ITS rDNA Sequence Data Research Articles

Additions to the genus Kirschsteiniothelia (Dothideomycetes); Three novel species and a new host record, based on morphology and phylogeny

During a survey of microfungi associated with forest plants, four specimens related to Kirschsteiniothelia were collected from decaying wood in Guizhou, Hainan and Yunnan Provinces, China. Kirschsteiniothelia species have sexual and asexual forms. They are commonly found as saprophytes on decaying wood and have been reported as disease-causing pathogens in humans as well. In this study, we introduce three novel Kirschsteiniothelia species (K. bulbosapicalis, K. dendryphioides and K. longirostrata) and describe a new host record for K. atra, based on morphology and multi-gene phylogenetic analyses of a concatenated ITS, LSU and SSU rDNA sequence data. These taxa produced a dendryphiopsis- or sporidesmium-like asexual morph and detailed descriptions and micromorphological illustrations are provided. Furthermore, we provide a checklist for the accepted Kirschsteiniothelia species, including detailed host information, habitat preferences, molecular data, existing morphological type, country of origin and corresponding references.

Molecular Epidemiology and Antifungal Susceptibility Profile in Nakaseomyces glabrata Species Complex: A 5-Year Countrywide Study.

The current study aimed to identify Iranian Nakaseomyces (Candida) glabrata complex species in the clinical isolates and determine their antifungal susceptibility profile. In total, 320 N. glabrata clinical isolates were collected from patients hospitalized in different geographical regions of Iran. The initial screening was performed by morphological characteristics on CHROMagar Candida. Each isolate was identified by targeting the D1/D2 rDNA using a multiplex-PCR method. To validate the mPCR method and determine genetic diversity, the ITS-rDNA region was randomly sequenced in 40 isolates. Additionally, antifungal susceptibility was evaluated against nine antifungal agents following the CLSI M27-A4 guidelines. All clinical isolates from Iran were identified as N. glabrata. The analysis of ITS-rDNA sequence data revealed the presence of eight distinct ITS clades and 10 haplotypes among the 40 isolates of N. glabrata. The predominant clades identified were Clades VII, V, and IV, which respectively accounted for 22.5%, 17.5%, and 17.5% isolates. The widest MIC ranges were observed for voriconazole (0.016-8 μg/mL) and isavuconazole (0.016-2 μg/mL), whereas the narrowest ranges were seen with itraconazole and amphotericin B (0.25-2 μg/mL). Haplotype diversity can be a valuable approach for studying the genetic diversity, transmission patterns, and epidemiology of the N. glabrata complex.

Morphological and molecular insights into the hidden edible mushroom diversity in Sri Lanka

With the increasing world population, identifying additional food sources is crucial. Mushrooms have long been considered a nutraceutical and are consumed globally, including in Sri Lanka. Due to its abundant biodiversity, there is a significant potential for diverse wild edible mushrooms in Sri Lanka. However, the identification of these mushrooms based on morphological characteristics can often be misleading. Molecular characterization ensures more accurate and reliable identification of mushrooms, yet it has not been conducted extensively in Sri Lanka. This study collected several wild edible mushroom samples from Colombo and Rathnapura Districts of Sri Lanka. The edibility of these wild edible mushrooms was confirmed based on traditional knowledge and later verified by published literature. Phylogenetic analyses based on maximum likelihood (ML) analyses of ITS rDNA sequence data and morphological characteristics were used to determine the identity of the collected mushrooms. In this study, six edible mushrooms, Calvatia candida, Pleurotus giganteus, Schizophyllum radiatum, Termitomyces heimii, T. microcarpus, and Volvariella volvacea were identified based on morphology and phylogeny. Calvatia candida and S. radiatum are reported as new records for Sri Lanka, while this is the first study conducted on molecular characterization of V. volvacea in Sri Lanka. Finally, this study aims to increase the research interest, cultivation, commercialization, and conservation of these identified edible mushrooms in Sri Lanka.

Untargeted Metabolomics Approach for the Differentiation between Panax vietnamensis var. vietnamensis and Panax vietnamensis var. fuscidiscus.

Panax vietnamensis var. vietnamensis (PVV) and Panax vietnamensis var. fuscidiscus (PVF) both belong to Panax vietnamensis species and are chemically and morphologically similar, making it hard to distinguish for the consumer. Herein, 42 PVF and 12 PVV samples were collected in Quang Nam and Lai Chau Province, respectively, and subsequently characterized by ITSr-DNA sequence data to verify their origins. Next, untargeted metabolomics combined with multivariate statistical analysis was developed to differentiate PVV and PVF. The metabolic profiles of PVV and PVF were found to be distinct and classified well using Partial Least-Squares Discriminant Analysis (PLS-DA) in the training set. Among them, seven ginsenosides were of high abundance in PVV, while six were of high abundance in PVF. Next, the test set was used to validate 13 putative differential markers found in the training set, illustrating a complete match with the expression patterns of these ginsenosides in the training set. Finally, PLS-DA and linear Support Vector Machine models both indicated distinct ginsenoside profiles of PVV and PVF without misclassification in the test set. Conclusively, the developed untargeted metabolomics approach might serve as a powerful tool for the authentication of PVV and PVF at the metabolome level.

Discovery of a New Lichtheimia (Lichtheimiaceae, Mucorales) from Invertebrate Niche and Its Phylogenetic Status and Physiological Characteristics.

Species of Lichtheimia are important opportunistic fungal pathogens in the order Mucorales that are isolated from various sources such as soil, indoor air, food products, feces, and decaying vegetables. In recent years, species of Lichtheimia have become an emerging causative agent of invasive mucormycosis. In Europe and USA, Lichtheimia are the second and third most common causal fungus of mucormycosis, respectively. Thus, the aim of this study was to survey the diversity of species of Lichtheimia hidden in poorly studied hosts, such as invertebrates, in Korea. Eight Lichtheimia strains were isolated from invertebrate samples. Based on morphology, physiology, and phylogenetic analyses of ITS and LSU rDNA sequence data, the strains were identified as L. hyalospora, L. ornata, L. ramosa, and a novel species, L. koreana sp. nov. Lichtheimia koreana is characterized by a variable columellae, sporangiophores arising solitarily or up to three at one place from stolons, and slow growth on MEA and PDA at all temperatures tested. The new species grows best at 30 and 35 °C and has a maximum growth temperature of 40 °C. Detailed descriptions, illustrations, and a phylogenetic tree are provided.

First Detection of Wilsonomyces carpophilus Causing Leaf Spot and Shot Hole Disease on Vitis vinifera

Grapevines ( Vitis spp.) are cultivated all over the world and viticulture is a horticultural industry. During the spring of 2020, leaf samples with leaf spot and shot-hole symptoms were collected from the vineyards of Urmia county, West Azerbaijan province, Iran. One fungal isolate was recovered from infected tissues. Based on cultural and morphological features, ITS rDNA sequence data, and a generated phylogenetic tree, the obtained fungal isolate was identified as Wilsonomyces carpophilus. Under in vitro conditions, the colony was white to cream colored, sporulation occurred only in a minor proportion of the colony, and the diameter of the colony on potato dextrose agar reached 44 mm after 14 days. To evaluate Koch's postulates, isolate NSS18 was inoculated on leaves of the Chafteh cultivar of grapevine and the same fungus was reisolated from developed symptoms. This is the first report of leaf spot and shot-hole disease on grapevine caused by W. carpophilus, and also Vitis vinifera is a new host for this species in the world.

Ulosarcina terrestrica gen. nov., sp. nov., a New Ulvophycean Sarcinoid Alga from the Russian Far East

Many filamentous and sarcinoid terrestrial or freshwater green algae that were previously assigned to the Chlorophyceae are members of lineages belonging to the class Ulvophyceae. One of these lineages is the Planophila-clade (Ulotrichales). Some genera in this clade share similar morphology: cell packages forming branched pseudofilaments, uniseriate or sometimes biseriate filaments, often embedded in common mucilage. During a study on soil algal diversity in the temperate monsoon climate zone in Russia (Primorsky Territory, Vladivostok), we isolated a strain of sarcinoid green alga and examined it using an integrative approach. SSU and ITS rDNA sequence data, morphological characteristics, and life cycle features differentiated this strain from closely related genera of the order Ulotrichales and led us to describe it as Ulosarcina terrestrica gen. et sp. nov.

First report on the morphological and molecular identification of Lactocollybia variicystis from Salahadin Governorate, North Central Iraq

A rare agaric, Lactocollybia variicystis D.A. Reid and Eicker (Family: Marasmiaceae) is identified as a new record to Iraqi macromycota, based on morphological characteristics and molecular analysis. Descriptions, color photographs, comments and phylogenetic analysis based on ITS rDNA sequence data are given.

Ganoderma multipileum and Tomophagus cattienensis— new records from Pakistan

New records of Ganoderma multipileum and Tomophagus cattienensis collected from Changa Manga Forest and Lahore, Pakistan, are presented based on morphological and nuclear rDNA ITS sequence data from fresh basidiomata. Specimens previously treated as G. lucidum from Pakistan were reviewed and found to represent different species, among them G. multipileum. Specimens of T. cattienensis determined for the first time from Pakistan presented morphological features similar to T. colossus but corresponded molecularly to T. cattienensis; the morphological description for T. cattienensis is expanded accordingly.

First Report of Downy Mildew on Sweet Basil Caused By Peronospora belbahrii in India.

Sweet basil (Ocimum basilicum L.; Family Lamiaceae) is an annual aromatic and medicinal plant grown in tropical and subtropical regions of the world. In India, it is cultivated as a commercial crop on ~8,000 ha. Aerial plant parts and essential oil of sweet basil are used in pharmaceutical, perfumery, food industries and in different formulations of traditional Ayurvedic and Unani medicines (Shahrajabian et al. 2020). The leaves have the highest concentrations of secondary metabolites such as terpenes and phenylpropanoids which provide the distinctive aroma (Viuda-Martos et al. 2011). During October 2020, severe foliar disease was observed in experimental fields of sweet basil at Council of Scientific and Industrial Research (CSIR)-Central Institute of Medicinal and Aromatic Plants (CIMAP) in Lucknow, India. Initial symptoms included large, interveinal chlorotic lesions on the adaxial surface of the leaves and black sporulation on the abaxial surface. Within a few days, the abaxial side of leaves turned necrotic, and leaf senescence and defoliation occurred on plants with severe symptoms. Disease incidence was 20 to 30% of plants. The pathogen was characterized morphologically using a light microscope. Sporangiophores were hyaline, dichotomously branched, 186.9 to 423.07 × 6.85 to 9.06 µm and, branched 3 to 5 times with each branch, terminating in two slightly curved branchlets, the longer one 7.05 to 25.31 µm and the shorter one 4.98 to 15.92 µm. Each branchlet had a single sporangium at the tip. Conidia were ellipsoidal to sub-globose, olive-brown in color, and typically measured 25.21 to 33.86 × 17.92 to 26.24 µm, each, without a pedicel. Based on these morphological characteristics, the foliar disease was identified as downy mildew was caused by Peronospora belbahrii (Thines et al. 2009). Eight symptomatic and two asymptomatic plant samples were collected from different locations in the field, and genomic DNA was extracted from the conidia of the eight naturally infected tissues of sweet basil samples as well as leaf tissues from two asymptomatic plants, using the CTAB method. The internal transcribed spacer region was amplified using ITS1 and ITS4 primers. Only eight infected samples amplified products of expected size (~ 700 bp) and two asymptomatic samples showed no amplification. Only five amplified PCR products were sequenced (White et al. 1990). All five sequences were identical and were a 98.1% match with five P. belbahrii isolates (MN450330.1, MN308051.1, MH620351.1, KJ960193, and MF693898). The consensus sequence was deposited into the NCBI database (GenBank Accession No. MW689257). Downy mildew caused by P. belbahrii previously has been reported on sweet basil from several countries (Wyenandt et al. 2015). To confirm the pathogenicity of these isolates on sweet basil (cv. CIM-Saumya), 25 - day-old sweet basil plants were sprayed with a suspension (1 × 105 sporangia/ml) of P. belbahrii. All plants were kept in a growth chamber with a 23/18°C diurnal cycle with 65 to 85% relative humidity for 24 h. Non-inoculated plants treated with sterile water served as a control treatment. After 8 days, typical symptoms of downy mildew appeared on all the inoculated plants while non-inoculated plants remained asymptomatic. Inoculated leaves with symptoms consistent of downy mildew were collected and the causal agent again identified as P. belbahrii on the basis of microscopic examination and ITS rDNA sequence data. To our knowledge, this is the first report of downy mildew caused by P. belbahrii on sweet basil in India. The pathogen has a broad host range and may pose a serious threat to the cultivation of this valuable crop in India. Thus, it is pertinent to develop effective control measures to avoid further spread and mitigate economic loss.

New Species of Chloroidium (Trebouxiophyceae, Chlorophyta) from East Asia.

Chlorella-like green algae that reproduce only asexually by immotile autospores or motile zoospores are the most typical inhabitants of non-aquatic environments. They have a simple morphology that hampers their differentiation, but algae of such habit represent a diverse array of lineages, which are mostly in the classes Chlorophyceae and Trebouxiophyceae. One of these lineages is the order Watanabeales (Watanabea clade; Trebouxiophyceae), which comprises 10 genera that share a distinct mode of reproduction through unequally sized autospores. Most of these genera account for a few species that are rarely recorded in nature. In contrast, the genus Chloroidium is one of the most species-rich and widely distributed members of the order. Three strains of coccoid green alga were isolated during a study of soil algae in the temperate monsoon climate zone of Asia. These strains are described here as a new species, Chloroidium orientalis. SSU and ITS rDNA sequence data, morphological characteristics, and life cycle features differentiate these strains from known members of the genus.

Revealing the endophytic mycoflora in tea (Camellia sinensis) leaves in Sri Lanka: the first comprehensive study

Endophytic fungi are a diverse group of microorganisms that live asymptomatically in healthy tissues of host and they have been reported from all kinds of plant tissues such as leaves, stems, roots, flowers, and fruits. In this study, fungal endophytes associated with tea leaves (Camellia sinensis) were collected from Kandy, Kegalle, and Nuwara Eliya districts in Sri Lanka and were isolated, characterized, and identified. A total of twenty endophytic fungal isolates belonging to five genera were recovered and ITS-rDNA sequence data were used to identify them. All isolated endophytic fungal strains belong to the phylum Ascomycota and the majority of these isolates were identified as Colletotrichum species. Phyllosticta capitalensis was the most commonly found fungal endophyte in tea leaves and was recorded in all three districts where the samples were collected. This is the very first investigation on fungal endophytes associated with C. sinensis in Sri Lanka based on molecular sequence data. In addition, a comprehensive account of known endophytic fungi reported worldwide on Camellia sinensis is provided.

Physico-Chemical Characteristics and Culturable Microbial Communities of Grape Berries Change Strongly during Noble Rot Development.

Botrytis cinerea is a well-known pathogen of grapevine. However, under certain microclimatic conditions, Botrytis infection results in noble rot, an essential process in the production of the world-known Tokaji aszú wines in Hungary. We investigated the physico-chemical characteristics and culturable microorganisms associated with grape berries through several noble rot phases in the two main cultivars grown in Tokaj: Vitis vinifera cv. “Furmint” and “Hárslevelű”. We measured physical and analytical parameters routinely tested in viticulture and analyzed the ITS rDNA sequence data of fungi isolated from the sampled berries. We observed significant differences in the physico-chemical parameters among the noble rot phases as well as sampling dates. The greatest variation in berry texture and microbial structure was observed in the initial phases, with variables converging as the noble rot progressed. By finding a bijection between the examined chemical properties and the factorial parameters (e.g., noble rot phase, collection time, cultivar), an appropriate sweet winemaking material can be designed. Fungal community differed significantly among cultivars, with higher number of species observed in Hárslevelű. Our results reveal that there is more to noble rot than only Botrytis cinerea and other microorganisms may play important roles in the aszú process.

Morphological and molecular characterization of Heterocapsa claromecoensis sp. nov. (Peridiniales, Dinophyceae) from Buenos Aires coastal waters (Argentina)

ABSTRACT A new species of the marine dinophyte genus Heterocapsa Stein is described. Two clonal strains originating from Argentinean coastal waters were examined with light and electron microscopy and LSU and ITS rDNA sequence data were obtained. Heterocapsa claromecoensis sp. nov. is described as a distinctive species with a conical epitheca, a similar sized and rounded hypotheca, a single reticulate chloroplast situated in the periphery of the cell, and a large pyrenoid in the episome, above an ellipsoidal to rounded nucleus in the hyposome. The plate tabulation pattern of Po (pore plate), cp (cover plate), X (X-plate or canal plate), 5ꞌ, 3a, 7ꞌꞌ, 6c, 5s, 5ꞌꞌꞌ, 2ꞌꞌꞌꞌ was as for most other species of Heterocapsa. H. claromecoensis sp. nov. differs from all other species of Heterocapsa by the microarchitecture of two different types of organic body scales, which uniquely were different in height. Phylogenetic trees based on LSU and ITS rDNA sequences placed H. claromecoensis in a separate branch, with a sequence assigned to H. orientalis being the closest match based on LSU rDNA.

Multi-locus phylogeny and taxonomy of an unresolved, heterogeneous species complex within the genus Golovinomyces (Ascomycota, Erysiphales), including G. ambrosiae, G. circumfusus and G. spadiceus

BackgroundPrevious phylogenetic analyses of species within the genus Golovinomyces (Ascomycota, Erysiphales), based on ITS and 28S rDNA sequence data, revealed a co-evolutionary relationship between powdery mildew species and hosts of certain tribes of the plant family Asteraceae. Golovinomyces growing on host plants belonging to the Heliantheae formed a single lineage, comprised of a morphologically differentiated complex of species, which included G. ambrosiae, G. circumfusus, and G. spadiceus. However, the lineage also encompassed sequences retrieved from Golovinomyces specimens on other Asteraceae tribes as well as other plant families, suggesting the involvement of a plurivorous species. A multilocus phylogenetic examination of this complex, using ITS, 28S, IGS (intergenic spacer), TUB2 (beta-tubulin), and CHS1 (chitin synthase I) sequence data was carried out to clarify the discrepancies between ITS and 28S rDNA sequence data and morphological differences. Furthermore, the circumscription of species and their host ranges were emended.ResultsThe phylogenetic and morphological analyses conducted in this study revealed three distinct species named, viz., (1) G. ambrosiae emend. (including G. spadiceus), a plurivorous species that occurs on a multitude of hosts including, Ambrosia spp., multiple species of the Heliantheae and plant species of other tribes of Asteraceae including the Asian species of Eupatorium; (2) G. latisporus comb. nov. (≡ Oidium latisporum), the closely related, but morphologically distinct species confined to hosts of the Heliantheae genera Helianthus, Zinnia, and most likely Rudbeckia; and (3) G. circumfusus confined to Eupatorium cannabinum in Europe.ConclusionsThe present results provide strong evidence that the combination of multi-locus phylogeny and morphological analysis is an effective way to identify species in the genus Golovinomyces.

New species and new genus of Pseudohaploporinae (Digenea): Pseudohaploporus pusitestis sp. n. and Parahaploporus elegantus n. g., sp. n. (Digenea: Pseudohaploporinae) from Vietnamese mullet fish

Two new species of Pseudohaploporinae, Pseudohaploporus pusitestis sp. n. and Parahaploporus elegantus n. g., sp. n., are described from intestines of the Vietnamese mullet fish Moolgarda seheli and Osteomugil cunnesius, respectively. Pseudohaploporus pusitestis sp. n. differs from two known Pseudohaploporus species, P. vietnamensis and P. planiliza, by the absence of a diverticulate hermaphroditic duct and muscular sphincters at the proximal end of the hermaphroditic sac. Metrically, P. pusitestis sp. n. is close to P. vietnamensis and differs from this species and from P. planilizum by lower maximum sizes of most parameters. Parahaploporus elegantus n. g., sp. differs from representatives of Pseudohaploporus by the presence of a single testis and the armament of hermaphroditic duct and is morphologically close to trematodes of the genus Haploporus. However, P. elegantus n. g differs from all known Haploporus species from mugilids of the Indo-West Pacific by the structure of the armament of the hermaphroditic duct and also by size of body, organs and eggs. The validity of designating two new species and a new genus of trematodes is supported by ITS and 28S rDNA sequence data. Phylogenetic reconstructions showed that the new trematodes belong to the Pseudohaploporinae, which formed a well-supported cluster within the monophyletic Haploporidae.

Golovinomyces bolayi causes powdery mildew on Persian speedwell (Veronica persica) in Iran

During October–November of 2017 and 2018, powdery fungal masses were observed on leaves of Persian speedwell (Veronica persica) on the campus of Tabriz University, East Azerbaijan Province, Iran. Infected leaves were collected and subjected to a taxonomic study. Based on morphological characteristics the fungus was identified as Golovinomyces orontii species complex. A phylogeny inferred based on ITS-rDNA sequence data clustered our isolate as Golovinomyces bolayi. Golovinomyces bolayi has recently been segregated from the Golovinomyces orontii species complex. Golovinomyces bolayi can be differentiated from other closely related species based on morphological characteristics of conidiophores and ITS-rDNA phylogeny. This is the first report of this species from Iran on Persian speedwell.

Lichens or endophytes? The enigmatic genus Leptosillia in the Leptosilliaceae fam. nov. (Xylariales), and Furfurella gen. nov. (Delonicicolaceae).

Based on DNA sequence data, the genus Leptosillia is shown to belong to the Xylariales. Molecular phylogenetic analyses of ITS-LSU rDNA sequence data and of a combined matrix of SSU-ITS-LSU rDNA, rpb1, rpb2, tef1 and tub2 reveal that the genera Cresporhaphis and Liberomyces are congeneric with Leptosillia. Coelosphaeria fusariospora, Leptorhaphis acerina, Leptorhaphis quercus f. macrospora, Leptorhaphis pinicola, Leptorhaphis wienkampii, Liberomyces pistaciae, Sphaeria muelleri and Zignoëlla slaptonensis are combined in Leptosillia, and all of these taxa except for C. fusariospora, L. pinicola and L. pistaciae are epitypified. Coelosphaeria fusariospora and Cresporhaphis rhoina are lectotypified. Liberomyces macrosporus and L. saliciphilus, which were isolated as phloem and sapwood endophytes, are shown to be synonyms of Leptosillia macrospora and L. wienkampii, respectively. All species formerly placed in Cresporhaphis that are now transferred to Leptosillia are revealed to be non-lichenized. Based on morphology and ecology, Cresporhaphis chibaensis is synonymised with Rhaphidicyrtis trichosporella, and C. rhoina is considered to be unrelated to the genus Leptosillia, but its generic affinities cannot be resolved in lack of DNA sequence data. Phylogenetic analyses place Leptosillia as sister taxon to Delonicicolaceae, and based on morphological and ecological differences, the new family Leptosilliaceae is established. Furfurella, a new genus with the three new species, F. luteostiolata, F. nigrescens and F. stromatica, growing on dead branches of mediterranean fabaceous shrubs from tribe Genisteae, is revealed to be the closest relative of Delonicicola in the family Delonicicolaceae, which is emended. ITS rDNA sequence data retrieved from GenBank demonstrate that the Leptosilliaceae were frequently isolated or sequenced as endophytes from temperate to tropical regions, and show that the genus Leptosillia represents a widely distributed component of endophyte communities of woody plants.

Tamhinispora saraburiensis sp. nov. (Tubeufiaceae, Dothideomycetes) on bamboo in Thailand based on morphology & phylogenetic analysis

Tamhinispora saraburiensis sp. nov. was collected on decaying culms of bamboo in Saraburi Province, Thailand. Tamhinispora saraburiensis is characterized as dark brown to black, obpyriform, and dictyoseptate and has conidia with long appendages arising from the conidial tip. Phylogenetic analyses of the combined ITS and LSU rDNA sequence data demonstrated that this species formed distinctly from other species and that it belongs in the family Tubeufiaceae. Tamhinispora saraburiensis is therefore introduced here with a description, morphological illustrations and molecular data. A key to the species of Tamhinispora is provided.

Revisiting a poorly known powdery mildew species: Morphology and molecular phylogeny of Erysiphe longissima

Abstract Erysiphe longissima is a species of Erysiphe sect. Microsphaera endemic to China. Its asexual morph is poorly known and DNA sequence data are unrecorded since its first description in 1977. In 2016 and 2017, two powdery mildew samples with asexual and sexual morphs were collected on Caragana rosea at Jilin Agricultural University, Jilin Province, China. The morphological observations suggested that the two samples belonged to E. longissima. To study the phylogenetic position of E. longissima, a phylogenetic tree was constructed based on the combined ITS and 28S rDNA sequence data. This fungus was close to E. palczewskii in phylogeny and clearly distinguished from other species of Erysiphe sect. Microsphaera. To our knowledge, this is the first detailed record of the asexual morph and molecular data of E. longissima.