| Root-knot nematodes do harm to the health of crops and have been considered as one of the important factors restricting the development of contemporary agriculture.Tomato plants infected by root-knot nematodes will form swollen structures of different sizes called root knots,resulting in impaired plant growth and yield.It is reported that root-knot nematode infestation can lead to changes in microbial composition and diversity of plant roots and rhizosphere soil,and some microbial flora are abundantly enriched in root-knot,indicating that microorganisms may play an important role during the occurrence and development of root-knot nematode disease.Based on this,this experiment took healthy and diseased tomato roots and rhizosphere soil as the research objects.Through metagenomic sequencing and other methods,the differences of flora structure and functional genes between different samples were explored,in order to reveal the role of microorganisms in root-knot nematode infestation and in the pathogenic process,to provide a basis for further exploration of root-knot nematode control strategies.Firstly,taking tomato root knot tissue as the research object,the enrichment technology of plant root microorganisms was optimized.The main results were as follows:The plant root tissue was treated with pectinase at a concentration of 1% for 36 h at room temperature.After fully grinding,the grinding liquid is coarsely filtered through four layers of gauze to remove most of the plant tissue.The filtrate was suction filtered through membranes with different pore sizes(100 μm,25 μm,12 μm and 8 μm),and the filtrate obtained by gradient suction filtration was centrifuged at high speed to remove the supernatant and to obtain the enriched plant root nodule microbial precipitation.PCR amplification was performed on the extracted plant root knot genomic DNA by using bacterial 16 S r DNA V5-V7 region primers,and the total amount of bacteria in the enriched pellet was preliminarily evaluated and detected.At the same time,small-scale metagenomic sequencing was performed to further evaluate the enrichment results.The analysis shows that after removing the host DNA of plants and nematodes,the obtained data of attributable microorganisms(mainly bacteria)account for 10.67% of the total sequencing data,which are nearly twice as high as the previous technology,and the structure and composition of bacterial flora are similar to the results based on previous 16 S r DNA flora.Based on the above-mentioned optimized tomato root microbiome enrichment technology,metagenomics sequencing and comparative analysis of the healthy and diseased tomato rhizosphere and root microbiomes were performed by the next-generation sequencing platform Illumina,and the third-generation sequencing platform Nanopore was used to further analyze the root microbiome,thus revealing the changes in the composition of microbial flora and functional genes during root-knot nematode infestation.The heatmap of correlation coefficient among samples and PCo A analysis show that there are differences in the compositional structure of healthy and diseased tomato roots and rhizosphere soils.The results show that the structure and functional composition of tomato rhizosphere soil and root endophyte are significantly different,mainly manifested in that the flora richness of rhizosphere soil was significantly higher than that of root tissue,and there are also significant differences between root-knot microorganisms and healthy and non-root-knot diseased roots.The healthy and diseased tomato rhizosphere soil and root endophytes mainly include Proteobacteria,Gemmatimonadetes,Acidobacteria,Firmicutes,Actinobacteria at the phylum level and Enterobacterales,Pseudomonadales,Burkholderiales,Sphingomonadales,Rhizobiales,Xanthomonadales at the order level.In the annotation results of tomato root-knot microbiome,a large number of genes and functional pathways related to biological nitrogen fixation and nitrogen cycling are found.The number of genes related to nitrogen metabolism pathways annotated in the KEGG database of root knot tissue are 134,including ammonification,nitrification,denitrification,nitrogen fixation and other metabolic pathways,indicating that there is a vigorous nitrogen cycle metabolism in the infection structure of root-knot nematodes.These metabolic processes related to nitrogen metabolism suggest that nitrogen utilization may be involved in root-knot nematode infestation.At the same time,the annotation results of the PHI database show that there are a large number of plant pathogenic genes in root knot microorganisms,which is related to the accumulation of a large number of Enterobacter and Agrobacterium in the root knot,indicating that the infestation of root knot nematodes leads to bacterial complex diseases.In addition,compared with soil microorganisms,a large number of enzymes related to lignocellulose degradation,as well as a large number of other genes related to Carbohydrate Binding Modules,Carbohydrate Esterases,Glycoside Hydrolases,Glycosyl Transferases and Polysaccharide Lyases are annotated in the tomato root microbiome.This may be related to the colonization of plant endophytes and the infestation as well as movement of root-knot nematodes within the plant root tissue.Previous studies have shown that the occurrence of plant root-knot nematode disease may be related to the biological nitrogen fixation process of nitrogen-fixing bacteria.Therefore,we further verified and analyzed the nitrogenase activity and nitrogenase gene abundance in healthy and diseased tomato roots and rhizosphere soil by enzyme-linked immunosorbent method(ELISA),combined with PCR,q PCR and other technologies.The results show that the nitrogenase activity of diseased tomato roots and rhizosphere soil is greater than that of healthy roots and rhizosphere soil.The abundance of nitrogenase genes in healthy roots and rhizosphere soil is higher than that in diseased roots and rhizosphere soil.In conclusion,the significant differences in nitrogenase activity and nitrogenase gene abundance of healthy and diseased tomato roots and rhizosphere soil may be caused by root-knot nematode infestation. |
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