Alan McKay

Development of a routine DNA-based testing service for soilborne diseases in Australia

A DNA-based soil testing service operates in Australia to assist grain growers in predicting the likely extent of losses from various soilborne diseases well before a crop is planted. Growers, therefore, have the option of changing cultivars or modifying cropping programs in situations where the risk of crop loss is high. The service was launched in 1997 and although the initial focus was on wheat and barley, pathogens of rotation crops are now included. Key features of the service include a unique high-throughput DNA extraction system to process 500-g soil samples and a series of specific real-time PCR assays that allow a range of fungal and nematode pathogens to be quantified in a single soil sample. Tests for Heterodera avenae, Pratylenchus neglectus, P. thornei, Gaeumannomyces graminis var. tritici, G. graminis var. avenae, Rhizoctonia solani AG-8, Fusarium pseudograminearum, F. culmorum and the pea pathogens Mycosphaerella pinodes and Phoma medicaginis var. pinodella are available at present, while tests for Bipolaris sorokiniana, Ditylenchus dipsaci and Pratylenchus teres are in development. This paper discusses issues that were addressed in establishing the service (e.g. sampling strategies, extraction of DNA from soil, development of specific tests, disease risk categories) and explains the training and accreditation programs that have been established to ensure that results are interpreted adequately at the farm level. It also outlines research being conducted to extend the service to horticulture.

Toward routine, DNA-based detection methods for marine pests.

Marine pest incursions can cause significant ongoing damage to aquaculture, biodiversity, fisheries habitat, infrastructure and social amenity. They represent a significant and ongoing economic burden. Marine pests can be introduced by several vectors including aquaculture, aquarium trading, commercial shipping, fishing, floating debris, mining activities and recreational boating. Despite the inherent risks, there is currently relatively little routine surveillance of marine pest species conducted in the majority of countries worldwide. Accurate and rapid identification of marine pest species is central to early detection and management. Traditional techniques (e.g. physical sampling and sorting), have limitations, which has motivated some progress towards the development of molecular diagnostic tools. This review provides a brief account of the techniques traditionally used for detection and describes developments in molecular-based methods for the detection and surveillance of marine pest species. Recent advances provide a platform for the development of practical, specific, sensitive and rapid diagnosis and surveillance tools for marine pests for use in effective prevention and control strategies.

Effect of soil water content, sampling method and sample storage on the quantification of root lesion nematodes (Pratylenchus spp.) by different methods

Quantification of root lesion nematodes (Pratylenchus thornei and P. neglectus) was evaluated using three different methods; the Whitehead tray method, the mister method and the commercially available quantitative DNA assay. These methods were compared to determine the effect of soil water content, sampling method and soil storage conditions on estimates of pre-sowing densities of nematodes. The Whitehead tray method, which is reliant on extraction of live nematodes, recovered fewer nematodes from dry soil than from moist soil and fewer from soil dried before storage. By contrast, the DNA assay was not influenced by soil water content at the time of sampling or drying of the soil after sampling

Combining an initial risk assessment process with DNA assays to improve prediction of soilborne diseases caused by root-knot nematode (Meloidogyne spp.) and Fusarium oxysporum f. sp. lycopersici in the Queensland tomato industry

A two-step process was used to assess the risk of losses from root-knot nematode and Fusarium wilt in fields to be planted to tomatoes. The first step involved deciding well before planting whether the risk of disease was high enough to justify collecting soil samples to determine pathogen inoculum density. This interim assessment was done using information on the major factors likely to affect disease risk (i.e. cropping history, disease history, soil texture and expected temperature during the growing season), in order to calculate a hazard index (score between 0 and 50). Its value as a predictive tool was validated by relating the hazard index to actual disease incidence and severity in representative tomato fields. The usefulness of the hazard index was often found to be limited by a lack of reliable information on disease history. Nevertheless, it had some predictive value, as all sites with moderate infestations of root-knot nematode had hazard indexes greater than 40, and most sites with more than 3% Fusarium wilt had hazard indexes greater than 35. The second step in the prediction process involved using DNA tests to estimate inoculum densities of Fusarium oxysporum f. sp. lycopersici and root-knot nematode in soil collected before planting. Experiments in pots and in the field confirmed that the incidence and severity of both diseases was related to pre-plant inoculum density. The DNA test for root-knot nematode was useful from a practical point of view as it detected nematode populations capable of causing economically damaging levels of galling at harvest. However, the test for F. oxysporum f. sp. lycopersici was not sensitive enough to always detect the pathogen in soils where 4–10% of plants were diseased.

Predicting Take-All Severity in Second-Year Wheat Using Soil DNA Concentrations of Gaeumannomyces graminis var. tritici Determined with qPCR

The lack of accurate detection of Gaeumannomyces graminis var. tritici inoculum in soil has hampered efforts to predict the risk of severe take-all for wheat growers. The current study used a molecular method to quantify soil G. graminis var. tritici concentrations in commercial wheat fields in New Zealand and to compare them with the proportion of crops surpassing the thresholds for visible and moderate to severe take-all over three growing seasons. The study evaluated a soil G. graminis var. tritici DNA-based take-all prediction system developed in Australia, with four take-all risk categories. These categories were found to be useful for predicting disease severity in second wheat but did not clearly separate risk between fields in medium- and high-risk categories. A sigmoidal relationship was identified between inoculum concentration and the proportion of fields exceeding the two disease thresholds. A logistic response curve was used to further examine this relationship and evaluate the boundaries between take-all risk categories. G. graminis var. tritici boundaries between medium- and high-risk categories were clustered near or within the upper plateau of the relationship. Alternative G. graminis var. tritici boundaries for a three-category system were identified that provided better separation of take-all risk between categories. This information could improve prediction of the risk of severe take-all.

A DNA-based method for studying root responses to drought in field-grown wheat genotypes

Root systems are critical for water and nutrient acquisition by crops. Current methods measuring root biomass and length are slow and labour-intensive for studying root responses to environmental stresses in the field. Here, we report the development of a method that measures changes in the root DNA concentration in soil and detects root responses to drought in controlled environment and field trials. To allow comparison of soil DNA concentrations from different wheat genotypes, we also developed a procedure for correcting genotypic differences in the copy number of the target DNA sequence. The new method eliminates the need for separation of roots from soil and permits large-scale phenotyping of root responses to drought or other environmental and disease stresses in the field.

Genetic Analysis of Plant Pathogenic Bacteria in the Genus Clavibacter Using Allozyme Electrophoresis

Summary: Allozyme electrophoresis was used to examine the relationships among species of Clavibacter, a genus of bacteria mostly causing gummosis in various graminaceous hosts. Allelic profiles were determined at 20 enzyme loci for 28 strains representing C. iranicum, C. rathayi, C. tritici, three subspecies of C. michiganense, Clavibacter sp. from Lolium rigidum, and “Corynebacterium agropyri”. The genetic differences between species were at least 70% and between strains within a species up to 35% except for the subspecies of C. michiganense, which differed by up to 75%. The implications of these results for the classification of these Clavibacter spp. is discussed.

Severity of root rot in mature subterranean clover and associated fungal pathogens in the wheatbelt of Western Australia

Pasture decline is considered to be a serious challenge to agricultural productivity of subterranean clover across southern Australia. Root disease is a significant contributing factor to pasture decline. However, root disease assessments are generally carried out in the early part of the growing season and in areas predominantly sown to permanent pastures. For this reason, in spring 2004, a survey was undertaken to determine the severity of root disease in mature subterranean clover plants in pastures located in the wheatbelt of Western Australia. DNA-based soil assays were used to estimate population density in the soil of a variety of soil-borne pathogens known to commonly occur in the Mediterranean-type environments of southern Australia. The relationships between severity of disease on tap and lateral roots and root diameter, root length, nodulation, and total rainfall were determined. The survey showed, for the first time, that severe root disease is widespread in spring across the wheatbelt of Western Australia. There was a positive correlation between rainfall and tap root disease, and between tap root disease and average root diameter of the entire root system. Despite the high levels of root disease present across the sites, the DNA of most root disease pathogens assayed was detected in trace concentrations. Only Pythium Clade F showed high DNA concentrations in the soil. DNA concentrations in the soil, in particular for Phytophthora clandestina and Rhizoctonia solani AG 2.1 and AG 2.2, were higher in the smaller autumn sampling in 2006. This study suggests that the productivity of subterranean clover-based pastures is severely compromised by root rot diseases throughout the growing season in the wheatbelt of Western Australia.

Dynamic root responses to drought and rewatering in two wheat (Triticum aestivum) genotypes

Background and aimsIn Mediterranean-type environments, effective capture of intermittent rainfall is important for crop drought tolerance. Two wheat genotypes RAC875 and Kukri differing in drought tolerance vary in several shoot traits and grain yield. Little is known about root traits contributing to drought tolerance. This study examined dynamic root responses to cyclic drought in these two contrasting genotypes.MethodsA pot experiment was conducted by exposing plants to moderate drought before heading, and then rewatering. Root responses were determined for coarse and fine root length density (RLD), root DNA concentration (RDC), nodal root properties, and metaxylem and protoxylem features of both primary and nodal roots in proximal and distal regions.ResultsModerate drought reduced fine RLD and inhibited new nodal root growth. Rewatering promoted new nodal root growth. Drought-tolerant RAC875 showed higher relative growth of newly emerged nodal roots than drought-intolerant Kukri after rewatering. RAC875 also had smaller diameter and total area of metaxylem vessels in nodal roots than Kukri, but higher number of metaxylem vessels and RDC independent of water regime.ConclusionsMultiple root traits identified could confer RAC875 advantage in drought tolerance under Mediterranean-type environments through conservative use of water and reduced risk of embolism, rapid responses to rainfall and root survival.

Genetic mapping and marker development for resistance of wheat against the root lesion nematode Pratylenchus neglectus.

BackgroundThe Rlnn1 locus, which resides on chromosome 7A of bread wheat (Triticum aestivum L.) confers moderate resistance against the root lesion nematode Pratylenchus neglectus. Prior to this research, the exact linkage relationships of Rlnn1 with other loci on chromosome 7A were not clear and there were no simple codominant markers available for selection of Rlnn1 in wheat breeding. The objectives of the research reported here were to (1) develop an improved genetic map of the Rlnn1 region of chromosome 7A and (2) develop molecular markers that could be used in marker-assisted selection to improve resistance of wheat against P. neglectus.ResultsA large-effect quantitative trait locus (QTL) for resistance against P. neglectus was genetically mapped using a population of Excalibur/Kukri doubled haploid lines. This QTL coincides in position with the rust resistance gene(s) Lr20/Sr15, the phytoene synthase gene Psy-A1 and 10 molecular markers, including five new markers designed using wheat-rice comparative genomics and wheat expressed sequence tags. Two of the new markers are suitable for use as molecular diagnostic tools to distinguish plants that carry Rlnn1 and Lr20/Sr15 from those that do not carry these resistance genes.ConclusionsThe genomic location of Rlnn1 was confirmed to be in the terminal region of the long arm of chromosome 7A. Molecular markers were developed that provide simple alternatives to costly phenotypic assessment of resistance against P. neglectus in wheat breeding. In Excalibur, genetic recombination seems to be completely suppressed in the Rlnn1 region.