Abstract: 【Objective】 Bacterial fruit blotch (BFB) caused by Acidovorax citrulli (Ac) is one of the most serious diseases in the world. Flagella are always considered as movement organ of bacteria and play an important role in their infection, and they have been reported that they could be controlled by flagellar protein gene fliS. The function of gene fliS in A. citrulli is still unclear. The objective of this study is to investigate the function of gene fliS of A. citrulli ineffecting on flagellum formation and pathogenicity. 【Method】 A set of primes were designed based on the genomic DNA No.1 of A. citrulli wild strain. The upstream and downstream fragments knocked out gene fliS were amplified by PCR, respectively. The gene knockout and complementary vectors were constructed through PCR amplification, recovery, digestion, connection and transformation. The strains with knockout gene fliS and complementation were constructed with the method of triparental hybridization. Morphological characteristics of flagella, pathogenicity, hypersensitive response, motility, quorum sensing, biofilm formation, growth rate, colonial morphology, etc have been tested among the wild type, the mutant and the complementary strains. In addition, the total RNA of the bacteria was extracted, and a real-time quantitative PCR (qRT-PCR) was carried out using glnA as an internal control for normalization. Then the expression of genes, flhD, fliE, fliC, flgK, flgM, fliD and fliA in the wild type, the mutant and the complement strains were compared.【Result】The deletion mutant and complementary strain were obtained successfully by screening of gentamicin resistance and PCR verifying, named as mutant 1-fliS and complementary strain 1-fliShb. The results showed that the deletion mutant had weakened motility and biofilm formation, and the complementary strain was almost recovered. Compared with the wild strain, the pathogenicity of the mutant on watermelon and melon was reduced and the complementary strain was recovered completely. The length of flagellum of the deletion mutant was 1/3-1/4 of that of the wild strain, and the complementary strain almost recovered and it was about 4/5 of that of the wild strain. The wild strain could form typical haloes obviously by bacteria migrating via twitching on NA medium, but the deletion mutant did not form haloes on NA medium and the complementary strain recovered partially. The growth rate of deletion mutant was slower than the wild strain and the complementary strain was not recovered. There were no difference among the wild, mutant and complement strains on hypersensitive response and quorum sensing. The results of qRT-PCR showed that in the mutant the expressions of gene flhD decreased obviously compared with the wild strain. In addition, the expressions of fliE, fliC and flgK increased obviously, the expressions of flgM and fliD increased slightly, the expressions of fliA was constant compared with the wild strain, the expressions of genes flhD, fliE and fliC recovered partially in complementary strain and, the expressions of flgK, flgM and fliD did not recover. 【Conclusion】 The flagellar gene fliS could regulate the flagellum formation, motility, biofilm formation, growth rate, colony morphology and pathogenicity of A. citrulli.

Key words: Acidovorax citrulli, bacterial fruit blotch; fliS, biological characteristics