Internal Transcribed Spacer Sequence Similarity Research Articles

Diversity, Phylogeny and Antagonistic Activity of Fungal Endophytes Associated with Endemic Species of Cycas (Cycadales) in China.

The culture-based approach was used to characterize the fungal endophytes associated with the coralloid roots of the endemic Cycas debaoensis and Cycas fairylakea from various population sites in China. We aim to determine if the assemblages of fungal endophytes inside these endemic plant hosts are distinct and could be explored for bioprospecting. The isolation method yielded a total of 284 culturable fungal strains. Identification based on the analysis of the internal transcribed spacer (ITS) rDNA showed that they belonged to two phyla, five classes, eight orders and 22 families. At least 33 known genera and 62 different species were confirmed based on >97% ITS sequence similarity. The most frequent and observed core taxa in the two host species regardless of their population origin were Talaromyces, Penicillium, Fusarium, Pochonia and Gliocladiopsis. Seventy percent was a rare component of the fungal communities with only one or two recorded isolates. Contrary to common notions, diversity and fungal richness were significantly higher in C. debaoensis and C. fairylakea collected from a botanical garden, while the lowest was observed in C. debaoensis from a natural habitat; this provides evidence that garden management, and to a minor extent, ex-situ conservation practice, could influence fungal endophyte communities. We further selected nineteen fungal isolates and screened for their antagonistic activities via a co-cultivation approach against the phytopathogens, Diaporthe sp. and Colletotrichum sp. Among these, five isolates with high ITS similarity matches with Hypoxylon vinosupulvinatum (GD019, 99.61%), Penicillium sp. (BD022, 100%), Penicillifer diparietisporus (GD008, 99.46%), Clonostachys rogersoniana (BF024, 99.46%) and C. rosea (BF011, 99.1%), which showed exceptional antagonistic activities against the phytopathogenic fungi with a significant inhibition rate of 70–80%. Taken together, our data presented the first and most comprehensive molecular work on culturable fungal endophytes associated with the coralloid roots of cycads. Our study also demonstrated that about 5% of fungal endophytes were not detected by the high-throughput sequencing approach, implying the equal importance of a culture-dependent approach to study fungal communities of cycads. We further highlighted the potential role of endemic and rare plants to discover and isolate unique plant-associated fungal taxa with excellent biocontrol properties.

A phylogenetic perspective on the association between ants (Hymenoptera: Formicidae) and black yeasts (Ascomycota: Chaetothyriales).

The frequency and the geographical extent of symbiotic associations between ants and fungi of the order Chaetothyriales have been highlighted only recently. Using a phylogenetic approach based on seven molecular markers, we showed that ant-associated Chaetothyriales are scattered through the phylogeny of this order. There was no clustering according to geographical origin or to the taxonomy of the ant host. However, strains tended to be clustered according to the type of association with ants: strains from ant-made carton and strains from plant cavities occupied by ants ('domatia') rarely clustered together. Defining molecular operational taxonomic units (MOTUs) with an internal transcribed spacer sequence similarity cut-off of 99% revealed that a single MOTU could be composed of strains collected from various ant species and from several continents. Some ant-associated MOTUs also contained strains isolated from habitats other than ant-associated structures. Altogether, our results suggest that the degree of specialization of the interactions between ants and their fungal partners is highly variable. A better knowledge of the ecology of these interactions and a more comprehensive sampling of the fungal order are needed to elucidate the evolutionary history of mutualistic symbioses between ants and Chaetothyriales.

Marine yeast biodiversity on seaweeds in New Zealand waters

ABSTRACTLittle is known about the biodiversity of marine yeast from New Zealand. Marine yeasts were isolated from various seaweed surfaces sampled at three locations in the Wellington Region. DNA sequencing of the internal transcribed spacer (ITS) regions was conducted, and the resultant sequence data were used in isolate identification. Yeasts isolated during this investigation were not unique; 71 isolates that are frequently detected in marine and terrestrial environments worldwide were identified from a range of seaweed genera. Furthermore, high ITS sequence similarity was observed between yeasts isolated during this investigation and those from geographically distant locations. These findings may indicate that marine yeasts are ubiquitous at a global level, although evidence is insufficient as to whether yeasts demonstrate biogeographic distribution patterns. Yeasts isolated during this investigation may have ecological implications in New Zealand's marine environment. Yeast isolates identified from seaweed genera have been demonstrated to have beneficial properties and applications and their potential should be explored.

Profundae diversitas: the uncharted genetic diversity in a newly studied group of fungal root endophytes

Endophytes associated with crops have potential as beneficial inoculants in agriculture, but little is known about their genetic diversity and phylogenetic relationships. We carried out the first ever ecological and phylogenetic survey of the culturable fungal root endophytes of a wild barley species. Fungal root endophytes were isolated from 10 populations of wall barley (Hordeum murinum), and 112 taxa of fungi were identified based on internal transcribed spacer sequence similarity. We found representatives from 8 orders, 12 families and 18 genera. Within this group, only 34 isolates (30% of the total) could be confidently assigned to a species, and 23 of the isolates (21% of the total) had no significant match to anything deposited in GenBank (based on <85% sequence similarity). These results suggest a high proportion of novel fungi, with 28% not assigned to a known fungal order. This includes three endophytes that have been shown to significantly improve agronomic traits in cultivated barley. This study has, therefore, revealed a profound diversity of fungal root endophytes in a single wild relative of barley. Extrapolating from this, the study highlights the largely unknown, hugely diverse and potentially useful resource of crop wild relative endophytes.

First Report of Colletotrichum phormii Causing Anthracnose on New Zealand Flax in the United States.

Phormium colensoi Hook.f. (syn. P. cookianum), New Zealand flax, (family Xanthorrhoeaceae) is popular in ornamental landscapes in the United States because of its sturdy blade-like foliage available in diverse colors. In February 2012, the Oregon State University Plant Clinic received three potted plants of P. colensoi 'Black Adder' from a commercial nursery in Santa Cruz County, California. The margins and midribs of several leaves had brown lesions that were variable in size, and fusiform to ellipsoidal in shape. Embedded in the lesions were black acervuli without setae that exuded salmon-colored spore masses under moist conditions. Conidia were hyaline, cylindrical to fusiform, straight to slightly curved, and 22.4 to 35.2 × 4.0 to 6.4 (average 24.7 × 4.9) μm. Based on morphology, the fungus was confirmed by USDA-APHIS National Identification Services to be Colletotrichum phormii (Henn.) D.F. Farr & Rossman (2). In March 2012, the California Department of Food and Agriculture Plant Pest Diagnostic Lab received additional samples from the same nursery lot (25% disease incidence) from which a similar fungus was recovered. rDNA sequences of the internal transcribed spacer (ITS) region from the California isolate (GenBank KC122681), amplified using primers ITS1 and ITS4 (2), were 100% identical to multiple species of Colletotrichum, including C. phormii by a BLAST query (JQ948446 through JQ948453). ITS sequence similarity alone is not sufficient to address Colletotrichum taxonomy and must be used in combination with host range and morphology (1). Pathogenicity of C. phormii (isolate CDFA986) was tested on three 'Black Adder' plants, which were inoculated with 6-mm agar plugs from a 14-day-old culture grown on half strength potato dextrose agar (PDA). Leaves were wound-inoculated along the midrib using colonized plugs (4). Five leaves per plant were inoculated with C. phormii plugs and five leaves per plant were treated with uncolonized PDA agar plugs as controls. Plants were sprayed with water and incubated in plastic bags at 22°C with a 12-h photoperiod. After 48 h, the bags and caps were removed and plants were kept under the same conditions. Two weeks later, water-soaked lesions had developed on the inoculated leaves. Lesions expanded along the midrib and became fusiform in shape after 21 to 28 days. C. phormii was isolated from lesion margins of all the inoculated leaves, but not from control leaves. This experiment was repeated once with similar results. Another Colletotrichum species, C. gloeosporiodes, also occurs on Phormium spp., but differs from C. phormii in morphology and symptom expression. Subsequent nursery and landscape surveys showed that anthracnose caused by C. phormii occurs on several P. colensoi cultivars as well as on P. tenax in five California counties including Santa Cruz, Yolo, Sacramento, San Luis Obispo, and Solano. C. phormii is also reported to infect P. colensoi and P. tenax in New Zealand, Europe, the United Kingdom, Australia, and South Africa (2,3). To our knowledge, this is the first report of C. phormii causing anthracnose on Phormium in North America. This disease could impact the American nursery trade and New Zealand flax production due to crop loss and increased production costs for pest management.

Relationships between 16S-23S rRNA gene internal transcribed spacer DNA and genomic DNA similarities in the taxonomy of phototrophic bacteria

Rapid and accurate identification of microbial species is essential task in microbiology and biotechnology. In prokaryotic systematics, genomic DNA-DNA hybridization is the ultimate tool to determine genetic relationships among bacterial strains at the species level. However, a practical problem in this assay is that the experimental procedure is laborious and time-consuming. In recent years, information on the 16S-23S rRNA gene internal transcribed spacer (ITS) region has been used to classify bacterial strains at the species and intraspecies levels. It is unclear how much information on the ITS region can reflect the genome that contain it. In this study, therefore, we evaluate the quantitative relationship between ITS DNA and entire genomic DNA similarities. For this, we determined ITS sequences of several species of anoxygenic phototrophic bacteria belonging to the order Rhizobiales, and compared with DNA-DNA relatedness among these species. There was a high correlation between the two genetic markers. Based on the regression analysis of this relationship, 70% DNA-DNA relatedness corresponded to 92% ITS sequence similarity. This suggests the usefulness of the ITS sequence similarity as a criterion for determining the genospecies of the phototrophic bacteria. To avoid the effects of polymorphism bias of ITS on similarities, PCR products from all loci of ITS were used directly as genetic probes for comparison. The results of ITS DNA-DNA hybridization coincided well with those of genomic DNA-DNA relatedness. These collective data indicate that the whole ITS DNA-DNA similarity can be used as an alternative to genomic DNA-DNA similarity.

Mycobacterium litorale sp. nov., a rapidly growing mycobacterium from soil

A gram-positive, acid-fast and rapidly growing rod, designated F4(T), was isolated from a soil sample of Haikou in China. The isolate shared 98.2 % 16S rRNA gene sequence similarity with Mycobacterium monacense B9-21-178(T), 96.2 % hsp65 sequence similarity with M. monacense FI-05352 and 79.6 % 16S-23S rRNA internal transcribed spacer sequence similarity with M. monacense B9-21-178(T). DNA-DNA relatedness between the isolate and M. monacense DSM 44395(T) was 43.5 %. The morphological analysis and physiological tests also showed that the isolate differed from any strain reported to date. The mycolic acid profile and the cellular fatty acid composition were also determined. On the basis of phenotypic and chemotaxonomic characteristics and phylogenetic data, it was concluded that strain F4(T) ( = CGMCC 4.5724(T) = JCM 17423(T)) merited classification as the type strain of a novel species, for which the name Mycobacterium litorale sp. nov. is proposed.

Mycobacterium shinjukuense sp. nov., a slowly growing, non-chromogenic species isolated from human clinical specimens

Seven isolates of a slowly growing, non-chromogenic Mycobacterium species were obtained from sputum and bronchial lavage fluid samples from elderly patients in different regions of Japan. These isolates were distinguished from related non-tuberculous species by colony morphology, positive results for Tween hydrolysis, catalase at 68 °C, nitrate reductase and pyrazinamidase and negative results for semi-quantitative catalase, urease and arylsulfatase. The mycolic acid pattern obtained by HPLC revealed a single cluster of late-eluting mycolic acids similar to but different from those of Mycobacterium malmoense ATCC 29571(T). The 16S rRNA gene, 16S-23S internal transcribed spacer (ITS), rpoB and hsp65 sequences were unique in comparison with those of other mycobacteria. Comparison of 16S rRNA gene sequences showed that the isolates were most closely related to Mycobacterium tuberculosis H37Rv(T) (21 base differences in 1508 bp; 98.6 % 16S rRNA gene sequence similarity). A representative strain, GTC 2738(T), showed 91.9 % rpoB sequence similarity with Mycobacterium marinum strain M, 95 % hsp65 sequence similarity with Mycobacterium kansasii CIP 104589(T) and 81.1 % 16S-23S ITS sequence similarity with Mycobacterium gordonae ATCC 14470(T). Phylogenetic analysis of concatenated sequences of the 16S rRNA, rpoB and hsp65 genes showed that strain GTC 2738(T) was located on a distinct clade adjacent to M. tuberculosis, M. ulcerans and M. marinum, with bootstrap values of 81 %. DNA-DNA hybridization demonstrated less than 70 % reassociation with type strains of genetically related species and supported the novel species status of the isolates. On the basis of this evidence, a novel species with the name Mycobacterium shinjukuense sp. nov. is proposed. The type strain, isolated from a sputum sample, is strain GTC 2738(T)( = JCM 14233(T) = CCUG 53584(T)).

Molecular phylogenetic biodiversity assessment of arctic and boreal ectomycorrhizal Lactarius Pers. (Russulales; Basidiomycota) in Alaska, based on soil and sporocarp DNA

Despite the critical roles fungi play in the functioning of ecosystems, especially as symbionts of plants and recyclers of organic matter, their biodiversity is poorly known in high-latitude regions. In this paper, we discuss the molecular diversity of one of the most diverse and abundant groups of ectomycorrhizal fungi: the genus Lactarius Pers. We analysed internal transcribed spacer rDNA sequences from both curated sporocarp collections and soil polymerase chain reaction clone libraries sampled in the arctic tundra and boreal forests of Alaska. Our genetic diversity assessment, based on various phylogenetic methods and operational taxonomic unit (OTU) delimitations, suggests that the genus Lactarius is diverse in Alaska, with at least 43 putative phylogroups, and 24 and 38 distinct OTUs based on 95% and 97% internal transcribed spacer sequence similarity, respectively. Some OTUs were identified to known species, while others were novel, previously unsequenced groups. Non-asymptotic species accumulation curves, the disparity between observed and estimated richness, and the high number of singleton OTUs indicated that many Lactarius species remain to be found and identified in Alaska. Many Lactarius taxa show strong habitat preference to one of the three major vegetation types in the sampled regions (arctic tundra, black spruce forests, and mixed birch-aspen-white spruce forests), as supported by statistical tests of UniFrac distances and principal coordinates analyses (PCoA). Together, our data robustly demonstrate great diversity and nonrandom ecological partitioning in an important boreal ectomycorrhizal genus within a relatively small geographical region. The observed diversity of Lactarius was much higher in either type of boreal forest than in the arctic tundra, supporting the widely recognized pattern of decreasing species richness with increasing latitude.

First Report of Damping-Off of Wild Rice in California Caused by Pythium torulosum.

During 1994, damping-off of wild rice (Zizania palustris L.) in a single field in eastern Shasta County, CA resulted in near total stand failure. Since then, the disease was observed in at least 11 other fields with varying levels of stand loss. In all cases, the affected wild rice was grown as a volunteer crop following one or more years of wild rice production. Symptoms included a dark red discoloration and necrosis of the primary root followed by seedling death. When the red discoloration was limited to secondary roots, the plants often survived. Pythium torulosum, readily recovered from symptomatic roots by isolation on PARP media, was identified by morphological structures produced on grass blades in water (homothallic with smooth-walled oogonia, plerotic oospores, monoclinous antheridia, and inflated filamentous sporangia) and a 99.2% internal transcribed spacer sequence similarity of the rDNA (1). To complete Koch's postulates, inoculum of two isolates of P. torulosum grown on moistened cornmeal/sand (2%/98% [v/v]) for 3 weeks at 25°C were combined and mixed into sterilized sandy loam soil at a rate of 30 cm3 inoculum per liter of soil. Sterilized noninfested soil was used as a control treatment. Twenty wild rice seeds (cv. Franklin) were sown in each of four replicate 20-cm-diameter pots in each treatment. Plants were submerged in water and maintained in a greenhouse at 18 to 25°C. After 8 weeks, plants stands were reduced 50% in infested pots; dry weights of infected plants were reduced by 45% relative to the controls. The fungus was reisolated from symptomatic plants but not from the plants grown in noninfested soil. The experiment was repeated with similar results. To our knowledge, this is the first report of damping-off of wild rice caused by P. torulosum. Reference: (1) C. A. Levesque and W. A. M. DeCock. Mycol. Res. 108:1363, 2004.

In Most Bradyrhizobium Groups Sequence Comparison of 16S-23S rDNA Internal Transcribed Spacer Regions Corroborates DNA-DNA Hybridizations

In an extension of a previous small-scale test to assess the use of 16S-23S rDNA internal transcribed spacer (ITS) sequences for rapid grouping of bradyrhizobia, we have sequenced the ITS region of 32 isolates of Bradyrhizobium that had previously been studied using AFLP and DNA-DNA hybridizations. We also included representatives of Afipia and Rhodopseudomonas. Our results indicate that ITS sequences are very diverse among bradyrhizobia. Nevertheless, for most of the bradyrhizobia, the grouping of ITS sequences was in line with AFLP results and DNA-DNA hybridization data. Strains that have at least 95.5% ITS sequence similarity belong to the same genospecies, i.e. they have more than 60% DNA-DNA hybridization values. The ITS sequences can therefore provide a relatively fast way to guide strain identification and aid selection of the reference groups that should be included in DNA-DNA hybridization experiments for precise genotypic identification. The Bradyrhizobium strains isolated from Aeschynomene species showed a much larger diversity in ITS sequences than other bradyrhizobia, possibly as a result of lateral exchange. The above ITS sequence similarity criterion for genospecies therefore does not apply to them, but they can easily be distinguished from other Bradyrhizobium genospecies because they have a distinct tRNA(ala) gene.