Screening And Fuction Analysis Of Pathogencity-Related Genes In Acidovorax Citrilli

Bacterial fruit blotch (BFB) caused by Acidovorax citrulli mainly damages the watermelon and melon fruit production all over the world. It may seriously harm the leaves and fruits, which is one of international stipulation quarantine plant diseases. So far, relatively little has been known on its pathogenicity. This study primarily explored the pathogenicity-related genes of Ac through genetic methods.The 1468 mutants were transplanted to culture media with 50μg/mL kanamycin and 100μg/mL rifampicin from mutant library of A. citrulli, which stored in minus 80 degree. After two-round inoculation on susceptible watermelon,215 mutants were identified to be complete or partial loss of virulence to host plant. Observing all the mutants cultured in NA and KB without antibiotics, the color of 131 mutants and the morphological characteristics of 160 mutants different from that of wild type strains were screened out respectively. According to the statistics,83.5% of the mutants transformed their color and 85.5% of mutants altered their shape from the 127 mutants which lost virulence completely. However, the another 88 mutants which pathogenicity were weakened were not obviously changed their color or shape. The 50 mutants lost their virulence completely or partially and changed their color or shape were chosen for melon seeding experiment. From the result the 16 out of 50 mutants were chosen further testing on watermelon with friction inoculation, then 11 mutants were finally screened out. By using subclone techonology, transposon insertion sites were identified and 9 related genes were got.The 5 complement strains were constructed by three hybrid technology. The defective genes were as following:transcriptional regulator AraC family, HrpB, hypothetical protein, DNA-3-methyladenine glycosylase I, L-threonine aldolase and pilus retraction ATPase PilT. Hypersensitive response(HR) in nonhost tobacco, pathogenicity in host, cell morphology, color visualize, growth ability, quorum sensing signal, biofilm formation and motility were determined among the wild type strains A13 and No.3, the mutants (195、462、944、X484、X522) and the complement strains (195hb、462hb、944hb、X484hb、X522hb). The results showed that 195hb generally recoverd its HR, pathogenity and biofilm formation, and strain X522hb only regained partial virulence, and strain 944hb renewed its motility. While the rest of the complement strains did not return to normal. It is indicated that HrpB and PUT are pathogenicity-related genes of Ac.