Identification of Pseudomonas aeruginosa Mutants Unable to Convert Ricinoleic Acid into 7,10,12-Trihydroxy-8(e)-Octadecenoic Acid (TOD) and a Survey of the Biological Activity Of TOD

Abstract

Rice blast, caused by Magnaporthe grisea, is a devastating problem in all rice producing areas of the world and is extremely difficult to prevent and/or to control. Therefore, it is critical to develop new methods to combat M. grisea which is estimated to cause the loss of enough rice to feed 60 million people each year. The gram-negative bacterium Pseudomonas aeruginosa has the ability to convert ricinoleic acid into 7,10,12-trihydroxy-8(E)-octadecenoic acid (TOD). Kuo et al. (2001) found TOD to have antifungal activity against Magnaporthe grisea, Monilinia fructicola, Phytophthora infestans, and Rhizoctonia solani. The long-term goal of this research project is to develop an efficient process for improving production of TOD from P. aeruginosa to mass-produce an economically competitive and environmentally friendly biological crop protection agent. As an initial step towards this goal, a genetic study was undertaken to identify the genes that are required for production of TOD in P. aeruginosa. Using transposon mutagenesis, Jessica Cofield isolated 15,168 independent insertion mutants. From initial genetic screens, 107 insertion mutants that appeared to be unable to convert ricinoleic acid into TOD were identified. In addition, a study was undertaken to assess the antimicrobial activity of TOD. Assays using 96 and 12-well microtiter plates were developed and utilized for the in vitro screening of 23 species of fungi, 17 species of bacteria, and 2 species of nematodes for sensitivity toward TOD. In the 96-well microtiter plate assay, M. grisea’s growth was inhibited by TOD at 150µg/ml. The additional 22 species of fungi screened were not inhibited by TOD at this concentration. Of the 17 bacterial species screened, growth of 15 were significantly inhibited by TOD compared to the ethanol control. The average zone of inhibition ranged from 1.50 cm to 2.01cm. Mobility and morphological development of the plant pathogenic nematodes Rotylenchulus reniformis and Meloidogyne incognita was not affected by TOD. The ability of these nematodes to infect cotton roots was also unaffected by TOD compared to the untreated control. Therefore, the antifungal activity of TOD appears to be specific against M. grisea although the data suggest a wider antibacterial activity. These data will be instrumental in assessing the efficacy of TOD in agricultural applications.