Study On The Induction Mechanism Of Casuarina Equisetifolia Tissues On The Early Infection Of Ralstonia Pseudosolanacearum

Ralstonia solanacearum species complex is a group of highly heterogeneous pathogenic strains,which can cause bacterial wilt of more than 200 crops and woody plants in the world,such as Casuarina equisetifolia.R.solanacearum can survive in the soil for a long time and start infection after activated by host plants.At present,the research on bacterial wilt mainly focuses on the infection mechanism on herbaceous crops,but less on the woody plants.As a soil-borne plant disease,the research on the mechanism of how its pathogens transform from a non-pathogenic state to a pathogenic state infecting host plants in the soil environment is of great significance for controlling the spread of the disease.In this paper,we studied the response pattern of the pathogen induced by the root and cladophyll tissues of Casuarina equisetifolia at the gene expression level,analyzed the differential expression patterns of the pathogen,screened the functional genes related to pathogenicity,and discovered the influence of host plant tissues on the early occurrence of the pathogen infection.These studies are helpful to develop new strategies for the prevention and control of C.equisetifolia wilt.The results are as follows:1、This experiment completed the genome sequencing of the pathogen YQ strain isolated from Casuarina equisetifolia with the symptom of bacterial wilt.The total length of the genome is 5.82 Mb,consisting of 3.78 Mb chromosomes and 2.04 Mb large plasmids.The average GC percentage of the genome was 66.93%,and 5104 protein sequences were predicted.Meanwhile,YQ and other 113 reference strains of R.solanacearum with different sequence types were analyzed and phylogenetic tree was constructed.The results showed that the sequence divergence of the partial egl genes between strain YQ and three sequevar 16 strains(UW1511,JS527 and SD53)was 0.1-0.3%,which was lower than the top limit of 1% variability to identify a typical sequevar.As a result,the YQ strain isolated from C.equisetifolia was identified as R.pseudosolanacearum phylotype I-sequevar 16(I-16),which is the first reported strain of I-16 to host woody plants.2、The biological verification of the effects of the host plant tissues on the pathogen.The results of the pathogen’s growth curve showed that,compared with the minimal medium(MG),there was no significant difference in the growth rate of the strain on the medium containing cladophyll(MGL)and root extract(MGR)within 24 hours.The swimming motility showed that the motility ability on MGL and MGR was significantly increased compared with MG,and the motility ability on MGL was more obvious than that of MGR within 48 hours.The pathogenicity test results showed that the pathogenicity of the strains treated with MGL and MGR was significantly stronger than that of the strain cultured in MG,and MGL caused faster wilting.This is consistent with the host tissues upregulating pathogenicity-related genes of the strain YQ on the transcriptomics analysis.3、Transcriptome sequencing was performed on the bacterial cultures of MG,MGL and MGR.The results showed that cladophyll tissue of C.equisetifolia had greater effects on the differentially expressed genes(DEGs)of the strain YQ than the root tissue compared with in the minimal medium.In MG vs.MGL,1482 genes were up-regulated and 428 genes were down-regulated,while only 67 up-regulated genes and 83 down-regulated genes in MG vs.MGR.According to the functional enrichment of differentially expressed genes between different treatments,the results show that the most differentially expressed genes of MG,MGL and MGR in the GO classification are concentrated in single biological process,cellular process and catalytic activity.KEGG richness analysis showed that the DEGs in MG vs.MGL were concentrated in flagellar assembly,and bacterial chemotaxis;the DEGs in MG vs.MGR were concentrated in flagellar assembly,sulfur metabolism,and RNA degradation.Further analysis of the function of DEGs revealed that most of them are related to the bacterial pathogenicity.Among the upregulated genes,most are genes encoding pathogenic bacterial motility,effectors,type Ⅲ secretion system,quorum sensing transcriptional regulators and plant cell wall degrading enzymes.In addition,it was also discovered that the bacterial wilt pathogenic genome contained 49 genes related to the second messenger cyclic dimeric guanosine monophosphate(c-di-GMP)production and degradation.Seven phosphodiesterase-related DEGs were upregulated in the cladophyll treatment,which suggests that the intracellular concentration of c-di-GMP is related to the pathogenicity of the bacterial wilt pathogen.In conclusion,our study reported the genome of a bacterial wilt pathogen from Casuarina equisetifolia for the first time,and we found that the host plant tissues could significantly induce the pathogenicity-related genes of the pathogen,and the induction effect of the cladophyll of C.equisetifolia was stronger than that of the root tissue,which was consistent with the results of biological verification.Therefore,it is suggested that litterfall management in C.equisetifolia forests,or even other plantations,should receive attention to prevent the induction of bacterial wilt disease caused by RSSC.