| Pseudomonas protegens SN15-2 which was isolated from tomato rhizosphere soil is a plant rhizosphere growth-promoting bacterium with great biocontrol potential Previous studies have shown that SN15-2 has a good antagonistic activity on various plant diseases such as tomato bacterial wilt,and its antagonistic mechanisms are mainly through the rhizosphere colonization;producing a variety of antibiotics and secondary metabolites;the secretion of siderophores and the induction of systemic resistance in plants,but research has found that its ability to resist external stress environments is poor so it is greatly consumed during the processing and production and extremely susceptible to inactivation.Therefore,it is crucial to study the genes and pathways related with biocontrol and stress resistance for the practical application of Pseudomonas protegens SN15-2.Since SN15-2 has a strong rhizosphere colonization ability in prevention and control of plant diseases,and the motility of the strain is closely related to the plant rhizosphere colonization ability,we have constructed a mutant strain ?kinB by swimming and swarming plates,biofilm formation assay,transmission electron microscope observation,plate antagonism test and chemotaxis assay were used to detect the motility(swimming and swarmming)ability,biofilm formation ability,flagella formation,antagonistic ability to plant pathogens and chemotactic ability to plant roots,stems and leaves.The results showed that the deletion of the kinB gene in Pseudomonas protegens SN15-2 increased the motility of the bacteria,and the diameter of swimming and swarmming increased by 10.3 and 9.6 mm,respectively;it impaired the ability of the strain to form biofilms,the position and numbers of flagella and the antagonistic ability to pathogen have no significant effect;the chemotactic ability of the strain to roots,stems and leaves were improved,especially to roots and reached to 2.88×109 CFU/mL while it was 5.47 × 108 CFU/mL for wildtype.In view of the poor resistance of SN15-2 in the preparation process,we constructed the deletion strains ?betA and ?betB related with stress resistance.Firstly,we screened the most appropriate choline concentration that can improve the survival rate under high osmotic stress,and then measured the growth curve of SN15-2 and mutant strains ?betA,?betB under hyperosmotic stress with choline addition,detecting the accumulation of celluar betaine through H-NMR qualitative and HPLC quantitative analysis and the expression of genes betA and betB under different treatments through qRT-PCR quantitative analysis.Finally,damage index assay(heat shock:50?,4min;cold stress:-20?,1h)were conducted between wildtype and mutant strains under different treatments.The results showed that the bacterial content of SN15-2 under hyperosmotic stress was 2.93 × 1010 CFU/mL.When the choline concentration was 8 mM,the bacterial content could reach to 1.03 × 1011 CFU/mL,which significantly improved survival rate under hyperosmotic stress.Adding a certain concentration of choline under hyperosmotic stress can effectively increase the growth of SN15-2,and also improve the growth of mutant strain ?betA,but it is far less than that of wildtype.However,choline inhibited the growth of ?betB under hyperosmotic stress.In the presence of the precursor choline,hyperosmotic stress can stimulate the accumulation of betaine(5.54 g/L)in SN15-2 greatly,while the content of betaine both in ?betA(3.44 g/L)and ?betB(2.68 g/L)were all significantly lower than that of wildtype.Choline alone could not induce the expression of betA and betB;hypertonic culture could not induce the expression of betA,but significantly induced the expression of betB;the combination of hypertonic culture and choline can significantly induce the expression of genes betA and betB,increased by 12-fold and 26-fold respectively compared with the control,and hyperosmotic stress combined with choline can significantly reduce the damage index of SN15-2 under heat shock and cold stress,decreased by 1.01 and 0.54 compared with control,respectively,while the damage index of two mutants were significantly higher than wildtype.This study successfully used the principle of homologous recombination to knock out three genes kinB?betA and betB seamlessly which were related with the biocontrol and stress resistance in Pseudomonas protegens SN15-2.Meanwhile,it revealed that kinB could regulate the ability of motility;biofilm formation and chemotaxis to plants and the gene betA and betB are two crucial genes in choline-betaine pathway in SN15-2.BetB seems to be more important than betA in choline-betaine pathway and hyperosmotic stress combined with choline could significantly increase the stress resistance of SN15-2.It laid a theoretical foundation for further study of the biological control and resistance mechanism,and provided techniques and methods for the practical application in Pseudomonas protegens SN15-2. |
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