Optimising nitrogen fixation of grain and forage legumes in the northern region

This project encompasses laboratory, glasshouse and field research to improve N fixation in grain and forage legumes in the northern region and assess compatability of rhizobial strains with current and new legume varieties.

Enhancing the performance of irrigated cotton and grain in Central Queensland

R&D to facilitate incorporation of grain and pulse crop phases in Central Queensland irrigated cotton monoculture systems and improve profitability of regional cropping systems.

Phosphine fumigation of cool grain

The biosecurity problem addressed was the need to understand and evaluate phosphine fumigation of cool grain (i.e. 20°C or less) as a means of controlling resistant biotypes of insect pests of stored grain which are major EPPs threatening the grain industry. The benefits of cooling and phosphine fumigation are that cooling preserves grain quality and reduces insect population growth, and phosphine kills insects and has a residue free status in all major markets. The research objectives were to: - conduct laboratory experiments on phosphine efficacy against resistant insects in cool grain, and determine times to population extinction. - conduct laboratory experiments on phosphine sorption in cool grain and quantify. - complete fumigation trials in three states (Queensland, WA and NSW) on cool grain stored insealed farm silos. - make recommendations for industry on effective phosphine fumigation of cool grain. Phosphine is used by growers and other stakeholders in the grain industry to meet domesticand international demands for insect-free grain. The project aim was to generate new information on the performance of phosphine fumigation of cool grain relevant to resistant biotypes. Effective control of resistant biotypes using phosphine to fumigate cool grain will benefit growers and other sectors of the grain industry, needing to fumigate grain in the cooler months of the year, or grain that has been cooled using aeration.

Flat grain beetle fumigation protocol

Flat grain beetle (FGB) is a major emergency plant pest (EPP) of stored grain in Australia. Populations of FGB have recently developed high level resistance to phosphine (the only viable fumigant available for non-quarantine use) resulting in control failures with current dosage regimes. As there is no practical alternative to phosphine, failure to control FGB with phosphine places at risk market access for Australian grain worth up to $7 billion in annual trade. Therefore there is an urgent need to develop appropriate phosphine fumigation protocols to eradicate outbreaks of strongly resistant FGB. Research outcomes: - Characterisation of high resistance to phosphine in flat grain beetles (FGB) for the first time internationally. - Establishment of fumigation protocols and an eradication strategy that will enable industry to eradicate infestations of phosphine-resistant flat grain beetle and prevent or delay further selection for resistance to phosphine. - Development of a rapid test to detect highly resistant FGB. -Facilitate continued market access of Australian grain.

CRC50089 Grain Insect Ecology

Resistance to phosphine in target pests threatens market access for Australian grain. While the grains industry is now attempting to develop an effective and sustainable strategy to manage this resistance, action is severely limited by significant gaps in our knowledge of the key ecological factors that influence the development of resistance. There is a need to research this information as a foundation for a rational approach to managing phosphine resistance in the Australian grains industry. Research outcomes: The project has provided critical research methodologies and preliminary data to fill the large gaps in our knowledge of the ecology of two key pests, Rhyzopertha dominica and Tribolium castaneum, and how this may drive the development of phosphine resistance. This information will contribute to the groundwork for future research needed to provide a scientific basis for a rational resistance management strategy.

AG15 (Sustainable Agricultural State-level Investment Program) natural resource management extension in irrigated cotton and grain

Natural Resource Management project developing reources and supporting best practice management for irrigated cotton and grain growers in Queensland.

Determining changes in the distribution and abundance of a Rhyzopertha dominica phosphine resistance allele in farm grain storages using a DNA marker

BACKGROUND: The lesser grain borer, Rhyzopertha dominica (F.), is a highly destructive pest of stored grain that is strongly resistant to the fumigant phosphine (PH3). Phosphine resistance is due to genetic variants at the rph2 locus that alter the function of the dihydrolipoamide dehydrogenase (DLD) gene. This discovery now enables direct detection of resistance variants at the rph2 locus in field populations. RESULTS: A genotype assay was developed for direct detection of changes in distribution and frequency of a phosphine resistance allele in field populations of R. dominica. Beetles were collected from ten farms in south-east Queensland in 2006 and resampled in 2011. Resistance allele frequency increased in the period from 2006 to 2011 on organic farms with no history of phosphine use, implying that migration of phosphine-resistant R. dominica had occurred from nearby storages. CONCLUSION: Increasing resistance allele frequencies on organic farms suggest local movement of beetles and dispersal of insects from areas where phosphine has been used. This research also highlighted for the first time the utility of a genetic DNA marker in accurate and rapid determination of the distribution of phosphine-resistant insects in the grain value chain. Extending this research over larger landscapes would help in identifying resistance problems and enable timely pest management decisions. © 2013 Society of Chemical Industry © 2013 Society of Chemical Industry 69 6 June 2013 10.1002/ps.3514 Rapid Report Rapid Report © 2013 Society of Chemical Industry.

Individual-based modelling of the efficacy of fumigation tactics to control lesser grain borer (Rhyzopertha dominica) in stored grain

Increasing resistance to phosphine (PH 3) in insect pests, including lesser grain borer (Rhyzopertha dominica) has become a critical issue, and development of effective and sustainable strategies to manage resistance is crucial. In practice, the same grain store may be fumigated multiple times, but usually for the same exposure period and concentration. Simulating a single fumigation allows us to look more closely at the effects of this standard treatment.We used an individual-based, two-locus model to investigate three key questions about the use of phosphine fumigant in relation to the development of PH 3 resistance. First, which is more effective for insect control; long exposure time with a low concentration or short exposure period with a high concentration? Our results showed that extending exposure duration is a much more efficient control tactic than increasing the phosphine concentration. Second, how long should the fumigation period be extended to deal with higher frequencies of resistant insects in the grain? Our results indicated that if the original frequency of resistant insects is increased n times, then the fumigation needs to be extended, at most, n days to achieve the same level of insect control. The third question is how does the presence of varying numbers of insects inside grain storages impact the effectiveness of phosphine fumigation? We found that, for a given fumigation, as the initial population number was increased, the final survival of resistant insects increased proportionally. To control initial populations of insects that were n times larger, it was necessary to increase the fumigation time by about n days. Our results indicate that, in a 2-gene mediated resistance where dilution of resistance gene frequencies through immigration of susceptibles has greater effect, extending fumigation times to reduce survival of homozygous resistant insects will have a significant impact on delaying the development of resistance. © 2012 Elsevier Ltd.

Dosage consistency is the key factor in avoiding evolution of resistance to phosphine and population increase in stored-grain pests

BACKGROUND Control of pests in stored grain and the evolution of resistance to pesticides are serious problems worldwide. A stochastic individual-based two-locus model was used to investigate the impact of two important issues, the consistency of pesticide dosage through the storage facility and the immigration rate of the adult pest, on overall population control and avoidance of evolution of resistance to the fumigant phosphine in an important pest of stored grain, the lesser grain borer. RESULTS A very consistent dosage maintained good control for all immigration rates, while an inconsistent dosage failed to maintain control in all cases. At intermediate dosage consistency, immigration rate became a critical factor in whether control was maintained or resistance emerged. CONCLUSION Achieving a consistent fumigant dosage is a key factor in avoiding evolution of resistance to phosphine and maintaining control of populations of stored-grain pests; when the dosage achieved is very inconsistent, there is likely to be a problem regardless of immigration rate. © 2012 Society of Chemical Industry

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.

Networks of stored grain diseases and pests: Strategies for sampling and mitigation

Pathogens and pests of stored grains move through complex dynamic networks linking fields, farms, and bulk storage facilities. Human transport and other forms of dispersal link the components of this network. A network model for pathogen and pest movement through stored grain systems is a first step toward new sampling and mitigation strategies that utilize information about the network structure. An understanding of network structure can be applied to identifying the key network components for pathogen or pest movement through the system. For example, it may be useful to identify a network node, such as a local grain storage facility, through which grain from a large number of fields will be accumulated and move through the network. This node may be particularly important for sampling and mitigation. In some cases more detailed information about network structure can identify key nodes that link two large sections of the network, such that management at the key nodes will greatly reduce the risk of spread between the two sections. In addition to the spread of particular species of pathogens and pests, we also evaluate the spread of problematic subpopulations, such as subpopulations with pesticide resistance. We present an analysis of stored grain pathogen and pest networks for Australia and the United States.

Formulation and characterization of drug-loaded microparticles using distiller’s dried grain kafirin

Kafirin, a protein extracted from sorghum grain has been formulated into microparticles, and proposed for use as a delivery system due to the resistance of kafirin to upper gastrointestinal digestion. However, extracting kafirin from sorghum “distiller’s dried grains with solubles” (DDGS) may be more efficient as the carbohydrate component has been removed by fermentation. This study investigated the properties and use of kafirin extracted from DDGS to formulate microparticles. Prednisolone, an anti-inflammatory drug that could benefit from a delayed and targeted delivery system to the colon, was loaded into DDGS kafirin microparticles by phase separation using sodium chloride. Scanning electron micrographs revealed that the empty and prednisolone-loaded microparticles were round in shape and varied in size. Surface binding studies indicated prednisolone was loaded within the microparticles rather than being solely bound on the surface. These findings demonstrate DDGS kafirin can be formulated into microparticles and loaded with medication. Future studies could investigate the potential applications of DDGS kafirin microparticles as an orally administered targeted drug-delivery system.

Formulation and characterization of drug-loaded microparticles using distiller’s dried grain kafirin

Kafirin, a protein extracted from sorghum grain has been formulated into microparticles, and proposed for use as a delivery system due to the resistance of kafirin to upper gastrointestinal digestion. However, extracting kafirin from sorghum distillers dried grains with solubles (DDGS) may be more efficient as the carbohydrate component has been removed by fermentation. This study investigated the properties and use of kafirin extracted from DDGS to formulate microparticles. Prednisolone, an anti-inflammatory drug that could benefit from a delayed and targeted delivery system to the colon, was loaded into DDGS kafirin microparticles by phase separation using sodium chloride. Scanning electron micrographs revealed that the empty and prednisolone-loaded microparticles were round in shape and varied in size. Surface binding studies indicated prednisolone was loaded within the microparticles rather than being solely bound on the surface. These findings demonstrate DDGS kafirin can be formulated into microparticles and loaded with medication. Future studies could investigate the potential applications of DDGS kafirin microparticles as an orally administered targeted drug-delivery system.

Genetic characterization of field-evolved resistance to phosphine in the rusty grain beetle, Cryptolestes ferrugineus (Laemophloeidae: Coleoptera)

Inheritance of resistance to phosphine fumigant was investigated in three field-collected strains of rusty grain beetle, Cryptolestes ferrugineus, Susceptible (S-strain), Weakly Resistant (Weak-R) and Strongly Resistant (Strong-R). The strains were purified for susceptibility, weak resistance and strong resistance to phosphine, respectively, to ensure homozygosity of resistance genotype. Crosses were established between S-strain × Weak-R, S-strain × Strong-R and Weak-R × Strong-R, and the dose mortality responses to phosphine of these strains and their F1, F2 and F1-backcross progeny were obtained. The fumigations were undertaken at 25 °C and 55% RH for 72 h. Weak-R and Strong-R showed resistance factors of 6.3 × and 505 × compared with S-strain at the LC50. Both weak and strong resistances were expressed as incompletely recessive with degrees of dominance of − 0.48 and − 0.43 at the LC50, respectively. Responses of F2 and F1-backcross progeny indicated the existence of one major gene in Weak-R, and at least two major genes in Strong-R, one of which was allelic with the major factor in Weak-R. Phenotypic variance analyses also estimated that the number of independently segregating genes conferring weak resistance was 1 (nE = 0.89) whereas there were two genes controlling strong resistance (nE = 1.2). The second gene, unique to Strong-R, interacted synergistically with the first gene to confer a very high level of resistance (~ 80 ×). Neither of the two major resistance genes was sex linked. Despite the similarity of the genetics of resistance to that previously observed in other pest species, a significant proportion (~ 15 to 30%) of F1 individuals survived at phosphine concentrations higher than predicted. Thus it is likely that additional dominant heritable factors, present in some individuals in the population, also influenced the resistance phenotype. Our results will help in understanding the process of selection for phosphine resistance in the field which will inform resistance management strategies. In addition, this information will provide a basis for the identification of the resistance genes.