| Plant Growth Promoting Bacteria(PGPB)is an important resource for biological control of plant diseases,and it is of great siginificance to reaserch the antimicrobial activity,environmental tolerance and the related genes for the mechainism of diseases prevention and growth promoting.In this study,the strain D-7 with highly antagonistic activity was obtained using the indicator pathogens from the rhizosphere soil of soybean.Based on the analysis of its species and biological characteristic,a random mutation library was constructed and analysis the biological characteristic and environmental tolerance of the wild-type strain and the mutant.The main resesrch results obtained are as follows:1.Screening of antagonistic strains and analysis of antagonistic activity.Fusarium oxysporum and Ralstonia solanacearum were used as indicator pathogens to screen an antagonistic strain which named D-7.The strain D-7 had a broad-spectrum of antimicrobial activity such as pathogenic fungi,bacteria,and oomycetes.The inhibition zone radius of D-7againt Pythium myriotylum and R.solanacearum were more than 1.5cm,while the inhibition zone radius of strain D-7 againt F.oxysporum,F.graminearum and Rhizoctonia solani were less than 1cm.2.Identification of strain D-7.Based on the phylogenetic analysis of the RecA gene in strain D-7,the strain D-7 is located on the same branch as Burkholderia cenocepacia DQ664181 and B.cenocepacia NC008060.1,which indicating that the strain D-7 belongs to B.cenocepacia.The accession number of the RecA gene of strain D-7 in NCBI is MF964231.3.Testing of virulence factors and pathogenicity of strain D-7.The toxicity of strain D-7on plants and animals was analyzed in terms of virulence genes,alfalfa model and onion pathogenicity determination.PCR detection revealed that the strain D-7 did not contain BCESM and cblA genes.Based on the alfalfa and onion pathogenicity tests,indicating the strain D-7 had no pathogenicity to related plants.4.Analysis of antimicrobiol mechanism of strain D-7.In order to understand the genes related to antibacterial substances of the strain D-7,a random mutation library of the strain D-7 was constructed using Tn5 transposon mutagenesis,and 10 mutants which had reducing antimicrobial activity to Fusarium oxysporum were obtained.By plamid rescue and sequenceanalysis,we found that the Tn5 insertion of the 10 mutants were inserted into the 6 genes.The insertion sites of 5 mutants,such as MT1393 and MT720,were located at different sites in the same gene.which encodes glucose-inhibited division protein A,while the insertion sites of the other 5 mutants were located on different gense,and the encoded proteins included glycosyl transferase,type II secretion system protein and so on.In order to analysis the relationship between the mutant genes and the antimicrobiol activity,the mutant MT1393 was selected for complementation analysis.Complementary expriments and the analysis of the antimicrobiol activity of complementary strain showed that the complementary strain MS1393 restored the antifugal activity against F.ocysporum,indicating that the GidA gene is associated with the antifugal activity of strain D-7.5.Analysis of biological characteristics of wild-type strain and mutant.The biofilm phenotypes of the wild-type strain,mutant and complementary strain were observed under the Leica Camera,showing that the the colonies of wile-type strain D-7 and complementary strain MS1393 have smooth surfaces,whereras the colony of mutant MT1393 is not smooth and have protruding points.The relative biofilm production of the three strains were measured,which found that the biofilm yield of MT1393 was significantly reduced in the rich sucrose medium,whereas the MT1393 formed biofilm similar to that formed by the wild-type strain in the presence of glucose,and the complementary strain recoved the ability to form biofilm.Analysis of the EPS produced by the wild-type strain,mutant and complementary strain,we found that whether in glucose-rich or rucrose-rich medium,the EPS produced by MT1393 was more than doubled than that produced by wild-type strain,and the EPS produced by complementary strain decreased to the level of wild-type strain.The motility of each strain was determined by the colony diameter method.After 3 days,the diameter of wile-type strain D-7 on 0.6% NY agar was 1.93 cm,while the diameter of MT1393 was 1.83 cm,and the complementary strain was 1.84 cm,indicating that the swarming ability could not restore to level of the wild-type strain.Wild-type strain D-7 had an average swim ring diameter of 2.35 cm,and mutant MT1393 had an average swim ring diameter of 1.81 cm,while the swimming ability of the complementary strain was 1.93 cm,and it did not recover to the level of the wild-type strain.Environmental tolerance is an important indicator of whether PGPB strains can adapt to complex environments and exert their biological control.The tolerances of the wild-type strain D-7,the mutant MT1393 and the complementary strain MS1393 were tested under 5environmental stresses.In the rich sucrose medium,the reduction of biofilm production leads to the reduced tolerence to high temperature,high NaCl and acid environment,but there was no siginificant differences under the treatment of SDS and UV and the complementary strain MS1393 partially recovered the tolerance under stress conditions.In the constrast,there is no significant differences between MT1393 and wild-type strain D-7 in the rich glucose medium.The data shows that the GidA gene is not only related to the antimicrobial activity of D-7,but also can participate in the biofilm formation and the tolerance to the environment. |
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