A. K. Lees

Conventional PCR and real-time quantitative PCR detection of Helminthosporium solani in soil and on potato tubers

Silver scurf is an economically important blemish disease of potato caused by the fungus Helminthosporium solani. Two sets of PCR primers, Hs1F1/Hs2R1 (outer) and Hs1NF1/Hs2NR1 (nested) were designed to unique sequences of the nuclear ribosomal internal transcribed spacer (ITS1 and ITS2) regions of H. solani. Nested PCR was used to increase the specificity and sensitivity of single round PCR. Each primer set amplified a single product of 447 bp and 371 bp respectively, with DNA from 71 European and North American isolates of H. solani, and the specificity of primers was confirmed by the absence of amplified product with DNA from other fungal and bacterial plant pathogens. A simple and rapid procedure for direct extraction of DNA from soils and potato tubers was modified and developed to yield DNA of a purity and quality suitable for PCR within 3 h. The sensitivity of PCR for the specific detection of H. solani in seeded soils was determined to be 1.5 spores g−1 of soil. H. solani was also detected by PCR in naturally infested soil and from peel and peel extract from infected and apparently healthy tubers. Specific primers and a TaqMan™ fluorogenic probe were designed using the original primer sequences to perform real-time quantitative (TaqMan™) PCR. The same levels of sensitivity for specific detection of H. solani in soil and tubers were obtained during first round mboxTaqMan-based PCR as with conventional nested PCR and gel electrophoresis. This rapid and quantitative PCR assay allows an accurate estimation of tuber and soil contamination by H. solani, thus providing a tool to study the ecology of the organism and to serve as a crucial component for disease risk assessments.

Detection and Quantification of Spongospora subterranea in Soil, Water and Plant Tissue Samples Using Real-Time PCR

A sensitive real-time polymerase chain reaction (PCR) assay was developed for the quantification of Spongospora subterranea, the cause of powdery scab and root galling in potato, and the vector of Potato mop top virus. A specific primer pair and a fluorogenic TaqMan® probe were designed to perform a quantitative assay for the detection of S. subterranea in soil, water and plant tissue samples. The assay was tested using DNA from cystosori, zoospores, plasmodia and zoosporangia of the pathogen. DNA was extracted directly from cystosori suspended in water and from clay soil with varying levels of added cystosori. DNA obtained from zoospores released into nutrient solution by cystosori in the presence of tomato bait plants was also tested, as was DNA from plasmodia and zoosporangia in infected tomato roots. In many cases, detection was successful even at low inoculum levels. This specific quantitative assay could therefore be a useful tool for studying the biology of S. subterranea, and for the optimisation of disease avoidance and control measures.

Mapping the R10 and R11 genes for resistance to late blight (Phytophthora infestans) present in the potato (Solanum tuberosum) R-gene differentials of Black

The R10 and R11 late blight differentials of Black (tetraploid clones 3681ad1 and 5008ab6) were crossed with the susceptible potato (Solanum tuberosum) cultivar Maris Piper and the progeny were assessed for blight resistance in a whole plant glasshouse test using race 1,2,3,4,6,7 of Phytophthora infestans. The disease scores for the R10 population displayed a continuous distribution whereas the progeny in the R11 population could be categorised as resistant or susceptible. A bulk segregant analysis using amplified fragment length polymorphism assays was done on the ten most resistant and ten most susceptible progeny in each population and two closely linked markers were found to be associated with resistance. R11 mapped to 8.5xa0cM from marker PAG/MAAG_172.3 and R10 mapped as a quantitative trait locus in which marker PAC/MATC_264.1 explained 56.9% of the variation in disease scores. The results were consistent with R10 and R11 being allelic versions of genes at the R3 locus on chromosome 11. The implications are discussed for mapping R-genes which fail to give complete immunity to a pathogen.

The Identification and Detection of Spongospora subterranea and Factors Affecting Infection and Disease

Powdery scab caused by Spongospora subterranea is a serious disease, affecting both the quality and quantity of ware and seed potatoes, particularly in northern Britain where soil conditions are conducive to disease development. To enable accurate quantification of S. subterranea DNA for epidemiological studies, a real-time PCR assay specific to this pathogen was developed. This assay could reliably detect and quantify DNA from sporeballs, zoospores and plasmodia/zoosporangia of S. subterranea. The assay was used to measure the viability of sporeballs in soil, was combined with a tomato bait plant technique in order to study zoospore release and was used to study infection levels in potato under various environmental conditions. Both the incidence and severity of powdery scab were found to be influenced by temperature, soil type and soil moisture regime when tested under controlled conditions. Soil inoculum level did not have a significant effect on infection and disease development and even low levels of inoculum were regularly found to result in severe disease symptoms. Evidence from A GB-wide trial and a five year survey in England and Wales confirmed that there is no simple or consistent relationship between powdery scab inoculum on seed and disease developing on the progeny crop. Disease risk appears to be more related to the relative conduciveness of environmental conditions rather than inoculum level. Integration of control measures is likely to be the most effective way for growers to reduce the impact of the disease.ResumenLa roña causada por Spongospora subterránea es una enfermedad seria que afecta tanto la calidad como la cantidad de la papa común y la papa semilla, particularmente en el norte de Gran Bretaña donde las condiciones de suelo son propicias para el desarrollo de la enfermedad. Para hacer posible una cuantificación exacta de ADN de S. subterránea para estudios epidemiológicos, se ha desarrollado una prueba PCR en tiempo real específica para este patógeno. Esta prueba puede detectar confiablemente y establecer la cantidad de ADN de las masas que contienen las esporas, zoosporas y plasmodio/zoosporangios de S. subterránea. La prueba fue usada para medir la viabilidad de las masas de esporas en el suelo, y fue combinada con la técnica de planta trampa de tomate, con el objeto de estudiar la liberación de las esporas y los niveles de infección en papa bajo diferentes condiciones ambientales. Se encontró que tanto la incidencia como la severidad de la roña son influenciadas por la temperatura, tipo de suelo y régimen de la humedad del suelo cuando se probó bajo condiciones controladas. El nivel de inóculo del suelo no tiene efecto significativo sobre la infección y desarrollo de la enfermedad y aún se encontró que niveles bajos de inoculo produjeron regularmente síntomas severos de infección de la enfermedad. La evidencia de una prueba en toda Gran Bretaña y cinco años de encuestas en Inglaterra y Gales confirmaron que no hay una simple o consistente relación entre el inóculo de roña en la semilla y el desarrollo de la enfermedad en el cultivo progenie. El riesgo de la enfermedad parece estar más relacionado a la relativa facilidad que dan las condiciones ambientales más que al nivel de inóculo. La integración de las medidas de control parece ser el camino más efectivo para que los agricultores reduzcan el impacto de la enfermedad.

Soft-rot resistance combined with other tuber characters in long day-adaptedSolanum phureja

SummaryOffspring were produced from a cross between two long-day-adaptedSolanum phureja clones which carried resistance to tuber soft rot (Erwinia carotovora subsp.atroseptica). In tests carried out on the produce of field-grown plants raised from tubers, over fifty per cent of the 173 offspring were found to be highly resistant. Assessments were also carried out of tuber yield, mean tuber weight, tuber number, shape, regularity, flesh colour, texture of the steamed flesh, fry colour, after-cooking blackening, sprout length after storage and overall dormancy. There were statistically significant differences between clones for all characters (P<0.001).Twelve of the clones were selected on the basis of high resistance, yield, tuber weight, regularity of shape and absence of after-cooking blackening. The value of resistant long-day-adapted diploid material for commercial breeding is discussed.

Inheritance of resistance toFusarium spp. and toPhytophthora infestans in crosses between Neotuberosum and Tuberosum potatoes estimated by seedling tests

SummaryNeotuberosum clones with differing levels of resistance toFusarium coeruleum andFurarium sulphureum, and putative resistance toPhytophthora infestans, were selected and used in crosses with Tuberosum clones. The resulting progenies were assessed for their resistance to each of these pathogens and for breeders preference. There was little correlation between disease scores for the twoFusarium species (r=0.21 and 0.34 for the Neotuberosum and hybrid clones respectively), indicating that resistance to each species is distinct. Statistical analyses revealed differences between the Neotuberosum parents and between the Tuberosum parents for all traits, but the Neotuberosum differences for late blight were not significant (P=0.10–0.05) when tested against the interaction between the two sets of parents. The interaction was significant forF. coeruleum and breeders preference, but notF. sulphureum. No reciprocal differences were found. The only statistically significant correlation between traits for the 72 progenies was a small one (r=0.33; P=0.01–0.001) between the twoFusarium species; for all other pairs of traits r was less than 0.10.It is concluded that there are good prospects for combinding resistances to the twoFusarium species from different sources and also for achieving high levels of other desirable characteristics.

Long-day-adapted Solanum phureja as a source of resistance to blackleg caused by Erwinia carotovora subsp. atroseptica

SummaryLong-day-adaptedSolanum phureja clones were assessed for resistance to blackleg caused byErwinia carotovora subsp.atroseptica under field and controlled environmental conditions over two years. In the field, twenty-two of the twenty-three clones ofS. phureja assessed were as resistant to blackleg as the commercial cultivar Ailsa, the most resistant control, and were significantly (P<0.001) more resistant than the intermediate and susceptible cultivars Wilja and Estima, respectively. Under controlled environmental conditions, resistance in commercial cultivars was more easily overcome. However, 18 of the 21S. phureja clones assessed were significantly more resistant to blackleg than these cultivars.

Inheritance of resistance toFusarium sulphureum in crosses betweenS. tuberosum potato cultivars measured on field and glasshouse grown tubers

SummarySolanum tuberosum potato cultivars with differing levels of resistance to dry rot caused byFusarium sulphureum were hybridised in a half diallel crossing programme. Glasshouse grown tubers from true seedlings and field-grown tubers of the resulting progenies were assessed for their resistance toF. sulphureum. Statistical analysis revealed differences between the progenies forF. sulphureum resistance on both field and glasshouse grown tubers. On the field grown tubers, differences between progenies were due to differences in the general combining ability (GCA) of the parents. Parental phenotypes and GCA values were highly correlated. Overall. GCA was consistent for resistance or susceptibility, and glasshouse grown progenies showed a good correlation for GCA between years (r=0.85***), and with the results of the field grown progenies (r=0.61* in 1998 and r=0.69** in 1999). It appears that resistance toF. sulphureum is heritable, and that this heritability is better assessed on field grown rather than glasshouse grown tubers, although the two are largely in agreement.

Testing the resistance to silver scurf in commercial potato cultivars under controlled environmental conditions.

SummaryOptimum storage conditions to identify resistance to silver scurf among commercially grown potato cultivars were determined in a series of experiments. Inoculation of field-grown tubers with a conidial suspension ofH. solani (concentration 104 conidia per ml of water) and incubation at 15°C with 95% RH for 1 month followed by 2 months at 85% RH produced the greatest differences in disease severity between potato cultivars. Results with glasshouse-grown tubers were comparable with those from field-grown tubers, indicating that the test could be used to identify resistance in wild species of potato and to understand the inheritance of this resistance.

The use of molecular diagnostics to investigate the epidemiology of potato diseases

In recent years, quantitative molecular diagnostic assays based on real-time PCR have been developed for many pests and pathogens of potato. In addition, simple sequence repeat markers have been developed and used to track isolates of Phytophthora infestans. These diagnostic assays are now being used as tools to study unresolved questions in the epidemiology of potato diseases including late blight, powdery scab and black dot. Examples of various investigations designed to examine the relative contribution of seed and soil-borne inoculum in causing black dot and powdery scab on progeny tubers, the effect of environmental factors on the incidence and severity of powdery scab and the survival of asexual and sexual inoculum of P. infestans in soil are described. Consideration is given to the development of appropriate diagnostic assays, their use in conjunction with relevant and robust sampling techniques, and the interpretation of results to inform disease risk assessment and control strategies through industry collaboration.