The meleagrid herpesvirus 1 genome is partially resistant to transposition

The propagation of herpesvirus genomes as infectious bacterial artificial chromosomes (iBAC) has enabled the application of highly efficient strategies to investigate gene function across the genome. One of these strategies, transposition, has been used successfully on a number of herpesvirus iBACs to generate libraries of gene disruption mutants. Gene deletion studies aimed at determining the dispensable gene repertoire of the Meleagrid herpesvirus 1 (MeHV-1) genome to enhance the utility of this virus as a vaccine vector have been conducted in this report. A MeHV-1 iBAC was used in combination with the Tn5 and MuA transposition systems in an attempt to generate MeHV-1 gene interruption libraries. However, these studies demonstrated that Tn5 transposition events into the MeHV-1 genome occurred at unexpectedly low frequencies. Furthermore, characterization of genomic locations of the rare Tn5 transposon insertion events indicated a nonrandom distribution within the viral genome, with seven of the 24 insertions occurring within the gene encoding infected cell protein 4. Although insertion events with the MuA system occurred at higher frequency compared with the Tn5 system, fewer insertion events were generated than has previously been reported with this system. The characterization and distribution of these MeHV-1 iBAC transposed mutants is discussed at both the nucleotide and genomic level, and the properties of the MeHV-1 genome that could influence transposition frequency are discussed. © American Association of Avian Pathologists.

A core metabolic enzyme mediates resistance to phosphine gas

Phosphine is a small redox-active gas that is used to protect global grain reserves, which are threatened by the emergence of phosphine resistance in pest insects. We find that polymorphisms responsible for genetic resistance cluster around the redox-active catalytic disulfide or the dimerization interface of dihydrolipoamide dehydrogenase (DLD) in insects (Rhyzopertha dominica and Tribolium castaneum) and nematodes (Caenorhabditis elegans). DLD is a core metabolic enzyme representing a new class of resistance factor for a redox-active metabolic toxin. It participates in four key steps of core metabolism, and metabolite profiles indicate that phosphine exposure in mutant and wild-type animals affects these steps differently. Mutation of DLD in C. elegans increases arsenite sensitivity. This specific vulnerability may be exploited to control phosphine-resistant insects and safeguard food security.

Sublethal Exposure to Phosphine Decreases Offspring Production in Strongly Phosphine Resistant Female Red Flour Beetles, Tribolium castaneum (Herbst)

The red flour beetle is a cosmopolitan pest of stored grain and stored grain products. The pest has developed resistance to phosphine, the primary chemical used for its control. The reproductive output of survivors from a phosphine treatment is an important element of resistance development but experimental data are lacking. We exposed mated resistant female beetles to 0.135 mg/L of phosphine for 48 h at 25°C. Following one week of recovery we provided two non-exposed males to half of the phosphine exposed females and to half of the non-exposed control females. Females that had been exposed produced significantly fewer offspring than non-exposed females. Females that remained isolated produced significantly fewer offspring than both exposed females with access to males and non-exposed controls (P<0.05). Some females were permanently damaged from exposure to phosphine and did not reproduce even when given access to males. We also examined the additional effects of starvation prior to phosphine exposure on offspring production. Non-exposed starved females experienced a small reduction in mean offspring production in the week following starvation, followed by a recovery in the second week. Females that were starved and exposed to phosphine demonstrated a very significant reduction in offspring production in the first week following exposure which remained significantly lower than that of starved non-exposed females (P<0.05). These results demonstrate a clear sublethal effect of phosphine acting on the female reproductive system and in some individuals this can lead to permanent reproductive damage. Pest population rebound after a fumigation may be slower than expected which may reduce the rate of phosphine resistance development. The results presented strongly suggest that phosphine resistance models should include sublethal effects. © 2012 Ridley et al.

Reduced fungicide use on a new Australian peanut cultivar, highly resistant to the late leaf spot and rust pathogens

Rust (caused by Puccinia arachidis) and late leaf spot (LLS, caused by Mycosphaerella berkeleyi) can cause significant yield losses in Australian peanut crops. Until recently, all commercial peanut varieties were highly susceptible to these pathogens, but the new Australian cultivar Sutherland has significantly higher levels of resistance than the older cultivars. Field trials were conducted at two sites in Queensland to (a) confirm the improved resistance of cv. Sutherland over another commercial cultivar, Menzies, (b) study the effects of timing of first spray, spray interval and cultivar on disease severity and yield, and (c) develop a suitable fungicide management program for cv. Sutherland. In the 2006 and 2007 trials, rust and LLS developed slower and had lower final disease ratings and AUDPC values on unsprayed plots of cv. Sutherland than on cv. Menzies. The timing of the first spray is critical in managing both rust and late leaf spot, with the results demonstrating that the first fungicide spray on cv. Sutherland should be applied as soon as rust and LLS are first seen on cv. Menzies. In most trials spray intervals of 14 days or 21 days were suitable to effectively control rust and LLS. In years with low disease pressure, few, if any, fungicide applications will be needed to manage the diseases, but in other years up to four sprays may be necessary. © Australasian Plant Pathology Society Inc. 2012.

Symptom development proceeds at different rates in susceptible and partially resistant cereal seedlings infected with Fusarium pseudograminearum

Variation in the reaction of cereal cultivars to crown rot caused by Fusarium spp., in particular Fusarium pseudograminearum, was identified over 50 yrs ago, however the parameters and pathways of infection by F. pseudograminearum remain poorly understood. Seedlings of wheat, barley and oat genotypes that differ in susceptibility to crown rot were inoculated with a mixture of F. pseudograminearum isolates. Seedlings were harvested from 7 to 42 days after inoculation and expanded plant parts were rated for severity of visible disease symptoms. Individual leaf sheaths were placed onto nutrient media and fungal colonies emerging from the leaf sheathes were counted to estimate the degree of fungal spread within the host tissue. Significant differences in both the timing and the severity of disease symptoms were observed in the leaf sheath tissues of different host genotypes. Across all genotypes and plant parts examined, the development of visible symptoms closely correlated with the spread of the fungus into that tissue. The degree of infection of the coleoptile and sub-crown internode varied between genotypes, but was unrelated to the putative resistance of the host. In contrast leaf sheath tissues of the susceptible barley cv. Tallon and bread wheat cv. Puseas scored higher disease ratings and consistently showed faster, earlier spread of the fungus into younger tissues than infections of the oat cv. Cleanleaf or the wheat lines 2-49 and CPI 133814. While initial infections usually spread upwards from near the base of the first leaf sheath, the pathogen did not appear to invade younger leaf sheaths only from the base, but rather spread laterally across from older leaf sheaths into younger, subtended leaf sheaths, particularly as disease progressed. Early in the infection of each leaf sheath, disease symptoms in the partially resistant genotypes were less severe than in susceptible genotypes, however as infected leaf sheaths aged, differences between genotypes lessened as disease symptoms approached maximum values. Hence, while visual scoring of disease symptoms on leaf sheaths is a reliable comparative measure of the degree of fungal infection, differences between genotypes in the development of disease symptoms are more reliably assessed using the most recently expanded leaf sheaths.

Differential recognition by tick-resistant cattle of the recombinantly expressed Rhipicephalus microplus serine protease inhibitor-3 (RMS-3)

Rhipicephalus micro plus is an important bovine ectoparasite, widely distributed in tropical and subtropical regions of the world causing large economic losses to the cattle industry. Its success as an ectoparasite is associated with its capacity to disarm the antihemostatic and anti-inflammatory reactions of the host. Serpins are protease inhibitors with an important role in the modulation of host-parasite interactions. The cDNA that encodes for a R. microplus serpin was isolated by RACE and subsequently cloned into the pPICZ alpha A vector. Sequence analysis of the cDNA and predicted amino acid showed that this cDNA has a conserved serpin domain. B- and T-cell epitopes were predicted using bioinformatics tools. The recombinant R. microplus serpin (rRMS-3) was secreted into the culture media of Pichia pastoris after methanol induction at 0.2 mg l(-1) qRT-PCR expression analysis of tissues and life cycle stages demonstrated that RMS-3 was mainly expressed in the salivary glands of female adult ticks. Immunological recognition of the rRMS-3 and predicted B-cell epitopes was tested using tick-resistant and susceptible cattle sera. Only sera from tick-resistant bovines recognized the B-cell epitope AHYNPPPPIEFT (Seq7). The recombinant RMS-3 was expressed in P. pastoris, and ELISA screening also showed higher recognition by tick-resistant bovine sera. The results obtained suggest that RMS-3 is highly and specifically secreted into the bite site of R. microplus feeding on tick-resistant bovines. Capillary feeding of semi-engorged ticks with anti-AHYNPPPPIEFT sheep sera led to an 81.16% reduction in the reproduction capacity of R. microplus. Therefore, it is possible to conclude that R. microplus serpin (RMS-3) has an important role in the host-parasite interaction to overcome the immune responses in resistant cattle. (C) 2012 Elsevier GmbH. All rights reserved.

Changes in weed species since the introduction of glyphosate-resistant cotton

Weed management practices in cotton systems that were based on frequent cultivation, residual herbicides, and some post-emergent herbicides have changed. The ability to use glyphosate as a knockdown before planting, in shielded sprayers, and now over-the-top in glyphosate-tolerant cotton has seen a significant reduction in the use of residual herbicides and cultivation. Glyphosate is now the dominant herbicide in both crop and fallow. This reliance increases the risk of shifts to glyphosate-tolerant species and the evolution of glyphosate-resistant weeds. Four surveys were undertaken in the 2008-09 and 2010-11 seasons. Surveys were conducted at the start of the summer cropping season (November-December) and at the end of the same season (March-April). Fifty fields previously surveyed in irrigated and non-irrigated cotton systems were re-surveyed. A major species shift towards Conyza bonariensis was observed. There was also a minor increase in the prevalence of Sonchus oleraceus. Several species were still present at the end of the season, indicating either poor control and/or late-season germinations. These included C. bonariensis, S. oleraceus, Hibiscus verdcourtii and Hibiscus tridactylites, Echinochloa colona, Convolvulus sp., Ipomea lonchophylla, Chamaesyce drummondii, Cullen sp., Amaranthus macrocarpus, and Chloris virgata. These species, with the exception of E. colona, H. verdcourtii, and H. tridactylites, have tolerance to glyphosate and therefore are likely candidates to either remain or increase in dominance in a glyphosate-based system.

Control by glyphosate and its alternatives of glyphosate-susceptible and glyphosate-resistant Echinochloa colona in the fallow phase of crop rotations in subtropical Australia

Echinochloa colona is the most common grass weed of summer fallows in the grain-cropping systems of the subtropical region of Australia. Glyphosate is the most commonly used herbicide for summer grass control in fallows in this region. The world's first population of glyphosate-resistant E. colona was confirmed in Australia in 2007 and, since then, >70 populations have been confirmed to be resistant in the subtropical region. The efficacy of alternative herbicides on glyphosate-susceptible populations was evaluated in three field experiments and on both glyphosate-susceptible and glyphosate-resistant populations in two pot experiments. The treatments were knockdown and pre-emergence herbicides that were applied as a single application (alone or in a mixture) or as part of a sequential application to weeds at different growth stages. Glyphosate at 720 g ai ha−1 provided good control of small glyphosate-susceptible plants (pre- to early tillering), but was not always effective on larger susceptible plants. Paraquat was effective and the most reliable when applied at 500 g ai ha−1 on small plants, irrespective of the glyphosate resistance status. The sequential application of glyphosate followed by paraquat provided 96–100% control across all experiments, irrespective of the growth stage, and the addition of metolachlor and metolachlor + atrazine to glyphosate or paraquat significantly reduced subsequent emergence. Herbicide treatments have been identified that provide excellent control of small E. colona plants, irrespective of their glyphosate resistance status. These tactics of knockdown herbicides, sequential applications and pre-emergence herbicides should be incorporated into an integrated weed management strategy in order to greatly improve E. colona control, reduce seed production by the sprayed survivors and to minimize the risk of the further development of glyphosate resistance.

Methods to determine resistance to anthelmintics when continuing larval development occurs

The in vivo faecal egg count reduction test (FECRT) is the most commonly used test to detect anthelmintic resistance (AR) in gastrointestinal nematodes (GIN) of ruminants in pasture based systems. However, there are several variations on the method, some more appropriate than others in specific circumstances. While in some cases labour and time can be saved by just collecting post-drench faecal worm egg counts (FEC) of treatment groups with controls, or pre- and post-drench FEC of a treatment group with no controls, there are circumstances when pre- and post-drench FEC of an untreated control group as well as from the treatment groups are necessary. Computer simulation techniques were used to determine the most appropriate of several methods for calculating AR when there is continuing larval development during the testing period, as often occurs when anthelmintic treatments against genera of GIN with high biotic potential or high re-infection rates, such as Haemonchus contortus of sheep and Cooperia punctata of cattle, are less than 100% efficacious. Three field FECRT experimental designs were investigated: (I) post-drench FEC of treatment and controls groups, (II) pre- and post-drench FEC of a treatment group only and (III) pre- and post-drench FEC of treatment and control groups. To investigate the performance of methods of indicating AR for each of these designs, simulated animal FEC were generated from negative binominal distributions with subsequent sampling from the binomial distributions to account for drench effect, with varying parameters for worm burden, larval development and drench resistance. Calculations of percent reductions and confidence limits were based on those of the Standing Committee for Agriculture (SCA) guidelines. For the two field methods with pre-drench FEC, confidence limits were also determined from cumulative inverse Beta distributions of FEC, for eggs per gram (epg) and the number of eggs counted at detection levels of 50 and 25. Two rules for determining AR: (1) %reduction (%R) < 95% and lower confidence limit <90%; and (2) upper confidence limit <95%, were also assessed. For each combination of worm burden, larval development and drench resistance parameters, 1000 simulations were run to determine the number of times the theoretical percent reduction fell within the estimated confidence limits and the number of times resistance would have been declared. When continuing larval development occurs during the testing period of the FECRT, the simulations showed AR should be calculated from pre- and post-drench worm egg counts of an untreated control group as well as from the treatment group. If the widely used resistance rule 1 is used to assess resistance, rule 2 should also be applied, especially when %R is in the range 90 to 95% and resistance is suspected.

Wheat biomass and yield increased when populations of the root-lesion nematode (Pratylenchus thornei) were reduced through sequential rotation of partially resistant winter and summer crops

The root-lesion nematode, Pratylenchus thornei, can reduce wheat yields by >50%. Although this nematode has a broad host range, crop rotation can be an effective tool for its management if the host status of crops and cultivars is known. The summer crops grown in the northern grain region of Australia are poorly characterised for their resistance to P. thornei and their role in crop sequencing to improve wheat yields. In a 4-year field experiment, we prepared plots with high or low populations of P. thornei by growing susceptible wheat or partially resistant canaryseed (Phalaris canariensis); after an 11-month, weed-free fallow, several cultivars of eight summer crops were grown. Following another 15-month, weed-free fallow, P. thornei-intolerant wheat cv. Strzelecki was grown. Populations of P. thornei were determined to 150 cm soil depth throughout the experiment. When two partially resistant crops were grown in succession, e.g. canaryseed followed by panicum (Setaria italica), P. thornei populations were <739/kg soil and subsequent wheat yields were 3245 kg/ha. In contrast, after two susceptible crops, e.g. wheat followed by soybean, P. thornei populations were 10 850/kg soil and subsequent wheat yields were just 1383 kg/ha. Regression analysis showed a linear, negative response of wheat biomass and grain yield with increasing P. thornei populations and a predicted loss of 77% for biomass and 62% for grain yield. The best predictor of wheat yield loss was P. thornei populations at 0-90 cm soil depth. Crop rotation can be used to reduce P. thornei populations and increase wheat yield, with greatest gains being made following two partially resistant crops grown sequentially.

Fluoroquinolone-resistant extraintestinal pathogenic Escherichia coli, including O25b-ST131, isolated from faeces of hospitalized dogs in an Australian veterinary referral centre

To determine rates of carriage of fluoroquinolone-resistant Escherichia coli and extraintestinal pathogenic E. coli (ExPEC) among dogs in a specialist referral hospital and to examine the population structure of the isolates. Fluoroquinolone-resistant faecal E. coli isolates (n232, from 23 of 123 dogs) recovered from hospitalized dogs in a veterinary referral centre in Sydney, Australia, over 140 days in 2009 were characterized by phylogenetic grouping, virulence genotyping and random amplified polymorphic DNA (RAPD) analysis. The RAPD dendrogram for representative isolates showed one group B2-associated cluster and three group D-associated clusters; each contained isolates with closely related ExPEC-associated virulence profiles. All group B2 faecal isolates represented the O25b-ST131 clonal group and were closely related to recent canine extraintestinal ST131 clinical isolates from the east coast of Australia by RAPD analysis. Hospitalized dogs may carry fluoroquinolone-resistant ExPEC in their faeces, including those representing O25b-ST131.

Managing patches of glyphosate resistant Echinochloa colona: can they be eradicated?

Glyphosate-resistant Echinochloa colona L. (Link) is becoming common in non-irrigated cotton systems. Echinochloa colona is a small seeded species that is not wind-blown and has a relatively short seed bank life. These characteristics make it a potential candidate to attempt to eradicate resistant populations when they are detected. A long term systems experiment was developed to determine the feasibility of attempting to eradicate glyphosate resistant populations in the field. To this point the established Best Management Practice (BMP) strategy of two non-glyphosate actions in crop and fallow have been sufficient to significantly reduce the numbers of plants emerging, and remaining at the end of the season. Additional eradication treatments showed slight improvement on the BMP strategy, however were not significant overall. The effects of additional eradication tactics are expected to be more noticeable as the seed bank gets driven down in subsequent seasons.

Developing effective fumigation protocols to manage strongly phosphine-resistant Cryptolestes ferrugineus (Stephens) (Coleoptera: Laemophloeidae)

BACKGROUND The emergence of high levels of resistance in Cryptolestes ferrugineus (Stephens) in recent years threatens the sustainability of phosphine, a key fumigant used worldwide to disinfest stored grain. We aimed at developing robust fumigation protocols that could be used in a range of practical situations to control this resistant pest. RESULTS Values of the lethal time to kill 99.9% (LT99.9, in days) of mixed-age populations, containing all life stages, of a susceptible and a strongly resistant C. ferrugineus population were established at three phosphine concentrations (1.0, 1.5 and 2.0 mg L−1) and three temperatures (25, 30 and 35 °C). Multiple linear regression analysis revealed that phosphine concentration and temperature both contributed significantly to the LT99.9 of a population (P < 0.003, R2 = 0.92), with concentration being the dominant variable, accounting for 75.9% of the variation. Across all concentrations, LT99.9 of the strongly resistant C. ferrugineus population was longest at the lowest temperature and shortest at the highest temperature. For example, 1.0 mg L−1 of phosphine is required for 20, 15 and 15 days, 1.5 mg L−1 for 12, 11 and 9 days and 2.0 mg L−1 for 10, 7 and 6 days at 25, 30 and 35 °C, respectively, to achieve 99.9% mortality of the strongly resistant C. ferrugineus population. We also observed that phosphine concentration is inversely proportional to fumigation period in regard to the population extinction of this pest. CONCLUSION The fumigation protocols developed in this study will be used in recommending changes to the currently registered rates of phosphine in Australia towards management of strongly resistant C. ferrugineus populations, and can be repeated in any country where this type of resistance appears.

Markers for seedling and adult plant crown rot resistance in four partially resistant bread wheat sources

Key message: QTLidentified for seedling and adult plant crown rot resistance in four partially resistant hexaploid wheat sources. PCR-based markers identified for use in marker-assisted selection. Abstract: Crown rot, caused by Fusarium pseudograminearum, is an important disease of wheat in many wheat-growing regions globally. Complete resistance to infection by F. pseudograminearum has not been observed in a wheat host, but germplasm with partial resistance to this pathogen has been identified. The partially resistant wheat hexaploid germplasm sources 2-49, Sunco, IRN497 and CPI133817 were investigated in both seedling and adult plant field trials to identify markers associated with the resistance which could be used in marker-assisted selection programs. Thirteen different quantitative trait loci (QTL) conditioning crown rot resistance were identified in the four different sources. Some QTL were only observed in seedling trials whereas others appeared to be adult plant specific. For example while the QTL on chromosomes 1AS, 1BS, and 4BS contributed by 2-49 and on 2BS contributed by Sunco were detected in both seedling and field trials, the QTL on 1DL present in 2-49 and the QTL on 3BL in IRN497 were only detected in seedling trials. Genetic correlations between field trials of the same population were strong, as were correlations between seedling trials of the same population. Low to moderate correlations were observed between seedling and field trials. Flanking markers, most of which are less than 10 cM apart, have now been identified for each of the regions associated with crown rot resistance.

Do Human Extraintestinal Escherichia coli Infections Resistant to Expanded-Spectrum Cephalosporins Originate From Food-Producing Animals? A Systematic Review

To find out whether food-producing animals (FPAs) are a source of extraintestinal expanded-spectrum cephalosporin-resistant Escherichia coli (ESCR-EC) infections in humans, Medline, Embase, and the Cochrane Database of Systematic Reviews were systematically reviewed. Thirty-four original, peer-reviewed publications were identified for inclusion. Six molecular epidemiology studies supported the transfer of resistance via whole bacterium transmission (WBT), which was best characterized among poultry in the Netherlands. Thirteen molecular epidemiology studies supported transmission of resistance via mobile genetic elements, which demonstrated greater diversity of geography and host FPA. Seventeen molecular epidemiology studies did not support WBT and two did not support mobile genetic element-mediated transmission. Four observational epidemiology studies were consistent with zoonotic transmission. Overall, there is evidence that a proportion of human extraintestinal ESCR-EC infections originate from FPAs. Poultry, in particular, is probably a source, but the quantitative and geographical extent of the problem is unclear and requires further investigation.