Identification of genetic and environmental factors stimulating excision from Streptomyces scabiei chromosome of the toxicogenic region responsible for pathogenicity

The genes conferring pathogenicity in Streptomyces turgidiscabies, a pathogen causing common scab of potato, are grouped together on a pathogenicity island (PAI), which has been found to be mobile and appears to transfer and disseminate like an integrative and conjugative element (ICE). However, in Streptomyces scabiei, another common scab‐inducing species, the pathogenicity genes are clustered in two regions: the toxicogenic region (TR) and the colonization region. The S. scabiei 87.22 genome was analysed to investigate the potential mobility of the TR. Attachment sites (att), short homologous sequences that delineate ICEs, were identified at both extremities of the TR. An internal att site was also found, suggesting that the TR has a composite structure (TR1 and TR2). Thaxtomin biosynthetic genes, essential for pathogenicity, were found in TR1, whereas candidate genes with known functions in recombination, replication and conjugal transfer were found in TR2. Excision of the TR1 or TR2 subregions alone, or of the entire TR region, was observed, although the excision frequency of TR was low. However, the excision frequency was considerably increased in the presence of either mitomycin C or Streptomyces coelicolor cells. A composite TR structure was not observed in all S. scabiei and Streptomyces acidiscabies strains tested. Of the ten strains analysed, seven lacked TR2 and no TR excision event could be detected in these strains, thus suggesting the implication of TR2 in the mobilization of S. scabiei TR.     

Effect of carbohydrates on the production of thaxtomin A by <Emphasis Type="Italic">Streptomyces acidiscabies</Emphasis>

Several Streptomyces species cause plant diseases, including S. scabies, S. acidiscabies and S. turgidiscabies, which produce common scab of potato and similar diseases of root crops. These species produce thaxtomins, dipeptide phytotoxins that are responsible for disease symptoms. Thaxtomins are produced in vivo on diseased potato tissue and in vitro in oat-based culture media, but the regulation of thaxtomin biosynthesis is not understood. S. acidiscabies was grown in a variety of media to assess the impact of medium components on thaxtomin A (ThxA) production. ThxA biosynthesis was not correlated with bacterial biomass, nor was it stimulated by α-solanine or α-chaconine, the two most prevalent potato glycoalkaloids. ThxA production was stimulated by oat bran broth, even after exhaustive extraction, suggesting that specific carbohydrates may influence ThxA biosynthesis. Oat bran contains high levels of xylans and glucans, and both of these carbohydrates, as well as xylans from wheat and tamarind, stimulated ThxA production, but not to the same extent as oat bran. Starches and simple sugars did not induce ThxA production. The data indicate that complex carbohydrates may act as environmental signals to plant pathogenic Streptomyces, allowing production of thaxtomin and enabling bacteria to colonize its host.     

Antimycin A-producing nonphytopathogenic Streptomyces turgidiscabies from potato

Aim:  To detect if substances with mammalian cell toxicity are produced by Streptomyces turgidiscabies and Streptomyces scabiei isolated from potato scab lesions. Methods and Results:  In vitro cultures of phytopathogenic and nonphytopathogenic strains of S. scabiei and S. turgidiscabies, isolated from scab lesions of potato tubers originating from nine different cultivars from Finland and Sweden, were tested for toxicity using the rapid spermatozoan motility inhibition assay, previously shown useful in the detection of many different Streptomyces toxins and antimicrobial compounds. Purified toxins were used as reference. Three nonphytopathogenic strains of S. turgidiscabies were found to produce antimycin A when cultured on solid medium. Conclusions:  Boar sperm-motility-inhibiting substances are produced by strains of S. turgidiscabies and S. scabiei. The most powerful inhibitory substance, produced by three nonphytopathogenic S. turgidiscabies strains, was identified as antimycin A. The phytotoxic compounds thaxtomin A and concanamycin A did not inhibit sperm motility even at high doses. Significance and Impact of the Study:  The presence of antimycin A-producing Streptomyces strains, nonpathogenic to potato, was unexpected but important, considering the high mammalian toxicity of this cytochrome bc-blocking antibiotic.     

What does it take to be a plant pathogen: genomic insights from <Emphasis Type="Italic">Streptomyces</Emphasis> species

Plant pathogenicity is rare in the genus Streptomyces, with only a dozen or so species possessing this trait out of the more than 900 species described. Nevertheless, such species have had a significant impact on agricultural economies throughout the world due to their ability to cause important crop diseases such as potato common scab, which is characterized by lesions that form on the potato tuber surface. All pathogenic species that cause common scab produce a family of phytotoxins called the thaxtomins, which function as cellulose synthesis inhibitors. In addition, the nec1 and tomA genes are conserved in several pathogenic streptomycetes, the former of which is predicted to function in the suppression of plant defense responses. Streptomyces scabies is the oldest plant pathogen described and has a world-wide distribution, whereas species such as S. turgidiscabies and S. acidiscabies are believed to be newly emergent pathogens found in more limited geographical locations. The genome sequence of S. scabies 87-22 was recently completed, and comparative genomic analyses with other sequenced microbial pathogens have revealed the presence of additional genes that may play a role in plant pathogenicity, an idea that is supported by functional analysis of one such putative virulence locus. In addition, the availability of multiple genome sequences for both pathogenic and nonpathogenic streptomycetes has provided an opportunity for comparative genomic analyses to identify the Streptomyces pathogenome. Such genomic analyses will contribute to the fundamental understanding of the mechanisms and evolution of plant pathogenicity and plant-microbe biology within this genus.     

A large, mobile pathogenicity island confers plant pathogenicity on Streptomyces species

Potato scab is a globally important disease caused by polyphyletic plant pathogenic Streptomyces species. Streptomyces acidiscabies, Streptomyces scabies and Streptomyces turgidiscabies possess a conserved biosynthetic pathway for the nitrated dipeptide phytotoxin thaxtomin. These pathogens also possess the nec1 gene which encodes a necrogenic protein that is an independent virulence factor. In this article we describe a large (325-660 kb) pathogenicity island (PAI) conserved among these three plant pathogenic Streptomyces species. A partial DNA sequence of this PAI revealed the thaxtomin biosynthetic pathway, nec1, a putative tomatinase gene, and many mobile genetic elements. In addition, the PAI from S. turgidiscabies contains a plant fasciation (fas) operon homologous to and colinear with the fas operon in the plant pathogen Rhodococcus fascians. The PAI was mobilized during mating from S. turgidiscabies to the non-pathogens Streptomyces coelicolor and Streptomyces diastatochromogenes on a 660 kb DNA element and integrated site-specifically into a putative integral membrane lipid kinase. Acquisition of the PAI conferred a pathogenic phenotype on S. diastatochromogenes but not on S. coelicolor. This PAI is the first to be described in a Gram-positive plant pathogenic bacterium and is responsible for the emergence of new plant pathogenic Streptomyces species in agricultural systems.     

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 FS70^T (=?PPPPB BD 2226^T?=?LMG 32103^T) 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).

     

Production of thaxtomin a by two species ofStreptomyces causing potato scab

A total of nine isolates of streptomycetes were isolated from scab lesions on potato tubers. Five of nine isolates were pathogenic on potato minitubers. Four of the pathogenic isolates produced thaxtomin A (ThxA) in infected tuber tissues. The lesion surface areas inducing ThxA were highest in treatment of the minitubers with an extract of OMB inoculated with S-66 and S-67, intermediate with that inoculated with S-64 and lowest with S-63. The pathogenic isolates were identified by gray aerial mycelia, melanin pigment productivity, the type of spore chain morphology and carbon utilization asS. scabies strains S-63, S-64 and S-68, andS. acidiscabies strains S-66 and S-67. Strains S-63 and S-64 produced 0.65 and 1.60 mg ThxA per L of OMB, respectively, strains S-66 and S-67 producing similar amounts,viz. 2.36 and 2.10 mg/L, respectively. The optimal temperature for production (by both species) was 28 °C. Production of ThxA byS. scabies strain S-64 andS. acidiscabies strain S-66 was suppressed at least 50-fold at 0.5 and 0.3 % of glucose, respectively. Fructose enhanced the production by both species.     

Rapid and Specific Method for Evaluating Streptomyces Competitive Dynamics in Complex Soil Communities

Quantifying target microbial populations in complex communities remains a barrier to studying species interactions in soil environments. Quantitative PCR (qPCR) assays were developed for quantifying pathogenic Streptomyces scabiei and antibiotic-producing Streptomyces lavendulae strains in complex soil communities. This assay will be useful for evaluating the competitive dynamics of streptomycetes in soil.Streptomyces spp. are ubiquitous soil bacteria that are noted for their capacity to produce a vast array of bioactive compounds, including antibiotics (10). Antibiotic-mediated species interactions are believed to be important to Streptomyces fitness and plant disease biocontrol in soil, and yet quantitative data on Streptomyces interactions in soil are limited. Moreover, because the impacts of one species on another can be mediated through interactions with other microbes in the community, detecting these impacts requires a sensitive and accurate method for quantifying the target populations within a complex community. Here, we describe a sensitive and specific assay that targets a short hypervariable region of the 16S rRNA gene to distinguish among Streptomyces organisms in complex soil communities. Streptomyces strains DL93 (Streptomyces lavendulae, an antibiotic producer that is effective in plant disease biocontrol [9]) and DL87 (Streptomyces scabiei, a plant pathogen) were studied in the present work. This approach has significant potential to shed light on the diversity and complexity of Streptomyces species interactions in soil.     

Thyme and suico essential oils: promising natural tools for potato common scab control

• Potato common scab is a worldwide disease mainly caused by Streptomyces scabiei. It seriously affects potato crops by decreasing tuber quality. Essential oils (EO) are natural products with recognised antimicrobial properties. In this research, the antibacterial activities of thyme, oregano, suico and mint EO against S. scabiei were analysed.
• Infected tubers and soil samples were used for bacterial isolation; the obtained isolates were genetically identified. The chemical composition of the EO was determined by GC‐MS. The broth microdilution method was used to analyse antibacterial properties of EO.
• Thirty‐one bacterial isolates were obtained. The isolate chosen for antibacterial assays was morpho‐physiologically and genetically identified as S. scabiei. Thyme EO was mainly composed of thymol and o‐cymene; suico EO of dihydrotagetone, trans‐tagetone and verbenone; oregano EO of trans‐sabinene hydrate, thymol and ?‐terpinene; and mint EO of menthone and menthol. All the EO tested were effective against S. scabiei, but thyme and suico EO were the most successful, with a minimum inhibitory concentration of 0.068 g·l^?1 and 0.147 g·l^?1, respectively, and a minimum bactericidal concentration of 0.137 g·l^?1 and 0.147 g·l^?1, respectively. Scanning electron microscopy showed similar damage caused by both thyme and suico EO to the bacterial envelope. Total phenolic content of EO was not related to their antibacterial activity.
• Thyme and suico EO are effective antibacterial agents against S. scabiei, impeding bacterial viability and disturbing the bacterial cell envelope. These EO are promising tools for control of potato common scab.

     

Multiple transporters are involved in natamycin efflux in Streptomyces chattanoogensis L10

Antibiotic‐producing microorganisms have evolved several self‐resistance mechanisms to prevent auto‐toxicity. Overexpression of specific transporters to improve the efflux of toxic antibiotics has been found one of the most important and intrinsic resistance strategies used by many Streptomyces strains. In this work, two ATP‐binding cassette (ABC) transporter‐encoding genes located in the natamycin biosynthetic gene cluster, scnA and scnB, were identified as the primary exporter genes for natamycin efflux in Streptomyces chattanoogensis L10. Two other transporters located outside the cluster, a major facilitator superfamily transporter Mfs1 and an ABC transporter NepI/II were found to play a complementary role in natamycin efflux. ScnA/ScnB and Mfs1 also participate in exporting the immediate precursor of natamycin, 4,5‐de‐epoxynatamycin, which is more toxic to S. chattanoogensis L10 than natamycin. As the major complementary exporter for natamycin efflux, Mfs1 is up‐regulated in response to intracellular accumulation of natamycin and 4,5‐de‐epoxynatamycin, suggesting a key role in the stress response for self‐resistance. This article discusses a novel antibiotic‐related efflux and response system in Streptomyces, as well as a self‐resistance mechanism in antibiotic‐producing strains.     

Development of a Genotyping Method for Potato Scab Pathogens Based on Multiplex PCR

Scab disease significantly damages potato and other root crops. Streptomyces scabiei, S. acidiscabiei, and S. turgidiscabiei are the best-known causal agents of this disease. We have developed a novel genotyping method for these potato scab pathogens using multiplex PCR, whose benefits include rapid and easy detection of multiple species. We designed a species-specific primer set (6 primers, 3 pairs) for the 16S rRNA genes and 16S–23S ITS regions of these potato scab pathogens. The specificity of the primer set was confirmed by testing 18 strains containing potato scab pathogens, other Streptomyces species, and strains of other genera. The application of the developed method to potato field soil and potato tissue samples resulted in the clear detection and identification of pathogens. Since this method is applicable to a large number of environmental samples, it is expected to be useful for a high-throughput analysis of soil and plant tissues of scab disease.     

Granaticins and their biosynthetic gene cluster from <Emphasis Type="Italic">Streptomyces vietnamensis</Emphasis>: evidence of horizontal gene transfer

Streptomyces vietnamensis, a recently designated species isolated from tropical forest soil, was found to be a new granaticin producer. The granaticin biosynthetic gene cluster (gra) and flanking genes from S. vietnamensis were cloned and sequenced by a sequential cloning strategy. All biosynthetic genes were found as expected. The high overall homology of the gra cluster from S. vietnamensis to that of Streptomyces violaceoruber Tü22 indicated a recent common ancestor of the two clusters. However, a flanking gene orf35 was missing from the gra cluster of S. vietnamensis, and high frequency of insertions and deletions of short fragment (shorter than 63 bp) were observed throughout the sequenced region compared to that of S. violaceoruber Tü22. These revealed a rapid evolution of the gra cluster and suggested that small insertions and deletions might be one of the basic evolution mechanisms for streptomycete genomes. The phylogenetic incongruence between 16S rDNA and the gra cluster and the scattered distribution of the granaticin producers within Streptomyces implicated horizontal gene transfer (HGT) being involved in the gra cluster dispersion. The remnants of orf35 found in S. vietnamensis present a scenario on how the antibiotic gene clusters evolved after HGT. The contemporary gra cluster residing in S. vietnamensis could be interpreted as a combination of HGT and highly variable vertical transmission.     

1‐Phenyl‐3‐pentanone,a Volatile Compound from the Edible Mushroom Mycoleptodonoides aitchisonii Active Against Some Phytopathogenic Fungi

Volatiles produced by mycelia of mushrooms with aromatic odour were investigated for their antifungal activity against plant‐pathogenic fungi. The results of the screening of 23 species of basidiomycetes revealed that volatile substances from mycelia of Mycoleptodonoides aitchisonii (TUFC10099), an edible mushroom, strongly inhibited the mycelial growth, spore germination and lesion formation on host leaves of some plant‐pathogenic fungi including Alternaria alternata, A. brassicicola, A. brassicae, Colletotrichum orbiculare and Corynespora cassiicola. The volatile compounds were isolated from the culture filtrate of M. aitchisonii, and 1‐phenyl‐3‐pentanone was identified as a major antifungal volatile. The compound had significantly inhibitory activity against plant‐pathogenic fungi at 35 ppm. This is the first report that the volatile compound produced by mycelia of M. aitchisonii has antifungal activity against plant‐pathogenic fungi.     

Ectophoma iranica sp. nov. and new hosts and records of Allophoma spp. in Iran

A survey was conducted to identify fungi associated with leaf diseases of ornamental herbs, including Christ plant, dracaena, rose geranium, rose periwinkle and treasure flower, in Ahvaz, southwestern Iran. Twenty‐five symptomatic herbs were collected and studied, which led to the isolation of seven Phoma‐like strains belonging to four taxa. These fungi were characterised based on DNA sequence data for the partial large subunit 28S nrDNA (LSU‐D1/D2), the internal transcribed spacer 1 and 2 and 5.8S nrDNA (ITS) and part of the β‐tubulin (tub2) gene regions. A multi‐locus‐based phylogeny, in combination with morphology, allowed for the identification of Allophoma hayatii, A. labilis, A. tropica and a novel species of the genus Ectophoma. Ectophoma iranica sp. nov. is morphologically and genetically distinctive from previously described species. In pathogenicity tests, the two strains of E. iranica (CBS 144681 and IRAN 3354C) caused leaf spot symptoms on leaves of Catharanthus roseus. New host plant species and records for A. hayatii, A. labilis and A. tropica are reported, plus further emended characterization of A. hayatii.     

A novel docking domain interface model predicting recombination between homoeologous modular biosynthetic gene clusters

An in silico model for homoeologous recombination between gene clusters encoding modular polyketide synthases (PKS) or non-ribosomal peptide synthetases (NRPS) was developed. This model was used to analyze recombination between 12 PKS clusters from Streptomyces species and related genera to predict if new clusters might give rise to new products. In many cases, there were only a limited number of recombination sites (about 13 per cluster pair), suggesting that recombination may pose constraints on the evolution of PKS clusters. Most recombination events occurred between pairs of ketosynthase (KS) domains, allowing the biosynthetic outcome of the recombinant modules to be predicted. About 30% of recombinants were predicted to produce polyketides. Four NRPS clusters from Streptomyces strains were also used for in silico recombination. They yielded a comparable number of recombinants to PKS clusters, but the adenylation (A) domains contained the largest proportion of recombination events; this might be a mechanism for producing new substrate specificities. The extreme G + C-content, the presence of linear chromosomes and plasmids, as well as the lack of a mutSL-mismatch repair system should favor production of recombinants in Streptomyces species.     

Cloning and characterization of a gene cluster from Streptomyces cyanogenus S136 probably involved in landomycin biosynthesis

From a cosmid library of Streptomyces cyanogenus S136, DNA fragments encompassing approximately 35 kb of the presumed landomycin biosynthetic gene cluster were identified and sequenced, revealing 32 open reading frames most of which could be assigned through data base comparison.     

Streptomyces associated with a marine sponge Haliclona sp.; biosynthetic genes for secondary metabolites and products

Terrestrial actinobacteria have served as a primary source of bioactive compounds; however, a rapid decrease in the discovery of new compounds strongly necessitates new investigational approaches. One approach is the screening of actinobacteria from marine habitats, especially the members of the genus Streptomyces. Presence of this genus in a marine sponge, Haliclona sp., was investigated using culture‐dependent and ‐independent techniques. 16S rRNA gene clone library analysis showed the presence of diverse Streptomyces in the sponge sample. In addition to the dominant genus Streptomyces, members of six different genera were isolated using four different media. Five phylogenetically new strains, each representing a novel species in the genus Streptomyces were also isolated. Polyphasic study suggesting the classification of two of these strains as novel species is presented. Searching the strains for the production of novel compounds and the presence of biosynthetic genes for secondary metabolites revealed seven novel compounds and biosynthetic genes with unique sequences. In these compounds, JBIR‐43 exhibited cytotoxic activity against cancer cell lines. JBIR‐34 and ‐35 were particularly interesting because of their unique chemical skeleton. To our knowledge, this is the first comprehensive study detailing the isolation of actinobacteria from a marine sponge and novel secondary metabolites from these strains.     

Identification of the New Pathogen (Stemphylium lycopersici) Causing Leaf Spot on Pepino (Solanum muricatum)

Pepino (Solanum muricatum var. pepino) plants were found affected by an extensive leaf spot caused by plant pathogenic fungi during a survey in the Cameron highlands, Pahang state, Malaysia. Symptomatic leaf samples were collected from infected pepino plants and cultivated on PDA medium, and the pathogen was isolated and purified; then, consequently, all isolates were identified as Stemphylium lycopersici on the basis of their cultural and morphological characteristics and combined sequences of the internal transcribed spacer (ITS) and glyceraldehyde‐3‐phosphate dehydrogenase (gpd) regions. A pathogenicity assay on detached leaves further confirmed that S. lycopersici causes leaf spot disease. To the best of our knowledge, this is the first report of S. lycopersici causing leaf spot on pepino in Malaysia and worldwide.