Large Subunit rDNA Research Articles

Morpho-phylogenetic evidence reveals novel species and new records of Nigrograna (Nigrogranaceae) associated with medicinal plants in Southwestern China

During a survey of saprobic fungal niches in Southwestern China, eighteen ascomycetous collections of Nigrograna (Nigrogranaceae, Pleosporales, Dothideomycetes) were found on dead branches of medicinal plants. These taxa were characterized and identified based on morphological and culture characteristics, and phylogenetic analyses of a combined the internal transcribed spacer region of rDNA (ITS), nuclear large subunit rDNA (28S, LSU), RNA polymerase second-largest subunit (rpb2), nuclear small subunit rDNA (18S, SSU), and translation elongation factor 1-alpha (tef1-α) sequence dataset also confirmed their placement. As a result, four novel species, namely Nigrograna camelliae, N. guttulata, N. longiorostiolata and N. neriicola were described. Additionally, four new host records of N. acericola, N. magnoliae, N. oleae and N. thymi were introduced. Furthermore, this study addresses the taxonomic status of N. trachycarpi, proposing its synonymy under N. oleae. Detailed illustrations, descriptions and informative notes for each newly identified taxon and novel host record are provided in this study.

Genetic variability of Myxobolus nagaraensis (Bivalvulida: Myxobolidae) infecting freshwater gobies Rhinogobius Gill 1859 (Gobiiformes: Oxudercidae) from rivers in Japan

Myxobolus nagaraensis is a myxozoan parasite first reported in freshwater gobies (Rhinogobius spp.) from the Nagara River, Gifu Prefecture, Japan. Myxospores of M. nagaraensis form plasmodia in the visceral cavities of gobies, commonly presenting as distended abdomens. Although Rhinogobius is a common fish genus in Japan, details of M. nagaraensis, including genetic information, remain unknown. We compared the nucleotide sequences of the ribosomal RNA gene (rDNA) of M. nagaraensis from three different host species (R. fluviatilis, R. nagoyae, and R. similis) caught in three different rivers in Japan (Sakai, Sagami, and Kaname). The ITS region (ITS-1, 5.8S rDNA, and ITS-2) and large subunit (LSU) rDNA exhibited 49 and 55 variable sites, respectively. The highest nucleotide diversity was observed in the ITS region (0.00962), whereas that of the LSU rDNA was 0.00187. Differences in host species, rather than rivers, were a significant factor for genetic variation in both the ITS region (62.58%; P < 0.001) and LSU rDNA (55.22%; P < 0.01). Significant genetic variation was observed in M. nagaraensis from R. similis compared to R. fluviatilis (P < 0.001) or R. nagoyae (P < 0.001) from the same river. Such details are valuable for understanding parasite dispersal and its ecological impact on Rhinogobius hosts.

Two novel members of Onygenales, Keratinophyton kautmanovae and K. keniense spp. nov. from soil

Two new Keratinophyton species, K. kautmanovae sp. nov. and K. keniense sp. nov., isolated from soil samples originating from two different geographical and environmental locations (Africa and Europe) are described and illustrated. Phylogenetically informative sequences obtained from the internal transcribed spacer (ITS) region and the nuclear large subunit (LSU) rDNA, as well as their unique phenotype, fully support novelty of these two fungi for this genus. Based on ITS and LSU combined phylogeny, both taxa are resolved in a cluster with eight accepted species, including K. alvearium, K. chongqingense, K. hubeiense, K. durum, K. lemmensii, K. siglerae, K. submersum, and K. sichuanense. The new taxon, K. kautmanovae, is characterized by clavate, smooth to coarsely verrucose conidia, absence of arthroconidia, slow growth at 25 °C, and no growth at 30 °C, while K. keniense is morphologically unique with a high diversity of conidial shapes (clavate, filiform, globose, cymbiform and rhomboid). Both species are described based on their asexual, a chrysosporium-like morph. While the majority of hitherto described Keratinophyton taxa came from Europe, India and China, the new species K. keniense represents the first reported taxonomic novelty for this genus from Africa.

An updated LSU database and pipeline for environmental DNA identification of arbuscular mycorrhizal fungi

Recent work established a backbone reference tree and phylogenetic placement pipeline for identification of arbuscular mycorrhizal fungal (AMF) large subunit (LSU) rDNA environmental sequences. Our previously published pipeline allowed any environmental sequence to be identified as putative AMF or within one of the major families. Despite this contribution, difficulties in implementation of the pipeline remain. Here, we present an updated database and pipeline with (1) an expanded backbone tree to include four newly described genera and (2) several changes to improve ease and consistency of implementation. In particular, packages required for the pipeline are now installed as a single folder (conda environment) and the pipeline has been tested across three university computing clusters. This updated backbone tree and pipeline will enable broadened adoption by the community, advancing our understanding of these ubiquitous and ecologically important fungi.

First Data on the Investigation of Gut Yeasts in Hermit Beetle (Osmoderma barnabita Motschulsky, 1845) Larvae in Lithuania.

In this study, yeasts from the gut of O. barnabita larvae were isolated and molecularly identified. It is worth noting that this research provides the first analysis of the gut yeast community in O. barnabita larvae in Lithuania, which is a significant contribution to the field. Two hermit-like L3-praepupa instars were collected from a decaying oak log in Lithuania. The isolation, morphology, biochemistry, and physiology of the yeast isolates were characterized using standards commonly employed in yeast taxonomy studies. The isolates were identified by sequencing the large subunit (26S) rDNA (D1/D2 domain of the LSU). All gut compartments were colonized by the yeast. A total of 45 yeast strains were obtained from the gut of both O. barnabita larvae, with 23 strains originating from Larva 1, 16 strains from Larva 2, and 6 strains from the galleries. According to our identification results of the 45 yeast strains, most of the species were related to Ascomycota, with most of them belonging to the Saccharomycetales order. Yeasts of the genera Candida, Debaryomyces, Meyerozyma, Priceomyces, Schwanniomyces, Spencermartinsiella, Trichomonascus, and Blastobotrys were present in gut of O. barnabita larvae. Species of the Trichosporonales order represented the Basidiomycota phylum.

First Report ofCoptis Wilt Disease Caused by Plectosphaerella cucumerina in China.

Coptis (Coptis chinensis) belongs to the Ranunculaceae family, the rhizomes used in traditional Chinese medicine. Since 2021, an uncommon stem and leaf wilt disease, with an average disease incidence of 70%~90%, has been observed in Guangdong and Guangxi provinces. The early wilt symptoms were observed on older leaves and stems, and the whole seedling wilted and died. The rhizome of the diseased seedlings changed in color, became necrotic, and rotted. Symptomatic roots and stems were surface-sterilized with 70% ethanol for 30 s, followed by 0.2% NaClO for 2-3 min, rinsed in sterile water three times, and then placed on potato dextrose agar (PDA) at 25℃for 14 days. Fungal growth was observed, and six isolates with similar morphology were obtained. The 14-day-old colonies on PDA were buff with few aerial hyphae and slimy surfaces. Aerial hyphae were sparse with simple or branched conidiophores. Conidia were hyaline, smooth, ovoid, septate or aseptate, and 5.77 to 9.53 × 2.15 to 3.32 µm (n = 50). Three of the six isolates were subjected to further analysis. The genomic DNA of three isolates (CCF1-1, CCF1-2, CCF1-3) was extracted using Axygen MAG-FRAG- I-50 (Axygen Bio-Tek) for molecular identification. Partial sequences of the internal transcribed spacer of rDNA (ITS) and large subunit rDNA (LSU) were amplified using the primers ITS1/4 and LR5F/LROR, respectively (Vilgalys and Hester 1990). Their sequences were aligned by MEGA X (Kumar et al., 2018), and the sequences of each region showed 100% sequence similarity among our isolates. A BLAST search of ITS and LSU sequences (accession nos. ON377369, ON428244) showed that both regions had the highest nucleotide similarities (99.43 to 99.89%) to the Plectosphaerella cucumerina strains. Based on morphological and molecular analyses, the isolates were identified as P. cucumerina (Palm et al. 1995). The pathogenicity of our isolates CCF1-1, CCF1-2, CCF1-3 was tested on ten 2-month-old healthy seedlings of coptis, respectively. For the seedlings, 30 ml of fungal conidial suspension (1×106 conidia/ml) or sterile water, as control, were poured into their root area. Conidia suspension were prepared from 14-day-old cultures on PDA by eluting with sterilized water. The seedlings were incubated at 25°C and 75% relative humidity under a 12-h/12-h light/dark cycle. The test was repeated three times. After 20 days, only seedlings inoculated with P. cucumerina exhibited symptoms similar to those diseased seedlings in the field. The control seedling had no symptoms. The morphologically similar fungus was re-isolated from the tested seedlings, thus fulfilling Koch's postulates. Based on molecular, morphological, and pathogenic properties, P. cucumerina is the causal fungal pathogen of coptis wilt disease. Previously, P. cucumerina has been related to wilt disease in strawberry and Chinese cabbage (Yang et al. 2023; Gao et al. 2022), but to our knowledge, this is the first report of P. cucumerina causing wilt disease on coptis in China. Coptis wilt disease tends to occur in a warm and rainy environment, and strengthening the detection and quarantine of seedlings is the key to preventing the occurrence and spread of the disease.

First Report of Rust Caused by Puccinia sasicola on Corylopsis coreana in South Korea.

Korean winter hazel (Corylopsis coreana) is an endemic species of the South Korea (Seo et al. 2016; Kim et al. 2021), which is cultivated as an ornamental plant in this country, but also in China and Japan (Yoon et al. 2016). In July 2022, typical symptoms of a rust disease were observed on C. coreana at Jirisan National Park (35°22'07.7"N 127°34'57.7"E) in Namwon, South Korea. Spermogonia were epiphyllous, densely grouped, pale brown or orange-yellow, round, and 0.23 - 0.38 × 0.19 - 0.41 mm in size. Aecia were hypophyllous, mostly densely grouped, yellow or pale orange, resembling small wart-like galls, and 0.04 - 0.06 × 0.89 - 1.68 mm in size. Aeciospores were hyaline, mostly angularly globose, ellipsoid or oblong-ellipsoid, and 17.8 - 25.2 × 15 - 26.5 μm (average 19.2 × 19.1 μm; n=50) in size. Aeciospore walls were echinulate-verrucose, and 1.1 - 2.2 µm (average 1.7 µm; n=50) in thickness. In December 2022, dark brown telia were observed on the lower surface of Sasa borealis leaves near C. coreana. Telia were mostly scattered but often compacted, brown to dark brown, round, and 1.5 - 1.95 × 1.24 - 1.55 mm in size. Teliospores were either one- or two-celled with a long tapering apex, and light brown to brown in color. One-celled teliospores were globose, and 95.1 - 186.5 × 20.5 - 36.4 μm (average 136.4 × 27.7 μm; n=50) in size. Two-celled teliospores were ellipsoid-cylindrical, and 111.4 - 180.3 × 13.5 - 32.6 μm (average 149 × 21.1 μm; n=50) in size. Side walls of teliospores were golden and 2.2 - 5.5 µm thick (average 3.5 µm; n=50), and pedicels were hyaline, measuring 150 - 300 μm long. Uredinial stage was not observed. Disease symptomology and pathogen morphology were mostly consistent with that of Puccinia sasicola reported in Japan (Hino. 1955). For phylogenetic analysis, genomic DNA was extracted from the aeciospores collected from C. coreana and the teliospores collected from S. borealis. The internal transcribed spacer (ITS) rDNA and the large subunit (LSU) rDNA regions were amplified using ITS5u/ITS4rust (Pfunder and Schürch 2001) and LRust1R/LRust3 (Beenken et al. 2012) primers, respectively. Both sequences were identical for the spores collected from the two different hosts. The sequences were deposited in GenBank (PP171665, PP174211 [ITS], PP171709, PP174356 [LSU]). A GenBank BLAST search revealed 89.53% and 96.78% similarity with Puccinia kusanoi (KX610657) and Puccinia sp. (MT7298241) for ITS and LSU sequences, respectively. In maximum-likelihood phylogenetic analysis of ITS and LSU sequences, the isolates from C. coreana and S. borealis formed a separate clade from other Puccinia species. To test Koch's postulates, leaf disks with telia from S. borealis were directly attached to the adaxial surface of six healthy C. coreana leaves with tape. As controls, healthy S. borealis leaf disks were attached to the adaxial surface of six C. coreana leaves. After four weeks, four inoculated leaves developed small yellow wart-like galls on the abaxial surface, while the control leaves remained symptom-free. The isolates obtained from the inoculated leaves had identical sequences to the original isolate. There are no publicly available sequences for P. sasicola, nor did we find any sequences that match our Puccinia samples. Nevertheless, based on morphological characteristics and life cycle, our isolates closely matched with the previous description of P. sasicola by Hino (1955). To our knowledge, this is the first report of P. sasicola causing leaf rust in C. coreana in South Korea.

First Report of Alternaria gossypina Causing Stem Base Rot on Passilora edulis in Guangxi, China.

Passion fruit (Passilora edulis), known as the "king of fruit juices", is popular in southern China (Yuan et al. 2019). Stem base rot is a devastating disease of passion fruit commonly caused by several Fusarium spp. (Zakaria, 2022). In July 2022, typical symptoms of stem base rot were observed in a poorly managed "Qinmi No. 9" Golden passion fruit orchard in Jingxi (23°13'10"N, 106°5'23"E). The disease incidence had reached 40% (n=200) in the survey. Symptoms included ulceration and mutilation at the stem base, making the plants prone to breakage when pulled, wilting and drooping leaves above ground, and severe cases leading to the entire plant withering and dying. Fourteen plants with obvious symptoms were collected. Thin sections of plant tissue were cut from junction of sickness and health of stem, sterilized with ethanol and sodium hypochlorite, and placed on PDA medium at 28°C. Sixty fungal strains were obtained, 90% of which was identified as Fusarium based on morphology. 80% of Fusarium were F. oxysporum species complex (FOSC), but pathogenicity experiment showed all FOSC were weakly pathogenic. However, two severely pathogenic fungi with similar morphology but distinct from Fusarium were identified from the same plant. The representative strain C11 was selected for further study. C11 demonstrated a rapid growth rate, reaching a 90 mm diameter colony on PDA cultured at 25°C for 7 days. The colony displaced a round, flat shape with an overall light brown front, and cottony gray or light brown mycelium, while the reverse side was dark brown. Conidia production was observed as typically occurred in multiple chains after 14 days culture on OA medium, with round, oval or straight rod-like brown conidia ranging in size from 5.74-23.42×14.67-67.22, featuring 1-8 transverse septa and 0-3 mediastinum (Figure S1). For molecular identification, the internal transcribed spacer (ITS, OR616614), translation elongation factor 1-alpha (TEF, OR633298), alternaria major allergen (Alt a1, OR633294) gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH, OR633295), RNA polymerase subunit II gene (RPB2, OR633297), 18S Small subunit rDNA (SSU, OR616608) , 28S Large Subunit rDNA (LSU, OR616615), the KOG1058 gene regions (OR633296) and an approximately segment of the anonymous noncoding region (OPA10-2, OR633299) were amplified from C11 (Liu et al. 1999, Li et al. 2023, Andrew et al. 2009), and deposited in GenBank with accession number shown in the brackets. Phylogenetic trees were constructed in MEGA11 after splicing by BioEdit (Figure S3). Combining morphology and molecular analyses, C11 was identified as Alternaria gossypina (Woudenberg et al. 2015). To test the pathogenicity, the base of the seedling stem (20cm in height) of 50 healthy "Tainong No. 1" variety of purple passion fruit, which was more susceptible to stem-base rot, was puncture wound with needles, inoculated with 6 mm diameter colonies of fungi, and then wrapped in wet cotton (Ángel et al. 2018). Ten healthy seedlings inoculated with PDA were used as controls. These plants were cultured in an artificial greenhouse at 30±5℃with 80±5% humidity. After 15 days, the plants inoculated with C11 exhibited symptoms similar to those in the field, whereas the controls remained healthy. A. gossypina was reisolated from the diseased plant stems, with the morphology and GAPDH sequence consistent with the inoculated (Figure S1, S2). This is the first report of passion fruit stem rot caused by A. gossypina. This finding will aid in the prevention and control of stem rot in passion fruit.

First Report of Neofusicoccum parvum Causing Seedpod Blight on Lotus (Nelumbo nucifera) in China.

Lotus (Nelumbo nucifera Gaertn.) is a widely cultivated plant in China, and the fruit lotus variety has a high economic value attributed to the exquisite flavor of its fresh seeds. During the summer of 2023, an unidentified blight was observed affecting lotus seedpods in Jiande City, Zhejiang province, with approximately 65% of seedpods impacted in a 130-hectare area. The initial symptoms included dark purple spots on the lotus seedpod surface, which gradually expanded over time. After 5 to 7 days, the entire seedpod turned black, withering, and rendering the lotus seeds inedible. To identify the causal agent, tissues from symptomatic seedpods were excised and disinfected in 75% ethanol for 60 s, and washed twice in sterile distilled water. The disinfected symptomatic tissues (5 × 5 mm) were plated on potato dextrose agar (PDA), incubated at 25 ℃, transferred hyphal tips to obtain pure isolates after 3 days. Fungal colonies exhibiting Botryosphaeriaceae morphology were isolated from 33% of the samples (n = 15). Pure cultures were grown on PDA for both morphological and molecular identification. The colonies displayed a white aerial mycelium, turning olivaceous grey after 7 days. Pycnidia were produced within 3 weeks on PDA with added sterilized healthy lotus seedpod pieces on the surface. Conidia were hyaline, unicellular, ellipsoidal, 12.65 to 20.72 × 3.92 to 9.38 μm in size (mean 16.67 × 6.24 μm, n = 100). To determine the fungal species, genomic DNA was extracted from one representative isolate (ZJUP1112-1), to amplify four gene loci through polymerase chain reactions (PCR): rDNA internal transcribed spacer (ITS) with primers ITS1/ITS4, rDNA large subunit (LSU) with LR0R/LR5, the translation elongation factor 1-alpha gene (tef1) with EF1-728F/EF1-986R, and β-tubulin gene (tub2) with Bt2a/Bt2b. The PCR products were Sanger sequenced in Zhejiang Shangya biotechnology co., LTD, and the resulting sequences were assembled and deposited in GenBank (ITS: OR740546; LSU: OR740547; tef1: OR776996; tub2: OR776997). BLAST searches indicated the highest nucleotide sequence identity with the reference strains of Neofusicoccum parvum CMW 9081 (ITS: 98.8%, AY236943; LSU: 100%, AY928045; tef1: 99.6%, AY236888; tub2: 99.3%, AY236917). Multi-locus phylogenetic analyses revealed that isolate ZJUP1112-1 formed a highly supported clade with N. parvum. Pathogenicity tests were performed on healthy lotus seedpods using mycelial plugs (5 mm diameter) from actively growing colonies of ZJUP1112-1 that were placed onto the front and side of the seedpods (6 each). Controls received PDA plugs. Treated seedpods were wrapped with parafilm and incubated at 25 ℃ and the experiment was repeated three times. After 5 days, dark purple lesions were observed on the inoculated seedpods, whereas controls remained symptomless. The same isolate was recovered from the margin of resulting lesions and confirmed by morphology, thus fulfilling Koch's postulates. N. parvum is a polyphagous pathogen causing blights and fruit rot on multiple economically important fruit crops, such as cacao (Puig et al. 2019), walnut (Chen et al. 2019), pistachio (Lopez-Moral et al. 2020), chestnut (Seddaiu et al. 2021), blueberry (Spetik et al. 2023) and mango (Polizzi et al. 2022), among others. To the best of our knowledge, this is the first report of N. parvum causing seedpod blight on lotus seedpods in China, which contributes to a better understanding of the pathogens affecting this plant species in China.

Description of Pauronemoides siddiqii n. gen., n. sp. (Tylenchomorpha, Paurodontidae), a morphological and molecular phylogenetic study

Summary Three populations of Pauronemoides siddiqii n. gen., n. sp. were recovered from three different locations in northwestern and northern Iran from bark and soil samples. The new genus was described and illustrated based upon morphological and molecular characteristics. Pauronemoides n. gen. belongs to the family Paurodontidae and is characterised by the medium-sized body, asymmetrical knobs in stylet base, stem-like extension into intestine, post-vulval uterine sac (PUS) absent or rudimentary, different tail tip shape between sexes, and bursa enveloping tail or reaching to the tail end. The type species, Pauronemoides siddiqii n. gen., n. sp., is mainly characterised by 752-1281 μm body length in females and 660-1085 μm in males, six equally-sized lips, stylet 9-11 μm long with asymmetrical knobs, six incisures in lateral fields forming five bands, three of which (two outers and one central) are wider, and variably lengthened stem-like extension projecting into the intestine. Vulva situated posteriorly (V = 87.7-91.7%), PUS absent. Conical tail with widely rounded end, having a small mucron at tip in females, males with conical tail ending to a narrower tip and bursa enveloping tail completely. The type species of the new genus was compared with all known species of the genera Prothallonema, Paurodontus, Paurodontoides and Paurodontella. Pauronemoides siddiqii n. gen., n. sp. resembles Prothallonema macrocellum and P. mucronatum by having the bulb extension penetrating into the intestine, dimorphism in tail shape of both sexes and bursa enveloping tail, and Paurodontella iranica by tail shape dimorphism between sexes and bursa ending near tail tip. Therefore, the above-mentioned species are transferred to the new genus as Pauronemoides macrocellus n. gen., n. comb. (= Prothallonema macrocellum), P. mucronatus n. gen., n. comb. (= Prothallonema mucronatum) and P. iranicus n. gen., n. comb. (= Paurodontella iranica). In addition to morphological comparisons, molecular phylogenetic analyses were performed using two sequences of small subunit (SSU), and three partial sequences of D2-D3 expansion segments of the large subunit (LSU) rDNA, and the resulting topologies were discussed.

First report of Puccinia xanthii causing rust disease on Xanthium orientale in Korea.

Puccinia xanthii Schw. is a microcyclic rust fungus, first found on Xanthium strumarium Lour in North Carolina, the United States. This rust fungus is native to the continental United States, Hawaii, Mexico, and the West Indies (Arthur 1934). It has become notoriously invasive and is now distributed in the Europe (Bulgaria, France, Hungary, Italy, Romania, Spain, and the former Yugoslavia), India, Indonesia, Australia, and South Africa (Parmelee 1969; Alcorn 1976; Wahyuno 2012). In East Asia, the fungus has been reported in Japan (Hiratsuka et al. 1992) and China (Zhao et al. 2014) but not in Korea. It has been reported mainly on the invasive weeds Xanthium and Ambrosia species. In addition, it rarely occurs on sunflowers (Helianthus spp.) in Australia (Alcorn 1976), South Africa (Pretorius et al. 2000), and North America (Gulya and Charlet 2002). In Korea, rust disease symptoms caused by a Puccinia fungus were first found on X. orientale L. at the roadside of Okcheon-gun, Chungcheongbuk-do (36 27'95.428"N 127 66'26.378"E) in October 2021 and were repeatedly observed in the same site in 2022. The similar symptom was additionally found on X. orientale in Yesan-gun, Oct. 2022. The symptoms were brown spots on round chlorotic haloes on the adaxial leaf surface and dark brown pustules on the abaxial leaf surface. Telia were brown to dark brown, round, mostly grouped, 0.28-0.61 mm in diameter, and mainly formed on the abaxial leaf surface but sometimes on the adaxial leaf surface. Teliospores were two-celled, pedicellate, and measured 37.6-110 × 12.4-21.5 μm in size; the wall was yellowish or almost colorless, smooth, 1.2-2.6 μm thick at the sides, and up to 7.4 μm thick at the apex. The morphological characteristics of the teliospores were identical to those of P. xanthii described by Arthur (1934) and Parmelee (1969). Based on phylogenetic analyses (e-Xtra 2) of the internal transcribed spacer (ITS) and partial large subunit (LSU) rDNA extracted from the teliospores, they were identified as P. xanthii. BLAST analysis showed that the sequences had high homologies (over 99.82%) with the reference strains of P. xanthii (EF635903 and KX999896). The representative specimens were preserved at the Animal and Plant Quarantine Agency Herbarium (PQK211005 for Okcheon-gun isolate and PQK220913 for Yesan-gun) and the sequences were deposited in GenBank (OR958716 and OR958692). A pathogenicity test was performed by dropping a suspension of germinating teliospores and basidiospores onto the adaxial leaf surfaces of apparently healthy X. orientale plants in Oct. 2022, using the isolate PQK220913 (OR958692). The three inoculated plants were placed together with three controls treated with only distilled water, in the dark at saturated humidity for 24 hours in an isolated greenhouse. After two weeks, typical rust symptoms were observed in the three infected plants, whereas no symptoms appeared in the control plants (e-Xtra 1). The causal fungus was identified as P. xanthii based on host relationships, successful experimental inoculation, morphological characteristics, and sequence similarity of partial DNA fragments. To our knowledge, this is the first report of P. xanthii on X. orientale in Korea. P. xanthii was additionally confirmed on X. orientale in Gumi-si, Boeun-gun, Seongju-gun, Naju-si, and Gunsan-si in 2023, indicating its wide distribution in Korea. It is expected that P. xanthii could be a candidate as a biological agent for controlling the invasive weed, X. orientale.

First report of Phaeosphaeria caricicola causing leaf spot on Belamcanda chinensis in China.

Belamcanda chinensis (L.) Redouté, a member of the Iridaceae family, is globally well-known for its medicinal value as clearing away heat, detoxifying, detumescence and pain (Qin 2000). In 2021, spots were observed on 40% B. chinensis leaves and about 28 disease index in Wanzhou District (30°32'N; 108°22'E) of Chongqing. Initial symptoms appeared as circular yellow white, sunken spots lesions, and then expanded into irregular lesions, the center of the spots was beige, external layer was light brown and surrounded by yellow halo. Symptomatic leaf tissues (5 × 5 mm) were cut from the infected margin, surface sterilized with 75% ethanol for 1 min, washed with 3% sodium hypochlorite for 3 min, rinsed three times with sterile water, placed on potato dextrose agar (PDA) medium incubated at 25°C for 7 days in the dark, forty isolates with similar morphology were obtained. Three isolates (SG9、SG20 and SG33) was selected for subsequent research. Colonies color changed from beige to light brown color after 14 days on PDA medium. Fungal colonies transformed from beige to brown at the edges after 28 days and light brown on top. Ascomata dark brown, ellipsoidal to globose 116.6 to 253.3 × 89.6 to 172.6 μm in diamensions. Asci stipitate, cylindrical with obtuse ends, and 69.1 to 114.7 × 10.2 to 24.1 μm (n = 30) in size, with eight overlapping linearly biseriate ascospores. Ascospores brown, narrowly fusiform, straight or slightly curved with three transversely septate, slightly constricted at septa, and 9.7 to 12.6 × 27.6 to 32.6 μm (n = 30). These characteristics are consistent with Phaeosphaeria sp. reported by Quaedvlieg et al in 2013. DNA was extracted from representative isolates. The internal transcribed spacer (ITS) region, the large subunit rDNA (LSU), the small subunit rDNA (SSU) and the RNA polymerase II second largest subunit (RPB2) genes were amplified for Polymerase chain reaction (PCR) by used ITS1/ITS4, LR5/LROR, NS1/NS4, and RPB2-5f2/RPB2-7cr primers (White et al. 1990; Vilgalys et al. 1990; Qi M W. et al. 2008; De G. J. et al. 1992). The sequences were submitted to NCBI GenBank: SG-G9 (ITS, OR701701; LSU, OR701699; SSU, OR701700; RPB2, OR738464); SG-G20 (ITS, OQ748032; LSU, OQ780728; SSU, OQ780723; RPB2, OQ779979); SG-G33 (ITS, OQ748033; LSU, OQ780729; SSU, OQ780722; RPB2, OQ779980). A phylogenetic analysis revealed a 99% similarity to the Phaeosphaeria caricicola CBS 603.86 (ITS, KF251182; LSU, GQ387590; SSU, GQ387529; RPB2, KF252189) sequences. Mycelial agar plugs (5-mm diameter) from a 7-day-old PDA culture of a fungal isolate were placed onto pinpricked leaves of three two-year-old B. chinensis plants. While the sterile PDA plugs inoculated in pinpricked leaves of B. chinensis as controls. Inoculated plants were placed in a greenhouse at 25°C and remained 95±1% relative humidity. The inoculated leaves of treatment developed symptoms after 20 days, whereas no symptoms occurred on controls, fulfilling Koch's postulates. The experiments were repeated three times. The fungus was re-isolated and was identical to original isolate by morphologically and molecularly. As far as we know, P. caricicola can cause diseases on carex plants and has been found in Switzerland. This is the first report of P. caricicola causing leaf spot on B. chinensis in China. Along with recording the occurrence of this disease, plant disease management strategies need to be established to reduce losses.

Unraveling the Life Cycle of Nyssopsora cedrelae: A Study of Rust Diseases on Aralia elata and Toona sinensis.

Rust disease poses a major threat to global agriculture and forestry. It is caused by types of Pucciniales, which often require alternate hosts for their life cycles. Nyssopsora cedrelae was previously identified as a rust pathogen on Toona sinensis in East and Southeast Asia. Although this species had been reported to be autoecious, completing its life cycle solely on T. sinensis, we hypothesized that it has a heteroecious life cycle, requiring an alternate host, since the spermogonial and aecial stages on Aralia elata, a plant native to East Asia, are frequently observed around the same area where N. cedrelae causes rust disease on T. sinensis. Upon collecting rust samples from both A. elata and T. sinensis, we confirmed that the rust species from both tree species exhibited matching internal transcribed spacer (ITS), large subunit (LSU) rDNA, and cytochrome oxidase subunit III (CO3) mtDNA sequences. Through cross-inoculations, we verified that aeciospores from A. elata produced a uredinial stage on T. sinensis. This study is the first report to clarify A. elata as an alternate host for N. cedrelae, thus providing initial evidence that the Nyssopsora species exhibits a heteroecious life cycle.

First Report of Leaf Spot Caused by Arthrinium arundinis on Amaranthus hybridus in Korea.

Amaranthus hybridus (=A. patulus), often called green amaranth, is an annual herbaceous plant of the Amaranthaceae. This plant is considered a harmful weed in the agricultural context of North America and has expanded its distribution to Asia and Europe. In Korea, it has become a problematic invasive issue, leading to economic losses due to reduced crop yields and rising weed management costs (Park et al., 2014), although its seeds and young leaves are edible and frequently consumed. In October 2020, we observed leaf spot symptoms on A. hybridus plants that were growing within perilla farms (Perilla frutescens var. japonica) located in Damyang (35°14'07"N, 126°59'40"E), Korea, with a disease incidence of 20 to 30% of the inspected plants. The initial signs appeared as grey to brown dots on the leaves, which gradually expanded into irregular, brown patches with a diameter of 2-3 cm. A single spore was isolated from the diseased leaf under a dissecting microscope, placed onto a 2% water agar plate, and incubated in darkness at 25°C for three days. Pure cultures were obtained by transferring single hyphal tips onto potato dextrose agar (PDA) plates. Five single-spore isolates were the same in the cultural and morphological examination, and a representative isolate (P309) was preserved at the Korean Agricultural Culture Collection (KACC49813), Korea. Colonies appeared light gray to white with regular margins and reached 4 to 5 cm in diameter after a week. After two weeks, black patches of spores were often visible in the aerial mycelia. Conidiophores were brown to pale brown, often branched, thick-walled, and measured 6.8 × 2.7 µm (n = 30). Conidia were single-celled, dark brown, globose to ellipsoid, and measured 6.8 × 5.0 µm (n = 50), with a ratio of length/width of 1.1 to 1.6 (n = 50). These morphological characteristics matched those of Arthrinium arundinis (Crous et al., 2013). For molecular identification, genomic DNA was extracted from conidia and mycelia on two-week-old PDA culture of the KACC49813. PCR was performed for the internal transcribed spacer (ITS) (primers ITS1/ITS4, White et al. 1990), the large subunit (LSU) rDNA (primers LROR/LR5, Vilgalys et al. 1990), the beta-tubulin gene (TUB) (primers T1/Bt-2b, O'Donnell and Cigelnik 1997), and the translation elongation factor 1-alpha (TEF) (primers EF1-728F/EF-2, Crous et al. 2013). A BLASTn search of the resulting sequences of ITS (560 bp; OL744431), LSU (881 bp; OL744432), TUB (790 bp; PP084934), and TEF (445 bp; PP084935) revealed 100 % similarity (e-value=0.0, coverage=100%) to previously reported sequences of Arthrinium arundinis (e.g. MF627422 for ITS, KF144930 for LSU, KF144973 for TUB, and KY705146 for TEF), confirming the identity of the Korean isolate. Pathogenicity assays were performed twice by spraying 1 ml of a conidial suspension (1.1 × 104 conidia per mL) onto the leaf surface of sixteen healthy A. hybridus plants. Sixteen control plants were sprayed with sterile water. All plants were kept in a growth chamber at 80% relative humidity and 23 °C with a 12-h light/dark cycle. Three weeks after the inoculation, initial symptoms mirroring the aforementioned ones appeared, while the control plants remained symptomless. Fungal isolates were successfully re-isolated from the inoculated leaves, and their identity as A. arundinis was confirmed by DNA sequencing, thus fulfilling Koch's postulates. To our knowledge, this is the first report of leaf spot caused by A. arundinis on Amaranthus hybridus, not only in Korea but globally. Arthrinium arundinis has also been reported as a plant pathogen on some agricultural crops (Ji et al. 2020; Liao et al. 2022; Farr and Rossman 2023), suggesting its polyphagous behavior. Then, this pathogen could represent a potential risk to the cultivation of edible amaranth in Korea and other crops where Amaranthus species are spread as weeds. For this reason, continuous monitoring is necessary to assess the impact of this fungus on Amaranthus and other crops.

Morpho-molecular and environmental evidence of the ocurrence of Karenia longicanalis (Dinophyceae: Kareniaceae) as a bloom former in the Eastern Pacific Ocean

Karenia longicanalis, an athecate dinoflagellate, was first described during a bloom in Victoria Harbour (Hong Kong, China). This study confirms the presence of K. longicanalis as a bloom former in the eastern Pacific Ocean. Specimens were collected in March 2019 at three sampling stations in Acapulco Bay, Mexico. Water temperature, salinity, dissolved oxygen, and pH were measured in situ at the time of sample collection. Cell morphology was examined by optical and scanning electron microscopy. A molecular analysis based on the amplification of the large subunit (LSU) rDNA region revealed that the LSU sequences formed a monophyletic group with other GenBank sequences belonging to K. longicanalis. The resulting phylogeny demonstrates that Karenia is closely related to Asterodinium, Gertia, and Shimiella. The morphology of the specimens was consistent with previous descriptions.

Alternaria tomentosae (Pleosporaceae, Dothideomycetes); novel endophytic species from Citrus grandis cv. ‘Tomentosa’ fruits in China

ABSTRACT Alternaria species show a wide range of life modes, viz. pathogens in plants, animals and human, saprobes and rarely endophytes. In the present study, three Alternaria isolates were obtained from healthy fruits of Citrus grandis cv. ‘Tomentosa’ in an industrial planting area in Guigang City, Guangxi Province, China. The nuclear internal transcribed spacer of the rDNA region (ITS), 28S large subunit rDNA (LSU), 18S small subunit rDNA (SSU), glyceraldehyde-3-phosphate dehydrogenase (gapdh), translation elongation factor 1 alpha (tef1-α), RNA polymerase second largest subunit (rpb2) and Alternaria major allergen (alt-a1) gene regions were sequenced and employed to construct phylogenetic trees. Based on the morphology and combined multi-genes phylogeny, our isolates were identified as a novel species, Alternaria tomentosae.

Notes of Five Wood-Decaying Fungi from Juwangsan National Park in Korea

Wood-decaying fungi are essential decomposers in forest ecosystems. They decompose wood substrates by producing various lignocellulolytic enzymes, which have significant industrial and medical applications. A survey was conducted at the Juwangsan National Park from 2018 to 2019 to determine the diversity of macrofungi in Korea. Five previously unrecorded wood-decaying polyporoid and corticioid fungi were identified among the collected specimens: Eichleriella sinensis, Hymenochaete anomala, Hyphoderma subsetigerum, Lyomyces orientalis, and Pseudowrightoporia crassihypha. These species were identified based on morphological, molecular, and phylogenetic analyses of the internal transcribed spacer (ITS) and nuclear large subunit rDNA (nLSU) region. In this study, we provide detailed macro- and micro-morphological figures with phylogenetic trees to support the discovery of five new species in Korea.

First report of Neophysopella vitis causing leaf rust on Parthenocissus semicordata in Guangdong Province, China.

In April 2022, leaf rust disease of Parthenocissus semicordata was discovered in the urban greenbelt in Guangzhou city, China (23.06°N, 113.16°E). The disease incidence was 70% and disease severity was 75%. Chlorotic spots and red-brown necrotic flecks were present on the upper surface of infected leaves, and orange uredinia were distributed on the lower surface (Fig. 1 a-c). Two representative disease plants were collected as voucher specimens and dried, then deposited in Mycological Herbarium of the Zhongkai University of Agriculture and Engineering (MHZU GR0413, MHZU GR0414). Microscopic examination of the pustules of the samples revealed the presence of uredial paraphyses and urediniospores (Fig.1 d-f). Uredial paraphyses were hyaline, incurved, length 20-25 μm and dorsal wall 4.5-8.5 μm thick. Urediniospores were subglobose to ovoid, 16-24.5 × 10.5-17 μm. The wall of the urediniospore was hyaline or pale yellow, echinulate, and 1.0-2.0 μm thick. Telial structures were not observed. The morphological characteristics of uredial paraphyses and urediniospores were consistent with Yoshitaka's description of Neophysopella vitis (Yoshitaka 2000). Ten uredinia were picked using a sterile tweezer and the FlaPure Plant DNA Extraction Kit (Genesand Biotech Co., Ltd) was used to extract genomic DNA. Primers ITS4-rust (5'-CAGATTACAAATTTGGGCT-3') /ITS 5u (5'-CAAGGTTTCTGTAGGTG-3') were used to amplify internal transcribed spacer (ITS) and Rust2inv (5'-GATGAAGAACACAGTGAAA-3') /LR6 (5'-CGCCAGTTCTGCTTACC-3') to amplify large subunit (LSU) rDNA regions (Zhao et al. 2021). The sequences were submitted to NCBI (ITS: OQ991182 OQ991183, LSU: OQ979612 OQ979613), and blastn analyses of ITS and LSU showed 99.81% and 99.84% identity with the sequences from N. vitis (ITS: OQ304336, LSU: OM420266), respectively, found in GenBank. ITS combined with LSU sequences from the current study and reference sequences from Zhao et al. (2021) were used to construct Maximum likelihood (ML) and Bayesian inference (BI) phylogenetic trees using CIPRES website. ML and BI phylogenetic trees showed that the sequences from the current study grouped with N. vitis, with bootstrap support and posterior probability of 98% and 1.0, respectively (Fig. 2). Morphological and molecular analyses confirmed that the rust fungus was N. vitis. In the pathogenicity test, urediniospores were picked from fresh diseased leaves with sterile tweezers, prepared into spore suspensions (1.0 × 106 urediniospores/ml), and sprayed on a one-week-old leaf from three healthy of potted plants of P. semicordata, while three other healthy leaves were sprayed with sterile water as control. Each treated leaf was wrapped in a plastic bag and incubated in the dark at 25℃ for 48 h after which plastic bags were removed and the plant were moved to a greenhouse at 25℃. At 15 days after inoculation, symptoms and the morphology of urediniospores were confirmed to be the same as those observed in the field collection and no symptoms or uredinia were observed in the control group (Fig.1 g, h). Yoshitaka first discovered leaf rust on P. semicordata caused by N. vitis in Nepal(Yoshitaka 2000). To our knowledge, this is the first report of N. vitis causing leaf rust on P. semicordata in China. P. semicordata is often used as an ornamental plant for exterior wall decoration in urban landscaping and court. The occurrence of this disease can lead to the decline of the P. semicordata leaves and the impairment of the plants aesthetically.

Tegumental topography and molecular characterisation of two trematodes (Platyhelminthes: Digenea) from Clarias gariepinus (Burchell, 1822) in Kenya

The discoveries of new taxonomic features of digenean species through the application of contemporary techniques, such as scanning electron microscopy (SEM) and molecular analysis are still growing. Two species of trematodes Glossidium pedatum and Tylodelphys mashonensis from the intestine and vitreous humour of Clarias gariepinus were recovered from Lake Ol’Bolossat, Kenya. The two endo-helminths were prepared for morphological examination using SEM and molecular characterisation. Additional morphological features were observed for G. pedatum such as domed papillae in the anterior extremity and a protruding cirrus which was unarmed, laterally folded and with a blunt tip as the first such observation for the genus and led to additional characteristics of the diagnosis of the genus. Tylodelphys mashonensis was characterised by a round oral sucker and tribocytic organ rounded with rows of papillae symmetrically arranged. The molecular analyses using ribosomal marker 28S large subunit (LSU) rDNA and mitochondrial (mtDNA) cytochrome c oxidase subunit 1 (cox1) for both G. pedatum and T. mashonensis confirmed the identity of the species and their phylogenetic relationship within the subclass Digenea. This study provides the first mitochondrial (mt)DNA sequence for G. pedatum and also extends the geographical record of two parasites to Kenya.

A phylogenetic assessment of a fungicolous lineage in Coniocybomycetes: Chaenotricha, a new genus of Trichaptum-inhabiting species.

The globally distributed genus Trichaptum is one of the most species-rich among polypores in terms of hosting other fungi. Among Trichaptum-associates, there is a group of mazaediate lichenized fungi (Coniocybomycetes, Ascomycota) that previously had an uncertain phylogenetic position. DNA sequences - mitochondrial small subunit (mtSSU), nuclear large subunit rDNA (nuLSU), and internal transcribed spacer (ITS) - were obtained from 29 specimens collected from Europe and North America. Maximum likelihood and Bayesian inference analyses of these three gene loci were used to infer phylogenetic position and relationships among lineages. Statistical tests were used to find which phenotypical characteristics distinguish species. The molecular sequence data provide evidence that the fungicolous specimens form a distinct lineage within Coniocybomycetes sister to the combined clade of Chaenotheca s. lat. and Sclerophora. Considering its phylogenetic placement and strict specialization, we describe a new genus - Chaenotricha. This fungicolous lineage contains three species based on molecular characteristics. Morphological characters mostly overlap except for spore size and stalk length of apothecia. We provide a new combination, Chaenotricha obscura, for the only previously described species for which we designate an epitype, and introduce a new species - Chaenotricha cilians. The third lineage remains undescribed because of a small sample size, which did not allow us to clearly delineate species boundaries. Citation: Suija A, McMullin RT, Lõhmus P (2023). A phylogenetic assessment of a fungicolous lineage in Coniocybomycetes: Chaenotricha, a new genus of Trichaptum-inhabiting species. Fungal Systematics and Evolution 12: 255-269. doi: 10.3114/fuse.2023.12.13.