Colonilization And Induction Of Plant Systemic Resistance By Biocontrol Agent Paenibacillus. Polymyxa SQR-21

In recent years, excessive use of chemical pesticides and germicides caused seriously environmental pollution. As a new, eco-friendly control way, biological control has got more and more attention world-wide since it emerged. Microbial agents, bioorganic fertilizer and other biological control agents have been widely. Bacillus species are important members of biological control agents since they possess several advantages such as antibiotics synthesis, spore formation and induction of plant system resistance. This study studied rhizosphere colonization, induction of plant systemic resistance and mechanism of Paenibacillus polymyxa SQR-21.Plasmid pPZZ84was isolated from wild antagonistic Bacillus pumilus ZZ84and sequenced. We described a simple and fast method to get full nucleotide sequence of plasmid by two-steps PCR. First, rep gene fragment was amplified by a pair of primer designed according to the conservation of rep and sequenced. Second, reverse primers were designed based on rep fragment obtained according to the circle structure of plasmid to got the rest nucleotide sequence of plasmid by PCR. Two fragments were combined to get full nucleotide sequence of plasmid pPZZ84. pPZZ84is6817bp in size with G+C36.7%. There are seven ORFs in pPZZ84. ORF1and ORF2encoded unknown protein respectively. ORF5and ORF7encoded Rap and Rep. There were no proteins homoloues to ORF4and ORF6. There were46copies of plasmid pPZZ84in Bacillus pumilus ZZ84analyzed by real-time PCR.In this paper, Two GFP-tagging vectors pHapⅡ (6838bp) and pHapⅢ (7235bp) have been constructed by the constitutively HapⅡ promoter, Kanamycin resistance gene, rep gene from pUB110and pPZZ84respectively. The pHAPII could be transformed into wild species of Bacillus subtilis, Bacillus pumilus, Bacillus amyloliquefaciens, Paenibacillus polymyxa, But pHapIII could be transformed only into wild species of Bacillus pumilus but not in other Bacillus species. The91%and89%pHAPII could remain in B. subtilus SQR-9, B. pumilus JK-001after growth of96generations. But the pHAPII disappeared completely in the P. polymyxa SQR21after growth of96generations.The SQR21/pHapII strain could be colonized on cucumber rhizosphere soil. The strain colonized on cucumber root surfaces. The number of SQR21/pHapII was about5.85log CFU-g-1root after4days of inoculation, then increased to about6.88log CFU-g-1root, after8days of inoculation. But the number of SQR21/pHapII slightly decreased at a relatively stable number (5.9log CFU·g-1root). The number of SQR21/pHapⅡ in rhizosphere soil was less than on root surface and maintained at a relatively stable level about (4.5log CFU·g-1root) from4days to16days after inoculation. GFP-marked cells also could be detected in roots (3log CFU-g-1root).This study studied biological control agents Paenibacillus polymyxa SQR-21, Bacillus mycoides BmJ and Bacillus mojavensis203-7(203-7) how to induce systemic resistance (ISR) using the cucumber/Glomerella cingulata var. orbiculare pathosystem. The pathogenic spores per mm2of lesion area in cucumber leaves after treated with SQR-21, BmJ and203-7could reduce69%,42%and58%respectively compared with that of control. Biological control agents SQR-21, BmJ and203-7could induce cucumber plants to rapidly express pathogenesis related (PR) genes PR1-1α, PR-3and APOX as measured by mRNA for these genes. PRl-1a was highly expressed at2dpi in cucumber lesion leaves if cucumber treated with SQR-21, BmJ and203-7. But PR1-1a was only less expressed at3dpi in control cucumber. At1,2and3dpi, PR-3were expressed significantly greater in cucumber treated with SQR-21, BmJ and203-7than in control cucumber (P<0.05). BmJ induced cucumber to express PR-3highest, followed by203-7and SQR-21(P<0.05). At1,2and3dpi only203-7treated cucumber had higher levels of APOX (P<0.05) than control.Cucumber treated with BmJ had significantly higher (P=0.05) chitinase activity than other treatments and showed an increase of enzyme activity by96%compared with controls. While chitinase activity in cucumber treated with203-7and SQR-21also increased by65%and72%respectively. The β-glucanase activity in cucumber treated with203-7was found to be163%of controls. Cucumbers induced with SQR-21and BmJ showed β-glucanase activity of118%and127%compared to controls.The effect of antagonistic bacteria on the colonization of in cucumber leaves was studied by real-time PCR method. The results showed that at3dpi DNA concentrations of Glomerella cingulata var. orbiculare in cucumber treated with SQR-21,203-7and BmJ were only1/3,1/2and2/3of that in control cucumber. The DNA concentration of pathogen Glomerella cingulata var. orbiculare increased28fold in control cucumber from3dpi to11dpi, but it was21fold in treated cucumber. The microscope observation showed that the number of appressoria and melanised setae of G. cingulata var. orbiculare in SQR-21treated cucumber were less than in control cucumber, indicating that SQR-21treated cucumber could inhibite the germination of spore and formation in the lesions of cucumber leaves. SQR-21also could induce cucumber to produce phytoalexin and inhibition of quorum sensing of bacteriaAn Arabidopsis thaliana mutant-Botrytis cinerea pathosystem was used to investigate the plant defense pathways activated by SQR-21. The incidence of disease reached80%in control, but the incidence of disease was only65%and54%in SQR-21-and BmJ-treated Arabidopsis thaliana, respectively. SQR-21was able to significantly decrease the lesions size in the leaves of arabidopsis thaliana. SQR-21could induce JA relative PR genes expression in the wide type Arabidopsis thaliana, but SQR-21did not decrease disease severity and induced PR genes expression in Arabidopsis thaliana mutant jar1-1, indicating that SQR-21induced plant systemic resistance was salicylic acid independent but jasmonic acid dependent.