Pathogenic Streptomyces Species Research Articles

Common scab disease: structural basis of elicitor recognition in pathogenic Streptomyces species.

In Streptomyces scabiei, the main causative agent of common scab disease of root and tuber crops, the interaction between the substrate-binding protein (SBP) CebE (CebEscab) and cellotriose released by the plant host (KD in the nanomolar range) is the first event for the onset of its pathogenic lifestyle. Here, we report the structure of CebEscab in complex with cellotriose at a resolution of 1.55 Å, adopting a general fold of the B subcluster of SBPs. The interaction between CebEscab and cellotriose involves multiple direct or water-mediated hydrogen bonds and hydrophobic interactions, with the glucose monomer at the non-reducing end occupying the most conserved part of the substrate-binding cleft. As main interactions between the two domains of CebE involve cellotriose itself, the closed conformational state of CebE is performed via an induced-fit ligand binding mechanism where cellotriose binding triggers the domain movement. Analysis of regulon predictions revealed that the signaling pathway from CebE-mediated cellotriose transport to the transcriptional activation of thaxtomin phytotoxin biosynthesis is conserved in Streptomyces spp. causing common scab, except for Streptomyces ipomoeae, which specifically colonizes sweet potatoes and responds to other and yet unknown virulence elicitors. Interestingly, strains belonging to the pathogenic species turgidiscabies and caniscabiei have a cellotriose-binding protein orthologous to the CebE protein of the saprophytic species Streptomyces reticuli with lower affinity for its substrate (KD in the micromolar range), suggesting higher cellotriose concentrations for perception of their host. Our work also provides the structural basis for the uptake of cellobiose and cellotriose by non-pathogenic cellulose-decomposing Streptomyces species.IMPORTANCECommon scab is a disease caused by a few Streptomyces species that affects important root and tuber crops including potato, beet, radish, and parsnip, resulting in major economic losses worldwide. In this work, we unveiled the molecular basis of host recognition by these pathogens by solving the structure of the sugar-binding protein CebE of Streptomyces scabiei in complex with cellotriose, the main elicitor of the pathogenic lifestyle of these bacteria. We further revealed that the signaling pathway from CebE-mediated transport of cellotriose is conserved in all pathogenic species except Streptomyces ipomoeae, which causes soft rot disease in sweet potatoes. Our work also provides the structural basis of the uptake of cellobiose and cellotriose in saprophytic Streptomyces species, the first step activating the expression of the enzymatic system degrading the most abundant polysaccharide on earth, cellulose.

Identification and Detection of a Virulence Gene of Streptomyces scabies Causing Potato Scab in Thailand

Potato scab is caused by several pathogenic Streptomyces species which diminish crop quality, quantity and marketability. In the present study, a 16S rRNA gene sequence was used to detect Streptomyces species in potato tuber lesions harvested in Chiang Mai, Thailand and to evaluate a virulence gene as a reliable marker for the detection of pathogenic Streptomyces species by PCR assays. Streptomyces isolates were isolated from potato scab lesions, of which one isolate was pathogenic on potato tubers. The pathogenic isolate MJ21 was identified as Streptomyces scabies based on 16S rRNA gene sequence and morphological characteristics. Subsequently, isolate MJ21 produced PCR products from the tomA and txtAB genes, which are related to the production of tomatinase enzyme and thaxtomin A, respectively. Moreover, when grown on nutrient agar (NA) with MJ21, eggplant seedlings showed severe stunting of the roots and shoots, and failed to germinate; by comparison, seedlings/seeds grown on NA plates without MJ21 exhibited no symptoms. This study reports that S. scabies MJ21 has a toxigenic region (TR) that is associated with the tomA and txtAB genes. Keywords: Pathogenicity tests, Potato scab, Streptomyces scabies, Toxigenic region, Virulence gene

Major Streptomyces species associated with fissure scab of potato in South Africa including description of Streptomyces solaniscabiei sp. nov.

Streptomyces species are the causal agents of several scab diseases on potato tubers. A new type of scab symptom, caused by Streptomyces species, was observed in South Africa from 2010 onwards. The disease was initially thought to be caused by a single Streptomyces species, however, subsequent isolations from similar symptoms on other potato tubers revealed diversity of the Streptomyces isolates. The objective of this study was to characterise these isolates in order to determine what are the major species involved in the disease. This was done by sequencing and phylogenetic analyses of the 16S rDNA as well as five housekeeping genes, investigation of growth on different culture media, standard phenotypic tests and scanning electron microscopy of culture morphology. The presence of the pathogenicity island (PAI) present in plant pathogenic Streptomyces species was also investigated. The genomes of eight isolates, selected from the three main clades identified, were sequenced and annotated to further clarify species boundaries. Three isolates of each of the three main clades were also inoculated onto susceptible potato cultivars in order to establish the pathogenicity of the species. The results of the phylogenetic and genome analyses revealed that there are three main species involved, namely, Streptomyces werraensis, Streptomyces pseudogriseolus and a novel Streptomyces species that is described here as Streptomyces solaniscabiei sp. nov., with strain FS70T (= PPPPB BD 2226T = LMG 32103T) as the type strain. The glasshouse trial results showed that all three of the Streptomyces species are capable of producing fissure scab symptoms. None of the Streptomyces isolates from fissure scab contained the full PAI and the mechanism of disease initiation still needs to be determined. Genomic comparisons also indicated that S. gancidicus Suzuki 1957 (Approved Lists 1980) is a later heterotypic synonym of S. pseudogriseolus Okami and Umezawa 1955 (Approved Lists 1980).

Genome-Wide Association Analysis of Common Scab Resistance and Expression Profiling of Tubers in Response to Thaxtomin A Treatment Underscore the Complexity of Common Scab Resistance in Tetraploid Potato

Common scab of potato caused by pathogenic Streptomyces species poses an economic burden to commercial potato cultivation. Despite decades of research, no molecular markers linked to this trait are currently available, making the development of scab resistant potato varieties inefficient. We evaluated common scab lesion type and tuber coverage in 198 individuals segregating for common scab resistance in replicated field trials with high natural disease pressure over two growing seasons. To identify genetic features associated with resistance, we genotyped the tetraploid (4x) population and parental lines (Kalkaska and Tundra) using the Illumina Infinium 8303 Potato Array and conducted genome-wide association analysis. Here, we report two significant single nucleotide polymorphism (SNP) markers on chromosome 1 associated with both scab traits in both years as well as a SNP marker on chromosome 2 associated with scab tuber coverage. Gene expression profiling of the parents of this population in response to Thaxtomin A treatment revealed differential expression of genes in tubers putatively involved in cell wall biosynthesis and hormone signaling.

Genetic background affects pathogenicity island function and pathogen emergence in Streptomyces.

With few exceptions, thaxtomin A (ThxA), a nitrated diketopiperazine, is the pathogenicity determinant for plant-pathogenic Streptomyces species. In Streptomyces scabiei (syn. S.scabies), the ThxA biosynthetic cluster is located within a 177-kb mobile pathogenicity island (PAI), called the toxicogenic region (TR). In S.turgidiscabies, the ThxA biosynthetic cluster is located within a 674-kb pathogenicity island (PAIst). The emergence of new plant pathogens occurs in this genus, but not frequently. This raises the question of whether the mobilization of these pathogenicity regions, through mating, is widespread and whether TR and PAIst can confer plant pathogenicity. We showed that ThxA biosynthetic clusters on TR and PAIst were transferred into strains from five non-pathogenic Streptomyces species through mating with S.scabiei and S.turgidiscabies. However, not all of the transconjugants produced ThxA and exhibited the virulence phenotype, indicating that the genetic background of the recipient strains affects the functionality of the ThxA biosynthetic cluster and therefore would be expected to affect the emergence of novel pathogenic Streptomyces species. Thxs have been patented as natural herbicides, but have yet to be commercialized. Our results also demonstrated the potential of the heterologous production of ThxA as a natural and biodegradable herbicide in non-pathogenic Streptomyces species.

Genotypic and Phenotypic Characterization of Streptomyces Species Causing Potato Common Scab in Uruguay.

Isolation and characterization of common scab (CS) pathogen Streptomyces spp. from Uruguayan potato tubers and soil samples were done in response to significant economic losses due to CS on potato in autumn 2010. Seventy of the 331 isolates were classified as pathogenic owing to their ability to induce necrosis on tuber disks and stunting of radish seedling. Streptomyces spp. causing CS on potato in Uruguay were found to represent a range of different species by virtue of their diverse morphological and physiological traits as well as rep-PCR, rpoB phylogenetic analysis, and multi-locus sequences analysis. We identified isolates primarily as Streptomyces scabiei, S. acidiscabies, and S. europaeiscabiei. However, some of the pathogenic isolates still remain to be identified at the species level. This highlights the need for improved methods for discrimination among pathogenic Streptomyces species. The presence of Streptomyces pathogenicity island (PAI) genes was analyzed, including genes encoding for thaxtomin synthetase (txtA, txtB), tomatinase (tomA), and a necrosis protein (nec1). Among the isolates that were pathogenic, 50% contained the four pathogenicity genes, 33% had an atypical composition of PAI marker genes, and 17% did not contain any genes. The absence of the genes reported to be involved in thaxtomin biosynthesis (txtA, txtB) was confirmed by whole-genome sequencing of two representative strains of this group. This finding suggests the participation of other virulence factors in plant pathogenicity.

Potato Common Scab: a Review of the Causal Pathogens, Management Practices, Varietal Resistance Screening Methods, and Host Resistance

Potato common scab is a widespread disease in which scab-like lesions develop on tubers. The disease is caused by pathogenic Streptomyces species, which synthesize the phytotoxin thaxtomin. The txtAB operon, responsible for thaxtomin production, can be used as a marker to identify pathogenic strains of the bacterium. Screening methods to assess scab susceptibility in breeding programs are time-consuming and can produce variable results. Management practices to control the disease vary and include crop rotation, tolerant varieties, monitoring soil pH, avoiding low soil moisture at tuber initiation, and application of soil- and/or seed-applied pesticides. There is a wide range in levels of tolerance among potato varieties. Many public research programs are committed to breeding for scab-tolerant varieties and evaluating management methods. Topics reviewed target readers focused on breeding and disease management objectives to reduce the incidence and severity of potato common scab.

Emergence of Novel Pathogenic Streptomyces Species by Site-Specific Accretion and cis-Mobilization of Pathogenicity Islands.

The main pathogenicity factor of Streptomyces species associated with the potato common scab disease is a nitrated diketopiperazine called thaxtomin A (ThxA). In Streptomyces scabiei (syn. S. scabies), which is thought to be the most ancient pathogenic Streptomyces species, the ThxA biosynthetic cluster is located within a mobile genomic island called the toxicogenic region (TR). Three attachment (att) sites further separate TR into two subregions (TR1 and TR2). TR1 contains the ThxA biosynthetic cluster and is conserved among several pathogenic Streptomyces species. However, TR2, an integrative and conjugative element, is missing in most pathogenic species. In our previous study, we demonstrated the mobilization of the whole TR element or TR2 alone between S. scabiei and nonpathogenic Streptomyces species. TR1 alone did not mobilize in these experiments. These data suggest that TR2 is required for the mobilization of TR1. Here, we show that TR2 can self mobilize to pathogenic Streptomyces species harboring only TR1 and integrate into the att site of TR1, leading to the tandem accretion of resident TR1 and incoming TR2. The incoming TR2 can further mobilize resident TR1 in cis and transfer to a new recipient cell. Our study demonstrated that TR1 is a nonautonomous cis-mobilizable element and that it can hijack TR2 recombination and conjugation machinery to excise, transfer, and integrate, leading to the dissemination of pathogenicity genes and emergence of novel pathogenic species. Additionally, comparative genomic analysis of 23 pathogenic Streptomyces isolates from ten species revealed that the composite pathogenicity island (PAI) formed by TR1 and TR2 is dynamic and various compositions of the island exist within the population of newly emerged pathogenic species, indicating the structural instability of this composite PAI.

Promiscuous Pathogenicity Islands and Phylogeny of Pathogenic Streptomyces spp.

Approximately 10 Streptomyces species cause disease on underground plant structures. The most economically important of these is potato scab, and the most studied of these pathogens is Streptomyces scabiei (syn. S. scabies). The main pathogenicity determinant of scab-causing Streptomyces species is a nitrated diketopiperazine, known as thaxtomin A (ThxA). In the pathogenic species Streptomyces turgidiscabies, ThxA biosynthetic genes reside on a mobile pathogenicity island (PAI). However, the mobilization of PAIs in other Streptomyces species remains uncharacterized. Here, we investigated the mobilization of the PAI of S. scabiei 87-22. Based on whole genome sequences, we inferred the evolutionary relationships of pathogenic Streptomyces species and discovered that Streptomyces sp. strain 96-12, a novel pathogenic species isolated from potatoes in Egypt, was phylogenetically grouped with nonpathogenic species rather than with known pathogenic species. We also found that Streptomyces sp. strain 96-12 contains a PAI that is almost identical to the PAI in S. scabiei 87-22, despite significant differences in their genome sequences. This suggested direct or indirect in vivo mobilization of the PAI between S. scabiei and nonpathogenic Streptomyces species. To test whether the S. scabiei 87-22 PAI could, indeed, be mobilized, S. scabiei 87-22 deletion mutants containing antibiotic resistance markers in the PAI were mated with Streptomyces diastatochromogenes, a nonpathogenic species. The PAI of S. scabiei was site-specifically inserted into the aviX1 gene of S. diastatochromogenes and conferred pathogenicity in radish seedling assays. Our results demonstrated that S. scabiei, the earliest described Streptomyces pathogen, could be the source of a PAI responsible for the emergence of novel pathogenic species.

Streptomyces – from Basic Microbiology to Role as a Plant Pathogen

Streptomycetes are spore-forming Gram-positive bacteria found in soil in large numbers world-wide. More than 600 Streptomyces species have been described. The complex developmental morphology and molecular genetics of Streptomyces is connected to production of a wealth of secondary products, including more than 60% of antibiotics in industrial and pharmaceutical use. Streptomycetes have important roles in soil ecology as decomposers, though specific roles in microbial community structure and plant health are poorly understood. Some species are used as biocontrol agents while others have specific associations with potatoes as endophytes, pathogens or as part of plant rhizosphere communities. Very few species are plant pathogenic (ca. 1%), causing common scab disease on underground tubers in potatoes and root diseases in a broad range of host plants. Several unique aspects of Streptomyces as a plant pathogen are that (a) there is a main dominant pathogenicity determinant (thaxtomin); (b) only the developing underground stems, stolons and tubers are susceptible to potato common scab (CS); (c) Streptomyces does not incite a plant defense response; and (d) CS is not easily managed. The best available control is the use of resistant potato cultivars and there is wide variation in resistance (tolerance) among potato cultivars, though none is completely resistant. New molecular genetic tools, including the complete genome sequences of a number of plant pathogenic Streptomyces species and association mapping using the potato genome sequence, promise greater understanding of the genetics of CS tolerance and of regulation of thaxtomin production and contributory pathogenicity factors for better management of potato CS.

A species-specific method for detecting pathogenic Streptomyces species from soil and potato tubers in Argentina

Potato common scab is caused by several soil-inhabiting pathogenic Streptomyces species. In the present study, a species-specific PCR method was used to detect Streptomyces species in potato tuber lesions and soils. Total genomic DNA from soil samples from six locations and tuber samples from four potato cultivars (Spunta, Shepody, Innovator and Russet Burbank) were assessed. Streptomyces scabies, Streptomyces acidiscabies, and Streptomyces turgidiscabies were detected in soybean, tobacco and potato soils and in all potato varieties except Russet Burbank. The phylogenetic analysis of the sequences obtained confirmed the identification. The method proposed proved to be time-saving and cost effective for the rapid detection of Streptomyces species. This is the first report of the detection of S. acidiscabies and S. turgidiscabies in soils and potato tubers from Argentina.

In Search of Better Management of Potato Common Scab

Common scab (CS) is an important disease and quality problem in potato crops worldwide. CS degrades the appearance of the potato tubers, thereby diminishing market value. Knowledge of CS has expanded considerably over recent years, enabling improved detection of the causal pathogens and increased understanding of mechanisms of pathogenicity, and providing potential methods of modulating pathogen response for disease resistance. However, effective control of this disease remains elusive, and will require increased understanding of both the host and the pathogen. Traditional control strategies such as irrigation and reduced soil pH are not sufficient and often fail. Optimizing environmental conditions for reduction of CS can also lead to favorable conditions for other diseases. The most desirable control method would be disease-resistant potato cultivars. However, no currently available commercial potato cultivar has been shown to be completely resistant to CS. In this review, we provide an overview of potato CS caused by plant pathogenic Streptomyces species, recent research on mechanisms and management of the disease, and knowledge gaps that limit successful control of this ubiquitous and troublesome disease.

Genetic and physiological determinants ofStreptomyces scabiespathogenicity

SUMMARY Common scab is a severe disease worldwide affecting tap root crops and potato tubers. It is caused by soil-borne filamentous bacteria belonging to the genus Streptomyces. Streptomycetes usually are saprophytic microorganisms, but a few species have acquired the ability to infect underground plant tissues. The predominant causal agent of potato scab worldwide is Streptomyces scabies. The production of phytotoxins called thaxtomins is essential for the virulence of common scab-causing agents. The genes involved in the biosynthetic pathway of thaxtomins and other virulence genes are clustered on a large pathogenicity island. The pathogenicity island can be mobilized and transferred to nonpathogenic relatives, leading to the emergence of new pathogenic streptomycetes. In most pathogenic Streptomyces species, thaxtomin A is the predominant form found. The regulation of thaxtomin A synthesis is complex. Although the plant-derived compound cellobiose is now recognized as the inducer of thaxtomin A synthesis at a genetic level, other molecules (including aromatic amino acids and some secondary metabolites) show inhibitory effects on the production of the toxin. This paper is an overview of common scab with a focus on S. scabies and its virulence mechanisms. Streptomyces scabies (Thaxt.) Lambert and Loria; Kingdom Bacteria; Phylum Actinobacteria; Class Actinomycetes; Order Actinomycetales; Family Streptomycetaceae; genus Streptomyces; species scabies or scabiei. Streptomyces scabies (syn. S. scabiei) has a broad host range comprising tuber vegetables and most tap root crops. Streptomyces scabies causes common scab on potato (Solanum tuberosum), beet (Beta vulgaris), carrot (Daucus carota), parsnip (Pastinaca sativa), radish (Raphanus sativus), rutabaga (Brassica napobrassica) and turnip (Brassica rapa). Disease symptoms: Common scab symptoms appear as randomly distributed shallow, raised or deep-pitted corky lesions. Their size and colour are quite variable, but lesions typically are brown with a diameter of a few millimetres. No above-ground symptoms disclose the presence of the disease as aerial tissues of scab-infected plants remain healthy. Streptomyces scabies also inhibits the growth of seedlings in monocot and dicot plants. http://www.sanger.ac.uk/Projects/S_scabies, http://www.potatodiseases.org/scab.html, http://www.uri.edu/ce/factsheets/sheets/potatoscab.html.

First Report of Potato Scab Caused by Streptomyces turgidiscabies in China.

Potato scab, caused by several plant pathogenic Streptomyces species, is known to occur in potato-planting areas worldwide. Symptoms of disease on potato tubers are shallow, raised, or pitted corky lesions (2). In 1998, Streptomyces turgidiscabies was reported as a new potato scab pathogen from Hokkaido, Japan (3). Potato scab has been observed in many potato-cultivation areas in China and incidence of the disease was approximately 6 to 10% in some fields in 2006 (in our survey). To investigate the casual agent of scab disease, isolations were made from scabby potato tubers collected from different areas using oatmeal agar. Identification of an isolate from Shaanxi Province was based on morphological and physiological characterization followed by 16S rRNA confirmation. Characteristics were gray, aerial hypha, rectiflexuous spore chains, a smooth spore surface, and spores that were 0.5 to 0.7 × 1.0 to 1.2 μm. The strain did not produce melanin on tyrosine-peptone-yeast extract agar media, did not produce diffusible pigments, used all the International Streptomyces Project (ISP) sugars (4) as single carbon sources, used l-hydroxyproline, l-tyrosine, and l-histidine as single nitrogen sources but not l-methionine, grew at pH 4.5, was susceptible to streptomycin (20 μg ml-1), phenol (0.1%), and penicillin (10 IU ml-1) but not to crystal violet (0.5 μg ml-1), and produced H2S. The identification was confirmed by comparison of its 16S rRNA sequence with the GenBank database using the BLAST program. The 16S rRNA sequence was amplified by PCR with primers S1: 5'-CATTCACGGAGAGTTTGATCC-3' and S2: 5'-AGAAAGGAGGTGATCCAGCC-3' and sequenced. BLASTn analysis of the sequence obtained showed the highest similarity (99.9%) with S. turgidiscabies type strain ATCC 700248 (GenBank Accession No. AB026221). The sequence was submitted to GenBank (Accession No. AM889495). Pathogenicity of the strain was tested in the greenhouse on potato tubers of cv. Favorita grown in pots (one plant per pot, three replicates). One hundred milliliters of inoculum (1 × 105 CFU ml-1) of the strain was mixed with sterile soil and vermiculite (1:1) in each pot. Potato plants were grown at 25°C and the soil was allowed to dry between waterings. The immature potato tubers were used to evaluate scab symptoms 10 weeks after planting. All tubers inoculated with the pathogen developed typical common scab symptoms consisting of erumpent, brown, corky lesions, which is different from the symptoms caused by S. reticuliscabiei (1). The noninoculated control tubers did not show scab symptoms. S. turgidiscabies was reisolated from lesions of diseased immature tubers. The pathogenicity test indicates that S. turgidiscabies caused scab disease on potato tubers. To our knowledge, this is the first report of S. turgidiscabies causing potato scab disease in China.

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.

Thaxtomin biosynthesis: the path to plant pathogenicity in the genus Streptomyces

Streptomyces species are best known for their ability to produce a wide array of medically and agriculturally important secondary metabolites. However, there is a growing number of species which, like Streptomyces scabies, can function as plant pathogens and cause scab disease on economically important crops such as potato. All of these species produce the phytotoxin thaxtomin, a nitrated dipeptide which inhibits cellulose synthesis in expanding plant tissue. The biosynthesis of thaxtomin involves conserved non-ribosomal peptide synthetases, P450 monooxygenases, and a nitric oxide synthase, the latter being required for nitration of the toxin. This nitric oxide synthase is also responsible for the production of diffusible nitric oxide by scab-causing streptomycetes at the host-pathogen interface, suggesting that nitric oxide production might play an additional role during the infection process. The thaxtomin biosynthetic genes are transcriptionally regulated by an AraC/XylS family regulator, TxtR, which is conserved in pathogenic streptomycetes and is encoded within the thaxtomin biosynthetic gene cluster. The TxtR protein specifically binds cellobiose, a known inducer of thaxtomin biosynthesis, and cellobiose is required for expression of the biosynthetic genes. A second virulence gene in pathogenic Streptomyces species, nec1, encodes a novel secreted protein that may suppress plant defence responses. The thaxtomin biosynthetic genes and nec1 are contained on a large mobilizable pathogenicity island; the transfer of this island to recipient streptomycetes likely explains the rapid emergence of new pathogenic species. The newly available genome sequence of S. scabies will provide further insight into the mechanisms utilized by pathogenic streptomycetes during plant-microbe interactions.

Using the TxtAB Operon to Quantify Pathogenic Streptomyces in Potato Tubers and Soil

The phytotoxin thaxtomin, produced by plant pathogenic Streptomyces species, is the only known pathogenicity determinant for common scab diseases of potato and other root and tuber crops. Genes encoding thaxtomin synthetase (txtAB) are found on a pathogenicity island characteristic of genetically diverse plant pathogenic Streptomyces species. In this study, an SYBR Green quantitative real-time polymerase chain reaction (PCR) assay using primers designed to anneal to the txtAB operon of Streptomyces was developed to quantify pathogenic bacterial populations in potatoes and soil. The real-time PCR assay was specific for pathogenic Streptomyces strains. The detection limit of the assay was 10 fg of the target DNA, or one genome equivalent. Cycle threshold (Ct) values were linearly correlated with the concentration of the target DNA (correlation coefficient R(2) = 0.99) and were not affected by the presence of plant DNA extracts, indicating the usefulness of the assay for quantitative analyses of the pathogenic bacteria in plant tissues. The amount of pathogenic Streptomyces DNA in total DNA extracts from 1 g asymptomatic and symptomatic tubers was quantified using the assay and ranged from 10(1) to 10(6) pg. A standard curve was established to quantify pathogenic Streptomyces in soil. Using the standard curve, numbers of pathogenic Streptomyces colony forming units were extrapolated to range from 10(3) to 10(6) per gram of soil from potato fields where common scab was found. This real-time PCR assay using primers designed from the txtAB operon allows rapid, accurate, and cost effective quantification of pathogenic Streptomyces strains in potato tubers and in soil.

Detection and characterization of Streptomyces causing potato common scab in Western Europe

A PCR‐based diagnostic method was developed for direct detection from tuber lesions of pathogenic Streptomyces causing common scab of potato. Primers were designed to amplify a fragment of the txtAB (txtA and txtB) genes, which are pathogenicity determinants in the main pathogenic Streptomyces species. The method was evaluated on 84 naturally infected potato samples, comprising 19 potato cultivars, harvested in the years from 2000 to 2004 in the Netherlands, the UK, France, Germany and Spain. Pathogenic Streptomyces in tuber lesions were detected by PCR in 70 samples and were also successfully isolated from these 70 samples. All pathogenic isolates showed the basic general phenotypic traits of the S. scabiei phenetic cluster. RFLP analysis of amplified rRNA sequences, together with carbon source utilization and repetitive BOX profiles, allowed most isolates to be assigned to S. europaeiscabiei, which emerged as the main cause of potato common scab in Western Europe.

A New Strain of Streptomyces Causing Common Scab in Potato.

Common scab is a serious disease of potatoes (Solanum tuberosum) and other root and tuber crops, affecting the quality and market value of these crops. The disease is caused by gram-positive soil bacteria in the genus Streptomyces. A new common scab-causing streptomycete was isolated from scabby potatoes originating in southeastern Idaho. Research has supported a model of horizontal transfer of pathogenicity determinants among streptomycetes, and the new strain has hallmarks of the recently characterized Streptomyces pathogenicity island (PAI); it has genes encoding the synthetase for the pathogenicity determinant thaxtomin and for a second pathogenicity factor, tomatinase, although it lacks a third gene characteristic of the Streptomyces PAI, the nec1 gene. The new strain has a unique 16s rDNA gene sequence closely related to those of other pathogenic Streptomyces species. This 16s rDNA sequence was also found in isolates lacking a PAI, suggesting that the new pathogenic strain arose by horizontal transfer of a PAI into a saprophytic streptomycete. Isolates of the new strain are pathogenic on radish and potato, and are more virulent than the S. scabies type strain. In addition to scab lesions on potato tubers, lesions were also seen on underground stems and stolons. This new strain represents additional complexity in the pathogenic strains causing plant disease in the United States.

Detection of the nec1 virulence gene and its correlation with pathogenicity in Streptomyces species on potato tubers and in soil using conventional and real-time PCR

To evaluate the virulence gene nec1 as a reliable marker for the detection of pathogenic Streptomyces species on potato tubers and in soil samples using conventional and real-time quantitative PCR assays. Two pairs of conventional primers (outer and nested) and one set of primers/probe for use in real-time PCR were designed to detect the necrogenic protein encoding nec1 gene of Streptomyces scabiei strain ATCC 49173(T). The conventional PCR primers were also incorporated into a multiplex PCR assay to simultaneously detect the nec1 gene in conjunction with the potato pathogens Helminthosporium solani and Colletotrichum coccodes. The specificity of each PCR assay was confirmed by testing 32 pathogenic and nonpathogenic reference strains of Streptomyces representing 12 different species and 74 uncharacterized streptomycete strains isolated from diseased tubers. A clear correlation between pathogenicity and the detection of nec1 by PCR was demonstrated. The sensitivity and specificity of both the conventional and real-time PCR assays allowed the detection of nec1 on potato tubers in the absence of visible symptoms of common scab, and in seeded soil down to a level equivalent to three S. scabiei spores per gram soil. Reliable and quantitative PCR techniques were developed in this study for the specific detection of the virulence gene nec1 of pathogenic Streptomyces species on potato tubers and in soil samples, and the data demonstrated a clear correlation between pathogenicity in Streptomyces species and the presence of the nec1 gene. Together with the DNA extraction protocols, these diagnostic methods will allow a rapid and accurate assessment of tuber and soil contamination by pathogenic Streptomyces species.