CRC Field Analysis

Provide diagnostic support for plant pathology projects, including the maintenance and preservation of reference specimens.

Limitations of soil microbial biomass carbon as an indicator of soil pollution in the field

The size of the soil microbial biomass carbon (SMBC) has been proposed as a sensitive indicator for measuring the adverse effects of contaminants on the soil microbial community. In this study of Australian agricultural systems, we demonstrated that field variability of SMBC measured using the fumigation-extraction procedure limited its use as a robust ecotoxicological endpoint. The SMBC varied up to 4-fold across control samples collected from a single field site, due to small-scale spatial heterogeneity in the soil physicochemical environment. Power analysis revealed that large numbers of replicates (3-93) were required to identify 20% or 50% decreases in the size of the SMBC of contaminated soil samples relative to their uncontaminated control samples at the 0.05% level of statistical significance. We question the value of the routine measurement of SMBC as an ecotoxicological endpoint at the field scale, and suggest more robust and predictive microbiological indicators.

Analysis of the serological variability of Lettuce mosaic virus using monoclonal antibodies and surface plasmon resonance technology

A panel of 19 monoclonal antibodies (mAbs) was used to study the immunological variability of Lettuce mosaic virus (LMV), a member of the genus Potyvirus, and to perform a first epitope characterization of this virus. Based on their specificity of recognition against a panel of 15 LMV isolates, the mAbs could be clustered in seven reactivity groups. Surface plasmon resonance analysis indicated the presence, on the LMV particles, of at least five independent recognition/binding regions, correlating with the seven mAbs reactivity groups. The results demonstrate that LMV shows significant serological variability and shed light on the LMV epitope structure. The various mAbs should prove a new and efficient tool for LMV diagnostic and field epidemiology studies.

High-resolution melt-curve analysis of random-amplified-polymorphic-DNA markers, for the characterisation of pathogenic Leptospira.

A new test for pathogenic Leptospira isolates, based on RAPD-PCR and high-resolution melt (HRM) analysis (which measures the melting temperature of amplicons in real time, using a fluorescent DNA-binding dye), has recently been developed. A characteristic profile of the amplicons can be used to define serovars or detect genotypes. Ten serovars, of leptospires from the species Leptospira interrogans (serovars Australis, Robinsoni, Hardjo, Pomona, Zanoni, Copenhageni and Szwajizak), L. borgpetersenii (serovar Arborea), L. kirschneri (serovar Cynopteri) and L. weilii (serovar Celledoni), were typed against 13 previously published RAPD primers, using a real-time cycler (the Corbett Life Science RotorGene 6000) and the optimised reagents from a commercial kit (Quantace SensiMix). RAPD-HRM at specific temperatures generated defining amplicon melt profiles for each of the tested serovars. These profiles were evaluated as difference-curve graphs generated using the RotorGene software package, with a cut-off of at least 8 'U' (plus or minus). The results demonstrated that RAPD-HRM can be used to measure serovar diversity and establish identity, with a high degree of stability. The characterisation of Leptospira serotypes using a DNA-based methodology is now possible. As an objective and relatively inexpensive and rapid method of serovar identification, at least for cultured isolates, RAPD-HRM assays show convincing potentia.

Analysis and Modelling of the Effects of Water Stress on Maize Growth and Yield in Dryland Conditions.

It is essential to provide experimental evidence and reliable predictions of the effects of water stress on crop production in the drier, less predictable environments. A field experiment undertaken in southeast Queensland, Australia with three water regimes (fully irrigated, rainfed and irrigated until late canopy expansion followed by rainfed) was used to compare effects of water stress on crop production in two maize (Zea mays L.) cultivars (Pioneer 34N43 and Pioneer 31H50). Water stress affected growth and yield more in Pioneer 34N43 than in Pioneer 31H50. A crop model APSIM-Maize, after having been calibrated for the two cultivars, was used to simulate maize growth and development under water stress. The predictions on leaf area index (LAI) dynamics, biomass growth and grain yield under rain fed and irrigated followed by rain fed treatments was reasonable, indicating that stress indices used by APSIM-Maize produced appropriate adjustments to crop growth and development in response to water stress. This study shows that Pioneer 31H50 is less sensitive to water stress and thus a preferred cultivar in dryland conditions, and that it is feasible to provide sound predictions and risk assessment for crop production in drier, more variable conditions using the APSIM-Maize model.

High-resolution melt-curve analysis of random amplified polymorphic DNA (RAPD-HRM) for the characterisation of pathogenic leptospires: intra-serovar divergence, interserovar convergence, and evidence of attenuation in Leptospira reference collections.

High-resolution melt-curve analysis of random amplified polymorphic DNA (RAPD-HRM) is a novel technology that has emerged as a possible method to characterise leptospires to serovar level. RAPD-HRM has recently been used to measure intra-serovar convergence between strains of the same serovar as well as inter-serovar divergence between strains of different serovars. The results indicate that intra-serovar heterogeneity and inter-serovar homogeneity may limit the application of RAPD-HRM in routine diagnostics. They also indicate that genetic attenuation of aged, high-passage-number isolates could undermine the use of RAPD-HRM or any other molecular technology. Such genetic attenuation may account for a general decrease seen in titres of rabbit hyperimmune antibodies over time. Before RAPD-HRM can be further advanced as a routine diagnostic tool, strains more representative of the wild-type serovars of a given region need to be identified. Further, RAPD-HRM analysis of reference strains indicates that the routine renewal of reference collections, with new isolates, may be needed to maintain the genetic integrity of the collections.

Identification and analysis in sweet corn

Identification and analysis of allelic variation in carotenoid biosynthesis genes present in sweet corn germplasm for eye health.

Corymbia leaf oils, latitude, hybrids and herbivory: a test using common-garden field trials

Foliar oils, particularly monoterpenes, can influence the susceptibility of plants to herbivory. In plants, including eucalypts, monoterpenes are often associated with plant defence. A recent analysis revealed an increase in foliar oil content with increasing latitudinal endemism, and we tested this pattern using three eucalypt taxa comprising a latitudinal replacement cline. We also examined the relative concentrations of two monoterpenes (alpha-pinene and 1,8-cineole), for which meta-analyses also showed latitudinal variation, using hybrids of these three taxa with Corymbia torelliana. These, and pure C. torelliana, were then assessed in common-garden field plots for the abundance and distribution of herbivory by four distinct herbivore taxa. Differing feeding strategies among these herbivores allowed us to test hypotheses regarding heritability of susceptibility and relationships to alpha-pinene and 1,8-cineole. We found no support for an increase in foliar oil content with increasing latitude, nor did our analysis support predictions for consistent variation in alpha-pinene and 1,8-cineole contents with latitude. However, herbivore species showed differential responses to different taxa and monoterpene contents. For example, eriophyid mites, the most monophagous of our censused herbivores, avoided the pure species, but fed on hybrid taxa, supporting hypotheses on hybrid susceptibility. The most polyphagous herbivore (leaf blister sawfly Phylacteophaga froggatti) showed no evidence of response to plant secondary metabolites, while the distribution and abundance patterns of Paropsis atomaria showed some relationship to monoterpene yields.

Recent emergence of the wheat Lr34 multi-pathogen resistance: insights from haplotype analysis in wheat, rice, sorghum and Aegilops tauschii

Spontaneous sequence changes and the selection of beneficial mutations are driving forces of gene diversification and key factors of evolution. In highly dynamic co-evolutionary processes such as plant-pathogen interactions, the plant's ability to rapidly adapt to newly emerging pathogens is paramount. The hexaploid wheat gene Lr34, which encodes an ATP-binding cassette (ABC) transporter, confers durable field resistance against four fungal diseases. Despite its extensive use in breeding and agriculture, no increase in virulence towards Lr34 has been described over the last century. The wheat genepool contains two predominant Lr34 alleles of which only one confers disease resistance. The two alleles, located on chromosome 7DS, differ by only two exon-polymorphisms. Putatively functional homoeologs and orthologs of Lr34 are found on the B-genome of wheat and in rice and sorghum, but not in maize, barley and Brachypodium. In this study we present a detailed haplotype analysis of homoeologous and orthologous Lr34 genes in genetically and geographically diverse selections of wheat, rice and sorghum accessions. We found that the resistant Lr34 haplotype is unique to the wheat D-genome and is not found in the B-genome of wheat or in rice and sorghum. Furthermore, we only found the susceptible Lr34 allele in a set of 252 Ae. tauschii genotypes, the progenitor of the wheat D-genome. These data provide compelling evidence that the Lr34 multi-pathogen resistance is the result of recent gene diversification occurring after the formation of hexaploid wheat about 8,000 years ago.

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.

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.

Identifying risk-efficient strategies using stochastic frontier analysis and simulation: An application to irrigated cropping in Australia

In irrigated cropping, as with any other industry, profit and risk are inter-dependent. An increase in profit would normally coincide with an increase in risk, and this means that risk can be traded for profit. It is desirable to manage a farm so that it achieves the maximum possible profit for the desired level of risk. This paper identifies risk-efficient cropping strategies that allocate land and water between crop enterprises for a case study of an irrigated farm in Southern Queensland, Australia. This is achieved by applying stochastic frontier analysis to the output of a simulation experiment. The simulation experiment involved changes to the levels of business risk by systematically varying the crop sowing rules in a bioeconomic model of the case study farm. This model utilises the multi-field capability of the process based Agricultural Production System Simulator (APSIM) and is parameterised using data collected from interviews with a collaborating farmer. We found sowing rules that increased the farm area sown to cotton caused the greatest increase in risk-efficiency. Increasing maize area also improved risk-efficiency but to a lesser extent than cotton. Sowing rules that increased the areas sown to wheat reduced the risk-efficiency of the farm business. Sowing rules were identified that had the potential to improve the expected farm profit by ca. $50,000 Annually, without significantly increasing risk. The concept of the shadow price of risk is discussed and an expression is derived from the estimated frontier equation that quantifies the trade-off between profit and risk.

A field investigation into a suspected outbreak of pyrrolizidine alkaloid toxicosis in horses in western Queensland

A disease outbreak investigation was conducted in western Queensland to investigate a rare suspected outbreak of pyrrolizidine alkaloid (PA) toxicosis in horses. Thirty five of 132 horses depastured on five properties on the Mitchell grass plains of western Queensland died in the first six months of 2010. Clinical–pathological findings were consistent with PA toxicosis. A local variety of Crotalaria medicaginea was the only hepatotoxic plant found growing on affected properties. Pathology reports and departure and arrival dates of two brood mares provided evidence of a pre wet season exposure period. All five affected properties experienced a very dry spring and early summer preceded by a large summer wet season. The outbreak was characterised as a point epidemic with a sudden peak of deaths in March followed by mortalities steadily declining until the end of June. The estimated morbidity (serum IGG > 50 IU/L) rate was 76%. Average crude mortality was 27% but higher in young horses (67%) and brood mares (44%). Logistic regression analysis showed that young horses and brood mares and those grazing denuded pastures in December were most strongly associated with dying whereas those fed hay and/or grain based supplements were less likely to die. This is the first detailed study of an outbreak of PA toxicosis in central western Queensland and the first to provide evidence that environmental determinants were associated with mortality, that the critical exposure period was towards the end of the dry season, that supplementary feeding is protective and that denuded pastures and the horses physiological protein requirement are risk factors.