Common Scab Pathogen Research Articles

Isolation and Characterization of Plant-PathogenicStreptomycesSpecies Associated with Common Scab-Infected Potato Tubers in Newfoundland

Potato common scab (CS) is an economically important crop disease that is caused by several members of the genus Streptomyces. In this study, we characterized the plant-pathogenic Streptomyces spp. associated with CS-infected potato tubers harvested in Newfoundland, Canada. A total of 17 pathogenic Streptomyces isolates were recovered from potato scab lesions, of which eight were determined to be most similar to the known CS pathogen S. europaeiscabiei. All eight S. europaeiscabiei isolates were found to produce the thaxtomin A phytotoxin and to harbor the nec1 virulence gene, and most also carry the putative virulence gene tomA. The remaining isolates appear to be novel pathogenic species that do not produce thaxtomin A, and only two of these isolates were determined to harbor the nec1 or tomA genes. Of the non-thaxtomin-producing isolates, strain 11-1-2 was shown to exhibit a severe pathogenic phenotype against different plant hosts and to produce a novel, secreted phytotoxic substance. This is the first report documenting the plant-pathogenic Streptomyces spp. associated with CS disease in Newfoundland. Furthermore, our findings provide further evidence that phytotoxins other than thaxtomin A may also contribute to the development of CS by Streptomyces spp.

HybProbes-based real-time PCR assay for specific identification of Streptomyces scabies and Streptomyces europaeiscabiei, the potato common scab pathogens.

The aim of this study was to develop and validate a HybProbes-based real-time PCR assay targeting the trpB gene for specific identification of Streptomyces scabies and Streptomyces europaeiscabiei. Four primer pairs and a fluorescent probe were designed and evaluated for specificity in identifying S.scabies and Streptomyces europaeiscabiei, the potato common scab pathogens. The specificity of the HybProbes-based real-time PCR assay was evaluated using 46 bacterial strains, 23 Streptomyces strains and 23 non-Streptomyces bacterial species. Specific and strong fluorescence signals were detected from all nine strains of S.scabies and Streptomyces europaeiscabiei. No fluorescence signal was detected from 14 strains of other Streptomyces species and all non-Streptomyces strains. The identification was corroborated by the melting curve analysis that was performed immediately after the amplification step. Eight of the nine S.scabies and S.europaeiscabiei strains exhibited a unique melting peak, at Tm of 69·1°C while one strain, Warba-6, had a melt peak at Tm of 65·4°C. This difference in Tm peaks could be attributed to a guanine to cytosine mutation in strain Warba-6 at the region spanning the donor HybProbe. The reported HybProbes assay provides a more specific tool for accurate identification of S.scabies and S.europaeiscabiei strains. This study reports a novel assay based on HybProbes chemistry for rapid and accurate identification of the potato common scab pathogens. Since the HybProbes chemistry requires two probes for positive identification, the assay is considered to be more specific than conventional PCR or TaqMan real-time PCR. The developed assay would be a useful tool with great potential in early diagnosis and detection of common scab pathogens of potatoes in infected plants or for surveillance of potatoes grown in soil environment.

Characterization of the Coronatine-Like Phytotoxins Produced by the Common Scab Pathogen Streptomyces scabies.

Streptomyces scabies is an important causative agent of common scab disease of potato tubers and other root crops. The primary virulence factor produced by this pathogen is a phytotoxic secondary metabolite called thaxtomin A, which is essential for disease development. In addition, the genome of S. scabies harbors a virulence-associated biosynthetic gene cluster called the coronafacic acid (CFA)-like gene cluster, which was previously predicted to produce metabolites that resemble the Pseudomonas syringae coronatine (COR) phytotoxin. COR consists of CFA linked to an ethylcyclopropyl amino acid called coronamic acid, which is derived from L-allo-isoleucine. Using a combination of genetic and chemical analyses, we show that the S. scabies CFA-like gene cluster is responsible for producing CFA-L-isoleucine as the major product as well as other minor COR-like metabolites. Production of the metabolites was shown to require the cfl gene, which is located within the CFA-like gene cluster and encodes an enzyme involved in ligating CFA to its amino acid partner. CFA-L-isoleucine purified from S. scabies cultures was shown to exhibit bioactivity similar to that of COR, though it was found to be less toxic than COR. This is the first report demonstrating the production of coronafacoyl phytotoxins by S. scabies, which is the most prevalent scab-causing pathogen in North America.

Regulation of coronafacoyl phytotoxin production by the PAS-LuxR family regulator CfaR in the common scab pathogen Streptomyces scabies.

Potato common scab is an economically important crop disease that is characterized by the formation of superficial, raised or pitted lesions on the potato tuber surface. The most widely distributed causative agent of the disease is Streptomyces scabies, which produces the phytotoxic secondary metabolite thaxtomin A that serves as a key virulence factor for the organism. Recently, it was demonstrated that S. scabies can also produce the phytotoxic secondary metabolite coronafacoyl-L-isoleucine (CFA-L-Ile) as well as other related metabolites in minor amounts. The expression of the biosynthetic genes for CFA-L-Ile production is dependent on a PAS-LuxR family transcriptional regulator, CfaR, which is encoded within the phytotoxin biosynthetic gene cluster in S. scabies. In this study, we show that CfaR activates coronafacoyl phytotoxin production by binding to a single site located immediately upstream of the putative -35 hexanucleotide box within the promoter region for the biosynthetic genes. The binding activity of CfaR was shown to require both the LuxR and PAS domains, the latter of which is involved in protein homodimer formation. We also show that CFA-L-Ile production is greatly enhanced in S. scabies by overexpression of both cfaR and a downstream co-transcribed gene, orf1. Our results provide important insight into the regulation of coronafacoyl phytotoxin production, which is thought to contribute to the virulence phenotype of S. scabies. Furthermore, we provide evidence that CfaR is a novel member of the PAS-LuxR family of regulators, members of which are widely distributed among actinomycete bacteria.

リアルタイム定量PCRと無底ポット栽培を利用したジャガイモそうか病菌の動態解析の試み

ジャガイモそうか病はStreptomyces spp.により引き起こされる病害である．これまで，そうか病菌の定量にはPAI遺伝子を標的としたリアルタイムPCR法が開発されている．しかし，そうか病菌は菌種により性質が異なり，特に，低pH耐性はそうか病の防除に直結する．効率的な防除対策のためには，菌種ごとの定量技術を開発することが必要である．そこで，国内で主要な菌種であるS. scabieiとS. turgidiscabiesについて，16S-23S rRNA internal transcribed spacerを標的としたリアルタイムPCR法（SYBR Green）による定量技術を開発した．本定量法はS. scabieiのJKタイプ（Ssj）およびS. turgidiscabies（St）に特異的であり，両菌種とも200 fgから20 ngまでのDNA濃度の対数値とCt値との間に高い直線回帰が認められた（Ssj：R2＝0.9985，St：R2＝0.9988）．また，両種が混在した状態の植物や土壌からも，特異的に両菌種の定量が可能であった．●さらに，無底ポット内の黒ボク土壌の異なるpH条件下（植え付け時のpH 4.4～5.2）で，ジャガイモ栽培を行い，S. turgidiscabies単独接種区，S. scabiei単独接種区，および両菌混合接種区を設けた．収穫時を中心に両菌の菌量およびサクストミン毒素（txtAB）量によりそうか病菌量を調査した結果，S. turgidiscabiesはS. scabieiよりも低いpH 4.7以上で土壌中の菌量が高くなった．また，両菌を接種した条件下では，土壌，根および病斑のすべてでS. turgidiscabiesが優先していた．

Optimal rates of 2,4‐dichlophenoxyacetic acid foliar application for control of common scab in potato

AbstractApplications of the synthetic auxin 2,4‐dichlorophenoxyacetic acid (2,4‐D) to the foliage of potato plants can reduce common scab, a tuber disease. However, in prior research effective applications at 200 mg L−1 2,4‐D resulted in phytotoxic side effects with reduced tuber yield and quality. This study showed that minimal significant threshold rates from 8.3 to 23.6 mg L−1 2,4‐D reduced disease incidence in pot trials, and from 10.8 to 41.0 mg L−1 minimised disease severity in both pot and field trials. In only one pot trial, significant phytotoxicity was found with rates of 100 mg L−1 or greater, reducing mean total tuber mass per plot and 38 mg L−1 or greater, reducing mean mass per tuber. Notably, within the field trial, a more reliable plant growth system for estimation of yield, no significant impacts were observed. Disease control was associated with decreased sensitivity of tubers to thaxtomin A, the phytotoxin produced by the common scab pathogen essential for disease induction. The amount of residual 2,4‐D in tubers at harvest varied with cultivar, Russet Burbank accumulating more 2,4‐D than Desiree. Application rates less than 100 mg L−1 resulted in levels of 2,4‐D below the Australian standard maximum residue limit. These data suggest that applications of 2,4‐D at low rates could provide a commercially suitable control strategy for common scab.

Global gene expression in the common scab pathogen, Streptomyces scabies, exposed to potato microtubers

AbstractStreptomyces scabies causes yield losses in potato by inducing common scab (CS) symptoms on tubers. Little information is available on global gene expression responses in CS‐causing streptomycetes upon interaction with host plants. In this study, expression of 8746 genes was analysed in the type strain of S. scabies (ATCC49173) using a custom‐designed, gene‐specific oligonucleotide microarray. Bacteria were grown in liquid yeast malt extract (YME) medium and 480 genes were found to be up‐regulated 24 h after supplementing the cultures with potato microtubers. Quantitative PCR analysis of eight putative or known virulence genes was consistent with the microarray data. Elevated expression of the thaxtomin synthesis gene txtA and txtR genes regulating synthesis of thaxomin A, the main pathogenicity factor of S. scabies, and production of thaxtomin A were detected in cultures containing microtubers. Unexpectedly, thaxtomin was also detected in cultures lacking microtubers, which indicated that growth in YME induces thaxtomin production in S. scabies. Many but not all microtubers incubated in the cultures of S. scabies for 48 h displayed symptoms, indicating that the microtubers were not equally susceptible even though they were produced under uniform in vitro growth conditions. These factors may have limited the differences in gene expression observed between S. scabies exposed to microtubers compared to controls. This study provides some of the first results on the transcriptome of S. scabies, a widely distributed and important potato pathogen, upon exposure to young developing potato tubers. The findings will inform future studies on interactions of CS‐inducing streptomycetes with potato and means to control the disease.

Potato MYB and bHLH transcription factors associated with anthocyanin intensity and common scab resistance

MYB and bHLH are large transcription factor families with largely uncharacterized biological functions. The patterns of expression of 42 MYB and 58 bHLH transcription factor genes were examined in potato clones that were demonstrated to have variation in anthocyanin intensity and common scab resistance to assess their possible involvement in regulating these traits. The control of expression of biosynthetic enzymes in regulation of anthocyanin intensity was also evaluated. The dihydroflavonol 4-reductase (DFR) gene, a key enzyme in the pathway, had the highest correlation with gene expression and anthocyanin intensity (quantified as levels of anthocyanidins). Expression of five uncharacterized MYB and three bHLH genes was also highly correlated with anthocyanin intensity, suggesting that they could be regulators of biosynthetic enzyme gene expression. The same potato clones were also demonstrated to have variation in resistance to Streptomyces scabiei, the causal pathogen of common scab in potato. Correlation analysis was used to show that anthocyanin intensity was not associated with common scab resistance. However, common scab resistance was correlated with expression of another two MYB and three bHLH genes, indicating that they might be involved in the regulation of the defense response of potato against the common scab pathogen.

Fissure Scab, a New Symptom Associated with Potato Common Scab Caused by a Streptomyces sp. in South Africa

Common scab caused by Streptomyces spp. is one of the major factors limiting successful potato (Solanum tuberosum) production in South Africa. Most potato cultivars are susceptible to the disease and huge losses are incurred due to a reduction in cosmetic value. Common scab symptoms that have been reported worldwide are variable, with circular, raised, tan-to-brown, corky lesions being the most common symptoms. However, a distinct atypical symptom was observed in isolated production regions (Western Free State and Mpumalanga) in South Africa since early 2010. Deep longitudinal fissures (3 to 12 mm) containing scab-like lesions were observed on the surface area of tubers from several potato cultivars (Mondial, BP1, and Buffelspoort). Lesions on Mondial were interesting since this cultivar, but not the susceptible BP1 and Buffelspoort cultivars, is tolerant to typical common scab in South Africa. Isolations were made from tuber lesions obtained from the two production regions and were plated onto yeast malt extract medium. Several pure culture strains were obtained that were positively identified as Streptomyces spp. based on morphology. Species identity of four strains was investigated using PCR primers targeting the 16S rRNA region of known species (3), which showed that the strains did not belong to any of the known pathogenic reference strains (S. scabiei, S. europascabiei, S. turgidiscabies, S. acidiscabies). Species identity of the strains was further investigated through sequencing of the 16S rRNA region (1.2 kb). The four strains had 100% sequence similarity (GenBank Accession. No. JQ241439) to each other and to 15 GenBank sequences that included several unknown Streptomyces spp., S. vinaceus, S. malachiticus, S. werraensis, S. cyaneus (the only published sequenced), and S. pseudogriseolus. The sequence of the isolates had only 95.5% identity to the most prevalent common scab pathogen, S. scabiei (GI154707840, [3]). The pathogenicity of the four strains, along with an S. scabiei reference isolate, was investigated using BP1 potato tubers and the double pot methodology (1). Fifteen-centimeter pots containing silica sand were each planted with one potato tuber and placed on top of a rectangular planting box (15 × 1.5 × 0.4 m) filled with native Hutton soil. The 15-cm pots were irrigated until the roots grew into the Hutton soil. Subsequently, irrigation was only applied to the Hutton soil, thus creating dry conditions in the tuber development area (15-cm pot), which is conducive for common scab development. The potato plants were inoculated with 10- to 14-day-old Streptomyces strains during the tuber initiation stage at a concentration of 1 × 106 spores/ml of sterile water, 10 ml per plant, and six replicates per strain. Ten weeks after inoculation, each of the four strains caused cracking and scabbing, similar to initial symptoms observed, on more than 90% of the tubers. The S. scabiei reference isolate caused typical circular, raised, brown, corky common scab lesions. Streptomyces was reisolated from the lesions, fulfilling Koch's postulates. To our knowledge, these results indicate that a previously unreported Streptomyces sp. is the causal agent of a new lesion type, fissure scab, on potato in South Africa that may lead to serious losses to the local potato industry.

Infection of Potato Tubers with the Common Scab Pathogen Streptomyces scabiei in a Soil-less System

A novel soil-less method was developed to define susceptibility of developing potato tubers accurately to infection with Streptomyces scabiei the causal agent of common scab disease. Hydroponic production enabled precise identification of individual tuber development. Direct inoculation of tubers with a spore suspension of S. scabiei resulted in disease development, demonstrating that infection could be initiated in a soil-less media. Tubers were most susceptible to infection between 3 and 20 days after tuber initiation, confirming that this early period of tuber formation is critical to disease development

Quantification of potato common scab pathogens in soil by quantitative competitive PCR with fluorescent quenching‐based probes

Common scab of potato tubers caused by pathogenic Streptomyces spp. is a cause of serious economic loss worldwide. For the rapid and accurate quantification of pathogenic Streptomyces spp. residing in soil, a new competitive real‐time PCR method using fluorescent quenching‐based probes (quantitative competitive quenching probe PCR: QCQP‐PCR) was developed. The virulence gene of pathogenic Streptomyces spp., nec1, was selected as the target for QCQP‐PCR. A specific primer set to amplify the nec1 gene, and a fluorescently labelled probe that specifically hybridizes with the nec1 amplicon were designed. For QCQP‐PCR, an internal standard DNA (IS DNA) that is identical to the nec1 amplicon but has a 4‐base mismatch in the probe‐hybridizing region, and a fluorescently labelled probe IS, which specifically hybridizes with IS DNA at the mutagenized region, were PCR‐synthesized. The target nec1 gene was co‐amplified with the known copy number of IS DNA by PCR using the same primer set in the presence of the specific probes. The PCR products were monitored in real‐time by measuring the fluorescence intensity (quenching) of each probe. The initial amount of the nec1 gene was quantified based on the ratio of the PCR products of the same PCR cycle. The results revealed that QCQP‐PCR could be used to precisely quantify the nec1 gene, even in the presence of PCR inhibitors in the soil samples examined. The lower limit of quantification was 20 copies per tube, which corresponded to 1500 copies per g dry soil. The quantification achieved by this method was completed within 5 h, i.e. the duration of the entire analysis. These results demonstrate the usefulness of the present method for monitoring pathogenic Streptomyces species in soil.

Precise, simple screening for resistance in potato varieties to common scab using paper pots

Resistance to common scab pathogen Streptomyces turgidiscabies of seven potato varieties was compared in the field with a newly developed paper pot method. Seedlings raised in soil in paper pots containing inocula at 1 × 103 to 107 cfu/g soil were transplanted into a scab-free field and grown for 3 months. The disease severity of the seven varieties in the field trials differed in iteration and from year to year, even though their resistance levels were approximately similar at the expected levels. With the paper pot method, the seven varieties had different resistance levels, which were almost completely consistent with the results of the field trials, at more than 1 × 105 cfu/g soil. Significant differences in disease severity between resistant and susceptible varieties were observed (P = 0.05) for 2 years, and the resistance level of the varieties was elucidated.

Responses of potato cultivars to the common scab pathogens, Streptomyces scabies and S. turgidiscabies

SummaryA glasshouse experiment was conducted to study the responses to Streptomyces scabies and S. turgidiscabies in potato cultivars Bellona, Matilda and Sabina (Solanum tuberosum). Potatoes were grown in a peat‐sand mixture inoculated with one of the two strains of either S. scabies or S. turgidiscabies. Logit models were used to analyse the data on disease incidence and severity, whereas the data on emergence and yield were tested by analysis of variance. S. turgidiscabies, a recently described potato pathogen in Finland, possessed a high ability to cause superficial, raised and pitted lesions on all three cultivars tested. Symptoms induced by S. turgidiscabies were similar to those of S. scabies, regardless of the cultivar, which suggests that the two causal organisms of common scab cannot be distinguished based on symptoms. Infection by S. turgidiscabies and S. scabies delayed emergence, had the tendency to decrease the yield, and increased the proportion of small tubers in the yield, regardless of the potato cultivar. Differences in the levels of resistance to common scab were evident between potato cultivars, since cvs. Matilda and Bellona showed higher disease incidence and more severe scab symptoms than cv. Sabina.

Host Selectivity of Actinophages Found in Soil Infected with Potato Common Scab Pathogens

Host Selectivity of Actinophages Found in Soil Infected with Potato Common Scab Pathogens

Production of potato common scab-antagonistic biofertilizer from swine feces with Streptomyces albidoflavus.

A biofertilizer, showing antagonistic activity against potato common scab in pot tests, was produced from swine feces with a newly isolated strain, CH-33, identified as Streptomyces albidoflavus. This strain characteristically grew on fresh swine feces at 20~35°C without sterili-zation or any additives, and produced an antibiotic substance against Streptomyces scabies, the common scab-pathogen, during composting. The addition of the biofertilizer at from 0.1 g to 1.6 g total nitrogen (N) per 600 g humic volcanic ash soil in a pot did not inhibit the growth of Brassica rapa var. perviridis but increased it, even at the highest nitrogen content tested. Common scab was completely inhibited when the biofertilizer was added at 0.1 g to 1.6g as nitrogen (N) per 4 kg of scab-infected soil in a pot. Thus a biofertilizer suppressing plant pathogenic microorganisms was developed.