Detection of the ectomycorrhizal fungusTricholoma matsutake and some related species with specific ITS primers

Oligonucleotide primers (Tm1 and Tm4) were designed to amplify a 447–448 base pair fragment, comprising sections of the rDNA internal transcribed spacers (ITS) and the entire 5.8S rDNA, ofTricholoma matsutake. PCR products of predicted size were produced for six of eight isolates ofT. matsutake from across its natural range in Asia, and for isolates of some closely related fungi includingT. bakamatsutake, T. magnivelare, andT. caligatum. The closely relatedT. robustum could be discriminated fromT. matsutake by PCR fragment size. No PCR products were produced where the primers were tested against 16 species of ectomycorrhizal fungi associated withPinus spp. in the Southern Hemisphere. The specific primers were also used successfully to produce PCR products from matsutake infected roots collected in natural forests in China and Japan, and from pure culture synthesisedPinus radiata-T. matsutake material. These primers will be useful in research directed at establishing matsutake in the Southern Hemisphere, and also have the potential to be applied to the study of matsutake within its natural range.     

Molecular phylogeny in Indian Tinospora species by DNA based molecular markers

The phylogenetic relationships among the three species of Tinospora found in India are poorly understood. Morphology does not fully help to resolve the phylogeny and therefore a fast approach using molecular analysis was explored. Two molecular approaches viz Random Amplified Polymorphic DNA (RAPD) assay and restriction digestion of ITS1-5.8S-ITS2 rDNA (PCR-RFLP) were used to evaluate the genetic similarities between 40 different accessions belonging to three species. Of the 38 random primers used only six generated the polymorphism, while as three out of 11 restriction enzymes used gave polymorphic restriction patterns. The average proportion of polymorphic markers across primers was 95%, however restriction endonucleases showed 92% polymorphism. RAPD alone was found suitable for the species diversions. In contrast PCR- RFLP showed bias in detecting exact species variation. The correlation between the two markers was performed by Jaccard's coefficient of similarity. A significant (r= 0.574) but not very high correlation was obtained. Further to authenticate the results obtained by two markers, sequence analysis of ITS region of ribosomal DNA (ITS1 and ITS2, including 5.8S rDNA) was performed. Three independent clones of each species T. cordifolia, T. malabarica and T. crispa were sequenced. Phylogenetic relationship inferred from ITS sequences is in agreement with RAPD data.     

ITS polymorphism within a single strain ofSclerotium rolfsii

Two morphologically distinct strains, 63–76 and 63H1, were isolated from a protoplast and a hyphal tip of the parentalSclerotium rolfsii strain S-63, respectively. Strains 63–76 and 63H1 showed reduced mycelial growth and lacked clamp connections on hyphae. The two strains also differed from each other and from their parent in RAPD patterns generated by several primers, suggesting that 63–76 and 63H1 were homokaryons isolated from the hetereokaryon S-63. Whereas the parent S-63 belonged to ITS-RFLP group 1, RFLP patterns of internal transcribed spacer (ITS) regions of rDNA of 63–76 and 63H1 were similar to those of ITS-RFLP groups 5 and 3, respectively. The sequence similarity of ITS regions were more than 99% between 63–76 and group 5 strains, 100% between 63H1 and the group 3 strain, and 96.3% between 63–76 and 63H1. Direct sequencing failed in the parental strain S-63. S-63 was considered to contain ITS types of groups 5 and 3.     

Identification of species in the genus Pichia by restriction of the internal transcribed spacers (ITS1 and ITS2) and the 5.8S ribosomal DNA gene

In this study, the variability within the ribosomal DNA region spanning the internal transcribed spacers ITS1 and ITS2 and the 5.8S gene (5.8S-ITS rDNA) was used to differentiate species in the genus Pichia. The 5.8S-ITS rDNA region was PCR-amplified and the PCR product digested with the enzymes CfoI, HinfI, and HaeIII. The variability in the size of the amplified product and in the restriction patterns enabled differentiation between species in the genus Pichia, and between Pichia species and yeast species from other genera in the Yeast-id database (). Moreover, the restriction fragment length polymorphism (RFLP) patterns of the 5.8S-ITS enabled misidentified strains to be detected and revealed genetic heterogeneity between strains within the Pichia membranifaciens and Pichia nakazawae species. Ultimately, the RFLP patterns of the 5.8S-ITS rDNA failed to differentiate between some Pichia and Candida species that could be distinguished on the basis of the sequence of the 5.8S-ITS rRNA region or the sequence of the D1/D2 domain of the 26S rDNA gene.     

Molecular phylogeny of ectomycorrhizal <Emphasis Type="Italic">Pisolithus</Emphasis> fungi associated with pine,dipterocarp, and eucalyptus trees in Thailand

The phylogenetic relationships among 135 Pisolithus basidiomes and two isolates collected from three pine forests, a pine-dipterocarp forest, two dipterocarp forests, and 29 eucalyptus plantations in Thailand were investigated. Internal transcribed spacer (ITS) polymorphism analyses, including terminal RFLP, divided them into 26 groups. The ITS in a representative basidiome of each group was sequenced, and a phylogenetic analysis was performed. The dendrogram suggested that at least three Pisolithus species are present in Thailand. Pisolithus basidiomes collected in the pine forests and under some Shorea roxburghii trees in a pine-dipterocarp forest corresponded to species 5 as previously described by Martin et al. in 2002. Those collected under S. roxburghii and Dipterocarp alatus trees in the dipterocarp forests did not match any previously reported species. Basidiomes collected from the eucalyptus plantations were all identified as Pisolithus albus.     

An ustilaginomycetous yeast,Pseudozyma graminicola sp. nov., isolated from the leaves of pasture plants

Two strains belonging to a novel anamorphic species, Pseudozyma graminicola, were isolated from the leaves of herbaceous plants in the Moscow region (Russia). This species was genetically distinct from all known Pseudozyma species, based on sequence divergence in the D1/D2 domains of the large subunit rDNA and the ITS region. It is related phylogenetically to species of the genus Sporisorium (Ustilaginaceae, Ustilaginales). Physiological characteristics distinguishing this novel species from the other species of the genus Pseudozyma are presented.     

Close sequence identity between ribosomal DNA episomes of the non-pathogenicEntamoeba dispar and pathogenicEntamoeba histolytica

Entamoeba dispar andEntamoeba histolytica are now recognized as two distinct species-the former being nonpathogenic to humans. We had earlier studied the organization of ribosomal RNA genes inE. histolytica. Here we report the analysis of ribosomal RNA genes inE. dispar. The rRNA genes ofE. dispar, like their counterpart inE. histolytica are located on a circular rDNA molecule. From restriction map analysis, the size ofE. dispar rDNA circle was estimated to be 24·4 kb. The size was also confirmed by linearizing the circle withBsaHI, and by limited DNAseI digestion. The restriction map of theE. dispar rDNA circle showed close similarity to EhR1, the rDNA circle ofE. histolytica strain HM-1:IMSS which has two rDNA units per circle. The various families of short tandem repeats found in the upstream and downstream intergenic spacers (IGS) of EhR1 were also present inE. dispar. Partial sequencing of the cloned fragments ofE. dispar rDNA and comparison with EhR1 revealed only 2·6% to 3·8% sequence divergence in the IGS. The region Tr and the adjoiningPvuI repeats in the IGS of EhR1, which are missing in thoseE. histolytica strains that have one rDNA unit per circle, were present in theE. dispar rDNA circle. Such close similarity in the overall organization and sequence of the IGS of rDNAs of two different species is uncommon. In fact the spacer sequences were only slightly more divergent than the 18S rRNA gene sequence which differs by 1·6% in the two species. The most divergent sequence betweenE. histolytica andE. dispar was the internal transcribed spacer, ITS2. Therefore, it was concluded that probes derived from the ITS1 and ITS 2 sequences would be more reliable and reproducible than probes from the IGS regions used earlier for identifying these species.     

Productivity of hydrolytic enzymes by mycorrhizal mushrooms

To survey the potential for production of extracellular hydrolytic enzymes by mycorrhizal mushrooms, productivities of these exo-enzymes from mycelia on potato-dextrose liquid medium were determined.Tricholoma matsutake produced relatively high levels of CM-cellulase and avicelase activities in all test strains. It also produced higher activity of acid proteinase than neutral proteinase. Its xylanase activities seemed to be higher than those of the other carbohydrases. The productivities ofLyophyllum shimeji strains were at similar levels to those ofT. matsutake strains. CM-cellulase and avicelase activities ofL. shimeji were higher than those ofT. matsutake. Neutral proteinase inL. shimeji strains showed higher activity levels than acid proteinase. The relative productivities of hydrolytic enzymes between the groups of mycorrhizal mushrooms and wood-rotting mushrooms were also examined.T. matsutake andL. shimeji both produce many kinds of hydrolytic enzymes in their culture broth, and the potential for production of hydrolytic enzymes by mycorrhizal mushrooms was judged to be relatively high.     

Characterization of the carbohydrase productions of an ectomycorrhizal fungus, Tricholoma matsutake

To evaluate the potential of the production of the ectomycorrhizal fungus Tricholoma matsutake to produce carbohydrases, (1) the distribution of carbohydrase activities among the different strains (18 strains) was investigated and (2) the abilities of T. matsutake and saprophytic fungi to produce β-glucosidase were compared. The results showed that the carbohydrase productions patterns of T. matsutake still resemble one another. Moreover, this fungus exhibited markedly higher β-glucosidase than did the saprophytic mushrooms. Tricholoma matsutake showed weak production of α-amylase and α-glucosidase in a static cultur filtrate. On the other hand, glucoamylase activity was not observed. Surprisingly, we discovered that β-glucosidase demonstrated strong activity. This finding suggests that this fungus has saprotrophic abilities. The carbohydrase production systems in T. matsutake were characterized from our experimental results. Also, we point out some weak points in the carbohydrase production systems of T. matsutake.     

Reproductive compatibility and genetic variation between two strains of Aphelinus albipodus (Hymenoptera: Aphelinidae), a parasitoid of the soybean aphid, Aphis glycines (Homoptera: Aphididae)

Aphelinus albipodus Hayat and Fatima is a potential biological control agent of the soybean aphid, Aphis glycines Matsumura, which is a newly introduced soybean pest in the United States. We compared the reproductive compatibility and molecular genetic variation between two geographic strains of A. albipodus. One strain was collected from soybean aphids in Japan and the other recovered from Russian wheat aphid, Diuraphis noxia (Mordvilko), in the western U.S., populations of which were established with parasitoids imported from Eurasia. We present results of crossing experiments between the two strains, genetic differences based on RAPD-PCR markers, rDNA ITS1 and ITS2 gene sequences, and presence of Wolbachia in the two strains using PCR amplification of the wsp gene. We found no reduction in the production of females in reciprocal crosses between strains, but a significant reduction in fecundity when F₁ females stemming from one of the reciprocal crosses were backcrossed to males from either source. The two strains differed by 3.4% in the rDNA ITS1 sequence and by presence/absence of one RAPD-PCR marker from a total of 20 RAPD primers screened, but their rDNA ITS2 sequences were identical. We used restriction enzyme analysis to separate the strains by differential digestion of the ITS1 PCR product. Wolbachia was present in 100% of males and females of both strains of A. albipodus.     

Intra- and inter-specific variations in the copy number of two types of retrotransposons from the ectomycorrhizal basidiomycete Tricholoma matsutake

To explore intra- and inter-specific variations of the ectomycorrhizal basidiomycete Tricholoma matsutake that produces the fruit body matsutake, we carried out real-time PCR analysis based on two types of retrotransposons, one designated marY1, which resembles a retrovirus carrying the long terminal repeat (LTR) and the other marY2N, which resembles mRNA carrying the polyadenylated tail. Calculation based on the average genome size of homobasidiomycetes (34 Mbp) shows that ca. 5.5% of the total genome of T. matsutake isolated from Asia is made up of these retrotransposons, whereas they occupy ca. 1.4% in the isolates from Morocco, ca. 0.8% in isolates from Mexico, and ca. 0.5% in Tricholoma magnivelare, the species which produces American matsutake. Other Tricholoma spp. that produce fruit bodies similar to those of T. matsutake, such as T. bakamatsutake, T. fulvocastaneum, and T. robustum, carry them in the region less than 0.05% of their total genome. Copy number of LTR of marY1 is consistently and markedly higher than that of the coding regions of marY1 and marY2N. Data suggest that retrotransposons are deeply involved in evolution of the ectomycorrhizal symbiont.     

Discrimination of <Emphasis Type="Italic">Tricholoma</Emphasis> species by species-specific ITS primers

The ITS region of ectomycorrhizal fungi was analyzed, and species-specific PCR primers were designed for 8 ectomycorrhizal Tricholoma species. Although a high degree of intraspecific homology was observed, interspecific variation was sufficient to design species-specific primers based on sequence of the ITS region. PCR amplification with the specific primers generated fragments of the expected sizes from DNA extracted from the strains of each species but gave no amplified products from the strains of the other 16 species in eight genera. These results suggest that sequence of the ITS region is appropriate to be used for species-level identification of ectomycorrhizal fungi.     

Intra-isolate heterogeneity of the ITS region of rDNA in Pythium helicoides

Heterogeneity of the rDNA ITS region in Pythium helicoides and the phylogenetic relationship between P. helicoides and closely related species were investigated. In PCR-RFLP analysis of the rDNA ITS region of six P. helicoides isolates investigated, including the type culture, intraspecific variation was found at the HhaI site. The total length of fragments was longer than before cutting, indicating sequence heterogeneity within isolates. Digestion of the cloned rDNA ITS region derived from seven isolates with HhaI revealed polymorphisms among and within single zoospore isolates, and variability of the region was also present among the clones derived from the same isolate. To test whether the rDNA ITS region of closely related species and other regions in the genome of P. helicoides are also variable, the rDNA ITS region of P. ultimum and the cytochrome oxydase II (cox II) gene encoded in mitochondria were sequenced. P. ultimum had little variation in the rDNA ITS region. The cox II gene sequences of both species revealed only a low intraspecific variability and no intra-isolate variation. In the phylogenic tree based on the rDNA ITS sequences, all clones of P. helicoides formed one large clade that was distinct from the clades comprising morphologically similar species, such as P. oedochilum and P. ostracodes, and was closely related to P. chamaehyphon rather than the other species.     

Molecular characterization and evaluation of mycorrhizal capacity of Suillus isolates from Central Spain for the selection of fungal inoculants

Suillus fungal specimens of pine forests from a Mediterranean area of central Spain (Madrid region) were studied based on molecular and physiological analysis of sporocarps to obtain fungal native inocula to produce mycorrhizal Pinus halepensis Miller in nursery. Variation within the internal transcribed spacer (ITS) region of the ribosomal RNA genes of Suillus was examined by restriction fragment length polymorphism (RFLP) and direct sequencing of polymerase chain reaction products. Ribosomal DNA (rDNA) spacers were amplified from pure cultures obtained from fruit bodies of a range of Suillus species: Suillus bellinii (Inzenga) Watling, Suillus bovinus (Pers.) Kuntze, Suillus collinitus (Fr.) Kuntze, Suillus granulatus (L.) Snell, Suillus mediterraneensis (Jacquet. & Blum) Redeuil, Suillus luteus L. (Gray), and Suillus variegatus (Sw.) Kuntze. Interspecific variation in the length and number of restriction sites of the amplified ITS region was observed. This variation was confirmed by sequencing, which allowed us to identify some isolates. This is the first time that the ITS sequence of S. mediterraneensis is completely described. No intraspecific rDNA variation was observed within isolates of S. collinitus, S. mediterraneensis, and S. luteus. The phylogenetic analysis established the close relationship among these Mediterranean fungal species. As a further step to characterize the different isolates and to understand the relation between genetic and functional diversity, some physiological variables were evaluated. Intraspecific variation in axenic fungal growth and in mycorrhizal capacities was detected, especially within S. collinitus isolates. The fungal isolates stimulated the growth of P. halepensis in different rates. These studies indicated that ITS analysis, in conjunction with mycorrhizal tests, provides suitable combined tools for the analysis of Suillus spp. in a small geographic area for selecting isolates with final afforestation purposes.     

Isolation ofTricholoma matsutake andT. bakamatsutake cultures from field-collected ectomycorrhizas

Tricholoma matsutake was isolated into pure cultures from field samples of ectomycorrhizas onPinus densiflora. The mycorrhizal tips were collected at different times of the year from a colony ofT. matsutake in aP. densiflora stand. The mycorrhizal tips were continuously washed with sterilized distilled water and diluted Tween 80 solution, surface-sterilized with calcium hypochlorite solution, and inoculated on several kinds of nutrient agar media. Most of the mycorrhizal tips collected in winter and spring produced colonies that were morphologically similar to cultures ofT. matsutake isolated from basidiocarps. The identity of isolates obtained from mycorrhizas was further confirmed to beT. matsutake based on fungal morphology and RFLP patterns of PCR amplified rDNA. The feasibility ofT. bakamatsutake isolation into pure culture from ectomycorrhizas onQuercus serrata was also confirmed. These results indicated that mycelium of matsutake mushrooms can be isolated into pure culture from ectomycorrhizas at different times of the year. Mycorrhizas of bothT. matsutake andT. bakamatsutake were not observed to have any specific association with soil fungi such asMortierella spp.     

The β-tubulin gene as a molecular phylogenetic marker for classification and discrimination of the <Emphasis Type="Italic">Saccharomyces</Emphasis> <Emphasis Type="Italic">sensu stricto</Emphasis> complex

The Saccharomyces sensu stricto complex comprises seven very closely related species. In this study, we compared the use of two different phylogenetic markers, the 26S rDNA and β-tubulin genes, for discriminating phylogenetic relationships among Saccharomyces sensu stricto strains using sequencing as well as RFLP methods. The average sequence similarity for the β-tubulin gene (90.0%) among seven strains was significantly less than that for 26S rDNA (98.6%). This result demonstrates that β-tubulin gene sequences provided higher resolution than 26S rDNA sequences. Species-specific restriction profiles of the Saccharomyces strains were obtained by cutting them with the Tsp509I enzyme. Our data indicate that phylogenetic relationships between these strains are best resolved using sequencing or RFLP analysis of the β-tubulin gene. The β-tubulin gene sequence data reported in this paper appear in the GenBank nucleotide sequence database with the following accession numbers: FJ238316–FJ238341.     

Polymorphism in the internal transcribed spacer (ITS) region of the ribosomal DNA among different Fusarium species

Fusarium species causing wilt diseases in different plants were characterised by comparing nonpathogenic and different pathogenic species using rDNA RFLP analysis. The ITS (internal transcribed spacer) region of 12 isolates belonging to the section Elegans, Laseola, Mortiella, Discolor, Gibbosum, Lateritium and Sporotrichiella were amplified by universal ITS primers (ITS-1 and ITS-4) using polymerase chain reaction (PCR). Amplified products, which ranged from 522 to 565 bp were obtained from all 12 Fusarium isolates. The amplified products were digested with seven restriction enzymes, and restriction fragment length polymorphism (RFLP) patterns were analysed. A dendrogram derived from PCR-RFLP analysis of the rDNA region divided the Fusarium isolates into three major groups. Assessment of molecular variability based on rDNA RFLP clearly indicated that Fusarium species are heterogeneous and most of the forma speciales have close evolutionary relationships.     

Natural Diversity of Nodular Microsymbionts of Alnus glutinosa in the Tormes River Basin

The genetic diversity of Frankia strains nodulating Alnus glutinosa along the basin of the Tormes River was studied on DNA extracted directly from nodules. Frankia strains inhabiting root nodules at 12 different locations, ranging in altitude from 409 to 1181 m, were characterized. For that, we amplified the whole IGS region between 16S–23S rDNA and performed a restriction fragment length polymorphism (RFLP) analysis with four restriction enzymes. Two different RFLP patterns (termed A and B) were obtained with HaeIII, indicating the existence of two different groups of Frankia strains. Three different nodule extracts from each of the two RFLP groups were selected for further analyses. Sequencing of the 16S–23S rDNA IGS showed a 100% of intragroup homology and also confirmed the difference (98.4% level of similarity) between the Frankia strains in the two nodule extract groups. The phylogenetic analyses based on the two 16S–23S rDNA IGS sequences obtained in this study and other previously published sequences indicated that Frankia strains TFAg5 and TFAg23 (chosen as representative of HaeIII–RFLP group A and B, respectively) are quite similar to other strains nodulating plants of A. rhombifolia and A. viridis in California (pairwise levels of similarity including gaps ranged from 97.8% to 98.6%), together with which they form a single group. To put the Frankia strains representative of each HaeIII–RFLP group in the context of overall Frankia diversity we amplified and sequenced the 16S rDNA and glnII gene from nodular DNA. An also remarkable fact found in this study was that Frankia strains belonging to the HaeIII–RFLP group A were distributed all along the river course, from the lowest site sampled to the highest, while Frankia strains placed into RFLP group B were restricted to the upper Tormes River, being exclusively found at altitudes of 946 m or higher.     

Internal Transcribed Spacer (ITS) of rDNA of appendaged and non-appendaged strains of Microsporum gypseum reveals Microsporum appendiculatum as its synonym

Recently a new taxon of geophilic dermatophytes was established as Microsporum appendiculatum Bhat and Mariam, based on the presence of appendaged macroconidia. However, such appendages are already known in the related species Microsporum gypseum. We conducted a survey of soil in central India as a part of a microbial biodiversity project and obtained two strains of M. gypseum with appendaged macroconidia. Using phenotypical characterization in combination with sequencing and restriction fragment length polymorphism (RFLP) of the Internal Transcribed Spacer (ITS) region of rDNA, we found that all strains of appendaged species are identical. Therefore M. appendiculatum is regarded as a synonym of M. gypseum.