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Phosphoric acid-containing amino acid pesticides, including glyphosate, glufosinate, and bialaphos, have been frequently encountered in human poisoning and suicidal cases, thus a quick and sensitive determination method for these compounds is very important. Herein we report a sensitive and rapid method for the determination of glyphosate, glufosinate, bialaphos and their metabolites, aminomethylphosphonic acid (AMPA) and 3-methylphosphinicopropionic acid (MPPA) in human blood samples. The method involves extraction using a modified Quick Polar Pesticides Extraction (QuPPe) method, cleanup with the PRiME HLB sorbents and detection with ultra performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS). The LODs were 0.02mg/L for glyphosate, glufosinate and AMPA, 0.01mg/L for bialaphos and MPPA, with the analysis time as short as ∼6min. To the best of our knowledge, the LODs are lower and the analysis time is shorter than previous reports on determination of these compounds in blood and serum samples. The method was successfully applied to the detection and quantification of glyphosate and AMPA for blood samples from real forensic cases. This extremely rapid determination method with high sensitivity is of significant importance, particularly for forensic cases where quick determination is vital.

Hairy roots of snapdragon (Antirrhinum ma-jus L.: Scrophulariaceae) induced by a wild-type strain of Agrobacterium rhizogenes were cultured on media containing various concentrations of a phosphinothricin-based herbicide, bialaphos, or plant growth regulators (PGRs). Adventitious shoot regeneration from hairy roots was observed with a low frequency (10%) on half-strength Murashige and Skoog medium. Addition of α-naphthalene-acetic acid in combination with 6-benzylaminopurine, thidiazuron, or zeatin to the medium had no effect on shoot regeneration from hairy roots. Although bialaphos at 0.9 mg l–1 or more was toxic to hairy roots, it significantly increased the shoot regeneration frequency up to 56% at 0.5 mg l–1. In contrast, non-transformed roots and leaves regenerated no shoots on media with or without bialaphos. Regenerated shoots detached from host roots readily developed roots on gellan-gum-solidified medium. Regenerated plants were successfully transferred to the greenhouse, but did not produce seed.

Natural products containing phosphonic or phosphinic acid functionalities often display potent biological activities with applications in medicine and agriculture. The herbicide phosphinothricin-tripeptide (PTT) was the first phosphinate natural product discovered, yet despite numerous studies, questions remain surrounding key transformations required for its biosynthesis. In particular, the enzymology required to convert phosphonoformate to carboxyphosphonoenolpyruvate and the mechanisms underlying phosphorus methylation remain poorly understood. In addition, the model for non-ribosomal peptide synthetase assembly of the intact tripeptide product has undergone numerous revisions that have yet to be experimentally tested. To further investigate the biosynthesis of this unusual natural product, we completely sequenced the PTT biosynthetic locus from Streptomyces hygroscopicus and compared it with the orthologous cluster from Streptomyces viridochromogenes. We also sequenced and analyzed the closely related phosalacine (PAL) biosynthetic locus from Kitasatospora phosalacinea. Using data drawn from the comparative analysis of the PTT and PAL pathways, we also evaluate three related recently discovered phosphonate biosynthetic loci from Streptomyces sviceus, Streptomyces sp. WM6386 and Frankia alni. Our observations address long-standing biosynthetic questions related to PTT and PAL production and suggest that additional members of this pharmacologically important class await discovery.

We constructed two series of Gateway binary vectors, pGWBs and R4pGWBs, possessing the bialaphos resistance gene (bar) as a selection marker for plant transformation. The reporters and tags employed in this system are sGFP, GUS, LUC, EYFP, ECFP, G3GFP, mRFP, TagRFP, 6xHis, FLAG, 3xHA, 4xMyc, 10xMyc, GST, T7 and TAP. Selection of Arabidopsis transformants with BASTA was successfully carried out using both plate-grown and soil-grown seedlings. Transformed rice calli and suspension-cultured tobacco cells were selected on plates containing BASTA or glufosinate-ammonium. These vectors are compatible with existing pGWB and R4pGWB vectors carrying kanamycin and hygromycin B resistance.

We wish to thank the European Commission for the finacial support. Thanks to Moses Mokoena, Rufus Khonoti, Gugulethu Ngwenya and Jeffrey Mathabe for their technical Support.

Transgenic herbicide-resistant snapdragon ( Antirrhinum majus L.: Scrophulariaceae) plants were obtained by co-cultivation of leaf explants with Agrobacterium rhizogenes harboring Ri plasmid and pARK5. The T-DNA region of pARK5 contains bar gene encoding phosphinothricin acetyltransferase under the control of the cauliflower mosaic virus 35S promoter, and nptII gene encoding neomycin phosphotransferase II under the control of nopaline synthase promoter. Transformed hairy roots induced from leaf explants inoculated with A. rhizogenes strain A13 (pARK5) could grow on 1 mg l −1 bialaphos-containing half-strength MS medium, although hairy roots transformed by the wild type strain of A. rhizogenes A13 could not survive at the same concentration of bialaphos. Shoot regeneration from the hairy roots integrating T-DNA of pARK5 was also stimulated at 1 mg l −1 bialaphos. Regenerated shoots readily developed roots on 2 g l −1 gellan gum-solidified half-strength MS medium. The transgenic nature of the plants was confirmed by opine, PCR and Southern analyses. Regenerated plantlets were successfully established in the growth chamber. Acclimatized plants exhibited altered phenotypes known as Ri syndrome such as reduced apical dominance with highly branched stems and short internodes, and also showed resistance to an applied standard level of commercial herbicides.

The antifungal activity of the herbicides bialaphos and glufosinate, the active moiety of bialaphos, was assessed. Bialaphos showed a higher level of in vitro antifungal activity againstRhizoctonia solani, Sclerotinia homoeocarpa, andPythium aphanidermatumthan glufosinate. Glufosinate suppressed the mycelial growth ofR. solaniandS. homoeocarpa, but it had no inhibitory effect onP. aphanidermatumup to the highest concentration in our testing regimes. Various concentrations of bialaphos solutions were applied to transgenic, bialaphos- and glufosinate-resistant creeping bentgrass inoculated with fungal pathogens. Bialaphos applications were able to significantly reduce symptomatic infection byR. solaniandS. homoeocarpaon transgenic plants. Bialaphos significantly inhibitedP. aphanidermatum, but not to the same degree thatR. solaniandS. homoeocarpawere inhibited. These results indicate that bialaphos may provide a means for the simultaneous control of weeds and fungal pathogens in turf areas with transgenic, bialaphos-resistant creeping bentgrass.

An enzymatic activity catalyzing P-methylation of N-acetyldemethylphosphinothricin, a biosynthetic intermediate of the herbicide bialaphos, was detected in a cell extract of Streptomyces hygroscopicus SF-1293, a bialaphos producing organism. The gene coding for this P-methylation enzyme in the bialaphos biosynthetic gene cluster was also expressed in Streptomyces lividans. The methyl donor of the reaction was determined to be methylcobalamin. The P-methylation enzyme utilized both N-acetyldemethylbialaphos and N-acetyldemethylphosphinothricin as substrates.

The biosynthetic step following the phosphoenolpyruvate (PEP) phosphomutase reaction which forms a C-P bond of bialaphos was proven by the identification of phosphonopyruvate (PnPy) and phosphonoacetaldehyde (PnAA) as intermediates in the culture broth of Streptomyces hygroscopicus, a producing organism of bialaphos, and by detection of enzymatic decarboxylation of PnPy to PnAA. Purified PnPy decarboxylase turned out to require thiamine diphosphate and Mg2+ as cofactors. PnPy decarboxylase drives the unfavorable forward reaction to form PnPy catalyzed by PEP phosphomutase and is suggested to be essential to C-P compound biosynthesis.

We are studying the possibility of altering the virulence and host range of a phytopathogen by transferring and expressing certain genes from the soil-dwelling saprophyte,Streptomyces hygroscopicus, in a plant pathogen model,Xanthomonas campestrispv.campestris(XCC). The genes, referred to herein as the “bialaphos genes,” encode the production of bialaphos, a potent glutamine-synthetase-inhibiting herbicide. This cluster of genes was originally isolated from several biosynthetically blocked mutants ofS. hygroscopicusand constructed into a plasmid vector, pBG9. We have transferred a fragment of the gene cluster into pLAFR3, a plasmid that functions in bothEscherichia coliand XCC and contains a tetracycline resistance marker. The resulting plasmid, named pIL-1, was used to transformE. coliand was incorporated into XCC by conjugation. The transfer of the fragment was confirmed by Southern analysis. The genes were maintained in XCC for about 47 generations in the absence of selection for tetracycline, and no changes in cultural phenotypes were seen in the transformed XCC (XCC/pIL-1). The XCC/pIL-1 cells were pathogenic to their natural hosts cabbage and broccoli, but induced an altered hypersensitive response in the nonhosts bean, pepper, sunflower, and tobacco. The pathogenic host-reaction, induced by the parent XCC, XCC/pLAFR3, and XCC/pIL-1, was a typical black rot disease in inoculated leaves of the two hosts. The nonhost reaction on the nonhost leaves was necrotic hypersensitivity, induced by XCC and XCC/pLAFR3, or the inhibition of hypersensitivity accompanied by only chlorosis at sites inoculated with XCC/pIL-1. We hypothesize that the altered hypersensitivity phenotype may be due to the transformed XCC becoming more compatible with the nonhosts, a step toward acquiring nonhost-virulence, or due to disruption of the normal expression of the hypersensitivity and pathogenicity genes in the transformed XCC. More work is needed to confirm that the introduced genes are being expressed in XCC. With further understanding, this approach may provide a useful model to study host range, virulence, and strain improvement of plant pathogens for biological control of weeds.

A plasmid pLC-bar containing the bialaphos resistance gene derived from Streptomyces hygroscopicus between the Lentinus edodes ras gene promoter and priA gene terminator was constructed. When protoplasts of Pleurotus ostreatus were mixed with the plasmid DNA in the presence of polyethylene glycol and CaCl2, bialaphos-resistant colonies were obtained. This indicated that transformation was successful. Southern blot analysis of total DNAs from transformants showed that the introduced plasmid DNA was integrated into the host chromosome and partly rearranged. A plasmid, pLC-GUS, containing the Escherichia coli beta-glucuronidase (GUS) gene under the control of the L. edodes ras gene promoter and priA gene terminator was constructed and introduced into protoplasts of P. ostreatus with pLC-bar by co-transformation. Two of 5 transformants obtained as bialaphos-resistant colonies showed two to twenty times higher specific activity of GUS than the recipient. Southern blot analysis of total DNAs from transformants indicated the presence of the GUS gene only in the two transformants. These results indicated that co-transformation of P. ostreatus was successful, and that the GUS gene was expressed in P. ostreatus. This transformation system will enable us to breed commercial strains of P. ostreatus at the molecular level.

The impact of glufosinate-ammonium and bialaphos on the zooplankton community in a small eutrophic lake was investigated using in situ enclosures. Concentrations inducing a 20% reduction in abundance (EC20), as interpolated from best-fit, nonlinear regression analyses were similar for both herbicides and ranged from 0.03 to 0.16 mg/L for various zooplankton taxa. Similarly, median effective concentrations (EC50) estimates ranged from 0.12 to 0.50 mg/L. Thus, toxicity endpoints overlapped and in some cases were well below expected environmental concentrations calculated for accidental direct overspray (1 mg/L) or drift events (0.25 mg/L). Significant concentration-dependent reductions were observed within the first 2 weeks following application and for a number of taxa persisted throughout the observation period (63 d posttreatment). At the highest treatment level, (10 mg/L), negative impacts were still apparent in the year following treatment. The results of this field study, which demonstrate significant negative effects on a variety of zooplankton taxa at environmentally relevant concentrations and relatively slow recovery therefrom, suggest a substantial risk of sustained adverse impacts on the zooplankton communities should these herbicides contaminate shallow lentic ecosystems. Extensive mitigative measures are required to protect such water bodies from potential impacts of phosphinothricin-based herbicides.

The effect of the microbially produced herbicide bialaphos on ammonium accumulation and photosynthesis of white spruce (Piceaglauca (Moench) Voss) and red pine (Pinusresinosa Ait.) seedlings was studied under controlled environmental conditions. Ammonium accumulation increased markedly during the first 48 h for seedlings treated in June and was greater in red pine than in white spruce. Ammonium accumulation also increased with higher doses of bialaphos. In contrast, the accumulation of ammonium following bialaphos application in August was minimal for both species and for doses up to an equivalent of 3.0 kg active ingredient (bialaphos) per hectare. Continuous measurements of photosynthesis for the first 12 h following bialaphos application showed a slight negative effect of the herbicide on photosynthetic activity within 2 h of treatment. The decrease was greater for red pine than for white spruce. Photosynthesis measurements taken under three different levels of photosynthetic photon flux density 24 h after bialaphos application showed a greater decline with increasing photosynthetic photon flux density and with higher doses of bialaphos. Furthermore, the decline was greater in red pine than it was in white spruce. When photosynthesis was followed over a 7-day period, a large decrease was observed during the first 24 h, after which time photosynthesis stayed relatively constant. The decrease was greater with higher doses and was greater for red pine than for white spruce. There was a strong negative correlation between photosynthetic rate at day 7 and both the dose of bialaphos and the development of chlorotic foliage. These results help provide a physiological basis for understanding previously reported morphological and growth responses.

Four species of boreal conifer seedlings used for reforestation in eastern Canada were tested for their tolerance to the microbially produced herbicide bialaphos under controlled conditions. The tolerance levels of white spruce (Piceaglauca (Moench) Voss), black spruce (Piceamariana (Mill.) B.S.R), Norway spruce (Piceaabies (L.) Karst.), and red pine (Pinusresinosa Ait.) were studied over two consecutive growing seasons using different morphological and growth criteria. Application rates were the equivalent of 0, 1.0, 1.5, 2.0, 2.5, and 3.0 kg active ingredient (bialaphos) per hectare in a 1400-L solution and were applied in July, August, or September. Survival rates were a linear function of the dose of bialaphos, but were greater than 95% for all treatments. Results indicated that there was no significant correlation between visible foliar injury and the amount of epicuticular wax on needles. There was also no significant correlation between visible foliar injury and initial seedling height. However, a significant negative correlation was observed between the degree of foliar injury and the relative growth rate following treatment. No additional foliar injury occurred during the second growing season. Doses of bialaphos that did not exceed the equivalent of 2.0 kg active ingredient per hectare had no effect on subsequent growth. Tolerance was greatest when the herbicide was applied in August and least when applied in July. These results, combined with the lower tolerance previously demonstrated for several species that compete with planted conifers in eastern Canada, suggest that bialaphos has a strong potential as an alternative to chemically synthesized herbicides for vegetation management in conifer plantations.