STM of a biocontrol Pseudomonas pseudoalcaligenes strain to identify genesinvolved in the interaction with Rosellinia necatrix

Abstract: To identify bacterial genes that could contribute to biological control of thephytopathogenic fungus Rosellinia necatrix, 1,104 mini-Tn5-tagged insertion mutants of thebiocontrol strain Pseudomonas pseudoalcaligenes AVO110 were screened in pools of ≤ 48 usingSignature Tagged Mutagenesis (STM). One hundred and twenty nine mutants were not recoveredfrom media containing R. necatrix exudates (BM-RE medium) 48 hours after inoculation,suggesting that transposon insertion in these mutants affected genes required for growth and/orsurvival in R. necatrix exudates. In a second round of STM screening, the number of mutants notrecovered from BM-RE was reduced to 25 mutants. To validate the screening results, these 25mutants were tested in BM-RE medium for competition with the wild type strain AVO110; sevenof them, were less competitive or resistant to R. necatrix exudates than the wild type strain andshowed a single insertion of the transposon. DNA fragments flanking the Tn5 insertions in theseseven mutants were cloned, sequenced and analysed using BLAST software. These genes arepredicted to be involved in central metabolism, regulation and bacterial protection againstenvironmental signals.