Development and Evaluation of a Loop-Mediated Isothermal Amplification (LAMP) Method for Detection of the Potato Powdery Scab Pathogen Spongospora subterranea.

Spongospora subterranea is the causal agent of powdery scab of potato (Solanum tuberosum), which can significantly reduce potato quality. In this study, we developed and evaluated a loop-mediated isothermal amplification (LAMP) method for the detection of S. subterranea. A set of LAMP primers named PS-LAMP was designed and tested for specificity and sensitivity. In the specificity test, in silico analysis using the NCBI Primer-BLAST tool indicated that PS-LAMP was specific to S. subterranea. The in vitro tests confirmed specificity, showing that PS-LAMP could produce positive signals from DNA isolated from each of three potato tubers with powdery scab symptoms but did not produce positive signals from DNA isolated from 38 nontarget plant pathogens. The sensitivity of PS-LAMP was tested on both gBlocks and DNA isolated from potato samples with powdery scab symptoms. On gBlocks, the lowest number of copies for a positive LAMP reaction was six, which was similar to results obtained via qPCR, but it was 10 times more sensitive than conventional PCR. On a DNA sample from S. subterranea-infected potato, the lowest amount of template DNA for a positive LAMP reaction was 2 pg, which was incomparable with the sensitivity of qPCR. Considering the convenience of the LAMP technique, as well as the high specificity and sensitivity, this assay can be very useful for plant pathology practitioners and diagnostic labs interested in rapid, accurate, and routine detection of S. subterranea and confirmation of powdery scab disease.

Comparative Proteomic Analysis of Potato Roots from Resistant and Susceptible Cultivars to Spongospora subterranea Zoospore Root Attachment In Vitro.

Potato (Solanum tuberosum L.) exhibits broad variations in cultivar resistance to tuber and root infections by the soilborne, obligate biotrophic pathogen Spongospora subterranea. Host resistance has been recognised as an important approach in potato disease management, whereas zoospore root attachment has been identified as an effective indicator for the host resistance to Spongospora root infection. However, the mechanism of host resistance to zoospore root attachment is currently not well understood. To identify the potential basis for host resistance to S. subterranea at the molecular level, twelve potato cultivars differing in host resistance to zoospore root attachment were used for comparative proteomic analysis. In total, 3723 proteins were quantified from root samples across the twelve cultivars using a data-independent acquisition mass spectrometry approach. Statistical analysis identified 454 proteins that were significantly more abundant in the resistant cultivars; 626 proteins were more abundant in the susceptible cultivars. In resistant cultivars, functional annotation of the proteomic data indicated that Gene Ontology terms related to the oxidative stress and metabolic processes were significantly over-represented. KEGG pathway analysis identified that the phenylpropanoid biosynthesis pathway was associated with the resistant cultivars, suggesting the potential role of lignin biosynthesis in the host resistance to S. subterranea. Several enzymes involved in pectin biosynthesis and remodelling, such as pectinesterase and pectin acetylesterase, were more abundant in the resistant cultivars. Further investigation of the potential role of root cell wall pectin revealed that the pectinase treatment of roots resulted in a significant reduction in zoospore root attachment in both resistant and susceptible cultivars. This study provides a comprehensive proteome-level overview of resistance to S. subterranea zoospore root attachment across twelve potato cultivars and has identified a potential role for cell wall pectin in regulating zoospore root attachment.

Multi-omics reveals mechanisms of resistance to potato root infection by Spongospora subterranea.

The pathogen Spongospora subterranea infects potato roots and developing tubers resulting in tuber yield and quality losses. Currently, there are no fully effective treatments for disease control. Host resistance is an important tool in disease management and understanding the molecular mechanisms of defence responses in roots of potato plants is required for the breeding of novel resistant cultivars. Here, we integrated transcriptomic and proteomic datasets to uncover these mechanisms underlying S. subterranea resistance in potato roots. This multi-omics approach identified upregulation of glutathione metabolism at the levels of RNA and protein in the resistant cultivar but not in the susceptible cultivar. Upregulation of the lignin metabolic process, which is an important component of plant defence, was also specific to the resistant cultivar at the transcriptome level. In addition, the inositol phosphate pathway was upregulated in the susceptible cultivar but downregulated in the resistant cultivar in response to S. subterranea infection. We provide large-scale multi-omics data of Spongospora-potato interaction and suggest an important role of glutathione metabolism in disease resistance.

Detection and Quantification of Spongospora subterranea Sporosori in Soil by Quantitative Real-Time PCR.

Powdery scab on potato tubers is caused by the obligate soilborne biotroph Spongospora subterranea and is known to cause substantial losses in potato production. The pathogen also infects roots of susceptible hosts, forming galls which can negatively affect root function. S. subterranea is also the vector of Potato mop-top virus, which causes a tuber necrosis disease that can, depending on temperature and cultivar, render potato tubers unmarketable. In this study, we adapted a published protocol to develop a sensitive and robust quantitative real-time PCR (qPCR) assay using specific primers and probes for detecting and quantifying S. subterranea sporosori in soil types that differ in physical properties, including organic matter content and soil pH. For the first time, an external control was utilized and applied directly to the soil prior to DNA extraction, which facilitated normalization of S. subterranea sporosori soil levels from sample to sample. The duplex qPCR protocol was demonstrated to be highly sensitive, capable of detecting and quantifying as few as 1 sporosorus/g of soil, with consistently high qPCR efficiency and the coefficient of determination (R2) values ranging from 94 to 99% and 0.98 to 0.99, respectively. The protocol was successfully implemented in enumerating S. subterranea sporosori in naturally infested field soil collected from several states and in artificial potting mixes with high organic matter content ranging from 64 to 71%. The qPCR method developed can be useful for potato growers to avoid agricultural soils highly infested with S. subterranea and in the development of risk assessment models in the future that incorporate cultivar susceptibility to powdery scab and soil infestation levels.

Population Biology and Genetic Variation of Spongospora subterranea f. sp. subterranea, the Causal Pathogen of Powdery Scab and Root Galls on Potatoes in South Africa.

Spongospora subterranea f. sp. subterranea, causal agent of powdery scab and root galls of potatoes, occurs worldwide and is responsible for quality and yield losses in potato production in South Africa. Despite being one of the most important potato pathogens in South Africa, little information is available on the genetic structure and diversity of S. subterranea f. sp. subterranea, which could provide insight into the factors shaping its evolution and the role of inoculum sources in disease development. A total of 172 samples were collected from four potato growing regions in South Africa. An additional 27 samples obtained from Colombia were included for comparative purposes. The samples were screened against six informative microsatellite (simple-sequence repeat) markers. Of the 172 samples obtained from potato growing regions in South Africa, there were 75 multilocus genotypes (MLGs), only 16 of which were shared between potato growing regions, indicating substantial gene flow and countrywide dispersal of the pathogen. The presence of common MLGs among the root- and tuber-derived samples indicated a lack of specialization of S. subterranea f. sp. subterranea to either tuber or root infection. Nei's unbiased estimates of gene diversity for the clone-corrected data were low and ranged from 0.24 to 0.38. Analysis of molecular variance and discriminant analysis of principal components showed no population differentiation between different potato growing regions in South Africa and between root- and tuber-derived genotypes. The presence of MLGs, high considerable genotypic diversity, and failure to reject the null hypothesis of random mating in most populations are indicative of some kind of recombination, either sexual or asexual, in these S. subterranea f. sp. subterranea populations. Information from this study provides new insights into the genetic structure and diversity of S. subterranea f. sp. subterranea in South Africa. Continuous monitoring of the pathogen population dynamics will be helpful in implementing effective region-specific management strategies for the pathogen, especially in the development of resistant potato cultivars.

Draft Genome Resource for the Potato Powdery Scab Pathogen Spongospora subterranea.

The Plasmodiophorida (Phytomyxea, Rhizaria) are a group of protists that infect plants. Of this group, Spongospora subterranea causes major problems for the potato industry by causing powdery scab and root galling of potatoes and as vector for the Potato mop-top virus (PMTV) (genus Pomovirus, family Virgaviridae). A single tuber isolate (SSUBK13) of this uncultivable protist was used to generate DNA for Illumina sequencing. The data were assembled to a draft genome of 28.08 Mb consisting of 2,340 contigs and an L50 of 280. A total of 10,778 genes were predicted and 93% of the BUSCO genes were detected. The presented genome assembly is only the second genome of a plasmodiophorid. The data will accelerate functional genomics to study poorly understood interaction of plasmodiophorids and their hosts.

Mitochondrial genome sequence of the potato powdery scab pathogen Spongospora subterranea.

Spongospora subterranea is a soil-borne obligate parasite responsible for potato powdery scab disease. S. subterranea is a member of the order Plasmodiophorida, a protist taxa that is related to Cercozoa and Foraminifera but the fine details of these relationships remain unresolved. Currently there is only one available complete mtDNA sequence of a cercozoan, Bigelowiella natans. In this work, the mitochondrial sequence of a S. subterranea isolate infecting an Andean variety of S. tuberosum ssp. andigena (Diacol-Capiro) is presented. The mtDNA codes for 16 proteins of the respiratory chain, 11 ribosomal proteins, 3 ribosomal RNAs, 24 tRNAs, a RNA processing RNaseP, a RNA-directed polymerase, and two proteins of unknown function. This is the first report of a mtDNA genome sequence from a plasmodiophorid and will be useful in clarifying the phylogenetic relationship of this group to other members in the supergroup Rhizaria once more mtDNA sequences are available.

Global genetics and invasion history of the potato powdery scab pathogen, Spongospora subterranea f.sp. subterranea.

Spongospora subterranea f. sp. subterranea (Sss) causes two diseases on potato (Solanum tuberosum), lesions on tubers and galls on roots, which are economically important worldwide. Knowledge of global genetic diversity and population structure of pathogens is essential for disease management including resistance breeding. A combination of microsatellite and DNA sequence data was used to investigate the structure and invasion history of Sss. South American populations (four countries, 132 samples) were consistently more diverse than those from all other regions (15 countries, 566 samples), in agreement with the hypothesis that Sss originated in South America where potato was domesticated. A substantial genetic differentiation was found between root and tuber-derived samples from South America. Estimates of past and recent gene flow suggested that Sss was probably introduced from South America into Europe. Subsequently, Europe is likely to have been the recent source of migrants of the pathogen, acting as a "bridgehead" for further global dissemination. Quarantine measures must continue to be focussed on maintaining low global genetic diversity and avoiding exchange of genetic material between the native and introduced regions. Nevertheless, the current low global genetic diversity of Sss allows potato breeders to select for resistance, which is likely to be durable.

Multiplex real-time PCR (TaqMan) assay for the simultaneous detection and discrimination of potato powdery and common scab diseases and pathogens.

AIMS: To develop a multiplex real-time PCR assay using TaqMan probes for the simultaneous detection and discrimination of potato powdery scab and common scab, two potato tuber diseases with similar symptoms, and the causal pathogens Spongospora subterranea and plant pathogenic Streptomyces spp. METHODS AND RESULTS: Real-time PCR primers and a probe for S. subterranea were designed based on the DNA sequence of the ribosomal RNA ITS2 region. Primers and a probe for pathogenic Streptomyces were designed based on the DNA sequence of the txtAB genes. The two sets of primer pairs and probes were used in a single real-time PCR assay. The multiplex real-time PCR assay was confirmed to be specific for S. subterranea and pathogenic Streptomyces. The assay detected DNA quantities of 100 fg for each of the two pathogens and linear responses and high correlation coefficients between the amount of DNA and C(t) values for each pathogen were achieved. The presence of two sets of primer pairs and probes and of plant extracts did not alter the sensitivity and efficiency of multiplex PCR amplification. Using the PCR assay, we could discriminate between powdery scab and common scab tubers with similar symptoms. Common scab and powdery scab were detected in some tubers with no visible symptoms. Mixed infections of common scab and powdery scab on single tubers were also revealed. CONCLUSIONS: This multiplex real-time PCR assay is a rapid, cost efficient, specific and sensitive tool for the simultaneous detection and discrimination of the two pathogens on infected potato tubers when visual symptoms are inconclusive or not present. SIGNIFICANCE AND IMPACT OF THE STUDY: Accurate and quick identification and discrimination of the cause of scab diseases on potatoes will provide critical information to potato growers and researchers for disease management. This is important because management strategies for common and powdery scab diseases are very different.

Genomics of biotrophic, plant-infecting plasmodiophorids using in vitro dual cultures.

The plasmodiophorids are a phylogenetically distinct group of parasitic protists that infect plants and stramenopiles, causing several important agricultural diseases. Because of the obligate intracellular part of their lifecycle, none of the plasmodiophorids has been axenically cultured. Further, the molecular biology of the plasmodiophorids is poorly understood because pure cultures are not available from any species. We report on an in-vitro dual culture system of the plasmodiophorids Plasmodiophora brassicae and Spongospora subterranea with their respective plant hosts, Brassica rapa and Solanum tuberosum. We show that these plasmodiophorids are capable of initiating and maintaining stable, long-term plant cell callus cultures in the absence of exogenous plant growth regulators. We show that callus cultures harbouring S. subterranea provide an excellent starting material for gene discovery from this organism by constructing a pilot-scale DNA library. Bioinformatic analysis of the sequences established that almost all of the DNA clones from this library were from S. subterranea rather than the plant host. The Spongospora genome was found to be rich in retrotransposable elements, and Spongospora protein-coding genes were shown to contain introns. The sequence of a near full-length non-LTR retrotransposon was obtained, the first transposable element reported from a cercozoan protist.