ITS Loci Research Articles

First report of Pyrrhoderma noxium from rice in northern Thailand

First report of Pyrrhoderma noxium from rice in northern Thailand

A taxonomic assessment of novel and remarkable fungal species in Didymosphaeriaceae (Pleosporales, Dothideomycetes) from plant litter.

Fungal taxonomy has a long history and changed significantly in the last few decades. Most recent studies have witnessed morphology combined with DNA-based molecular analyses as the main research tool for fungal species identification. During field surveys, some interesting Didymosphaeriaceae species were found from plant litter in China and Thailand. Morphology combined with phylogenetic analyses (Bayesian and maximum likelihood) of ITS, LSU, SSU, tef1-α, and tub2 loci was used to identify fungal taxa. In this article, three new species and six new host records are described. The new species, Montagnula acaciae, Paraconiothyrium zingiberacearum, and Paraphaeosphaeria brachiariae, can be distinguished from other species of the respective genera based on their distinct size differences (ascomata, asci, and ascospores) and DNA sequence data. The new host records, Montagnula jonesii, Paraconiothyrium fuckelii, Spegazzinia deightonii, and S. tessarthra are reported from Ficus benjamina, Dimocarpus longan, Hedychium coronarium, and Acacia auriculiformis respectively, for the first time. Also, Paraconiothyrium archidendri and P. brasiliense are reported for the first time from Magnolia sp. in China. Moreover, Paraconiothyrium rosae is synonymized under P. fuckelii based on close phylogeny affinities and morphological characteristics. In-depth morphological descriptions, micrographs, and phylogenetic trees are provided to show the placement of new taxa.

Molecular characterization and phylogenetic relationships in Asiatic Vigna using ITS and cpDNA loci

Molecular characterization and phylogenetic relationships in Asiatic Vigna using ITS and cpDNA loci

Metagenomics of Toenail Onychomycosis in Three Victorian Regions of Australia.

Onychomycosis is a fungal disease of the nail that is found worldwide and is difficult to diagnose accurately. This study used metagenomics to investigate the microbiology of 18 clinically diagnosed mycotic nails and two normal nails for fungi and bacteria using the ITS2 and 16S loci. Four mycotic nails were from Bass Coast, six from Melbourne Metropolitan and eight from Shepparton, Victoria, Australia. The mycotic nails were photographed and metagenomically analysed. The ITS2 sequences for T. rubrum and T. interdigitale/mentagrophytes averaged over 90% of hits in 14/18 nails. The high abundance of sequences of a single dermatophyte, compared to all other fungi in a single nail, made it the most likely infecting agents (MLIA). Trichophyton rubrum and T. interdigitale/mentagrophytes were found in Bass Coast and Shepparton while only T. interdigitale/mentagrophytes was found in Melbourne. Two nails with T. interdigitale/mentagrophytes mixed with high abundance non-dermatophyte moulds (NDMs) (Aspergillus versicolor, Acremonium sclerotigenum) were also observed. The two control nails contained chiefly Fusarium oxysporum and Malassezia slooffiae. For bacteria, Staphylococcus epidermidis was in every nail and was the most abundant, including the control nails, with an overall mean rate of 66.01%. Rothia koreensis, Corynebacterium tuberculostearicum, and Brevibacterium sediminis also featured.

New species and records of Neomassaria, Oxydothis and Roussoella (Pezizomycotina, Ascomycota) associated with palm and bamboo from China.

Several micro fungi were gathered from bamboo and palm in Guizhou Province, China. In morphology, these taxa resemble Neomassaria, Roussoella and Oxydothis. Multi-gene phylogenetic analyses based on combined ITS, LSU, SSU, rpb2 and tef1 loci confirmed that two are new geographical records for China, (viz. Roussoellasiamensis, Neomassariafabacearum), while two of them are new to science (viz. Oxydothisfortunei sp. nov. and Roussoellabambusarum sp. nov.). The stromata of Roussoellabambusarum are similar to those of R.thailandica, but its ascospores are larger. In addition, multi-gene phylogenetic analyses show that Oxydothisfortunei is closely related to O.inaequalis, but the J- ascus subapical ring as well as the ascospores of O.inaequalis are smaller. Morphological descriptions and illustrations of all species are provided.

Fusarium falciforme and F. oxysporum causing postharvest fruit rot of watermelon (Citrullus lanatus) in Malaysia: A first report

Watermelon is one of the fruit crops being produced in large scale (9000 ha in the year of 2021) in Malaysia. However, fungal diseases are a major factor in suppressing yield. In November 2019, field surveys in the states of Kelantan, Terengganu, and Penang found harvested watermelon showing fruit rot disease symptoms. In the present study, we delineated the pathogens causing these symptoms using morphological observations, molecular methods, and pathogenicity tests. Morphological characterization, DNA sequencing and phylogenetic analysis using ITS and TEF loci revealed that two isolates, S9 and S11, isolated from symptomatic fruits were Fusarium falciforme and F. oxysporum, respectively. Pathogenicity tests confirmed the both Fusarium species caused the observed symptoms, fulfilling Koch's postulates. This is the first report of these Fusarium species as the pathogens of watermelon fruit rot in Malaysia. Findings from the present study contribute to the knowledge of watermelon diseases in Malaysia, provide an essential foundation for development of effective pre-harvest and postharvest disease management strategies to curb disease spread.

DNA-metabarcoding analyses of the grapevine wood fungal community in the Krasnodar Region and Crimea

Grapes are an economically important agricultural plant. Studies of the Grapevine microbiota and rhizosphere have become increasingly important in the last decade. The importance of such research is also supported by the fact that grapes are a perennial, long-used crop.
 In this work, we present the results of a DNA-metabarcoding analysis of the fungal community of grape wood, collected from vineyards in the Krasnodar Territory and the Southern Coast of Crimea, and considered approaches to the analysis of DNA-metabarcoding data. Classifier is Nave base (sklearn) based on machine learning is more informative metagenomic data classifier than BLAST+ (local alignment) and Vsearch (global alignment). Analysis of the ITS locus revealed the largest number of taxa, which was confirmed for all types of classifiers used in the study. Primers for the ITS locus showed a high specificity of fungal DNA in comparison with the LSU and SSU loci. The most common genera in the fungal community are Acidea, Alternaria, Cladosporium and Fusarium. Significant differences were revealed in the assessment of alpha and beta diversity in the analysis of samples from different regions. This article presents an analysis of the wood grapevine fungal community and ways to ASV classification. This study is the first to describe the endophytic fungal communities of the Krasnodar Territory and the Crimea vines using the analysis of DNA metabarcoding data.

Multigene phylogeny, morphology, and pathogenicity trials reveal novel Cytospora species involved in perennial canker disease of apple trees in Iran

In recent years, canker and die-back diseases have become a growing threat for the productivity and longevity of apple orchards in Iran. In this study, 131 Cytospora isolates were recovered from symptomatic tissues of apple trees in apple orchards of Iran. Multigene phylogenetic inference based on combined sequence data of ITS, act, rpb2, and tef1-α loci, supplemented with morphological characteristics and pathogenicity assay revealed four novel Cytospora species which were described as C. avicennae, C. azerbaijanica, C. ershadii, and C. iranica, and four known species, namely C. chrysosperma, C. parasitica, C. paratranslucens, and C. sorbicola. Also, C. sorbicola is newly reported on apple trees worldwide. Koch's postulates were fulfilled to confirm that all eight species in this study were pathogenic on apple trees in Iran, among which C. sorbicola was the most intensive species. The results of this study further highlight rich diversity among Cytospora species occurring on apple trees, revealing several novel Cytospora species on this host. The host range, geographical distribution, and economic significance of novel species on apple industry remains to be studied.

Yeast Diversity in the Qaidam Basin Desert in China with the Description of Five New Yeast Species.

The Qaidam Basin is the highest and one of the largest and driest deserts on Earth. It is considered a mars analog area in China. In contrast to numerous studies concerning its geology, geophysical, and chemistry, relatively few studies have reported microbial diversity and distribution in this area. Here, we investigated culturable yeast diversity in the northeast Qaidam Basin. A total of 194 yeast strains were isolated, and 12 genera and 21 species were identified, among which 19 were basidiomycetous yeasts. Naganishia albida, N. adeliensis, and Filobasidium magnum were the three most dominant species and were distributed in thirteen samples from eight locations. Five new species (Filobasidium chaidanensis, Kondoa globosum, Symmetrospora salmoneus, Teunia nitrariae, and Vishniacozyma pseudodimennae) were found and described based on ITS and D1D2 gene loci together with phenotypic characteristics and physiochemical analysis. Representative strains from each species were chosen for the salt-tolerant test, in which species showed different responses to different levels of NaCl concentrations. Further, the strain from soil can adapt well to the higher salt stress compared to those from plants or lichens. Our study represents the first report of the yeast diversity in the Qaidam Basin, including five new species, and also provides further information on the halotolerance of yeasts from the saline environment in mars analog.

Individual-level specialisation and interspecific resource partitioning in bees revealed by pollen DNA metabarcoding.

It is increasingly recognised that intraspecific variation in traits, such as morphology, behaviour, or diet is both ubiquitous and ecologically important. While many species of predators and herbivores are known to display high levels of between-individual diet variation, there is a lack of studies on pollinators. It is important to fill in this gap because individual-level specialisation of flower-visiting insects is expected to affect their efficiency as pollinators with consequences for plant reproduction. Accordingly, the aim of our study was to quantify the level of individual-level specialisation and foraging preferences, as well as interspecific resource partitioning, in three co-occurring species of bees of the genus Ceratina (Hymenoptera: Apidae: Xylocopinae), C. chalybea, C. nigrolabiata, and C. cucurbitina. We conducted a field experiment where we provided artificial nesting opportunities for the bees and combined a short-term mark-recapture study with the dissection of the bees’ nests to obtain repeated samples from individual foraging females and complete pollen provisions from their nests. We used DNA metabarcoding based on the ITS2 locus to identify the composition of the pollen samples. We found that the composition of pollen carried on the bodies of female bees and stored in the brood provisions in their nests significantly differed among the three co-occurring species. At the intraspecific level, individual females consistently differed in their level of specialisation and in the composition of pollen carried on their bodies and stored in their nests. We also demonstrate that higher generalisation at the species level stemmed from larger among-individual variation in diets, as observed in other types of consumers, such as predators. Our study thus reveals how specialisation and foraging preferences of bees change from the scale of individual foraging bouts to complete pollen provisions accumulated in their nests over many days. Such a multi-scale view of foraging behaviour is necessary to improve our understanding of the functioning of plant-flower visitor communities.

Fungi associated with dead branches of Magnolia grandiflora: A case study from Qujing, China.

As a result of an ongoing survey of microfungi associated with garden and ornamental plants in Qijing, Yunnan, China, several saprobic fungal taxa were isolated from Magnolia grandiflora. Both morphological and combined SSU, LSU, ITS, tef1, and rpb2 locus phylogenetic analyses (maximum-likelihood and Bayesian analyses) were carried out to identify the fungal taxa. Three new species are introduced in Pleosporales, viz., Lonicericola qujingensis (Parabambusicolaceae), Phragmocamarosporium magnoliae, and Periacma qujingensis (Lentitheciaceae). Botryosphaeria dothidea, Diplodia mutila, and Diplodia seriata (in Botryosphaeriaceae) are reported from Magnolia grandiflora for the first time in China. Angustimassarina populi (Amorosiaceae) is reported for the first time on M. grandiflora from China, and this is the first report of a member of this genus outside Europe. Shearia formosa is also reported for the first time on M. grandiflora from China.

The root-associated arbuscular mycorrhizal fungal assemblages of exotic alien plants are simplified in invaded distribution ranges, but dominant species are retained: A trans-continental perspective.

Arbuscular mycorrhizal fungi (AMF) provide crucial support for the establishment of plants in novel environments. We hypothesized that the OTU/genus richness and diversity of soil- and root-associated AMF associated with alien plant species in their exotic ranges are lower than those in their native ranges. We examined the root-associated and soil-dwelling AMF of 11 invasive plant species in their native and exotic ranges in the United States and Europe by DNA sequencing of the ITS2 locus. Examined root-associated AMF assemblages were simplified, which manifested as the loss of several AMF genera in the exotic ranges of the plants. These fungal assemblages were also characterized by greater dominance and simplification of the fungal assemblages. The dominant fungal genera were present regardless of whether their host plants were in their native or exotic ranges. Interestingly, both the native and invaded soils hosted diverse local AMF assemblages. Therefore, alien plant invasions were not limited to soils with low AMF diversity. Some AMF taxa could be context-dependent passengers rather than drivers of alien plant invasions. Further studies should identify functions of AMF missing or less abundant in roots of plants growing in exotic ranges.

Genomic Characteristics and Comparative Genomics Analysis of Parafenestella ontariensis sp. nov.

A new ascomycetous species of Parafenestella was isolated from Acer negundo during the survey of diseased trees in Southern Ontario, Canada. The species is morphologically similar to other taxa of Cucurbitariacea (Pleosporales). The new species is different from the extant species in the morphology of ascospores, culture characteristics and molecular data. The novel species is described as Parafenestella ontariensis sp. nov. based on morphological and multi-gene phylogenetic analyses using a combined set of ITS, LSU, tef1 and tub2 loci. Additionally, the genome of P. ontariensis was sequenced and analyzed. The phylogenomic analysis confirmed the close relationship of the species to the fenestelloid clades of Cucurbitariaceae. The comparative genomics analysis revealed that the species lifestyle appears to be multitrophic (necrotrophic or hemi-biotrophic) with a capability to turn pathogenic on a corresponding plant host.

Bithyniid snails as hosts of Opisthorchiidae and Notocotylidae in the south of Western Siberia, Russia.

Opisthorchiasis and related infections are persistent and substantial public health burdens from eastern Europe to southeastern and northern Asia. Snails of the family Bithyniidae act as first intermediate hosts not only for the trematodes of the family Opisthorchiidae but also for those of the family Notocotylidae. There are certain morphological similarities between the aforementioned trematode cercariae. In order to find natural local foci of opisthorchiasis, which are established primarily according to the presence of infected bithyniid snails at the area under examination, it is crucial to correctly identify the rediae and cercariae of the trematodes. The aim of our investigation was to evaluate the role of bithyniid snails in the transmission of Opisthorchiidae and Notocotylidae in ecosystems in the south of Western Siberia. We have been studying the process of bithyniid snail dissemination in Western Siberia and examining their infection by trematodes from 1994 until now. A total of 16,213 bithyniid snails in 23 water bodies (in 28 localities) of four major basins situated in the Novosibirsk region were inspected for trematode infestation. Long-term research has been conducted in the Kargat river estuary and the Ob river floodplain for 15 and 25years, respectively. In both water bodies, the prevalence of notocotylid and opisthorchiid trematodes was positively correlated with the global land-ocean temperature index. Trematode parthenitae were identified if there were mature cercariae capable of leaving the shell of the host snail on their own. Identification to the species of opisthorchiid cercariae was verified using molecular genetic analysis methods targeting ITS2 locus. Opisthorchis felineus and Metorchis bilis, two opisthorchiid species that are potentially perilous to human health, were found in bithyniids in the Novosibirsk region. The mean prevalence of infection with notocotylid trematodes in bithyniid snails was higher than the corresponding prevalence of infection with opisthorchiid trematodes. The results of this study can be used to identify and predict natural foci of epidemiologically and/or epizootically dangerous diseases.

Molecular Characterization of 18S rDNA, ITS-1, ITS-2, and COI from Eimeria christenseni and E. arloingi in Goats from Shaanxi Province, Northwestern China.

Simple SummaryThis study first determined the 18S rDNA, ITS-1, ITS-2, and COI sequences of E. christenseni and E. arloingi. The lengths of the 18S rDNA, ITS-1, ITS-2, and COI were 1790 bp, 403 bp, 584 bp, and 1268 bp for E. arloingi and 1796 bp, 386 bp, 565 bp, and 1268 bp for E. christenseni, respectively. Furthermore, phylogenetic analyses based on the 18S rDNA, ITS-1, ITS-2, and COI loci were conducted to assess the relationship of E. christenseni, E. arloingi, and other Eimeria species in ruminants.Coccidiosis caused by Eimeria is one of the most common and significant diseases in goats, leading to serious economic losses in the development of the goat industry. Although several genetic loci, such as 18S rDNA, ITS-1, ITS-2, and COI, have been applied in the molecular characterization of Eimeria in chicken, rabbits, turkey, and wildlife, little is known about these molecular markers of Eimeria in goats. In the present study, we isolated purified oocysts of highly pathogenic Eimeria arloingi and Eimeria christenseni from fecal samples of goats in Shaanxi province, China, and then subjected these purified oocysts to genomic DNA isolation, PCR amplification, and sequencing of 18S rDNA, ITS-1, ITS-2, and COI loci of Eimeria arloingi and Eimeria christenseni. Finally, the obtained sequences were used for phylogenetic analysis of Eimeria species in goats and other livestock. The lengths of the 18S rDNA, ITS-1, ITS-2, and COI were 1790 bp, 403 bp, 584 bp, and 1268 bp for E. arloingi and 1796 bp, 386 bp, 565 bp, and 1268 bp for E. christenseni, respectively. The phylogenetical analysis based on 18S rDNA indicated that E. christenseni and E. arloingi were the most closely related to ovine Eimeria, followed by E. bovis, E. ellipsoidalis, and E. zuernii from cattle. The phylogenetical analysis based on ITS-1 and ITS-2 could not effectively distinguish ovine Eimeria from caprine Eimeria. The phylogenetical analysis based on the COI locus could effectively distinguish between Eimeria species from goats and cattle, but it was ineffective in distinguishing between Eimeria species from sheep and goats. To the best of our knowledge, this is the first characterization of 18S rDNA, ITS-1, ITS-2, and COI in E. arloingi and E. christenseni; it can provide useful genetic markers for molecular epidemiological and population genetic studies on E. arloingi and E. christenseni in goats and contribute to the prevention and control of goat coccidiosis.

Taxonomic and Phylogenetic Characterizations Reveal Four New Species, Two New Asexual Morph Reports, and Six New Country Records of Bambusicolous Roussoella from China.

During the ongoing investigation of bambusicolous ascomycetous fungi in Yunnan, China, 24 specimens belonging to the family Roussoellaceae were collected and identified based on morphological features and phylogenetic support. Maximum-likelihood (ML) analyses and Bayesian analyses were generated based on the combined data set of ITS, LSU, tef1, and rpb2 loci. The phylogenetic analyses revealed four novel lineages in Roussoella s. str.; thus, we introduced four new species viz., Roussoella multiloculate sp. nov., R. papillate sp. nov., R. sinensis sp. nov., and R. uniloculata sp. nov. Their morphological characters were compared with the known Roussoella taxa, which lack sequence data in the GenBank. Asexual morphs of R. kunmingensis and R. padinae were recorded from dead bamboo culms in China (from the natural substrates) for the first time. Neoroussoella bambusae, Roussoella japanensis, R. nitidula, R. padinae, R. scabrispora, and R. tuberculate were also reported as the first records from China. All new taxa are described and illustrated in detail. Plates are provided for new reports.

The Diversity of Lichenized Trentepohlioid Algal (Ulvophyceae) Communities is Driven by Fungal Taxonomy and Ecological Factors.

Trentepohliales are a group of both free-living and lichenized algae, with most diversity occurring in tropical regions. Recent studies showed that the abundance of lichens with a trentepohlioid photobiont has been increasing in temperate habitats, probably because of global warming, which makes them an interesting study case. A detailed molecular study of the diversity of lichenized Trentepohliales, epiphytic as well as epilithic, was performed in three forests of north-western Europe. Additional samples of lichens of the Arthoniales order (associating essentially with a trentepohlioid photobiont) from other European regions and from other continents were also sequenced. A total of 195 algal sequences were obtained. Phylogenetic analyses with rbcL and ITS loci were performed and associations between phylogenetic distances of photobionts and ecological factors (substratum, climate or Wirth indices, mycobiont taxonomy, and geographic location) were tested by variation partitioning and phylogenetic signal analyses. The high number of rbcL algal haplotypes found in some lichens or on different substrata revealed that the Trentepohliales diversity in extratropical regions was underestimated. The phylogenetic patterns showed selectivity of some photobionts in their fungal partner choice and vice-versa, while others were linked with several haplotypes. Photobionts seemed to be less selective than mycobionts. The main factors influencing lichenized algal community were climate and mycobiont species. Coevolution between mycobionts and photobionts as well as switching between free living and lichenized lifestyles appeared to drive the evolution of Trentepohliales and might explain the high cryptic diversity observed, which might be changing in some regions due to climate change.

Metagenomic analysis of fungal phytopathogens in birch planting material

The data of the is presented. The technology allows to identify the entire spectrum of organisms in an experimental sample per one cycle, while separate study for each potentially contained component previously was required. An integrated approach is submitted as a method for early diagnosis of causative agents of the main fungal diseases during of silver birch (Betula pendula Roth) and downy birch (Betula pubescens Ehrh.) plants. It based on an estimation of the size of internal transcribed spacers (ITS1 and ITS2) in the 18S-5.8S-28S rDNA gene cluster. Identification of the dominant fungi species phytopathogenic for birch under in planta conditions is possible because of the the specificity of size of the rDNA operon internal transcribed spacers, which is constant for most micromycete species, as well as the possibility of electrophoretic analysis of the ITS1 and ITS2 loci.

Brunneosporopsis yunnanensis gen. et sp. nov. and Allocryptovalsa xishuangbanica sp. nov., New Terrestrial Sordariomycetes from Southwest China.

Three fungal taxa were collected on dead branches of wood during fieldwork in Sichuan and Yunnan Provinces, China. The new generic name Brunneosporopsis gen. nov. and species B. yunnanensis sp. nov. are introduced for a novel taxon characterized by globose to subglobose and dark olivacous-brown conidia. Phylogenetic analyses based on combined LSU, SSU and tef1-α loci strongly support the monophyly of this taxon and place it in the subclass Diaporthomycetidae. It could not be assigned to any currently recognized families in the subclass and was, therefore, placed in the Diaporthomycetidae genera incertae sedis. A second taxon represents a new species in Allocryptovalsa based on an analysis of the sequence datasets of ITS and btub loci of the novel, brown-spored sexual morphic species. This taxon is described here as A. xishuangbanica sp. nov. An interesting hypocrealean fungus producing synnemata, Stilbocrea gracilipes, was collected from dead wood of an unknown host from Sichuan Province and is reported here, with asexual morph from both the host and culture as well as LSU, ITS, tef1-α, rpb2 and rpb1 sequence data.

Multi-methodological approach for the Quality assessment of Senecionis scandentis Herba (Qianliguang) in the herbal market.

We set forth to assess the quality of an herbal medicine sold in Hong Kong called Qianliguang by employing a multi-methodological approach. The quality is set by its identity, chemical composition, and bioactivities, among others. Qianliguang (Senecionis scandentis Herba, Senecio scandens Buch.-Ham. ex D.Don) has known antibacterial properties. However, it is poisonous and overconsumption can result in liver damage. Eighteen Qianliguang samples were purchased from herbal shops at various districts in Hong Kong. Samples were first authenticated organoleptically. DNA barcoding at the psbA-trnH, ITS2, and rbcL loci was then conducted to confirm the species. HPLC-UV was performed to screen for the presence of the chemical compounds and to quantify the flavonoid hyperoside. UPLC-MS was used to quantify the amount of the toxic pyrrolizidine alkaloid (PA) adonifoline. Microdilution assay was performed to show the antibacterial effect on Streptococcus aureus and S. pneumoniae. Results showed that five samples were found to be substituted by species belonging to the genus Lespedeza; four samples were mixtures containing not only Qianliguang but also Achyranthes aspera L., Lonicera confusa DC., or Solanum nigrum L. HPLC-UV showed that only ten contained enough hyperoside to meet the standard requirement. In addition, nine samples had adonifoline that exceeded the toxicity standard requirement. In the microdilution assay, samples containing Qianliguang showed inhibition on S. aureus and S. pneumoniae, while among the five Lespedeza sp. samples the antibacterial effects on S. aureus were not detectable; only one sample showed inhibition to S. pneumoniae. Our study illustrated the necessity of using a multi-methodological approach for herbal medicine quality assessment. We also showed that Qianliguang samples in the Hong Kong market were either toxic or adulterated. It is therefore essential to improve the quality control of Qianliguang and probably other herbs in the herbal market.