rRNA Internal Transcribed Spacer Region Research Articles

Skin lesion-associated pathogens from Octopus vulgaris: first detection of Photobacterium swingsii, Lactococcus garvieae and betanodavirus.

The common octopus Octopus vulgaris Cuvier, 1798 is extremely important in fisheries and is a useful protein source in most Mediterranean countries. Here we investigated pathogens associated with skin lesions in 9 naturally deceased specimens that included both cultured and wild common octopus. Within 30 min after death, each octopus was stored at 4°C and microbiologically examined within 24 h. Bacterial colonies, cultured from swabs taken from the lesions, were examined using taxonomical and biochemical analyses. Vibrio alginolyticus and V. parahaemolyticus were only isolated from cultured animals. A conventional PCR targeting the 16S ribosomal RNA (rRNA) gene and sequencing were performed on 2 bacterial isolates that remained unidentified after taxonomical and biochemical analysis. The sequence results indicated that the bacteria had a 99% identity with Lactococcus garvieae and Photobacterium swingsii. L. garvieae was confirmed using a specific PCR based on the 16S-23S rRNA internal transcribed spacer region, while P. swingsii was confirmed by phylogenetic analyses. Although all animals examined were found to be infected by the protozoan species Aggregata octopiana localised in the intestines, it was also present in skin lesions of 2 of the animals. Betanodavirus was detected in both cultured and wild individuals by cell culture, PCR and electron microscopy. These findings are the first report of L. garvieae and betanodavirus from skin lesions of common octopus and the first identification of P. swingsii both in octopus skin lesions and in marine invertebrates in Italy.

Genotypes of ITS region of rRNA in Microcystis (Cyanobacteria) populations in Erhai Lake (China) and their correlation with eutrophication level.

Previous studies on spatiotemporal changes of Microcystis genotypes have shown that the existence and succession of dominant genotypes always occur in eutrophicated freshwater bodies. However, few studies have focused on the correlation between genotype composition and eutrophication level. In the present study, clone libraries of the internal transcribed spacer (ITS) of rrn operon were sequenced from Microcystis populations in Erhai Lake, a subtropical plateau lake in the preliminary eutrophication stage. The genotype composition of the Microcystis populations was highly variable at spatiotemporal scales, and 473 ITS genotypes were identified from the 800 ITS sequences obtained. However, no significantly dominant ITS genotypes existed in the lake. Comparison of Erhai Lake with four major lakes in China, namely, Taihu, Chaohu, Gucheng, and Shijiu Lakes, showed that the Microcystis ITS genotypes and genetic diversity were negatively correlated with eutrophication level. Extensive comparison of the Microcystis ITS genotypes from waters worldwide revealed that 440 ITS genotypes were unique to Erhai Lake, and no obvious phylogenetic correlations can be detected among the dominant genotypes from different water bodies. The high genetic diversity of the Microcystis populations in Erhai Lake may have resulted from the effect of the early stage of eutrophication.

Wickerhamiella siamensis f.a., sp. nov., an endophytic and epiphytic yeast species isolated from sugar cane leaf.

Six strains representing a novel yeast species were isolated from tissue (DMKU-SE106(T), DMKU-SE110, DMKU-SE112 and DMKU-SE132) and the external surface (DMKU-SP335 and DMKU-SP406) of sugar cane leaves collected in Thailand. On the basis of morphological, biochemical, physiological and chemotaxonomic characteristics, and sequence analysis of the D1/D2 region of the LSU rRNA gene and the internal transcribed spacer (ITS) region, the six strains were found to represent a single novel species of the genus Wickerhamiella although the formation of ascospores was not observed. The sequences of the D1/D2 region of the LSU rRNA gene and ITS region of the six strains differed from each other by 0-2 and 2-3 nt substitutions, respectively. The novel species was related most closely to Candida infanticola but with 4.5-4.6% nucleotide substitutions in the D1/D2 region of the LSU rRNA gene and 6.6-7.1% nucleotide substitutions in the ITS region. The name Wickerhamiella siamensis f.a., sp. nov. is proposed. The type strain is DMKU-SE106(T) ( =BCC 61185(T) =NBRC 109697(T) =CBS 13331(T)).

Characterization and phylogenetic diversity of Agrobacterium vitis from Serbia based on sequence analysis of 16S-23S rRNA internal transcribed spacer (ITS) region

Serious outbreaks of grapevine crown gall disease were observed in major Serbian viticultural regions during the last five years. Tumorigenic Agrobacterium vitis was identified as a causal agent by using conventional bacteriological and molecular tests. The 36 studied strains of A. vitis showed homogeneous biochemical and physiological characteristics, but were heterogeneous in their pathogenic properties, especially on tomato and sunflower. Furthermore, genetic differences related to chromosomal and plasmid DNA were observed. The Ti plasmid of 35 strains was classified as the octopine/cucumopine (O/C) type, whereas one was classified as the vitopine (V) type. The O/C strains were further divided into O/C-1 and O/C-2 groups based on PCR analysis. Moreover, the sequence analysis of the 16S-23S rRNA ITS region provided robust and precise delineation of studied strains. Although a high level of genetic diversity in A. vitis strains from Serbia was revealed by using this approach, their genotypic relatedness with the strains from other countries suggested their common origin. Also, association between the chromosomal and plasmid DNA was determined for some phylogenetic groups and clusters.

Papiliotrema siamense f.a., sp. nov., a yeast species isolated from plant leaves.

Two strains representing a novel species were isolated from the external surface of a sugar cane leaf (DMKU-SP85(T)) and tissue of a rice leaf (DMKU-RE97) collected in Thailand. On the basis of morphological, biochemical, physiological and chemotaxonomic characteristics, and sequence analysis of the D1/D2 region of the large subunit (LSU) rRNA gene and the internal transcribed spacer (ITS) region, the two strains were determined to represent a novel species of the genus Papiliotrema although sexual reproduction was not observed. The sequences of the D1/D2 region of the LSU rRNA gene and ITS region of the two strains were identical, but differed from those of the type strain of Cryptococcus nemorosus by 0.6 % nucleotide substitutions (four nucleotide substitutions out of 597 nucleotides) in the D1/D2 region of the LSU rRNA gene and 1.8 % nucleotide substitutions (nine nucleotide substitutions out of 499 nucleotides) in the ITS region. The name Papiliotrema siamense f.a., sp. nov. is proposed. The type strain is DMKU-SP85(T)( = BCC 69499(T) = CBS 13330(T)).

Isolation and molecular characterization of Rhizoctonia-like fungi associated with orchid roots in the Quadrilátero Ferrífero and Zona da Mata regions of the state of Minas Gerais, Brazil

Mycorrhizal associations can be considered required for orchids, which depend on the fungi for germination and establishment in natural conditions. Knowledge of the mycorrhizal fungi is important for programs aimed at the reintroduction, conservation and management of orchid species. The objective of this study was the molecular characterization of Rhizoctonia-like fungi from orchids in the Quadrilatero Ferrifero (Iron Quadrangle) and Zona da Mata (Forest Zone) regions of the state of Minas Gerais, Brazil. The affinities of these fungi were studied by comparing the rRNA internal transcribed spacer region with that of other isolates and sequences in GenBank. Three isolates had an affinity for Epulorhiza repens, and one was the holotypeof E. epiphytica.

Halophytophthora fluviatilissp. nov. from freshwater in Virginia

Halophytophthora fluviatilis, a novel species from inland freshwater in Virginia, is characterized and described in this study. This homothallic species produced ovoid to globose sporangia, which release zoospores directly through exit pores. It grew well in a relatively wide range of salinity from 1.8 to 19.0 parts per thousand. Sequence analysis of the rRNA internal transcribed spacer region placed this new species in the Halophytophthora sensu stricto clade. Description of this new species expanded the habitat to include geographically distinct inland freshwater ecosystems for the genus Halophytophthora, challenging the notion that this genus is marine or brackish. The need to construct a molecular-based taxonomy for the genus Halophytophthora is also discussed.

Atypical bacterial rRNA operon structure is prevalent within the Lachnospiraceae, and use of the 16S-23S rRNA internal transcribed spacer region for the rapid identification of ruminal Butyrivibrio and Pseudobutyrivibrio strains

The rumen is the fermentative forestomach of ruminant animals, and is host to a wide range of anaerobic bacteria whose primary function is to facilitate forage degradation. Butyrivibrio and Pseudobutyrivibrio are closely related proteolytic and fibrolytic genera within the family Lachnospiraceae, and are commonly isolated from the rumens of animals fed fibrous diets. The ribosomal RNA (rRNA) operon is an important phylogenetically informative locus that is present in multiple copies in bacterial genomes. Ribosomal RNA genes are typically arranged in the order 16S-23S-5S, with internal transcribed spacer (ITS) regions located between the genes. However, in the rumen bacterium, Butyrivibrio proteoclasticus B316, rRNA operons have a 16S-5S-23S rRNA gene arrangement, and analysis of bacterial genome projects revealed that this configuration was present in all publicly available complete genomes from members of the family Lachnospiraceae. The 16S-23S ITS region is commonly used to identify bacterial strains, thus we sought to determine the utility of this region from rumen Butyrivibrio and Pseudobutyrivibrio isolates for their rapid molecular identification. Polymerase chain reaction was used to amplify 16S-23S ITS regions, which were assessed for length polymorphism (ITS-LP), and restriction fragment length polymorphism (ITS-RFLP) using AluI, HaeIII and HhaI on a panel of 13 Butyrivibrio and Pseudobutyrivibrio reference strains. Cluster analysis of the resulting banding patterns revealed that while the ITS-LP method did not group the strains according to major Butyrivibrio and Pseudobutyrivibrio clades identified via 16S rRNA gene sequences, ITS-RFLP was more discriminative, and able to rapidly delineate the strains into these clades.

Novel Species Including Mycobacterium fukienense sp. Is Found from Tuberculosis Patients in Fujian Province, China, Using Phylogenetic Analysis of Mycobacterium chelonae/abscessus Complex

ObjectiveTo identify the novel species ‘Mycobacterium fukienense’ sp. nov of Mycobacterium chelonae/abscessus complex from tuberculosis patients in Fujian Province, China. MethodsFive of 27 clinical Mycobacterium isolates (Cls) were previously identified as M. chelonae/abscessus complex by sequencing the hsp65, rpoB, 16S-23S rRNA internal transcribed spacer region (its), recA and sodA house-keeping genes commonly used to describe the molecular characteristics of Mycobacterium. Clinical Mycobacterium isolates were classified according to the gene sequence using a clustering analysis program. Sequence similarity within clusters and diversity between clusters were analyzed. ResultsThe 5 isolates were identified with distinct sequences exhibiting 99.8% homology in the hsp65 gene. However, a complete lack of homology was observed among the sequences of the rpoB, 16S-23S rRNA internal transcribed spacer region (its), sodA, and recA genes as compared with the M. abscessus. Furthermore, no match for rpoB, sodA, and recA genes was identified among the published sequences. ConclusionThe novel species, Mycobacterium fukienense, is identified from tuberculosis patients in Fujian Province, China, which does not belong to any existing subspecies of M. chelonea/abscessus complex.

Use of restriction enzyme fragment length polymorphism (RFLP) of the 16S–23S rRNA internal transcribed spacer region (ITS) for identification of fish mycobacteria

PCR targeting the 16S–23S rRNA gene internally transcribed spacer (ITS) region has been proposed as a rapid and reliable method for the detection of Mycobacterium species in human clinical specimens. Because of variation in ITS sequences amongst Mycobacterium species, a single PCR amplification can be used to differentiate slowly growing and rapidly growing species within this genus. In the present study, analysis by ITS-PCR and ITS-restriction fragment length polymorphism (RFLP) was found to be a useful, simple and rapid method compared to current molecular and phenotypic techniques. The ITS was amplified from 13 reference strains and 59 fish isolated mycobacteria using a set of published PCR primers. After PCR, the banding patterns generated allowed slowly growing mycobacteria to be differentiated from all other rapidly growing species, with the exception of Mycobacterium conceptionense. HaeIII was selected as one of two restriction enzymes that, together with the knowledge about amplicon sizes, would produce an acceptable level of discrimination in the resulting RLFP patterns, especially in the rapidly growing group of mycobacteria. After digestion with Sau96I, the amplified products of most isolates of Mycobacterium fortuitum, including subtypes II and V and those 2 isolates with new patterns (220, 100bp), presented identical or very similar patterns as obtained by HaeIII digestion. All isolates of Mycobacterium marinum, Mycobacterium chelonae and Mycobacterium gordonae, whose PCR products were not digested with HaeIII, produced two well-defined fragments with the Sau96I restriction enzyme.

Specific detection and identification of [Actinobacillus] muris by PCR using primers targeting the 16S–23S rRNA internal transcribed spacer regions

[Actinobacillus] muris represents along with [Pasteurella] pneumotropica the most prevalent Pasteurellaceae species isolated from the laboratory mouse. Despite the biological and economic importance of Pasteurellaceae in relation to experimental animals, no molecular based methods for the identification of [A.] muris are available. The aim of the present investigation was to develop a PCR method allowing detection and identification of [A.] muris. In this assay, a Pasteurellaceae common forward primer based on a conserved region of the 16S rRNA gene was used in conjunction with two different reverse primers specific for [A.] muris, targeting the 16S–23S internal transcribed spacer sequences. The specificity of the assay was tested against 78 reference and clinical isolates of Pasteurellaceae, including 37 strains of [A.] muris. In addition, eight other mice associated bacterial species which could pose a diagnostic problem were included. The assay showed 100% sensitivity and 97.95% specificity. Identification of the clinical isolates was validated by ITS profiling and when necessary by 16S rRNA sequencing. This multiplex PCR represents the first molecular tool able to detect [A.] muris and may become a reliable alternative to the present diagnostic methods.

CutaneousMycobacterium shigaenseInfection in Immunocompetent Woman, China

Cutaneous<i>Mycobacterium shigaense</i>Infection in Immunocompetent Woman, China

Pierce's Disease of Grapevines in Taiwan: Isolation, Cultivation and Pathogenicity of Xylella fastidiosa

AbstractCharacteristic symptoms of Pierce's disease (PD) in grapevines (Vitis vinifera L.) were observed in 2002 in the major grape production fields of central Taiwan. Disease severity in vineyards varied, and all investigated grape cultivars were affected. Diseased tissues were collected from fields for subsequent isolation and characterization of the causal agent of the disease (Xylella fastidiosa). Koch's postulates were fulfilled by artificially inoculating two purified PD bacteria to grape cultivars Kyoho, Honey Red and Golden Muscat. The inoculated plants developed typical leaf‐scorching symptoms, and similar disease severity developed in the three cultivars from which the bacterium was readily re‐isolated, proving that the leaf scorch of grapevines in Taiwan is caused by the fastidious X. fastidiosa. This confirmed PD of grapevines is also the first report from the Asian Continent. Phylogenetic analyses were performed by comparing the 16S rRNA gene and 16S‐23S rRNA internal transcribed spacer region (16S‐23S ITS) of 12 PD strains from Taiwan with the sequences of 13 X. fastidiosa strains from different hosts and different geographical areas. Results showed that the PD strains of Taiwan were closely related to the American X. fastidiosa grape strains but not to the pear strains of Taiwan, suggesting that the X. fastidiosa grape and pear strains of Taiwan may have evolved independently from each other.

Phylogenetic diversity of indigenous soya bean bradyrhizobia from different agro-climatic regions in Myanmar

Soya bean (Glycine max Merr.) is the most important legume crop in the world and an important cash crop in Myanmar. In the present study, we characterized 43 isolates of indigenous soya bean rhizobia from Myanmar based on the sequence analysis of the bacterial 16S-23S rRNA internal transcribed spacer region. The sequence analysis confirmed that all isolates were categorized and identified as the genus Bradyrhizobium and they were conspecific with B. japonicum, B. elkanii, B. yuanmingense, and Bradyrhizobium sp. The highest genetic diversity of Bradyrhizobium strains was observed in the Shan State soya bean growing area. B. elkanii was distributed throughout the cultivation areas and was recorded as the dominant soya bean-nodulating strain in Myanmar. The present study revealed that there were two Rj-genotypes as Rj4- and non-Rj-genotypes among the collected soya bean cultivars from Myanmar. These findings indicate a high compatibility between Rj4-genotypes soya beans and nodulation type B strains in Shan State and Yangon region. In Mandalay region, the compatibility between non-Rj-genotype soya beans and nodulation type A strains was clearly investigated. This is the first report describing B. yuanmingense from nodules of soya bean and the geographical distribution of indigenous bradyrhizobia of soya bean from different regions in Myanmar.

&amp;lt;i&amp;gt;SAP&amp;lt;/i&amp;gt; Expression in &amp;lt;i&amp;gt;Candida albicans&amp;lt;/i&amp;gt; Strains Isolated from Mexican Patients with Vaginal Candidosis

To determine the frequency and expression of the ten SAP (secreted aspartyl protease) genes in a group of Candida albicans strains isolated from Mexican women suffering from vaginal candidosis, a group of 264 women (age 18 - 57 years) with vaginal infections, predisposed by diabetes mellitus or contraceptive consumption, were evaluated. C. albicans was identified using PCR to amplify the rRNA internal transcribed spacer regions ITS1 and ITS2. The presence of the SAP genes was determined using conventional PCR, and their expression levels were determined using real-time PCR after the C. albicans strains had been grown in reconstituted human vaginal epithelium (RHVE). C. albicans was identified in the samples from 50 women (18.9%). The genotyping frequencies of the SAP genes were as follows: SAP1, 94%; SAP2, 98%; SAP3, 80%; SAP4, 100%; SAP5, 100%; SAP6, 100%; SAP7, 63%; SAP8, 96%; SAP9, 70%; and SAP10, 88%. The most frequently expressed genes in the strains harboring all of the genes were SAP1, 90%; SAP2, 90%; SAP3, 90%; SAP4, 100%; SAP5, 90%; SAP6, 90%; SAP7, 100%; SAP8, 90%; SAP9, 100%; and SAP10, 100%. SAP genes were expressed in the RHVE, suggesting that the Sap proteins play an important role in the pathogenesis of infection.

Isolation and characterization of the grain mold fungiCochliobolusandAlternariaspp. from sorghum using semiselective media and DNA sequence analyses

Mold diseases, caused by fungal complexes including Alternaria, Cochliobolus, and Fusarium species, limit sorghum grain production. Media were tested by plating Fusarium thapsinum, Alternaria sp., and Curvularia lunata, individually and competitively. Dichloran chloramphenicol rose bengal (DRBC) and modified V8 juice (ModV8) agars, found to be useful, were compared with commonly used agar media, dichloran chloramphenicol peptone (DCPA) and pentachloronitrobenzene (PCNB). Radial growth, starting with mycelia or single-conidia and hyphal tips, demonstrated an effect of media. For isolation of grain fungi, DRBC and ModV8 were similar or superior to DCPA and PCNB. When seedlings were inoculated with conidia of C. lunata, Alternaria sp., F. thapsinum, or mixtures, the percentage of root infection ranged from 28% to 77%. For mixed inoculations, shoot weights, lesion lengths, and percentage of root infections were similar to F. thapsinum inoculations; most colonies recovered from roots were F. thapsinum. For Alternaria grain isolates, 5 morphological types, including Alternaria alternata, were distinguished by colony morphologies and conidial dimensions. Sequence analysis using a portion of the endo-polygalacturonase gene was able to further distinguish isolates. Cochliobolus isolates were identified morphologically as C. lunata, Curvularia sorghina, and Bipolaris sorghicola. Multiple molecular genotypes were apparent from rRNA internal transcribed spacer region sequences from Cochliobolus grain isolates.

Accuracy of protist diversity assessments: morphology compared with cloning and direct pyrosequencing of 18S rRNA genes and ITS regions using the conspicuous tintinnid ciliates as a case study

Deep-sequencing technologies are becoming nearly routine to describe microbial community composition in environmental samples. The 18S ribosomal DNA (rDNA) pyrosequencing has revealed a vast diversity of infrequent sequences, leading to the proposition of the existence of an extremely diverse microbial 'rare biosphere'. Although rare microbes no doubt exist, critical views suggest that many rare sequences may actually be artifacts. However, information about how diversity revealed by molecular methods relates to that revealed by classical morphology approaches is practically nonexistent. To address this issue, we used different approaches to assess the diversity of tintinnid ciliates, a species-rich group in which species can be easily distinguished morphologically. We studied two Mediterranean marine samples with different patterns of tintinnid diversity. We estimated tintinnid diversity in these samples employing morphological observations and both classical cloning and sequencing and pyrosequencing of two different markers, the 18S rDNA and the internal transcribed spacer (ITS) regions, applying a variety of computational approaches currently used to analyze pyrosequence reads. We found that both molecular approaches were efficient in detecting the tintinnid species observed by microscopy and revealed similar phylogenetic structures of the tintinnid community at the species level. However, depending on the method used to analyze the pyrosequencing results, we observed discrepancies with the morphology-based assessments up to several orders of magnitude. In several cases, the inferred number of operational taxonomic units (OTUs) largely exceeded the total number of tintinnid cells in the samples. Such inflation of the OTU numbers corresponded to 'rare biosphere' taxa, composed largely of artifacts. Our results suggest that a careful and rigorous analysis of pyrosequencing data sets, including data denoising and sequence clustering with well-adjusted parameters, is necessary to accurately describe microbial biodiversity using this molecular approach.

Antibiotic resistance patterns of Acinetobacter calcoaceticus–A. baumannii complex species from Colombian hospitals

IntroductionOnly automated phenotypic methods are currently used in Colombian hospitals for identifying isolates of the Acinetobacter calcoaceticus–A. baumannii complex (ACB). The phenotypical similarities in these species mean that they cannot be differentiated by manual or automated methods, thereby leading to their identification as A. baumannii, or ACB complex in clinical settings. Our objective was to identify to the species level 60 isolates, from four hospitals, evaluate their antibiotic susceptibility, and detect resistance-related genes. Methods16S–23S rRNA internal transcribed spacer (ITS) region and rpoB gene partial sequences were amplified. Resistance genes for cephalosporin, carbapenem and aminoglycoside were detected by PCR. Possible mutations in the quinolone resistance-determining region (QRDR) were evaluated. The association of ISAba-1 with blaOXA and blaADC genes was determined by PCR. Amplification products of ITS region, rpoB gene and some resistance genes were sequenced and compared using the BLAST tool. Results16S–23S rRNA ITS region and partial rpoB gene sequence analysis allowed 51isolates to be identified as A. baumannii, 8 as A. nosocomialis, and 1 isolate as A. pitti. A. baumannii isolates were highly resistant to all antibiotics tested, while the others were susceptible to ciprofloxacin and ampicillin/sulbactam. Quinolone resistance, found only in A. baumannii, was associated with mutations in the QRDR region of gyrA and parC genes. ConclusionThis is the first investigation in Colombia that has identified ACB complex species using molecular methods, and determined differences in antibiotic resistance and resistance genes among the species. It is of the highest importance to identify isolates to the species level for future resistance and epidemiology studies in our region.

First Report of Flower Anthracnose Caused by Colletotrichum karstii in White Phalaenopsis Orchids in the United States.

In October 2010, a Colletotrichum species was isolated from white Phalaenopsis flowers growing in a greenhouse in San Francisco, CA. This Phalaenopsis is a common commercial orchid hybrid generated mostly likely from Phalaenopsis amabilis and P. aphrodite. The white petals showed anthracnose-like lesions where necrotic tissue is surrounded by a ring of green tissue. The green halo tissues around the necrotic tissue contain functional chloroplasts. One-centimeter disks were cut around the necrotic sites and surface-sterilized with 95% ethanol and 0.6% sodium hypochlorite. The disks were placed on potato dextrose agar (PDA) medium to establish cultures. Pure cultures were obtained by subculturing hyphal tips onto fresh PDA plates. The generated colonies had white aerial mycelia and orange conidial mass. The color of the reverse colony varies between colorless and pale orange. Microscopic observations identified the conidia as cylindrical, straight, and rounded at both ends. In addition, the conidia were approximately 15.0 to 18.0 μm long and 5.0 to 6.5 μm in diameter. These observed morphological features suggested that these isolates possessed the same characteristics as previously described for Colletotrichum karstii, a species considered as part of the C. boninense species complex (1). Four putative independent Colletotrichum isolates were recovered (DED9596, DED9597, DED9598, and DED9599). To confirm the Colletotrichum isolates as the causative pathogen, healthy white Phalaenopsis flowers (five total) in a whole plant were sprayed with a conidial suspension (approximately 1.2 × 106 conidia/ml) of the isolates and incubated at 20°C and 100% relative humidity with cycles of 16 h light and 8 h of darkness. Approximately 1 ml of conidial suspension solution was used for each flower. The plants were watered regularly and flowers were sprayed with sterile double-distilled water daily. As negative controls, five flowers in a whole plant were sprayed with water. Fifteen to twenty days after inoculation, lesions started to form on the petals sprayed with the putative Colletotrichum isolates. All controls remained healthy. The Colletotrichum-inoculated flowers remained alive and did not die as a result of the infection. This same experiment was repeated and the same results were obtained. DNA was extracted from the necrotic regions of the petals infected by the pure cultures of the four isolates and used to sequence the 18S rRNA ITS (internal transcribed spacer) region. All four isolates gave identical ITS sequences. Analysis of the obtained representative sequences (GenBank Accession No. JQ277352) suggested that the isolated pathogen as C. karstii. Using the published ITS data for the C. boninense species complex (1), a phylogenetic tree was generated via the maximum likelihood method. This created tree places the isolates in the same group as C. karstii. This type of C. karstii infection in Phalaenopsis orchid petals was not documented in the U.S. before, although it has been reported in China and Thailand (2). To our knowledge, this is the first report of infection and green island formation caused by C. karstii on orchid flower in the United States.

Analysis of 16S–23S rRNA internal transcribed spacer regions in Pasteurellaceae isolated from laboratory rodents

The internal transcribed spacer (ITS) regions of members of Pasteurellaceae isolated from rodents, including the [Pasteurella] pneumotropica biotypes Jawetz and Heyl, [Actinobacillus] muris, “Hemophilus influenzaemurium” and Bisgaard taxon 17 were studied and their feasibility to discriminate these species was analyzed. The reference strains of all species analyzed showed unique species‐specific ITS patterns which were further present in 49 clinical isolates of [P.] pneumotropica biotypes Jawetz and Heyl and [A.] muris allowing their identification by comparison to the reference strains pattern. Sequence analysis of the amplified fragments revealed in all species, with exception of “H. influenzaemurium”, a larger ITSile+ala which contained the genes for tRNAIle(GAU) and tRNAAla(UGC) and a smaller ITSglu with the tRNAGlu(UUC) gene. “H. influenzaemurium” revealed two each of the larger and respectively the smaller ITS fragments. Both the length and the sequence of each ITS type were highly conserved within the [P.] pneumotropica biotypes Jawetz and Heyl and [A.] muris strains tested. On the contrary, ITS sequences revealed significant interspecies variations with identity levels ranging from 61.2 to 89.5% for ITSile+ala and 56.5 to 86.8% for ITSglu. Sequences regions with significant interspecies variation but highly conserved within the species were identified and might be used to design probes for the identification of rodent Pasteurellaceae to the species level.