The presence of two different target-site resistance mechanisms in individual plants of Alopecurus myosuroides Huds., identified using a quick molecular test for the characterisation of six ALS and seven ACCase SNPs.

BACKGROUND: Target-site resistance to ALS- and ACCase-inhibiting herbicides in the grass weed Alopecurus myosuroides is associated with well-characterised allelic variants encoding ALS- and ACCase-based resistance. The potential for combined ALS and ACCase resistance presents a threat to future control, given the extent to which these herbicides are used. The authors present a primer extension method for rapid detection of known resistance-conferring substitutions. RESULTS: Individuals showing combined resistance to field-rate mesosulfuron + iodosulfuron and cycloxydim were identified in four field-collected populations, with proportions ranging from 30 to 100%. Genotyping with the SNaPshot primer extension kit showed the T197 and L574 ALS and L1781 ACCase isoforms to be associated with ALS and ACCase resistance whenever they occurred. CONCLUSION: Combined ALS and ACCase target-site resistance threatens future control of A. myosuroides. The SNaPshot extension assay provides a reliable new multiplexable method for characterising known allelic variants of the ALS and ACCase genes of A. myosuroides. The method offers significant advantages over both CAPS/dCAPS and PASA in that full genotyping can be accomplished at any nucleotide position using a single extension primer.

Serological evidence of West Nile virus, Usutu virus and Sindbis virus infection of birds in the UK.

The introduction and rapid dispersal of the African flavivirus West Nile virus (WNV) throughout North America, and the high fatality rate due to encephalitis in birds, horses, other wildlife species and humans, has attracted major attention worldwide. Usutu virus, another flavivirus, came to prominence in 2001, when it was identified as the agent responsible for a drop in the bird population in Austria; previously this encephalitic virus was found only in birds and mosquitoes in Africa. Sindbis virus, a pathogenic alphavirus that causes arthritis, is widespread throughout Africa, Europe, Asia and Australia, infecting a range of arthropods and vertebrates and is genetically related to encephalitic viruses in North America. Currently there is no evidence that any of these viruses cause disease in the UK. Here the presence of virus-specific neutralizing antibodies is reported in the sera of resident and migrant birds in the UK, implying that each of these viruses is being introduced to UK birds, possibly by mosquitoes. This is supported by nucleotide sequencing that identified three slightly different sequences of WNV RNA in tissues of magpies and a blackbird. The detection of specific neutralizing antibodies to WNV in birds provides a plausible explanation for the lack of evidence of a decrease in the bird population in the UK compared with North America. The potential health risk posed to humans and animals by these viruses circulating in the UK is discussed.

Graminicide insensitivity correlates with herbicide-binding co-operativity on acetyl-CoA carboxylase isoforms.

The sensitivity of grass species to important classes of graminicide herbicides inhibiting ACCase (acetyl-CoA carboxylase) is associated with a specific inhibition of the multifunctional ACCase located in the plastids of grasses. In contrast, the multisubunit form of ACCase found in the chloroplasts of dicotyledonous plants is insensitive and the minor cytosolic multifunctional isoforms of the enzyme in both types of plants are also less sensitive to inhibition. We have isolated, separated and characterized the multifunctional ACCase isoforms found in exceptional examples of grasses that are either inherently insensitive to these graminicides, or from biotypes showing acquired resistance to their use. Major and minor multifunctional enzymes were isolated from cell suspension cultures of Festuca rubra and the 'Notts A1'-resistant biotype of Alopecurus myosuroides, and their properties compared with those isolated from cells of wild-type sensitive A. myosuroides or from sensitive maize. Purifications of up to 300-fold were necessary to separate the two isoforms. The molecular masses (200-230 kDa) and K(m) values for all three substrates (ATP, bicarbonate and acetyl-CoA) were similar for the different ACCases, irrespective of their graminicide sensitivity. Moreover, we found no correlation between the ability of isoforms to carboxylate propionyl-CoA and their sensitivity to graminicides. However, insensitive purified forms of ACCase were characterized by herbicide-binding co-operativity, whereas, in contrast, sensitive forms of the enzymes were not. Our studies on isolated individual isoforms of ACCase from grasses support and extend previous indications that herbicide binding co-operativity is the only kinetic property that differentiates naturally or selected insensitive enzymes from the typical sensitive forms usually found in grasses.

Characterisation of target-site resistance to ACCase-inhibiting herbicides in the weed Alopecurus myosuroides (black-grass).

Resistance to aryloxyphenoxypropionate (AOPP), cyclohexanedione (CHD) and phenylurea herbicides was determined in UK populations of Alopecurus myosuroides Huds. Two populations (Oxford AA1, Notts. A1) were highly resistant (Resistance indices 13-->1000) to the AOPP and CHD herbicides fenoxaprop, diclofop, fluazifop-P and sethoxydim, but only marginally resistant to the phenylurea, chlorotoluron. Analyses of acetyl coenzyme A carboxylase (ACCase) activity showed that an insensitive ACCase conferred resistance to all the AOPP/CHD herbicides investigated. Another population, Oxford S1, showed no resistance to sethoxydim at the population level, but contained a small proportion of plants (<10%) with an insensitive ACCase. Genetic studies on the Notts A1 and Oxford S1 populations demonstrated that target site resistance conferred by an insensitive ACCase is monogenic, nuclearly inherited with the resistant allele showing complete dominance. Investigations of the molecular basis of resistance in the Notts A1 population showed that sethoxydim resistance in A myosuroides was associated with the substitution of an isoleucine in susceptible with a leucine in resistant plants, which has also been found in three other resistant grass-weed species (Setaria viridis (L) Beauv, Avena fatua L, Lolium rigidum Gaud).

Herbicidal action of 2-hydroxy-3-alkyl-1,4-naphthoquinones.

The main mode of herbicidal activity of 2-hydroxy-3-alkyl-1,4-naphthoquinones is shown to be inhibition of photosystem II (PSII). The herbicidal and in vitro activities have been measured and correlated with their (Log)octanol/water partition coefficients (Log Ko/w). The length of the 3-n-alkyl substituent for optimal activity differed between herbicidal and in vitro activity. The maximum in vitro activity was given by the nonyl to dodecyl homologues (Log Ko/w between 6.54 and 8.12), whereas herbicidal activity peaked with the n-hexyl compound (Log Ko/w = 4.95). The effect of chain branching was also investigated using isomeric pentyl analogues substituted at position 3. All exhibited similar levels of in vitro activities but herbicidal activities differed, albeit moderately, with the exception of one analogue that was much less phytotoxic. Other modes of action were also investigated using two representative compounds. They did not show any activity on photosystem I or mitochondrial complex I, or generate toxic oxygen radicals by redox cycling reactions. Only moderate activity was found against mitochondrial complex III from plants, in contrast to much higher corresponding activity using an insect enzyme.