Feasiblity Study into fungal biocontrol of small hive beetle

Investigation of potential for fungal control of small hive beetles.

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.

Stem injection: a control technique often overlooked for exotic woody weeds

In the rangelands of northern Australia, basal bark, cut stump, hand applied residual herbicides and foliar spraying have traditionally been the main herbicide techniques for control of individual exotic woody weeds growing within scattered to medium density infestations. In this paper we report on the preliminary results of stem injection as an alternate technique for the control of yellow oleander ( Cascabela thevetia (L.) Lippold), a woody weed that is difficult to kill. A randomised complete block experiment comprising 12 herbicide treatments (including a control) and three replicates was undertaken. Two rates of triclopyr + picloram, hexazinone, glyphosate, 2,4- D + picloram and metsufuron methyl and one rate of imazapyr were tested. At 15 months after application, triclopyr + picloram, glyphosate, 2,4-D + picloram and imazapyr all recorded high mortality (>90%) for at least one application rate. These results suggest that stem injection warrants further investigation as a control technique for other exotic woody weeds growing in rangelands.

Potential of the neonicotinoid imidacloprid and the oxadiazine indoxacarb for controlling five coleopteran pests of stored grain.

The potential for using imidacloprid (a neonicotinoid) and indoxacarb (an oxadiazine) as grain protectants was investigated in bioassays against resistant strains of five stored grain beetles. The species investigated were Rhyzopertha dominica (F.) (the lesser grain borer), Sitophilus oryzae (L.) (the rice weevil), Tribolium castaneum (Herbst) (the rust-red flour beetle), Oryzaephilus surinamensis (L.) (the saw tooth flour beetle), and Cryptolestes ferrugineus (Stephens) (the flat grain beetle). Each of these species has developed resistance to one or more protectants, including organophosphorus insecticides, synthetic pyrethroids and the juvenile hormone analogue methoprene. Mortality and reproduction after a 2-week exposure of adults to treated wheat depended on species, dose and insecticide. Imidacloprid had no effect on S. oryzae at any dose, but none of the other species produced any live progeny at 10 mg/kg. Indoxacarb had no effect on T. castaneum at any dose, but none of the other species produced any live progeny at 5 mg/kg. The results show that although both imidacloprid and indoxacarb can control at least four of the five key pests tested at doses comparable to those used for organophosphorus protectants, more potent neonicotinoid or oxadiazine insecticides would be needed than either of these to provide broad spectrum protection of stored grain.

Rotary veneering of plantation-grown spotted gum (Corymbia citriodora subsp. variegata) and Dunn's white gum (Eucalyptus dunnii)

This report evaluates the wood and veneer properties of plantation-grown spotted gum (Corymbia citriodora subsp. variegata, or CCV) and Dunn's white gum (Eucalyptus dunnii), grown at different stockings, in thinning trials near Ellangowan in north-east New South Wales (mean annual rainfall 1050 mm) and Kingaroy in south-east Queensland (mean annual rainfall 873 mm). Thinning trials were established at age seven years. Both species showed a significant increase in stem diameter growth of the dominant trees in response to thinning. At age 10 years, trees from the unthinned (950–1270 stems ha-1) and 300 stems ha-1 treatments were selected for veneering. Five dominant trees were felled from each combination of species x sites x thinning treatment. Diameter at breast height over bark of the selected trees ranged from 20 cm to 27 cm at Ellangowan, and 19 cm to 26 cm at Kingaroy. From each tree, 1.5 m long billets were removed at two positions: a butt billet from 0.3–1.8 m above ground and a top billet from approximately 5.5–7.0 m. Log end splitting was assessed 24 hours after harvesting and again after steaming, approximately four days after harvesting. Disks from just above both billets were collected for assessment of wood properties. Billets were peeled on a spindleless veneer lathe to produce a full veneer ribbon with a target green thickness of 2.8 to 3.0 mm. The 1.55 m wide (tangential dimension) veneer sheets were dried and graded according to AS/NZ Standard 2269:2008, which describes four veneer grades. Veneer samples taken along the length of the veneer ribbon, at regular intervals of 1.55 m, were tested for stiffness, shrinkage and density. Veneer length measurements were used to calculate the radial distance of each sample from the central axis of the billet. Overall veneer gross recoveries ranged from 50% to 70%. They were significantly lower at the Kingaroy site, for both species. The veneer recoveries achieved were 2–3 times higher than typical green off saw recoveries from small plantation hardwood logs of similar diameter. Most of the veneer recovered was classified as D-grade. CCV trees from the Ellangowan site yielded up to 38% of the better C-grade and higher grade veneers. The main limiting factors that prevented veneer from meeting higher grades were the presence of kino defects and encased knots. Splits in E. dunnii veneer also contributed to reduced grade quality. Log end splits were higher for E. dunnii than for CCV, and logs from Ellangowan exhibited more severe splitting. Split index was generally higher for top than for butt billets. Split index was strongly correlated with the average veneer grade from corresponding billets. The Ellangowan site, where rainfall was higher and trees grew faster, yielded significantly denser and stiffer veneers than did the drier sites near Kingaroy, where tree growth was slower. The difference was more pronounced for E. dunnii than for CCV. Differences in measured wood properties between thinned and unthinned treatments were generally small and not significant. On average, 10% of billet volume was lost during the peeling rounding-up process. Much of the wood laid down following thinning was removed during rounding-up, meaning the effect of thinning on veneer properties could not be effectively assessed. CCV was confirmed as having high veneer density and very good veneer stiffness, exceeding 15 GPa, making it very suitable for structural products. E. dunnii also demonstrated good potential as a useful structural plywood resource, achieving stiffness above 10 GPa. Veneer stiffness and density in CCV increased from pith to bark at both sites, while for E. dunnii there was a radial increase in these properties at the Ellangowan site only. At the drier Kingaroy site, veneer stiffness and density declined from mid-radius to the log periphery. This may be associated with prolonged drought from 2005 to 2009, corresponding to the later years of tree growth at the Kingaroy site. CCV appeared to be less sensitive to drought conditions. Standing tree acoustic velocity, determined by the Fakopp time-of-flight method, provided a reliable prediction of average veneer stiffness for both species (R2=0.78 for CCV and R2=0.90 for E. dunnii) suggesting that the Fakopp method may be a useful indicator of tree and stand quality, in terms of veneer stiffness in standing trees.

Insect induced bud fall in cultivated hibiscus and aspects of the biology of Macroura concolor (Macleay) (Coleoptera: Nitidulidae)

The influence of insect attack on bud fall and subsequent poor flowering in cultivated hibiscus (Hibiscus rosa-sinensis) was studied in cages and in the field in southern Queensland. Three species of Hemiptera (most importantly Aulacosternum nigrorubrum but also Nezara viridula and Tectocoris diophthalmus) caused some bud fall in 2 plantations studied. Adults of Macroura concolor suppressed flowering for long periods in spring and summer. Data from white funnel traps and counts in flowers showed that M. concolor was most active in these seasons. Methiocarb (0.75 g a.i./litre) reduced beetle numbers and increased flowering. When 15 or more adults of M. concolor occurred per bud (or flower) most buds fell and few flowers were produced, but when beetles declined to 10 or fewer many buds survived and widespread flowering occurred. Larvae fed in fallen buds and flowers and the mean duration of development of the combined immature stages was 14 days at 26 deg C. The preference of adults of M. concolor for pale coloured flowers was examined. Hibiscus plants produced most buds from December to June with lower numbers in winter and spring (July to November). Bud production in spring and early summer (September-December) varied greatly and probably contributed to poor flowering, however, even when large numbers of buds occurred very few flowers were produced because of the activities of M. concolor.

Australian spore-feeding thrips of the genus Phaulothrips (Thysanoptera, Idolothripinae)

Breeding by several of the spore-feeding species in the genus Phaulothrips is shown to be associated with abandoned tunnels of bees and scolytid beetles, as well as with the dead seed capsules of Eucalyptus species. The breeding sites for other species in the genus remain unknown, but 16 species are here recognised from Australia, of which the following six are newly described: P. daguilaris, P. flindersi, P. kingae, P. kranzae, P. oakeyi, P. whyallae. Copyright © 2013 Magnolia Press.

Short-range resource location by Tribolium castaneum Herbst (Coleoptera: Tenebrionidae) demonstrates a strong preference for fungi associated with cotton seed

Tribolium castaneum Herbst (Coleoptera: Tenebrionidae) is a common stored grain pest for which a wide range of suitable resources has been recorded. These beetles are facultatively fungivorous and their resource range may extend to fungi associated with non-grain resources (e.g. cotton seed) and other decaying plant matter. Little is known with respect to fungi in terms of resource location by these beetles in the field. We, therefore, conducted a series of experiments in laboratory arenas, glasshouse cages and the field to determine how beetles respond to grain resources in relation to cotton seed (together with its lint stubble and associated fungal flora). Results from the tests conducted in relatively small arenas and cages in the laboratory and glasshouse reveal that the responses of T. castaneum adults to food resources were twice as strong when walking as when flying (as measured by the proportion of the released beetles that were trapped). Also, a clear preference for linted cotton seeds was evident in walking T. castaneum, especially in small-scale arenas in the laboratory, where at least 60% of beetles released preferred linted cotton seeds over wheat and sorghum. Similarly, in cages (1 m3) they responded five times more strongly to linted cotton seed than to conventional grain resources. However, this pattern was not consistent with those obtained from field trapping over 20 m and the beetles did not show any particular preference to any of the resources tested above. Our results suggest a focus on walking beetles in trapping studies for population estimations and, for developing effective food-based trapping lures, the potential use of active volatiles from the fungi associated with linted cotton seed. © 2012 Elsevier Ltd.

Responses of tribolium castaneum to olfactory cues from cotton seeds, the fungi associated with cotton seeds, and cereals

We tested, in an olfactometer, whether or not Tribolium castaneum Herbst (Coleoptera: Tenebrionidae) responds preferentially to the volatiles that emanate from the fungi associated with cotton [Gossypium hirsutum L. (Malvaceae)] seed over those that emanate from cereals, because cereals are usually portrayed as the primary resources of these beetles. Pairwise comparisons were conducted between cotton seed, wheat (Triticum aestivum L.), and sorghum [Sorghum bicolor (L.) Moench] (both Poaceae); volatiles were tested from intact seeds and from both water and ethanol extracts. The results demonstrate that T. castaneum is attracted more strongly to cotton seeds with its lint contaminated with fungi, than to the conventional resources of this species (i.e., wheat and sorghum). Further tests prove that it is the fungus on the lint that produces the active volatiles, because the beetles did not respond to sterilized cotton lint (i.e., without the fungi typically associated with it when cotton seed is stored). Tests with five fungal cultures (each representing an unidentified species that was isolated from the field-collected cotton lint) were variable across the cultures, with only one of them being significantly attractive to the beetles. The others were not attractive and one may even have repulsed the beetles. The results are consistent with the beetles having a strong ecological association with fungi and suggest it would be worth investigating the ecology of T. castaneum from this perspective. © 2012 The Netherlands Entomological Society.

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.

Constructing a new individual-based model of phosphine resistance in lesser grain borer (Rhyzopertha dominica): do we need to include two loci rather than one?

In this article, we describe and compare two individual-based models constructed to investigate how genetic factors influence the development of phosphine resistance in lesser grain borer (R. dominica). One model is based on the simplifying assumption that resistance is conferred by alleles at a single locus, while the other is based on the more realistic assumption that resistance is conferred by alleles at two separate loci. We simulated the population dynamic of R. dominica in the absence of phosphine fumigation, and under high and low dose phosphine treatments, and found important differences between the predictions of the two models in all three cases. In the absence of fumigation, starting from the same initial frequencies of genotypes, the two models tended to different stable frequencies, although both reached Hardy-Weinberg equilibrium. The one-locus model exaggerated the equilibrium proportion of strongly resistant beetles by 3.6 times, compared to the aggregated predictions of the two-locus model. Under a low dose treatment the one-locus model overestimated the proportion of strongly resistant individuals within the population and underestimated the total population numbers compared to the two-locus model. These results show the importance of basing resistance evolution models on realistic genetics and that using oversimplified one-locus models to develop pest control strategies runs the risk of not correctly identifying tactics to minimise the incidence of pest infestation.

Field Assessments of Control Agents for Lesser Mealworm (Coleoptera: Tenebrionidae) Using Litter Sampling

Spinosad, diatomaceous earth, and cyfluthrin were assessed on two broiler farms at Gleneagle and Gatton in southeastern Queensland, Australia in 2004-2005 and 2007-2009, respectively to determine their effectiveness in controlling lesser mealworm, Alphitobius diaperinus (Panzer) (Coleoptera: Tenebrionidae). Insecticide treatments were applied mostly to earth or 'hard' cement floors of broiler houses before the placement of new bedding. Efficacy of each agent was assessed by regular sampling of litter and counting of immature stages and adult beetles, and comparing insect counts in treatments to counts in untreated houses. Generally, the lowest numbers of lesser mealworm were recorded in the house with hard floors, these numbers equalling the most effective spinosad applications. The most effective treatment was a strategic application of spinosad under feed supply lines on a hard floor. In compacted earth floor houses, mean numbers of lesser mealworms for two under-feed-line spinosad treatments (i.e., 2-m-wide application at 0.18 g of active insecticide (g [AI]) in 100-ml water/m(2), and 1-m-wide application at 0.11 g ([AI] in 33-ml water/m(2)), and an entire floor spinosad treatment (0.07 g [AI] in 86-ml water/m2) were significantly lower (i.e., better control) than those numbers for cyfluthrin, and no treatment (controls). The 1-m-wide under-feed-line treatment was the most cost-effective dose, providing similar control to the other two most effective spinosad treatments, but using less than half the active component per broiler house. No efficacy was demonstrated when spinosad was applied to the surface of bedding in relatively large volumes of water. All applications of diatomaceous earth, applied with and without spinosad, and cyfluthrin at the label rate of 0.02 g (AI)/100-ml water/m(2) showed no effect, with insect counts not significantly different to untreated controls. Overall, the results of this field assessment indicate that cyfluthrin (the Australian industry standard) and diatomaceous earth were ineffective on these two farms and that spinosad can be a viable alternative for broiler house use.

Response of Tribolium castaneum and Rhyzopertha dominica to various resources, near and far from grain storage

Tribolium castaneum (Herbst) and Rhyzopertha dominica (F.) are common cosmopolitan pests of stored grain and grain products. We evaluated the relative attraction of T.castaneum and R.dominica to wheat, sorghum and cotton seeds in the field, near grain storage facilities and well away from storages in southern and central Queensland using multiple trapping techniques. The results show that T.castaneum is more strongly attracted to linted cotton seed relative to wheat, whereas R.dominica did not respond to cotton seed at all and was attracted only to wheat. Significantly more adults of T.castaneum (10-15 times) were attracted to traps placed on the ground, near grain storage, than to equivalent traps that were suspended (1.5m above the ground) nearby. These results suggest that Tribolium beetles detect and respond to resources towards the end of their dispersal flight, after which they localize resources while walking. By contrast R.dominica was captured only in suspended traps, which suggests they fly directly onto resources as they localize them. The ability of both species to colonize and reproduce in isolated resource patches within the relatively short time of 1month is illustrated by the returns from the traps deployed in the field (at least 1km from the nearest stored grain) even though they caught only a few beetles. The results presented here provide novel insights about the resource location behaviours of both T.castaneum and R.dominica. In particular, the relationship of T.castaneum with non-cereal resources that are not conventionally associated with this species suggests an emphasis on these other resources in investigating the resource location behaviour of these beetles. This new perspective on the ecology of T. castaneum highlights the potential role of non-cereal resources (such as the lint on cotton seed) in the spread of grain pest infestations.

Corymbia phloem phenolics, tannins and terpenoids: interactions with a cerambycid borer

Plant secondary chemistry mediates the ability of herbivores to locate, accept and survive on potential host plants. We examined the relationship between attack by the cerambycid beetle Phoracantha solida and the chemistry of the secondary phloem (inner bark) of two differentially attacked plantation forestry taxa, Corymbia variegata and its hybrid with C. torelliana. We hypothesised that this differential rate of attack may have to do with differences in secondary chemistry between the taxa. We found differences in the bark chemistry of the taxa, both with respect to phenolic compounds and terpenoids. We could detect no difference between bored and non-bored C. variegata trees (the less preferred, but co-evolved host). Hybrid trees were not different in levels of total polyphenols, flavanols or terpenes according to attack status, but acetone extracts were significantly different between bored and non-bored trees. We propose that variations in the bark chemistry explain the differential attack rate between C. variegata and the hybrid hosts.

Rapid genome wide mapping of phosphine resistance loci by a simple regional averaging analysis in the red flour beetle, Tribolium castaneum

Background Next-generation sequencing technology is an important tool for the rapid, genome-wide identification of genetic variations. However, it is difficult to resolve the ‘signal’ of variations of interest and the ‘noise’ of stochastic sequencing and bioinformatic errors in the large datasets that are generated. We report a simple approach to identify regional linkage to a trait that requires only two pools of DNA to be sequenced from progeny of a defined genetic cross (i.e. bulk segregant analysis) at low coverage (<10×) and without parentage assignment of individual SNPs. The analysis relies on regional averaging of pooled SNP frequencies to rapidly scan polymorphisms across the genome for differential regional homozygosity, which is then displayed graphically. Results Progeny from defined genetic crosses of Tribolium castaneum (F4 and F19) segregating for the phosphine resistance trait were exposed to phosphine to select for the resistance trait while the remainders were left unexposed. Next generation sequencing was then carried out on the genomic DNA from each pool of selected and unselected insects from each generation. The reads were mapped against the annotated T. castaneum genome from NCBI (v3.0) and analysed for SNP variations. Since it is difficult to accurately call individual SNP frequencies when the depth of sequence coverage is low, variant frequencies were averaged across larger regions. Results from regional SNP frequency averaging identified two loci, tc_rph1 on chromosome 8 and tc_rph2 on chromosome 9, which together are responsible for high level resistance. Identification of the two loci was possible with only 5-7× average coverage of the genome per dataset. These loci were subsequently confirmed by direct SNP marker analysis and fine-scale mapping. Individually, homozygosity of tc_rph1 or tc_rph2 results in only weak resistance to phosphine (estimated at up to 1.5-2.5× and 3-5× respectively), whereas in combination they interact synergistically to provide a high-level resistance >200×. The tc_rph2 resistance allele resulted in a significant fitness cost relative to the wild type allele in unselected beetles over eighteen generations. Conclusion We have validated the technique of linkage mapping by low-coverage sequencing of progeny from a simple genetic cross. The approach relied on regional averaging of SNP frequencies and was used to successfully identify candidate gene loci for phosphine resistance in T. castaneum. This is a relatively simple and rapid approach to identifying genomic regions associated with traits in defined genetic crosses that does not require any specialised statistical analysis.