Potato blackleg: Epidemiology,host-pathogen interaction and control

Blackleg is caused byErwinia carotovora subsp.atroseptica (Eca) andE. chrysanthemi (Echr) in cool and hot climates respectively. The bacteria are opportunistic pathogens and rely on their strong pectolytic character to infect plants when conditions favor their multiplication. Blackleg is a seedborne disease and the bacteria can survive in a quiescent form in lenticels and wounds during storage. The contaminated mother tuber and not the blackleg plant is the main source of progeny tuber contamination. Other sources of the pathogen are airborne (insects and aerosols) erwinias deposited on leaves and from there to the soil and progeny tubers, and erwinias in rotting tubers smeared into wounds incurred during mechanical crop handling.Most seed tubers are contaminated but blackleg incidence is related to seed contamination level modulated by soil water status. Competitiveness of the erwinias in the rotting mother tuber is affected by temperature, Eca is favored at <25°C and Echr at higher temperatures. The ubiquitousE. carotovora subsp.carotovora apparently fails to compete successfully with the other erwinias and saprophytic pectolytic bacteria in mother tubers and therefore does not cause blackleg.Disease control measures are based on avoiding tuber contamination by cultural means (early harvesting), reducing tuber contamination level (dry storage and hot water treatment) and planting ‘clean’ seed identified by quantifying tuber contamination rather than by visual crop inspection. Finally, recently identified highly resistant, even under anaerobic conditions, wildSolanum spp. could be used to breed for resistant cultivars by conventional methods or by genetic engineering.     

Detection of <Emphasis Type="Italic">Potato virus Y</Emphasis> in industrial quantities of seed potatoes by TaqMan Real Time PCR

Potato (Solanum tuberosum) is the largest crop in Israel. Production is based on the import of seed tubers from Europe for the spring crop. Imported tubers are generally free from virus infection. The most important virus infecting potato is Potato virus Y (PVY), which may cause severe damage to marketable yields. In Israel, tubers from the spring harvest are stored over the summer for planting in the autumn. It is important to be able to determine the infection rate of seed tuber lots from the spring harvest prior to storage. Commonly, infection is measured by sprouting tubers and measuring virus titre in the leaves using ELISA (the “Growing-On test”), which takes at least 6 weeks to give results. There is a need for a faster method to produce results, such as Taqman Real Time PCR (qPCR), for direct analysis of viral infection in tubers at harvest. To use qPCR as a diagnostic tool, it is necessary to demonstrate that both techniques give comparable results on batches of field-grown tubers. Such a comparison was performed on potential seed tuber lots of 14 different cultivars over three Israeli spring harvests (2013–2015). The agreement between the results of the two techniques was not of high statistical significance. However, the qPCR technique can distinguish well, by binary classification, between tuber lots with a low PVY infection rate (<5% by Growing On test; suitable for seed) and those unsuitable for seed (≥5% by Growing On test). Therefore, qPCR is an appropriate technique for determination of the PVY infection rate of seed tuber lots in Israel.     

Factors affecting the temporal progress of stem canker (Rhizoctonia solani ) on potatoes (Solanum tuberosum)

The effects of different densities of tuber-borne inoculum, selected agronomic treatments (date of planting, irrigation and size of seed tubers) and their interactions on the temporal progress of stem canker ( Rhizoctonia solani ) on potato plants ( Solanum tuberosum ) were investigated in a multifactorial experiment. Data comprising stem number and the incidence and severity of stem canker from planting until tuber initiation in two consecutive growing seasons were analysed using linear contrasts, quadratic contrasts and the area under the disease progress or host growth curve. Differences in the incidence and severity of stem canker were dominated by the effect of different densities of tuber-borne inoculum. The majority of disease progress curves were nonmonotonic for the incidence and severity of stem canker with a rapid rise in disease up to stem emergence and a decline thereafter. Most treatments affected the area under the curve and to a lesser extent the average rate of increase in disease. Of the agronomic treatments, later dates of planting and pre-emergence irrigation reduced the levels of stem canker whereas size of seed tubers did not affect the progress of disease. Little additional information was revealed by scoring for the severity rather than the incidence of stem canker.     

A new quantitative real-time PCR assay for Rhizoctonia solani AG3-PT and the detection of AGs of Rhizoctonia solani associated with potato in soil and tuber samples in Great Britain

Rhizoctonia solani is an important pathogen of potatoes causing stem canker and black scurf. The fungus is a species complex comprised of 13 known anastomosis groups (AGs). AG3-PT is the anastomosis group frequently associated with disease in potatoes. A real-time PCR assay was designed to the rDNA ITS region of AG3-PT isolates to enable the pathogen to be detected directly in tuber and soil samples. The resulting assay was highly specific for AG3-PT, and did not amplify DNA from isolates from other AGs or subgroups of AG3. Using a bulk DNA extraction method capable of extracting from up to 250 g of soil, the assay could detect one individual sclerotium of AG3-PT (weighing 200 μg) in 250 g of soil. The AG3-PT assay was used, with assays for AG2-1, AG5 and AG8 to determine the prevalence of those AGs in UK potato soils and tubers. AG2-1 and AG3-PT were the predominant groups in tubers and soils, although AG3-PT was more frequently isolated from tubers, highlighting its importance as a potato pathogen. AG3-PT was also detected in more than half of the tuber samples tested suggesting the importance of seed borne inoculum.     

Effect of inoculum source on root and tuber infection by potato blemish disease fungi

The incidence of potato pathogens on healthy roots of micropropagated (MP) and seed tuber (ST) plants was examined on successive dates during the growing season in two field experiments. Microplants were grown in a glasshouse for 4–5 weeks in perlite or peal-based substrates, and exposed or not to natural inoculum before planting in the field. The seed tubers originated from stocks of visually clean or moderately blemished tubers and were surface-sterilized or not before planting. Polyscytalum pustulans and Helminthosporium solani only infected roots of ST plants and inoculated MP plants. The incidence of P. pustulans was affected by seed tuber-borne inoculum and, in I year, by the substrate. H. solani was detected infrequently on roots. Rhizoctonia solani was present at low frequencies in most root samples, and more ST than MP plant roots were colonized; there were no substrate effects. In 1 year, increased inoculum levels increased root infection, but only in MP roots. Colletotrichum coccodes occurred at high frequencies and was most common in roots of ST plants. Progeny tubers showed some treatment effects when tested in September and after storage for 6 months, but there were no consistent relationships between root and progeny tuber infection.     

PCR-based determination of colonization patterns during potato tuber infection by single and multiple pathogens

Potato tubers piled in storage are prone to infection by numerous pathogens. Each pathogen can cause damage alone, but severe losses often arise when more than one pathogen is involved. Currently, only a visual diagnosis is practiced on potato tubers before storing them, which does not allow any prediction of further disease spread. The aim of the present study was to determine differences in patterns of tissue colonization by several tuber decay pathogens and how late blight infection affects further tuber colonization by other important tuber pathogens. This study was conducted using artificial inoculation of potato tubers and PCR to provide an early and accurate diagnosis of disease development for major potato tuber rots, and to assess potential synergism/antagonism between Phytophthora infestans and other pathogens in stored tubers. In order to accurately follow the progress of each pathogen in tuber tissues, samples were collected over time from both the surface (peel, 0–2 mm depth) and internal tissues (flesh, depth > 2 mm) of the tubers at various distances from the inoculation site, at 3, 5, 7, 10, 12, 14, 17, and 19 days after inoculation. Successful detection of single or multiple pathogens was achieved using specific PCR-primers for each pathogen. Pathogens were always detected several centimeters ahead of the visible lesions. This tracking enabled us to determine the extent of colonization both on the tuber’s surface and in internal tissues by each tested pathogen, either after single or multiple infections involving P. infestans as the primary pathogen. The presence of P. infestans was shown to enhance the development of Pectobacterium atrosepticum and to slow down that of P. erythrospetica and Pythium ultimum. No noticeable effect on further tuber colonization by F. sambucinum, V. dahliae or V. albo-atrum was observed in the presence of P. infestans. This approach involving more than one pathogen is more realistic than classical studies considering single pathogens, and may be helpful in monitoring the sanitary status of stored tubers. Our results make the outcome of certain combinations of pathogens in potato tubers more predictable and may result in more efficient preventive measures.     

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.     

Relationships between stem canker, stolon canker, black scurf (Rhizoctonia solani) and yield of potato (Solanum tuberosum) under different agronomic conditions

Relationships between diseases caused by Rhizoctonia solani on different parts of potato plants ( Solanum tuberosum ) at different stages of crop growth were investigated under selected agronomic conditions. The effects of different densities of tuber-borne inoculum, date of planting, irrigation, size of seed tubers and their interactions on the incidence of stem and stolon canker during crop growth, the incidence and severity of black scurf and the yield of progeny tubers at harvest were quantified in a multifactorial experiment. Differences in the incidence of stem canker, stolon canker and black scurf were dominated by the effect of density of inoculum on seed tubers at planting. Highly positive correlations between the disease variables indicated a close relationship between the incidence of disease at each stage of crop growth although the degree of association between variables measured at an early growth stage and those measured at progressively later stages of crop growth weakened as the time interval increased. Total yield of progeny tubers was not affected by the density of tuber-borne inoculum although there was a shift in the size distribution, with a decrease in the yield of main-sized tubers and an increase in the yield of baker- and oversized tubers at the higher density of inoculum. Of the remaining factors, the effect of season tended to be more pronounced than any of the agronomic treatments although the use of irrigation and later dates of planting did influence the incidence of infection to a limited extent.     

<Emphasis Type="Italic">Aphelenchoides gorganensis</Emphasis> n. sp. (Nematoda: Aphelenchoididae)<Emphasis Type="Italic">,</Emphasis> a new species from Iran

Phytophthora capsici infection of chili pepper seedlings can cause substantial losses due to damping-off and collar rot diseases. Chemical control is no longer effective due to reported resistance development, on top of the related environmental concerns and the consumer demands for reduced use of fungicides. Biological control is a sustainable option, with several agents having been reported to be effective against this pathogen. This research focused on optimizing the application of strain THSW13 of Trichoderma hamatum and a bacterial isolate BJ10–86 with the objectives of improving chili pepper seed germination, reduce damping-off disease incidence, and improve the growth of the seedlings. Bacterial isolate BJ10–86 was subjected to molecular identification and found to be Pseudomonas aeruginosa. Chili pepper seeds treated with the biocontrol agents, individually or in combination, were seeded into commercial nursery media that had been pre-inoculated with P. capsici zoospores. Over a period of 35 days the chili pepper seed treatments significantly (P = 0.008) reduced the disease incidence of seedlings damping-off. Combined application of T. hamatum and P. aeruginosa was the best biocontrol treatment with an area under disease curve of only 36.61 units compared to 92.87 units for the control treatment. Similar results were observed in vitro where T. hamatum and P. aeruginosa synergistically inhibited P. capsici growth by 73.2 %. The inhibition activity of this treatment was similar to mefenoxam treatment, which implies that it is an effective and sustainable alternative for chili pepper seed treatment. The biocontrol seed treatment had no effect on seed germination and seedling growth.     

Biocontrol of powdery scab of potato by seed tuber application of an antagonistic fungus, <Emphasis Type="Italic">Aspergillus versicolor</Emphasis>, isolated from potato roots

An environmentally friendly measure to control potato powdery scab caused by a protozoan pathogen Spongospora subterranea f.sp. subterranea (Sss) was developed by focusing on antagonistic microorganisms that were considered compatible with potato root. Five hundred and eight soil fungi, isolated from potato root cultivated in soil suspensions from four potato fields in Hokkaido, were screened for suppressiveness of root infection by Sss in a hydroponic culture system and for powdery scab severity in greenhouse and field experiments. Antagonistic isolate Im6-50, identified as Aspergillus versicolor, was selected as a potent biological control agent. In a 3-year field test, A. versicolor Im6-50 suppressed powdery scab with a protection value of 54–70 (100?=?complete protection) when applied directly on seed tubers compared with a protection value of 77–93 by the synthetic fungicide fluazinam. A. versicolor Im6-50 was detected from the surface of daughter tubers and from the soil in which the inoculated seed tubers were cultivated by PCR using species-specific primers. The establishment of A. versicolor Im6-50 on the stolon of inoculated potato plants and in the rhizosphere is considered to contribute to the mechanism for disease suppression.     

Evaluation of seedborne bacterial pathogens on common bean cultivars grown in central Anatolia region,Turkey

Bacterial diseases of bean cause economically important losses worldwide. The most important method for managing bacterial diseases on bean is the use of pathogen-free seed. In this study, 198 different dry bean seed samples of six different cultivars including Dermason, Cali, Sira, Battal, Bombay and Seker, were collected from 12 provinces of Central Anatolia Region of Turkey. All were tested to investigate the seedlots as primary inoculum sources of the major bacterial diseases. The data revealed that 22,72 %, 13,63 %, 11,11 %, 1,51 % and 0.5 % of seed samples tested were contaminated with five seedborne bacterial pathogens, Pseudomonas savastanoi pv. phaseolicola (Psp), Pseudomonas syringae pv. syringae (Pss), Xanthomonas axonopodis pv. phaseoli (Xap), X. axonopodis pv. phaseoli var. fuscans (Xapf) and Curtobacterium flaccumfaciens pv. flaccumfaciens (Cff), respectively. All bacterial strains isolated were identified based on morphological, physiological, biochemical, molecular and pathogenicity tests. The results showed that Psp and Pss were found together on cv. Cali; Psp and Xap on cv. Dermason and cv. Sira; and Pss and Xap on cv. Seker, cv. Dermason, and cv. Cali. Therefore, the results in the present study suggested that evaluation and selection of pathogen-free seeds are very important for preventing the spread of pathogens and effective management of seed borne bacterial diseases prevalent in bean growing regions; in addition to implementation of integrated crop production strategies such as crop rotation, sanitation, seed treatment, tolerant/resistant cultivar selection and proper bactericide application.     

Increased susceptibility of potato to <Emphasis Type="Italic">Rhizoctonia</Emphasis> diseases in <Emphasis Type="Italic">Potato leafroll virus</Emphasis>-infected plants

In Hokkaido potato fields, tubers produced from the plants with leaf curl symptoms caused by potato leaf roll virus (PLRV) were noted to be more densely covered with Rhizoctonia sclerotia. This observation led us to hypothesize that potato infected with PLRV would have an increased susceptibility to Rhizoctonia solani. To test this hypothesis, in a pot experiment, we inoculated PLRV-infected mother tubers with Rhizoctonia. As a result, PLRV-infected plants produced significantly fewer and smaller tubers than virus-free plants did, suggesting that PLRV-infected plants are more susceptible than virus-free plants to R. solani. Virus-free seed tubers should thus be used to reduce Rhizoctonia diseases.     

Detection of spore balls of Spongospora subterranea on potato tubers by enzyme-linked immunosorbent assay

A rabbit was immunized with a homogenate of spore balls (cystosori) of Spongospora subterranea which also contained some potato tuber debris. The resultant polyclonal antiserum contained antibodies which reacted against extracts of spore balls and healthy tubers. Antibodies to the tuber debris were removed by incubating the blood serum with an extract from a S. subterranea -free tuber. In enzyme-linked immunosorbent assay (ELISA), the γ-globulin fraction of the absorbed antiserum reacted with dilute tuber extracts containing the equivalent of as little as 0·08 spore balls per ml. The reaction with spore balls was inhibited in high concentrations of tuber sap. Extracts of peel from symptomless tubers which had been stored in contact with scabbed tubers also reacted with the γ-globulin, presumably because the symptomless tubers became contaminated with spore balls. However, spore balls in soil were only weakly detected when high numbers were present. The γ-globulin did not react with the plasmodial stage of the pathogen, or with 15 other micro-organisms tested, including resting spores of Plasmodiophora brassicae.     

Disease Assessment Concepts and the Advancements Made in Improving the Accuracy and Precision of Plant Disease Data

New concepts in phytopathometry continue to emerge, such as the evolution of the concept of pathogen intensity versus the well-established concept of disease intensity. The concept of pathogen severity, defined as the quantitative measurement of the amount of pathogen per sampling unit has also emerged in response to the now commonplace development of quantitative molecular detection tools. Although the concept of disease severity, i.e., the amount of disease per sampling unit, is a well-established concept, the accuracy and precision of visual estimates of disease severity is often questioned. This article will review disease assessment concepts, as well as the methods and assessment aides currently available to improve the accuracy and precision of visually-based disease severity data. The accuracy and precision of visual disease severity assessments can be improved by quantitatively measuring and comparing the accuracy and precision of rates and/or assessment methods using linear regression, by using computer-based disease assessment training programmes, and by developing and using diagrammatic keys (standard area diagrams).     

Dickeya species: an emerging problem for potato production in Europe

Dickeya species (formerly Erwinia chrysanthemi) cause diseases on numerous crop and ornamental plants world‐wide. Dickeya spp. (probably D. dianthicola) were first reported on potato in the Netherlands in the 1970s and have since been detected in many other European countries. However, since 2004–5 a new pathogen, with the proposed name ‘D. solani’, has been spreading across Europe via trade in seed tubers and is causing increasing economic losses. Although disease symptoms are often indistinguishable from those of the more established blackleg pathogen Pectobacterium spp., Dickeya spp. can initiate disease from lower inoculum levels, have a greater ability to spread through the plant’s vascular tissue, are considerably more aggressive, and have higher optimal temperatures for disease development (the latter potentially leading to increased disease problems as Europe’s climate warms). However, they also appear to be less hardy than Pectobacterium spp. in soil and other environments outside the plant. Scotland is currently the only country in Europe to enforce zero tolerance for Dickeya spp. in its potato crop in an attempt to keep its seed tuber industry free from disease. However, there are a number of other ways to control the disease, including seed tuber certification, on‐farm methods and the use of diagnostics. For diagnostics, new genomics‐based approaches are now being employed to develop D. dianthicola‐ and ‘D. solani’‐specific PCR‐based tests for rapid detection and identification. It is hoped that these diagnostics, together with other aspects of ongoing research, will provide invaluable tools and information for controlling this serious threat to potato production.     

Disease risk assessment of sugar beet root rot using quantitative real-time PCR analysis of <Emphasis Type="Italic">Aphanomyces cochlioides</Emphasis> in naturally infested soil samples

Sugar beet root rot, caused by the oomycete Aphanomyces cochlioides, is a serious and economically important disease of sugar beets world-wide. Today, disease risk assessment consists of a time-consuming greenhouse bioassay using bait plants. In the present study, a real-time quantitative PCR (qPCR) assay for determination of A. cochlioides DNA in field-infested soil samples was developed and validated using the standard bioassay. The qPCR assay proved to be species-specific and was optimized to give high amplification efficiency suitable for target copy quantification. A high correlation (R² > 0.98, p < 0.001) with pathogen inoculum density was shown, demonstrating the suitability for monitoring soil samples. The limit of detection (LOD) was evaluated in several different soil types and varied between 1 and 50 oospores/g soil, depending on clay content. Soils with a high LOD were characterised as having a low clay content and high content of sand. Varying levels of the A. cochlioides target sequence were detected in 20 of the 61 naturally infested soil samples. Discrepancies between the bioassay and the qPCR assay were found in soils from low- and medium-risk fields. However, the qPCR diagnostic assay provides a potentially valuable new tool in disease risk assessment, enabling sugar beet growers to identify high-risk fields.     

Endophytic bacterial communities in the periderm of potato tubers and their potential to improve resistance to soil-borne plant pathogens

To evaluate whether the location of bacterial endophyte communities contributes to disease resistance in potato tubers ( Solanum tuberosum ), the population density, biodiversity and antibiotic activity of endophytic bacteria was examined from the tuber peel (periderm plus top 3 mm of tissue) of four cultivars (Russet Burbank, Kennebec, Butte and Shepody). There were no significant differences for population density of bacteria among the layers of peel examined and no cultivar × peel layer interaction. Endophytic bacteria from several layers of peel were challenged in in vitro bioassays to the soil-borne plant pathogens Fusarium sambucinum , Fusarium avenaceum , Fusarium oxysporum and Phytophthora infestans (mating types A1 and A2). In general, antibiosis of bacterial endophytes against these pathogens was significantly higher ( P  = 0.01) in isolates recovered from the outermost layer of tuber peel and decreased progressively toward the centre of the tuber. Antibiosis against P. infestans was variable, with a progressive decrease in antibiotic activity from outer to inner layers of peel occurring in cvs Russet Burbank and Kennebec only. For antibiosis there were significant cultivar × peel, and cultivar × pathogen interactions ( P  = 0.01). In all cases the inhibitory activity of endophytic bacteria was significantly greater ( P  = 0.01) against the A1 than the A2 mating type of P. infestans . In four of seven cases, where the same species of bacteria were recovered from all three peel layers, antibiosis to pathogens decreased significantly ( P  = 0.01) with depth of recovery (from the periderm to inside the tuber), indicating that in certain communities of endophytic bacteria, defence against pathogens may be related to bacterial adaptation to location within a host and may be tissue-type and tissue-site specific.     

Visual detection of <Emphasis Type="Italic">Didymella bryoniae</Emphasis> in cucurbit seeds using a loop-mediated isothermal amplification assay

A method was developed using a Loop-mediated isothermal amplification assay (LAMP) for detecting Didymella bryoniae in cucurbit seeds. The LAMP primers were designed based on the DNA-dependent RNA polymerase II RPB140 gene (RPB2) from D. bryoniae. Calcein was used as an indicator for the endpoint visual detection of DNA amplification. The LAMP assay was conducted in isothermal (65 °C) conditions within 1 h. The detection threshold of the LAMP assay was 10 pg of genomic DNA and D. bryoniae was detected in 100 % of artificially infested seedlots with 0.05 % infestation or greater. With the LAMP assay, 16 of 60 watermelon and muskmelon seedlots collected from Xinjang province were determined to be positive for D. bryoniae. In contrast, a real-time PCR assay determined that 11 of the 60 seedlots from Xinjiang province were positive for the pathogen. These results showed that the LAMP technique was simple, rapid and well suited for detecting D. bryoniae DNA, especially in seed health testing.     

An investigation of the reliability of ELISA as a practical test for detecting potato leaf roll virus and potato virus Y in tubers

Enzyme-linked immunosorbent assay (ELISA), involving mechanical sampling of tubers, was compared with a growing-on test in which virus was assessed visually, and with ELISA on leaves. The percentage infection of potato leaf roll virus and potato virus Y on samples of potential seed tubers sent for advisory test during the winter, was determined by each method. The tuber ELISA underestimated the incidence of both viruses, and was less accurate than the growing-on or leaf ELISA. The effect on the reliability of tuber ELISA of the amount and distribution of virus in the tuber, the compromises made to make the test fast and simple, and the quality of anti-serum and composition of the extraction buffer are discussed.