Spongospora Research Articles

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.

Actin and ubiquitin protein sequences support a cercozoan/foraminiferan ancestry for the plasmodiophorid plant pathogens.

The plasmodiophorids are a group of eukaryotic intracellular parasites that cause disease in a variety of economically significant crops. Plasmodiophorids have traditionally been considered fungi but have more recently been suggested to be members of the Cercozoa, a morphologically diverse group of amoeboid, flagellate, and amoeboflagellate protists. The recognition that Cercozoa constitute a monophyletic lineage has come from phylogenetic analyses of small subunit ribosomal RNA genes. Protein sequence data have suggested that the closest relatives of Cercozoa are the Foraminifera. To further test a cercozoan origin for the plasmodiophorids, we isolated actin genes from Plasmodiophora brassicae, Sorosphaera veronicae, and Spongospora subterranea, and polyubiquitin gene fragments from P. brassicae and S. subterranea. We also isolated actin genes from the chlorarachniophyte Lotharella globosa. In protein phylogenies of actin, the plasmodiophorid sequences consistently branch with Cercozoa and Foraminifera, and weakly branch as the sister group to the foraminiferans. The plasmodiophorid polyubiquitin sequences contain a single amino acid residue insertion at the functionally important processing point between ubiquitin monomers, the same place in which an otherwise unique insertion exists in the cercozoan and foraminiferan proteins. Taken together, these results indicate that plasmodiophorids are indeed related to Cercozoa and Foraminifera, although the relationships amongst these groups remain unresolved.

Occurrence of Potato Powdery Scab Caused by Spongospora subterranea in Korea

Potato tubers with scab lesions were found in several regions of Gangwon province in Korea during disease surveys in 2000 and 2001. The lesions appeared as shallow scabs with torn and raised skin around the periphery of the lesion. Sometimes the lesions expanded on tuber surface and formed hollowed-out areas. The causal organism was identified as Spongospora subterranea based on morphological characteristics using light and electron microscope. Ten cultivars of potato were evaluated to select resistant cultivars against powdery scab. Among the 10 cultivars, Jowon, Superior, and Atlantic were resistant, while Sephody, Gawon, and Irish Cobbler were susceptible to powdery scab. In the pathogenicity test, typical symptoms of powdery scab were formed on potato tubers, and galls developed on root hairs. This is the first report of powdery scab of potato caused by S. subterranea in Korea.

First Report of Potato mop-top virus on Potato from the United States.

Potato mop-top virus (PMTV) is a tripartite pomovirus vectored by the powdery scab plasmodiophoromycete Spongospora subterranea pv. subterranea (1). PMTV occurs on potato (Solanum tuberosum) in Europe, the Andes, Asia, and Canada. Internal necrotic arc and fleck tuber symptoms ("spraing") may reduce commercial acceptance of some cultivars (3). PMTV symptoms were discovered in 'Shepody' tubers at the Aroostook Research Farm, Presque Isle, ME in May 2002 and subsequently in 'Russet Burbank' tubers in commercial storage from the 2001 Maine crop. Symptomatic tubers exhibited single or multiple concentric necrotic arcs that were partial or complete, but exhibited no distinct external symptoms. The presence of PMTV in eight 'Shepody' tubers was indicated by positive enzyme-linked immunosorbent assay (ELISA; Adgen, Ltd., Auchincruive, Ayr, Scotland) and confirmed by reverse transcription polymerase chain reaction (RT-PCR). 'Russet Burbank' potatoes were visually diagnosed, and the corresponding halves of 128 symptomatic tubers were forwarded to the University of Maine and APHIS (Beltsville, MD). Of these, ELISA readings in Maine were strongly positive (>3 × background) for 88, ambiguous (1.5-3 × background) for 13, and negative for 27. Subsamples from these three categories were positive by PCR in 17 of 17, 9 of 9, and 12 of 14 cases, respectively. A similar rating, positive or ambiguous, in ELISA testing was identical for all but one case at Beltsville. Confirmation of PMTV required PCR testing, resulting in a characteristic PCR product of 401 bp that was generated from the coat protein coding region on RNA 2 (2) using the primer pair PMTV 1 5'-GCAGCCGTCGAGAATAGATA-3' (RNA nucleotides 316-335) and PMTV 4 5'-GCGAGTTGATGTGCC ACATT-3' (complementary to RNA 2 nucleotides 716-697). An immunocapture RT-PCR using this primer set and the coating antibody from the Adgen ELISA kit was also successful in detecting PMTV. In separate reactions, a second product of 646 bp was generated from the triple gene block on RNA 3 (4) using the primer pair PMTV 5 5'-GGTGAACACGAGGACAAGGT-3' (RNA 3 nucleotides 1417-1436) and PMTV 7 5'-AACAGTCCGGTCTTGTGAAC-3' (complementary to RNA 3 nucleotides 2063-2044). The sequence of these products was 98 to 100% identical to PMTV published sequences. The discovery of this virus will result in adjustments to U.S. and Canadian seed potato certification standards and symptom characterization for common North American cultivars.

Susceptibility of potato (Solanum tuberosum) cultivars to powdery scab (caused by Spongospora subterranea f. sp. subterranea), and relationships between tuber and root infection

Ninety-nine potato (Solanum tuberosum) cultivars and 13 breeding lines were assessed in field trials for susceptibility to powdery scab (caused by Spongospora subterranea f. sp. subterranea). The trials wer e carried out over 11 growing seasons (each cultivar/line tested in at least two growing seasons) in soil inoculated with S. subterranea and regularly irrigated during crop growth. The cultivars and lines were categorised as ‘very resistant’ (21%). ‘moderately resistant’ (28%). ‘moderately susceptible’ (33%) or ‘very susceptible’ ( 19%). Scaled average severity scores for the cultivars and lines followed a continuum between very resistant and very susceptible, suggesting that resistance to powdery scab is quantitative. In a glasshouse experiment where small plants were inoculated with S. subterranea sporosori, 15 cultivars selected to cover the spectrum of field-assessed susceptibility were assessed for intensity of infection by S. subterranea zoosporangium in roots and for numbers of root galls on root systems. All of the cultivars developed zoosporangia and root galls (i.e. no cultivar was immune from infection), and root infection was usually related to tuber infection. Field-resistant cultivars (tuber infection) generally had low numbers of root zoosporangia and root galls in the glass house experiment, and cultivars that were very susceptible to tuber infection in the field had high levels of root infection in the glasshouse experiment. An exception was the early maturing cv. Swift, which had very low levels of tuber infection in the field, but had high numbers of root zoosporangia and root galls in the glasshouse. These results demonstrate the relative reaction of a large number of potato cultivars to powdery scab and reaffirm the potential for using plant resistance as a powdery scab management strategy. Development of S. subterranea in host roots, even in cultivars with resistance to tuber infection, is likely to be important in the epidemiology of powdery scab.

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.

The effect of irrigation and nitrogen on powdery scab and yield of potatoes

Powdery scab, caused by the fungusSpongospora subterranea, has developed in the Cappodocia region of Turkey because of changes in agronomic practices, such as excessive irrigation and nitrogen fertilizer use and growth of highly susceptible cultivars. The most suitable irrigation and nitrogen application levels were established to maintain powdery scab at minimum levels without affecting potato yield. Three irrigation levels were used in 1998 (673, 897, 1121 mm) and five in 1999 (329, 494, 658, 823, 987 mm). Nitrogen levels were applied 150, 300, 450, 600 kg N ha−1. In 1998, least powdery scab occurred with 673 mm irrigation, which yielded 41 t ha−1 potatoes. The lowest disease severity occurred in 1999 with 494 mm irrigation and 150 kg N ha−1. At these levels, tuber yield was 34 t ha−1 which was not significantly different from the highest yield measured. In 1999 and at two of the three irrigation levels inl998, nitrogen increased disease severity.

Occurrence of Potato Powdery Scab Caused by Spongospora subterranea f. sp. subterranea in Costa Rica.

Powdery scab of potatoes, caused by Spongospora subterranea (Wallr.) Lagerheim f. sp. subterranea Tomlinson, is important worldwide due to its effect on tuber quality and transmission of Potato mop-top virus. Although powdery scab-like lesions have been observed on potato in Costa Rica (1), the presence of the pathogen has not been confirmed. During a survey in 2001, powdery scab-on was observed from a field and a greenhouse in the high elevation zone of the main potato-producing area of Costa Rica. Commercial potatoes with scab-like lesions were also obtained at a farmers' market. Scraping the lesions, and observing spore balls or cystosori with a honey-comb-like structure under light microscopy confirmed the identity of S. subterranea. The identity of the pathogen was also confirmed by enzyme-linked immunosorbent assay using monoclonal antibodies specific for S. subterranea (BioReba Ag, Reinach, Switzerland). Pathogenicity of S. subterranea was confirmed by a bioassay on tomato plants grown in nutrient solution culture (2). Tomato cv. Supermarmande plants were grown from seed in pots filled with quartz and watered with nutrient solution. Three weeks after planting, the roots were trimmed to 60 mm, and the plants were transferred to the nutrient solution for additional growth. After growing for 1 week in the nutrient solution, tomato seedlings were inoculated by replacing the nutrient solution with nutrient solution containing cystosori (20 mg/liter, wt/vol) that were scraped from the scab lesions. Zoosporangia of S. subterranea were observed in root hairs and epidermal cells of the seedlings 2 weeks after inoculation. To our knowledge, this is the first report that confirms the presence of S. subterranea on potato in Costa Rica.

Effect of Seed or Soil Treatment with Fungicides on the Control of Powdery Scab of Potato

Effect of Seed or Soil Treatment with Fungicides on the Control of Powdery Scab of Potato

Development of a PCR‐based diagnostic test for Spongospora subterranea f.sp. nasturtii, the causal agent of crook root of watercress (Rorippa nasturtium‐aquaticum)

The polymerase chain reaction (PCR) technique was utilized to obtain internal transcribed spacer ribosomal DNA (ITS rDNA) and small‐subunit (18S) rDNA sequences from UK isolates of Spongospora subterranea f.sp. nasturtii, a plasmodiophorid pathogen of watercress (Rorippa nasturtium‐aquaticum). ITS sequence data obtained from S. subterranea isolated from a range of UK sites were found to be identical. PCR primers were designed using these sequences and were shown to be capable of specific amplification of S. subterranea f.sp. nasturtii DNA from plant tissue and from water samples containing zoospores of the pathogen. As little as 5 ng total genomic DNA from infected plant material, or 1000 zoospores, was required for consistently successful amplification of DNA. A filtration‐based method for obtaining pathogen DNA for PCR from watercress‐bed water was developed.

Initial infection of roots and leaves reveals different resistance phenotypes associated with coat protein gene-mediated resistance to Potato mop-top virus.

Resistance to the pomovirus Potato mop-top virus (PMTV) was studied in potato (Solanum tuberosum cv. Saturna) and Nicotiana benthamiana transformed with the coat protein (CP) gene of PMTV. The incidence of PMTV infections was reduced in tubers of the CP-transgenic potatoes grown in the field in soil infested with the viruliferous vector, Spongospora subterranea. However, in those tubers that were infected, all three virus RNAs were detected and virus titres were high. The CP-transgenic N. benthamiana plants were inoculated with PMTV using two methods. Following mechanical inoculation of leaves, no RNA 3 (the CP-encoding RNA homologous to the transgene) was detected in leaves, but in some plants low amounts of RNA 3 were detected in roots; RNA 2 was readily detected in leaves and roots of several plants. Inoculation of roots using viruliferous S. subterranea resulted in infection of roots in all plants and the three PMTV RNAs were detected. However, no systemic movement of PMTV from roots to the above-ground parts was observed, indicating a novel expression of resistance. These data indicate that the CP gene-mediated resistance to PMTV specifically restricts accumulation of PMTV RNA 3, and is more effective in leaves than roots. Furthermore, expression of resistance is different depending on whether leaves or roots are inoculated. Data do not exclude the possibility that both a protein-mediated and an RNA-mediated resistance mechanism are involved.

Evaluation of some North and South American potato breeding lines for resistance toPotato mop-top virus in Sweden

The necrotic symptoms (spraing) in tubers caused byPotato mop-top pomovirus (PMTV) have become a severe problem for potato production in Scandinavia. PMTV is transmitted by the powdery scab pathogen (Spongospora subterranea) and no methods for control are currently available. Two breeding lines (NY99 and NY103) from the Cornell University potato breeding program, twotuberosum ×acaule lines (TA3.8.3.3 and TA3.5.3.7) from the International Potato Center (CIP), and one line (IvP35) ofSolarium phureja were tested for resistance to PMTV in field trials in Sweden in 1998 and 1999. In both years, NY99 had a low incidence of PMTV infection in tubers, and the infected tubers showed a low accumulation of PMTV, indicating that NY99 is a promising new source of resistance to PMTV for breeding programs. Furthermore, it may be suitable for cultivation in Scandinavia, which, however, needs to be evaluated in more extensive field trials.

Studies on amoebae and cysts associated with the isolation of Spongospora subterranea f.sp. subterranea in vitro

New evidence is presented to support the contention that the amoeba/cyst colonies isolated from surface‐sterilized Spongospora subterranea f.sp. subterranea‐infected potato tubers and spore balls have a saprophytic phase but are contaminants and not S. subterranea. Amoebae isolated from infected tissues and spore balls formed colonies associated with bacteria on 1% water agar at 18°C and encysted after 5–7 days. These cysts were morphologically distinct from the resting spores of S. subterranea and were formed singly or in a layer, unlike the spore ball (cystosorus) of S. subterranea. Amoebae, cysts and mixtures of amoebae and cysts in primary, secondary and tertiary subcultures failed to infect tomato roots. PCR amplification of DNA from amoebae, cysts and spore balls using the S. subterranea‐specific primer pair SsF/R generated a 434‐bp product from S. subterranea spore balls only and not from amoebae or cysts. When an amoeba/cyst‐specific primer pair AmF/R was designed and used for PCR amplification, a single 411‐bp product was generated from DNA of amoebae and cysts, but not from DNA of S. subterranea spore balls. These results are discussed in relation to earlier reports claiming the successful isolation of S. subterranea and other plasmodiophorids in vitro.

‘Dawn'—a new early‐maincrop fresh market/crisping potato cultivar

‘Dawn’ is a new, high‐yielding, early‐maincrop potato (Solanum tuberosum L.) cultivar suited to the fresh market and crisp manufacture. Tubers are round‐oval in shape and have white flesh. This variety has moderate resistance to late blight (Phytophthora infestans) and to powdery scab (Spongospora subterranea).

Genetic diversity within commercial populations of watercress (Rorippa nasturtium-aquaticum), and between allied Brassicaceae inferred from RAPD-PCR

Commercial productivity of watercress (Rorippa nasturtium-aquaticum) can be adversely affected by the pathogenic crook-root fungus, Spongospora subterranea f.sp. nasturti, and watercress viruses. As there are no effective control measures for these diseases, attempts have been made to breed varieties resistant to the crook-root pathogen. This work has been hindered by a lack of knowledge of the genetic base of commercial watercress, and the genetic distance between watercress and allied Brassicaceae which have been identified as candidates for hybridisation programmes. We measured the diversity within these two groups using the RAPD-PCR fingerprinting technique and analysed the data by both distance methods and principal co-ordinate analysis. Little genetic diversity was found within commercial watercress populations. However, watercress formed a unique cluster genetically distinct from other Rorippa species, but equidistant to Cardamine species. It was placed closer to Barbarea verna.

Detection and quantification of fungal and bacterial potato pathogens in plants and soil*

We have developed polymerase chain reaction (PCR)‐based diagnostic assays for fungal and bacterial diseases of potato together with rapid methods of DNA extraction and purification from soil and plant samples. Pathogen detection and quantification is a crucial step in the control of crop diseases. PCR primers were designed to unique sequences for specific detection of the potato blemish complex of pathogens: Spongospora subterranea (powdery scab), Streptomyces scabies (common scab), Helminthosporium solani (silver scurf), Rhizoctonia solani AG‐3 (black scurf) and Colletotrichum coccodes (black dot). A multiplex PCR test was designed with a high level of sensitivity to detect H. solani, C. coccodes, and S. scabies simultaneously in one reaction, and individual quantitative PCR assays were also developed to detect these pathogens and S. subterranea in soil and tubers. PCR‐based detection systems for the major storage‐rot pathogens of potato (Fusarium spp., Phoma foveata, Pythium ultimum and Phytophthora erythroseptica) are also under development. The potential of these techniques to detect and monitor these pathogen populations in soil and potato tubers for epidemiological studies is discussed.

Serological Methods for Detection of Polymyxa graminis, an Obligate Root Parasite and Vector of Plant Viruses

A purification procedure was developed to separate Polymyxa graminisresting spores from sorghum root materials. The spores were used as im-munogen to produce a polyclonal antiserum. In a direct antigen coating enzyme-linked immunosorbent assay (DAC ELISA), the antiserum could detect one sporosorus per well of the ELISA plate. In spiked root samples, the procedure detected one sporosorus per mg of dried sorghum roots. The majority of isolates of P. graminis from Europe, North America, and India reacted strongly with the antiserum. Interestingly, P. graminis isolates from the state of Rajasthan (northern India), from Pakistan, and an isolate from Senegal (West Africa) reacted weakly with the antiserum. The cross-reactivity of the serum with P. betae isolates from Belgium and Turkey was about 40% of that observed for the homologous isolate. There was no reaction with common fungi infecting roots or with the obligate parasite Olpidium brassicae. However, two isolates of Spongospora sub-terranea gave an absorbance similar to that observed with the homologous antigen. The DAC ELISA procedure was successfully used to detect various stages in the life cycle of P. graminis and to detect infection that occurred under natural and controlled environments. A simple procedure to conjugate antibodies to fluorescein 5-isothiocyanate (FITC) is described. Resting spores could be detected in root sections by using FITC-labeled antibodies. The potential for application of the two serological techniques for studying the epidemiology of peanut clump disease and for the characterization of Polymyxa isolates from various geographical origins is discussed.

Specific polyclonal antibodies for the obligate plant parasite Polymyxa— a targeted recombinant DNA approach

Highly specific rabbit polyclonal antibodies for the obligate sugar‐beet root parasite, Polymyxa betae, were produced using a novel recombinant DNA approach. Parasite cDNA was selectively isolated from infected roots, expressed in vitro, and the purified protein used to raise antibodies. This produced clean, precisely targeted antibodies, and allowed for rigorous screening of candidate genes and their products at the molecular level prior to animal immunization. This approach selects for genes whose products are highly expressed by the parasite in planta, and five such candidate genes from Polymyxa betae were identified and cloned. Polyclonal antiserum developed using the product of one such gene was found to react specifically with P. betae in sugar‐beet roots and with the closely related Polymyxa graminis in barley roots, and to cross‐react with Plasmodiophora brassicae in cabbage roots, without the need for further purification. No cross‐reaction was detected with protein extracts from potato roots infected by the plasmodiophoromycete Spongospora subterranea. In all cases, there was no interaction with proteins from host plants, or from other microorganisms found in association with uninoculated sugar‐beet, barley, cabbage and potato roots.

Suppression on Powdery Scab Disease of Potato by a Special Fertilization Method

Suppression on Powdery Scab Disease of Potato by a Special Fertilization Method

Soilless production of potato minitubers using a hydroponic technique

Potato minituber production is a classic way to multiply or acclimatize in vitro material before its use in the open field. This production is generally performed in a substrate of vegetable origin (based on peat) but it involves the risk of an infection by different pathogens present in the substrate, such asStreptomyces scabies (common scab),Spongospora subterranea (powdery scab), orErwinia spp. (bacterial soft rot). Another drawback associated with this system of production is its relatively high cost due to the initial cost of the in vitro material and the low multiplication rates.