Dissertation
Dissertation > Agricultural Sciences > Plant Protection > A variety of control methods > Biological control > The use of microbial pathogens

Analysis of Gene Ycze in Bacillus and Biocontrol Efficacy of Genetically Engineered Bacillus Expressed Harpin on Rice Bacterial Leaf Blight

Author TianDaWei
Tutor GaoXueWen
School Nanjing Agricultural College
Course Plant Pathology
Keywords Bacillus yczE swarming Harpin protein genetic engineering bacteria biocontrol
CLC S476.1
Type Master's thesis
Year 2011
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The genus Bacillus is comprised of Gram-positive, spore-forming, and rod-shaped organisms with an aerobic or facultatively anaerobic metabolism, are ubiquitous in many environments. They are well-known and extensively-used beneficial rhizobacteria for biocontrol of plant diseases. Their interest arises from their ability to generate a broad array of bioactive metabolites among which three families of cyclic lipopeptides (CLPs). These CLPs display a huge diversity of structures and physico-chemical and biological properties which probably account for an important part of the biocontrol potential of the producing strains. Beside their antimicrobial properties, they are also involved in colonization and motility as well as in the systemic stimulation of immune system of the host plant. We summarize here the current knowledge of CLPs activities and focus on the recent findings in the context of biocontrol.In this article, OKB105ΔyczE and FZB42ΔyczE were constructed using the method of double-crossing homologous recombination to study the function of yczE gene. Lipopepetides have important activities of hemolysis and bacteriostasis. Hemolysis and plates antagonistic of Bacillus on the Xanthomonas oryzae pv. Oryzae revealed that these two mutations have no obvious differences with their original strains, it showed that the absent of yczE had little effect on the synthesis of lipopepetides. At the same time, the results also showed that the tobacoo dealt with four Bacillus grow better compared with the control one, especially FZB42 has the best effectiveness, but two mutations have no significant differences against their original ones, yczE doesn’t have the direct influence on growth-promoting. Then in Bacillus, what role does this conserved gene play? A series of phenotype experiments were done and we found that yczE related to swarming inBacillus.Swarming plays essential role in the synthesis of biofilm and colonization in the surface of roots. Activity detection of swarming revealed that OKB105 and OKB105ΔyczE showed similar phenotype. However, yczE gene affects swarming of FZB42 remarkably. The radius of FZB42 andB42ΔyczE on swarming were measured on time.FZB42 swarming more rapidly than FZB42ΔyczE, differences were firstly observed in 4 h and obvious from 6 h. Meanwhile, outermost layer of swarming were observed in the electron microscope, cells of FZB42 arrayed more closely and have much more flagella compared with FZB42ΔyczE. Two strains of Bacillus on the ability of biofilm formation revealed that FZB42 has more dense、strong and stable biofilm than FZB42ΔyczE.In this research, we analyzed molecular mechanism of swarming using qRT-PCR. Flagella plays essential role in swarming,so we chose swrA、flgB、efp and cheB releated to the synthesis of flagella.During strain OKB105, beacause of the absence of swrA, it’s ability of swarming weak obvious compared with FZB42.Analyzing swarming in 6h, with yczE gene mutated, swrA、flgB and efp were obviously down regulated 2.58、6.176 and 12.832, respectively. YczE effect swarming indirectly by regulating expression of these three genes on mRNA.In this study, Bacillus OKB 105、FZB42 and their genetically engineered bacteria OKBHF and FZBHarpin, which expressed and secreted Harpin proteins were used to check biocontrol efficacy on rice bacterial leaf blight. In greenhouse, controlling effect of all the four strains OKB105、FZB42、OKBHF and FZBHarpin were recorded as 31.7%、23.6%、43.2% and 56.7%, respectively; while 29.8%、21.8%、40.4% and 43.1% in the field, respectively. Genetically engineered bacteria showed the higher biocontrol efficacy compared with their original strains, especially FZBHarpin. In addition, they owned the ability of growth-promotion having the same potential as biocontrol, and expressed much more obvious effectiveness. On promoting rice growth, FZBHarpin increased the plant length and reached up to 15.86%. The results also revealed the yield of rice dealt with OKBHF reached up to 8869kg·ha-1 with 14% increment. Thus, genetically engineered bacteria had higher ability on biocontrol efficacy and growth-promotion compared with the original once, which would have a prospective application in agriculture.

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