Characterisation of chicken Campylobacter jejuni isolates using resolution optimised single nucleotide polymorphisms and binary gene markers

The principal objective of this study was to determine if Campylobacter jejuni genotyping methods based upon resolution optimised sets of single nucleotide polymorphisms (SNPs) and binary genetic markers were capable of identifying epidemiologically linked clusters of chicken-derived isolates. Eighty-eight C. jejuni isolates of known flaA RFLP type were included in the study. They encompassed three groups of ten isolates that were obtained at the same time and place and possessed the same flaA type. These were regarded as being epidemiologically linked. Twenty-six unlinked C. jejuni flaA type I isolates were included to test the ability of SNP and binary typing to resolve isolates that were not resolved by flaA RFLP. The remaining isolates were of different flaA types. All isolates were typed by real-time PCR interrogation of the resolution optimised sets of SNPs and binary markers. According to each typing method, the three epidemiologically linked clusters were three different clones that were well resolved from the other isolates. The 26 unlinked C. jejuni flaA type I isolates were resolved into 14 SNP-binary types, indicating that flaA typing can be unreliable for revealing epidemiological linkage. Comparison of the data with data from a fully typed set of isolates associated with human infection revealed that abundant lineages in the chicken isolates that were also found in the human isolates belonged to clonal complex (CC) -21 and CC-353, with the usually rare C-353 member ST-524 being especially abundant in the chicken collection. The chicken isolates selected to be diverse according to flaA were also diverse according to SNP and binary typing. It was observed that CC-48 was absent in the chicken isolates, despite being very common in Australian human infection isolates, indicating that this may be a major cause of human disease that is not chicken associated.

Campylobacter jejuni and Campylobacter coli Genotyping by High-Resolution Melting Analysis of a flaA Fragment.

The highly variable flagellin-encoding flaA gene has long been used for genotyping Campylobacter jejuni and Campylobacter coli. High-resolution melting (HRM) analysis is emerging as an efficient and robust method for discriminating DNA sequence variants. The objective of this study was to apply HRM analysis to flaA-based genotyping. The initial aim was to identify a suitable flaA fragment. It was found that the PCR primers commonly used to amplify the flaA short variable repeat (SVR) yielded a mixed PCR product unsuitable for HRM analysis. However, a PCR primer set composed of the upstream primer used to amplify the fragment used for flaA restriction fragment length polymorphism (RFLP) analysis and the downstream primer used for flaA SVR amplification generated a very pure PCR product, and this primer set was used for the remainder of the study. Eighty-seven C. jejuni and 15 C. coli isolates were analyzed by flaA HRM and also partial flaA sequencing. There were 47 flaA sequence variants, and all were resolved by HRM analysis. The isolates used had previously also been genotyped using single-nucleotide polymorphisms (SNPs), binary markers, CRISPR HRM, and flaA RFLP.flaA HRM analysis provided resolving power multiplicative to the SNPs, binary markers, and CRISPR HRM and largely concordant with the flaA RFLP. It was concluded that HRM analysis is a promising approach to genotyping based on highly variable genes.

Measuring nitrous oxide emissions from conventional and controlled release fertilisers in south-east Queensland pineapple production

Nitrous oxide (N2O) is a potent greenhouse gas with a global warming potential 298 times higher than carbon dioxide. Soils are a natural source of N2O, contributing 65% of global emissions. This paper is the first in Australia to measure and compare N2O emissions from pre-plant controlled release (CR) and conventional granular (CV) fertilisers in pineapple production using static PVC chambers to capture N2O emissions. Farm 1 cumulative emissions from the CR fertiliser were 3.22 kg ha-1 compared to 6.09 kg ha-1 produced by the CV. At farm 2 the CV blend emitted 2.36 kg ha-1 in comparison to the CR blend of 2.92 kg ha-1. Daily N2O flux rates showed a relationship of direct response to rainfall and soil moisture availability. High emissions were observed for wheel tracks where increased N2O emissions may be linked to soil compaction and waterlogging that creates anaerobic conditions after rain events. Emission measurements over three months highlighted the inconsistencies found in other studies relative to reducing emissions through controlled release nitrogen. More investigations are required to verify the benefits associated with controlled release fertiliser use in pineapples, placement and seasonal timing to address N2O emissions in pineapples.

Premature fruit softening, a major physiological problem of persimmon in subtropical Australia.

Premature or abnormal softening of persimmon fruit within 3-7 days after harvest is a major physiological problem of non-astringent persimmon cultivars grown in subtropical regions of Australia. Up to 30% of consignments may soften rapidly frequently overnight, often resulting in the flesh becoming very soft, completely translucent, and impossible to handle. Incidence of premature soft fruit can vary with season and production location. To study the incidence of this problem, we conducted surveys of fruit harvested from five environmentally-diverse regions of Australia over a two-year period. We found wide variation in the rate of both premature softening and normal softening with differences of up 37 days between orchards in percentage of fruit reaching 50% soft. We found that the rate of fruit softening was exacerbated by lower calcium concentrations at fruit set, shorter fruit development periods and heavier rainfall during the fruit development period. The implications of our findings, in terms of orchard management, export and domestic marketing strategies are discussed.

Driving better vegetable irrigation through profitable practice change

The availability and quality of irrigation water has become an issue limiting productivity in many Australian vegetable regions. Production is also under competitive pressure from supply chain forces. Producers look to new technologies, including changing irrigation infrastructure, exploring new water sources, and more complex irrigation management, to survive these stresses. Often there is little objective information investigating which improvements could improve outcomes for vegetable producers, and external communities (e.g. meeting NRM targets). This has led to investment in inappropriate technologies, and costly repetition of errors, as business independently discover the worth of technologies by personal experience. In our project, we investigated technology improvements for vegetable irrigation. Through engagement with industry and other researchers, we identified technologies most applicable to growers, particularly those that addressed priority issues. We developed analytical tools for ‘what if’ scenario testing of technologies. We conducted nine detailed experiments in the Lockyer Valley and Riverina vegetable growing districts, as well as case studies on grower properties in southern Queensland. We investigated root zone monitoring tools (FullStop™ wetting front detectors and Soil Solution Extraction Tubes - SSET), drip system layout, fertigation equipment, and altering planting arrangements. Our project team developed and validated models for broccoli, sweet corn, green beans and lettuce, and spreadsheets for evaluating economic risks associated with new technologies. We presented project outcomes at over 100 extension events, including irrigation showcases, conferences, field days, farm walks and workshops. The FullStops™ were excellent for monitoring root zone conditions (EC, nitrate levels), and managing irrigation with poor quality water. They were easier to interpret than the SSET. The SSET were simpler to install, but required wet soil to be reliable. SSET were an option for monitoring deeper soil zones, unsuitable for FullStop™ installations. Because these root zone tools require expertise, and are labour intensive, we recommend they be used to address specific problems, or as a periodic auditing strategy, not for routine monitoring. In our research, we routinely found high residual N in horticultural soils, with subsequently little crop yield response to additional nitrogen fertiliser. With improved irrigation efficiency (and less leaching), it may be timely to re-examine nitrogen budgets and recommendations for vegetable crops. Where the drip irrigation tube was located close to the crop row (i.e. within 5-8 cm), management of irrigation was easier. It improved nitrogen uptake, water use efficiency, and reduced the risk of poor crop performance through moisture stress, particularly in the early crop establishment phases. Close proximity of the drip tube to the crop row gives the producer more options for managing salty water, and more flexibility in taking risks with forecast rain. In many vegetable crops, proximate drip systems may not be cost-effective. The next best alternative is to push crop rows closer to the drip tube (leading to an asymmetric row structure). The vegetable crop models are good at predicting crop phenology (development stages, time to harvest), input use (water, fertiliser), environmental impacts (nutrient, salt movement) and total yields. The two immediate applications for the models are understanding/predicting/manipulating harvest dates and nitrogen movements in vegetable cropping systems. From the economic tools, the major influences on accumulated profit are price and yield. In doing ‘what if’ analyses, it is very important to be as accurate as possible in ascertaining what the assumed yield and price ranges are. In most vegetable production systems, lowering the required inputs (e.g. irrigation requirement, fertiliser requirement) is unlikely to have a major influence on accumulated profit. However, if a resource is constraining (e.g. available irrigation water), it is usually most profitable to maximise return per unit of that resource.

First report of Vialaea minutella in Australia, its association with mango branch dieback and systematic placement of Vialaea in the Xylariales

Vialaea minutella was consistently isolated from infected mango trees showing branch dieback symptoms in northern Queensland. The fungus was identified by morphology and confirmed with molecular sequence data. This is the first report of V. minutella in Australia. The systematic position of Vialaea was confirmed to be in the Xylariales based on reconstructed LSU sequence data.