Submitted to: International Congress of Plant Pathology Abstracts and Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: January 31, 2003
Publication Date: February 1, 2003
Citation: Mills, D., Armstrong, D., Azevedo, M.D., Banowetz, G.M. 2003. A novel bioherbicide for control of grassy weeds. International Congress of Plant Pathology Abstracts and Proceedings. p. 213-219. Interpretive Summary: Bacteria that produce compounds that inhibit the germination of the seeds of certain weeds were isolated from soils in western Oregon. The weeds that are inhibited include serious pests in grass seed and wheat production including annual bluegrass, cheatgrass, and jointed goatgrass. The compounds produced by these bacteria only inhibit seed germination; they do not affect the vegetative growth of plants after the seed is germinated. We isolated three genes that are involved in the production or secretion of the seed germination inhibiting compounds and showed that one is a previously undescribed sequence. We also conducted characterization studies on the substance that is responsible for the inhibition of germination. These characterization studies will facilitate the identification and eventual synthesis of the compound, essential steps in transfer of this technology to farmers.
Technical Abstract: Pseudomonas spp. were isolated from the rhizosphere of Poa, Triticale, Triticum, Hordeum, and Lolium species. These bacterial isolates were initially screened for the ability of live cultures to cause stunting of the roots and shoots of young seedlings of the grassy weed known as annual bluegrass (Poa annua). Isolates selected in this manner were evaluated further to determine whether live cultures arrested the germination of P. annua seeds.. Twelve isolates were active in this test, and culture filtrates prepared from these isolates were also active in arresting P. annua seed germination.. When culture filtrates of these twelve isolates were diluted to 25% of their original concentration, filtrates from five isolates still provided complete arrest of germination . We concluded that these isolates produced and secreted a putative Germination-Arrest Factor (GAF). Five of the most active isolates (WH6, E34, AD31, AH4 and WH19), identified as Pseudomonas fluorescens or P. putida with fatty acid methyl ester analysis, were selected for further study. Bacteria-free culture filtrates of P. fluorescens isolates WH6 and E34 irreversibly arrest the germination of seeds of P. annua at a stage immediately following emergence of the coleorhiza and plumule. Successive dilutions of the culture filtrates elicit a decreasing array of developmental disturbances that can be scored and used to estimate the relative concentration of GAF responsible for these effects. GAF was shown to be both developmentally- and species-specific. Foliar applications of GAF extracts or root immersion in GAF solutions had little or no effect on growth at later stages of seedling development. The effect of GAF treatment on seed germination is irreversible in a relatively short period of time with permanent arrest achieved in less than 24 hours. The species-specificity of the respective GAF activities present in culture filtrates from isolates WH6 and E34 were evaluated in tests of the seeds from 14 species of graminacious weeds and crop plants. The GAF activity from both isolates arrested the germination of seeds of Aegilops cylindrica (jointed goatgrass), Bromus tectorum (downey brome or cheat grass), Vulpia myuros (rattail fescue), and six perennial and annual species of Poa. Seed germination was arrested in tall fescue and perennial ryegrass regardless of whether the grass cultivars used were infected with the fungal endophytes Neotyphodium coenophialum or N. loliae (e.g. cultivars Titan and Cutter) or free of these endophytes (e.g. cultivars A.U. Triumph and Linn). Seed germination of spring wheat, barley, maize, clover and Arabidopsis was unaffected by GAF treatment. The discovery of genes that control production or secretion of GAF was accomplished by transposon mutagenesis. Transposon Tn5, which confers tetracycline resistance, was introduced into P. fluorescence wild-type strain WH6 on plasmid pUTmini-Tn5gfp (Tn5gfp). Of 1,214 tetracycline-resistant transformants screened, three lacked GAF activity in culture filtrates. Fragments containing Tn5 were cloned from each of the three mutants and used as molecular probes to identify wild-type fragments by sequence homology. These wild-type fragments were cloned into pBlueScript for sequencing and into pME4510 for genetic complementation studies to identify genes that would restore GAF activity in the respective Tn5 mutants. The three Tn5 mutants, designated gaf1, gaf2 and gaf3, were complemented with wild-type DNA fragments. The nucleotide sequences of all three genes have varying degrees of homology with genes already sequenced and entered into the genome data bases, and their putative activities can be tentatively inferred. The complemented mutants exhibit restored GAF activity, establishing that the DNA sequences in question actually code for products essential for the expression of GAF activity in the bacterial culture filtrate. GAF produced by strain WH6 is a hydrophilic molecule that is insoluble, or sparingly soluble, in all organic solvents tested to date. It has a molecular weight that is less than 3,000 daltons and it contains at least one ionizable group. GAF activity associated with culture filtrates appears to be stable when stored under sterile conditions at 4oC or at room temperature. GAF activity is not measurably affected by heating to temperatures up to 55oC for periods of at least 1 hour, but some loss of activity is evident at 75oC, and activity is destroyed in boiling water (100oC) or by autoclaving. GAF activity from WH6 culture filtrates is not affected by exposure to acid pH (down to pH 2) for periods of up to 3 hours and activity does not appear to be affected by exposure to weakly alkaline pH (up to pH 9) for 3 hours, but exposure to more extreme alkaline pH values (pH 11 and above) results in some lost of activity. GAF reacts with ninhydrin, and it can be visualized as a ninhydrin-positive band that chromatographs with characteristic Rf values in defined thin-layer chromatography systems. This band is produced by five? strains that exhibit GAF activity. Positive identification of GAF as the compound responsible for the specific ninhydrin-positive band in question has been accomplished by mutational genetic analysis. gaf1, gaf2 and gaf3 mutants fail to produce the band defined by thin layer chromatography, while the band is restored in each mutant transformed with the respective wild type gene.