Diverse environmental bacteria displaying activity against Phakopsora pachyrhizi, the cause of soybean rust.

The management of soybean rust (SBR) caused by the obligate fungus Phakopsora pachyrhizi mostly relies on the use of synthetic fungicides, especially in areas where the disease inflicts serious yield losses. The reliance on synthetic fungicides to manage this disease has resulted in resistance of P. pachyrhizi populations to most fungicides. In this study, bacteria isolated from diverse environments were evaluated for their biocontrol potential against P. pachyrhizi using soybean detached-leaf method and on-plant in the growth chamber, greenhouse, and field. Among 998 bacterial isolates evaluated using the detached-leaf method; 58% were isolated from plant-related materials, 27% from soil, 10% from insects, and 5% from other environments. Of the isolates screened, 73 were active (they had ⪖ 75% rust reduction) with an active rate of 7.3%. From the active isolates, 65 isolates were re-tested on-plant in the growth chamber for activity confirmation. In the confirmation test, 49 bacteria isolated from plant-related materials maintained their activity with a confirmation rate of 75%. The majority of bacteria with confirmed activity belonged to the taxonomic classes Bacilli and Gammaproteobacteria (70%). Active isolates were prioritized for greenhouse and field testing based on activity in the initial screen and confirmation test. Six bacterial isolates AFS000009 (Pseudomonas_E chlororaphis), AFS032321 (Bacillus subtilis), AFS042929 (Bacillus_C megaterium), AFS065981 (Bacillus_X simplex_A), AFS090698 (Bacillus_A thuringiensis_S), and AFS097295 (Bacillus_A toyonensis) were selected from those bacteria that maintained activity in the confirmation test and were evaluated in the greenhouse, and five among them were evaluated in the field. From the Alabama field evaluation, all bacterial isolates reduced rust infection as well as azoxystrobin (Quadris® at 0.3 L/ha) used as the fungicide control (P > 0.05). Moreover, the scanning electron micrographs demonstrated evidence of antagonistic activity of AFS000009 and AFS032321 against P. pachyrhizi urediniospores. Bacterial isolates that consistently showed activity comparable to that of azoxystrobin can be improved through fermentation and formulation optimization, developed, and deployed. These bacteria strains would provide a valuable alternative to the synthetic fungicides and could play a useful role in integrated disease management programs for this disease.

Characterization and plant expression of a glyphosate-tolerant enolpyruvylshikimate phosphate synthase.

BACKGROUND: Glyphosate tolerance is a dominant trait in modern biotech crops. RESULTS: A gene encoding a glyphosate-tolerant EPSP synthase (aroA(1398)) from bacterial strain ATX1398 was cloned and characterized. The protein is initiated at a GTG translational start codon to produce a protein that provides robust glyphosate resistance in Escherichia coli (Mig) Cast & Chalm. The aroA(1398) protein was expressed and purified from E. coli, and key kinetic values were determined (K(i) = 161 microM; K(m)(PEP) = 11.3 microM; k(cat) = 28.3 s(-1)). The full-length enzyme is 800-fold more resistant to glyphosate than the maize EPSP synthase while retaining high affinity for the substrate phosphoenol pyruvate. To evaluate further the potential of aroA(1398), transgenic maize events expressing the aroA(1398) protein were generated. T(0) plants were screened for tolerance to glyphosate sprays at 1.3x commercial spray rates, and T(1) plants were selected that completely resisted glyphosate sprays at 1x, 2x and 4x recommended spray rates in field trials. CONCLUSION: These data suggest that aroA(1398) is a suitable candidate for conferring glyphosate tolerance in transgenic crop plants.

Biocatalytic conversion of avermectin to 4"-oxo-avermectin: characterization of biocatalytically active bacterial strains and of cytochrome p450 monooxygenase enzymes and their genes.

4"-Oxo-avermectin is a key intermediate in the manufacture of the agriculturally important insecticide emamectin benzoate from the natural product avermectin. Seventeen biocatalytically active Streptomyces strains with the ability to oxidize avermectin to 4"-oxo-avermectin in a regioselective manner have been discovered in a screen of 3,334 microorganisms. The enzymes responsible for this oxidation reaction in these biocatalytically active strains were found to be cytochrome P450 monooxygenases (CYPs) and were termed Ema1 to Ema17. The genes for Ema1 to Ema17 have been cloned, sequenced, and compared to reveal a new subfamily of CYPs. Ema1 to Ema16 have been overexpressed in Escherichia coli and purified as His-tagged recombinant proteins, and their basic enzyme kinetic parameters have been determined.

Biocatalytic conversion of avermectin to 4"-oxo-avermectin: heterologous expression of the ema1 cytochrome P450 monooxygenase.

The cytochrome P450 monooxygenase Ema1 from Streptomyces tubercidicus R-922 and its homologs from closely related Streptomyces strains are able to catalyze the regioselective oxidation of avermectin into 4"-oxo-avermectin, a key intermediate in the manufacture of the agriculturally important insecticide emamectin benzoate (V. Jungmann, I. Molnár, P. E. Hammer, D. S. Hill, R. Zirkle, T. G. Buckel, D. Buckel, J. M. Ligon, and J. P. Pachlatko, Appl. Environ. Microbiol. 71:6968-6976, 2005). The gene for Ema1 has been expressed in Streptomyces lividans, Streptomyces avermitilis, and solvent-tolerant Pseudomonas putida strains using different promoters and vectors to provide biocatalytically competent cells. Replacing the extremely rare TTA codon with the more frequent CTG codon to encode Leu4 in Ema1 increased the biocatalytic activities of S. lividans strains producing this enzyme. Ferredoxins and ferredoxin reductases were also cloned from Streptomyces coelicolor and biocatalytic Streptomyces strains and tested in ema1 coexpression systems to optimize the electron transport towards Ema1.

2,5-dialkylresorcinol biosynthesis in Pseudomonas aurantiaca: novel head-to-head condensation of two fatty acid-derived precursors.

2-Hexyl-5-propylresorcinol is the predominant analog of several dialkylresorcinols produced by Pseudomonas aurantiaca (Pseudomonas fluorescens BL915). We isolated and characterized three biosynthetic genes that encode an acyl carrier protein, a beta-ketoacyl-acyl carrier protein synthase III, and a protein of unknown function, all of which collectively allow heterologous production of 2-hexyl-5-propylresorcinol in Escherichia coli. Two regulatory genes exhibiting similarity to members of the AraC family of transcriptional regulators are also present in the identified gene cluster. Based on the deduced functions of the proteins encoded by the gene cluster and the observed incorporation of labeled carbons from octanoic acid into 2-hexyl-5-propylresorcinol, we propose that dialkylresorcinols are derived from medium-chain-length fatty acids by an unusual head-to-head condensation of beta-ketoacyl thioester intermediates. Genomic evidence suggests that there is a similar pathway for the biosynthesis of the flexirubin-type pigments in certain bacteria belonging to the order Cytophagales.