The effect of post‐harvest storage conditions on the development of black dot (Colletotrichum coccodes) on potato in crops grown for different durations

The effects of post‐harvest curing and storage temperature on severity of black dot, caused by Colletotrichum coccodes, were investigated for potato crops grown for different crop durations (days from 50% emergence to harvest) in soils that posed a low, medium and high risk of disease. In field trials over four growing seasons (2005–8), black dot severity at harvest increased with increasing crop duration, within the range 103–146 days from 50% emergence to harvest (P < 0.05). In field trials over three growing seasons (2006–8), black dot severity on tubers at harvest increased significantly with increasing soil inoculum in each year, within the range 43–4787 pg C. coccodes DNA/g soil (P < 0.05). Storage trials were conducted to measure the influence of accumulated post‐harvest temperature on black dot. In 2005, no difference in black dot severity was observed on tubers stored for 20 weeks at 2.5 and 3.5 °C. In 2006 (but not 2007), increasing the duration of curing after harvest from 4 to 14 days increased black dot severity on tubers from 8.9 to 11.2% (P < 0.01) in long duration crops (>131 days after 50% emergence) grown under high (>1000 pg C. coccodes DNA/g soil) soil inoculum. The number of days of curing did not affect disease severity for shorter duration crops grown at high soil inoculum, or on crops grown at medium or low (100–1000 and <100 pg C. coccodes DNA/g soil, respectively) soil inoculum concentrations. Soil inoculum and crop duration together provided a reasonable prediction of black dot severity at harvest and after a 20‐week storage period.     

The importance of the infected seed tuber and soil inoculum in transmitting Potato mop‐top virus to potato plants

Potato mop‐top virus (PMTV), the cause of spraing in potato tubers, is transmitted by Spongospora subterranea, the cause of powdery scab, and by planting infected seed tubers. This study was undertaken to determine the relative importance of these sources of infection in seed potato production in Scotland. The transmission of PMTV from tested seed tubers to daughter plants was examined over 2 years and six cultivars. The development of foliar symptoms varied with year and cultivar. Infection of daughter tubers derived from PMTV‐infected seed tubers was more prevalent on plants affected by foliar symptoms than those without symptoms. The rate of transmission of PMTV from infected seed tubers to daughter tubers ranged from 18 to 54%. Transmission was affected by cultivar and by origin of seed tubers used for a cultivar, but not by a cultivar's sensitivity to PMTV infection. The incidence of PMTV in daughter tubers of cv. Cara grown from seed potatoes from one source (common origin) by more than 25 seed producers was examined over two successive generations. The incidence of PMTV in daughter tubers was not correlated with that in the seed tubers but appeared to be strongly associated with soil inoculum. The incidence of PMTV was correlated with powdery scab in those crops in which both were present. There was some evidence from soil tests conducted in 2006 using a tomato bait plant and real‐time RT‐PCR that planting PMTV‐infected seed potatoes could increase the risk of introducing the virus into land not infested by PMTV.     

Temporal association of potato tuber development with susceptibility to common scab and Streptomyces scabiei‐induced responses in the potato periderm

Using hydroponics and novel non‐destructive pot culture systems which enable inoculation at specific tuber development stages, the dynamics of common scab infection patterns in potato were studied in order to provide more precise identification of tuber physiological factors associated with susceptibility. At the whole‐tuber level, infection percentages were greatest when Streptomyces scabiei inoculation occurred early; at 2 weeks after tuberization (WAT) 68% of tubers became infected, contrasting with late inoculation (8 WAT), when only 4% infection occurred. The first‐formed internodes were most susceptible to infection, whilst later‐forming and slower‐expanding internodes were less susceptible. Detailed tuber physiological examination of internode 2 showed that pathogen‐induced changes, including increased phellem (periderm) thickness, cell layers and phellem suberization (key physiological features believed critical to S. scabiei infection) were promoted through S. scabiei inoculation. Sequential harvesting showed enhanced phellem suberization (28% greater than the control) within 7 days of pathogen exposure, while phellem thickness and layer responses were also initiated early in the infection process (10–14 days after pathogen exposure) and these responses were independent of symptom expression. Differences in cultivar response were observed, with greater phellem suberization observed 10 days after tuberization (DAT) in the common‐scab‐tolerant cv. Russet Burbank than in the susceptible cv. Desiree. Likewise, Russet Burbank had thicker and more numerous cell layers in the phellem (up to eight cell layers) during early tuber growth (20–30 DAT) than Desiree (up to six cell layers).     

Influence of abiotic factors,inoculum source,and cultivar susceptibility on the potato tuber blemish diseases black dot (Colletotrichum coccodes) and silver scurf (Helminthosporium solani)

Black dot and silver scurf are potato blemish diseases whose economic impact has increased in recent years. Because their symptomatology on tubers is visually similar, disease assessment does not usually differentiate between the two pathogens, which share the same ecological niche. The epidemiology of black dot has been extensively studied, especially in the UK, but the factors that influence silver scurf have been less investigated. In this study, the influence of cultivar, source of inoculum, and environmental conditions on both diseases was studied in field trials over a three-year period (2016–2018) in Switzerland. Planting minitubers did not prevent either disease in daughter tubers, indicating the contribution of soil as an inoculum reservoir. An arbitrary threshold of Colletotrichum coccodes soil inoculum could be set to discriminate between low and high disease risk. For the first time, Helminthosporium solani DNA was detected in stolons, and infections appeared earlier in stolons than in tubers. H. solani stolon and tuber infections usually appeared later in the season than those of C. coccodes. Black dot severity correlated positively with precipitation, while silver scurf severity correlated positively with temperature. Table potato cultivars commonly grown in Switzerland exhibited significant differences in susceptibility to both diseases, and cultivars with low susceptibility to both silver scurf and black dot were identified. These results gave new insights into understanding the factors driving the epidemiology of potato blemish diseases and may contribute to building a risk assessment scheme to manage both diseases simultaneously.     

Detection of the root lesion nematode Pratylenchus penetrans in potato tubers

The root lesion nematode Pratylenchus penetrans parasitizes a wide range of economically important crops, including potato (Solanum tuberosum). Damage by P. penetrans impacts not only the potato yield but can also reduce the tuber quality. Detailed information on tuber infection by P. penetrans is scarce for most cultivars and molecular detection of nematodes from infected tubers is needed. The objective of this study was to assess tuber symptomatology due to P. penetrans infection in 10 potato cultivars and to provide an accurate molecular methodology for nematode detection using tuber peels. Sprouts of certified potato seed from cultivars Agata, Agria, Camel, Désirée, Dirosso, Kennebec, Laura, Picasso, Royata, and Stemster were planted in 2 L pots, and soil was inoculated with 4 P. penetrans/g of soil. Sixty days after inoculation, tubers were harvested, inspected for lesions, and the number of nematodes/g of potato peel assessed. Observations of tubers with symptoms showed the presence of P. penetrans in superficial layers of peels around the lenticels and injured necrotic tissue. Different nematode stages were detected in tubers of all inoculated cultivars, varying from 4 to 46 nematodes/g of potato peel. Species-specific primers showed suitable sensitivity and reproducibility for the detection of P. penetrans in tuber potato peel samples. The molecular detection of P. penetrans directly from tuber peels can facilitate routine nematode inspections of potato seed tubers or cull potatoes for nematode detection, and prevent further dissemination of this species.     

Aggressive and mild Potato virus Y isolates trigger different specific responses in susceptible potato plants

Differences in the early responses of two potato cultivars, Igor and Nadine, to two isolates of Potato virus Y (PVY), the aggressive PVY^NTN and the mild PVY^N, were monitored. Microarray and quantitative real‐time PCR analyses were carried out to identify differentially expressed genes after inoculation with each virus isolate. Additionally, symptom severity and development was observed and the amount of virus isolate accumulated in systemically infected leaves was evaluated, where a significantly higher amount of PVY^NTN was detected. Microarray analysis revealed 572, 1288 and 1706 differentially expressed genes at 0·5, 12 and 48 h post‐inoculation, respectively in cv. Igor, with a similar pattern observed in cv. Nadine. Microarray and quantitative real‐time PCR results implied an earlier accumulation of sugars and lower photosynthesis in leaves inoculated with the aggressive isolate than in leaves inoculated with the mild isolate. The PVY^NTN isolate did not activate early differential expression of the Fe‐superoxide dismutase and pectin methylesterase inhibitor (PMEI) genes, indicating a delay in plant response relative to that following PVY^N inoculation. Differences in the expression of the β‐glucanase‐I gene were also observed in early plant responses to inoculation with each virus isolate.     

Detection and characterization of Streptomyces causing potato common scab in Western Europe

A PCR-based diagnostic method was developed for direct detection from tuber lesions of pathogenic Streptomyces causing common scab of potato. Primers were designed to amplify a fragment of the txtAB ( txtA and txtB ) genes, which are pathogenicity determinants in the main pathogenic Streptomyces species. The method was evaluated on 84 naturally infected potato samples, comprising 19 potato cultivars, harvested in the years from 2000 to 2004 in the Netherlands, the UK, France, Germany and Spain. Pathogenic Streptomyces in tuber lesions were detected by PCR in 70 samples and were also successfully isolated from these 70 samples. All pathogenic isolates showed the basic general phenotypic traits of the S. scabiei phenetic cluster. RFLP analysis of amplified rRNA sequences, together with carbon source utilization and repetitive BOX profiles, allowed most isolates to be assigned to S. europaeiscabiei , which emerged as the main cause of potato common scab in Western Europe.     

Development of a quantitative real‐time PCR assay for Phytophthora infestans and its applicability to leaf,tuber and soil samples

A sensitive real‐time polymerase chain reaction (PCR) assay was developed for the quantification of Phytophthora infestans, the cause of foliar and tuber late blight in potato. A primer pair (PinfTQF/PinfTQR) and a fluorogenic probe (PinfTQPR) were designed to perform a quantitative assay for the detection of P. infestans in leaves, tubers and soils. The assay was shown to be specific to P. infestans and the very closely taxonomically related non‐potato pathogen species P. mirabilis, P. phaseoli and P. ipomoea, but did not detect the potato pathogens P. erythroseptica and P. nicotianae. The assay was able to reliably detect P. infestans DNA at 100 fg per reaction and was effective in quantifying P. infestans in infected leaf tissue from 24 h after inoculation and also in infected symptomless tubers and diseased tubers. Attempts to detect oospores of P. infestans in naturally and artificially infested soil samples are described and compared with baiting tests and previous literature. It was not possible to detect oospores in soil samples due to problems with DNA extraction from the oospores themselves. However, the assay was shown to detect even very low levels of asexual inoculum (sporangia and mycelium) in soil. This work assembles all the necessary features of a quantitative P. infestans assay, which have previously been somewhat disparate: the sensitivity, specificity and quantitation are fully validated, the assay is shown to work in common applications in leaf and tuber tissue and the problems with P. infestans oospore detection are explored and tested experimentally.     

Relative importance of different types of inoculum to the establishment of Mycosphaerella graminicola in wheat crops in north‐west Europe

The contribution of wheat debris to the early stages of septoria leaf blotch epidemics was assessed in a 3‐year field experiment. First lesions were detected very early (December) in the case of an early sowing (mid‐October), showing that the first contamination could occur as soon as the seedlings emerge. The tested debris management options (chopped debris, removal of debris followed by tillage, or tillage in absence of debris) had a strong effect, although transient, on the epidemic dynamic: the more debris present on the soil surface, the more severe initial disease was. The magnitude of differences between treatments differed substantially between years. The relative production of pycnidiospores and ascospores was measured on the chopped debris. Peaks in pycnidiospore and ascospore production coincided in October–November. Both types of spores can be involved as primary inoculum in north‐west European conditions. The local amount of pycnidiospores available on debris in the field, estimated per square metre, was 1000‐fold the local ascospore production. Moreover, inoculum production was quantified on debris exposed to different environmental conditions. Autumnal conditions, characterized by moderate temperature with alternating wet and dry periods, were favourable for the production of both pycnidiospores and ascospores, as shown by the high inoculum production on debris exposed to field or outdoor conditions. By late autumn, the canopy became the most important source of pycnidiospores, and this period, characterized by the decreasing role of debris as a local source of inoculum compared to distant potential sources, can be considered as the end of the early epidemic stages.     

Detection and quantification of airborne inoculum of Hymenoscyphus pseudoalbidus using real‐time PCR assays

A method based on real‐time polymerase chain reaction (PCR) and the use of rotating‐arm spore traps was developed for quantifying airborne Hymenoscyphus pseudoalbidus ascospores. The method was sensitive and reproducible, and the collection efficiency was 10% of the spores present in the air. The temporal ascospore dispersal pattern was studied over 3 years by collecting spores every 15 days for a 24 h air‐sampling period during the ash‐growing season. The highest production was detected from the end of June to the beginning of September. The overall ascospore production did not differ significantly among stands within a specific year but there were differences from year to year. There was a positive correlation between air temperature and the number of ascospores trapped, with most of the positive samples being observed at temperatures above 12°C. The vertical profile of ascospore dispersal showed a strong decrease in ascospore density within a height of 3 m, regardless of date of collection. An analysis of the spore traps installed at increasing distances from an infected stand showed that most of the ascospores were deposited downwind within 50 m of the stand. These data are discussed in context of the epidemiology of the disease.     

Detection of Dickeya spp. latent infection in potato seed tubers using PCR or ELISA and correlation with disease incidence in commercial field crops under hot‐climate conditions

Over a 5‐year period (2006–2010), 277 certified, visually healthy potato seed lots, imported from Europe to Israel for commercial use, were tested for Dickeya spp. latent infection by PCR analysis (277 seed lots) and ELISA (154 seed lots). Seeds from these lots were grown in commercial potato fields which were inspected twice a season by Plant Protection and Inspection Services (PPIS). Stem samples were tested for the presence of Dickeya spp. by PCR analysis. PCR and ELISA results from seed lot testing correlated with disease expression in 74 and 83·8% of the cases, respectively. Positive laboratory results with no disease symptoms in the field (‘+lab/?field’ results) comprised 24·7 and 9·7% of the PCR and ELISA analyses, respectively, whereas negative laboratory results with disease symptoms in the field results (‘?lab/+field’) were obtained in 1·3 and 6·5%, of cases respectively. Maximum disease incidence, as well as the number of cultivars expressing disease symptoms, increased over the years of this study, indicating an increase in the prevalence of the disease. Severe disease incidence was observed on cvs Dita, Rodeo, Desiree, Mondial, Tomensa and Jelly. Of the 55 imported seed lots from which disease was recorded in the field, 49 originated from the Netherlands, four from Germany and two from France. None originated from Scotland.     

Effects of strip intercropping of potatoes with non‐hosts on late blight severity and tuber yield in organic production

The effects of strip cropping of potatoes with cereals or a grass‐clover mix in and perpendicular to the main wind direction on foliar late blight severity and tuber yield were studied in large‐scale field experiments in Germany. Disease progress was assessed in 12–18 sections per plot and yields determined from the same sections. In 2000, plot size was 3 × 10 m and there were no disease reductions apparently due to interplot interference. In 2001 and 2002, with plot sizes of 6 × 18 and 6 × 36 m in strip‐cropped potatoes, disease was significantly reduced by 9–20% and 4–12%, respectively, compared to pure stands of potato, with the greatest reductions in plots planted perpendicular to the wind and neighboured by grass‐clover. The most important factors contributing to disease reduction were loss of inoculum outside of the plots and barrier effects of neighbouring non‐potato hosts. Only 0–20% of the overall yield variation could be explained by the area under the disease progress curve, depending on cultivar and year. In one year disease effects on the yield of a moderately resistant cultivar were higher than on the yield of a susceptible cultivar. This was probably caused by differences in bulking behaviour. Nutrient limitation appeared to be more important than disease in reducing yields. Yields in the edge potato rows directly neighboured by cereals were significantly reduced in all 3 years, but competition by cereals did not change the disease–yield‐loss relationship. Strip intercropping might be a useful component in an overall management strategy to reduce incoming late blight inoculum.     

Attract‐and‐kill: a new strategy for the management of the potato tuber moths Phthorimaea operculella (Zeller) and Symmetrischema tangolias (Gyen) in potato: laboratory experiments towards optimising pheromone and insecticide concentration

BACKGROUND: Sex pheromones of the potato tuber moths Phthorimaea operculella (Zeller) and Symmetrischema tangolias (Gyen) are ideal tools to monitor pest flight activity but are not used as means of control. The aim of the present study was to test the suitability of an attract‐and‐kill strategy consisting of pure pheromones and the contact insecticide cyfluthrin as the active ingredient, formulated with plant oils and ultraviolet absorbers, and applied in droplet sizes of 100 µL. RESULTS: Cyfluthrin at a concentration of 5 g L^?1 resulted in the highest and fastest killing of males after 48 h, with a 100% mortality after 3–4 days. In contrast, control males survived for 13 days. In olfactometer experiments, the pheromone concentration of 0.5 g L^?1 was significantly most attractive against eight virgin females. At controlled conditions (20 °C), no reduction in efficacy of the attract‐and‐kill formulation was observed for a minimum period of 36 days, whereas under natural environmental conditions the efficacy reduced gradually after day 6 of exposure. The longer the droplet was exposed, the longer was the time to reach 100% mortality of males. CONCLUSIONS: Compared with attract‐and‐kill studies for other pest species, the results are promising as a means of achieving highly effective control of potato tuber moths under field conditions. Copyright © 2010 Society of Chemical Industry     

Quantification of potato common scab pathogens in soil by quantitative competitive PCR with fluorescent quenching‐based probes

Common scab of potato tubers caused by pathogenic Streptomyces spp. is a cause of serious economic loss worldwide. For the rapid and accurate quantification of pathogenic Streptomyces spp. residing in soil, a new competitive real‐time PCR method using fluorescent quenching‐based probes (quantitative competitive quenching probe PCR: QCQP‐PCR) was developed. The virulence gene of pathogenic Streptomyces spp., nec1, was selected as the target for QCQP‐PCR. A specific primer set to amplify the nec1 gene, and a fluorescently labelled probe that specifically hybridizes with the nec1 amplicon were designed. For QCQP‐PCR, an internal standard DNA (IS DNA) that is identical to the nec1 amplicon but has a 4‐base mismatch in the probe‐hybridizing region, and a fluorescently labelled probe IS, which specifically hybridizes with IS DNA at the mutagenized region, were PCR‐synthesized. The target nec1 gene was co‐amplified with the known copy number of IS DNA by PCR using the same primer set in the presence of the specific probes. The PCR products were monitored in real‐time by measuring the fluorescence intensity (quenching) of each probe. The initial amount of the nec1 gene was quantified based on the ratio of the PCR products of the same PCR cycle. The results revealed that QCQP‐PCR could be used to precisely quantify the nec1 gene, even in the presence of PCR inhibitors in the soil samples examined. The lower limit of quantification was 20 copies per tube, which corresponded to 1500 copies per g dry soil. The quantification achieved by this method was completed within 5 h, i.e. the duration of the entire analysis. These results demonstrate the usefulness of the present method for monitoring pathogenic Streptomyces species in soil.     

The use of digital image analysis and real‐time PCR fine‐tunes bioassays for quantification of Cercospora leaf spot disease in sugar beet breeding

Cercospora leaf spot, caused by the fungus Cercospora beticola, is a major fungal sugar beet disease worldwide and the cause of significant yield losses. The disease is most successfully countered by the introduction of genetic tolerance into elite sugar beet hybrids. To this end, breeding programmes require high quality biological assays allowing discrimination of minor differences between plants within a segregating population. This study describes the successful implementation of image analysis software in the bioassays for quantification of necrotic lesions at different stages of C. beticola infection, allowing selection on minor phenotypic differences during the sugar beet breeding process for C. beticola resistance. In addition, a real‐time PCR assay was developed for the quantification of C. beticola pathogen biomass in infected beet canopy. The use of both techniques, even in an early stage of infection, fine‐tunes current bioassays, allowing more accurate and efficient selection of resistant breeding material.     

The role of seeds and airborne inoculum in the initiation of leaf blotch (Rhynchosporium secalis) epidemics in winter barley

Both airborne spores of Rhynchosporium secalis and seed infection have been implied as major sources of primary inoculum for barley leaf blotch (scald) epidemics in fields without previous history of barley cropping. However, little is known about their relative importance in the onset of disease. Results from both quantitative real‐time PCR and visual assessments indicated that seed infection was the main source of inoculum in the field trial conducted in this study. Glasshouse studies established that the pathogen can be transmitted from infected seeds into roots, shoots and leaves without causing symptoms. Plants in the field trial remained symptomless for approximately four months before symptoms were observed in the crop. Covering the crop during part of the growing season was shown to prevent pathogen growth, despite the use of infected seed, indicating that changes in the physiological condition of the plant and/or environmental conditions may trigger disease development. However, once the disease appeared in the field it quickly became uniform throughout the cropping area. Only small amounts of R. secalis DNA were measured in 24 h spore‐trap tape samples using PCR. Inoculum levels equivalent to spore concentrations between 30 and 60 spores per m³ of air were only detected on three occasions during the growing season. The temporal pattern and level of detection of R. secalis DNA in spore tape samples indicated that airborne inoculum was limited and most likely represented rain‐splashed conidia rather than putative ascospores.     

High‐throughput detection of highly benzimidazole‐resistant allele E198A with mismatch primers in allele‐specific real‐time polymerase chain reaction

BACKGROUND: A point mutation often confers resistance of organisms against medical drugs and agricultural pesticides. Allele‐specific nucleotide polymerase chain reaction (ASPCR) and allele‐specific quantitative real‐time PCR using SYBR Green (ASQPCR) are widely and effectively applied to detect and monitor this type of resistance. However, the former is unsuitable for high‐throughput detection, and the latter often reduces the accuracy of detection. RESULTS: In order to decrease background amplification, a rapid and high‐throughput genotyping method with mismatch primers was developed (ASQPCR‐MP) and applied specifically to survey the frequency of the highly benzimidazole‐resistant MBC^HR mutation (E198A) in the β‐tubulin gene of Sclerotinia sclerotiorum (Lib.) de Bary populations. Genomic DNA from 223 sclerotia was analysed. Similar genotype results were also obtained using ASPCR with mismatch primers and a mycelial growth inhibition assay. It was found that ASQPCR‐MP clearly differentiated MBC^HR and benzimidazole‐sensitive MBC^S phenotypes. Moreover, ASQPCR‐MP took less than 6 h to complete. CONCLUSION: ASQPCR‐MP appears suitable for large epidemiological studies involving resistant genotypes and requiring high‐throughout formats. Copyright © 2009 Society of Chemical Industry