Screening Of Bacillus Strains Against Tomato Bacterial Wilt And Research On Biocontrol Efficiency

Bacterial wilt is a very serious soil-borne disease caused by Ralstonia solanacearum.In 2012,the bacterial wilt caused by R.solanacearum has become the second largest bacterial disease,and the damage is becoming more serious.According to statistics,R.solanacearum can infect 54 families and more than 250 plants,including the important economic crops such as tomatoes and potatoes.It is very difficult to control.There are many pathogenic factors in R.solanacearum,including the type III effectors secreted by the T3 SS,the cell wall degrading enzymes secreted by the T2 SS,and the extracellular polysaccharide.The T3 Es can inject into the plants and interact with the cells and inhibit the host's immune defense.R.solanacearum can secrete cell wall degrading enzymes to help R.solanacearum infect host cells.R.solanacearum secretes extracellular polysaccharide,which can block the plant's ducts and sieve tubes and eventually cause the plant to wither and die.In recent years,the incidence of bacterial wilt has rapidly expanded,which makes the prevention of bacterial wilt disease more and more important.Bacillus is an important biocontrol bacteria that can be used to control a variety of plant diseases.For example,Bacillus subtilis can be used to control diseases such as rice sheath blight,rice panicle neck blight and bacterial wilt.They have been reported to have a variety of functions,such as increasing crop resistance,nitrogen fixation and promoting plant growth.Bacillus megaterium has the good effect on degrading organic phosphorus in the soil.In addition,Bacillus has various physiological properties such as heat resistance,acid and alkali resistance and salt tolerance,they are advantageous for storage and transportation,and they are getting more and more attention.This project aims to control tomato bacterial wilt,and we screened the Bacillus against R.solanacearum based on the abundant Bacillus in our laboratory.Next we obtained 270 strains against R.solanacearum from 1428 strains,and measured the antagonistic activities.Subsequently,we found 16 strains that can control tomato bacterial wilts by the pot experiment,and the biocontrol efficiency of 2014WK-N25,2014WK-N18,2014WK-N14 and 2014WK-N60 were 56.55%,55.57%,55.80% and 58.90% respectively.Then,we analyzed their 16 S r RNA sequence,the Bacillus 2014WK-N25,2014WK-N14,2014WK-N18 and 2014WK-N60 were identified as Bacillus velezensis.Next,we tested the effect of promoting tomato.Bacillus 2014WK-N14 was superior to other experimental groups in promoting the growth of tomato roots.Finally,we combined these strains according to the biocontrol efficiency,the type of the strains,the biological characteristics and growth-promoting factors.It was found that the combination of six of biocontrol efficiency was obvious,and the biocontrol efficiency reached 58.57%.Among the above 16 strains,2014LJL-54 is a Bacillus thuringiensis,and we have obtained its genome sequence.A complete antibiotic gene cluster was found in the strain2014LJL-54 through the bioinformatics prediction.We found that the gene cluster contains three modular core synthetic genes and other modified genes and regulatory genes.Then we compared the gene cluster sequence with the reported gene clusters sequence,we found that the high homology gene clusters in evolutionary relationship were nostophycin and pelgipeptin,and the sequence coverage reached 71% and 72%,respectively,the sequence similarity reached 43% and 38%,respectively,which suggested that the synthetic product may be a novel non-ribosomal peptide.Subsequently,we tested the different culture conditions of the activity against R.solanacearum.Finally,we found that the activity with NB solid medium to cultivate and extracting with ethyl acetate was high.Then we analysised the crude extract by HPLC: we found that the collection with the retention time of 4.5min-6.5min had obvious antagonistic activity against R.solanacearum with agar diffusion method.In summary,we screened 270 strains with antagonistic activity against R.solanacearum,and then obtained 16 strains,which were resistant to tomato bacterial wilt.Then we combined these strains according to the biocontrol efficiency,the type of the strain,and the biological characteristics and growth-promoting factors.Next we found a new non-ribosomal peptide synthetic gene cluster through the bioinformatics prediction,and the active substances were initially isolated.This project provides the potential biocontrol strains for the control of tomato bacterial wilt.It provides a new controlstrategy for the control of tomato bacterial wilt in the agricultural production of important economic crop tomatoes.