|Kang, Beom Ryong|
|Kim, Kil Yong|
|Kim, Young Cheol|
Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/28/2006
Publication Date: 1/11/2007
Citation: Krishnan, H.B., Kang, B., Krishnan, A.H., Kim, K., Kim, Y. 2007. Rhizobium etli usda9032 engineered to produce a phenazine antibiotic inhibits the growth of fungal pathogens but is impaired in symbiotic performance. Applied and Environmental Microbiology. 73:327-330. Interpretive Summary: The growing use of chemical fertilizers and pesticides in agricultural production has become a concern with respect to their impact on human health and the environment. Growing health awareness among the general public is encouraging the production of pesticide free food crops. Plant growth-promoting bacteria (PGPB) have been shown to play a major role in controlling plant pathogens and improving the soil health. Rhizobia are abundant in soils of both temperate and tropical soils and are intimately associated with legume roots. Because of their ubiquitous occurrence and rhizosphere competence, we have evaluated the use of rhizobia as potential biocontrol agents of plant diseases. In this study, we have engineered rhizobia to produce antibiotics such as phenazine. Phenazine producing rhizobia was able to inhibit fungal growth but was impaired in their symbiotic ability with legumes. Information obtained from this study demonstrates that phenazine-producing soil bacteria have potential as biocontrol agents. Utilization of this potential could reduce the use of undesirable synthetic chemicals and promote maximum yield of important vegetable crops.
Technical Abstract: Phenazine antibiotics produced by Pseudomonas spp. play a major role in preventing various plant diseases. In this study, the phenazine biosynthesis locus of P. chlororaphis O6, a plant growth-promoting rhizobacteria (PGPR), was introduced into several symbiotic bacteria belonging to the family Rhizobiace. Transconjugants of Rhizobium etli USDA9032 and R. leguminosarum USDA2370 harboring the phz locus of P. chlororaphis O6 produced yellow pigmentation. When R. etli carrying the phz locus was grown in broth culture, the appearance of yellow pigmentation was first observed during late exponential growth and intensified during stationary growth. Northern blot analysis revealed phzABCD transcripts in R. etli harboring the phz locus during the stationary growth. UV-visible absorption spectra of the yellow pigment exhibited a characteristic peak at 365 nm. Analysis of concentrated cell-free culture supernatants by thin-layer chromatography revealed the production of phenazine by R. etli carrying the phz locus. Production of phenazine by R. etli had a negative effect on the growth of the rhizobia. Phenazine-producing R. etli was able to inhibit the growth of Botrytis cinerea and Fusarium oxysporum in vitro. Black bean inoculated with phenazine-producing R. etli produced brownish fix nodules and plants appeared nitrogen-starved as reflected by leaf chlorosis. Light microscopy of bean nodules initiated by the phenazine-producing strain revealed cells that were mostly vacuolated which contrasted to the bacteroid filled wild-type R. etli elicited nodules. Transmission electron microscope observation of nodules formed by phenazine-producing R. etli revealed the presence of disintegrating bacteroids containing prominent polyhydroxybutryrate inclusions enclosed in sac-like vacuolar structures. The cellular integrity was lost as evidenced by the presence of numerous dilated tubular structures and mitochondria of various sizes and shapes. The observed ultrastructural changes seen in the nodules are presumably caused by the production of phenazine antibiotic which are known to undergo oxidation-reduction transformations resulting in the accumulation of damaging superoxide radicals.