Molecular diversity of fungi from ericoid mycorrhizal roots

In order to investigate the diversity of fungal endophytes in ericoid mycorrhizal roots, about 150 mycelia were isolated from surface-sterilized roots of 10 plants of Calluna vulgaris. Each mycelium was reinoculated to C. vulgaris seedlings under axenic conditions, and the phenotype of the plant-fungus association assessed by light and electron microscopy. Many isolates that were able in vitro to produce typical ericoid mycorrhizae did not form reproductive structures under our culture conditions, whereas others could be identified as belonging to the species Oidiodendron maius. Morphological and molecular analysis of the fungal isolates showed that the root system of a single plant of C. vulgaris is a complex mosaic of several populations of mycorrhizal and non mycorrhizal fungi. PCR-RFLP techniques, used to investigate the mycorrhizal endophytes, revealed up to four groups of fungi with different PCR-RFLP patterns of the ITS ribosomal region from a single plant. Some of the mycorrhizal fungi sharing the same PCR-RFLP pattern showed high degree of genetic polymorphism when analysed with the more sensitive RAPD technique; this technique may prove a useful tool to trace the spread of individual mycorrhizal mycelia, as it has allowed us to identify isolates with identical RAPD fingerprints on different plants.     

Ceratobasidiaceae mycorrhizal fungi isolated from nonphotosynthetic orchid <Emphasis Type="Italic">Chamaegastrodia sikokiana</Emphasis>

Mycorrhizal fungi were isolated from the nonphotosynthetic orchid Chamaegastrodia sikokiana and identified as members of Ceratobasidiaceae by phylogenetic analysis of the internal transcribed spacer (ITS) region of ribosomal deoxyribonucleic acid. The ITS sequences were similar among geographically separated samples obtained from Mt. Kiyosumi in Chiba Prefecture and Mt. Yokokura in Kochi Prefecture. One of the isolated fungi, KI1-2, formed ectomycorrhiza on seedlings of Abies firma in pot culture, suggesting that tripartite symbiosis exists among C. sikokiana, mycorrhizal fungi, and A. firma in nature, and carbon compounds are supplied from A. firma to C. sikokiana through the hyphae of the mycorrhizal fungi. To our knowledge, this is the second study to suggest the involvement of Ceratobasidiaceae fungi in tripartite symbiosis with achlorophyllous orchids and photosynthetic host plants.     

进境美国加州脐橙中丁香疫霉Phytophthora syringae的截获

从产自美国加利福尼亚州的新鲜脐橙样品中发现多个腐烂病果,通过分离培养得到3个疑似丁香疫霉Phytophthora syringae菌株,对3个菌株进行形态学研究、致病性测定和分子序列比对分析。结果表明病菌在V8A培养基上菌落稀疏、平铺,呈星状,菌丝紧贴培养基生长或埋于基质内生长;在PDA培养基上菌落呈菊花花瓣状,菌丝致密,乳白色;游动孢子囊和菌丝膨大体在无菌水和土壤浸出液中黑暗条件下48h后产生;菌株为同宗配合,卵孢子在带有新鲜脐橙果实组织或杜鹃叶片的V8A培养基中大量产生;创伤接种脐橙果实,7d后接种脐橙出现典型的褐腐症状;通用引物ITS1/ITS4扩增测序,Blastn分析表明序列与GenBank中P. syringae序列相似性为99%。依据上述研究结果,将分离获得的3株菌鉴定为丁香疫霉Phytophthora syringae,系国内首次截获的一种植物检疫性真菌病害。     

Colonization of roots of cultivated Solanum lycopersicum by dark septate and other ascomycetous endophytes

Tomato (Solanum lycopersicum L.) roots from four different crop sites in Colombia were surface sterilized and 51 fungal isolates were obtained and conserved for further analysis. Based on microscopical observations and growth characteristics, 20 fungal isolates corresponded to genus Fusarium, six presented asexual conidia different from Fusarium, eight were sterile mycelia, seven of which had dark septate hyphae and 17 did not continue to grow on plates after being recovered from conservation. Growth on different media, detailed morphological characterization and ITS region sequencing of the six sporulating and eight sterile isolates revealed that they belonged to different orders of Ascomycota and that the sterile dark septate endophytes did not correspond to the well known Phialocephala group. Interactions of nine isolates with tomato plantlets were assessed in vitro. No effect on shoot development was revealed, but three isolates caused brown spots in roots. Colonization patterns as analyzed by confocal microscopy differed among the isolates and ranged from epidermal to cortical penetration. Altogether 11 new isolates from root endophytic fungi were obtained, seven of which showed features of dark septate endophytes. Four known morphotypes were represented by five isolates, while six isolates belonged to five morphotypes of putative new unknown species.     

Distribution and characterisation of entomopathogenic fungi from Korean soils

We investigated the occurrence of entomopathogenic fungi in 1080 soil samples representing multiple locations and conditions in Korea. Entomopathogenic fungi were isolated from soils using a selective medium containing dodine and antibiotics. Following an initial identification based on morphology, the fungal isolates were more precisely identified by the sequence of their nuclear ribosomal RNA (rRNA) internal transcribed spacer (ITS) regions. As a result, entomopathogenic fungi were found to occur in 32% (342 isolates) of the soil samples studied. The most abundant species were Beauveria spp. (125 isolates) and Metarhizium spp. (82 isolates). Entomopathogenic fungi were more often recovered from natural mountain and riparian soils than from agricultural habitats. The pathogenicity of isolated fungi was evaluated by using wax moth Galleria mellonella L. (Lepidoptera: Pyralidae) larvae. It was determined that 60% (207 isolates) of the isolates were pathogenic using this model. These entomopathogenic fungi may, therefore, have potential use against a variety of agricultural pests. This is the first study of the isolation and distribution of entomopathogenic fungi in representative sampling locations throughout Korea.     

Identification and Characterization of the Causal Agent of Gummy Stem Blight from Muskmelon and Watermelon in East China

Gummy stem blight (GSB) is one of the most destructive foliar diseases of cucurbits, worldwide. To identify and characterize the pathogen which causes GSB on watermelon and muskmelon in East China, morphological characteristics, pathogenicity assays as well as sequence characterization of the rDNA internal transcribed spacer (ITS) were performed on 41 isolates collected from Jiangsu, Zhejiang, Anhui and Jiangxi provinces. The mycelia of all the isolates were white on top and olivaceous green to black with concentric circles at the bottom on potato‐dextrose agar medium. The isolates differed significantly on aggressiveness based on pathogenicity assays. rDNA‐ITS sequences and phylogenetic analysis confirmed the isolates as Didymella bryoniae. The isolates were found to be highly identical with the exception of 13 isolates, which had a guanine substitution instead of adenine at position 131 of the ITS.     

MYCELIUM OF LIMB RUST FUNGI

Limb rust is a killing disease of hard pines caused by Peridermium spp. Study of tissue sections shows that growth of limb rust fungi differs from growth of other plant rusts: (1) longitudinal spread is mainly by hyphal growth within host tracheids; (2) hyphae grow radially in mature xylem, deep in host sapwood; (3) in larger stems mycelia avoid (rather than concentrate in) bark and outer xylem rings; and (4) mycelia become much larger than any previously described for rust fungi. Antibiotics being tested as therapeutants are unlikely to be effective against such deep-seated mycelia.     

Characterization of mycorrhizal fungi isolated from the threatened Cypripedium macranthos in a northern island of Japan: two phylogenetically distinct fungi associated with the orchid

We isolated Rhizoctonia-like fungi from populations of the threatened orchid Cypripedium macranthos. In ultrastructural observations of the septa, the isolates had a flattened imperforate parenthesome consisting of two electron-dense membranes bordered by an internal electron-lucent zone, identical to the septal ultrastructure of Rhizoctonia repens (teleomorph Tulasnella), a mycorrhizal fungus of many orchid species. However, hyphae of the isolates did not fuse with those of known tester strains of R. repens and grew less than half as fast as those of R. repens. In phylogenetic analyses, sequences for rDNA and internal transcribed spacer (ITS) regions of the isolates were distinct from those of the taxonomically identified species of Tulasnella. On the basis of the ITS sequences, the isolates clustered into two groups that corresponded exactly with the clades demonstrated for other Cypripedium spp. from Eurasia and North America despite the geographical separation, suggesting high specificity in the Cypripedium–fungus association. In addition, the two phylogenetic groups corresponded to two different plant clones at different developmental stages. The fungi from one clone constituted one group and did not belong to the other fungal group isolated from the other clone. The possibility of switching to a new mycorrhizal partner during the orchid’s lifetime is discussed.     

Ascoma genotyping and mating type analyses of mycorrhizas and soil mycelia of Tuber borchii in a truffle orchard established by mycelial inoculated plants

Tuber borchii (the Bianchetto truffle) is a heterothallic Ascomycete living in symbiotic association with trees and shrubs. Maternal and paternal genotype dynamics have already been studied for the black truffles Tuber melanosporum and Tuber aestivum but not yet for T. borchii. In this study, we analysed maternal and paternal genotypes in the first truffle orchard realized with plants inoculated with five different T. borchii mycelia. Our aims were to test the persistence of the inoculated mycelia, if maternal and/or paternal genotypes correspond to inoculated mycelia and to assess the hermaphroditism of T. borchii. The mating type of each isolate as well as those of mycorrhizas, ascomata and extraradical soil mycelia was determined. Moreover, simple sequence repeat (SSR) profiles of maternal and paternal genotypes were assessed in 18 fruiting bodies to investigate the sexual behaviour of this truffle. The maternal genotypes of the fruiting bodies corresponded to those of the inoculated mycelia with only two exceptions. This confirmed that the inoculated mycelia persisted 9 years after plantation. As regards paternal partner, only two had the same genotype as those of the inoculated mycelia, suggesting hermaphroditism. Most of the new paternal genotypes originated from a recombination of those of inoculated mycelia.     

Identification of Armillaria species associated with Polyporus umbellatus using ITS sequences of nuclear ribosomal DNA

The sclerotia of Polyporus umbellatus were collected from three locations in Japan and three locations in China. All the collected sclerotia were adhered to by rhizomorphs of the symbionts. When the sclerotium of P. umbellatus was cross sectioned, the internal part of the sclerotium was cream colored, and many black regions surrounding the invading rhizomorphs were observed. The surrounding zone contained string-like, gelatinous masses composed of hyphae, and its outside was brown in color. All isolates were similar in colony morphology and grew well on PDA medium with well-developed rhizomorphs. All the isolates showed typical morphology of Armillaria. The isolated fungi were identified via the ITS region of the nuclear ribosomal DNA sequence. Phylogenetic analysis based on the neighbor-joining method showed that all the isolates clustered with fungi belonging to Armillaria species. Among them, five species (A. sinapina, A. calvescens, A. gallica, A. cepistipes, and A. nabsnona) and the symbiont formed a highly supported clade. We report on the case where Armillaria has a relationship in the sclerotium of Polyporus umbellatus.     

Molecular Identification of Fomitiporia mediterranea and Eutypa lata/Libertella blepharis in Platanus × acerifolia

The studies of woody organs‐affecting diseases of Platanus × acerifolia can be hampered by the finding of fungi whose identification is difficult with mycological techniques. In a previous study on Platanus × acerifolia affected by severe cankers, Fomitiporia mediterranea/punctata‐like fungi, not associated with fruit bodies and Libertella sp. were recovered. Due to the severity of the associated symptoms, a characterization of fungal ribosomal DNA genes was undertaken in the present study, aimed to specific identification of the pathogens. From DNA of Fomitiporia‐vegetative isolates and Fomitiporia‐fruit body isolates, included in the study for comparison (from fruit body on plane trees typical of F. mediterranea/punctata), and Libertella sp., DNA fragments were amplified in polymerase chain reaction (PCR) with the use of ITS5/ITS4 and 5.8S‐R/LR7 primer pairs. Sequence alignments showed that Fomitiporia‐vegetative/fruit body isolates had highly homologous ITS5/ITS4 sequences, with a nucleotide identity of 98–100%. All the Fomitiporia isolates from plane tree showed 97–100% and 91–94% of nucleotide identity respectively with ITS5/ITS4 sequences of known strains of F. mediterranea and F. punctata, extracted from GenBank. The strong similarity of these identity ranges with those obtained within F. mediterranea (98–100%) and between the two Fomitiporia species (90–94%) confirms the identification of the isolates from plane tree as F. mediterranea. Sequence comparison between Libertella sp. from plane tree and known strains of Eutypa lata/L. blepharis showed 94–99% of nucleotide identity. The comparison with eight additional species of Eutypa showed 90–93% of nucleotide identity. As previously reported for the different taxa within Diatrypaceae, also ITS5/ITS4 sequence of Libertella sp. from plane tree exhibited 11‐bp tandem repeats motifs. Results of sequence alignments, of phylogenetic trees, and of the putative restriction map, identify the Fomitiporia isolates of this work as F. mediterranea, and the isolates of Libertella sp. as E. lata/L. blepharis. For comparative purposes, ITS5/ITS4 sequences of Spongipellis pachyodon and Fomes fomentarius from plane tree, were also obtained in this work.     

Differentiation and grouping of isolates of the Ganoderma lucidum complex by random amplified polymorphic DNA-PCR compared with grouping on the basis of internal transcribed spacer sequences.

Laccate polypores of the Ganoderma lucidum species complex are widespread white rot fungi of economic importance, but isolates cannot be identified by traditional taxonomic methods. Parsimony analysis of nucleotide sequences from the internal transcribed spacers (ITS) of the ribosomal gene (rDNA) distinguished six lineages in this species complex. Each ITS lineage may represent one or more putative species. While some isolates have identical ITS sequences, all of them could be clearly differentiated by genetic fingerprinting using random amplified polymorphic DNA (RAPD). To investigate the suitability of RAPD markers for taxonomic identification and grouping of isolates of the G. lucidum complex, RAPD fragments (RAPDs) were used as phenotypic characters in numerical and parsimony analyses. Results show that data from RAPDS do not distinguish the same clades as ITS data do. Groupings based on analysis of RAPD data were very sensitive to the choice of the grouping method used, and no consistent grouping of isolates could be proposed. However, analysis with RAPDs did resolve several robust terminal clades containing putatively conspecific isolates, suggesting that RAPDs might be helpful for systematics at the lower taxonomic levels that are unresolved by ITS sequence data. The limitations of RAPDs for systematics are briefly discussed. The conclusion of this study is that ITS sequences can be used to identify isolates of the G. lucidum complex, whereas RAPDs can be used to differentiate between isolates having identical ITS sequences. The practical implications of these results are briefly illustrated.     

NUCLEAR RIBOSOMAL DNA INTERNAL TRANSCRIBED SPACER REGIONS (ITS1 AND ITS2) DEFINE DISCRETE BIOGEOGRAPHIC GROUPS IN CLADOPHORA ALBIDA (CHLOROPHYTA)1

Nucleotide sequences of the nuclear ribosomal DNA internal transcribed spacers (ITS1 and ITS2), the 5.8S, and short stretches of the adjacent 18S and 26S coding regions were determined in isolates from four disjunct Cladophora albida (Huds.) Kütz. populations (NE-America, W-Europe, Japan, and W-Australia). The two Pacific isolates share nearly identical ITS sequences as do the two Atlantic isolates. In contrast, interoceanic comparisons exhibit a 21% sequence difference. Variation within ITS regions is useful for identification of population groups on a regional or oceanic scale. However, both spacers are characterized by numerous repeat motifs as well as point mutations, which result in alignment problems at the interspecific level within Cladophora.     

UNITE: a database providing web-based methods for the molecular identification of ectomycorrhizal fungi

Identification of ectomycorrhizal (ECM) fungi is often achieved through comparisons of ribosomal DNA internal transcribed spacer (ITS) sequences with accessioned sequences deposited in public databases. A major problem encountered is that annotation of the sequences in these databases is not always complete or trustworthy. In order to overcome this deficiency, we report on UNITE, an open-access database. UNITE comprises well annotated fungal ITS sequences from well defined herbarium specimens that include full herbarium reference identification data, collector/source and ecological data. At present UNITE contains 758 ITS sequences from 455 species and 67 genera of ECM fungi. UNITE can be searched by taxon name, via sequence similarity using blastn, and via phylogenetic sequence identification using galaxie. Following implementation, galaxie performs a phylogenetic analysis of the query sequence after alignment either to pre-existing generic alignments, or to matches retrieved from a blast search on the UNITE data. It should be noted that the current version of UNITE is dedicated to the reliable identification of ECM fungi. The UNITE database is accessible through the URL http://unite.zbi.ee     

Postharvest Fruit rot of Persimmon (Diospyros kaki) in Spain Caused by Lasiodiplodia theobromae and Neofusicoccum spp.

Three different fungi (isolates IVIA QCV‐1, IVIA QCV‐3 and IVIA QCV‐4) were isolated as potential causal agents of postharvest decay losses observed on sweet persimmons (Diospyros kaki L.) cv. ‘Rojo Brillante’ from commercial packinghouses in the Valencia area (Spain). Disease symptoms were irregular brownish and soft lesions mainly located under and surrounding the fruit calyx (stem‐end) that expanded rapidly at room temperature and turned to dark brown or black colour producing apparent and in some cases abundant white to grey mycelium. The identification of Lasiodiplodia theobromae, Neofusicoccum mediterraneum and Neofusicoccum luteum by macroscopic and microscopic morphological observations was confirmed with the amplification and subsequent sequencing of the ribosomal DNA ITS2 region. Representative nucleotide sequences were deposited in GenBank. Pathogenicity of all three isolates was demonstrated by fulfilling Koch's postulates.     

The phylogenetic relationship and non‐specific symbiotic habit of mycorrhiza fungi from a terrestrial orchid (Cymbidium)

To investigate the specificity of the symbiotic relationship between Cymbidium plants and their mycorrhiza fungi, thirty mycorrhiza fungi were isolated from roots of six terrestrial Cymbidium species. The internal transcribed spacer (ITS) region of nuclear ribosomal DNA (rDNA) were amplified by polymerase chain reaction (PCR) with universal fungal primers ITS1/ITS4. All fungal strains isolated from natural roots of orchids were inoculated into the rhizomes of in vitro Cymbidium goeringii. Phylogenetic analysis indicated fungal isolates of different cluster could be obtained from a special terrestrial Cymbidium species. Observation of light microscope and scanning electron microscope showed that fungi entered the cortical tissue by destroying cell wall of epidermal cells, where they formed hyphal knots in the cortical cells and were digested gradually. A large number of small protuberances were visible on cross sections of the rhizome. There was no strict inter‐species specificity between the isolated mycorrhiza fungi and terrestrial Cymbidium.     

Two new Ophiostoma species with Sporothrix anamorphs from Austria and Azerbaijan

The genus Ophiostoma includes numerous species of primarily insect-vectored, wood-staining fungi. Several anamorph genera that differ in their micronematous or macronematous conidiogenous cells have been associated with Ophiostoma species. Among the former group, Sporothrix is associated with many species and is characterized by conidiogenous cells that arise laterally or terminally from any place on the hyphae and produce nonseptate conidia on sympodially developing denticles. The purpose of this study was to characterize ophiostomatoid isolates with Sporothrix anamorphs recently collected in Austria and Azerbaijan. The isolates were characterized based on comparisons of rDNA and β-tubulin sequence data. Morphology, growth in culture, and sexual reproductive mode were also considered. Phylogenetic analyses of the combined sequence data showed that the isolates formed two distinct groups, one including isolates from Austria and the other isolates from Austria and Azerbaijan. Growth at 25 C and morphology revealed some differences between the two groups, and supported the view that they represent two new species, which we describe here as Ophiostoma fusiforme sp. nov. and Ophiostoma lunatum sp. nov. Both these groups phylogenetically were related to, but distinct from, Ophiostoma stenoceras.     

Genetic Diversity of Amylomyces rouxii from Ragi tapai in Java Island Based on Ribosomal Regions ITS1/ITS2 and D1/D2

Amylomyces rouxii is commonly found as amylolytic fungi in tapai fermentation. However, its diversity is rarely reported despite being often used for food production in Southeast Asia. This research aims to analyze the genetic diversity and the distribution pattern of A. rouxii from Ragi tapai in Java Island, Indonesia. We isolated the fungus from samples obtained from Ragi tapai producing centers in Bandung, Sumedang, Muntilan, Blora, Yogyakarta, and Bondowoso. The obtained isolates were molecularly identified based on the ribosomal regions ITS1/ITS2 and D1/D2, then analyzed for phylogenetic tree reconstruction, genetic distance, genetic variation, and haplotype networking. Six isolates showed specific morphological traits of A. rouxii. However, phylogenetic tree reconstruction on the ribosomal genes showed that the isolates were grouped into two different clades related to two species. Clade A included BDG, SMD, and MTL isolates related to A. rouxii, whereas clade B included YOG, BLR, and BDS isolates related to Mucor indicus. The genetic distances between clades for ITS1/ITS2 and D1/D2 were 0.6145 and 0.1556, respectively. In conclusion, we confirmed the genetic diversity of molds from Ragi tapai in Java Island and showed that the isolates are not only related to A. rouxii as reported before.     

Sequence variation in nuclear ribosomal small subunit,internal transcribed spacer and large subunit regions of Rhizophagus irregularis and Gigaspora margarita is high and isolate‐dependent

Arbuscular mycorrhizal (AM) fungi are known to exhibit high intra‐organism genetic variation. However, information about intra‐ vs. interspecific variation among the genes commonly used in diversity surveys is limited. Here, the nuclear small subunit (SSU) rRNA gene, internal transcribed spacer (ITS) region and large subunit (LSU) rRNA gene portions were sequenced from 3 to 5 individual spores from each of two isolates of Rhizophagus irregularis and Gigaspora margarita. A total of 1482 Sanger sequences (0.5 Mb) from 239 clones were obtained, spanning ~4370 bp of the ribosomal operon when concatenated. Intrasporal and intra‐isolate sequence variation was high for all three regions even though variant numbers were not exhausted by sequencing 12–40 clones per isolate. Intra‐isolate nucleotide variation levels followed the expected order of ITS > LSU > SSU, but the values were strongly dependent on isolate identity. Single nucleotide polymorphism (SNP) densities over 4 SNP/kb in the ribosomal operon were detected in all four isolates. Automated operational taxonomic unit picking within the sequence set of known identity overestimated species richness with almost all cut‐off levels, markers and isolates. Average intraspecific sequence similarity values were 99%, 96% and 94% for amplicons in SSU, LSU and ITS, respectively. The suitability of the central part of the SSU as a marker for AM fungal community surveys was further supported by its level of nucleotide variation, which is similar to that of the ITS region; its alignability across the entire phylum; its appropriate length for next‐generation sequencing; and its ease of amplification in single‐step PCR.     

Fungal endophyte communities in four Rhizophoraceae mangrove species on the south coast of China

This study investigated the taxonomic identities and diversity of fungal endophytes isolated from four Rhizophoraceae mangrove plant species, Ceriops tagal, Rhizophora apiculata, R. stylosa and Bruguiera sexangula var. rhynchopetala, using a combination of morphological and molecular approaches. Two hundred ninety-five isolates were classified into 38 taxa by morphological characteristics. The representative 38 isolates from each taxa were selected for further molecular identification using nuclear ribosomal DNA sequences, including both the internal transcribed spacers (ITS1 and ITS2) and the 5.8S gene region. The 38 representative endophytes were identified to various taxonomic levels. These results suggest that Pestalotiopsis and Phomopsis were the most frequent endophytes in the four host species. Some of the endophytes exhibit host and tissue specificity. The colonization frequencies of endophytic fungi in the stems of the four host plants are evidently higher than in the roots. The four Rhizophoraceae mangrove species have low similarities of endophyte communities.