Kathy Ophel-Keller

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.

Fungal community structure in disease suppressive soils assessed by 28S LSU gene sequencing.

Natural biological suppression of soil-borne diseases is a function of the activity and composition of soil microbial communities. Soil microbe and phytopathogen interactions can occur prior to crop sowing and/or in the rhizosphere, subsequently influencing both plant growth and productivity. Research on suppressive microbial communities has concentrated on bacteria although fungi can also influence soil-borne disease. Fungi were analyzed in co-located soils ‘suppressive’ or ‘non-suppressive’ for disease caused by Rhizoctonia solani AG 8 at two sites in South Australia using 454 pyrosequencing targeting the fungal 28S LSU rRNA gene. DNA was extracted from a minimum of 125 g of soil per replicate to reduce the micro-scale community variability, and from soil samples taken at sowing and from the rhizosphere at 7 weeks to cover the peak Rhizoctonia infection period. A total of ∼994,000 reads were classified into 917 genera covering 54% of the RDP Fungal Classifier database, a high diversity for an alkaline, low organic matter soil. Statistical analyses and community ordinations revealed significant differences in fungal community composition between suppressive and non-suppressive soil and between soil type/location. The majority of differences associated with suppressive soils were attributed to less than 40 genera including a number of endophytic species with plant pathogen suppression potentials and mycoparasites such as Xylaria spp. Non-suppressive soils were dominated by Alternaria, Gibberella and Penicillum. Pyrosequencing generated a detailed description of fungal community structure and identified candidate taxa that may influence pathogen-plant interactions in stable disease suppression.

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.

Persistence of DNA of Gaeumannomyces graminis var. tritici in soil as measured by a DNA-based assay

There are an increasing number of assays available for fungal plant pathogens based on DNA technology. We have developed such an assay for Gaeumannomyces graminis var. tritici (Ggt) in soil, using slot-blot hybridisation. To ensure the validity of DNA-based soil assays for the fungus, it is important to determine the stability of Ggt DNA in soil. This study was undertaken to quantify the DNA degradation of dead Ggt in soil using a DNA-based assay. Mycelia were killed using various treatments, then DNA was extracted and estimated by a slot-blot hybridisation technique using the specific Ggt DNA probe, pG158. Mycelia were also killed using a fungicide (triadimefon) at a concentration of 150-250 microg ml(-1). The amount of detectable DNA of Ggt, killed using triadimefon, declined by 82-93%. Inoculum in the form of diseased wheat roots, artificially inoculated ryegrass seed, particulate soil organic matter and whole soil was killed using heat-treatment. The amount of detectable DNA of Ggt declined markedly (90%) in both heat-treated roots and inoculated ryegrass seeds, and declined by 50% in both treated soil and soil organic matter. The rate of DNA degradation of Ggt in soil varied with the type of inoculum. The amount of detectable DNA of Ggt in dead mycelia declined by 99.8% after 4 days of incubation in soil. No DNA was detected after 8 days of incubation. In contrast, Ggt DNA in live mycelia declined by 70% after 8 days of incubation and declined to 10% of original DNA level after 32 days. In ground ryegrass seed inoculum, DNA in both killed and live Ggt declined by 50% after 8 days. In diseased roots, DNA from both live and killed Ggt did not appear to decline over 16 days. Estimates of the amount of Ggt in the soil using a DNA-based assay reflect both live and dead populations of the fungus. The rate of breakdown of DNA of the dead fungus is very high and the presence of dead fungi in roots probably a rare event so the DNA from dead fungus probably contributes little to the total DNA level.

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.

Survival of a lacZY‐marked strain of Pseudomonas corrugata following a field release

Abstract Pseudomonas corrugata, strain 2140, a biological control agent of take-all disease of wheat, was originally isolated from an acidic red-brown earth soil in New South Wales, Australia. A spontaneous rifampicin-resistant mutant of this bacterium was marked with the disarmed transposon, Tn7::lacZY. This marked strain (2140RlacZY) was introduced into a calcareous sandy loam soil (pH 8) in South Australia. Up to 4 years after its release, P. corrugata 2140RlacZY cells were re-isolated, single colony purified and stored at -80 degrees C. Re-isolated bacteria, including re-isolates obtained 3 (22 re-isolates) and 4 (3 re-isolates) years after release, were examined for stability of the lacZY insert site and for gross chromosomal changes. Hybridization of a cloned lacZY fragment to DNA extracted from the soil re-isolates did not reveal any major changes to the lacZY insert site. Gross chromosomal changes were further examined by restriction endonuclease fingerprinting and PCR based on repetitive sequences (repetitive extragenic palindromic-, enterobacterial repetitive intergeneric consensus- and BOX-PCR). MspI digests distinguished the lacZY-marked strain from the parental strain. None of the genetic techniques used revealed any polymorphisms between the original 2140RlacZY-marked strain and the soil re-isolates. The results demonstrated that the chromosomal landscape within and around the insertion site of the lacZY construct had not altered in the re-isolated bacteria during the 4 years the organism had been in the field.

Developing and Testing a Diagnostic Probe for Grape Phylloxera Applicable to Soil Samples

Abstract Grape phylloxera, Daktulosphaira vitifoliae (Fitch) (Hemiptera Phylloxeridae) is a damaging pest of grapevines (Vitis spp.) around the world, and the management of this pest requires early detection of infestations. Here, we describe the development and validation of a sensitive DNA test for grape phylloxera that can be applied to soil. Species-specific primers were developed for grape phylloxera in the internal transcribed space region 2, and their specificity was confirmed after thorough screening by using a wide range of vineyard organisms and aphid genera. Preliminary testing of the detection limits of the grape phylloxera-specific primers was conducted using field-sourced soil types spiked with a known number of grape phylloxera. The assay was converted to a real-time polymerase chain reaction format (TaqMan MGB). This assay, in combination with DNA extraction from soil, can detect phylloxera crawlers added to soil. The assay was evaluated in the field at a recently detected grape phylloxera infestation site from the Yarra Valley in Victoria, Australia. The DNA assay proved to be substantially more sensitive than a standard ground survey for detecting grape phylloxera presence on vine roots in the infested vineyard. Moreover, unlike the ground survey, the assay provided quantitative information on grape phylloxera infestations, because grape phylloxera DNA concentrations in samples from vines closely matched the numbers of grape phylloxera crawlers collected with emergence traps placed at the base of vines. Unlike other detection techniques, the method can be applied at any time of the year, and it can be potentially modified to provide specific information on the virulence levels of the particular grape phylloxera genotypes responsible for any new infestations.

Quantitative molecular assays for evaluating changes in broiler gut microbiota linked with diet and performance

Changes in the levels of specific gut bacteria have been linked to improved broiler feed efficiency. Quantitative polymerase chain reaction (qPCR) assays were developed to five potential performance-related bacteria (Lactobacillus salivarius, L. crispatus, L. aviarius, Gallibacterium anatis and Escherichia coli) and generic eubacteria. These were used to screen broiler gut samples from four geographically diverse Australian feeding trials showing significant treatment-related differences in feed efficiency. It was our aim to validate the association of particular bacteria with broiler feed efficiency across a broad range of environmental and dietary conditions, and hence to evaluate their predictive potential for monitoring broiler performance. Across trials L. salivarius, L. crispatus, L. aviarius, E. coli and total eubacterial numbers were significantly altered by diet, environment (litter), and/or sex of birds. Furthermore, changes in the numbers of these gut bacteria were significantly linked to broiler performance. Lactobacilli and total eubacteria were significantly decreased in birds that were more feed efficient. E. coli was not consistently linked with either improved or decreased performance and these discrepancies may be due to differences at the strain level which were not detectable using our assays. G. anatis was detected only in two of the four trials and found not to be significantly linked with broiler performance. These qPCR assays have been useful in either validating or disproving previous reported findings for the association of specific gut bacteria with broiler feed efficiency. This qPCR format can be easily expanded to include other organisms and used as a quantitative screening tool in evaluating dietary additives for improved broiler production.