Rapid discrimination of potato scab-causing <Emphasis Type="Italic">Streptomyces</Emphasis> species based on the RNase P RNA gene sequences

Scab disease significantly damages potatoes and other root crops. Some Streptomyces species have been reported as potato-scab pathogens. Identification of the phytopathogenic Streptomyces is mainly done on the basis of the 16S rRNA gene, but use of this gene has some limitations for discriminating these strains because they share high similarities of 16S rRNA gene sequences. We tested the RNase P RNA (rnpB) gene as a taxonomic marker to clarify the relationship among closely related scab-causing Streptomyces strains. The rnpB genes were analyzed for 41 strains including 9 isolates from Jeju, Korea. There were 4 highly variable regions including nucleotide gaps in the rnpB genes. Interspecies similarity of the rnpB gene in tested Streptomyces strains was lower than about 97%, while the intraspecies similarity was higher than about 98%. Phylogenetic analysis demonstrated that the rnpB tree has similar topology to the 16S rRNA gene tree, but produces a more divergent phyletic lineage. These results revealed that the rnpB gene could be used as a powerful taxonomic tool for rapid differentiation of closely related Streptomyces species. In addition, it was also suggested that the variable regions marked as α, β, γ, and δ in the rnpB gene could be useful markers for the detection of specific Streptomyces species.     

Investigation of actinomycete diversity in the tropical rainforests of Singapore

Five thousand actinomycetes were isolated from soil samples collected from rainforests in Singapore and the generic identities of these isolates were determined by using a procedure that combined morphological, chemotaxonomic and 16S rDNA sequence-based phylogenetic analyses. Actinomycetes belonging to a total of 36 genera were identified. The most abundant isolates are members of Streptomyces, Micromonospora, Actinoplanes, Actinomadura, Nonomuria, Nocardia and Streptosporangium. By phylogenetic analysis of 16S rDNA sequences of our isolates together with those of known actinomycete species, we also evaluated the species diversity of several genera including Streptomyces, Micromonospora, Nonomuria, and Actinomadura. We found that: first, the tropical isolates are present in most clades represented by known species; and second, many tropical isolates form new clades distant from the known species, indicating the presence of unidentified taxa at both species and genus levels. Based on these results, we conclude that actinomycete diversity in the tropical rainforest is very great and should represent an excellent source for discovery of novel bioactive compounds. Received 17 March 1999/ Accepted in revised form 24 June 1999     

Presence and Diversity of <Emphasis Type="Italic">Streptomyces</Emphasis> in <Emphasis Type="Italic">Dendroctonus</Emphasis> and Sympatric Bark Beetle Galleries Across North America

Recent studies have revealed several examples of intimate associations between insects and Actinobacteria, including the Southern Pine Beetle Dendroctonus frontalis and the Spruce Beetle Dendroctonus rufipennis. Here, we surveyed Streptomyces Actinobacteria co-occurring with 10 species of Dendroctonus bark beetles across the United States, using both phylogenetic and community ecology approaches. From these 10 species, and 19 other scolytine beetles that occur in the same trees, we obtained 154 Streptomyces-like isolates and generated 16S sequences from 134 of those. Confirmed 16S sequences of Streptomyces were binned into 36 distinct strains using a threshold of 0.2% sequence divergence. The 16S rDNA phylogeny of all isolates does not correlate with the distribution of strains among beetle species, localities, or parts of the beetles or their galleries. However, we identified three Streptomyces strains occurring repeatedly on Dendroctonus beetles and in their galleries. Identity of these isolates was corroborated using a house-keeping gene sequence (efTu). These strains are not confined to a certain species of beetle, locality, or part of the beetle or their galleries. However, their role as residents in the woodboring insect niche is supported by the repeated association of their 16S and efTu from across the continent, and also having been reported in studies of other subcortical insects.     

The Biodiversity of Actinomycetes in Lake Baikal

The taxonomic analysis of 107 actinomycete strains isolated from the bottom sediments and water of Lake Baikal showed that most of the water isolates belong to the genus Streptomyces and most of the sediment isolates belong to the genus Micromonospora. In the sediments, the number of actinomycetes increased with depth (down to 200 m). Eight Streptomyces isolates were identified to a species level.     

Identification of Actinomycetes Producing Phospholipase D with High Transphosphatidylation Activity

Previously we isolated six actinomycetes strains, 9-4, 10-1, 10-2, 10-3, 10-6, and 21-4, that produce phospholipase D (PLD) with high transphosphatidylation activity. In this study, we identified these strains, and the PLD activities were compared with those of reference strains. 16S rDNA sequences and DNA–DNA hybridization tests indicated taxonomic affiliations of strain 9-6 with Streptomyces senoensis, strains 10-1 and 10-6 with S. vinaceus, and strains 10-2 and 10-3 with S. racemochromogenes. Strain 21-4, though identified as a Streptomyces sp., could not be identified with any known species. Meanwhile, most of the culture supernatants of reference strains demonstrated no or very weak PLD activity, while those of our strains exhibited significantly higher activity. All of the strains in this study were identified as Streptomyces species. The PLD activity of our strains exceeded most of the reference Streptomyces strains. The findings in this study imply that the Streptomyces strains, although they are members of the same species, can produce different quantities of PLD enzyme.     

Culturable Actinobacteria from the Marine Sponge Hymeniacidon perleve: Isolation and Phylogenetic Diversity by 16S rRNA gene-RFLP Analysis

A total of 106 actinobacteria associated with the marine sponge Hymeniacidon perleve collected from the Yellow Sea, China were isolated using eight different media. The number of species and genera of actinobacteria recovered from the different media varied significantly, underlining the importance of optimizing the isolation conditions. The phylogenetic diversity of the actinobacteria isolates was assessed using 16S rRNA gene amplification–restriction fragment length polymorphism (RFLP) analysis of the 106 strains with different morphologies. The RFLP fingerprinting of selected strains by HhaI-digestion of the 16S rRNA genes resulted in 11 different patterns. The HhaI-RFLP analysis gave good resolution for the identification of the actinobacteria isolates at the genus level. A phylogenetic analysis using 16S rRNA gene sequences revealed that the isolates belonged to seven genera of culturable actinobacteria including Actinoalloteichus, Micromonospora, Nocardia, Nocardiopsis, Pseudonocardia, Rhodococcus, and Streptomyces. The dominant genus was Streptomyces, which represented 74% of the isolates. Three of the strains identified are candidates for new species.     

大连海域沉积物中可培养海洋链霉菌活性及其多样性

以大肠杆菌、金黄色葡萄球菌和尖孢镰刀枯萎病菌作为测试靶目标,采用9种分离培养基从大连海域13个不同采样点的海洋沉积物样品中分离到165株海洋链霉菌。从165株海洋放线菌中筛选到对金黄色葡萄球菌具有抑制活性的菌株85株,占总菌株数的51.5%;对大肠杆菌具有抑制活性的菌株27株,占总菌株数的16.4%;对尖孢镰刀枯萎病菌具有抑制活性的菌株仅有6株,占总菌株数的3.6%。因此,海洋链霉菌的活性更多地表现为对细菌的抗性,尤其对革兰氏阳性细菌具有更高的抑制活性。对其中具有抑制活性或形态独特的菌株进行了16S r DNA序列分析,并构建系统发育树,显示活性海洋链霉菌具有丰富的种类多样性和广谱抗菌活性。同种海洋链霉菌与土壤链霉菌活性比较结果也表明,海洋链霉菌多表现抗革兰氏阳性细菌活性。     

Classification of novel soil streptomycetes as Streptomyces aureus sp. nov., Streptomyces laceyi sp. nov. and Streptomyces sanglieri sp. nov

The taxonomic positions of soil isolates known as Streptomyces groups A, B and C were clarified. Comparative 16S rDNA sequence studies indicated that representatives of all three taxa formed distinct phyletic lines within the Streptomyces tree though the group A strains were shown to be related to Streptomyces griseus and associated validly described species. The taxonomic integrity of all three groups was highlighted by DNA:DNA relatedness and ribotype data though the group A strains encompassed a higher degree of genetic variation than the group B and C strains. In light of these and earlier phenotypic data it is proposed that Streptomyces groups A, B and C be given species status as Streptomyces sanglieri sp. nov., Streptomyces aureus sp. nov. and Streptomyces laceyi sp. nov., respectively. This revised version was published online in June 2006 with corrections to the Cover Date.     

Studies on Xylanase from Microorganisms

As xylanase-producing microorganisms, 64 strains belonging to the genus Streptomyces were isolated from the barn-yard manures, silages and litters collected in Hokkaido district. Among these isolates the strain 102–1–4, which was found to be a new species under taxonomical studies and named Streptomyces xylophagus nov. sp., had the most outstanding ability for the enzyme production. In addition to the isolates, 38 strains of Streptomyces and 480 strains of filamentous fungi which have been preserved in our culture collection were also examined on their ability to produce the enzyme. 1) Among the strains of Streptomyces tested, only two strains, St. albogriseolus IAM 0031 and St. olivaceus IAM 0025 were found to have the ability, but their abilities were less than that of St. xylophagus nov. sp. 2) Out of 480 strains of fungi tested, Chaetomium, Schyzophyllum, Trametes, Echinodontium, Alternaria, Cepharosporium, Cercospora, Gibberella, Glomerella and Macrosporium produced the enzyme. Especially, Ch. trilateral 2264 was the most excellent.     

Biological Control of Phytophthora Root Rots on Alfalfa and Soybean with Streptomyces

A collection of 53 antibiotic-producing Streptomyces isolated from soils from Minnesota, Nebraska, and Washington were evaluated for their ability to inhibit plant pathogenic Phytophthora medicaginis and Phytophthora sojae in vitro. Eight isolates having the greatest pathogen-inhibitory capabilities were subsequently tested for their ability to control Phytophthora root rots on alfalfa and soybean in sterilized vermiculite and naturally infested field soil. The Streptomyces isolates tested significantly reduced root rot severity in alfalfa and soybean caused by P. medicaginis and P. sojae, respectively (P < 0.05). On alfalfa, isolates varied in their effect on plant disease severity, percentage dead plants, and plant biomass in the presence of the pathogen. The same eight isolates of Streptomyces were also tested for inhibitory activities against each other and against three strains of Bradyrhizobium japonicum and two strains of Sinorhizobium meliloti isolated from soybean and alfalfa, respectively. Streptomyces isolates clustered into two major compatibility groups: isolates within the same group were noninhibitory toward one another in vitro. The compatibility groups corresponded with groupings obtained based upon inhibition of B. japonicum and S. meliloti strains.     

Recovery of soil streptomycetes from arid habitats in Jordan and their potential to inhibit multi-drug resistant <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> pathogens

A total of 161 different Streptomyces isolates were recovered from 5 soil samples representing the driest habitats of Jordan. These were then characterized and assessed for their antagonistic activity against four clinical multi-drug resistant Pseudomonas aeruginosa test pathogens. Results indicated that only 3 strains out of 139 and 6 out of 22 isolated at 27°C and 45°C, respectively, were active against at least three strains of pathogenic Pseudomonas. However, three Streptomyces strains (J_2b, J₄, and J₁₂) that were isolated at 45°C inhibited all of the tested pathogens with an inhibition zone ranging between 5 and 16 mm in diameter. Data obtained from comparing the inhibition activity of these unique Streptomyces strains toward multi-resistant Pseudomonas pathogens with standard used antibiotics revealed that these isolates produce possible different inhibitory bioactive compounds other than the standard antibiotics.     

Diversity of the culturable lichen-derived actinobacteria and the taxonomy of Streptomyces parmotrematis sp. nov.

A total of 37 actinobacteria were isolated from eighteen lichen samples collected in Thailand. Based on the 16S rRNA gene sequences, they were identified into five genera including Actinoplanes (1 strain), Actinomadura (1 strain), Pseudosporangium (1 strain), Wangella (1 strain) and Streptomyces (33 strains). Among these isolates, strain Ptm05^T, Ptm01 and Ptm12 showed low 16S rRNA gene similarity and was selected for the further taxonomic study using the polyphasic approach. These strains showed the highest 16S rRNA gene sequence similarity with Streptomyces sparsogenes ATCC 25498^T (97.44–97.72%). Strain Ptm05^T was selected for the type strain. The chemical cell composition of the strain was similar to the members of Streptomyces genus. LL-diaminopimelic acids were detected in the peptidoglycan. Menaquinones were MK-9(H₈) and MK-9(H₆). Phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannoside, one unidentified phospholipid, one unidentified glycolipid and one unidentified lipid were detected as the polar lipids. The predominant cellular fatty acids are anteiso-C_15:0, iso-C_15:0, iso-C_16:0, iso-C_17:0 and C_16:0. The dDNA-DNA hybridization values among strain Ptm05^T and its closely related Streptomyces type strains were 17.2–18.0%. In addition, the ANIb and ANIm between strain Ptm05^T and related Streptomyces type strains were ranged from 75.69 to 76.13% and 85.21 to 85.35%, respectively. Based on phenotypic and genomic evidence, strain Ptm05^T (=?TBRC 14546^T?=?NBRC 115203^T) represents the novel species of the genus Streptomyces for which the name Streptomyces parmotrematis sp. nov. is proposed. This study showed that the lichens are the promising source of the novel actinobacterial taxa.

     

Rapid identification of Streptomyces isolates by MALDI-TOF MS

The recent emergence of multidrug-resistant bacteria over the last decade has led to a renewal in the discovery of new antimicrobial drugs. Streptomyces members are practically unlimited sources of new antibiotics. However, the identification of Streptomyces species is difficult and time-consuming. Therefore, there is a need for alternative methods for their rapid identification. In this study, an efficient protocol of identification using Matrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF MS) was developed and applied for the rapid identification of Streptomyces isolates from the El Kala lakes in northeastern Algeria. A collection of 48 Streptomyces isolates were used for this study. The optimized procedure allowed us to obtain specific and reproducible protein spectra for each Streptomyces isolate tested. The spectra generated were used to build a preliminary local database based on their initial 16S rRNA identification. The blind test used for the identification of 20 Streptomyces strains already available in our created database and 20 unknown Streptomyces isolates showed that all (100%) of the Streptomyces strains listed in the database were rapidly (<30 min) identified with high scores of up to 2.8. Here, for the first time we showed that MALDI-TOF MS could be used as a cost-effective tool for the rapid identification of Streptomyces isolates.     

Control of potato soft rot caused by Pectobacterium carotovorum and Pectobacterium atrosepticum by Moroccan actinobacteria isolates

Pectobacterium carotovorum and Pectobacterium atrosepticum are dreadful causal agents of potato soft rot. Actually, there are no efficient bactericides used to protect potato against Pectobacterium spp. Biological control using actinobacteria could be an interesting approach to manage this disease. Thus, two hundred actinobacteria isolated from Moroccan habitats were tested for their ability to inhibit in vitro 4 environmental Pectobacterium strains and the two reference strains (P. carotovorum CFBP 5890 and P. atrosepticum CFBP 5889). Eight percent of these isolates were active against at least one of the tested pathogens and only 2% exhibited an antimicrobial activity against all tested Pectobacterium strains. Four bioactive isolates having the greatest pathogen inhibitory capabilities and classified as belonging to the genus Streptomyces species through 16S rDNA analysis were subsequently tested for their ability to reduce in vivo soft rot symptoms on potato slices of Bintje, Yukon Gold, Russet and Norland cultivars caused by the two pathogens P. carotovorum and P. atrosepticum. This test was carried out by using biomass inoculums and culture filtrate of the isolates as treatment. Among these, strain Streptomyces sp. OE7, reduced by 65–94% symptom severity caused by the two pathogens on potato slices. Streptomyces OE7 showed a potential for controlling soft rot on potato slices and could be useful in an integrated control program against potato soft rot pathogens in the objective to reduce treatments with chemical compounds.     

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).

     

Dereplication of<Emphasis Type="Italic"> Streptomyces</Emphasis> soil isolates and detection of specific biosynthetic genes using an automated ribotyping instrument

The discrimination of distinct cultures among morphologically similar Streptomyces soil isolates (dereplication) and the detection of specific biosynthetic pathways in these strains are important steps in the selection of microorganisms to include in a natural products library. We have developed methods for analysis of actinomycetes using the RiboPrinter microbial characterization system, an automated instrument that performs ribotyping on bacterial samples. To evaluate our dereplication method, 26 Streptomyces isolates, obtained from soil samples collected in Maui, Hawaii, were ribotyped and compared with each other, using the RiboPrinter. The strains were also compared by 16S rDNA sequence analysis, MIDI fatty acid analysis, and LC-MS profiling of fermentation extracts. The RiboPrinter was able to identify closely related isolates and to discriminate between morphologically similar isolates with unique genetic, fatty acid and fermentation profiles. For the detection of biosynthetic genes, a 1,006-bp probe containing a portion of an adenylation domain of a non-ribosomal peptide synthetase (NRPS) was employed. Using this alternate probe in place of the standard ribosomal probe, the RiboPrinter was able to detect NRPS genes in several strains of Streptomyces. These results demonstrate that the RiboPrinter has multiple applications in a natural products research program.     

Characterization of Bacillus thuringiensis ser. balearica (Serotype H48) and ser. navarrensis (Serotype H50): Two Novel Serovars Isolated in Spain

The novel strains of Bacillus thuringiensis PM9 and NA69, isolated from soil samples in Spain, were classified and characterized in terms of their crystal proteins, plasmid profile, cry genes content, and their toxicological properties against several species of Lepidoptera, Coleoptera, and Diptera. Both strains share morphological and biochemical characteristics with previously described B. thuringiensis strains, although their unique H antigens identify them as two new serotypes. Two new serovar names, B. thuringiensis serovar balearica (H serotype 48) and B. thuringiensis serovar navarrensis (H serotype 50) are proposed for the type strains PM9 and NA69, respectively. Received: 22 June 1999 / Accepted: 2 August 1999     

Investigation of structure and antigenic capacities of Thermococcales cell envelopes and reclassification of "Caldococcus litoralis" Z-1301 as Thermococcus litoralis Z-1301

Fourteen strains of hyperthermophilic organotrophic anaerobic marine Archaea were isolated from shallow water and deep-sea hot vents, and four of them were characterized. These isolates, eight previously published strains, and six type strains of species of the order Thermococcales were selected for the study of cell wall components by means of thin sectioning or freeze-etching electron microscopy. The cell envelopes of most isolates were shown to consist of regularly arrayed surface protein layers, either single or double, with hexagonal lattice (p6) symmetry, as the exclusive constituents outside the cytoplasmic membrane. The S-layers studied differed in center-to-center spacing and molecular mass of the constituent protein subunits. Polyclonal antisera raised against the cells of 10 species were found to be species-specific and allowed 12 new isolates from shallow water hot vents to be identified as representatives of the species Thermococcus litoralis, Thermococcus stetteri, Thermococcus chitonophagus, and Thermococcus pacificus. Of the 7 deep-sea isolates, only 1 was identified as a T. litoralis strain. Thus, hyperthermophilic marine organotrophic isolates obtained from deep-sea hot vents showed greater diversity with regard to their S-layer proteins than shallow water isolates. Received: February 5, 1999 / Accepted: May 11, 1999     

A comparative study on the phylogenetic diversity of culturable actinobacteria isolated from five marine sponge species

A cultivation-based approach was employed to compare the culturable actinobacterial diversity associated with five marine sponge species (Craniella australiensis, Halichondria rugosa, Reniochalina sp., Sponge sp., and Stelletta tenuis). The phylogenetic affiliation of the actinobacterial isolates was assessed by 16S rDNA-RFLP analysis. A total of 181 actinobacterial strains were isolated using five different culture media (denoted as M1–M5). The type of medium exhibited significant effects on the number of actinobacteria recovered, with the highest number of isolates on M3 (63 isolates) and the lowest on M1 (12 isolates). The genera isolated were also different, with the recovery of three genera on M2 and M3, and only a single genus on M1. The number of actinobacteria isolated from the five sponge species was significantly different, with a count of 83, 36, 30, 17, and 15 isolates from S. tenuis, H. rugosa, Sponge sp., Reniochalina sp., and C. australiensis, respectively. M3 was the best isolation medium for recovery of actinobacteria from S. tenuis, H. rugosa, and Sponge sp., while no specific medium preference was observed for the recovery of actinobacteria from Reniochalina sp., and C. australiensis. The RFLP fingerprinting of 16S rDNA genes digested with HhaI revealed six different patterns, in which 16 representative 16S rDNAs were fully sequenced. Phylogenetic analysis indicated that 12 strains belong to the group Streptomyces, three strains belong to Pseudonocardia, and one strain belongs to Nocardia. Two strains C14 (from C. australiensis) and N13 (from Sponge sp.) have only 96.26% and 96.27% similarity to earlier published sequences, and are therefore potential candidates for new species. The highest diversity of three actinobacteria genera was obtained from Sponge sp., though the number of isolates was low. Two genera of actinobacteria, Streptomyces, and Pseudonocardia, were isolated from both S. tenuis and C. australiensis. Only the genus of Streptomyces was isolated from H. rugosa and Reniochalina sp. Sponge species have been demonstrated here to vary as sources of culturable actinobacterial diversity, and the methods for sampling such diversity presented may be useful for improved sampling of such diversity.     

Rapid detection and high-resolution discrimination of the genus Streptomyces based on 16S–23S rDNA spacer region and denaturing gradient gel electrophoresis

As the leading source of antibiotics, Streptomyces species are the subject of widespread investigation. Many approaches have been tried to aid in the classification of Streptomyces isolates to the genus, species, and strain levels. Genetic methods are more rapid and convenient than classification methods based on phenotypic characteristics, but a method that is universal in detecting all Streptomyces yet selective in detecting only Streptomyces is needed. The highly conserved nature of the 16S rRNA gene (16S rDNA) combined with the need to discriminate between closely related strains results in analyses of ribosomal intergenic spacer (RIS) regions being more productive than analyses of 16S rRNA genes. PCR primers were designed to amplify the RIS region as well as a sufficient length of the 16S rRNA gene to enable phylogenetic analyses of Streptomyces. Improved selectivity and specificity for the amplification of RIS sequences from Streptomyces with environmental samples was demonstrated. The use of RIS–PCR and denaturing gradient gel electrophoresis (DGGE) was shown to be a convenient means to obtain unique genetic “fingerprints” of Streptomyces cultures allowing them to be accurately identified at species, and even strain classification levels. These RIS–PCR and DGGE approaches show potential for the rapid characterization of environmental Streptomyces populations.