Strong resistance to phosphine in the rusty grain beetle, Cryptolestes ferrugineus (Stephens) (Coleoptera: Laemophloeidae): its characterisation, a rapid assay for diagnosis and its distribution in Australia

BACKGROUND: The recent development of very high resistance to phosphine in rusty grain beetle, Cryptolestes ferrugineus (Stephens), seriously threatens stored-grain biosecurity. The aim was to characterise this resistance, to develop a rapid bioassay for its diagnosis to support pest management and to document the distribution of resistance in Australia in 20072011. RESULTS: Bioassays of purified laboratory reference strains and field-collected samples revealed three phenotypes: susceptible, weakly resistant and strongly resistant. With resistance factors of > 1000 x , resistance to phosphine expressed by the strong resistance phenotype was higher than reported for any stored-product insect species. The new time-to-knockdown assay rapidly and accurately diagnosed each resistance phenotype within 6 h. Although less frequent in western Australia, weak resistance was detected throughout all grain production regions. Strong resistance occurred predominantly in central storages in eastern Australia. CONCLUSION: Resistance to phosphine in the rusty grain beetle is expressed through two identifiable phenotypes: weak and strong. Strong resistance requires urgent changes to current fumigation dosages. The development of a rapid assay for diagnosis of resistance enables the provision of same-day advice to expedite resistance management decisions. (c) 2012 Commonwealth of Australia. Published by John Wiley & Sons, Ltd.

Microbial indicators related to yield and disease and changes in soil microbial community structure with ginger farm management practices

TRFLP (terminal restriction fragment length polymorphism) was used to assess whether management practices that improved disease suppression and/or yield in a 4-year ginger field trial were related to changes in soil microbial community structure. Bacterial and fungal community profiles were defined by presence and abundance of terminal restriction fragments (TRFs), where each TRF represents one or more species. Results indicated inclusion of an organic amendment and minimum tillage increased the relative diversity of dominant fungal populations in a system dependant way. Inclusion of an organic amendment increased bacterial species richness in the pasture treatment. Redundancy analysis showed shifts in microbial community structure associated with different management practices and treatments grouped according to TRF abundance in relation to yield and disease incidence. ANOVA also indicated the abundance of certain TRFs was significantly affected by farming system management practices, and a number of these TRFs were also correlated with yield or disease suppression. Further analyses are required to determine whether identified TRFs can be used as general or soil-type specific bio-indicators of productivity (increased and decreased) and Pythium myriotylum suppressiveness.

Pericarps retained in the tree canopy and stomatal abundance are components of resistance to husk spot caused by Pseudocercospora macadamiae in macadamia

Pseudocercospora macadamiae Beilharz, Mayers and Pascoe infects macadamia fruit via stomata causing husk spot disease. Information on the variability of fruit stomatal abundance, its association with diseased fruit pericarps (sticktights) that are retained in the tree canopy, and its influence on the husk spot intensity (incidence, severity and lesion number) among macadamia genotypes is lacking. We examined a total of 230 macadamia trees comprising 19 cultivars, 56 wild germplasm accessions and 40 breeding progeny, for the prevalence of sticktights and husk spot intensity over three production seasons. We observed a strong association between the prevalence of sticktights and disease intensity indicating its usefulness as a predictor of husk spot and as a useful phenotypic trait for husk spot resistance selection in breeding programmes. Similarly, stomatal abundance varied among macadamia genotypes, and a significant linear relationship (P < 0.001; 93%) was observed between fruit stomatal abundance and husk spot for all the macadamia genotypes analysed, confirming the utility of that trait for disease resistance screening. The genotypes were grouped into disease resistance groups. Correlations between fruit stomatal abundance, disease intensity and prevalence of sticktights revealed that the numbers of sticktights, and relative stomatal abundance were the main factors influencing the intensity of husk spot among macadamia genotypes. This is the first comprehensive study of natural variation of stomatal abundance in Macadamia species that reveals genetic variation, and provides relevant relationships with disease intensity and the prevalence of sticktights. The phenotypic plant traits indentified in this study may serve as selection tools for disease resistance screening in macadamia breeding programmes.

Origin of leaf rust adult plant resistance gene Rph20 in barley

Rph20 is the only reported, simply inherited gene conferring moderate to high levels of adult plant resistance (APR) to leaf rust (Puccinia hordei Otth) in barley (Hordeum vulgare L.). Key parental genotypes were examined to determine the origin of Rph20 in two-rowed barley. The Dutch cultivar 'Vada' (released in the 1950s) and parents, 'Hordeum laevigatum' and 'Gull' ('Gold'), along with the related cultivar 'Emir' (a derivative of 'Delta'), were assessed for APR to P. hordei in a disease screening nursery. The marker bPb-0837-PCR, co-located with Rph20 on the short arm of chromosome 5H (5HS), was used to screen genotypes for the resistance allele, Rph20.ai. Results from phenotypic assessment and DNA analysis confirmed that Rph20 originated from the landrace 'H. laevigatum' (i.e., Hordeum vulgare subsp. vulgare). Tracing back this gene through the pedigrees of two-rowed barley cultivars, indicated that Rph20 has contributed APR to P. hordei for more than 60 years. Although there have been no reports of an Rph20-virulent pathotype, the search for alternative sources of APR should continue to avoid widespread reliance upon a single resistance factor.

Mapping Quantitative Trait Loci for Partial Resistance to Powdery Mildew in an Australian Barley Population

Genomic regions influencing resistance to powdery mildew [Blumeria graminis (DC.) E.O. Speer f. sp. hordei Em. Marchal] were detected in a doubled haploid (DH) barley (Hordeum vulgare L.) population derived from a cross between the breeding line ND24260 and cultivar Flagship when evaluated across four field environments in Australia and Uruguay. Significant quantitative trait loci (OIL) for resistance to B. graminis were detected on six of the seven chromosomes (1H, 2H, 3H, 4H, 5H, and 7H). A QTL with large effect donated by ND24260 mapped to the short arm of chromosome 1H (1 HS) conferring near immunity to B. graminis in Australia but was ineffective in Uruguay. Three OIL donated by Flagship contributed partial resistance to B. graminis and were detected in at least two environments. These OIL were mapped to chromosomes 3H, 4H, and 5H (5HS) accounting for up to 18.6, 3.4, and 8.8% phenotypic variation, respectively. The 5HS QTL contributed partial resistance to B. graminis in all field environments in both Australia and Uruguay and aligned with the genomic region of Rph20, a gene conferring adult plant resistance (APR) to leaf rust (Puccinia hordei Otth), which is found in some cultivars having Vada' or 'Emir' in their parentage. Selection for favorable marker haplotypes within the 3H, 4H, and 5H QTL regions can be performed even in the presence of single (major) gene resistance. Pyramiding such QTL may provide an effective and potentially durable form of resistance to B. graminis.

Rapid phenotyping for adult-plant resistance to stripe rust in wheat

Stripe or yellow rust (YR) is a significant problem in wheat crops worldwide. The deployment of adult-plant resistance (APR) genes in wheat cultivars is considered a sustainable management strategy, as these genes confer partial resistance that is usually non-race specific. Screening for APR typically involves assessment of adult plants in the field, where expression may be influenced by environmental factors. We report a high-throughput screening method for YR APR that can be used to assess fixed lines or segregating populations grown under controlled environmental conditions (CEC). Inoculation of 3-week-old wheat plants from lines with known APR responses to YR, when grown under constant light and temperature, provided disease responses typical of adult plants. Two F-2 populations ('H45' x 'ST93' and 'Wyalkatchem' x 'ST93') segregating for APR were assessed under both CEC and field conditions. These populations showed similar variation in disease response and lines assessed in both environments attained similar rankings. Phenotypic screening using CEC and continuous light provides an opportunity to accelerate the development of new wheat cultivars with durable resistance.

Phosphine Resistance in the Rust Red Flour Beetle, Tribolium castaneum (Coleoptera: Tenebrionidae): Inheritance, Gene Interactions and Fitness Costs

The recent emergence of heritable high level resistance to phosphine in stored grain pests is a serious concern among major grain growing countries around the world. Here we describe the genetics of phosphine resistance in the rust red flour beetle Tribolium castaneum (Herbst), a pest of stored grain as well as a genetic model organism. We investigated three field collected strains of T. castaneum viz., susceptible (QTC4), weakly resistant (QTC1012) and strongly resistant (QTC931) to phosphine. The dose-mortality responses of their test- and inter-cross progeny revealed that most resistance was conferred by a single major resistance gene in the weakly (3.2x) resistant strain. This gene was also found in the strongly resistant (431x) strain, together with a second major resistance gene and additional minor factors. The second major gene by itself confers only 12-206x resistance, suggesting that a strong synergistic epistatic interaction between the genes is responsible for the high level of resistance (431x) observed in the strongly resistant strain. Phosphine resistance is not sex linked and is inherited as an incompletely recessive, autosomal trait. The analysis of the phenotypic fitness response of a population derived from a single pair inter-strain cross between the susceptible and strongly resistant strains indicated the changes in the level of response in the strong resistance phenotype; however this effect was not consistent and apparently masked by the genetic background of the weakly resistant strain. The results from this work will inform phosphine resistance management strategies and provide a basis for the identification of the resistance genes.

Interaction between production characteristics and postharvest performance and practices for fresh fruit

Significant interactions have been demonstrated between production factors and postharvest quality of fresh fruit. Accordingly, there is an attendant need for adaptive postharvest actions to modulate preharvest effects. The most significant preharvest effects appear to be mediated through mineral nutrition influences on the physical characteristics of fruit. Examples of specific influencers include fertilisers, water availability, rootstock, and crop load effects on fruit quality attributes such as skin colour, susceptibility to diseases and physiological disorders, and fruit nutritional composition. Also, rainfall before and during harvest can markedly affect fruit susceptibility to skin blemishes, physical damage, and diseases. Knowledge of preharvest-postharvest interactions can help determine the basis for variability in postharvest performance and thereby allow refinement of postharvest practices to minimise quality loss after harvest. This knowledge can be utilised in predictive management systems. Such systems can benefit from characterisation of fruit nutritional status, particularly minerals, several months before and/or at harvest to allow informed decisions on postharvest handling and marketing options. Other examples of proactive management practices include adjusting harvesting and packing systems to account for rainfall effects before and/or during harvest. Improved understanding of preharvest-postharvest interactions is contributing to the delivery of consistently higher quality of fruit to consumers. This paper focuses on the state of knowledge for sub-tropical and tropical fruits, in particular avocado and mango.

The rph2 Gene Is Responsible for High Level Resistance to Phosphine in Independent Field Strains of Rhyzopertha dominica

The lesser grain borer Rhyzopertha dominica (F.) is one of the most destructive insect pests of stored grain. This pest has been controlled successfully by fumigation with phosphine for the last several decades, though strong resistance to phosphine in many countries has raised concern about the long term usefulness of this control method. Previous genetic analysis of strongly resistant (SR) R. dominica from three widely geographically dispersed regions of Australia, Queensland (SRQLD), New South Wales (SRNSW) and South Australia (SRSA), revealed a resistance allele in the rph1 gene in all three strains. The present study confirms that the rph1 gene contributes to resistance in a fourth strongly resistant strain, SR2(QLD), also from Queensland. The previously described rph2 gene, which interacts synergistically with rph1 gene, confers strong resistance on SRQLD and SRNSW. We now provide strong circumstantial evidence that weak alleles of rph2, together with rph1, contribute to the strong resistance phenotypes of SRSA and SR2(QLD). To test the notion that rph1 and rph2 are solely responsible for the strong resistance phenotype of all resistant R. dominica, we created a strain derived by hybridising the four strongly resistant lines. Following repeated selection for survival at extreme rates of phosphine exposure, we found only slightly enhanced resistance. This suggests that a single sequence of genetic changes was responsible for the development of resistance in these insects.

The rph1 Gene Is a Common Contributor to the Evolution of Phosphine Resistance in Independent Field Isolates of Rhyzopertha Dominica

Phosphine is the only economically viable fumigant for routine control of insect pests of stored food products, but its continued use is now threatened by the world-wide emergence of high-level resistance in key pest species. Phosphine has a unique mode of action relative to well-characterised contact pesticides. Similarly, the selective pressures that lead to resistance against field sprays differ dramatically from those encountered during fumigation. The consequences of these differences have not been investigated adequately. We determine the genetic basis of phosphine resistance in Rhyzopertha dominica strains collected from New South Wales and South Australia and compare this with resistance in a previously characterised strain from Queensland. The resistance levels range from 225 and 100 times the baseline response of a sensitive reference strain. Moreover, molecular and phenotypic data indicate that high-level resistance was derived independently in each of the three widely separated geographical regions. Despite the independent origins, resistance was due to two interacting genes in each instance. Furthermore, complementation analysis reveals that all three strains contain an incompletely recessive resistance allele of the autosomal rph1 resistance gene. This is particularly noteworthy as a resistance allele at rph1 was previously proposed to be a necessary first step in the evolution of high-level resistance. Despite the capacity of phosphine to disrupt a wide range of enzymes and biological processes, it is remarkable that the initial step in the selection of resistance is so similar in isolated outbreaks.

Description of the pig production systems, biosecurity practices and herd health providers in two provinces with high swine density in the Philippines

A cross-sectional study was conducted between October 2011 and March 2012 in two major pig producing provinces in the Philippines. Four hundred and seventy one pig farms slaughtering finisher pigs at government operated abattoirs participated in this study. The objectives of this study were to group: (a) smallholder (S) and commercial (C) production systems into patterns according to their herd health providers (HHPs), and obtain descriptive information about the grouped S and C production systems; and (b) identify key HHPs within each production system using social network analysis. On-farm veterinarians, private consultants, pharmaceutical company representatives, government veterinarians, livestock and agricultural technicians, and agricultural supply stores were found to be actively interacting with pig farmers. Four clusters were identified based on production system and their choice of HHPs. Differences in management and biosecurity practices were found between S and C clusters. Private HHPs provided a service to larger C and some larger S farms, and have little or no interaction with the other HHPs. Government HHPs provided herd health service mainly to S farms and small C farms. Agricultural supply stores were identified as a dominant solitary HHP and provided herd health services to the majority of farmers. Increased knowledge of the routine management and biosecurity practices of S and C farmers and the key HHPs that are likely to be associated with those practices would be of value as this information could be used to inform a risk-based approach to disease surveillance and control. © 2014 Elsevier B.V.

The persistence of biofilm-associated antibiotic resistance of Staphylococcus aureus isolated from clinical bovine mastitis cases in Australia

The aim of this investigation was to determine the persistence of biofilm-associated antibiotic resistance developed by methicillin-sensitive Staphylococcus aureus (MSSA), of different capsular types, during biofilm formation. Because of superiority of the tissue culture plate (TCP) over the Congo Red Agar (CRA) method for measuring biofilm formation, it was used to determine the persistence of the antibiotic resistance developed by the isolates in biofilms. The antibiotic resistance was found to persist for 3-4 wk post-propagation as planktonic subcultures. Interestingly, some strains even developed resistance to vancomycin and/or teicoplanin. However, no association of either biofilm formation or persistent antibiotic resistance with the major capsular phenotype was observed. These observations highlight the potential significance of (a) determining the antibiograms of S. aureus subcultured from biofilms developed in vitro using the TCP method as well as from planktonic cultures for formulation of an optimal therapeutic strategy, and (b) continuing to identify predominant non-capsular antigens contributing to biofilm formation, regardless of the capsular phenotype for the development of an effective potentially broad-spectrum vaccine for prevention of bovine mastitis caused by S. aureus.

Implications of methoprene resistance for managing Rhyzopertha dominica (F.) in stored grain

Commercial formulations of methoprene have been used in a number of countries such as Australia and the USA to provide long-term protection to grain from a range of storage pests. The level of resistance in Rhyzopertha dominica (F.), the lesser grain borer, was investigated in laboratory experiments by direct exposure of adults on treated wheat. Adults of a reference homozygous resistant strain of R.dominica were exposed to treatments of 0, 2, 3, 5, 10, 20 and 40kg-1 of s-methoprene for 7 days. Mortality and progeny production were dose dependent with 98.7% mortality and complete progeny suppression at 40mgkg-1, which is 67 times the registered rate at which s-methoprene is applied as a grain protectant in Australia (0.6mgkg-1) and eight times the rate which has been used in the USA (5mgkg-1). This strain was also tested by adding adults to wheat treated at 0, 1, 3, 10 and 30mgkg-1 and determining the number of adults (progeny plus original parental adults) after 6, 8, 10 or 12 weeks of continuous exposure. The effect of s-methoprene was consistent regardless of the number of weeks of continuous exposure and average population suppression was 99.5% at 30mgkg-1. Screening of 162 field samples collected from southeast Australia in 2009 showed that 93% of samples reproduced when exposed to wheat treated with the Australian registered rate of 0.6mgkg-1. When four unselected resistant field samples from this region were tested, progeny production but not mortality was dose dependent, and all four samples produced live progeny at the highest dose of 30mgkg-1. Our results show that methoprene resistance is a serious threat to the management of R.dominica, and that strategies need to be developed to minimise the further development and spread of resistance. © 2013.

A Bayesian hurdle model for analysis of an insect resistance monitoring database

Motivated by the analysis of the Australian Grain Insect Resistance Database (AGIRD), we develop a Bayesian hurdle modelling approach to assess trends in strong resistance of stored grain insects to phosphine over time. The binary response variable from AGIRD indicating presence or absence of strong resistance is characterized by a majority of absence observations and the hurdle model is a two step approach that is useful when analyzing such a binary response dataset. The proposed hurdle model utilizes Bayesian classification trees to firstly identify covariates and covariate levels pertaining to possible presence or absence of strong resistance. Secondly, generalized additive models (GAMs) with spike and slab priors for variable selection are fitted to the subset of the dataset identified from the Bayesian classification tree indicating possibility of presence of strong resistance. From the GAM we assess trends, biosecurity issues and site specific variables influencing the presence of strong resistance using a variable selection approach. The proposed Bayesian hurdle model is compared to its frequentist counterpart, and also to a naive Bayesian approach which fits a GAM to the entire dataset. The Bayesian hurdle model has the benefit of providing a set of good trees for use in the first step and appears to provide enough flexibility to represent the influence of variables on strong resistance compared to the frequentist model, but also captures the subtle changes in the trend that are missed by the frequentist and naive Bayesian models. © 2014 Springer Science+Business Media New York.