Root infection of potato by Spongospora subterranea: knowledge review and evidence for decreased plant productivity

Information is reviewed on root infection of potato by the plasmodiophorid Spongospora subterranea f. sp. subterranea. This pathogen has long been recognized as the cause of root galls (hyperplasia) and the economically important disease powdery scab on tubers (modified stolons). The significance for plant productivity of the zoosporangium stages of the pathogen in potato roots has only recently begun to be documented. Two experiments are described that assessed effects of S. subterranea root infection on potato plant root function and productivity. A greenhouse experiment measured root function and plant parameters for eight potato cultivars with markedly different susceptibilities to tuber powdery scab. Water uptake and plant growth were reduced by S. subterranea inoculation in all eight cultivars. The magnitudes of these negative effects, and intensities of root hyperplasia, differed among the cultivars, but were not related to respective susceptibilities to tuber powdery scab. A field trial assessed root function and plant productivity for a cultivar (Iwa) that is very susceptible to Spongospora tuber and root diseases. Soil water content beneath uninoculated plants was consistently less than for inoculated plants, indicating that inoculation reduced water uptake (root function). Inoculation reduced shoot and root dry weights, and reduced weight of tubers per plant by 42%. Spongospora subterranea causes three diseases of potato: root membrane dysfunction, root hyperplasia and tuber powdery scab. The root diseases caused by the pathogen are likely to be important both for powdery scab management and for deleterious effects on potato crop yields.     

Somaclonal selection in potato for resistance to common scab provides concurrent resistance to powdery scab

Variant somaclones of potato cultivar Russet Burbank, selected for resistance to common scab using in vitro cell selection techniques, were tested for resistance to powdery scab, another important disease of potato caused by Spongospora subterranea. This pathogen also invades roots, producing root galls. Most variants consistently showed increased resistance to powdery scab, both in field and glasshouse challenge, when compared to the parental cultivar, several significantly so. On average, the best variant reduced powdery scab incidence by 51% and severity (tuber surface coverage) by 64%. In contrast, no improvement in the extent of root infection and root galling was seen. These results suggest host interactions during root and tuber infection are distinct. Correlation analyses of disease indices amongst the selected variants showed no association between Sp. subterranea root infection and gall scores, nor between root infection and tuber disease severity. However, a weak positive association was found between root gall score and tuber disease, and a strong correlation between tuber disease incidence and severity scores. Interestingly, positive correlations were also found between the extent of powdery and common scab resistance expressed and both incidence and severity of these diseases within the variants, suggesting a common defence mechanism. The role of thaxtomin A in selecting for concurrent resistance to both diseases is discussed.     

Identification of differentially expressed genes in tolerant and susceptible potato cultivars in response to Spongospora subterranea f. sp. subterranea tuber infection

Powdery scab caused by Spongospora subterranea f. sp. subterranea (Sss) has recently become one of the most devastating potato diseases of economic importance in South Africa. The use of resistant cultivars has long been considered the most effective and sustainable strategy to manage the pathogen. However, little is known about the molecular mechanisms underlying resistance of potato tubers to Sss. Using RNA-sequencing (RNA-seq), 2058 differentially expressed genes (DEGs) were identified from two potato cultivars (tolerant and susceptible) in response to Sss infection. Analysis of the expression patterns of 10 selected defence-response genes was carried out at two different stages of tuber growth using RT-qPCR to validate the RNA-seq data. Several defence-related genes showed contrasting expression patterns between the tolerant and susceptible cultivars, including marker genes involved in the salicylic acid hormonal response pathway (StMRNA, StUDP and StWRKY6). Induction of six defence-related genes (StWRKY6, StTOSB, StSN2, StLOX, StUDP and StSN1) persisted until harvest of the tubers, while three other genes (StNBS, StMRNA and StPRF) were highly up-regulated during the initial stages of disease development. The results of this preliminary study suggest that the tolerant potato cultivar employs quantitative resistance and salicylic acid pathway hormonal responses against tuber infection by Sss. The identified genes have the potential to be used in the development of molecular markers for selection of powdery scab resistant potato lines in marker-assisted breeding programmes.     

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.     

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.     

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.     

Characterization of weeds and rotational crops as alternative hosts of Spongospora subterranea,the causal agent of powdery scab in Israel

Alternative hosts of Spongospora subterranea may allow multiplication and survival of the pathogen over time; thus, host range is important from an epidemiological aspect. Weeds and rotational crops, such as wheat and barley, were sampled from potato fields with a history of powdery scab (PS) and examined for the presence of S. subterranea by root staining followed by microscopic observations and by qPCR analysis after DNA extraction. The pathogen was detected in plants of 16 weed species from eight families and in volunteer plants of potato and wheat. The ability of the pathogen to infect weeds and rotational crops was further examined by artificial inoculations with sporosori in pot experiments. Successful inoculations occurred with 13 weed species from eight families and with 12 rotational crops from five families. The findings of this study indicate a wide host range in Israel; the families Malvaceae and Zygophyllaceae and the following species are reported for the first time as S. subterranea hosts: Solanum elaeagnifolium, Triticum aestivum, Cynodon dactylon, Phalaris paradoxa, Phalaris minor, Setaria verticillata, Rostaria cristata, Sinapis nigra, Arachis hypogaea, Medicago sativa, Astragalus hauraensis, Amaranthus albus, Chenopodium murale, Chenopodium opulifolium, Salsola soda, Malva nicaeensis, Chrysanthemum segetum, Verbesina encelioides, Ammi majus and Tribulus terrestris. Controlling weeds and avoiding the relevant rotational crops observed to be S. subterranea-positive and thus potential hosts, should be taken into consideration in the management of PS, to reduce pathogen inoculum build-up.     

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).     

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.     

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.     

<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.     

Quantifying resistance to Plasmodiophora brassicae in Brassica hosts

Plasmodiophora brassicae causes clubroot of crucifers. A quantitative PCR (qPCR)‐based protocol was developed to measure P. brassicae DNA in the roots of susceptible, intermediately susceptible, intermediately resistant and resistant Brassica hosts, and the non‐host wheat, at 5, 10, 15, 20 and 42 days post‐inoculation (dpi). The final reaction of each plant genotype was recorded as an index of disease at 42 dpi. Plasmodiophora brassicae DNA showed an increase in susceptible and moderately resistant hosts from 5 to 42 dpi, in contrast to a decrease in a highly resistant host and the non‐host wheat over the same period. Index of disease was significantly positively correlated with the amount of P. brassicae DNA in the roots at 5, 15, 20 and 42 dpi in one experiment, and at 10, 15, 20 and 42 dpi in a repeated experiment. Significant positive correlations also existed between the amounts of P. brassicae DNA in the roots at 42 dpi and those at 5, 10, 15 and 20 dpi in one experiment, and those at 10, 15 and 20 dpi in a repeated experiment. The results generated by the qPCR assay were validated by microscopic examination of roots inoculated with P. brassicae. The qPCR‐based protocol developed in this study allows for the accurate quantification of P. brassicae DNA in host root tissues as early as 5 dpi, and may serve as a useful tool to evaluate pathogen proliferation and development in the roots.     

Powdery scab resistance in Solanum tuberosum: an assessment of cultivar × environment effect

Powdery scab of potato caused by Spongospora subterranea is one of the main disease problems in many potato production regions of the world. However, no efficient and economically sound control method is currently available. Host resistance will be a key component of the integrated management of powdery scab, but there are discrepancies in published powdery scab resistance ratings of cultivars between countries. In order to identify the main factors causing such discrepancies, 10 reference cultivars thought to have a range of susceptibility to powdery scab and potato mop‐top virus were cropped over 4 years in four to six locations across Europe and disease levels on roots and tubers were assessed using standardized scoring scales. Soil contamination was tested using real‐time PCR and ELISA. The cultivars performed as expected according to previous characterization, with one exception. No relationship was found between tuber and root susceptibility. Assessment of powdery scab symptoms 1 month before harvest gave results comparable to those assessed 2 months after harvest. Neither real‐time PCR nor ELISA soil test results were closely related to disease index data. The field trial results indicate that different scoring methods are the main factor for the discrepancy in resistance ratings, and that environmental conditions and/or soil inoculum level play a minor role. Furthermore, there was either no difference between the pathogen populations in each location or the resistance of most of the cultivars is polygenic.     

云南马铃薯粉痂病发生情况初步研究

马铃薯粉痂病由Spongospora subterranea f.sp.subterranea引起,是典型的土传病害,主要危害块茎和根部。2004年调查了昭通市和会泽县7个乡镇,8个马铃薯品种的粉痂病发生情况。在调查的品种中,会-2发病最轻,平均发病率27%。会泽县马铃薯粉痂病平均发病率59%,昭通市马铃薯粉痂病平均发病率35%。分析土壤中氮、磷、钾、有机质含量及pH与发病率的关系。PB06发病率与土壤中氮、有机质呈正相关,与磷、钾、pH呈负相关,其中与钾含量相关系数为0.693。会-2发病率与土壤pH、钾含量呈负相关,相关系数分别为0.384、0.858,其中与钾含量相关性显著;与磷、有机质含量呈正相关,相关系数分别为0.727、0.323;与氮含量呈正相关,相关系数为0.993,相关性显著。     

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).     

Control of potato powdery scab caused by Spongospora subterranea by foliage cover and soil application of chemicals under field conditions with naturally infested soil

Powdery scab (PS), caused by Spongospora subterranea, reduces the quality and marketability of potatoes worldwide. Disease symptoms include lesions on the tuber surface and root galling, which may lead to yield losses. In the current study we report a sustainable approach to reduce PS by manipulating soil temperature during tuber initiation. Plant cover with nonwoven fabric significantly reduced PS on tubers by 54%–69% in 2017 and 84%–93% in 2019, compared to the control, and root galling by 96% in 2019, due to an increased average minimum and maximum soil temperature of 1.8 and 4.2 °C in respective years. Additional preplanting soil treatments were also evaluated in naturally infested soil. In 2017, disease incidence and severity were significantly reduced using 2.5 or 5 L a.i./ha fluazinam in broadcast application or in-furrow, and by 0.375 or 0.75 L a.i./ha flusulphamide applied in-furrow. In 2019, disease incidence and severity were significantly reduced by the broadcast application of fluazinam, and 75 kg/ha calcium cyanamide, but the latter had a negative impact on yield. Soil fumigation with metam sodium resulted in a 98% reduction in PS. Root galling was significantly reduced by calcium cyanamide, metam sodium, and fluazinam in the 2019 trial only. Foliar application of resistance-inducing phosphonates combined with fluazinam application had no additive effect on PS incidence and severity. Integrated approaches such as tolerant cultivars, soil testing, preplanting fungicide application, and sustainable means of control such as foliage cover for a short period may be implemented in order to control the disease and minimize damage.     

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.     

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.     

Colonization of lettuce cultivars and rotation crops by Fusarium oxysporum f. sp. lactucae,the cause of fusarium wilt of lettuce

The severity of fusarium wilt is affected by inoculum density in soil, which is expected to decline during intervals when a non‐susceptible crop is grown. However, the anticipated benefits of crop rotation may not be realized if the pathogen can colonize and produce inoculum on a resistant cultivar or rotation crop. The present study documented colonization of roots of broccoli, cauliflower and spinach by Fusarium oxysporum f. sp. lactucae, the cause of fusarium wilt of lettuce. The frequency of infection was significantly lower on all three rotation crops than on a susceptible lettuce cultivar, and the pathogen was restricted to the cortex of roots of broccoli. However, F. oxysporum f. sp. lactucae was isolated from the root vascular stele of 7·4% of cauliflower plants and 50% of spinach plants that were sampled, indicating a greater potential for colonization and production of inoculum on these crops. The pathogen was also recovered from the root vascular stele of five fusarium wilt‐resistant lettuce cultivars. Thus, disease‐resistant plants may support growth of the pathogen and thereby contribute to an increase in soil inoculum density. Cultivars that were indistinguishable based on above‐ground symptoms, differed significantly in the extent to which they were colonized by F. oxysporum f. sp. lactucae. Less extensively colonized cultivars may prove to be superior sources of resistance to fusarium wilt for use in breeding programmes.