Detection of Spongospora subterranea using monoclonal antibodies in ELISA

Five hybridoma cell lines secreting antibodies (MAbs) recognizing zoospores of S. subterranea were raised from splenocytes of mice. One MAb also weakly recognized plasmodia/zoosporangia and cystosori of S. subterranea , and another recognized only plasmodia/zoosporangia in plate-trapped antigen ELISA. Polymyxa graminis was recognized most strongly out of 26 micro-organisms other than S. subterranea against which the MAbs were tested. Most were recognized only weakly or not at all. The MAb that recognized zoospores of S. subterranea most strongly detected as few as three zoospores per microtitre plate well when 12 replicate wells per treatment were arranged randomly on plates and absorbance values subjected to analysis of variance. The sensitivity of detection was not improved by mixing antibodies, using a biotin-streptavidin amplification system, or by using a double antibody sandwich system. Zoospores of S. subterranea flushed from soil were detected only after unrealistically large numbers of cystosori had been added. They were not detected in samples of naturally infested soil removed from a field shortly after a severely scabbed potato crop had been harvested.     

Detection and Quantification of Spongospora subterranea f. sp. subterranea in Soils and on Tubers Using Specific PCR Primers

PCR-based methods were developed for the detection and quantification of the potato pathogen Spongospora subterranea f. sp. subterranea (S. subterranea) in peel, tuber washings and soil. A partial sequence was obtained for S. subterranea ribosomal DNA and specific PCR primers (Sps1 and Sps2) were chosen from the internal transcribed spacer regions. These primers amplified a 391bp product from S. subterranea DNA but did not amplify DNA from potato or a range of soil-borne microbes, including related species. Diluted S. subterranea DNA was detected at a concentration equivalent to 25×10^–5 cystosori or 1 zoospore per PCR. Amplification was detected from peel and washings of infected and apparently healthy tubers, but not from peel of Scottish classified seed potatoes or axenically micropropagated potatoes. A rapid method for extracting S. subterranea DNA from soils was developed. This yielded DNA pure enough for PCR within 3h and facilitated the detection of 1–5 cystosori per gram of soil. A PCR quantification technique was developed involving comparison of product ratios obtained after co-amplification of S. subterranea DNA along with an internal standard (competitor DNA fragment). This quantitative technique was also adapted for use in soil. PCR detection of S. subterranea in soil was considerably more sensitive than previously reported immunoassays and was quicker and easier than conventional bait plant bioassays. Such an assay could be useful for developing disease risk assessments for field soils and seed potato stocks and for future studies on the ecology and control of S. subterranea.     

Improved immunological detection of S<Emphasis Type="Italic">pongospora subterranea</Emphasis>

The genus Spongospora has two members which are important pathogens of vegetables, S. subterranea f.sp. subterranea (Sss) and S. subterranea f.sp. nasturtii (Ssn). The close taxonomic relationship of these formae speciales is based on similar cystosori morphology. The potato disease powdery scab, caused by Sss, is difficult to control. The key control measure is avoidance, aimed at planting clean seed in clean soil. For the development of routine tests for the presence of the pathogen on tubers and in soil, a monoclonal antibody (MAb) was developed using Sss cystosori as immunogen. It detected less than one Sss cystosorus and recognised Sss material from many parts of the world. No cross-reactions with other Plasmodiophoromycetes including Plasmodiophora brassicae, Polymyxa betae, Polymyxa graminis and different Streptomyces species causing common and netted scab of potatoes were observed. A novel tuber sample test method was developed using a kitchen peeling machine. This detected two tubers with one powdery scab lesion each in a sample including eighteen uninfected tubers. When soil samples spiked with cystosori were tested with the MAb, different Sss infestation levels could be discriminated. Ssn cystosori gave absorbance values in ELISA as high as Sss cystosori, whereas fresh crook roots of watercress containing Ssnzoosporangia and plasmodia or mud from an Ssninfected watercress bed gave low absorbance values or no reaction. The potential of these findings for the development of a disease control management are discussed.     

Germinate to exterminate: chemical stimulation of Spongospora subterranea resting spore germination and its potential to diminish soil inoculum

Hoagland's solution (HS), a defined nutrient supplement for plants, has been previously reported to stimulate zoospore release from resting spores of the potato pathogen Spongospora subterranea f. sp. subterranea. This study obtained direct empirical evidence for an increase in zoospore release with HS treatment, and identified Fe‐EDTA as the stimulant component of HS. Stimulation of resting spores by HS and Fe‐EDTA resulted in greater and earlier zoospore release compared to a distilled water control, and in the presence of a susceptible tomato host plant resulted in enhanced root infection. Given the labile nature of S. subterranea zoospores, it was postulated that stimulation of premature release of zoospores from the dormant resting spores in absence of susceptible hosts could reduce soil inoculum levels. In two glasshouse trials in the absence of host plants, both Fe‐EDTA and HS soil treatments reduced S. subterranea soil inoculum levels, providing proof of concept for the ‘germinate to exterminate’ approach to inoculum management.     

The potential for the Rapid Screening of Potato Cultivars (Solanum tuberosum) for Resistance to Powdery Scab (Spongospora subterranea) using a Laboratory Bioassay

Powdery scab of potato, once established in a field, is difficult to control because of the longevity of the resting spores (cystosori) of the causal organism, Spongospora subterranea f.sp. subterranea. Host resistance is likely to be the most efficient in a long-term control strategy for preventing build-up of field inoculum and spread of the disease. Resistance screening of potato cultivars is mostly done in laborious field trials where disease development is likely to be unpredictable. A bioassay with potato tissue cultured plantlets and cystosori as inoculum is described and was tested for its potential to screen potato cultivars at an early stage for their relative susceptibility to powdery scab by comparing the lab results with field data. With cystosori inoculum of Swiss origin, the laboratory test showed clear differences between the potato cultivars in the severity of zoosporangial root infection which correlated better with ranked tuber infection data, compared to root galling. There are apparent differences in the relative trends in susceptibility between roots and tubers of five selected cultivars when using naturally infested soil instead of prepared cystosori as inoculum in the lab bioassay. Furthermore, differences in the severity of zoosporangial root infection of two selected cultivars were found when cystosori from different countries where used as inoculum. A possible host genotype × pathogen interaction is discussed. The bioassay has the potential to screen and select for resistant material at an early breeding stage thus making field trials not unnecessary but more economical. It will allow the use of a standard set of pathogen collections and facilitate testing for inoculum virulence in infested soils.     

Effect of fungicides on stages of the life cycle of Phytophthora cinnamomi

An assessment was made of the effects of several fungicides on the formation of zoosporangia, the release of zoospores from zoosporangia, zoospore motility, germ tube formation and mycelial growth, by Phytophthora cinnamomi Rands. Compounds active against mycelial growth (such as metalaxyl and furalaxyl), as well as those active against zoospores (such as captafol and zineb), could be detected by a lupin (Lupinus angustifolius L.) assay which has been developed. This assay may be useful as a screening procedure to detect new fungicides.     

Infectivity,inoculum density and germination of Spongospora subterranea resting spores: a solution-culture test system1

Spongospora subterranea, causal agent of powdery scab of potatoes and vector of potato mop-top furovirus, survives in the soil as balls of resting spores (cystosori). So far, the factors affecting longevity, germination and infectivity of cystosori have not been investigated. A rapid and versatile bioassay with tomatoes as bait plants has been developed to quantify the infectivity of cystosorus inoculum or infested soil. The intensity of root infection, as a measure of infectivity, was determined by evaluating the quantity of zoosporangia present in epidermal cells and root hairs of the whole, stained root system. A correlation was obtained between the intensity of root infection and the cystosorus inoculum density in nutrient solution. Sterile soil suppressed the inoculum potential of pure cystosori. Infectivity of untreated soil decreased with increasing time of storage. Root infection was not influenced by the pH level of the nutrient solution.     

Identification of barley yellow mosaic virus by immuno-electron microscopy in barley but not in Polymyxa graminis or Lagena radicicola

Samenvatting Gerstegeelmozaïekvirus (BaYMV) werd in een perceel wintergerst (cv. Igri) gevonden by Wittem in Limburg, Nederland. Het virus werd met immuno-elektronen-microscopie geïdentificeerd in bladweefsel als BaYMV. In de wortels van geïnfecteerde planten werden alle vormen die van de schimmel-vector (Polymyxa graminis Led.) bekend zijn aangetroffen nl. cystosori, plasmodia en zoosporangia. Inwendig kon geen BaYMV worden aangetoond of waargenomen inPolymyxa ofLagena spp.Cooperative investigation of the ARS, USDA, the Nebraska Agricultural Experiment Station, and the Plant Protection Service, Wageningen. Research conducted under Project 21-12. Published as Paper No. 7787, Journal Series, Nebraska Agricultural experiment Station. Mention of a trademark or proprietary product does not constitute a guarantee or warranty by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable.     

Evaluation of the <Emphasis Type="BoldItalic">Sss</Emphasis> AgriStrip rapid diagnostic test for the detection of <Emphasis Type="BoldItalic">Spongospora subterranea</Emphasis> on potato tubers

Spongospora subterranea, f.sp. subterranea (Sss), which causes powdery scab, is mainly spread through infected seed tubers and survives in contaminated soil for many years. The visual assessment of tuber lots by inspectors carries the risk of misidentification due to the difficulty of distinguishing lesions caused by either Sss or Streptomyces spp.. To avoid this, the “Sss AgriStrip”, a rapid and lab-independent test tool based on a lateral flow immunoassay has been developed, and we assessed its accuracy and sensitivity for detecting Sss. The Sss AgriStrip performed as well as other lab-based identification methods. The Sss AgriStrip, microscopy, ELISA, PCR, and real-time PCR techniques identified infection with S. subterranea in all tubers with typical powdery scab lesions. When lots with tubers showing a mixture of typical and atypical (suspicious) symptoms were tested, the presence of S. subterranea was confirmed in all lesions by all methods. The DNA content was generally lower in atypical than in typical lesions. Diverse and suspicious symptoms, which were difficult to assign to either powdery or common scab, tested negative with Sss AgriStrip and the other methods. This was despite microscopic observation of sporosori-like structures in some samples. Isolation and molecular identification confirmed that these lesions were mostly caused by Streptomyces spp. The Sss AgriStrip is as sensitive as DAS-ELISA with a detection limit between 1 and 10 sporosori per ml buffer. It is ideal for rapid and selective detection of Sss on farms and border inspection points to prevent spread of the pathogen.     

Spongospora subterranea root infection assessed in two potato cultivars differing in susceptibility to tuber powdery scab

Infection by Spongospora subterranea of roots of two potato (Solanum tuberosum) cultivars, either very resistant or very susceptible to powdery scab on their tubers, was studied in a glasshouse experiment. Plants grown in sand/nutrient solution culture were inoculated with S. subterranea sporosori 2 weeks after planting. Plant parameters, the intensity of zoosporangium infection in roots, numbers of Spongospora root galls and amounts of Spongospora DNA in roots, measured using quantitative PCR (qPCR), were assessed at sequential harvests. Inoculation with S. subterranea reduced water use (56 days after planting) by 26% in the tuber resistant cultivar compared with uninoculated plants, and by 60% in the susceptible cultivar. Inoculation did not affect growth of the resistant cultivar, nor shoot mass of the susceptible cultivar, but caused a 38% reduction in root mass of the susceptible cultivar. The intensities of zoosporangium development in both cultivars were similar. The susceptible cultivar had approximately four times more Spongospora root galls g^?1 root mass than the resistant cultivar. Quantitative PCR detected S. subterranea DNA in roots 1 week after inoculation, and indicated a twofold greater amount of pathogen DNA in roots of the susceptible than the resistant cultivar. This study suggests that the S. subterranea zoosporangium stage in host roots is affected differently by host resistance factors than the sporosorus (root gall and tuber scab) stages. The study has also demonstrated the usefulness of qPCR for sensitive and consistent detection of S. subterranea across the duration of potato root infection.     

Sequence Analysis and Detection of Ralstonia solanacearum by Multiplex PCR Amplification of 16S–23S Ribosomal Intergenic Spacer Region with Internal Positive Control

Polymerase chain reaction (PCR) methods for detection and differentiation of Ralstonia solanacearum strains were compared. The 16S–23S rRNA gene ITS sequence data revealed the two main sequence clusters (divisions I and II) of R. solanacearum and further subclusters of division II. Based on this sequence data, primers were designed which differentiated divisions I and II. Furthermore, to improve reliability of the PCR assay for routine detection of R. solanacearum in host plants, a novel multiplex PCR assay was developed in which the pathogen-specific sequences are coamplified with host plant DNA as an internal PCR control (IPC). The assay was validated during routine testing of potato samples submitted in official surveys. Of 4300 samples from 143 cultivars, 13 tested positive in both multiplex PCR and immunofluorescence (IF) assays and could be confirmed by bioassay in tomato seedlings and reisolation of the pathogen. The IPC was successfully amplified from all samples tested. A further 12 samples gave positive IF results which were not confirmed by either the multiplex PCR or tomato bioassay, indicating a greater specificity of the latter two assays.     

<Emphasis Type="Italic">Spongospora subterranea</Emphasis> soil contamination and its relationship to severity of powdery scab on potatoes

We investigated soil contamination by Spongospora subterranea f. sp. subterranea (Sss) and disease severity of powdery scab in 29 potato fields in Hokkaido, Japan, using a hydroponic culture method with tomato seedlings as bait plants. The quantity of Sss infection on the roots of bait plants was evaluated using the polymerase chain reaction (PCR) and expressed in terms of the infection potential in the soil. The infection potential was positively correlated with the disease severity of harvested tubers, whereas the spore ball density determined using PCR had an indistinct relationship with disease severity. The infection potential can be useful in evaluating soil contamination and in applying countermeasures against powdery scab.     

Relationship between Spongospora subterranea f. sp. subterranea soil inoculum level,host resistance and powdery scab on potato tubers in the field

The relationship between initial soil inoculum level of Spongospora subterranea f. sp. subterranea (Sss) and the incidence and severity of powdery scab on potato tubers at harvest was investigated. In all experiments soil inoculum level of Sss (sporeballs/g soil) was measured using a quantitative real‐time PCR assay. Of 113 commercial potato fields across the UK, soil inoculum was detected in 75%, ranging from 0 to 148 Sss sporeballs/g soil. When arbitrary soil inoculum threshold values of 0, <10 and >10 sporeballs/g soil were set, it was observed that the number of progeny crops developing powdery scab increased with the level of inoculum quantified in the field soil preplanting. In four field trials carried out to investigate the link between the amount of inoculum added to the soil and disease development, disease incidence and severity on progeny tubers was found to be significantly (P < 0·01) greater in plots with increasing levels of inoculum incorporated. There was a cultivar effect in all years, with disease incidence and severity scores being significantly greater in cvs Agria and Estima than in Nicola (P < 0·01).     

Partial stem and leaf resistance against the fungal pathogen <Emphasis Type="Italic">Botrytis cinerea</Emphasis> in wild relatives of tomato

Tomato (Solanum lycopersicum) is one of many greenhouse crops that can be infected by the necrotrophic ascomycete Botrytis cinerea. Commercial cultivation of tomato is hampered by the lack of resistance. Quantitative resistance has been reported in wild tomato relatives, mostly based on leaf assays. We aimed to identify wild tomato relatives with resistance to B. cinerea based on quantitative assays both on leaves and stem segments, monitoring infection frequency and disease expansion rate as parameters. A quantitative tomato stem segment assay was developed. This stem assay and a previously described leaf assay were used to screen a collection of 22 Solanum accessions. Significant differences in disease parameters were observed among accessions. Resistance to B. cinerea was observed in a number of wild Solanum species, including accessions of S. chilense, S. habrochaites and S. neorickii, both in the leaf assay and the stem segment assay. A number of resistant and susceptible accessions were evaluated as adult plants under greenhouse conditions. The data obtained in greenhouse assays confirmed the leaf and stem disease data. The expression of several defence-related genes was studied in a subset of accessions. There was no apparent correlation between the expression levels of the genes tested and the quantitative resistance level to B. cinerea. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Acidic soil conditions suppress zoospore release from zoosporangia in <Emphasis Type="Italic">Olpidium virulentus</Emphasis>

Lettuce big-vein disease, caused by Mirafiori lettuce big-vein virus and Lettuce big-vein associated virus, is suppressed when the pH of field soil becomes acidic. Therefore, we evaluated the effect of soil pH on the activities of Olpidium virulentus, the vector of the viruses. We found that acidic soil, pH less than 6.0, significantly reduced O. virulentus infection of the root and influenced the detection rate of zoospores released in the surrounding water. We concluded that acidic soil suppresses zoospore release from zoosporangia.     

Development of PCR-based Detection Methods for the Quarantine Phytopathogen <Emphasis Type="Italic">Synchytrium endobioticum</Emphasis>, Causal Agent of Potato Wart Disease

PCR-based methods were developed for the detection and quantification of the potato pathogen Synchytrium endobioticum in soil extracts and in planta. PCR primers, based on the internal transcribed spacer region of the multi-copy gene rDNA were tested for specificity, sensitivity and reproducibility in conventional and real-time PCR assays. Soil extraction procedures compared included the Hendrickx centrifugation (HC) procedure, nested wet sieving (NWS) and a method used by the Plant Protection Service (PPS). The primers amplified a 472 bp product from S. endobioticum DNA, but did not amplify DNA from other potato pathogens, other plant pathogens, and related species. Standard cell disruption and DNA extraction and purification methods were optimized for amplification of S. endobioticum DNA from resting sporangia. DNA was successfully amplified from a single sporangium and equivalent DNA preparations from soil extracts. Low levels of target DNA in water did not amplify, possibly due to DNA loss during final purification steps. A real-time PCR assay, developed for soil-based extracts using primers and probe based on the rDNA gene sequences, involved co-amplification of target DNA along with an internal DNA fragment. Both conventional and real-time PCR methods performed well with HC- and NWS-extracts having a threshold sensitivity of 10 sporangia per PCR assay. Of the three soil extraction methods, only with the HC method could 100 g soil samples be efficiently processed in one single PCR assay. Such a high capacity assay could be useful for routine soil analysis in respect to disease risk assessments and to secure de-scheduling according to EPPO guidelines.     

Improved real-time PCR assay for detection of the quarantine potato pathogen, <Emphasis Type="Italic">Synchytrium endobioticum</Emphasis>, in zonal centrifuge extracts from soil and in plants

Real-time PCR was used for quantitative detection of the potato pathogen, Synchytrium endobioticum, in different substrates: zonal centrifuge extracts, warts and different plant parts of potato. Specific primers and a TaqMan probe, designed from the internal transcribed spacer region of the multi-copy rDNA gene were tested in extracts from artificially and naturally infested soil. Co-amplification of target DNA along with an internal competitor DNA fragment made the diagnostic assay more reliable by guarding against false negative results. A calibrations curve was created by spiking zonal centrifuge fractions of clean soil samples with a dilution series of winter spores. The Taqman assay was also performed on infected potato plant material (stolons) along with the detection of the cytochrome oxidase gene as a potato endogenous control. Sensitivity of the TaqMan assay was improved at least 100-fold and proved to be reliable for accurate diagnosis of the disease.     

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.     

Detection and quantification of Fusarium commune in host tissue and infested soil using real‐time PCR

Fusarium wilt caused by Fusarium commune is a major limiting factor for Chinese water chestnut (Eleocharis dulcis) production in China. A SYBR Green I real‐time quantitative polymerase chain reaction (qPCR) assay was developed based on the mitochondrial small subunit rDNA of F. commune. Assay specificity of the FO1/FO2 primer set was tested on 41 fungal isolates, and only a single PCR band of c. 178 bp from F. commune was amplified. The detection limits of the assay were 1 fg μL^?1 pure F. commune genomic DNA, 1 pg μL^?1 F. commune genomic DNA mixed with host plant genomic DNA (0·5 ng μL^?1), and 1000 conidia/g soil (artificially inoculated). The amount of F. commune DNA in stem tissues detected by qPCR was significantly correlated with the disease severity (DS) ratings; however, the qPCR assay showed no significant positive correlation between spore densities in soil of different fusarium wilt DS groupings and the DS ratings. The qPCR assay was further applied to 76 soil samples collected from commercial fields of E. dulcis during the 2011 and 2012 growing seasons. The spore density of F. commune detected was positively correlated with disease index in the 2012 growing season but not in 2011. The qPCR method can be used for rapid and specific detection of F. commune in plant and soil samples, which will facilitate monitoring of the pathogen and improvement of disease management.     

Effect of temperature on primary infection by Plasmodiophora brassicae and initiation of clubroot symptoms

A study was conducted to assess the effect of temperature on infection and development of Plasmodiophora brassicae in root hairs of Shanghai pak choi (Brassica rapa subsp. chinensis) and on initiation of clubroot symptoms. Ten‐day‐old seedlings were grown in liquid‐sand culture, inoculated with resting spores and maintained in growth cabinets at 10, 15, 20, 25 and 30°C. Seedlings were harvested at 2‐day intervals, starting 2 days after inoculation (DAI) and continuing until swelling of the tap root was observed (maximum 28 days). Roots were assessed for root hair infection (RHI), stage of development of infection (primary plasmodia, zoosporangia, release of zoospores, secondary plasmodia), symptom development, and for clubroot severity at 24 DAI. Temperature affected every stage of clubroot development; RHI was highest and visual symptoms initiated earliest at 25°C, intermediate at 20 and 30°C, and lowest and latest at 15 and 10°C. Root hair infection was observed at every temperature, but clubroot symptoms developed only above 15°C. A substantial delay in the development of the pathogen was observed at 10 and 15°C. No symptoms were observed at 28 DAI in plants grown at 10°C. Swelling of the tap root was visible at 28 DAI in plants at 15°C, 14 DAI at 20 and 30°C, and 10 DAI at 25°C. These results support and explain the observation in companion studies that cool temperatures result in slower development of clubroot symptoms in brassica crops.