Diagnostic Molecular Markers for Phosphine Resistance in U.S. Populations of Tribolium castaneum and Rhyzopertha dominica

Stored product beetles that are resistant to the fumigant pesticide phosphine (hydrogen phosphide) gas have been reported for more than 40 years in many places worldwide. Traditionally, determination of phosphine resistance in stored product beetles is based on a discriminating dose bioassay that can take up to two weeks to evaluate. We developed a diagnostic cleaved amplified polymorphic sequence method, CAPS, to detect individuals with alleles for strong resistance to phosphine in populations of the red flour beetle, Tribolium castaneum, and the lesser grain borer, Rhyzopertha dominica, according to a single nucleotide mutation in the dihydrolipoamide dehydrogenase (DLD) gene. We initially isolated and sequenced the DLD genes from susceptible and strongly resistant populations of both species. The corresponding amino acid sequences were then deduced. A single amino acid mutation in DLD in populations of T.castaneum and R.dominica with strong resistance was identified as P45S in T.castaneum and P49S in R.dominica, both collected from northern Oklahoma, USA. PCR products containing these mutations were digested by the restriction enzymes MboI and BstNI, which revealed presence or absence, respectively of the resistant (R) allele and allowed inference of genotypes with that allele. Seven populations of T.castaneum from Kansas were subjected to discriminating dose bioassays for the weak and strong resistance phenotypes. Application of CAPS to these seven populations confirmed the R allele was in high frequency in the strongly resistant populations, and was absent or at a lower frequency in populations with weak resistance, which suggests that these populations with a low frequency of the R allele have the potential for selection of the strong resistance phenotype. CAPS markers for strong phosphine resistance will help to detect and confirm resistant beetles and can facilitate resistance management actions against a given pest population.

Prevalence and potential fitness cost of weak phosphine resistance in Tribolium castaneum (Herbst) in eastern Australia

The prevalence of resistance to phosphine in the rust-red flour beetle, Tribolium castaneum, from eastern Australia was investigated, as well as the potential fitness cost of this type of resistance. Discriminating dose tests on 115 population samples collected from farms from 2006 to 2010 showed that populations containing insects with the weakly resistant phenotype are common in eastern Australia (65.2 of samples), although the frequency of resistant phenotypes within samples was typically low (median of 2.3). The population cage approach was used to investigate the possibility that carrying the alleles for weak resistance incurs a fitness cost. Hybridized populations were initiated using a resistant strain and either of two different susceptible strains. There was no evidence of a fitness cost based on the frequency of susceptible phenotypes in hybridized populations that were reared for seven generations without exposure to phosphine. This suggests that resistant alleles will tend to persist in field populations that have undergone selection even if selection pressure is removed. The prevalence of resistance is a warning that this species has been subject to considerable selection pressure and that effective resistance management practices are needed to address this problem. The resistance prevalence data also provide a basis against which to measure management success.

Commercial-scale Alternaria brown spot resistance screening as the first step in breeding new mandarins for Australia

Rapid screening tests and an appreciation of the simple genetic control of Alternaria brown spot (ABS) susceptibility have existed for many years, and yet the application of this knowledge to commercial-scale breeding programs has been limited. Detached leaf assays were first demonstrated more than 40 years ago and reliable data suggesting a single gene determining susceptibility has been emerging for at least 20 years. However it is only recently that the requirement for genetic resistance in new hybrids has become a priority, following increased disease prevalence in Australian mandarin production areas previously considered too dry for the pathogen. Almost all of the high-fruit-quality parents developed so far by the Queensland-based breeding program are susceptible to ABS necessitating the screening of their progeny to avoid commercialisation of susceptible hybrids. This is done effectively and efficiently by spraying 3-6 month old hybrid seedlings with a spore suspension derived from a toxin-producing field isolate of Alternaria alternate, then incubating these seedlings in a cool room at 25°C and high humidity for 5 days. Susceptible seedlings show clear disease symptoms and are discarded. Analysis of observed and expected segregation ratios loosely support the hypothesis for a single dominant gene for susceptibility, but do not rule out the possibility of alternative genetic models. After implementing the routine screening for ABS resistance for three seasons we now have more than 20,000 hybrids growing in field progeny blocks that have been screened for resistance to the ABS disease.

Inheritance and Characterization of Strong Resistance to Phosphine in Sitophilus oryzae(L.)

Sitophilus oryzae (Linnaeus) is a major pest of stored grain across Southeast Asia and is of increasing concern in other regions due to the advent of strong resistance to phosphine, the fumigant used to protect stored grain from pest insects. We investigated the inheritance of genes controlling resistance to phosphine in a strongly resistant S. oryzae strain (NNSO7525) collected in Australia and find that the trait is autosomally inherited and incompletely recessive with a degree of dominance of -0.66. The strongly resistant strain has an LC50 52 times greater than a susceptible reference strain (LS2) and 9 times greater than a weakly resistant strain (QSO335). Analysis of F2 and backcross progeny indicates that two or more genes are responsible for strong resistance, and that one of these genes, designated Sorph1, not only contributes to strong resistance, but is also responsible for the weak resistance phenotype of strain QSO335. These results demonstrate that the genetic mechanism of phosphine resistance in Soryzae is similar to that of other stored product insect pests. A unique observation is that a subset of the progeny of an F1 backcross generation are more strongly resistant to phosphine than the parental strongly resistant strain, which may be caused by multiple alleles of one of the resistance genes.

Efficacy of acibenzolar-S-methyl (Bion®) treatment of Australian commercial passionfruit, Passiflora edulis f. sp. flavicarpa, on resistance to Passionfruit woodiness virus (PWV) and activities of chitinase & β-1,3-glucanase

This greenhouse study investigated the efficacy of acibenzolar-S-methyl (Bion®) treatment of lower leaves of passionfruit, (Passiflora edulis f. sp. flavicarpa), on Passionfruit woodiness disease and activities of two pathogenesis-related proteins, chitinase and β-1,3-glucanase after inoculation with passionfruit woodiness virus (PWV). All Bion® concentrations reduced disease symptoms, but the concentration of 0.025 g active ingredient (a.i.)/l was the most effective, reducing disease severity in systemic leaves by 23, 29 and 30 compared with water-treated controls at 30, 40 and 50 days post inoculation (dpi) with PWV, respectively. Correspondingly, relative virus concentration as determined by DAS-ELISA in the upper, untreated leaves (new growth) above the site of inoculation at 50 dpi was reduced by 17 and 22 in plants treated with 0.025 and 0.05 g a.i./l, respectively. Bion® treatment and subsequent inoculation with PWV increased chitinase and β-1,3-glucanase activities in the new leaves above the site of inoculation at 30 dpi with PWV. It was concluded that optimal protective Bion® treatment concentrations were 0.025 and 0.05 g a.i./l.

Cereal cultivars can be ranked consistently for resistance to root-lesion nematodes (Pratylenchus thornei & P. neglectus) using diverse procedures

The root-lesion nematodes (RLN) Pratylenchus thornei and P. neglectus are widely distributed in Australian grain producing regions and can reduce the yield of intolerant wheat cultivars by up to 65 , costing the industry ~123 M AUD/year. Consequently, researchers in the northern, southern and western regions have independently developed procedures to evaluate the resistance of cereal cultivars to RLN. To compare results, each of the three laboratories phenotyped a set of 26 and 36 cereal cultivars for relative resistance/susceptibility to P. thornei and P. neglectus respectively. The northern and southern regions also investigated the effects of planting time and experiment duration on RLN reproduction and cultivar ranking. Results show the genetic correlation between cultivars tested using the northern and southern procedures evaluating P. thornei resistance was 0.93. Genetic correlations between experiments using the same procedure, but with different planting times, were 0.99 for both northern and southern procedures. The genetic correlation between cultivars tested using the northern, southern and western procedures evaluating P. neglectus resistance ranged from 0.71 to 0.95. Genetic correlations between experiments using the same procedure but with different planting times ranged from 0.91 to 0.99. This study established that, even though experiments were conducted in different geographic locations and with different trial management practices, the diverse nematode resistance screening procedures ranked cultivars similarly. Consequently, RLN resistance data can be pooled across regions to provide national consensus ratings of cultivars.

Optimising initial population density, growth time and nitrogen nutrition for assessing resistance of wheat cultivars to root-lesion nematode (Pratylenchus thornei)

Pratylenchus thornei is a major pathogen of wheat in Australia. Two glasshouse experiments with four wheat cultivars that had different final populations (Pf) of P. thornei in the field were used to optimise conditions for assessing resistance. With different initial populations (Pi) ranging up to 5250 P. thornei/kg soil, Pf of P. thornei increased to 16 weeks after sowing, and then decreased at 20 weeks in some cultivar x Pi combinations. The population dynamics of P. thornei up to 16 weeks were best described by a modified exponential equation P f (t) = aP i e kt where P f (t) is the final population density at time t, P i is the initial population density, a is the proportion of P i that initiates population development, and k is the intrinsic rate of increase of the population. The cultivar GS50a had very low k values at Pi of 5250 and 1050 indicating its resistance, Suneca and Potam had high k values indicating susceptibility, whereas intolerant Gatcher had a low value at the higher Pi and a high value at the lower Pi. Nitrate fertiliser increased plant growth and Pf values of susceptible cultivars, but in unplanted soil it decreased Pf. Nematicide (aldicarb 5 mg/kg soil) killed P. thornei more effectively in planted than in unplanted soil and increased plant growth particularly in the presence of N fertiliser. In both experiments, the wheat cultivars Suneca and Potam were more susceptible than the cultivar GS50a reflecting field results. The method chosen to discriminate wheat cultivars was to assess Pf after growth for 16 weeks in soil with Pi ~1050–5250 P. thornei/kg soil and fertilised with 200 mg NO3–N/kg soil.

A boat hitchhiker’s guide to survival: Cabomba caroliniana desiccation resistance and survival ability

Cabomba caroliniana is a submersed macrophyte that has become a serious invader. Cabomba predominantly spreads by stem fragments, in particular through unintentional transport on boat trailers (‘hitch hiking’). Desiccation resistance affects the potential dispersal radius. Therefore, knowledge of maximum survival times allows predicting future dispersal. Experiments were conducted to assess desiccation resistance and survival ability of cabomba fragments under various environmental scenarios. Cabomba fragments were highly tolerant of desiccation. However, even relatively low wind speeds resulted in rapid mass loss, indicating a low survival rate of fragments exposed to air currents, such as fragments transported on a boat trailer. The experiments indicated that cabomba could survive at least 3 h of overland transport if exposed to wind. However, even small clumps of cabomba could potentially survive up to 42 h. Thus, targeting the transport of clumps of macrophytes should receive high priority in management. The high resilience of cabomba to desiccation demonstrates the risk of continuing spread. Because of the high probability of fragment viability on arrival, preventing fragment uptake on boat trailers is paramount to reduce the risk of further spread. These findings will assist improving models that predict the spread of aquatic invasive macrophytes.

Pre-emptive breeding to combine superior eating quality in tropical super sweet corn with resistance to major diseases

This report presents the process and outcomes of a five year project, which employed genetics and breeding approach for integrating disease resistance,agronomy and quality traits that enhances sustainable productivity improvement in sweet corn production. The report outlines a molecular markers based approach to introgress quantitative traits loci that are believed to contribute to resistance to downy mildew, a potentially devastating disease that threatens sweet corn and other similar crops. It also details the approach followed to integrate resistances for other major diseases such as southern rust (caused by Puccinia polysora Underw), Northern Corn Leaf Blight (Exserohilum turcicum) with improved agronomy and eating quality. The report explains the importance of heterosis (hybrid vigour) and combining ability in the development of useful sweet corn hybrids. It also explains the relevance of parental performance to predict its breeding value and the performance of its hybrids.

Pen parameters for improving the performance of sheep imported from Australia to Persian Gulf feedlots

Summer in the Persian Gulf region presents physiological challenges for Australian sheep that are part of the live export supply chain coming from the Australian winter. Many feedlots throughout the Gulf have very high numbers of animals during June to August in order to cater for the increased demand for religious festivals. From an animal welfare perspective it is important to understand the necessary requirements of feed and water trough allowances, and the amount of pen space required, to cope with exposure to these types of climatic conditions. This study addresses parameters that are pertinent to the wellbeing of animals arriving in the Persian Gulf all year round. Three experiments were conducted in a feedlot in the Persian Gulf between March 2010 and February 2012, totalling 44 replicate pens each with 60 or 100 sheep. The applied treatments covered animal densities, feed-bunk lengths and water trough lengths. Weights, carcass attributes and health status were the key recorded variables. Weight change results showed superior performance for animal densities of ≥1.2 m2/head during hot conditions (24-h average temperatures greater than 33 °C, or a diurnal range of around 29–37 °C). However the space allowance for animals can be decreased, with no demonstrated detrimental effect, to 0.6 m2/head under milder conditions. A feed-bunk length of ≥5 cm/head is needed, as 2 cm/head showed significantly poorer animal performance. When feeding at 90 ad libitum 10 cm/head was optimal, however under a maintenance feeding regime (1 kg/head/day) 5 cm/head was adequate. A minimum water trough allowance of 1 cm/head is required. However, this experiment was conducted during milder conditions, and it may well be expected that larger water trough lengths would be needed in hotter conditions. Carcass weights were determined mainly by weights at feedlot entry and subsequent weight gains, while dressing percentage was not significantly affected by any of the applied treatments. There was no demonstrated effect of any of the treatments on the number of animals that died, or were classified as unwell. However, across all the treatments, these animals lost significantly more weight than the healthy animals, so the above recommendations, which are aimed at maintaining weight, should also be applicable for good animal health and welfare. Therefore, best practice guidelines for managing Australian sheep in Persian Gulf feedlots in the hottest months (June–August) which present the greatest environmental and physical challenge is to allow feed-bunk length 5 cm/head on a maintenance-feeding program and 10 cm/head for 90 ad libitum feeding, and the space allowance per animal should be ≥1.2 m2/head. Water trough allocation should be at least 1 cm/head with provision for more in the summer when water intake potentially doubles.

Post-head-emergence frost in wheat and barley: defining the problem, assessing the damage, and identifying resistance

Radiant frost is a significant production constraint to wheat (Triticum aestivum) and barley (Hordeum vulgare), particularly in regions where spring-habit cereals are grown through winter, maturing in spring. However, damage to winter-habit cereals in reproductive stages is also reported. Crops are particularly susceptible to frost once awns or spikes emerge from the protection of the flag leaf sheath. Post-head-emergence frost (PHEF) is a problem distinct from other cold-mediated production constraints. To date, useful increased PHEF resistance in cereals has not been identified. Given the renewed interest in reproductive frost damage in cereals, it is timely to review the problem. Here we update the extent and impacts of PHEF and document current management options to combat this challenge. We clarify terminology useful for discussing PHEF in relation to chilling and other freezing stresses. We discuss problems characterizing radiant frost, the environmental conditions leading to PHEF damage, and the effects of frost at different growth stages. PHEF resistant cultivars would be highly desirable, to both reduce the incidence of direct frost damage and to allow the timing of crop maturity to be managed to maximize yield potential. A framework of potential adaptation mechanisms is outlined. Clarification of these critical issues will sharpen research focus, improving opportunities to identify genetic sources for improved PHEF resistance.

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.

Pen parameters for improving the performance of sheep imported from Australia to Persian Gulf feedlots

Summer in the Persian Gulf region presents physiological challenges for Australian sheep that are part of the live export supply chain coming from the Australian winter. Many feedlots throughout the Gulf have very high numbers of animals during June to August in order to cater for the increased demand for religious festivals. From an animal welfare perspective it is important to understand the necessary requirements of feed and water trough allowances, and the amount of pen space required, to cope with exposure to these types of climatic conditions. This study addresses parameters that are pertinent to the wellbeing of animals arriving in the Persian Gulf all year round. Three experiments were conducted in a feedlot in the Persian Gulf between March 2010 and February 2012, totalling 44 replicate pens each with 60 or 100 sheep. The applied treatments covered animal densities, feed-bunk lengths and water trough lengths. Weights, carcass attributes and health status were the key recorded variables. Weight change results showed superior performance for animal densities of ≥1.2 m2/head during hot conditions (24-h average temperatures greater than 33 °C, or a diurnal range of around 29–37 °C). However the space allowance for animals can be decreased, with no demonstrated detrimental effect, to 0.6 m2/head under milder conditions. A feed-bunk length of ≥5 cm/head is needed, as 2 cm/head showed significantly poorer animal performance. When feeding at 90% ad libitum 10 cm/head was optimal, however under a maintenance feeding regime (1 kg/head/day) 5 cm/head was adequate. A minimum water trough allowance of 1 cm/head is required. However, this experiment was conducted during milder conditions, and it may well be expected that larger water trough lengths would be needed in hotter conditions. Carcass weights were determined mainly by weights at feedlot entry and subsequent weight gains, while dressing percentage was not significantly affected by any of the applied treatments. There was no demonstrated effect of any of the treatments on the number of animals that died, or were classified as unwell. However, across all the treatments, these animals lost significantly more weight than the healthy animals, so the above recommendations, which are aimed at maintaining weight, should also be applicable for good animal health and welfare. Therefore, best practice guidelines for managing Australian sheep in Persian Gulf feedlots in the hottest months (June–August) which present the greatest environmental and physical challenge is to allow feed-bunk length 5 cm/head on a maintenance-feeding program and 10 cm/head for 90% ad libitum feeding, and the space allowance per animal should be ≥1.2 m2/head. Water trough allocation should be at least 1 cm/head with provision for more in the summer when water intake potentially doubles.