and fermentation process optimization,at the same time increasing the bacterial nematicidal active substance and exploring the process and technology for preparing nematicidal fungicides. |
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