Integrated weed management (IWM): will it reduce herbicide use?

The Sustainable Use of Pesticides Directive (2009/128/EC), part of the EU Thematic Strategy for Pesticides, requires Integrated Pest Management (IPM) to be actively promoted. A key objective is to give greater priority to non-chemical methods of plant protection to reduce the impact of pesticides on human health and the environment. Integrated Weed Management (IWM) can be considered part of IPM, and many non-chemical methods are available. For example, a recent review of methods for control of Alopecurus myosuroides (black-grass) in winter wheat found the following mean annual levels of control: ploughing 67%; delayed drilling 37%; fallowing 70%; higher seed rates 30%; competitive cultivars 27%. In comparison with herbicides these efficacy levels are mediocre, and A. myosuroides would be classified as resistant (R) or moderately resistant (MR) to all these methods if the criteria used by the Chemicals Regulation Directorate in the UK for assigning ratings to herbicide efficacy were used. It is, therefore, not surprising that farmers are reluctant to embrace IWM and continue to place greater.reliance on herbicides as a more reliable and cost effective method of weed control. While non-chemical methods will not replace herbicides on most farms, reduced reliance on herbicides will be necessary both for practical (increasing resistance, lack of new herbicides) and political reasons (complying with EU legislation). Farmers will use nonchemical control methods when they have a major weed problem, and have no alternative, but they must be encouraged to adopt IWM at an earlier stage. Research into IWM must be relevant and practical, and not simply conducted as some sort of 'academic' exercise. More effective knowledge transfer is vital, and this is a challenge due to the decline in independent, state funded, advisory services in many European countries. The question arises; is it possible to achieve reductions in pesticide use by simply promoting non-chemical methods of weed control, or will statutory limits on pesticides be needed to achieve this goal?

Control of Alopecurus myosuroides (black-grass) resistant to mesosulfuron+iodosulfuron.

Resistance to the ALS inhibitor mesosulfuron+iodosulfuron ('Atlantis') had been identified in 293 populations of A. myosuroides in the UK by 2008. Two field trials were conducted in winter wheat crops where ALS target site resistance (Pro197Thr) occurred. Mesosulfuron+iodosulfuron (12+2.4 g a.i. ha(-1)) gave 73% - 79% reductions in head numbers in Town Mead field, but only -7% - 5% reductions in Long Covert. Mixtures and sequences improved overall control. Pre-emergence flufenacet+pendimethalin (240+1200 g a.i. ha(-1)) followed by mesosulfuron+ iodosulfuron plus pendimethalin (1320 g a.i. ha(-1)) or clodinafop+trifluralin (30+960 g a.i. ha(-1)) gave 93 - 98% reductions in Town Mead but only 60 - 73% reductions in Long Covert. A non-ALS treatment of pre-emergence flufenacet+pendimethalin followed by isoproturon+pendimethalin (1500+1320 g a.i. ha(-1)) in late October and clodinafop+trifluralin in November or February achieved 97% and 93% reductions in the two trials. Seed samples collected from surviving plants were evaluated in glasshouse assays to quantify any changes in the incidence of resistance. There was an increase in the proportion of plants resistant to mesosulfuron+iodosulfuron regardless of whether it was used alone, in mixture or sequence. No such changes occured with non-ALS treatments. The trials highlight the difficulty of achieving adequate control with alternative herbicides, especially as isoproturon and trifluralin will not be available for use in the UK after 2009.

Recombination in rabbit haemorrhagic disease virus: possible impact on evolution and epidemiology.

Rabbit haemorrhagic disease (RHD) was first recognised in 1984 following the introduction of apparently healthy rabbits into China from Germany. The aetiological agent Rabbit haemorrhagic disease virus (RHDV) has subsequently killed hundreds of millions of domestic and wild rabbits particularly in Europe, China and Australia. Previously, using phylogenetic analysis we have attempted to understand the underlying factors that determine why this virus emerged, and why it has such an unpredictable epidemiology. Here we report the use of tree congruency supported by bootscanning analysis to detect recombination amongst both closely related, and widely divergent strains of RHDV. We show that recombination occurs commonly and in several different regions of the RHDV genome. Moreover, the first identified strain of RHDV, i.e. from China in 1984, showed evidence of recombination in the capsid gene, with a virus or viruses containing lineages in German strains. These observations imply that recombination may play a significant role in the evolution, epidemiology and diversity of RHDV.

Resistance to ACCase-inhibiting herbicides in the weed Lolium multiflorum.

Resistance to five ACCase-inhibiting herbicides (dictofop, fluazifop, tralkoxydim, cycloxydim and pinoxaden) was investigated in 55 Lolium multiflorum populations from 50 farms in England. Resistance screening experiments at whole plant level showed differences in the response to each herbicide suggesting different resistance mechanisms for different herbicides, despite all of them having the same mode of action. Plants from populations resistant to at least one herbicide were analysed at the molecular level using the derived Cleaved Amplified Polymorphic Sequence method (dCAPS). Three single-site point mutations (ile-1781-leu, asp-2078-gly and cys-2088-arg) known to confer resistance to ACCase inhibitors were found in 13 populations. In the remaining resistant populations a more specific mutation or an enhanced metabolism mechanism might be involved.

Herbicide-resistance to mesosulfuron + iodosulfuron in Alopecurus myosuroides (black-grass).

A formulated mixture of two sulfonylurea herbicides, mesosulfuron and iodosulfuron, combined with the safener mefenpyr-diethyl ('Atlantis') is being used extensively in Europe and there is concern that resistance will evolve in Alopecurus myosuroides (black-grass). Glasshouse screening bioassays showed that the best single discriminating dose for detecting resistance is the UK field rate of 12 g mesosulfuron + 2.4 g iodosulfuron ha(-1) applied at the 3 leaf stage, with herbicidal effects recorded 4 weeks later. Using this methodology with 466 UK seed samples, resistance was confirmed on a total of 24 farms in 11 counties by 2005, 81 farms in 19 counties by 2006 and 133 farms in 21 counties by 2007. Cultural histories for 10 resistant (R) and 7 susceptible (S) fields were obtained. Winter cereals were grown in 73% R/ 71% S years and a mean of 3.0 grass-weed active ingredients applied per year in both R and S fields. Four herbicide classes dominated, comprising almost 80% of all applications: ALS inhibitors 17% R/ 21% S; ACCase inhibitors 19% R/ 17% S; substituted ureas 18% R/ 10% S; dinitroanilines 25% R/ 30% S. Consequently, ALS inhibitor use was not excessive and field histories were not a reliable indicator of resistance risk. DNA sequencing of the ALS gene from resistant and susceptible individuals of nine populations was used to identify resistance mechanisms. All highly resistant individuals from seven populations showed a single nucleotide polymorphism in the first position of the Pro197 codon of an A. myosuroides ALS gene. One population showed resistant individuals with a single nucleotide polymorphism in the second position of the Trp574 codon. Consequently ALS target site resistance was confirmed in eight of the nine populations studied in detail.

Phylogenetic analysis of rabbit haemorrhagic disease virus strains from the Arabian Peninsula: did RHDV emerge simultaneously in Europe and Asia?

Rabbit haemorrhagic disease virus (RHDV) emerged in 1984 in China and subsequently a single strain apparently dispersed worldwide killing millions of rabbits. Two isolates that caused outbreaks in Saudi Arabia and Bahrain have been sequenced and analysed phylogenetically. The Saudi Arabian lineage is directly descended from the Chinese strain, but the Bahrain isolate occupies a distinct and more divergent lineage than the Chinese virus implying that epidemic RHDV strains have emerged at least twice during the past 20 years and are co-circulating in both domestic and wild rabbits.

Epidemiology of rabbit haemorrhagic disease virus in the United Kingdom: evidence for seasonal transmission by both virulent and avirulent modes of infection.

Rabbit haemorrhagic disease virus (RHDV) has killed many millions of wild rabbits in Europe and Australia, but has had little impact in the United Kingdom, despite outbreaks having occurred since 1994. High seroprevalence detected in the absence of associated mortality had suggested the presence of an endemic non-pathogenic strain which may be 'protecting' UK populations. Following the first detailed field study of RHDV epidemiology in the United Kingdom, using mark-recapture with serum sampling, we report that RHDV caused highly prevalent persistent infection in seropositive rabbits in the absence of associated mortality. Furthermore the virus strains responsible could not be distinguished phylogenetically from known pathogenic isolates, and were clearly very different from the only previously identified non-pathogenic strain of RHDV. These findings suggest that many--perhaps most--strains of RHDV may be propagated through both 'pathogenic' and 'non-pathogenic' modes of behaviour. Transmission occurred predominantly during and just after the breeding season.

Evolution and dispersal of encephalitic flaviviruses.

There are two major groups of encephalitic flaviviruses, those that infect and are transmitted by ticks, particularly Ixodes spp. and those that infect and are transmitted by mosquitoes, particularly Culex spp. The tick-borne encephalitic flaviviruses exhibit evolutionary characteristics that are largely determined by the protracted life cycle of the tick, its habitat and the prevailing climatic conditions. These viruses appear to have evolved gradually from non-encephalitic viruses that radiated eastwards and north eastwards out of Africa into Asia and the southern islands, then northwards to far east Asia and finally westwards across Eurasia to western Europe, during the past two to four thousand years. Only one of these recognized species has found its way to North America viz. Powassan virus. In contrast, the evolution of the recognized mosquito-borne encephalitic flaviviruses reflects the wide range of mosquito species that they infect. They emerged out of Africa relatively recently and at roughly the same time, i.e., probably during the past few centuries. Although many of these mosquito-borne viruses are geographically widely dispersed, with the exception of West Nile virus, they are found either in the Old World or the New World, never in both, and we are now beginning to understand the reasons. Phylogenetic trees will be used here to describe the evolution, epidemiology and dispersal characteristics of these viruses, taking into account the importance of virus persistence and recombination.

Herbicide-resistant weeds in Europe: the wider implications.

Herbicide-resistance occurs in 55 weed species in 21 European countries. 91% of cases are associated with just four herbicide mode of action groups: ACCase and ALS inhibitors, and triazine and urea/amide photosynthetic inhibitors. There are also a few cases of resistance to bypiridiliums, dinitroanilnes and synthetic auxins. Resistance to ALS inhibitors tends to be less prevalent in Europe than elsewhere, but is likely to increase. A small scale survey showed that Alopecurus myosuroides is considered to be the most important herbicide-resistant weed in Europe at present. Lolium spp., and to a lesser extent Papaver rhoeas and Avena spp., were also highlighted as being of major importance in many countries. One consequence of the ongoing EC review of pesticides may be a reduction in the range of modes of action available to European farmers. This may reduce the opportunities for rotating different modes of action as a method of reducing resistance risk. Greater dependence on high resistance risk herbicides, such as ACCase and ALS inhibitors, because of lack of alternative modes of action, is likely to increase the incidence of resistance in grass-weeds.

Long-term survival of New Zealand rabbit haemorrhagic disease virus RNA in wild rabbits, revealed by RT-PCR and phylogenetic analysis.

Because Rabbit haemorrhagic disease virus (RHDV) is highly pathogenic for rabbits, farmers illegally introduced it as a bio-control agent onto New Zealand farms in 1997. The virus was dispersed rapidly, initially causing high fatality rates in rabbits. Nevertheless, many survived and these surviving rabbits have been investigated for evidence of infection by RHDV. Livers from healthy rabbits contained RHDV-specific RNA, as shown by nested RT-PCR sequencing. The sequences of the viral capsids were related closely to the released Czech strain of RHDV, although the sequence from one rabbit was related most closely to a Spanish strain of RHDV. Phylogenetic analysis of the capsid sequences of 38 samples implied that there have been at least two introductions of the Czech virus into New Zealand, probably corresponding firstly to the original illegal introduction by farmers and secondly to the introduction of the same virus under governmental control. Genomic length sequence of two samples was obtained, suggesting that they may have retained the potential to be infectious, although this has not yet been demonstrated. The detection of genomic-length RNA in the liver of healthy rabbits suggests that even though a highly virulent virus was introduced into New Zealand, it rapidly established persistent or latent infections in a proportion of rabbits. This might account for their ability to survive in the face of virulent released virus. Moreover, the co-circulation of other strains of RHDV in the same rabbit population, such as the Spanish strain, might also impact on their susceptibility to the bio-control agent.

Cytotoxic T-cell activity is not detectable in Venezuelan equine encephalitis virus-infected mice.

Previously published research has established that the immune response to the Venezuelan equine encephalitis virus (VEEV) vaccine strain TC-83 is Th 1-mediated, with local activation of both CD4+ and CD8+ T cells. This suggests that cytotoxic lymphocytes CTL may play a role in protection against virulent VEEV. Studies involving a variety of immunisation schedules with either TC-83 or strain CAAR 508 (serogroup 5) of VEEV, and six different haplotypes of mice, failed to reveal functional CTL activity against VEEV-infected targets in secondary antigen-stimulated lymphocyte cultures from either the draining lymph nodes (LN) or spleen. Nor were VEEV-specific CTL detected after immunisation of mice (three haplotypes) with recombinant vaccinia viruses (VV) expressing either the non-structural (nsP1-4) or the structural (C-E3-E2-6K-E1) genes of TC-83. Reciprocal experiments in which mice were immunised with TC-83, and their lymphocytes tested against VV recombinant-infected targets also failed to detect CTL activity. These data suggest that VEEV infection of mice does not elicit detectable CTL activity, and that CTL are unlikely to play a role in protection against virulent VEEV.

Molecular epidemiology of Rabbit haemorrhagic disease virus.

Millions of domestic and wild European rabbits (Oryctolagus cuniculus) have died in Europe, Asia, Australia and New Zealand during the past 17 years following infection by Rabbit haemorrhagic disease virus (RHDV). This highly contagious and deadly disease was first identified in China in 1984. Epidemics of RHDV then radiated across Europe until the virus apparently appeared in Britain in 1992. However, this concept of radiation of a new and virulent virus from China is not entirely consistent with serological and molecular evidence. This study shows, using RT-PCR and nucleotide sequencing of RNA obtained from the serum of healthy rabbits stored at 4 degrees C for nearly 50 years, that, contrary to previous opinions, RHDV circulated as an apparently avirulent virus throughout Britain more than 50 years ago and more than 30 years before the disease itself was identified. Based on molecular phylogenetic analysis of British and European RHDV sequences, it is concluded that RHDV has almost certainly circulated harmlessly in Britain and Europe for centuries rather than decades. Moreover, analysis of partial capsid sequences did not reveal significant differences between RHDV isolates that came from either healthy rabbits or animals that had died with typical haemorrhagic disease. The high stability of RHDV RNA is also demonstrated by showing that it can be amplified and sequenced from rabbit bone marrow samples collected at least 7 weeks after the animal has died.

Resistance to ACCase-inhibiting herbicides and isoproturon in UK populations of Lolium multiflorum: mechanisms of resistance and implications for control.

Herbicide-resistant Lolium multiflorum (Italian rye-grass) was first reported in the UK in 1993 and had been confirmed on 25 farms by 1999. In this study, resistance to five herbicides belonging to the aryloxyphenoxypropionate, cyclohexanedione and phenyl-urea classes was determined in six populations of L multiflorum from the UK under glasshouse and simulated field conditions. Glasshouse conditions tended to exaggerate the degree of resistance, but experiments performed in both environments detected resistance in four populations of L multiflorum. Four populations (Essex A1, Lincs A1, Wilts B1, Yorks A2) were resistant to diclofop-methyl, fluazifop-P-butyl, tralkoxydim and partially resistant to isoproturon, but only the population from Yorkshire (Yorks A2) showed resistance to cycloxydim. Biochemical analyses of acetyl coenzyme A carboxylase (ACCase) activity, oxygen consumption by thylakoids, diclofop metabolism and glutathione S-transferase activity showed that, in three of the resistant populations, an enhanced rate of herbicide metabolism conferred resistance. This is the first report world-wide of an enhanced metabolism mechanism of diclofop resistance in L multiflorum. In the Yorks A2 population, an insensitive ACCase was detected (target-site resistance) which also conferred cross-resistance to all of the other ACCase inhibitors investigated.

Structure of Broadhaven virus by cryoelectron microscopy: correlation of structural and antigenic properties of Broadhaven virus and bluetongue virus outer capsid proteins.

The three-dimensional structure of Broadhaven virus (BRDV) has been determined to 23 A resolution by cryoelectron microscopy and image processing. As predicted from sequence homology, the BRDV structure resembles that of bluetongue virus (BTV) with the notable exception of one of the outer shell proteins. The cores of BRDV and BTV are identical at medium resolution; they have a diameter of 710 A and the VP7 trimers are arranged on a T = 13 icosahedral lattice. The outer shell proteins, VP5 of BRDV and BTV, have roughly the same molecular weight while VP4 of BRDV is only half the molecular weight of the corresponding VP2 of BTV. This size difference allows unambiguous determination of the identity of the triskelion shape as trimers of VP4 of BRDV (VP2 of BTV). The VP4 of BRDV sits on the VP7 trimers and projects outwards 40 A, giving the capsid an overall diameter of 790 A. This contrasts with VP2 of BTV, which projects outwards 95 A to give the capsid a diameter of 900 A. The difference in accessibility of the outer shell proteins of BRDV and BTV correlates with the difference in antigenic properties of these viral proteins. The shape of the BRDV VP5 indicates that it too is a trimer, thus implying that there are 360 copies of VP5 and 180 copies of VP4 per virion.

Immunization with VP2 is sufficient for protection against lethal challenge with African horsesickness virus Type 4.

Horses were immunized by inoculation with a vaccinia construct containing a full-length cDNA corresponding to the L2 gene segment of African horsesickness virus type 4(AHSV-4). All immunized horses developed serum neutralizing antibodies prior to challenge with virulent AHSV-4. No ELISA-reactive antibodies were present prior to challenge. A group of four seronegative control horses died after developing clinical signs and lesions typical of the pulmonary form of African horsesickness while the immunized horses were clinically normal. Increases in serum neutralizing and ELISA-reactive antibody titers following challenge indicate that at least some replication of challenge virus occurred in immunized horses. These results demonstrate that AHSV VP2 alone is sufficient to induce a protective immune response in horses and indicate the usefulness of ELISA-reactive antibodies for differentiation of vaccinated and naturally exposed horses.

Comparison of the non-structural protein, NS1, of tick-borne and insect-borne orbiviruses.

The nucleotide sequence of RNA segment 6 of Broadhaven virus (BRDV), a tick-borne orbivirus, was determined principally from two overlapping cDNA clones and RNA end sequence analysis. The genome segment is 1714 base pairs in length and has a coding capacity for a protein of 537 amino acids, having a net charge of +4.0 at neutral pH. Comparison of the predicted amino acid sequence of BRDV RNA segment 6 with the NS1 sequence of insect-borne orbiviruses, bluetongue virus (BTV), African horse sickness virus (AHSV) and epizootic haemorrhagic disease virus (EHDV) of deer, revealed amino acid identities of 21, 22 and 21%, respectively. This compares with amino acid identities of 31 to 50% between the NS1 proteins of these gnat-transmitted orbiviruses. A recombinant baculovirus was produced containing a full-length clone of BRDV segment 6, which expressed a protein of 61 kD in infected Spodoptera frugiperda cells. Like other orbivirus NS1 proteins the expressed protein formed tubules similar to those produced in BRDV-infected BHK21 cells.

Subcore- and core-like particles of Broadhaven virus (BRDV), a tick-borne orbivirus, synthesized from baculovirus expressed VP2 and VP7, the major core proteins of BRDV.

The genes encoding the two major core proteins (VP2 and VP7) of Broadhaven (BRD) virus, a tick-borne orbivirus, were inserted into the genome of Autographa californica nuclear polyhedrosis virus (AcNPV) under the control of copies of the AcNPV polyhedrin promoter to produce two recombinant baculoviruses. Infection of Spodoptera frugiperda (Sf) cells with a recombinant AcNPV that synthesized BRDV VP2 produced large numbers of BRDV subcore-like particles. Co-infection of cells with the two recombinants that made either BRDV VP2 or VP7 produced core-like particles similar in appearance to authentic BRDV cores. No evidence was obtained for the formation of core-like particles between the major core proteins of BRDV and those of bluetongue virus (BTV) following the co-expression of BRDV VP2 and BTV VP7, or BRDV VP7 and BTV VP3, indicating that in this respect the proteins of these two orbiviruses are incompatible, unlike the situation previously described for epizootic haemorrhagic disease virus and BTV core proteins.

The disposition and metabolic fate of 14C-meropenem in man.

1. The metabolism and pharmacokinetics of 14C-meropenem were studied in five volunteers who received 0.5 g (40 microCi) of the radiolabelled drug by i.v. infusion. 2. The maximum concentration of drug in plasma was 27 +/- 2 micrograms/ml (70 microM) corresponding to 98% of plasma radioactivity at the end of a 30 min infusion. The elimination half-life for meropenem in plasma was 1 h and meropenem remained the major radioactive component up to 6 h, but represented a decreasing proportion of the plasma radioactivity with time. One metabolite (the ring-open lactam) accounted for most of the remaining plasma radioactivity. The maximum concentration of metabolite was 1 +/- 0.1 micrograms/ml and the concentration of total radioactivity decreased to 2% of the peak value by 8 h. 3. Over the 5 days of the study, urinary excretion of radioactivity accounted for 99 +/- 0.5% dose, most of which was recovered in the first 8 h. There was negligible excretion in faeces. 4. Structural confirmation of the drug-related components in urine was accomplished by h.p.l.c.-mass spectrometry. Meropenem accounted for 71 +/- 2% dose of 14C and the ring-open lactam metabolite for most of the remainder, no other metabolites were detected. 5. Meropenem was the major radioactive component in urine up to 8 h after dosing and is therefore remarkably stable to human renal dehydropeptidase (DHP-1) compared with other carbapenems in clinical use.

Comparison of the major structural core proteins of tick-borne and Culicoides-borne orbiviruses.

Comparison of sequence data for Broadhaven (BRD) virus, a tick-borne orbivirus, and bluetongue virus (BTV), the type species of the genus, indicated that RNA segments 2 and 7 of BRD virus encode the two structural core proteins, VP2 and VP7, respectively. Segment 2 is 2792 nucleotides in length with a coding capacity for a protein (VP2) of 908 amino acids and a net charge of +8.5 at neutral pH. Segment 7 is 1174 nucleotides in length with a coding capacity for a protein (VP7) of 356 amino acids and a net charge of +11.5 at neutral pH. Comparison of the two sequences with BTV serotype 10 revealed amino acid identity of 35% between the product of segment 2 and BTV VP3, and 21% between the product of segment 7 and BTV VP7. The core proteins therefore show evidence of significant evolutionary divergence compared with that shown between different insect-borne orbiviruses. In particular, the amino terminus of BRD virus VP7 differed markedly from the equivalent region in VP7 of BTV and African horse sickness virus. This region is thought to interact with the outer capsid layer of insect-borne orbiviruses.

Enhanced neurovirulence of tick-borne orbiviruses resulting from genetic modulation.

The genome of orbiviruses (Reoviridae family) comprises 10 segments of double-stranded RNA. The fourth largest segment of the tick-borne Kemerovo (KEM) group orbiviruses is the genetic determinant of neurovirulence in experimentally infected mice, and segment 6 determines serotype. Reassortant viruses derived from a cross between two KEM-related viruses, Great Island (GI) and Wexford (WEX), that had the heterotypic gene combination W4G6 (segment 4 of WEX virus and segment 6 from GI virus) were nonpathogenic in mice. This apparent genetic modulation of neurovirulence may have resulted from steric interaction between the two outer capsid proteins of nonpathogenic reassortants. Further data are consistent with this hypothesis. Reassortants generated from additional KEM group viruses showed various degrees of enhanced neurovirulence in terms of their PFU/LD50 (ratio of infectivity in cell culture and in mice) and ASTmax (the average survival time at the highest virus dilution resulting in 100% mortality). Some reassortants were more pathogenic than either of their parental viruses. The results indicate that the gene determining neurovirulence dictates ASTmax, and the PFU/LD50 is a measure of the interaction between the products of the gene determining neurovirulence and that determining serotype. The nonpathogenic phenotype of a low passage isolate (St. Abb's 84-34 virus), derived from a single tick, generated neurovirulent reassortants. This result indicates that genetic modulation of KEM group viruses may occur in nature.