Internal Transcribed Spacer Restriction Fragment Length Polymorphism Research Articles

Up-regulation of CDR1 and MDR1 efflux pump genes and fluconazole resistance are involved in recurrence in Candida albicans-induced vulvovaginal candidiasis

Recurrent vulvovaginal candidiasis (RVVC) due to fluconazole resistance in Candida albicans isolates causes a wide range of complications. A number of 63 Candida albicans isolates obtained from vulvovaginal candidiasis (VVC) were identified by Internal Transcribed Spacer-Restriction Fragment Length Polymorphism (ITS-RFLP). Antifungal susceptibility testing was performed by broth microdilution method according to the CLSI protocol. The role of CDR1 and MDR1 genes in progress of VVC to RVVC was examined and the activity of virulence-related enzymes was assessed. Candida albicans was diagnosed in 62.4 % cases, of which 22.2 % were confirmed as RVVC. Voriconazole was the most active drug among five tested antifungals. The mean expression level of CDR1 and MDR1 was higher in RVVC isolates compared to multidrug azole-resistant VVC isolates. Our results demonstrated that the expression of CDR1 and MDR1 and the level of phospholipase and proteinase activities could be quite important to induce fluconazole resistance in C. albicans and to progress of VVC to become RVVC in involved patients.

Diversity and technological characterization of Pediococcus pentosaceus strains isolated from Nigerian traditional fermented foods

The diversity and technological properties of Pediococcus pentosaceus strains, isolated from some traditional fermented foods in Nigeria, were studied with the aim of selecting suitable strains as a starter and/or adjunct cultures. Eighteen lactic acid bacteria isolates from ogi, gari and fufu were presumptively identified as Pediococcus species using the conventional methods, these isolates were screened for technological and safety properties such as exopolysaccharide production, acidifying activity, antimicrobial activity, and biogenic amine production. The LAB isolates were further differentiated by using internal transcribed spacer-restriction fragment length polymorphism (ITS-RFLP), randomly amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) and pulsed-field gel electrophoresis (PFGE) molecular typing techniques. The Pediococcus sp. displayed antimicrobial activities against Bacillus cereus, Staphylococcus aureus, Listeria monocytogenes, and Enterococcus faecium. They all had high acidifying activities and were able to reduce the pH to lower than 5.20 after 24 h. Pediococcus pentosaceus OF31 showed interesting potential technological characteristics due to its antimicrobial activity and exopolysaccharide production. The combination of RAPD-PCR (using primers OPA20 and M13) and PFGE-SfiI genotypic fingerprinting technique allows successful intraspecific differentiation. The present study confirmed the presence of exopolysaccharide-producing strain of P. pentosaceus in indigenous fermented foods which could be used as autochthonous starter culture to impart more functional attributes to fermented foods.

Breeding and fruiting evaluation of new strains by the interspecific mating of the high commercial potential mushroom Pleurotus tuoliensis with its closely related P. eryngii

Pleurotus tuoliensis (Bailinggu) is a precious mushroom species cultivated industrially in eastern Asia. The long cropping cycle, high deformity rate, and low yield are the major bottlenecks for its widespread cultivation. There is thus a need for improved P. tuoliensis cultivars. In the present study, interspecific crossing of monokaryons between P. tuoliensis and P. eryngii was carried out. Six out of nine pairings were observed to result in clamp connections. Dikaryotized regions within a mating Petri dish varied considerably with the compatible pairings. A total of nine hybrids were isolated within the junction zone and on the periphery at either sides of the paired colonies from three selected crosses. The internal transcribed spacer (ITS) restriction fragment length polymorphism (ITS-RFLP) profiles, together with ITS sequencing revealed significant genetic differences between the nine selected hybrids with parental strains. The polymerase chain reaction (PCR) amplification revealed that the inheritance of mitochondrial DNA was uniparental between these two species. Of nine crossbred strains, six were selected for fruiting test. Compared with their parental P. tuoliensis strains, all the six tested hybrids fruited faster, and three hybrids showed improved biological efficiency compared to their parents, either P. tuoliensis or P. eryngii. Two hybrids (Ht-26 and He-26) formed a white-colored fruit body, with improved biological efficiency (62.5 %–64.2 %) compared to parents P. tuoliensis (41.8 %) and P. eryngii (52.1 %), and thus hold great potential as new cultivars for the commercial production of P. tuoliensis.

Polyphasic analysis reveals correlation between phenotypic and genotypic analysis in soybean bradyrhizobia (Bradyrhizobium spp.)

Soybean bradyrhizobia (Bradyrhizobium spp.) are bacteria that fix atmospheric nitrogen within the root nodules of soybean, a crop critical for meeting global nutritional protein demand. Members of this group differ in symbiotic effectiveness, and historically both phenotypic and genotypic approaches have been used to assess bradyrhizobial diversity. However, agreement between various approaches of assessment is poorly known. A collection (n=382) of soybean bradyrhizobia (Bradyrhizobium japonicum, B. diazoefficiens, and B. elkanii) were characterized by Internal Transcribed Spacer – Restriction Fragment Length Polymorphism (ITS-RFLP), cellular fatty acid composition (fatty acid methyl esters, FAME), and serological reactions to assess agreement between phenotypic and genotypic methods. Overall, 76% of the accessions demonstrated identical clustering with each of these techniques. FAME was able to identify all 382 accessions, whereas 14% were non-reactive serologically. One ITS-RFLP group, containing 36 Delaware isolates, produced multiple ITS amplicons indicating they possess multiple ribosomal RNA (rrn) operons. Cloning and sequencing revealed that these strains contained as many as three heterogenous rrn operons, a trait previously unknown in bradyrhizobia. A representative subset of 96 isolates was further characterized using 16S rRNA and Internal Transcribed Spacer (ITS) amplicon sequencing. ITS sequences showed better inter- and intra-species discrimination (65–99% identity) than 16S sequences (96–99% identity). This study shows that phenotypic and genotypic approaches are strongly correlated at the species level but should be approached with caution. We also suggest using combined 16S and ITS genotyping data to obtain better inter- and intra-species resolution in bradyrhizobia classification.

Effects of protoplast fusion on the antifungal activity of Trichoderma strains and their molecular characterisation

The main purposes of this study were to examine the effects of protoplast fusion on chitinase production, antifungal activity and major antifungal compounds in parental strains and fusants and to investigate them using the internal transcribed spacer-restriction fragment length polymorphism (ITS-RFLP) method. The chitinase levels of eight fusants named Fu1-Fu8, formed by protoplast fusion between endophytic Trichoderma harzianum strains, were compared. Antifungal activities of the eight fusants were tested against Rhizoctonia solani, Sclerotinia sclerotiorum and Aspergillus flavus. Pathogens were significantly controlled by the fusant Fu3. Isolation and purification of the compounds from the fusant Fu3 and parental strains were done based on the preparative thin layer chromatography method. After an antifungal activity assessment of isolated fractions, Fr5 and Fr2Fr2 fractions from fusant Fu3 and parental strains were injected into liquid chromatography-mass spectrometry (LC-MS). Harzianopyridone and chrysophanic acid compounds were identified in the Fr2Fr2 and Fr5 fractions which showed the greatest effect on S. sclerotiorum, R. solani and A. flavus, respectively.

Morphological and Molecular Characterization of Globodera Populations from Oregon and Idaho

An unusual population of cyst nematode was found in soils collected from a Powell Butte, OR field with a cropping history including potato, wheat, other crops, and significant weed presence. These nematodes could not be placed with certainty into any known species and exhibited some unique morphological features in some specimens. Compared with Globodera pallida, the cyst body length was slightly longer and the second-stage juvenile stylet length was slightly shorter. In some individuals, the J2 stylet knob height was greater and the tail annules were more prominent than in G. pallida, and the tail abruptly narrowed, with a slight constriction near the posterior third of the hyaline terminus. Compared with G. rostochiensis, the hyaline tail terminus had a larger number of refractive bodies, and cysts of this population had a smaller Granek's ratio and fewer cuticular ridges between the anus and vulva. In some individuals, the tail termini of second-stage juveniles were more bluntly pointed, and the stylet knobs were more anteriorly directed with greater height. Unlike G. tabacum, the cyst wall often lacked a network-like pattern and, in some individuals, the juvenile tail terminus distinctly narrowed after a constriction. Molecularly, the population was distinct from G. pallida, G. rostochiensis, and G. tabacum. Multiplex polymerase chain reaction of the internal transcribed spacer (ITS) rDNA region gave results similar to G. tabacum; however, ITS restriction fragment length polymorphism patterns were observed to have individual bands in common with G. rostochiensis and G. pallida. Phylogenetic analysis based on ITS1 and -2 rDNA sequences showed greatest similarity to populations from Argentina and Chile; together, they form a moderately supported clade, distinct from G. rostochiensis, G. tabacum, G. "mexicana," European type G. pallida, and several G. pallida populations from South America.

Determination of genetic diversity among Indian isolates of Rhizoctonia bataticola causing dry root rot of chickpea

Genetic diversity among 27 isolates (23 from chickpea and 4 from other host crops) of Rhizoctonia bataticola representing 11 different states of India was determined by random amplified polymorphic DNA (RAPD), internal transcribed spacer restriction fragment length polymorphism (ITSRFLP) and ITS sequencing. The isolates showed variability in virulence test. Unweighted paired group method with arithmetic average cluster analysis was used to group the isolates into distinct clusters. The clusters generated by RAPD grouped all the isolates into six categories at 40% genetic similarity. High level of diversity was observed among the isolates of different as well as same state. Some of the RAPD (OPN 4, OPN 12, and OPN 20) markers clearly distinguished majority of the isolates into the area specific groups. The ITS I, 5.8 rDNA and ITS II regions of 11 isolates representing different RAPD groups were amplified with primers ITS 1 and ITS 4 and digested with seven restriction enzymes. The restriction enzymes DraI, MboI, RsaI, and AluI were found to be suitable for differentiating the isolates into five categories by showing isolate specific ITS-RFLP patterns. The isolates were variable in their nucleotide sequences of the ITS regions. This is the first study on genetic diversity among chickpea isolates of R. bataticola.

Diversity of root-associated fungal endophytes in Rhododendron fortunei in subtropical forests of China

To investigate the diversity of root endophytes in Rhododendron fortunei, fungal strains were isolated from the hair roots of plants from four habitats in subtropical forests of China. In total, 220 slow-growing fungal isolates were isolated from the hair roots of R. fortunei. The isolates were initially grouped into 17 types based on the results of internal transcribed spacer-restriction fragment length polymorphism (ITS-RFLP) analysis. ITS sequences were obtained for representative isolates from each RFLP type and compared phylogenetically with known sequences of ericoid mycorrhizal endophytes and selected ascomycetes or basidiomycetes. Based on phylogenetic analysis of the ITS sequences in GenBank, 15 RFLP types were confirmed as ascomycetes, and two as basidiomycetes; nine of these were shown to be ericoid mycorrhizal endophytes in experimental cultures. The only common endophytes of R. fortunei were identified as Oidiodendron maius at four sites, although the isolation frequency (3-65%) differed sharply according to habitat. Phialocephala fortinii strains were isolated most abundantly from two habitats which related to the more acidic soil and pine mixed forests. A number of less common mycorrhizal RFLP types were isolated from R. fortunei at three, two, or one of the sites. Most of these appeared to have strong affinities for some unidentified root endophytes from Ericaceae hosts in Australian forests. We concluded that the endophyte population isolated from R. fortunei is composed mainly of ascomycete, as well as a few basidiomycete strains. In addition, one basidiomycete strain was confirmed as a putative ericoid mycorrhizal fungus.

First Report of the European Type of Bursaphelenchus mucronatus on Korean Pine (Pinus koraiensis) in Korea.

More than 70 species of Bursaphelenchus have been reported and they are morphologically very similar. The pine wood nematode (Bursaphelenchus xylophilus) is a causal agent of pine wilt and accurate identification is essential for diagnosis of the disease. However, many other saprophytic nematodes are found in dead trees and some of them have high morphological similarity to B. xylophilus. B. mucronatus is commonly found in dead pine trees and is distinguished from B. xylophilus only by the presence of a distinct mucro in the female tail (3). However, natural populations of B. xylophilus show variation in tail morphology and some of them have a small lump on the end of the tail that looks like a short mucro, which could lead to misidentification. B. mucronatus has been reported to have two genotypes known as the East Asian type and the European type (2). The European type has a more rounded tail with a mucro, which is much more similar to B. xylophilus than the East Asian type. Molecular identification by internal transcribed spacer restriction fragment length polymorphism (ITS-RFLP) can distinguish these two genotypes clearly by using five different restriction enzymes (2). In Korea, we collected wood samples from dead Pinus koraiensis located at Pocheon, Gyeonggi Province in April, 2007 and nematodes were extracted by a modified Baermann funnel method. After 24 h, nematodes were collected and morphology was observed under an inverted light microscope (Leica DE/DMI 3000B, Wetzlar, Germany). Morphology was characterized by a typical Aphelenchoid-type esophagus, head constriction, female vulva flap, post uterine sac, female tail with mucro, and males with arched-shaped spicules. The tail was rounded with a long mucro >4 μm. For molecular diagnosis, DNA was extracted from single individual nematodes by using a DNeasy Kit (Qiagen, Valencia, CA) and the ITS 1 and 2 and 5.8S regions in rDNA were amplified by PCR (US/PTC-0220; Bio-Rad, Hercules, CA). ITS-RFLP patterns were analyzed and nucleotide sequences were compared with sequences of B. mucronatus already reported in GenBank (Accession Nos. AM179514, BMU93554, and DQ841162). The ITS-RFLP pattern of individuals matched the European type of B. mucronatus and was differentiated from the East Asian type by RsaI and HaeIII digestion (2). The ITS DNA sequence of nematodes from Korean pines was >98% identical to B. mucronatus in GenBank. The European type of B. mucronatus was consistently isolated from dead trees, but the pathogenicity of the European type of B. mucronatus on Korean pine is not yet clear. To our knowledge, this is the first report of the European type of B. mucronatus species on P. koraiensis in Korea, but it is already distributed in certain locations of Japan (4), China, and Europe (1).

Molecular typing of Australian Scedosporium isolates showing genetic variability and numerous S. aurantiacum.

One hundred clinical isolates from a prospective nationwide study of scedosporiosis in Australia (2003-2005) and 46 additional isolates were genotyped by internal transcribed spacer-restriction fragment length polymorphism (ITS-RFLP) analysis, ITS sequencing, and M13 PCR fingerprinting. ITS-RFLP and PCR fingerprinting identified 3 distinct genetic groups. The first group corresponded to Scedosporium prolificans (n = 83), and the other 2 comprised isolates previously identified as S. apiospermum: one of these corresponded to S. apiospermum (n = 33) and the other to the newly described species S. aurantiacum (n = 30). Intraspecies variation was highest for S. apiospermum (58%), followed by S. prolificans (45%) and S. aurantiacum (28%) as determined by PCR fingerprinting. ITS sequence variation of 2.2% was observed among S. apiospermum isolates. No correlation was found between genotype of strains and their geographic origin, body site from which they were cultured, or colonization versus invasive disease. Twelve S. prolificans isolates from 2 suspected case clusters were examined by amplified fragment length polymorphism analysis. No specific clusters were confirmed.

Fungal Genetic Diversity within Decomposing Woody Conifer Roots in Oregon, U.S.A

Internal Transcribed Spacer - Restriction Fragment Length Polymorphism (ITS-RFLP) and sequencing techniques were used to compare similarity of fungi decomposing roots of Picea sitchensis (Sitka spruce), Pinus contorta (lodgepole pine), Pinus ponderosa (ponderosa pine), Pseudotsuga menziesii (Douglas-fir), and Tsuga heterophylla (western hemlock). Fungal DNA from hyphae growing on coarse roots, cultures isolated from roots, herbarium specimens, culture collections, and field collected samples was extracted, amplified, and digested with endonucleases. Over two hundred ITS-RFLP patterns from fungal samples were separated. Linearly increasing ITS-RFLP sampling intensity curves indicate high total genetic diversity of fungi. Nei and Li similarity index analysis showed similarities of fungi on roots as measured by ITS-RFLP patterns samples ranged from 0 to 1 (i.e., 0% to 100% similar) depending upon the root species, location, and harvest stand age. Sequences were obtained for the most common ITS-RFLP patterns. Results indicate that zygomycete fungi were most prominent in decomposing woody conifer roots and that the similarity of fungi was low between tree species and locations.

Water transfer via ectomycorrhizal fungal hyphae to conifer seedlings

Little is known about water transfer via mycorrhizal hyphae to plants, despite its potential importance in seedling establishment and plant community development, especially in arid environments. Therefore, this process was investigated in the study reported in this paper in laboratory-based tripartite mesocosms containing the shrub Arctostaphylos viscida (manzanita) and young seedlings of sugar pine (Pinus lambertiana) and Douglas-fir (Pseudotsuga menziesii). The objectives were to determine whether water could be transported through mycorrhizal symbionts shared by establishing conifers and A. viscida and to compare the results obtained using two tracers: the stable isotope deuterium and the dye lucifer yellow carbohydrazide. Water containing the tracers was added to the central compartment containing single manzanita shrubs. The fungal hyphae were then collected as well as plant roots from coniferous seedlings in the other two compartments to determine whether water was transferred via fungal hyphae. In addition, the length of the hyphae and degree of mycorrhizal colonisation were determined. Internal transcribed spacer-restriction fragment length polymorphism (ITS-RFLP) analysis was used to identify the fungal species involved in dye (water) transfer. Results of the stable isotope analysis showed that water is transferred via mycorrhizal hyphae, but isotopically labelled water was only detected in Douglas-fir roots, not in sugar pine roots. In contrast, the fluorescent dye was transported via mycorrhizal hyphae to both Douglas-fir and sugar pine seedlings. Only 1 of 15 fungal morphotypes (identified as Atheliaceae) growing in the mesocosms transferred the dye. Differences were detected in the water transfer patterns indicated by the deuterium and fluorescent dye tracers, suggesting that the two labels are transported by different mechanisms in the same hyphae and/or that different fungal taxa transfer them via different routes to host plants. We conclude that both tracers can provide information on resource transfer between fungi and plants, but we cannot be sure that the dye transfer data provide accurate indications of water transfer rates and patterns. The isotopic tracer provides more direct indications of water movement and is therefore more suitable than the dye for studying water relations of plants and their associated mycorrhizal fungi.

Ectomycorrhizal fungal communities of nursery-inoculated seedlings outplanted on clear-cut sites in northern Alberta

Seedlings from three conifer species (Pinus contorta Doug. ex Loud. var. latifolia Englem., Picea glauca (Moench) Voss, and Picea mariana (Mill.) BSP) were planted on two clear-cut sites in Alberta, Canada, after inoculation in the nursery with strains of six different ectomycorrhizal species (Hebeloma longicaudum, Laccaria bicolor,Paxillus involutus,Pisolithus tinctorius,Rhizopogon vinicolor, and Suillus tomentosus). Five and 6 years after planting, morphological characterization and molecular typing techniques (internal transcribed spacer – restriction fragment length polymorphism (ITS-RFLP) and simple sequence repeat (SSR) markers) were used to identify the ectomycorrhizal fungal communities and to assess the occurrence of the inoculated ectomycorrhizal fungi on host roots. Ectomy corrhi zae recovered from the roots of the planted trees on each of the two sites showed little diversity, with a total of 16 and 19 ITS-RFLP patterns corresponding to 11 and 13 ectomycorrhizal taxa, respectively. The most abundant ectomycorrhizal fungi found on colonized roots were ascomycetes and the widespread basidiomycete Amphinema byssoides. Amongst the six introduced fungal strains, only L. bicolor UAMH 8232 was detected on one site after 5 and 6 years, as determined using six SSR markers. Although not detected after 5 years, some of the introduced strains might have had a positive effect on the early growth of the trees before their replacement by competing species, because significant differences in plot volume index were detected between inoculation and control treatments.

Mycelial incompatibility operative in pairings between single basidiospore isolates of Helicobasidium mompa

Helicobasidium mompa is a clampless basidiomycete and binucleate both in secondary and primary hyphae. Single basidiospore isolates were paired in sibling and non-sibling combinations. We did not observe any alterations in colony morphology, such as tuft formation, but mycelial incompatibility, as indicated by the presence of a dark demarcation line between colonies, occurred in a unifactorial manner. Techniques using DNA molecular markers, such as internal transcribed spacer-restriction fragment-length polymorphism (ITS-RFLP), inter-simple-sequence-repeat polymerase chain reaction (PCR), and universally primed PCR, failed to identify nuclear migration to opposite single basidiospore isolates in all but one of the 92 pairings. These results imply that single basidiospore isolates of H. mompa may be incompetent to mate under laboratory conditions and that a single mycelial incompatibility factor operates in single basidiospore isolates.

ITS-RFLP and ITS sequence analysis of a foliar endophytic Phyllosticta from different tropical trees

Different isolates of a foliar endophytic species of Phyllosticta were isolated from different tropical tree species in India to examine genetic variation among the isolates. Internal transcribed spacer-restriction fragment length polymorphism (ITS-RFLP) analysis did not detect any variation among the isolates, suggesting that they all belong to the same species. Sequence data of the ITS region of ribosomal DNA (ITS1 and ITS2, including 5.8S rDNA) supported the identity of the present fungal isolates as P. capitalensis. These results show that P. capitalensis (teleomorph Guignardia endophyllicola?) is an ubiquitous foliar endophyte that can infect tree hosts from different families and habitats.

Dominance of the mycorrhizal fungus Rhizopogon rubescens in a plantation of Pinus pinea seedlings inoculated with Suillus collinitus

We examined the below-ground mycorrhizal diversity of P. pinea seedlings inoculated with S. collinitus six years after out- planting in a disturbed site, located at La Petite-Camargue (Gard, France). This was performed using the polymerase chain reaction (PCR), the restriction fragment length polymorphism (RFLP) and the sequencing of the nuclear ribosomal internal transcribed spacer (ITS). Seven and one plants corresponding, respectively, to inoculated and non-inoculated (control) treatments were chosen randomly. Examinations were carried out directly from ectomycorrhizae. A total number of 233 root tips was examined. Five ITS RFLP taxa were detected. The ITS taxon I corresponded to the ectomycorrhizal species Rhizopogon rubescens. This fungus was abundant (55%) in the P. pinea ectomycorrhizal community. The other ITS taxa were rare and remained unidentified. The current P. pinea plantation showed a restricted diversity of the ectomycorrhizal community which is typical of ectomycorrhizal communities in young plantations established in disturbed stands.

Evidence of Two Formae Speciales in Venturia inaequalis, Responsible for Apple and Pyracantha Scab

ABSTRACT Genetic relationships, mating crosses, and host specificity of Venturia inaequalis isolates from Malus spp. and of Spilocaea pyracanthae isolates from Pyracantha spp. were evaluated. Sequence analysis of the complete internal transcribed spacer (ITS) region (ITS1-5.8S to ITS2) revealed a total similarity between these two putative species. ITS restriction fragment length polymorphism carried out with five restriction enzymes on a collection of 28 isolates confirmed a lack of diversity in this region between and within these two populations. Additional isolates from three related species (V. pirina, V. nashicola, and S. eriobotryae) were divided into two distinct monophyletic groups in a phylogenetic tree using ITS sequence comparison. These groups were related to their anamorph (i.e., Spilocaea or Fusicladium). When inoculated on their host of origin, fields isolates caused typical symptoms of scab disease, and a host specificity was demonstrated by cross pathogenicity of isolates from Malus x domestica and Pyracantha spp. Mating on dried leaves in vitro between one isolate of each putative species led to production of numerous perithecia. Ninety-six sporulating monoascosporic progenies were isolated from this cross. Based on these genetic and pathogenic data, we proposed that pathogens responsible for scab on Malus spp. and Pyracantha spp. are considered as two formae speciales belonging to V. inaequalis.

Characterization of Pinus ectomycorrhizas from mixed conifer and pygmy forests using morphotyping and molecular methods

We used morphotyping and molecular methods to characterize ectomycorrhizas of bishop pine (Pinus muricata D. Don) and Bolander pine (Pinus contorta ssp. bolanderi (Parl.) Critchf.) from mixed conifer and hydric pygmy forests on the northern California coast. Sixteen ectomycorrhizal morphotypes were described, producing 15 internal transcribed spacer restriction fragment length polymorphism (ITS-RFLP) types, and 12 were identified via ITS sequencing. From a given site, all root tips of a specific morphotype produced identical ITS-RFLP patterns. However, sometimes two morphotypes produced the same ITS-RFLP type, and sometimes samples of the same morphotype from two different sites produced two different ITS-RFLP types. These results indicate that surveys of ectomycorrhizal fungi based on morphology alone are not sufficient, and that grouping morphotypes prior to molecular analysis can expedite the process. Ectomycorrhizas from mixed conifer included Russuloid sp., Tomentella sublilacina (Ellis & Holw.) Wakef., Tuber sp., and two Thelephoroid species. Ectomycorrhizas from hydric pygmy included two Dermocybe spp., a Cortinarius sp., two Thelephoroid spp., and Suillus tomentosus (Kauffman) Singer. Both plant communities contained Cenococcum geophilum Fr.:Fr. The hydric pygmy sites were more similar to each other than to the mixed conifer site (Jaccard similarity). The presence of ectomycorrhizal taxa in one plant community type may reflect biotic (host specificity) or abiotic (soil fertility or hydrology) adaptation.Key words: ectomycorrhiza, bishop pine, Pinus muricata, Bolander pine, Pinus contorta ssp. bolanderi, morphotyping, ITS-RFLP.

Characterization of <i>Pinus</i> ectomycorrhizas from mixed conifer and pygmy forests using morphotyping and molecular methods

We used morphotyping and molecular methods to characterize ectomycorrhizas of bishop pine (Pinus muricata D. Don) and Bolander pine (Pinus contorta ssp. bolanderi (Parl.) Critchf.) from mixed conifer and hydric pygmy forests on the northern California coast. Sixteen ectomycorrhizal morphotypes were described, producing 15 in- ternal transcribed spacer restriction fragment length polymorphism (ITS-RFLP) types, and 12 were identified via ITS sequencing. From a given site, all root tips of a specific morphotype produced identical ITS-RFLP patterns. However, sometimes two morphotypes produced the same ITS-RFLP type, and sometimes samples of the same morphotype from two different sites produced two different ITS-RFLP types. These results indicate that surveys of ectomycorrhizal fungi based on morphology alone are not sufficient, and that grouping morphotypes prior to molecular analysis can expedite the process. Ectomycorrhizas from mixed conifer included Russuloid sp., Tomentella sublilacina (Ellis & Holw.) Wakef., Tuber sp., and two Thelephoroid species. Ectomycorrhizas from hydric pygmy included two Dermocybe spp., a Cortinarius sp., two Thelephoroid spp., and Suillus tomentosus (Kauffman) Singer. Both plant communities contained Cenococcum geophilumFr.:Fr. The hydric pygmy sites were more similar to each other than to the mixed conifer site (Jaccard similarity). The presence of ectomycorrhizal taxa in one plant community type may reflect biotic (host speci- ficity) or abiotic (soil fertility or hydrology) adaptation.

Ectomycorrhizal fungal communities in late‐successional Swedish boreal forests, and their composition following wildfire

This study was conducted to evaluate the effects of wildfires on ectomycorrhizal (EM) fungal communities in Scots pine (Pinus sylvestris) stands. Below‐ and above‐ground communities were analysed in terms of species richness and evenness by examining mycorrhizas and sporocarps in a chronosequence of burned stands in comparison with adjacent unburned late‐successional stands. The internal transcribed spacer (ITS)‐region (rDNA) of mycobionts from single mycorrhizas was digested with three restriction enzymes and compared with an ITS–restriction fragment length polymorphism (RFLP) reference database of EM sporocarps. Spatial variation seemed to be more prominent than the effects of fire on the EM fungal species composition. Most of the common species tended to be found in all sites, suggesting that EM fungal communities show a high degree of continuity following low‐intensity wildfires. Species richness was not affected by fire, whereas the evenness of species distributions of mycorrhizas was lower in the burned stands. The diversity of EM fungi was relatively high considering that there were only three EM tree species present in the stands. In total, 135 EM taxa were identified on the basis of their RFLP patterns; 66 species were recorded as sporocarps, but only 11 of these were also recorded as mycorrhizas. The species composition of the below‐ground community of EM fungi did not reflect that of the sporocarps produced. EM fungal species present in our ITS–RFLP reference database accounted for 54–99% of the total sporocarp production in the stands, but only 0–32% of the mycorrhizal abundance.