Mechanism Research Of Bacillus On Growth Promoting And Preventing Tomato Yellow Leaf Curl Disease

Bacillus spp is often employed as important biocontrol agent.It is not clear whether the regulation of Bacillus spp on soil microbial community is related to its biocontrol effect.In recent years,there have been outbreaks of tomato yellow leaf curl virus?TYLCV?with increasing severity in many countries,and the biocontrol effect of Bacillus on tomato yellow leaf curl disease?TYLCD?has not been reported.In this study,11 strains of Bacillus preserved in the laboratory were screened for potential biocontrol Bacillus strains with excellent growth-promoting and antagonistic effects by petri dish germination and antagonism test,and the excellent Bacillus strains were identified by morphological,physiological,biochemical and molecular biology methords.The growth pormoting effects of potential biocontrol Bacillus strains on tomato growth and the soil rhizosphere microorganisms were determined by Petri dish germination test and pot experiments.At the same time,we explored the biocontrol effect and mechanism of Bacillus spp.strains on yellow leaf curl disease?TYLCV?.The main results are as follows:?1?Four strains of Bacillus spp.Bs10,Ba13,Ba12 and Bl10 with obvious effect of promoting and antagonizing were screened from 11 Bacillus strains by seed growth-promoting test and antagonistic test in the dish.?1?Together,Ba13 was the best excellent strain with growth promotion and antagonism characteristics.Ba13 exhibited antagonistic effect on all 13 plant pathogens whose antagonistic effect is most prominent.The Ba13-promoting effect is second only to Bs10,and the simple vigour index of cabbage and tomato treated with culture filtrate of Ba13 increased by 26.3%and 40.8%,respectively.?2?The strain Bs10 has no antagonistic effect on the target pathogens,but it has the most significant effect on the radicle and hypocotyls growth of cabbage and tomato seeds.The simple vigour index of cabbage and tomato treated with culture filtrate of Bs10 increased by 29.1%and 43.0%,respectively.?3?Ba12 and Bl10also showed plant growth-promoting and antagonistic effect more or less.Ba12 exhibits antagonistic effects on 10 of 13 plant pathogenic fungi,but Bl10 has no antagonistic effect on pathogenic.Based on the above screening results,Ba13 and Bs10 was the potential superior biocontrol strains for further research.Ba12 and Bl10 are less effective,and corresponding research is conducted.?2?The taxonomic status of strains Bs10,Ba13,Ba12,and Bl10 were determined through 16S rDNA.Bs10 was Bacillus subtilis andthe sequence identity was 99.70%with Bacillus subtilis KCTC 13429?GCA000332645?,Ba13 was Bacillus amyloliquefaci ens RD7-7?CP016913?and the sequence identity was 99.90%with Bacillus amyloliqu efaciens DSM 7?000196735.1?,Bl10 was Bacillus sonorensis and the sequence identit y was 99.81%with Bacillus sonorensis NBRC?AYTN01000016?.The morphology and physiological characteristics of Bs10 and Ba13 were further studied.?3?The effects of Bacillus Bs10,Ba12 and Bl10 on tomato growth and the soil rhizosphere microorganisms were determined by Petri dish germination test and pot experiments.The results showed that,?1?The three Bacillus spp.have a significant effect on the growth of tomato plants.The length,surface area and volume of tomato roots increased increased by104.5%²5.6%,76.2%¹0.4%and 63.3%⁷.9%,respectively.Among them,Bs10 exhibits the most obvious effect of promoting growth,and each indicator has reached a significant level.?2?Bacillus can improve the culturable microbial community structure in tomato rhizosphere.The B/F of tomato rhizosphere and rhizoplane increased by 213.0%and 271.0%,115.0%and236.0%,and 54.0%and 106.0%respectively,after inoculation with Bs10,Ba12 and Bl10.Specially,increase in B/F indicates that the improvement of soil disease resistance?3?The numbers of the dominant bacteria and fungi were changed.The dominant bacteria such as Bacillus velezensis B₃ increased significantly,and the pathogenic fungi F solani and F oxysporum decreased significantly.Further research found that the dominant bacteria B.methylotrophicus also had a significant promoting effect on pakchoi.These results suggested that the addition of Bs10,Ba12 and Bl10 could improve the micro-ecosystem of the root domain of tomato,which might play an important role in growth promoting and disease prevention of effect on tomato plants.?4?Tomato plants in pots were inoculated with Ba13 followed by natural infection with whiteflies.We measured plant growth,physiological parameters,disease severity,and leaf virus quantity.We also assayed resistance-related gene expression,defense enzyme activity,and rhizosphere microbial community structure.Strain Ba13 lowered the infection rate,disease severity,and leaf virus quantity in tomato plants,with the control rate of 48%⁵2%.Root development and shoot biomass markedly improved in tomato following Ba13 treatment.Ba13elevated expression of pathogenesis-related PR1,PR2,and PR3 genes,and activities of phenylalanine ammonialyase,polyphenol oxidase,peroxidase,?-1,3 glucanase,and chitinase in tomato leaves.Ba13 improved the community structure of cultivable rhizosphere microbes by increasing the bacteria-to-fungi ratio and the number of beneficial bacteria microbes?e.g.,B.methylotrophicus B3?,and decreasing the number of pathogenic fungi?e.g.,Fusarium solani F₁ and F.oxysporum F₂?.Further analysis revealed the potential of a dominant rhizosphere bacterium,B.methylotrophicus B₃,to promote plant growth and improve systemic resistance in tomato.B.amyloliquefaciens Ba13 can enhance plant resistance through direct induction of systemic resistance and also by increasing the number of beneficial microbes in the rhizosphere,thereby achieving control of TYLCD.In summary,4 Bacillus strains Bs10,Ba13,Ba12 and Bl10 with excellent growth-promoting and antagonistic effects were obtained.Bs10,Ba12 and Bl10 had significant growth promoting effects on tomato and improved the microbial community structure of tomato rhizosphere.Ba13 can induce the plant system resistance by directlyaffectinging systemic resistance and increasing the number of beneficial bacteria in the rhizosphere,which led to a common result that resistance to TYLCV.This study provides excellent strains for biological control of plant diseases and presents new insight into the biocontrol mechanism of beneficial microbes.