Large Subunit Ribosomal RNA Gene Research Articles

First Report of Monosporascus cannonballus causing Root Rot and Vine Decline in Watermelon (Citrullus lanatus) in China.

In June 2023, a sudden outbreak root rot and vine decline symptoms was observed during a watermelon (Citrullus lanatus T.) variety demonstration trial located in Taizhou City, Zhejiang Province, China, with an incidence rate ranging from 75% to 100% and an affected area of nearly 2,000 square meters. The disease initially appeared with a rapid and alarming invasion of root rot and vine decline symptoms within watermelon plants. Affected plants exhibited rapid deterioration, showing symptoms of wilting, yellowing and eventual demise, predominantly during the pre-harvest stage. Notably, numerous black, spherical, erumpent perithecia were clearly visible on the watermelon's root epidermis, a characteristic trait of the disease. Symptomatic plant samples were rigorously disinfected with 75% ethanol, and plated on potato dextrose agar medium for incubation at 25°C, successfully isolate two potential strains. These isolates were inoculated in oatmeal agar and incubated in a 25℃ light incubator. After 30 days, mature perithecia, the same as those found on the watermelon's root epidermis, reached a diameter of 500 μm. Each perithecium contained several pear-shaped asci, 56 to 108.5 μm in length and 30.5 to 46.4 μm in width, typically holding 1, rarely 2 ascospores. These characteristics align precisely with the typical strains of Monosporascus cannonballus Pollack and Uecker (1974). Additionally, sequencing the internal transcribed spacer region of ribosomal DNA (ITS) gene (White et al., 1990), large subunit ribosomal RNA (LSU) gene (Rehner and Samuels 1995), and beta-tubulin (TUB) gene (Glass and Donaldson, 1995) were performed. BLAST analysis indicated the highest nucleotide sequence identity with M. cannonballus CBS 586.93 reference sequence (ITS: 100%, JQ771930; TUB: 98.99%, JQ907292). Representative sequences of isolate ZJUP0990-2 from these regions were deposited in GenBank (Accession No.: OR357656 for ITS; OR474500 for LSU; OR365762 for TUB). A multigene phylogenomic analysis (ITS-LSU-TUB) was undertaken to ascertain the exact phylogenetic position of M. cannonballus within the genus Monosporascus. The amalgamation of both morphological and molecular insights consistently reaffirmed the accurate classification of the causative agent as M. cannonballus. To validate the pathogenicity of M. cannonballus, a controlled greenhouse experiment was conducted using watermelon (cv. Nabite) as the subject. Mycelium fragments, harvested from the edge of the colony ZJUP0990-2, were inoculated into oat liquid medium and cultivated under dark conditions at a consistent temperature of 30°C for 7 days. After 20 days, the inoculated plants exhibited root rot and wilting, mirroring the symptoms observed during the field outbreak. In contrast, the control plants did not exhibit any signs of disease. M. cannonballus was successfully re-isolated from the symptomatic roots of the inoculated plants, satisfying Koch's postulates. This experiment was repeated three times. This pathogenic fungus has previously been documented as a menace to melons in various regions including Mexico (Chew-Madinaveitia et al., 2012) and Brazil (Sales et al., 2004), as well as watermelons in Brazil (Sales et al., 2010), northern Mexico (Gaytan-Mascorro et al., 2012), and Saudi Arabia (Karlatti et al., 1997). To our knowledge, this is the first reported presence of M. cannonballus on watermelons in China. This new disease poses a serious threat to watermelon production, potentially leading to severe economic losses and impacting food security.

First Discovery of Stereostratum corticioides Causing Rust on the Culm of the Bamboo Pseudosasa japonica

A fungus strain <i>Stereostratum corticioides</i> PKVL1, belonging to the family <i>Pucciniaceae</i> that causes rust in plants, was discovered on the sheath of the bamboo <i>Pseudosasa japonica</i> leading to the death of the infected bamboo in the following year. Microscopic observation of the yellow fungal mass revealed teliospores with an oval, one-septate (two-celled) structure. The average length and width of teliospores were 31.83±3.57 μm and 20.74±1.72 μm, respectively. The large-subunit ribosomal RNA gene was amplified using the LR0R and LR7 primers, showing that the strain PKVL1 had a similarity of 99.34% to previously reported <i>S. corticioides</i>. In particular, the two <i>Stereostratum</i> strains form a separate cluster among the fungi in the family <i>Pucciniaceae</i>. This is the first report in the Republic of Korea of fungal rust occurring on the culm of bamboo rather than on the leaves.

Phylogenetic insights into the terrestrial snails Helicoidei (Gastropoda: Stylommatophora) with special emphasis on the Camaenidae

Abstract The superfamily Helicoidei are a widely distributed and diverse infraorder of land snails and slugs. Here, we constructed a phylogenetic tree based on sequences of the large subunit ribosomal RNA gene to investigate the relationships within the Helicoidei. Based on our phylogenetic tree, the family Camaenidae, as currently recognized, is not monophyletic, with the Polygyridae falling within the group. Likewise, the family Hygromiidae as currently defined is not monophyletic and incorporates the Geomitridae. The family Xanthoncyhidae form a monophyletic group that is sister to the Pleurodontidae. Likewise, the family Helicidae form a monophyletic group; it is sister to the branch incorporating Hygromiidae, Geomitridae, Pleurodontidae, Helicodontidae, and Xanthonychidae. The Solaropsidae are the sister group to a clade comprising other Sagdoidea. Our findings suggest that the Hygromiidae s.l. families should be restored to their previous name, Hygromiidae. A new genus of Bradybaeninae, Jiaoliaous Zhang gen. nov., is described here, with a simple genital system, single dart sac with multi-branched mucous glands, and a penis with a penial sheath.

Richness of yeast community associated with apple fruits in Estonia

Yeasts are single-celled fungi that are widespread around the globe. They are part of a community of microorganisms that use a wide variety of habitats, including fruit surfaces. This study aimed to characterise the culturable epiphytic yeasts associated with apple fruits. The isolated yeast strains were identified by sequencing the 5.8S-ITS region and D1/D2 region of the large subunit ribosomal RNA gene and maintained for long-term storage. A total of 230 yeast isolates belonging to 33 species were recovered. Most of the collected isolates belonged to the phylum Basidiomycota. Members of genera Vishniacozyma, Filobasidium, and Rhodotorula were most frequently isolated. Over half of the species were isolated on only one to three occasions. In seven of the species obtained, the isolates were considerably divergent from their closest relatives and may therefore represent new distinct species. The results of this study demonstrate a high diversity of yeast species associated with apple fruits.

First report of branch dieback caused by Neoscytalidium dimidiatum on Styphnolobium japonicum (L.) Schott in China.

Styphnolobium japonicum (L.) Schott (family Fabaceae Juss.) also called pagoda tree, is widely planted in northern China in landscape plantings, for erosion control and forestry. In recent years, symptoms of branch dieback were observed on S. japonicum in the southern part of Xinjiang province, China. From 2019 to 2022, in total ca. 1000 ha area was surveyed in Korla (41.68°N, 86.06°E), Bohu (41.95°N, 86.53°E) and Alaer (41.15°N, 80.29°E). Typical symptoms were observed in 70% of the surveyed branches. To identify the cause, we collected 50 symptomatic branches. Symptoms were initially observed on green current-year twigs, which turned grayish white in color. In the later stages of disease development, a large number of nacked black conidia formed under epidermis of perennial branches, causing visible black protrusions (pycnidia) on branch surface. The disease occurred throughout the entire growing season of S. japonicum. Symptoms also occurred on the inflorescence, fruit, and twigs. In some cases, infection resulted in tree mortality. Isolations were made from the margin between healthy and diseased tissues. Small pieces were excised, surface disinfested (75% ethanol 30 s, 1% NaClO solution 5 mins), cut into pieces (5 to 10 mm2), and incubated on PDA medium at 28℃ for 3 days. A total of 16 isolates (GH01-GH16) with similar colony morphology were obtained. The colonies were initially white, gradually turning to olive-green on the surface and black on the underside after 7 days. Microscopically, the conidia were aseptate, 1-septate, two-septate, and muriform, 2.6-4.5 × 2.9-27.6 μm (n=50). Pycnidia ranged in size from 120.2 to 135.5 × 112.4 to 118.6 µm (n=20). Those morphological characters matched the descriptions of Neoscytalidium dimidiatum (previously N. novaehollandiae) (Alizadeh et al. 2022; Pavlic et al. 2008). For molecular identification, genomic DNA of GH01-GH16 were extracted from fresh mycelia. The internal transcribed spacer (ITS), large subunit ribosomal RNA gene (LSU), and translation elongation factor 1-alpha (EF1-α) gene were amplified using the primer sets ITS1/ITS4 (White 1990), LRoR/LR5 (Vilgalys and Hester 1990) and EF1-728F/EF1-986R (Carbone and Kohn 1999). The sequences were deposited in GenBank (accession No. OP379832, OQ096643-OQ096657 for ITS, OP389048, OQ127403-OQ127417 for LSU, and OQ136617, OQ586044-OQ586058 for EF1-α). The ITS sequence had 100% identity (505/505 bp) to MT362600. Similarly, the LSU and EF1-α sequences were found to be identical to MW883823 (100%, 821/821 bp) and KX464763(99%, 256/258 bp), respectively. Pathogenicity was tested on one-year-old healthy S. japonicum seedlings. Spores of representative isolate GH01 were produced on PDA by incubating for 7-days at 28℃. Conidia were washed with sterile water. Five trees were inoculated with 1 × 106 conidia/ml conidial suspensions and five trees were sprayed with sterile water. All trees were covered with plastic bags for 24 h and kept at 25°C in a greenhouse. Signs and symptoms were similar to those observed in field collections one month after inoculation, while no symptoms occurred on the controls. The original fungus was successfully reisolated from the inoculated trees and was identified as N. dimidiatum following the methods described above. N. dimidiatum has been reported in many Asian country such as Malaysia, India, Turkey, and Iran(Akgül et al. 2019; Alizadeh et al. 2022; Khoo et al. 2023; Salunkhe et al. 2023). To our knowledge, this is the first report of N. dimidiatum associated with branch dieback of S. japonicum in China. Our findings have expanded the host range of N. dimidiatum in China and provides a theoretical basis for the diagnosis and treatment of the disease.

Kudoa tanakai n. sp. (Myxozoa: Myxosporea: Multivalvulida), a new kudoid species with spheroid myxospores from the scalpel sawtail (Actinopterygii: Prionurus scalparum) from western Japan.

We describe a new kudoid species, Kudoa tanakai n. sp., in the scalpel sawfish, Prionurus scalprum (Actinopterygii: Acanthuriformes: Acanthuridae), from the natural water around western Japan. The plasmodia were filamentous, localized in pseudocysts in the myofibers of the trunk muscles. The occurrence of plasmodia in the trunk muscle showed no site preference. Its myxospores were spheroid, measuring 6.6-7.6 (7.0) µm by 5.8-6.9 (6.3) µm in apical view (width) and 5.7-6.6 (6.2) in length (n = 30), with four shell valves and a corresponding number of spheroid polar capsules. Shell valves lacked apical protrusions, but scanning electron microscopy revealed that one of the four shell valves had two semi-lunar flaps at its apical terminus. Nucleotide sequencing of the small and large subunit ribosomal RNA genes of the present isolate showed phylogenetic affinities to kudoid species characterized by spheroid myxospores, such as K. musculoliquefaciens, K. hemiscylli, and K. carcharhini, but was molecularly and morphometrically distinct from these and other kudoid species. For direct comparison, Kudoa hemiscylli was collected from the Pacific spadenose shark, Scoliodon macrorhynchos (Elasmobranchii: Carcharhiniformes: Carcharhinidae), in the South China Sea off Guangdong Province, China, and the myxospore surface of the species was observed using scanning electron microscopy. Our study describes the new host and distribution record of this kudoid species originally described from a variety of elasmobranchs in the Australian Coral Sea.

First report of Sphaerulina azaleae causing leaf spot on Rhododendron pulchrum Sweet in China.

Rhododendron pulchrum Sweet is a popular ornamental evergreen shrub, renowned for its exquisite flowers. In November 2020, leaf spot disease was observed at Qujing Normal University (25.527°N; 103.744°E), Qujing, Yunnan, China. Symptoms were observed on 25-35 % leaves. This led to early leaf drop, significantly reducing its ornamental value. The infected leaves exhibited irregular, dark brown to blackish spots, with random distribution. To investigate the causal agent, ten symptomatic leaves were collected for pathogen isolation. Leaf spots bearing ascomata were picked with a sterile needle, placed on water agar, and then incubated at 26 ℃ for 24 h. Individual germinated spores were transferred onto PDA for further purification and morphological study. Two pure isolates (chl01 and chl02) were obtained. The colonies on PDA were circular, raised, pink-white at the center, dark brown at the margin, and dark brown to black from reverse after four weeks at 26 °C. Moreover, colonies on PDA sporulated following a 5-hour UV light exposure in a laminar flow hood and a subsequent 14 h light: 10 h dark (14:10) cycle for 5 days at 26 °C. The conidiophores are reduced to conidiogenous cells which are 1.5-3 × 1.5-2.5 µm (n = 10), integrated, hyaline, proliferating sympodially near the apex, cylindrical, and widest at the base. Conidia are hyaline, slightly verruculose, guttulate, cylindrical, straight or irregularly curved, subobtuse at the apex, truncate at the base, and 19-28 × 3-4 µm (n= 30), with 1-3 septa. The morphological features were similar to the genus Sphaerulina (Quaedvlieg et al. 2013). To confirm the candidate pathogen, we performed PCR and gene sequencing using specific primers ACT-512F/ACT2Rd, EF1-728F/EF2, ITS4/ITS5, LSU1Fd/LR5 and fRPB2-5F/fRPB2-414R for actin (ACT), the partial translation elongation factor 1-alpha (TEF1), internal transcribed spacer region (ITS), large subunit ribosomal RNA gene (LSU), and RNA polymerase II subunit (RPB2) genes, respectively (Choi et al. 2020). Sequences were deposited in GenBank with accession numbers OR359635 (ACT), OR359637 (TEF1), OR335339 (ITS), OR335299 (LSU) and OR359636 (RPB2). BLAST searches showed 97.34% (ACT), 99.47% (TEF1), 100% (ITS), 100% (LSU) and 100% (RPB2) identity with those of Sphaerulina azaleae CBS 128605 from GenBank, respectively. A multilocus phylogenetic tree was generated using maximum likelihood (ML) based on the concatenated ACT-TEF-ITS-LSU-RPB2 sequence, placing our isolates within the S. azaleae group with high confidence. To fulfill Koch's postulates, three R. pulchrum plants in the landscape (three leaves per plant) were inoculated by pipetting 0.2 ml spore suspension (1 × 106 conidia/ml) onto the surface of each leaf (Khoo et al. 2022). Three additional plants in the landscape were inoculated with sterile water on similar aged leaves. The leaves were enclosed in plastic bags for 72 hours. At 7 days postinoculation, infected leaves exhibited the symptoms as described above, whereas the controls showed no symptoms. This experiment was repeated twice. The same fungus was reisolated from the infected leaves and identified based on morphology and Sanger sequencing. It is worth noting that S. azaleae can cause leaf spot on Rhododendron yedoense f. poukhanense (Choi et al. 2020). To our knowledge, this is the first report of S. azaleae causing leaf spot on R. pulchrum Sweet in China, indicating that appropriate management strategies are needed.

First report of powdery mildew on Vigna unguiculata caused by Podosphaera xanthii in China.

Vigna unguiculata belongs to the Legume family, and is an annual twining, herbaceous vine plant, which is native to Africa. V. unguiculata is the most economically beneficial type of off-season vegetables in Hainan, China because of its rich in nutrients such as protein, minerals, dietary fiber, and vitamins (Jayathilake et al. 2018). In April 2022, typical powdery mildew infection was observed on V. unguiculata leaves in Haikou, Hainan Province, China (20°3'40.428"N, 110°19'45.217"E). More than 70% leaves of 13 V. unguiculata plants displayed severe powdery mildew disease. The diseased leaves at first exhibited white rounded irregular patches, which gradually enlarged, fused and covered all the leaf as well as stems. Edges of the infected leaves crinkled upwards, and the leaves often fell off the plants at the late infection stage. On the infected leaves, many conidiophores and dense mycelium were observed by microscopic analysis. Hyphae were septate, branched, epigenous, and flexuous to straight. Hyphal appressoria were indistinct or slightly nipple-shaped, and the haustoria developed were in the shape of oval-sphere and 9 to 11μm long. Foot cells of conidiophores were straight, cylindrical, and measured 43 to 70 × 10 to 12 μm. The conidiophores arising straightly from a hyphal cell, were measured 168 to 252 (mean = 204) μm in length and produced 6 to 9 immature conidia in each chain. Conidia were ovate, pyriform or barrel-shaped, with fibrosin bodies, and measured 26 to 32 (mean = 29.4) × 16 to 20 (mean = 18.6) μm. The chasmothecia was not found in all samples. These morphological characteristics were typical of the conidial stage of the powdery mildew Podosphaera xanthii of genus Podosphaera (Braun and Cook 2012). To further confirm the identity of this causal fungus, the internal transcribed spacer (ITS) region, and the partial sequence of large subunit ribosomal RNA gene (28S rRNA), were amplified with primer pairs ITS1/ ITS4 (White et al. 1990) and NL1/NLP2 (Mori et al. 2000) from extracted genomic DNA. The obtained 563-bp ITS region and 715-bp 28S rRNA gene sequences were deposited in GenBank (ITS, OQ415534; 28S rRNA, OQ415545.1), and were compared with BLAST analysis in the GenBank nr database. The results revealed that the ITS region sequence was 99.82% identity with P. xanthii isolate HUVU-08 (MH143485.1), and the 28S rRNA gene partial sequence was 100% identity with P. xanthii isolate XHL1 (MK357442.1). On the basis of the morphological characteristics and sequence analysis, this fungus was identified as P. xanthii. Pathogenicity tests were performed by gently brushing conidia onto the leaves of six healthy potted V. unguiculata plants. Six non-inoculated plants were used as control. All plants were maintained in a greenhouse at 26 ± 2°C. After 2 weeks inoculation, similar symptoms were observed in the inoculated plants, whereas no symptoms occurred on the control plants. By microscopic observation, the fungus present on the inoculated plants was morphologically identical to those on originally diseased plants. Furthermore, ITS and 28S rRNA sequences of the re-isolated fungus individually displayed 100% identity with OQ415534 and OQ415545.1. So far, although powdery mildew disease caused by P. xanthii on different plants including Sigesbeckia orientalis (Mukhtar et al. 2022), Vigna radiata (Sheu et al. 2021), Cosmos bipinnatus (Kong et al. 2023), Verbena brasiliensis (Luecke et al. 2020), Cucurbita ficifolia (Choi et al. 2022), Glandularia tenera (Pei et al. 2023) and Verbena bonariensis (Choi et al. 2023) have been reported, to our knowledge, this is the first report of powdery mildew caused by P. xanthii on V. unguiculata in Hainan, China, which seriously threatens the utilization of V. unguiculata on off-season vegetables industry.

Nakazawaea atacamensis f.a., sp. nov. a novel nonconventional fermentative ascomycetous yeast species from the Atacama Desert.

In this study, we describe Nakazawaea atacamensis f. a., sp. nov., a novel species obtained from Neltuma chilensis plant samples in Chile's hyperarid Atacama Desert. In total, three strains of N. atacamensis were obtained from independent N. chilensis samples (synonym Prosopis chilensis, Algarrobo). Two strains were obtained from bark samples, while the third strain was obtained from bark-exuded gum from another tree. The novel species was defined using molecular characteristics and subsequently characterized with respect to morphological, physiological, and biochemical properties. A neighbor-joining analysis using the sequences of the D1/D2 domains of the large subunit ribosomal RNA gene revealed that N. atacamensis clustered with Nakazawaea pomicola. The sequence of N. atacamensis differed from closely related species by 1.3%-5.2% in the D1/D2 domains. A phylogenomic analysis based on single-nucleotide polymorphism's data confirms that the novel species belongs to the genus Nakazawaea, where N. atacamensis clustered with N. peltata. Phenotypic comparisons demonstrated that N. atacamensis exhibited distinct carbon assimilation patterns compared to its related species. Genome sequencing of the strain ATA-11A-BT revealed a genome size of approximately 12.4 Mbp, similar to other Nakazawaea species, with 5116 protein-coding genes annotated using InterProScan. In addition, N. atacamensis exhibited the capacity to ferment synthetic wine must, representing a potential new yeast for mono or co-culture wine fermentations. This comprehensive study expands our understanding of the genus Nakazawaea and highlights the ecological and industrial potential of N. atacamensis in fermentation processes. The holotype of N. atacamensis sp. nov. is CBS 18375T . The Mycobank number is MB 849680.

Plasmopara echinaceae, a new species of downy mildew affecting cone flowers (Echinacea purpurea) in the United States.

Downy mildew is one of the most important diseases of commercial sunflower and other Asteraceae hosts, including ornamental Rudbeckia. Plasmopara halstedii has historically been identified as the causal agent of this disease, considered a complex of species affecting nearly 35 genera in various tribes. However, with the use of molecular DNA characters for phylogenetic studies, distinct lineages of P. halstedii in the Asteraceae have been identified, confirmed as distinct or segregated as new species. During August of 2022, a downy mildew was observed on potted Echinacea purpurea grown in a retail greenhouse in Jefferson County, Wisconsin, USA. Phylogenetic analyses of the cytochrome c oxidase subunit 2 (cox2) and nuclear large subunit ribosomal RNA (nc LSU rDNA) gene regions indicated these Plasmopara sp. isolates are not conspecific with P. halstedii. Based on phylogenetic evidence and new host association, the Plasmopara isolates from E. purpurea are here described as Plasmopara echinaceae. Diagnostic morphological characters for this new species were not observed when compared with other isolates of P. halstedii or other Plasmopara species found on Asteraceae hosts, and therefore a list of species-specific substitutions in the cox2 region are provided as diagnostic characters. As this study corresponds to the first observation of downy mildew in cone flowers, it is recommended to follow the required disease prevention guidelines to prevent outbreaks and the establishment of this plant pathogen in production sites. Citation: Salgado-Salazar C, Romberg MK, Hudelson B (2023). Plasmopara echinaceae, a new species of downy mildew affecting cone flowers (Echinacea purpurea) in the United States. Fungal Systematics and Evolution 12: 203-217. doi: 10.3114/fuse.2023.12.10.

Complete mitochondrial genome analyses confirm that bat Polychromophilus and ungulate Plasmodium constitute a distinct clade independent of other Plasmodium species

In recent phylogenetic studies, bat Polychromophilus and ungulate Plasmodium, two relatively understudied haemosporidian parasites within the Apicomplexa phylum, have often been overlooked. Instead, the focus has been primarily on haemosporidian parasites in primates, rodents, and birds. Several phylogenetic analyses of bat Polychromophilus have relied on limited datasets and short informative DNA sequences. As a result of these inherent limitations, the substantiation of their evolutionary stance has encountered a diminished degree of robust validation. This study successfully obtained complete mitochondrial genome sequences from 11 Polychromophilus parasites originating from Hipposideros gentilis and Myotis siligoensis bats for the first time. Additionally, the authors have sequenced the apicoplast caseinolytic protease C genes from Polychromophilus murinus and a potentially new Polychromophilus species. These mitochondrial genomes range in length from 5994 to 6001 bp and consist of three protein-coding genes (PCGs), seven small subunit ribosomal RNA genes (SSU rRNA), 12 large subunit ribosomal RNA genes (LSU rRNA), and seven miscellaneous RNA genes. Phylogenetic analyses using Bayesian Inference and Maximum Likelihood methods indicated robust support for the grouping of ungulate Plasmodium and bat Polychromophilus in a single clade separate from other Plasmodium spp., confirming previous reports, albeit with stronger evidence in this study. The divergence between Polychromophilus in bats and Plasmodium in ungulates occurred approximately 29.61 to 55.77 million years ago (Mya), with a node age estimated at 40.63 Mya. These findings highlight that the genus Plasmodium, which includes species found in ungulates, birds, reptiles, and other mammals, does not form a monophyletic group. By incorporating Polychromophilus in bats and Plasmodium in ungulates, this study contributes significantly to understanding the phylogenetic relationships within the Haemosporida order. It provides valuable insights into the evolutionary history and interconnections among these diverse parasites, thereby expanding knowledge in this field.

Genetic Diversity within Scorpio Genus (Scorpion: Scorpionidae) from Iran: Preliminary Evidence based on 16srRNA Sequence.

The yellow digger scorpion, Scorpio maurus, is a medically important scorpion for which little is known about its genetic diversity. Polymerase chain reaction products of 16srRNA gene fragments were generated from scorpion specimens named SmKh1 and SmKh2. These sequences showed high similarity with the only partial sequence of S. maurus isolate SCA1 large subunit ribosomal RNA gene available in the Genbank database. The drawing of the phylogeny tree showed two clusters, A and B. The two specimens (SmKh1 and SmKh2), which are placed in sub-cluster A2, were provided from Behbahan, Iran, and they have the closest relationship with the only sequence of S. maurus (MW281771), which is also collected from Behbahan. It is noteworthy that the two sequences obtained from S. maurus scorpions recorded from Miandoab (MK170444) and Mahabad (KU705354), which are in sub-cluster A1, are more similar to the scorpions isolated from the Mediterranean basin than those collected from Behbahan. This issue is probably due to the fact that patterns of genetic diversity are a reflection of variation in gene flow, which is also influenced by factors such as territorial barriers and geographical distances. We conclude that the scorpions of this study accompanied by similar scorpions in the Mediterranean basin, belong to the same species despite the insignificant differences.

First Report of Cobweb Disease in Auricularia cornea var. Li. Caused by Hypomyces mycophilus in China.

Auricularia cornea var. Li. is a natural mutation strain of A. cornea which has been preferred by consumers for its white colour, good taste and pharmacological properties (Rebecca et al. 2020). In October 2021, a disease with symptoms similar to that of cobweb disease (Carrasco et al. 2017) was observed in A. cornea var. Li. in four mushroom farms in Fangshan District (115.83°E, 39.55°N), Beijing, China, infecting 20% of the fruiting bodies (Fig. 1A-D). White cottony mycelia formed typically on the casing soil and they gradually spread to the stipes and pileus, covering the whole fruiting body, which eventually died and lost commodity value. Cultures were obtained by aseptically transferring the diseased fruiting bodies onto potato dextrose agar (PDA); they were deposited in the culture collection (ID: JZBQA3) of the Beijing Academy of Agriculture and Forestry Sciences, China. The colonies were floccose with aerial mycelium white. Purplish grey diffusing pigments occasionally formed on the reverse side of the plate at 25 °C (Fig. 2A-B). Conidiophores arising in aerial mycelium, indefinite in length, branches septate, each cell producing denticulate conidiogenous loci, each denticle bearing a single conidium. Conidia mostly oblong to ellipsoidal, smooth, (9.0-)9.9-17.0(-18.0)×(6.0-)6.9-10.2 μm (n = 60), 0~1 septate (Fig. 2C-E). Chlamydospores forming as lateral branches of hyphae were commonly observed, globose, ellipsoid or oblong, 14.8-22×14.7-19.6 μm, l/w = 1.0-1.3 (Fig. 2F-G). The morphological characteristics were consistent with that of Hypomyces mycophilus, whose anamorph was Cladobotryum polypori (Rogerson et al. 1993, Zeng et al. 2017). For further species identification, genomic DNA was extracted using the DNeasy Plant Mini Kit (Qiagen, USA). The internal transcribed spacer (ITS) regions and large subunit ribosomal RNA (LSU) genes were amplified using the primer pairs ITS1/ITS4 (White et al. 1990) and LROR/LR5 (Bhattacharya et al. 2000), respectively. The sequences were deposited in GenBank with accession numbers OP430530 and OP430531. BLAST nucleotide searches showed more than 99% homology with corresponding sequences of Hypomyces mycophilus HMAS 275554 and CBS 175.56. Phylogenetic trees based on ITS and LSU revealed that the strain JZBQA3 was grouped with H. mycophilus with high support value (Fig. 3). A in vivo pathogenicity test was performed using eight mushroom sticks with healthy fruiting bodies in triplicate. Each four sticks were sprayed with conidial suspension (108 spores/mL) of strain JZBQA3 or sterile distilled water, respectively, and maintained in an artificial climate chamber at 25-26°C. Cobweb-like characteristics were observed on the fruiting bodies treated with the JZBQA3 conidial suspension 2-3 days after inoculation, while those treated with sterile distilled water remained symptomless (Fig. 4 A-B). The same pathogen was re-isolated and confirmed from the infected fruiting bodies by integrated analysis of morphological characteristics and gene sequencing data, fulfilling Koch's postulates. Hypomyces mycophilus was first reported on Trametes versicolor in North Carolina (Rogerson et al. 1993), and is the causal agent of cobweb diseases on Auricularia heimuer (Zhang et al. 2023). To our knowledge, this is the first report of cobweb disease caused by H. mycophilus in A. cornea var. Li. This finding is a valuable contribution to the knowledge of cobweb disease development in edible fungi.

New evidence of consistency between phylogeny and morphology for two taxa in ciliated protists, the subclasses Oligotrichia and Choreotrichia (Protista, Ciliophora)

Marine planktonic ciliates are largely oligotrichs and choreotrichs, which are two subclasses of the class Spirotrichea. The current phylogenetic assignments of oligotrichs and choreotrichs are inconsistent with previous results based on morphological features, probably hindered by the limited information from a single gene locus. Here we provide 53 new sequences from small subunit ribosomal RNA (SSU rDNA), ITS1-5.8S rDNA-ITS2, and large subunit ribosomal RNA (LSU rDNA) gene loci in 25 oligotrich and choreotrich species. We also predict RNA secondary structures for the ITS2 regions in 55 species, 48 species of which are reported for the first time. Based on these novel data, we make a more comprehensive phylogenetic reconstruction, revealing consistency between morphological taxonomy and an updated phylogenetic system for oligotrichs and choreotrichs. With the addition of data from ciliature patterns and genes, the phylogenetic analysis of the subclass Oligotrichia suggests three evolutionary trajectories, among which: 1) Novistrombidium asserts an ancestral ciliary pattern in Oligotrichia; 2) the subgenera division of Novistrombidium and Parallelostrombidium are fully supported; 3) the three families (Tontoniidae, Pelagostrombidiidae and Cyrtostrombidiidae) all evolved from the most diverse family Strombidiidae, which explains why strombidiids consistently form polyphyletic clades. In the subclass Choreotrichia, Strombidinopsis likely possesses an ancestral position to other choreotrichs, and both phylogenetic analysis and RNA secondary structure prediction support the hypothesis that tintinnids may have evolved from Strombidinopsis. The results presented here offer an updated hypothesis for the evolutionary history of oligotrichs and choreotrichs based on new evidence obtained by expanding sampling of molecular information across multiple gene loci.

First Report of Athelia rolfsii Causing Damping-Off and Leaf Blight on Basella alba in Malaysia.

Basella alba is an evergreen perennial vine that grows as an edible leafy vegetable in Malaysia (Nordin et al. 2007). During January 2021, a cottony white hypha associated with aggregates of white to brown sclerotia and symptoms of damping-off were visualized on the stem base of B. alba at the soil surface in an isolated field (~0.03 ha) located in the district of Penampang, Sabah province, Malaysia (5°56'51.0"N 116°04'31.8"E). Yellowing and wilting of leaves, and defoliation were observed after four days of the development of damping-off. Survey was conducted on 100 plants which 30 were found infected. The disease severity (90%) on stems was estimated using IMAGEJ. Symptomatic stem tissues were surface sterilized with 75% of ethanol for 1 min, washed with 2% of sodium hypochlorite solution for 1 min, rinsed thrice with sterile distilled water, air dried and plated on potato dextrose agar (PDA). Plates were incubated for 7 days at 25°C in the dark. After 7 days, fungi were isolated; colony color was white and had a cottony appearance. On day 14, white to brown sclerotia 1.0 to 2.2 mm in diameter were produced. Hyaline septate hyphae with clamp connections and multiple nuclei were seen. Conidia and conidiophores were absent from the colony on PDA. Genomic DNA of fungi was extracted based on Khoo et al. (2022a and 2022b). PCR amplification (Khoo et al. 2022b) was performed using primer set ITS1/ITS4, EF983/EF2218 and LR0R/LR05 to amplify the internal transcribed spacer (ITS) region of rDNA, partial translation elongation factor 1 alpha (TEF-1α) gene and partial large subunit ribosomal RNA (LSU rRNA) gene, respectively (Vilgalys and Hester 1990; White et al. 1990; Carbone and Kohn, 1999; Rehner 2001). Phylogenetic analysis indicated that the isolates formed a supported clade to the related Athelia rolfsii sequences. The sequencing result (GenBank Accession Nos. OK465460, OP809607, OP857217) had a 99% identity over 625 bp, 941 bp, and 1,101 bp with the corresponding gene sequence of A. rolfsii (GenBank Accession Nos. MN622806, AY635773, MW322687) after analysis in BLASTn program. Pathogenicity test was performed based on Le (2011). Three 8-week-old B. alba plants cultivated on sterilized soil were inoculated with 5-mm mycelia plugs from 7-day-old culture. A plug was put on the upper soil surface layer 2 cm away from the base of the stem of B. alba plant before fully covered with a layer of sterilized soil. Plants that were inoculated with sterile PDA plugs served as the control treatment. Plastic bags were used to cover the plants after inoculation for 24 h before keeping them in a glasshouse under ambient conditions. Water-soaked and brown lesions were visualized on the stem base of all inoculated plants after four days of inoculation. Symptom of damping-off and leaf blight was observed after another 3 days. No symptoms developed on the mock controls. The pathogenicity test was repeated twice. Re-isolation was performed from the symptomatic tissues of inoculated plants and mock controls. The isolates reisolated from the symptomatic tissues were verified as A. rolfsii based on morphology and molecular characterization, thus fulfilling Koch's postulates. No pathogens were isolated from the mock controls. To our knowledge, this is the first report of A. rolfsii causing damping-off and leaf blight on B. alba in Malaysia, as well as worldwide. Our findings documented the wider geographical and host range of A. rolfsii and indicate its potential threat to B. alba production in Malaysia.

Evaluation of MALDI-TOF MS, sequencing of D2 LSU rRNA and internal transcribed spacer regions (ITS) for the identification of filamentous fungi isolated from a pharmaceutical facility

The identification of filamentous fungi through culture characterization may be hampered by phenotypic variability. Information obtained from the identification of microorganisms are important for investigation of sources of contamination of a product or process. The aim of this study was to identify filamentous fungal strains (n = 50) isolated from a pharmaceutical facility by using Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), as well as D2 domain of the large-subunit (LSU) ribosomal RNA gene and internal transcribed spacer regions (ITS) sequencing. MALDI-TOF MS system only identified five strains at the species level, while 45 were not identified. The analysis through GenBank allowed the identification of up to 19 strains at the species level, while MycoBank allowed the identification of up to nine strains at the species level. The databases identified up to 11 genera: Penicillium, Aspergillus, Cladosporium, Chaetomium, Coniochaeta, Curvularia, Diaporthe, Fusarium, Trichoderma, Rhizopus and Microdochium. MALDI-TOF MS showed an insufficient database to identify the species of fungi. DNA sequencing was the best methodology to identify to the genus level but was unable to differentiate between closely related species. Therefore further methods for the identification of filamentous fungi from pharmaceutical areas at species level need to be developed.

Body size of fungus-growing termites infers on the volume and density of their fungal cultivar

The body size of an animal plays a crucial role in determining its trophic level and position within the food web, as well as its interactions with other species. In the symbiosis between Termitomyces and fungus-growing termites, termites rely on nutrition of fungal nodules produced by Termitomyces . To understand whether the size of termites and fungal nodules are related to their partner specificity, we quantified the size of termite farmer caste, and the size and density of nodules in termite nests of four genera of fungus-growing termites, and identified their cultivated Termitomyces fungus species based on internal transcribed spacer regions and partial large subunit ribosomal RNA gene sequences. The results showed that the size and density of fungal nodules were different among Termitomyces clades and revealed a constant trade-off between size and density among clades. The nodule size of each clade has low variation and fits normal distribution, indicating that size is a stabilized trait. Moreover, we found larger termite genera cultivated Termitomyces with larger but less numerous nodules. Based on these results, we concluded that there is a size specificity between Termitomyces and fungus-growing termites, which may lead to diversification of Termitomyces as adaptations to different termite genera.

Schizosaccharomyces lindneri sp. nov., a fission yeast occurring in honey.

Two strains of fission yeast were isolated from honey. They differ from the type strain of Schizosaccharomyces octosporus by three substitutions in the D1/D2 domain of the nuclear 26S large subunit ribosomal RNA(rRNA) gene sequence, resulting in a 99.5% identity. In the internal transcribed spacer (ITS) region (consisting of ITS1, 5.8S rDNA, and ITS2), the strains differ from S. octosporus by 16 gaps and 91 substitutions, which is equivalent to an identity of 88.1%. Genome sequencing on one of the new strains revealed that the average nucleotide identity (ANI) between its genome and the reference genome of S. octosporus is 90.43% and there exist major genome rearrangements between the two genomes. Mating analysis revealed that S. octosporus and one of the new strains are completely reproductively separated. A strong prezygotic barrier exists and the few mating products consist of diploid hybrids that do not form recombinant ascospores. In the new strains, asci are either zygotic, arising from conjugation, or they develop without conjugation from asexual cells (azygotic). Compared to the currently recognized Schizosaccharomyces species, the spectrum of nutrients that are assimilated by the new strains is restricted. Of the 43 carbohydrates that were included in the physiological standard tests, only 7 were assimilated. According to the results of the genome sequence analysis, the mating trials, and the phenotypic characterization, the new species Schizosaccharomyces lindneri is described to accommodate the two strains(holotype: CBS 18203T and ex-type: MUCL 58363; MycoBank no.: MB 847838).

Genetic Diversity within Scorpio Genus (Scorpion: Scorpionidae) from Iran: Preliminary Evidence based on 16srRNA Sequence

The yellow digger scorpion, Scorpio maurus, is a medically important scorpion for which little is known about its genetic diversity. Polymerase chain reaction products of 16srRNA gene fragments were generated from scorpion specimens named SmKh1 and SmKh2. These sequences showed high similarity with the only partial sequence of S. maurus isolate SCA1 large subunit ribosomal RNA gene available in the Genbank database. The drawing of the phylogeny tree showed two clusters, A and B. The two specimens (SmKh1 and SmKh2), which are placed in sub-cluster A2, were provided from Behbahan, Iran, and they have the closest relationship with the only sequence of S. maurus (MW281771), which is also collected from Behbahan. It is noteworthy that the two sequences obtained from S. maurus scorpions recorded from Miandoab (MK170444) and Mahabad (KU705354), which are in sub-cluster A1, are more similar to the scorpions isolated from the Mediterranean basin than those collected from Behbahan. This issue is probably due to the fact that patterns of genetic diversity are a reflection of variation in gene flow, which is also influenced by factors such as territorial barriers and geographical distances. We conclude that the scorpions of this study accompanied by similar scorpions in the Mediterranean basin, belong to the same species despite the insignificant differences.

First report of Planomicrobium okeanokoites associated with Himantothallus grandifolius (Desmarestiales, Phaeophyta) from Southern Hemisphere.

Gram-positive, aerobic, motile, rod-shaped, mesophilic epiphytic bacterium Planomicrobium okeanokoites was isolated from the surface of endemic species Himantothallus grandifolius in Larsemann Hills, Eastern Antarctica. The diversity of epiphytic bacterial communities living on marine algae remains primarily unexplored; virtually no reports from Antarctic seaweeds. The present study used morpho-molecular approaches for the macroalgae and epiphytic bacterium characterization. Phylogenetic analysis was performed using mitochondrial genome encoded COX1 gene; chloroplast genome encodes rbcL; nuclear genome encoded large subunit ribosomal RNA gene (LSU rRNA) for Himantothallus grandifolius and ribosomal encoded 16S rRNA for Planomicrobium okeanokoites. Morphological and molecular data revealed that the isolate is identified as Himantothallus grandifolius, which belongs to Family Desmarestiaceae of Order Desmarestiales in Class Phaeophyceae showing 99.8% similarity to the sequences of Himantothallus grandifolius, from King George Island, Antarctica (HE866853). The isolated bacterial strain was identified on the basis of chemotaxonomic, morpho-phylogenetic, and biochemical assays. A phylogenetic study based on 16S rRNA gene sequences revealed that the epiphytic bacterial strain SLA-357 was closest related to the Planomicrobium okeanokoites showing 98.7% sequence similarity. The study revealed the first report of this species from the Southern Hemisphere to date. Also, there has been no report regarding the association between the Planomicrobium okeanokoites and Himantothallus grandifolius; however, there are some reports on this bacterium isolated from sediments, soils, and lakes from Northern Hemisphere. This study may open a gateway for further research to know about the mode of interactions and how they affect the physiology and metabolism of each other.