R. Hull

Detection of episomal banana streak badnavirus by IC-PCR.

A polymerase chain reaction (PCR) based strategy to detect episomal banana streak badnavirus (BSV) in banana and plantain plants that carry integrated BSV sequences was developed. Antisera used in immuno-capture polymerase chain reaction (IC-PCR) are capable of binding a large number of BSV serotypes. The primers used for PCR are capable of annealing to and amplifying across the aspartic protease-reverse transcriptase domain boundaries of both episomal and integrated BSV sequences and result in similar or identical sequence size fragments from either template. However, we show that under the conditions selected for IC-PCR, nuclear, mitochondrial or chloroplast genomic sequences are not amplified and thus only captured episomal BSV is amplified. IC-PCR is suitable for the large-scale screening of Musa for episomal BSV which is necessary for germplasm movement.

Sequence analysis of rice hoja blanca virus RNA 3

RNA 3 of rice hoja blanca tenuivirus (RHBV) has 2299 nucleotides and resembles RNA 3 of other tenuiviruses such as maize stripe (MStV) and rice stripe (RStV) viruses in potentially coding with an ambisense strategy for two proteins. Both the viral-sense protein of 23K and the complementary-sense protein of 35K have about 46% amino acid identity with the analogous proteins encoded by RNA 3 of MStV and RStV. As the proteins of MStV and RStV have about 65% identity between themselves, RHBV cannot be a South and Central American strain of the Asian RStV. The intergenic region resembles those of other tenuiviruses, being rich in A and U residues, but its predicted folding pattern is unlike those of other tenuiviruses. Instead, the predicted folding of the intergenic region was indistinguishable from that of the coding regions and there was no evidence for a distinct hairpin-loop structure. The significance to the evolution of tenuiviruses of the similarities that the two proteins have with their analogues in other tenuiviruses is discussed.

Comparison of ELISA and RT-PCR for the detection of beet yellows closterovirus in plants and aphids

A reverse-transcription polymerase chain reaction (RT-PCR) was developed to detect beet yellows closterovirus (BYV) in plants and single aphids using primers spanning the conserved regions of the published sequences of the coat protein gene and open reading frames 7 and 8. Three total RNA extraction procedures were examined and all were found to produce RNA of sufficient quality for RT-PCR, although the RNA extraction kit supplied by Flowgen was found to be the most versatile for the extraction of BYV from individual aphids. When 60 aphids, which had fed on virus infected sugar beet were tested by RT-PCR, 55% of individuals were found to contain BYV. Two groups of 36 individual aphids were tested by TAS-ELISA using a specific BYV monoclonal antibody; 53% gave positive absorbance values when a substrate amplification system was used, but none was found to contain virus when the system was replaced with the substrate p-nitrophenyl phosphate. An immunocapture RT-PCR method was shown to detect BYV regardless of whether the RNA had been extracted from the trapped particles or the reverse transcription and PCR mixtures were added to the wells containing intact particles. The RT-PCR method is now used to determine the numbers of BYV carrying aphids migrating into sugar-beet crops.

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.

Rapid, small scale purification of rice hoja blanca and Echinochloa hoja blanca tenuivirus ribonucleoprotein

Highly purified tenuivirus ribonucleoprotein was obtained from small amounts of leaf tissue by sedimenting the ribonucleoprotein particles from debris-free plant extract into a 30% sucrose cushion, in 1.5-mL microfuge tubes. Using this protocol, significant size differences were discovered in the double-stranded forms of the viral genomic RNAs of rice hoja blanca tenuivirus and a tenuivirus isolated from Echinochloa colonum, a common weed of rice cultivation.

Measuring the effectiveness of management interventions at regional scales by integrating ecological monitoring and modelling

Abstract BACKGROUND Because of site‐specific effects and outcomes, it is often difficult to know whether a management strategy for the control of pests has worked or not. Population dynamics of pests are typically spatially and temporally variable. Moreover, interventions at the scale of individual fields or farms are essentially unreplicated experiments; a decrease in a target population following management cannot safely be interpreted as success because, for example, it might simply be a poor year for that species. Here, we argue that if large‐scale data are available, population models can be used to measure outcomes against the prevailing mean and variance. We apply this approach to the problem of rotational management of the weed Alopecurus myosuroides. RESULTS We derived density‐structured population models for a set of fields that were not subject to rotational management (continuous winter wheat) and another group that were (rotated into spring barley to control A. myosuroides). We used these models to construct means and variances of the outcomes of management for given starting conditions, and to conduct transient growth analysis. We show that, overall, this management strategy is successful in reducing densities of weeds, albeit with considerable variance. However, we also show that one variant (rotation to spring barley along with variable sowing) shows little evidence for additional control. CONCLUSION Our results suggest that rotational strategies can be effective in the control of this weed, but also that strategies require careful evaluation against a background of spatiotemporal variation.

The First Cases of Evolving Glyphosate Resistance in UK Poverty Brome (Bromus sterilis) Populations

Abstract Poverty brome (Bromus sterilis L.) [sterile or barren brome, syn. Anisantha sterilis (L.) Nevski] is a problematic UK arable weed. There are currently no confirmed cases of glyphosate resistance in any weed species in the United Kingdom or in B. sterilis worldwide. However, there are reports of poor control by glyphosate in this species. Here, we report experiments to confirm the suspected on-farm resistance of B. sterilis populations to glyphosate. Glyphosate screening and dose–response experiments established that glyphosate sensitivity of three UK B. sterilis populations exhibiting poor field control is outside the normal range of sensitivity of 30 sensitive populations and adjacent unexposed populations. Control of sensitive populations ranged from 49% to 82% and for suspected resistant populations from 21% to 30%. Dose–response ED50 values of sensitive populations ranged between 241 and 313 g ai ha − 1; corresponding values of suspected resistant populations ranged between 420 and 810 g ha − 1, and resistance indices ranged from 1.55 to 4.5. Suspected resistant populations were incompletely controlled at the recommended field rate of glyphosate (540 g ha − 1), while adjacent unexposed populations were completely controlled. We conclude that some UK populations of B. sterilis have reduced glyphosate sensitivity and are in the process of evolving resistance. This is the first reported case of reduced glyphosate sensitivity in any UK weed species and in B. sterilis worldwide. This, coupled with increasing glyphosate use, highlights the need for increased vigilance and monitoring for glyphosate resistance in the United Kingdom.

Changes in the proteome of the problem weed blackgrass correlating with multiple herbicide resistance

Summary Herbicide resistance in grass weeds is now one of the greatest threats to sustainable cereal production in Northern Europe. Multiple‐herbicide resistance (MHR), a poorly understood multigenic and quantitative trait, is particularly problematic as it provides tolerance to most classes of chemistries currently used for post‐emergence weed control. Using a combination of transcriptomics and proteomics, the evolution of MHR in populations of the weed blackgrass (Alopecurus myosuroides) has been investigated. While over 4500 genes showed perturbation in their expression in MHR versus herbicide sensitive (HS) plants, only a small group of proteins showed >2‐fold changes in abundance, with a mere eight proteins consistently associated with this class of resistance. Of the eight, orthologues of three of these proteins are also known to be associated with multiple drug resistance (MDR) in humans, suggesting a cross‐phyla conservation in evolved tolerance to chemical agents. Proteomics revealed that MHR could be classified into three sub‐types based on the association with resistance to herbicides with differing modes of action (MoA), being either global, specific to diverse chemistries acting on one MoA, or herbicide specific. Furthermore, the proteome of MHR plants were distinct from that of HS plants exposed to a range of biotic (insect feeding, plant–microbe interaction) and abiotic (N‐limitation, osmotic, heat, herbicide safening) challenges commonly encountered in the field. It was concluded that MHR in blackgrass is a uniquely evolving trait(s), associated with changes in the proteome that are distinct from responses to conventional plant stresses, but sharing common features with MDR in humans.

The implications of spatially variable pre‐emergence herbicide efficacy for weed management

Abstract BACKGROUND The efficacy of pre‐emergence herbicides within fields is spatially variable as a consequence of soil heterogeneity. We quantified the effect of soil organic matter on the efficacy of two pre‐emergence herbicides, flufenacet and pendimethalin, against Alopecurus myosuroides and investigated the implications of variation in organic matter for weed management using a crop–weed competition model. RESULTS Soil organic matter played a critical role in determining the level of control achieved. The high organic matter soil had more surviving weeds with higher biomass than the low organic matter soil. In the absence of competition, surviving plants recovered to produce the same amount of seed as if no herbicide had been applied. The competition model predicted that weeds surviving pre‐emergence herbicides could compensate for sublethal effects even when competing with the crop. The ED50 (median effective dose) was higher for weed seed production than seedling mortality or biomass. This difference was greatest on high organic matter soil. CONCLUSION These results show that the application rate of herbicides should be adjusted to account for within‐field variation in soil organic matter. The results from the modelling emphasised the importance of crop competition in limiting the capacity of weeds surviving pre‐emergence herbicides to compensate and replenish the seedbank.

Extraction and aphid transmission of beet western yellows luteovirus from air dried leaf tissue

A protocol is described for the recovery of viable, aphid transmissible beet western yellows luteovirus from air-dried field material. The advantages of the protocol for long-term and cross-border field sampling are discussed.