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.

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.

Field evaluation of transgenic pineapple (Ananas comosus (L.) Merr.) cv. ‘Smooth Cayenne’ for resistance to blackheart under subtropical conditions

A comparative analysis of transgenic pineapple lines transformed with a polyphenol oxidase (PPO) gene (ppo) and the untransformed cultivar ‘Smooth Cayenne’ was made from plants grown in a series of field trials under cool subtropical conditions in southeast Queensland. In the four field trials where blackheart was recorded, all of the control lines expressed blackheart on each occasion and exhibited the greatest incidence (50%) and severity (34%) of symptoms. Irrespective of the gene transfer method or the gene construct used, 38% of the lines produced were regarded as blackheart resistant, having no blackheart symptoms in two or more trials. Five blackheart resistant transgenic lines consistently performed as well as or better than control plants in terms of fruit characteristics and quality.

Screening Corymbia populations for resistance to Puccinia psidii

The exotic rust pathogen Puccinia psidii is now widespread along the east coast of Australia from temperate Victoria to tropical far north Queensland, with a current host range exceeding 200 species from 37 myrtaceous genera. To determine the threat P. psidii poses to plantation and native eucalypts, artificial inoculation was used to screen germplasm of spotted gum (Corymbia spp.) for resistance to the biotype of P. psidii that has become established in Australia. The objective was to characterize resistance to P. psidii within the Corymbia species complex so that management strategies for the deployment of germplasm from existing breeding programmes of these spotted gum species could be developed. Symptom development initiated 7 days after inoculation, with resistant and susceptible seedlings identified within all species, provenances and families. Inter- and intraspecific variability in rust resistance was observed among spotted gum species. There was no apparent relationship between climatic conditions at the provenance origin and disease resistance. The heritability estimates for all assessments are moderate to high and indicate a significant level of additive genetic variance for rust resistance within the populations. The results of this study clearly identify potential to select for resistance at the family level within the tested populations. While the potential for P. psidii to detrimentally impact upon Corymbia in the nursery and in young plantations was demonstrated, estimations of the heritability of resistance suggest that efforts to enhance this trait through breeding have reasonable prospects for success.

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.

Temporal and spatial patterns of soil water extraction and drought resistance among genotypes of a perennial C-4 grass

The objective of this study was to investigate patterns of soil water extraction and drought resistance among genotypes of bermudagrass (Cynodon spp.) a perennial C-4 grass. Four wild Australian ecotypes (1-1, 25a1, 40-1, and 81-1) and four cultivars (CT2, Grand Prix, Legend, and Wintergreen) were examined in field experiments with rainfall excluded to monitor soil water extraction at 30-190 cm depths. In the study we defined drought resistance as the ability to maintain green canopy cover under drought. The most drought resistant genotypes (40-1 and 25a1) maintained more green cover (55-85% vs 5-10%) during water deficit and extracted more soil water (120-160 mm vs 77-107 mm) than drought sensitive genotypes, especially at depths from 50 to 110 cm, though all genotypes extracted water to 190 cm. The maintenance of green cover and higher soil water extraction were associated with higher stomatal conductance, photosynthetic rate and relative water content. For all genotypes, the pattern of water use as a percentage of total water use was similar across depth and time We propose the observed genetic variation was related to different root characteristics (root length density, hydraulic conductivity, root activity) although shoot sensitivity to drying soil cannot be ruled out.

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.

Resistance to Fusarium oxysporum f. sp fragariae and Predicted Breeding Values in Strawberry

Fusarium wilt of strawberry, incited by Fusarium oxysporum f. sp. fragariae (Fof), is a major disease of the cultivated strawberry (Fragaria xananassa) worldwide. An increase in disease outbreaks of the pathogen in Western Australia and Queensland plus the search for alternative disease management strategies place emphasis on the development of resistant cultivars. In response, a partial incomplete diallel cross involving four parents was performed for use in glasshouse resistance screenings. The resulting progeny were evaluated for their susceptibility to Fof. Best-performing progeny and suitability of progenies as parents were determined using data from disease severity ratings and analyzed using a linear mixed model incorporating a pedigree to produce best linear unbiased predictions of breeding values. Variation in disease response, ranging from highly susceptible to resistant, indicates a quantitative effect. The estimate of the narrow-sense heritability was 0.49 +/- 0.04 (SE), suggesting the population should be responsive to phenotypic recurrent selection. Several progeny genotypes have predicted breeding values higher than any of the parents. Knowledge of Fof resistance derived from this study can help select best parents for future crosses for the development of new strawberry cultivars with Fof resistance.

Growth, yield and Fusarium wilt resistance of six FHIA tetraploid bananas (Musa spp.) grown in the Australian subtropics

Six tetraploid hybrids from Fundación Hondureña de Investigación Agrícola (FHIA) were evaluated in Australia over a five year period. They included three AAAA hybrids (FHIA-02, FHIA-17 and FHIA-23) and three AAAB hybrids (FHIA-01, FHIA-18 and SH-3640.10) and they were compared with industry standards, ‘Williams’ (AAA, Cavendish subgroup) and ‘Lady Finger’ (AAB, Pome subgroup). They were screened for their resistance to Fusarium wilt race 1 and subtropical race 4 caused by the pathogen Fusarium oxysporum f.sp. cubense and they were also grown for several cycles on farms not infested with Fusarium wilt to record their agronomic characteristics. The AAAB hybrids, all derived from female parent ‘Prata Anã’ (AAB, Pome subgroup) were the most resistant to both races of Fusarium wilt and were very productive in the subtropics. They were significantly more productive than ‘Lady Finger’, which was susceptible to both races of Fusarium wilt. The AAAA hybrids, with the exception of FHIA-02 which was very susceptible to Fusarium wilt and displayed the poorest agronomic traits of the six hybrids, produced bunch weights as good as Cavendish but were significantly slower to cycle. FHIA-17 and FHIA-23, both derived from the female parent ‘Highgate’ (AAA, Gros Michel subgroup), were also significantly more resistant to Fusarium wilt than ‘Gros Michel’, while FHIA-17 demonstrated a level of resistance similar to ‘Williams’ and FHIA-23 was intermediate between ‘Lady Finger’ and ‘Williams’

Foliar herbicide control of sticky florestina (Florestina tripteris DC.)

Sticky florestina (Florestina tripteris DC.) is an annual exotic weed that has become naturalised near the townships of Tambo and Barcaldine in central western Queensland, Australia. Three experiments conducted near Barcaldine identified foliar herbicides effective in killing sticky florestina plants and in providing residual activity to reduce recruitment from the soil seed bank. An initial chemical screening experiment evaluated the efficacy of 28 herbicide treatments. The most promising herbicides were then further evaluated in two response-rate experiments. Overall, 2,4-D/picloram, aminopyralid/fluroxypyr, clopyralid, metsulfuron-methyl and triclopyr/picloram proved to be the most effective selective herbicides. Two of these, metsulfuron-methyl at 18 g active ingredient (a.i) ha–1 and 2,4-D + picloram at 900 g a.i. ha–1 + 225 g a.i. ha–1 have now been included in a minor use permit (PER11920) with the Australian Pesticides and Veterinary Medicines Authority (APVMA) for the control of sticky florestina in pasture, stock route, roadside and non-crop situations using both spot and boom-spray applications (APVMA 2010). The permit also allows the use of 2,4-D amine for the control of seedlings only.

Resistance gene analogues in sugarcane and sorghum and their association with quantitative trait loci for rust resistance

Fifty-four different sugarcane resistance gene analogue (RGA) sequences were isolated, characterized, and used to identify molecular markers linked to major disease-resistance loci in sugarcane. Ten RGAs were identified from a sugarcane stem expressed sequence tag (EST) library; the remaining 44 were isolated from sugarcane stem, leaf, and root tissue using primers designed to conserved RGA motifs. The map location of 31 of the RGAs was determined in sugarcane and compared with the location of quantitative trait loci (QTL) for brown rust resistance. After 2 years of phenotyping, 3 RGAs were shown to generate markers that were significantly associated with resistance to this disease. To assist in the understanding of the complex genetic structure of sugarcane, 17 of the 31 RGAs were also mapped in sorghum. Comparative mapping between sugarcane and sorghum revealed syntenic localization of several RGA clusters. The 3 brown rust associated RGAs were shown to map to the same linkage group (LG) in sorghum with 2 mapping to one region and the third to a region previously shown to contain a major rust-resistance QTL in sorghum. These results illustrate the value of using RGAs for the identification of markers linked to disease resistance loci and the value of simultaneous mapping in sugarcane and sorghum.

Screening and breeding for genetic resistance to anthracnose in mango

Postharvest diseases remain a significant constraint to the transport, storage and marketing of mangoes. The two main ones are anthracnose and stem end rot. Anthracnose caused by Colletotrichum gloeosporioides is the more wide-spread of the two. Varieties within Mangifera indica are known to vary in their level of reactions to anthracnose; however, the best tolerance in current commercial cultivars is not sufficient to eliminate the need for pre- and postharvest fungicides treatments. A screening program was initiated in mango accessions in the Australian National Mango Genebank to look for any significant resistance to C. gloeosporioides in fruit as they ripened. Screening was conducted by rating reactions to natural infection of anthracnose and reactions to artificially inoculating fruit with virulent isolates of C. gloeosporioides. A range of reactions to the pathogen were identified, with strong resistance found in one accession of the species M. laurina. This accession was used as the pollen parent in a controlled crossing program with a M. indica hybrid from the Australian Mango Breeding Program (AMBP). Sixty successful hybrids between the species have been generated. The hybrid population will be screened for resistance to anthracnose and used for gene discovery investigations to identify markers for anthracnose resistance.

Do Phosphine Resistance Genes Influence Movement and Dispersal Under Starvation?

Phosphine resistance alleles might be expected to negatively affect energy demanding activities such as walking and flying, because of the inverse relationship between phosphine resistance and respiration. We used an activity monitoring system to quantify walking of Rhyzopertha dominica (F.) and a flight chamber to estimate their propensity for flight initiation. No significant difference in the duration of walking was observed between the strongly resistant, weakly resistant, and susceptible strains of R. dominica we tested, and females walked significantly more than males regardless of genotype. The walking activity monitor revealed no pattern of movement across the day and no particular time of peak activity despite reports of peak activity of R. dominica and Tribolium castaneum (Herbst) under field conditions during dawn and dusk. Flight initiation was significantly higher for all strains at 28 degrees C and 55% relative humidity than at 25, 30, 32, and 35 degrees C in the first 24 h of placing beetles in the flight chamber. Food deprivation and genotype had no significant effect on flight initiation. Our results suggest that known resistance alleles in R. dominica do not affect insect mobility and should therefore not inhibit the dispersal of resistant insects in the field.