Synergistic Mechanism Of Saisentong And Bacillus Velezensis B-001 In Managing Bacterial Wilt Of Tomato

Bacterial wilt of tomato,caused by Ralstonia solanacearum,was the most damaging disease in tomato production,which has strong transmission and survival ability.In order to control the occurrence of bacterial wilt,overuse of bactericides has caused environmentally destructive and increased risk of resistance.The biological control is greatly affected by surrounding and the control effect is unstable.It was that has brought new challenges to agricultural production and disease prevention and control.Bacillus velezensis B-001 was a biocontrol bacterium isolated from plants.In previous study,it was found that it has a good synergistic effect with Saisentong to control tomato bacterial wilt.The study analyzed the compatibility mechanism of bacteria and bactericides from the transcription level,analyzed the colonization level of biocontrol bacteria in tomato after the synergistic treatment of B.velezensis B-001 and Saisentong by means of fluorescent labeling.Then q PCR was used to elucidated the molecular regulatory mechanism of synergistic effect between Saisentong and B.velezensis B-001.The main results were as follows:Using the Nanopore sequencing platform to obtain B.velezensis B-001 genome sequence,the genome size was 4,067,009 bp and a total of 3,968 coding genes were predicted.Through the analysis of evolutionary relationships of B.velezensis B-001 was determined as Bacillus velezensis.The analysis of secondary metabolic gene clusters showed that B-001 was a strain with strong biocontrol ability,with multiple secondary metabolic gene clusters and the presence of genes expressed to inhibit the growth of fungi and bacteria.Meanwhile,the analysis of gene annotation information revealed the presence of genes related to restriction modification system in B-001.The gene expression of B.velezensis B-001 was studied under the stress of Saisentong using transcriptomics.After analyzing the functional enrichment results of differentially expressed genes,most of the genes were enriched in functions such as budding spore formation and cell wall repaired during logarithmic growth phase.And most of the genes were enriched in functions such as motility and metabolite secretion and degradation during stable growth phase.The B-001 in the above periods were observed by electron microscope.The results showed that was more sensitive to 100mg/L Saisentong than 50 mg/L Saisentong and cell shrinkage appeared and spore formation in the logarithmic growth phase of strain B-001.However,as the growth continued,the cell morphology of strain B-001 gradually recovered and more upregulated genes were enriched for motility and secondary metabolite production and digestion functions.Through the comparison of Rebase database and PCR amplification verification,it was found that four sets of restriction modification systems in B.velezensis B-001.The simulation test of the restriction modification system in vitro showed that the cell fluid of strain B-001 could digest the exogenous plasmids.Subsequently,through homologous recombination and gene replacement methods,the four methylated genes in the restriction modification system of strain B-001 were successfully integrated into the genome of B.subtilis WB600,and the homologous methylation simulation of strain B-001 was established,which achieved methylation modification of exogenous plasmids.B.velezensis B-001 was labeled with GFP and the growth kinetics after labeling was consistent with that of the wild type,which could antagonize R.solanacearum and maintain good compatibility with Saisentong.R.solanacearum GQ-1 was labeled with m Cherry,and the virulence verification test showed that it could still infect tomato with a high level.The colonization of strains under the synergistic treatment of B-001 and Saisentong was observed by fluorescence confocal analysis,showing that B-001 mainly colonized in tomato root epidermis,vessels,stem epidermis,vascular bundles and leaves.Synergistic treatment of tomato with Saisentong and B-001 can significantly improve the colonization ability of B-001 in these parts.At the same time,when R.solanacearum infects tomato,the invasion and colonization ability of R.solanacearum was significantly decreased after co-treatment with Saisentong and B-001.Expression of tomato SA signaling pathway regulatory genes PR-1b and PR-P2 and JA/ET signaling pathway Lap A,Jar1,Etr1,Osmotin-like were determined by q PCR technique.To investigate the induction of tomato SAR and ISR resistance systems by synergistic treatment of Saisentong with B.velezensis B-001.The results showed that the expression of genes related to SA and JA/ET pathway in tomato was significantly up-regulated after the synergistic using of Saisentong and B.velezensis B-001 when the pathogen invaded tomato compared with the single fungicide or agent treatment groups,indicating that the synergistic use of fungicides and agents could promote the expression of tomato resistance genes and induce SAR and ISR resistance in tomato.The above results showed that B.velezensis B-001 was a bacterium with good biocontrol ability,which could self-repair and adapt to chemical stress,resulting in compatibility characteristics.Saisentong could promote the colonization ability of biocontrol bacteria in tomato after synergistic using bacteria B-001.When encountering the attack of Ralstonia solanacearum,it occupied an ecological niche to prevent further attack of pathogenic bacteria.Otherwise,the synergistic use of Saisentong and biocontrol bacteria B-001 could improve tomato self-induced resistance and systemic resistance,and enhanced the level of resistance to pathogenic microorganisms.The above research on the synergistic mechanism of Saisentong and B.velezensis B-001 provided a theoretical basis for the synergistic use of biological pesticides and chemical pesticides to control plant diseases.