ITS Analysis Research Articles

Three New Species of Gongronella (Cunninghamellaceae, Mucorales) from Soil in Hainan, China Based on Morphology and Molecular Phylogeny.

The genus Gongronella is important in agriculture and industry by secreting various natural bioactive metabolites such as chitosanases and organic acids. During the most recent 8 years, a total of 14 new species have been described, remarkably enriching the diversity of this genus. In this study, we added three more new species to this valuable genus, based on a combination of morphological traits and phylogenetic information. Six strains of the genus Gongronella were isolated from soil collected in Hainan Province, China. Phylogenetic analyses of ITS and LSU rDNA sequences grouped these strains into three independent clades. According to their unique morphological characteristics, they were classified as G. multiramosa sp. nov., G. qichaensis sp. nov. and G. oleae sp. nov. The G. multiramosa was characterized by multiple branched sporangiophores and was closely related to G. pedratalhadensis. The G. qichaensis was characterized by obscure collars and closely related to G. butleri, G. hydei and G. banzhaoae. The G. oleae was characterized by the presence of oil droplets in the sporangiospores and was closely related to G. chlamydospora and G. multispora. Their descriptions and illustrations were provided, and their differences from morphological allies and phylogenetic-related species are discussed.

Alternaria alternata and Alternaria koreana, the Causal Agents of Leaf Spot in Celtis sinensis and Their Sensitivity to Fungicides

Celtis sinensis is a highly versatile species that is commonly cultivated in the southern regions of China. In June 2022, leaf spot disease was detected in C. sinensis in Nanjing, Jiangsu, China. Based on morphological characteristics, three isolates were determined to be of the Alternaria species. A phylogenetic analysis of combined ITS, GAPDH, TEF1-α, RPB2, and Alt a 1 sequences identified the three isolates we obtained as Alternaria alternata and A. koreana. We determined the pathogenicity of A. alternata and A. koreana in C. sinensis leaves using inoculation through in vivo experiments. Symptoms of inoculation onset in indoor pots were in accordance with those observed in the open country. A. alternata and A. koreana can grow at 15–35 °C, with the best growth occurring at 25 °C. The findings from fungicide susceptibility experiments demonstrated that A. alternata and A. koreana were the most sensitive to prochloraz, which could offer an effective approach for future prevention and control measures against A. alternata and A. koreana. This study provides the initial step toward further research on A. alternata and A. koreana as pathogens of C. sinensis and provides the stage for future control strategy development.

Tetraploa lignicola, a new freshwater fungal species from Yunnan Province, China

Two fungal strains were isolated from submerged decaying woods in the freshwater habitats, which are characterized by cylindrical, septate, verrucose, brown conidia, composed of four vertical columns of cells, 3-septate in each column, with four appendages at the base which are slightly tapering toward the apex and each with 5–10-septate. Multigene phylogenetic analyses of combined ITS, LSU, and SSU sequences data showed that the two isolates clustered into a separated clade in Tetraploa. Herein, we report it as a new hyphomycetous species, Tetraploa lignicola. The descriptions, illustrations, and a phylogenetic tree show the placement of T. lignicola, the comparisons between the new species and other morphologically similar taxa in Tetraploa are also provided.

Exploration of ITS region as DNA barcode for Kakothrips priesneri Pelikan phylogeny

The Thripidae family is a diverse group of insects with global distribution and significant economic importance as pests of agricultural crops. Accurate identification and classification of Thripidae species are critical for their effective management and control. To aid in this effort, DNA barcoding using the ITS gene region has proven to be an efficient and reliable tool for identifying and differentiating Thripidae species. The high variability rate of the ITS region makes it particularly effective for identifying and classifying closely related species, providing valuable insight for understanding and preserving the taxonomic diversity of the Thripidae family. The present study aims to explore the effectiveness of the ITS gene region for DNA barcoding of Kakothrips priesneri Pelikan species. For the first time, the ITS data obtained from this study showed the placement of Kakothrips priesneri in both common methods, using DNA barcoding (BLAST and MEGA), produced positive results for the ITS analysis, indicating that the region may be suitable for the Kakothrips species.

Evaluation of parasitic infections with histological and molecular detection in Anoles from St. Kitts

Anoles are a widely distributed group of reptiles that are also increasing in popularity as a pet animal. These species have been described as paratenic and intermediate hosts for many parasites of veterinary and medical importance. However, while human and animals such as cats and dogs may often interact with anoles, little research has been conducted in terms of assessing the full potential of anoles to harbor parasites of public and veterinary health significance. The current study analyzed the parasites of anoles captured and dissected in St. Kitts, West Indies, to describe the parasitic population within these reptiles and evaluate the potential role of anoles as intermediate/paratenic hosts. From March until May of 2019, samples from 11 anoles were collected following humane euthanasia and evaluated grossly, histologically, and molecularly for the presence of parasites. Histologic evaluation revealed adult ascarids, oxyurids, and spirurids within the stomach, as well as intraluminal adult ascarids within the intestine, and encysted larval trematodes within the intestinal wall. Molecular analysis based on 18S rRNA and ITS2 DNA markers for nematodes and trematodes, respectively, identified Physaloptera sp. (99.4–99.8% identical with Physaloptera turgida, Physaloptera mirandai, Physaloptera retusa, Physaloptera rara) and Platynosomum illiciens (99.7% identical). Phylogenetic analysis of ITS 2 sequences of P. illiciens from this study cladded monophyletically with the same species from different geographic areas and hosts. Our study highlights the importance of understanding the role anoles play in the life cycles of less well-studied parasites, and the potential of these animals to act as intermediate or paratenic hosts.

Myrmecridium hydei, a novel marine species from Thailand

Myrmecridium hydei was isolated from decaying branch of Areca species submerged in seawater from Prachuap Khiri Khan Province, Thailand. The new species is supported by phylogenetic analysis of combined ITS and LSU sequence data. Myrmecridium hydei formed a distinct clade from M. phragmitigenum, M. phragmiticola and M. sambuci with moderate statistical support. The species also differs from other Myrmecridium species by having a longer sympodial, straight or flexuous to geniculate-sinuous conidiophore and obovoid or fusoid to irregular, guttulate conidia without a wing-like sheath. Myrmecridium hydei is described as a novel species based on morphology and phylogeny.

Taxonomy and evolution history of two new litter-decomposing Ciliochorella (Amphisphaeriales, Sporocadaceae).

The genus Ciliochorella is a group of pestalotioid fungi, which typically occurs in subtropical and tropical areas. Species from the Ciliochorella genus play important roles in the decomposition of litter. In this study, we introduce two new species (Ciliochorellachinensissp. nov. and C.savannicasp. nov.) that were found on leaf litter collected from savanna-like vegetation in hot dry valleys of southwestern China. Phylogenetic analyses of combined LSU, ITS and tub2 sequence datasets indicated that C.chinensis and C.savannica respectively form a distinct clade within the Ciliochorella genus. The comparison of the morphological characteristics indicated that the two new species are well differentiated within this genus species. Analysis of the evolutionary history suggests that Ciliochorella originated from the Eurasian continent during the Paleogene (38 Mya). Further, we find that both new species can produce cellulase and laccase, playing a decomposer role.

Ecological, morphological and phylogenetic survey of Fomes fomentarius and F. inzengae (Agaricomycetes, Polyporaceae) co-occurring in the same geographic area in Central Europe

The phenomenon of cryptic species is widespread among various fungal lineages. Fomes inzengae (Ces. & De Not.) Cooke has been recently recognized as a South European kin of wood-decay basidiomycete F. fomentarius (L.) Fr. due to the problematic morphological identification of both species, their taxonomic status has been disputed. The aim of this research is to examine the distribution, host preferences, morphological characters, and phylogenetic relationships between F. fomentarius and F. inzengae in the South Moravian region in Czechia (Central Europe), where both species occur sympatrically. The results revealed the ecological preferences of Fomes spp. along an altitudinal gradient, while F. inzengae is a lowland taxon, F. fomentarius dominates at higher altitudes in forests with abundant Fagus sylvatica. The main contact zone of the two taxa is located in the upper-colline vegetation belt (elevation ca. 400‒550 m a.s.l.). The morphological analysis revealed that the basidiospore size, the width of skeletal hyphae in basidiomes, and the linear density of pores of both taxa are almost identical and can not be used for the identification of the two species. Multigene sequence analyses of ITS, LSU, RPB1, RPB2, and TEF1 markers confirmed that F. fomentarius and F. inzengae are phylogenetically distinct species. The relationship of F. inzengae and F. fomentarius to Globifomes graveolens and Hexagonia spp. is discussed.

Morpho-Phylogenetic Evidence Reveals Novel Species and New Records of Botryosphaeriaceae in China and Thailand.

Species in the Botryosphaeriaceae are common plant pathogens, endophytes, and saprobes found on a variety of mainly woody hosts. Botryosphaeriaceae is a high-profile fungal family whose genera have been subjected to continuous revisions in recent years. Surveys conducted during 2019 and 2020 on several decaying woody hosts (from dead arial twigs, branches, stems, bark, and seed pods) in China and Thailand revealed a high diversity of Botryosphaeriaceae fungi. Identification of 16 Botryosphaeriaceae isolates was carried out based on both morphological characteristics and phylogenetic analyses of combined ITS, LSU, tef1-α, and tub2 sequence data. Four novel species (Dothiorella ovata, Do. rosacearum, Do. septata, and Lasiodiplodia delonicis) and seven previously known species (Botryosphaeria fujianensis, Diplodia mutila, Di. seriata, L. crassispora, L. mahajangana, Macrophomina euphorbiicola and Sphaeropsis eucalypticola) were identified while new hosts and geographical records were reported. This study indicates that the fungal family Botryosphaeriaceae seems to be common and widespread on a broad range of hosts in China and Thailand.

Computed Tomography-Based Quantitative Texture Analysis and Gut Microbial Community Signatures Predict Survival in Non-Small Cell Lung Cancer.

This study aims to combine computed tomography (CT)-based texture analysis (QTA) and a microbiome-based biomarker signature to predict the overall survival (OS) of immune checkpoint inhibitor (ICI)-treated non-small cell lung cancer (NSCLC) patients by analyzing their CT scans (n = 129) and fecal microbiome (n = 58). One hundred and five continuous CT parameters were obtained, where principal component analysis (PCA) identified seven major components that explained 80% of the data variation. Shotgun metagenomics (MG) and ITS analysis were performed to reveal the abundance of bacterial and fungal species. The relative abundance of Bacteroides dorei and Parabacteroides distasonis was associated with long OS (>6 mo), whereas the bacteria Clostridium perfringens and Enterococcus faecium and the fungal taxa Cortinarius davemallochii, Helotiales, Chaetosphaeriales, and Tremellomycetes were associated with short OS (≤6 mo). Hymenoscyphus immutabilis and Clavulinopsis fusiformis were more abundant in patients with high (≥50%) PD-L1-expressing tumors, whereas Thelephoraceae and Lachnospiraceae bacterium were enriched in patients with ICI-related toxicities. An artificial intelligence (AI) approach based on extreme gradient boosting evaluated the associations between the outcomes and various clinicopathological parameters. AI identified MG signatures for patients with a favorable ICI response and high PD-L1 expression, with 84% and 79% accuracy, respectively. The combination of QTA parameters and MG had a positive predictive value of 90% for both therapeutic response and OS. According to our hypothesis, the QTA parameters and gut microbiome signatures can predict OS, the response to therapy, the PD-L1 expression, and toxicity in NSCLC patients treated with ICI, and a machine learning approach can combine these variables to create a reliable predictive model, as we suggest in this research.

Taxonomy and phylogeny of Paradictyoarthrinium salsipaludicola sp. nov. (Paradictyoarthriniaceae, Pleosporales) from mangroves

A new fungal species found on decaying wood in a mangrove habitat is described. The analysis of combined LSU, ITS, and rpb2 sequence data revealed that it belongs to the family Paradictyoarthriniaceae (Pleosporales). Maximum likelihood and Bayesian analyses were used to determine the taxon’s phylogenetic affinities. The novel species is typical of Paradictyoarthrinium in its gregarious, black, powdery colonies with macronematous conidiophores and unevenly dictyoseptate, muriform, green to brown conidia. The new taxon is compared to other Paradictyoarthrinium species, and a detailed morphological description and illustration are provided.

Colletotrichum truncatum causing anthracnose of Catharanhthus roseus in Brazil

Catharanthus roseus is an important plant group used for landscaping purposes and is cultivated in several locations in Brazil. However, anthracnose is compromising its development. Despite the importance of the disease, it is not known which Colletotrichum species is responsible for causing anthracnose. This study aimed to identify Colletotrichum isolates associated with anthracnose of vinca. It evaluated morphological markers and performed multilocus phylogenetic analysis. According to the morphological analyses and multilocus phylogeny analysis of ACT, GAPDH, ITS, and TUB gene regions, we identified the isolates as Colletotrichum truncatum. This study is the first to report C. truncatum in C. roseus worldwide.

Perenniporiopsis srijayewardenepurana sp. nov. and validation of Pyrrhoderma nigrum in Sri Lanka

Polypores, a diverse group of macrofungi belonging to Basidiomycota, are widespread in tropical and subtropical regions worldwide. In this study, four polyporoid specimens were collected in the Western Province of Sri Lanka. Based on multilocus phylogenetic analyses of combined ITS and nLSU sequence data, along with morphological characteristics, the specimens were identified as Pyrrhoderma nigrum (Pyrrhoderma “nigra”) and a novel species of a recently described genus Perenniporiopsis. The new species was named P. srijayewardenepurana, while P. nigrum, which has not been validly published previously, is validated in this publication.

Trolliella Herzog, a new synonym of Pylaisia Schimp. (Pylaisiaceae, Bryophyta) and Pylaisia euendostoma (Herzog) Frank Müll., Jan Kučera & S.He, comb. nov.

ABSTRACT Introduction: Trolliella euendostoma Herzog is the single species of a monospecific genus and we know of no molecular study since its description and we know of only one report since its discovery. Therefore, we decided to compare a recently collected Myanmar specimen with the type material deposited in JE and obtain molecular data from the new material with which to explore the phylogenetic affinities of this species. Methods: In addition to a morphological examination, an analysis of ITS and trnF–trnS sequence data from the newly collected Myanmar specimen of Trolliella euendostoma was carried out. Phylogenetic affinities were assessed using Bayesian inference and maximum likelihood analyses. Results and discussion: Molecular affinities and morphological features of Trolliella euendostoma suggest that the species should be excluded from the Sematophyllaceae and transferred to Pylaisia Schimp. in the Pylaisiaceae. Consequently, we propose the new combination Pylaisia euendostoma (Herzog) Frank Müll., Jan Kučera & S.He, select the lectotype for T. euendostoma, and additionally synonymise Pylaisia kunisawae (Ando) Ochyra (≡ Pylaisiella kunisawae Ando) with P. euendostoma.

Vanderbylia kashmiriana sp. nov. (Polyporales, Polyporaceae) from Azad Jammu & Kashmir, Pakistan

A novel wood rotting species of genus Vanderbylia occurring at the base of Zanthoxylum armatum is described from mixed vegetation of District Bhimber, Azad Jammu & Kashmir, based on phylogenetic analyses of ITS and LSU sequences. Further, distinct morpho-anatomical characters confirmed the novelty of the species as Vanderbylia kashmiriana. The taxon is characterized by pileate and corky basidiomata, a dimitic hyphal system with peculiar, extensively branched arboriform hyphae, globose to sub-globose to ellipsoid, hyaline, apiculate, strongly dextrinoid basidiospores, sub-clavate to obovoid basidia with the variable number of sterigmata (1–4), clavate to obclavate to ventricose to tubular, apically encrusted, granular, clamped cystidia. This is the first described species of the genus Vanderbylia from Pakistan.

Wongia suae sp. nov., a lignicolous freshwater fungus from Yuanjiang (Red River) Basin, China

Diversity of lignicolous freshwater fungi in Yuanjiang (Red River) basin, Yunnan, China is currently being studied. Three fresh collections of Papulosaceae were collected. Wongia suae sp. nov. and W. fusiformis were identified based on morphology and phylogenetic analysis of combined ITS, LSU, rpb2, SSU and tef1-α sequence data. The detailed descriptions and illustrations of these two species are provided, as well as the morphological comparison with similar taxa.

Lignicolous Freshwater Fungi from Plateau Lakes in China (I): Morphological and Phylogenetic Analyses Reveal Eight Species of Lentitheciaceae, Including New Genus, New Species and New Records.

During the investigation of lignicolous freshwater fungi in plateau lakes in Yunnan Province, China, eight Lentitheciaceae species were collected from five lakes viz. Luguhu, Qiluhu, Xingyunhu, Cibihu, and Xihu lake. Based on morphological characters and phylogenetic analysis of combined ITS, LSU, SSU, and tef 1-α sequence data, a new genus Paralentithecium, two new species (Paralentithecium suae, and Setoseptoria suae), three new records (Halobyssothecium phragmitis, H. unicellulare, and Lentithecium yunnanensis) and three known species viz. Halobyssothecium aquifusiforme, Lentithecium pseudoclioninum, and Setoseptoria bambusae are reported.

Trichoderma species from rhizosphere of Oxalis corymbosa release volatile organic compounds inhibiting the seed germination and growth of Echinochloa colona

Trichoderma spp. have the potential to act as biocontrol agents for regulating plant growth. In this study, Trichoderma spp. were isolated from the rhizosphere soil of Oxalis corymbosa in sugarcane fields, and six strains were identified as T. koningiopsis, T. afroharzianum, T. atroviride, T. virens and T. asperelloides through morphology and phylogenetic analysis of ITS and tef 1 gene sequences. The activity of volatile organic compounds (VOCs) emitted by Trichoderma spp. was determined using the double-plate chamber method, revealing that five strains significantly inhibited the seed germination and growth of Echinochloa colona. Subsequently, SMPE-GC–MS and Venn analyses unveiled the presence of five common VOCs, namely phenylethyl alcohol, 2,4,6-trichloroanisole, hexadecanoic acid ethyl ester, 10(E),12(Z)-conjugated linoleic acid, and linoleic acid ethyl ester, within Trichoderma spp.. Independently, five compounds were evaluated for their inhibitory effect on the shoot growth of E. colona. Additionally, the results demonstrated that linoleic acid ethyl ester exhibited the highest inhibitory activity, while hexadecanoic acid ethyl ester was the least effective. The inhibitory effect of 2,4,6-trichloroanisole increased with concentration. Hormone analysis results indicated that VOCs influenced the concentration of abscisic acid, resulting in the maintenance of dormancy in E. colona. This study provides compelling evidence for the potential use of Trichoderma as bioherbicides.

First Report of Nigrospora Species Causing Leaf Spot on Olive (Olea europaea L.)

Leaf spot symptoms were spotted in two olive orchards in Istria and in Kvarner Gulf, Croatia. Fungal species from three representative isolates (P13 LECIII, R18 BI, JA20 NP) have been morphologically characterized based on the colony and conidial characteristics. Several techniques were performed for inducing the sporulation of the JA20 NP isolate. Only PDA + banana medium was successful. PCR was conducted for ITS, TUB, and EF1α gene regions. Phylogenetic analyses were performed using internal transcribed spacer, beta-tubulin, and translation elongation factor 1-alpha sequence data. Three types of tests were conducted: a pathogenicity test on detached leaves, on detached and scratched leaves, and on olive seedlings. Ultimately, from the morphological characterizations, DNA sequence analysis of ITS, TUB, and EF1α gene regions, and phylogenetic analysis, these species were identified as Nigrospora gorlenkoana Novobr., Nigrospora osmanthi Mei Wang & L. Cai, and Nigrospora philosophiae-doctoris M. Raza, Qian Chen & L. Cai. This is the first report of Nigrospora species causing leaf spot on olive trees and the first report of Nigrospora philosophiae-doctoris as a plant pathogen. Fungal leaf diseases in conditions that are favorable for infection and disease development can lead to a decrease in the yield and olive oil quality. Therefore, it is necessary to conduct further research and the monitoring of fungal leaf diseases.

First Report of Cladosporium cladosporioides Causing Leaf Spot of Aralia cordata var. continentalis in Korea.

Aralia cordata var. continentalis (Kitag), commonly known as Japanese spikenard, is an upright herbaceous perennial medicinal plant effective in relieving pain. It is also consumed as a leafy vegetable. Leaf spots and blight symptoms on A. cordata resulting in defoliation were observed in July 2021 from a research field with a disease incidence of nearly 40-50% from 80 plants in Yeongju, Korea. Brown spots with chlorotic halos first appear on the upper leaf surface (Fig. 1A). In the later stage, spots enlarge and coalesce; resulting in the leaves to dry-off (Fig. 1B). To isolate the causal agent, small pieces of diseased leaves displaying the lesion were surface-sterilized by 70% ethanol for 30 s and rinsed twice with sterile distilled water (SDW). Later, the tissues were crushed in a sterile 2.0-ml Eppendorf tube with a rubber pestle in SDW. The suspension was serially diluted and spread on potato dextrose agar (PDA) medium, incubated at 25°C for 3 days. A total of 3 isolates were obtained from the infected leaves. Pure cultures were obtained by the monosporic culture technique (Choi et al. 1999). After 2 to 3 days of incubation with a 12-h photoperiod, the fungus initially produced gray mold colonies in olive color, and the edges of the mold appeared white with a velvety texture after 20 days (Fig. 1C). Microscopic observations revealed small, single-celled, rounded, and pointed conidia that measured 6.67 ± 0.23 µm × 4.18 ± 0.12 µm (length × width) (n=40 spores) (Fig. 1D). On the basis of its morphology, the causal organism was identified as Cladosporium cladosporioides (Torres et al. 2017). For molecular identification, pure colonies of three single-spore isolates were used for DNA extraction. A fragment of the ITS, ACT, and TEF1-α were amplified using the primers ITS1/ITS4 (Zarrin et al. 2016), ACT-512F/ACT-783R, and EF1-728F/EF1-986R, respectively, by PCR (Carbone et al. 1999). The DNA sequences from all three isolates (GYUN-10727, GYUN-10776, and GYUN-10777) were identical. The resulting ITS (ON005144), ACT (ON014518), and TEF1-α (OQ286396) sequences from the representative isolate GYUN-10727 were 99 to 100% identical to the C. cladosporioides (ITS: KX664404, MF077224; ACT: HM148509; TEF1-α: HM148268, HM148266). The phylogenetic dendrogram was constructed from the comparative analysis of ITS, ACT, and TEF1-α gene sequences, showing the relationship between Cladosporium cladosporioides and related Cladosporium species (Fig. 2). The isolate GYUN-10727 has been deposited in Korean Agricultural Culture Collection (KACC 410009), and used as a representative strain in this study. For the pathogenicity test, healthy fresh leaves (3 leaves per plant) of 3-months-old A. cordata plants in pots were spray inoculated with conidial suspensions (1 × 10⁴ conidia/mL) of GYUN-10727, which was obtained from a 7-day-old PDA culture. Leaves sprayed with SDW were considered as control. After 15 days of incubation at 25°C ± 5°C under greenhouse conditions, necrotic lesions were observed on the inoculated A. cordata leaves, while control leaves did not develop any disease symptoms. The experiment was performed twice with three replicates (pots) per treatment. The pathogen was re-isolated from the symptomatic A. cordata leaves, but not from control plants, to fulfill Koch's postulates. The re-isolated pathogen was identified by PCR. Cladosporium cladosporioides has been reported to cause diseases in sweet pepper (Krasnow et al. 2022) and garden peas (Gubler et al. 1999). To our knowledge, this is the first report of C. cladosporioides causing leaf spots of A. cordata in Korea. The identification of this pathogen will help develop strategies to efficiently control the disease in A. cordata.