Four new Ophiostoma species associated with hardwood-infesting bark beetles in Norway and Poland

Ophiostoma spp. (Ophiostomatales, Ascomycota) are well-known fungi associated with bark and ambrosia beetles (Curculionidae: Scolytinae, Platypodinae). Fungi in the Ophiostomatales include serious tree pathogens as well as agents of timber blue-stain. Although these fungi have been extensively studied in the northern hemisphere, very little is known regarding their occurrence on hardwoods in Europe. The aims of the present study were to identify and characterize new Ophiostoma spp. associated with bark and ambrosia beetles infesting hardwoods in Norway and Poland, and to resolve phylogenetic relationships of Ophiostoma spp. related to the Norwegian and Polish isolates, using multigene phylogenetic analyses. Results obtained from five gene regions (ITS, LSU, β-tubulin, calmodulin, translation elongation factor 1-α) revealed four new Ophiostoma spp. These include Ophiostoma hylesinum sp. nov., O. signatum sp. nov., and O. villosum sp. nov. that phylogenetically are positioned within the Ophiostoma ulmi complex. The other new species, Ophiostoma pseudokarelicum sp. nov. reside along with Ophiostoma karelicum in a discrete, well-supported phylogenetic group in Ophiostoma s. stricto. The results of this study clearly show that the diversity and ecology of Ophiostoma spp. on hardwoods in Europe is poorly understood and that further studies are required to enrich our knowledge about these fungi.     

Diversity and ubiquity of xylariaceous endophytes in live and dead leaves of temperate forest trees

To test the hypothesis that xylariaceous endophytes were ubiquitous on live and dead leaves of various tree species in the field, xylariaceous fungi were isolated from live leaves and bleached and nonbleached portions of dead leaves of a total of 94 tree species in a cool temperate forest in Japan. The biodiversity of xylariaceous endophytes was evaluated as the richness of operational taxonomic units (OTUs) determined by phylogenetic analysis of the nucleotide sequence of the D1/D2 region of the LSU rDNA of fungal isolates. A total of 326 isolates of xylariaceous fungi were isolated from live and dead leaves and classified into 15 OTUs. The three major OTUs, Xylaria sp.1, Nemania sp., and Biscogniauxia sp., accounted for 94% (308 isolates) of the total number of isolates, and were isolated from various live and dead leaves. Xylaria sp.1 was frequently encountered on bleached portions (which were produced due to the selective decomposition of lignin) of dead leaves of broad-leaved deciduous tree species. The results suggest that xylariaceous endophytes did not show host specificity and had a saprobic phase on dead leaves in their life cycles and that Xylaria sp.1 was capable of decomposing lignin in the field conditions.     

ITS2 sequence–structure phylogeny reveals diverse endophytic <Emphasis Type="Italic">Pseudocercospora</Emphasis> fungi on poplars

For matching the new fungal nomenclature to abolish pleomorphic names for a fungus, a genus Pseudocercospora s. str. was suggested to host holomorphic Pseudocercosproa fungi. But the Pseudocercosproa fungi need extra phylogenetic loci to clarify their taxonomy and diversity for their existing and coming species. Internal transcribed spacer 2 (ITS2) secondary structures have been promising in charactering species phylogeny in plants, animals and fungi. In present study, a conserved model of ITS2 secondary structures was confirmed on fungi in Pseudocercospora s. str. genus using RNAshape program. The model has a typical eukaryotic four-helix ITS2 secondary structure. But a single U base occurred in conserved motif of U-U mismatch in Helix 2, and a UG emerged in UGGU motif in Helix 3 to Pseudocercospora fungi. The phylogeny analyses based on the ITS2 sequence–secondary structures with compensatory base change characterizations are able to delimit more species for Pseudocercospora s. str. than phylogenic inferences of traditional multi-loci alignments do. The model was employed to explore the diversity of endophytic Pseudocercospora fungi in poplar trees. The analysis results also showed that endophytic Pseudocercospora fungi were diverse in species and evolved a specific lineage in poplar trees. This work suggested that ITS2 sequence–structures could become as additionally significant loci for species phylogenetic and taxonomic studies on Pseudocerospora fungi, and that Pseudocercospora endophytes could be important roles to Pseudocercospora fungi’s evolution and function in ecology.     

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.     

The ITS2 Database

The internal transcribed spacer 2 (ITS2) has been used as a phylogenetic marker for more than two decades. As ITS2 research mainly focused on the very variable ITS2 sequence, it confined this marker to low-level phylogenetics only. However, the combination of the ITS2 sequence and its highly conserved secondary structure improves the phylogenetic resolution¹ and allows phylogenetic inference at multiple taxonomic ranks, including species delimitation^2-8.The ITS2 Database⁹ presents an exhaustive dataset of internal transcribed spacer 2 sequences from NCBI GenBank¹¹ accurately reannotated¹⁰. Following an annotation by profile Hidden Markov Models (HMMs), the secondary structure of each sequence is predicted. First, it is tested whether a minimum energy based fold¹² (direct fold) results in a correct, four helix conformation. If this is not the case, the structure is predicted by homology modeling¹³. In homology modeling, an already known secondary structure is transferred to another ITS2 sequence, whose secondary structure was not able to fold correctly in a direct fold.The ITS2 Database is not only a database for storage and retrieval of ITS2 sequence-structures. It also provides several tools to process your own ITS2 sequences, including annotation, structural prediction, motif detection and BLAST¹⁴ search on the combined sequence-structure information. Moreover, it integrates trimmed versions of 4SALE^15,16 and ProfDistS¹⁷ for multiple sequence-structure alignment calculation and Neighbor Joining¹⁸ tree reconstruction. Together they form a coherent analysis pipeline from an initial set of sequences to a phylogeny based on sequence and secondary structure.In a nutshell, this workbench simplifies first phylogenetic analyses to only a few mouse-clicks, while additionally providing tools and data for comprehensive large-scale analyses.     

Molecular phylogeny of 21 tropical bamboo species reconstructed by integrating non-coding internal transcribed spacer (ITS1 and 2) sequences and their consensus secondary structure

The unavailability of the reproductive structure and unpredictability of vegetative characters for the identification and phylogenetic study of bamboo prompted the application of molecular techniques for greater resolution and consensus. We first employed internal transcribed spacer (ITS1, 5.8S rRNA and ITS2) sequences to construct the phylogenetic tree of 21 tropical bamboo species. While the sequence alone could grossly reconstruct the traditional phylogeny amongst the 21-tropical species studied, some anomalies were encountered that prompted a further refinement of the phylogenetic analyses. Therefore, we integrated the secondary structure of the ITS sequences to derive individual sequence-structure matrix to gain more resolution on the phylogenetic reconstruction. The results showed that ITS sequence-structure is the reliable alternative to the conventional phenotypic method for the identification of bamboo species. The best-fit topology obtained by the sequence-structure based phylogeny over the sole sequence based one underscores closer clustering of all the studied Bambusa species (Sub-tribe Bambusinae), while Melocanna baccifera, which belongs to Sub-Tribe Melocanneae, disjointedly clustered as an out-group within the consensus phylogenetic tree. In this study, we demonstrated the dependability of the combined (ITS sequence+structure-based) approach over the only sequence-based analysis for phylogenetic relationship assessment of bamboo.     

A Brevibacillus sp. antagonistic to mycotoxigenic Fusarium spp.

Antagonistic microbes were isolated from soils to control mycotoxin contamination of cereals by limiting the growth of mycotoxigenic Fusarium species. In total, 341 bacterial isolates were examined for antifungal activity against eight mycotoxigenic Fusarium species using dual culture assays. The screening identified 11 isolates that inhibited mycelial growth of all Fusarium species tested. The culture filtrates of 2 of the 11 isolates completely inhibited germination of conidia up to 21 days of incubation. These two isolates exhibited identical activity toward the fungi tested and were identified as Brevibacillus spp. based on 16S rRNA sequence homology. The most closely related species based on phylogenetic analysis was Brevibacillus reuszeri. Additional dual culturing using further fungal species showed that the antagonistic Brevibacillus inhibited the growth of most Fusarium species tested (39 of 46 species), two Epicoccum spp., one Alternaria sp., three Aspergillus spp. (3 of 11), and three Penicillium spp. (3 of 8). The in vivo assay was performed to test the efficacy of antagonistic Brevibacillus isolates on maize ears and revealed that the application of microbes suppressed ear rot (ANOVA, p = 0.0020). This Brevibacillus sp. may be an antagonist of the majority of Fusarium species, including mycotoxigenic species.     

Ribosomal ITS1 sequence-based diversity analysis of anaerobic rumen fungi in cattle fed on high fiber diet

Fiber degradation in the ruminant digestive process is a major activity accomplished by rumen microbes, a process in which the role of fungi is important. Therefore, the present study was conducted to establish the community structure of anaerobic rumen fungi in cattle fed on a high fiber diet using molecular approaches. Total community DNA was extracted, and the ribosomal internal transcribed spacer (ITS) 1 region was amplified, cloned, and sequenced. The resulting nucleotide sequences were used to construct a phylogenetic tree. A total of 52 clones were analyzed, revealing 31 different ITS1 gene phylotypes. Of these, 12 belonged to the genus Orpinomyces (48 % of clones), followed by uncultured Neocallimastigale clones (29 %), Cyllamyces spp. (9 %) and Anaeromyces spp. (8 %). Our results indicate that genus Orpinomyces dominates the rumen fungal community in Indian crossbred Karan Fries cattle.     

Diversity and Taxonomy of Endophytic Xylariaceous Fungi from Medicinal Plants of Dendrobium (Orchidaceae)

Dendrobium spp. are traditional Chinese medicinal plants, and the main effective ingredients (polysaccharides and alkaloids) have pharmacologic effects on gastritis infection, cancer, and anti-aging. Previously, we confirmed endophytic xylariaceous fungi as the dominant fungi in several Dendrobium species of tropical regions from China. In the present study, the diversity, taxonomy, and distribution of culturable endophytic xylariaceous fungi associated with seven medicinal species of Dendrobium (Orchidaceae) were investigated. Among the 961 endophytes newly isolated, 217 xylariaceous fungi (morphotaxa) were identified using morphological and molecular methods. The phylogenetic tree constructed using nuclear ribosomal internal transcribed spacer (ITS), large subunit of ribosomal DNA (LSU), and beta-tubulin sequences divided these anamorphic xylariaceous isolates into at least 18 operational taxonomic units (OTUs). The diversity of the endophytic xylariaceous fungi in these seven Dendrobium species was estimated using Shannon and evenness indices, with the results indicating that the dominant Xylariaceae taxa in each Dendrobium species were greatly different, though common xylariaceous fungi were found in several Dendrobium species. These findings implied that different host plants in the same habitats exhibit a preference and selectivity for their fungal partners. Using culture-dependent approaches, these xylariaceous isolates may be important sources for the future screening of new natural products and drug discovery.     

锦绣杜鹃菌根真菌rDNA ITS序列分析及接种效应研究

利用rDNA ITS序列对锦绣杜鹃菌根真菌的16个菌株进行了分类分析。根据菌株ITS序列全长计算各菌株间序列相似度和遗传距离,并与GenBank中最相似菌株序列构建系统发育树。结果表明：16个菌株在系统树上聚为3个大分支。其中7个菌株在支持率为100%的基础上与树粉孢属真菌Oidiodendron sp.聚为一类;2个菌株与未鉴定的杜鹃花科植物根系真菌unidentified root associated fungi聚为一类,支持率为100%;其他7个菌株在98%的支持率上与几种未命名的欧石楠类菌根真菌     

Secondary structure prediction of ITS rRNA region and molecular phylogeny: an integrated approach for the precise speciation of <Emphasis Type="Italic">Muscodor</Emphasis> species

Muscodor is a non-sporulating, volatile organic compounds producing endophytic fungi that has been extensively explored as a bio-fumigant and bio-preservative. Novel species of this genus have been mainly identified using ITS sequences. However, the ITS hyper-variability hinders the creation of reproducible alignments and stable phylogenetic trees. Conserved structural data of the ITS region represents as a vital auxiliary information for accurate speciation of fungi. In the present study, secondary structural data of ITS1, 5.8S, and ITS2 region of all Muscodor species were generated using LocaRNA web server. The predicted secondary structural data displayed greater variability in ITS1 region in comparison to ITS2. The structural data of all sequences exhibited characteristic conserved features of eukaryotic rRNA. Evolutionary conserved motifs were found among all 5.8S and ITS2 sequences. Profile neighbor joining (PNJ) tree based on combined sequence-structural information of ITS region was generated in ProfDists. The PNJ tree resolved into four major groups whereby M. fengyangenesis and M. albus species formed monophyletic clades. However, three M. albus species along with other Muscodor species emerged as sister branches to the existing clades, thereby, improving the precision of phylogenetic analysis for identification of novel species of Muscodor genus. Hence, the results indicated that structural analysis along with primary sequence information can provide new insights for precise identification of Muscodor species.     

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 of two ovine Babesia based on the ribosomal DNA internal transcribed spacer (ITS) sequences

The first and second internal transcribed spacers (ITS1, ITS2) as well as the intervening 5.8S coding region of the rRNA gene for six Babesia spp. isolated from different geographic origins were characterized. Varying degrees of ITS1 and ITS2 intra- and inter-species sequence polymorphism were found among these isolates. Phylogenetic analysis of the ITS1-5.8S gene-ITS2 region clearly separated the isolates into two clusters. One held an unidentified Babesia sp. transmitted by Hyalomma anatolicum anatolicum. The second held five other isolates, which were considered to be Babesia motasi. Each Babesia species cluster possessed ITS1 and ITS2 of unique size(s) and species specific nucleotide sequences. The results showed that ITS1, ITS2 and the complete ITS1-5.8S-ITS2 region could be used to discriminate these ovine Babesia spp. effectively.     

Diversimorbus metrosiderotis gen. et sp. nov. and three new species of Holocryphia (Cryphonectriaceae) associated with cankers on native Metrosideros angustifolia trees in South Africa

The Cryphonectriaceae includes important tree pathogens, especially on the Myrtales. During a routine disease survey in the Western Cape Province of South Africa, a fungus resembling the Eucalyptus pathogen Holocryphia eucalypti was observed on native Metrosideros angustifolia (Myrtales). The aims of this study were to identify the fungus and to expand surveys for fungi in the Cryphonectriaceae on M. angustifolia. Fungi were identified based on DNA sequence comparisons and morphological features, and their pathogenicity was tested on M. angustifolia under field conditions. Based on morphology and multigene phylogenetic analyses of DNA sequence data from six gene regions, we describe a new genus including a single species and three new species of Holocryphia (Cryphonectriaceae) from M. angustifolia. These fungi are provided with the names Diversimorbus metrosiderotis gen. et sp. nov., Holocryphia capensis sp. nov., Holocryphia gleniana sp. nov., and Holocryphia mzansi sp. nov. We also revise H. eucalypti, the type of the genus, to include only isolates from Eucalyptus in South Africa. Research results indicated that H. mzansi may undergo host shifts between different tree genera in the Myrtaceae. Inoculation tests showed that isolates of all the newly described species can cause lesions on the branches of M. angustifolia, indicating that they are all pathogens of this tree.     

ITS-2 secondary structures and phylogeny of Anopheles culicifacies species

Background: Second internal transcribed spacer (ITS2) has proven to contain useful biological information at higher taxonomic levels. Objectives: This study was carried out to unravel the biological information in the ITS2 region of An. culicifacies and the internal relationships between the five species of Anopheles culicifacies. Methodology: In achieving these objectives, twenty two ITS2 sequences (~370bp) of An. culicifacies species were retrieved from GenBank and secondary structures were generated. For the refinement of the primary structures, i.e. nucleotide sequence of ITS2 sequences, generated secondary structures were used. The improved ITS2 primary structures sequences were then aligned and used for the construction of phylogenetic trees. Results and discussions: ITS2 secondary structures of culicifacies closely resembled near universal eukaryotes secondary structure and had three helices, and the structures of helix II and distal region of helix III of ITS2 of An. culicifacies were strikingly similar to those regions of other organisms strengthening possible involvement of these regions in rRNA biogenesis. Phylogenetic analysis of improved ITS2 sequences revealed two main clades one representing sibling B, C and E and A and D in the other. Conclusions: Near sequence identity of ITS2 regions of the members in a particular clade indicate that this region is undergoing parallel evolution to perform clade specific RNA biogenesis. The divergence of certain isolates of An. culicifacies from main clades in phylogenetic analyses suggests the possible existence of camouflaged sub-species within the complex of culicifacies. Using the fixed nucleotide differences, we estimate that these two clades have diverged nearly 3.3 million years ago, while the sibling species in clade 2 are under less evolutionary pressure, which may have evolved much later than the members in clade 1.     

Molecular phylogenetic analyses reveal a close relationship between powdery mildew fungi on some tropical trees and Erysiphe alphitoides, an oak powdery mildew

To investigate the phylogenetic relationships among the powdery mildew fungi of some economically important tropical trees belonging to Oidium subgenus Pseudoidium, we conducted molecular phylogenetic analyses using 30 DNA sequences of the rDNA internal transcribed spacer (ITS) regions and 26 sequences of the domains D1 and D2 of the 28S rDNA obtained from the powdery mildews on Hevea brasiliensis (para rubber tree), Anacardium occidentale (cashew), Bixa orellana, Citrus spp., Mangifera indica (mango), and Acacia spp. The results indicate that the powdery mildew fungi isolated from these tropical trees are closely related to one another. These powdery mildews are also closely related to E. alphitoides (including Erysiphe sp. on Quercus phillyraeoides). Because of the obligate biotrophic nature of the powdery mildew fungi, the relationship between powdery mildews and their host plants is conservative. However, the present study suggests that a particular powdery mildew species has expanded its host ranges on a wide range of the tropical trees. This article also suggests that a powdery mildew fungus distributed in temperate regions of the Northern Hemisphere expanded its host ranges onto tropical plants and may be a good example of how geographical and host range expansion has occurred in the Erysiphales.     

A new wilt and die-back disease of Acacia mangium associated with Ceratocystis manginecans and C. acaciivora sp. nov. in Indonesia

Species of Ceratocystis are well-known wound related pathogens of many tree species, including commercially planted Acacia spp. Recently, several Ceratocystis isolates were collected from wilting A. mangium in plantations in Indonesia. The aim of this study was to identify these Ceratocystis isolates and to investigate their ability to cause disease on two plantation-grown Acacia spp. using greenhouse and field inoculation experiments. For identification, morphological characteristics and comparisons of DNA sequence data for the ITS, β-tubulin and TEF 1-α gene regions, was used. Ceratocystis isolates were identified as C. manginecans, a serious pathogen of mango trees in Oman and Pakistan and a previously undescribed species, described here as C. acaciivora sp. nov. Both fungi produced significant lesions in inoculation experiments on A. mangium and A. crassicarpa, however, C. acaciivora was most pathogenic suggesting that this fungus is the primary cause of the death of trees under natural conditions.     

Molecular authentication of Anthemis deserti Boiss. (Asteraceae) based on ITS2 region of nrDNA gene sequence

The dried plant material of medicinally important Anthemis deserti Boiss. (family: Asteraceae) especially when it remains in the powdered form often look similar to Anthemis melampodina Del.; and therefore, difficult to distinguish, finally lead to chances of adulteration. The adulteration in medicinal plants effects on the efficacy of the drugs. The molecular authentication of herbal plant materials such as based on the internal transcribed spacer 2 (ITS2) sequences of nuclear ribosomal DNA (nrDNA) is considered as more reliable method compared to other the biochemical or histological methods. The present study aims to molecular authentication ofA. deserti based on molecular phylogenetic analyses of ITS2 gene sequence of nrDNA region. The ITS2 region of nrDNA of A. deserti were sequenced, and the molecular phylogenetic analyses were performed together with the GenBank sequences. The Maximum Parsimony tree revealed the close relationships of A. deserti with A. melampodina; however, the Neighbor-Joining and Maximum Likelihood tree clearly revealed that A. deserti is distinct from A. melampodina, which is also supported by the differences in nucleotides at five diffident positions (i.e. 22, 28, 87, 175 and 198) in the DNA sequence alignment.