Internal Transcribed Spacer rDNA Research Articles

First report of leaf blight caused by Stemphylium lycopersici on Salvia splendens in China.

Salvia splendens is a popular ornamental plant in China with extensive potentials, including value in traditional Chinese medicine and in environmental restoration function (Li et al. 2008). In September 2019, leaf blight disease was observed on road side plants of S. splendens in Bayi park, Nanchang city, Jiangxi province, China. The typical symptoms appeared as irregular necrotic spots or leaf blight, accompanied by extensive scorch necrosis or ultimately defoliation. Small segments cut from diseased leaves were surface sterilized in a 2% sodium hypochlorite solution for 2 min and rinsed three times with sterile distilled water. Then, the samples were placed on potato dextrose agar (PDA) plates incubated at 25°C in darkness. Pure cultures were obtained by the hyphal tip method. Morphologically, all 11 colonies were identical to each other on PDA. Two strains, YZU 191468 and YZU 191481, were selected for further study and deposited in the Fungal Herbarium of Yangtze University (YZU), Jingzhou, Hubei, China. The 7-day-old colonies were circular, 53 to 56 mm in diameter, and consisted of white mycelium with a buff margin, and were cinnamon colored in the center of the reverse side. To examine conidial morphology, the mycelium was transferred onto potato carrot agar (PCA) and incubated at 23°C with a period of 8 h light/16 h dark for 7 days. Conidia were normally solitary or two in a chain, ellipsoid or long ellipsoid, beakless, 10 to 23×30 to 60 µm in size (n=50). Based on morphology, the isolates were consistent with Stemphylium lycopersici (Yamamoto 1960). To confirm the identification, genomic DNA was extracted from both isolates and used to amplify the internal transcribed spacer rDNA region (ITS), glyceraldehydes-3-phosphate dehydrogenase (GAPDH) and calmodulin (CAL) genes with primer pairs ITS5/ITS4, gpd1/gpd2, and CALDF1/CALDR2, respectively (Woudenberg et al. 2017). Sequences were deposited in GenBank with accession numbers OP564983 and OP564984 (ITS), OP892529 and OP892530 (GAPDH), OP584970 and OP584971 (CAL). A neighbor-joining tree was constructed with Mega 7.0 based on the combined dataset with 1,000 bootstrap replicates. The resulting phylogenetic tree showed that the strains from S. splendens clustered with S. lycopersici (CBS 122639 and CBS 124980) supported with 100% bootstrap values. The molecular analyses confirmed that the species causing leaf blight symptoms was S. lycopersici. To test pathogenicity, healthy leaves of S. splendens were surface sterilized and inoculated by mycelium blocks (6 mm in diameter) and spore suspension (1×106 spore/mL) of representative strains YZU 191468 and YZU 191481, respectively. Controls were inoculated with blocks of PDA and sterile water. Each strain was inoculated on three leaves of a plant. One clean plant was used as control. The test was replicated three times. After inoculation, the plants were covered with plastic bags and incubated in a greenhouse (25℃, 80 % relative humidity, 8 h light/16 h dark). After 5 days, the inoculated leaves exhibited dark brown spots with white mycelium, followed by withering of necrotic tissues. There were no symptoms observed on the controls. The fungal isolates inoculated leaves had the same morphological characteristics as the strains used for inoculation. S. lycopersici has been found on eggplant and Zinnia elegans in China (He et al. 2019; Yang et al. 2017). To the best of our knowledge, this is the first report of S. lycopersici causing leaf blight on S. splendens in China. This finding offers a new reference for the management and control of S. splendens leaf diseases in China.

First Report of Rust Disease on Alcea rosea caused by Puccinia modiolae in China.

Alcea rosea, belonging to the Alcea genus in the Malvaceae family, originated from China, but it is now grown worldwide. A. rosea has been widely used in traditional Chinese medicine to alleviate constipation, pain, swelling, and sores. In February 2023, typical symptoms of fungal infection were observed on A. rosea at Guizhou Normal University in Guiyang, Guizhou Province, China. The disease incidence was over 90% (n = 100) for the surveyed A. rosea plants, and the disease severity range from 30% to 90%. The initial symptoms of A. rosea rust were the appearance of chlorotic spots on the leaves. Subsequently, numerous reddish to dark-brown erumpent pustules (telia) were observed. Gradually, the entire plant was covered by rust and the center of each lesion turned brown, necrotic, and ruptured over times, eventually causing defoliation. Voucher specimens of infected A. rosea leaves as representative samples have been deposited at Guizhou Normal University (GNU2023LS008). Telia are round in shape, mostly aggregated in mass, with a diameter of 0.28-0.78 mm (0.46 mm, n = 20). They range in color from reddish-brown to dark brown, and are mainly hypophyllous but occasionally formed on the adaxial leaf surface. The teliospores are fusoid with dimensions of 31.3-93.8 × 10.9-21.3 μm (57.5 × 15.1 µm average, n = 50), hyaline or yellowish to light-brown in color, mostly two-celled, with a smooth wall (1.5-3.0 µm) and a thickened apex (3.0-9.0 µm). However, teliospores which are one-, three-, or four-celled with a notch at the apex, are rarely observed. The morphological characteristics of host symptoms and teliospores were similar to those of Puccinia modiolae (Aime and Abbasi 2018; Albu et al. 2019). For phylogenetic analysis, genomic DNA was extracted from the teliospores of infected leaves. To confirm the species-level identification, PCR was performed on the extracted DNA to amplify the ribosomal DNA internal transcribed spacer (ITS) and large subunit (LSU) regions using primer pairs ITS1/ITS4 (Schoch et al. 2012) and NL1/NL4 (Ziemiecki et al. 1990), respectively. The resulting ITS DNA sequence (GenBank accession no. OR607960) showed 100% identity with P. modiolae sequences (OP369291.1), when the query coverage was 100%. The LSU DNA sequence obtained (OR607961.2) shared 99.85% similarity with P. modiolae (MK458702.1). A phylogenetic tree was constructed using MEGA7.0 and the maximum likelihood method based on the ITS and LSU sequences. The fungal isolates collected in this study and several reference sequences of P. modiolae were grouped within a clade that included the isolates reported on A. rosea in Korea (Ryu et al. 2023), with 100% bootstrap support. Pathogenicity testing was conducted by gently pressing spore powder of naturally diseased leaves onto young leaves of three healthy A. rosea plants, with three noninoculated healthy plants serving as controls. The inoculated and noninoculated plants were kept in a growth chamber at the 26°C with a 12 hour light/dark cycle and 80% humidity. After 2 weeks, all inoculated A. rosea plants showed characteristic disease symptoms of rust infection and telia of P. modiolae, while control plants remained symptomless. The pathogen was identical to that observed on the original diseased leaves. The study results indicate that the causal fungus responsible for the disease is P. modiolae, which has been previously reported on Malvaceae plants (Farr and Rossman 2022). To the best of our knowledge, this is the first report of P. modiolae on A. rosea in China. This study will contribute to an increased understanding of the host range of Puccinia modiolae.

Gastropod invasions in anthropogenically impacted impoundments in South Africa: Tracing their origins and exploring field evidence of parasite spillback and amplification

Invasive snails are associated with ecological problems in freshwater bodies worldwide. However, their impact on the transmission of digenean infections remain underreported. In the present study, 1708 specimens representing four snail species were sampled from four impoundments in the Limpopo River system in South Africa. Gyraulus chinensis (Planorbidae), Physella acuta (Physidae) and Pseudosuccinea columella (Lymnaeidae), which are invasive, were found in all the sampling sites. In contrast, the native lymnaeid Radix natalensis occurred at only one study site. Digeneans were observed only from R. natalensis (prevalence = 49%) and Ps. columella (prevalence = 23%). Morphological and genetic analyses revealed four digeneans: Fasciola nyanzae, Orientocreadium sp., Petasiger sp. and Patagifer vioscai. Pseudosuccinea columella was infected by the four digeneans while R. natalensis harboured only Orientocreadium sp. and Petasiger sp. Partial sequences of Orientocreadium sp. from the current study differed from congeners whose DNA data are available on GenBank, by p-distances of at least 1.84 and 2.2% for 28S and the internal transcribed spacer (ITS) rDNA, respectively. Phylogenetic analyses demonstrated that the present species is sister to Orientocreadium batrachoides. Genetic and phylogenetic data based on 28S and ITS rDNA suggested that Petasiger sp. from the present study and isolates of three unidentified Petasiger spp. from Kenya, Hungary and Australia, were representatives of the same species. This is the first known report of Orientocreadium, Petasiger and Patagifer from Ps. columella. The occurrence of F. nyanzae in Ps. columella indicates spillback from R. natalensis. These findings echo the concerns raised in previous studies about the potential role of Ps. columella in the amplification of digenean diseases in its introduced range. Phylogenetic analyses of partial sequences of the cytochrome c oxidase subunit 1 mitochondrial gene (cox1) showed multiple lineages of Ps. columella in North and South America. Pseudosuccinea columella specimens from the present study belong to an invasive genotype that has spread globally and has been reported from Zimbabwe, Egypt, Portugal, Australia, Argentina, Colombia and New Mexico (USA). Physella acuta from the current study had a stronger genetic relationship with isolates from Canada and Iceland, than with isolates from other parts of Africa, suggesting several invasion routes into Africa. This is the first known DNA characterisation of G. chinensis from Africa. Phylogenetic reconstruction indicated multiple exit events of G. chinensis from Asia into Europe and Africa. South African isolates clustered in a recent branch containing isolates from the Czech Republic and Hong Kong, China. Considering the presence of invasive snails in all the sampling sites in the present study, it is necessary to investigate the factors that enhance their establishment and to monitor their effects on the native snail populations.

Leaf Spot of Hedychium coronarium Caused by Colletotrichum gloeosporioides in China

Hedychium coronarium (white ginger) is widely cultivated for garden decoration and folk medicine. Since 2020, symptomatic leaves showed brown necrosis and yellow borders on H. coronarium in the field (approximately 200 m2) at Southwest University, Rongchang District, Chongqing City. Small brown-yellow spots gradually enlarged and caused withering in severe cases with a mortality rate of around 10%. Disease incidence and severity varied from 55 to 65% and from 30 to 40%, respectively. Infected tissues (5 mm in diameter) were cut from lesion margins, surface sterilized in 70% ethanol for 10 s and 0.1% acidic mercuric chloride for 3 min, followed by rinsing in sterile water three times, and then were cultured onto potato dextrose agar (PDA) at 25 °C. Five isolates were transferred onto fresh PDA and purified by single-spore culturing. The colonies were initially white and turned hoary, and the diameter reached 32.95 to 38.37 mm × 32.42 to 38.61 mm after 3 days of incubation. Pale gray abundant fluffy aerial mycelia were arranged irregularly and densely. Hyphae were septate and branched, 2 to 5 µm in width. Conidiophores were pale brown, septate, branched, cylindrical to ampulliform. Conidia were hyaline, smooth-walled, cylindrical with obtuse ends, and 8.1 to 13.3 μm × 2.4 to 5.8 μm (n = 50) in size. Appressoria were medium brown to dark brown, aseptate, in irregular shape, solitary or in groups, and measured 1.5 to 12.5 μm × 2.1 to 13.3 μm. Morphological characteristics of isolates agreed with the description of Colletotrichum (Liu et al. 2015). Genomic DNA was extracted from fungal colonies incubated on PDA for 7 days following the instructions from the PlantGen DNA Kit (CWBIO, China). The internal transcribed spacer (ITS) region, and fragments of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), beta-tubulin (TUB2) and large subunit ribosomal DNA (LSU rDNA) genes were amplified by primer pairs ITS1/ ITS4, GDF1/GDR1, ACT512F/ACT783R (Naz et al. 2017), T1/Bt2b (Glass et al. 1995) and LROR/LR7 (Castlebury et al. 2002), respectively. The sequence of representative isolate CG-H (GenBank accession nos. OM010355, OM238213, OM238214, OM045778 and OM010358 for ITS, GAPDH, ACT, TUB2 and LSU rDNA, respectively) exhibited 99 to 100% identity to Colletotrichum gloeosporioides. A multi-locus phylogenetic tree with concatenated sequences of ITS, GAPDH, ACT and TUB regions was constructed using the maximum likelihood method by MEGA7, which revealed that strain CG-H was grouped with C. gloeosporioides. To confirm the pathogenicity, six healthy H. coronarium plants were surface sterilized, and conidial suspension (1 × 106 conidia/mL) was sprayed onto the leaves. Six plants were inoculated with sterile distilled water as controls. All the plants grew in a greenhouse at 25 °C under 12 h/12 h photoperiod. The experiment was repeated four times. Yellow lesions appeared after 7 days of inoculation, irregular-shaped brown spots were formed and slightly sunken within 14 days, and the whole leaf gradually became withered in 50 days. All inoculated plants exhibited leaf spot symptoms while the control plants remained asymptomatic. C. gloeosporioides was re-isolated from lesions of leaves and identified by morphology and sequence analysis, fully confirming Koch’s postulates. This is the first report of C. gloeosporioides associated with H. coronarium leaf spot in China and worldwide. Further studies will be conducted on the sensitivity of C. gloeosporioides to various fungicides.

First Report of Charcoal Rot on Soybean Caused by Macrophomina phaseolina in Manitoba, Canada.

Macrophomina phaseolina (Tassi) Goid. is a soilborne necrotrophic fungal pathogen causing charcoal rot on approximately 500 plant species worldwide (Mengistu et al. 2015). Charcoal rot occurs in eastern Canada and many regions of the USA, causing substantial yield losses in soybean [Glycine max (L.) Merr.] (Allen et al. 2017; Bradley et al. 2021; Wrather et al. 2001). However, it has not been reported in soybean in western Canada. Manitoba is the second largest soybean producer in Canada, comprising 31% of total seeded areas with 2.29 M acres in 2017 (Statistics Canada 2022). Still, soybean is a relatively new crop to Manitoba and annual surveys of soybean root diseases began in 2012. In August 2020, randomly selected soybean fields were surveyed for root diseases at 63 different locations in south-central and southwest Manitoba. A total of thirty diseased plants were sampled in a zigzag pattern at three random sites in each field and all samples were brought to the laboratory and rated for disease severity. All plants showed symptoms of root rot, and some samples exhibited wilting with yellowing-brown leaves attached to the stems by the petioles; when the taproot was sectioned longitudinally, black streaking could be observed. In the laboratory, 600 roots from 40 selected fields were processed for pathogen isolation and identification. A 1 cm section from each root was surface-sterilized in a 95% EtOH:5.25% NaOCl solution for 30 sec, rinsed in sterile water for 60 sec, and air-dried on sterilized filter paper in a laminar flow hood. Root tissues with two replicates were placed on potato dextrose agar (PDA) plates amended with streptomycin sulfate (2 mg/mL) and incubated at room temperature. Black microsclerotia were observed in cultures from three different fields and three individual fungal isolates were obtained from each field through isolation of a single microsclerotium and subsequent hyphal tip transfer. The mycelia were initially hyaline and turned gray to dark brown or black, forming numerous microsclerotia ranging in size from 13 to 61 µm long and 12 to 32 µm wide, based on measurements of approximately 100 microsclerotia per isolate using a Zeiss Axio Imager A2 microscope equipped with an AxioCam HRc (Carl Zeiss, Jena, Germany) and AxioVision software. The color of the microsclerotia was jet black and the shape was round to oblong or irregular, as described by Mengistu et al. (2015). Based on morphological characteristics and microscopic examination, three fungal isolates were identified as M. phaseolina (Mengistu et al. 2015). For molecular identification, genomic DNA was extracted from 10 to 14-day old mycelia and microsclerotia of each isolate using a ZymoBIOMICS™ DNA Miniprep Kit (Zymo Research Corp., Irvine, CA, USA) according to the manufacturer's instructions. The internal transcribed spacer (ITS) region, translation elongation factor-1α (TEF-1α), and calmodulin (CAL) genes were amplified using the primer sets ITS1/ITS4 (White et al. 1990), MpTefF/MpTefR, and MpCalF/MpCalR (Santos et al. 2020), respectively, according to the original reaction conditions. Subsequently, PCR products were sequenced at Eurofins Genomics (Louisville, KY, USA). BLASTn analysis in GenBank showed that the nucleotide sequences of these regions of the three isolates (NSRR20-MB-24, NSRR20-MB-34, and NSRR20-MB-40) matched multiple isolates of M. phaseolina with 100% query cover and 100% identity. Sequences were deposited in GenBank for the ITS (OK127887, OK142725, OK128266), TEF-1α (OR363103, OR363104, OR363105), and CAL (OR357627, OR357628, OR357629) regions. In addition, the ITS and TEF-1α sequences of the three novel isolates were further aligned with multiple previously reported isolates of M. phaseolina, M. pseudophaseolina, and M. euphorbiicola (Chen et al. 2013; Machado et al. 2019; Sarr et al. 2014) using Muscle and trimmed (Edgar 2004). Alignments were concatenated to generate a maximum likelihood tree. Once concatenated, sequences were re-aligned. The obtained alignments were employed to construct a phylogenetic tree using the max likelihood method and Tamura-Nei model (Tamura and Nei 1993) with 10,000 bootstrap replicates using MEGA 11 (Tamura et al. 2021). The ITS and TEF-1α analysis indicated that the isolates were grouped in three differentiated clades (Figure 1). Macrophomina phaseolina isolates clustered in the same clade at 98% similarity, with the three novel soybean isolates NSRR20-MB-24, NSRR20-MB-34, and NSRR20-MB-40 grouped closely in the cluster at 98% similarity and identified as M. phaseolina. In contrast, isolates of M. euphorbiicola formed another clade at 87% similarity and M. pseudophaseolina isolates grouped in a clade at 99%. The pathogenicity of the three isolates was evaluated under controlled conditions. Given that no information on charcoal rot resistance in soybean has been reported in Canada, one of the commonly grown varieties in Manitoba, "TH 32004", was selected for the pathogenicity test. Surface-sterilized soybean seeds, which had been pre-germinated for three days, were sown in a sterilized soilless growing mix (Sunshine #5) together with 5 g (approx. 1 × 105 microsclerotia) of macerated 10 to 14-day old inoculum grown on PDA-streptomycin agar medium at room temperature and applied using an inoculum layering technique. For the non-inoculated control, macerated PDA-streptomycin agar without mycelia was used. Twenty plants per treatment were maintained in a walk-in plant growth chamber with a 16 h photoperiod at 25/20 °C ± 1 °C (day/night) and 50% relative humidity. Plants were watered weekly but were subjected to water stress. Symptoms of charcoal rot were observed in the root systems of all inoculated soybean plants after 28 days, while no symptoms were observed in the control plants (Figure S1). There was production of microsclerotia on the roots inoculated with each isolate (data not shown). Three isolates of M. phaseolina were re-isolated from the inoculated plants and found to be identical to the inoculated isolates with respect to morphological characteristics in culture, as well as with respect to the ITS, TEF-1α and CAL DNA sequences. For each isolate and non-inoculated control, five seeds of 'TH 32004' were seeded per pot, and four pots were used for the inoculated and control treatments. The experiment was repeated twice in a randomized complete block design with similar results, fulfilling Koch's postulates. To our knowledge, this is the first report of charcoal rot caused by M. phaseolina on soybean in Manitoba, Canada.

Molecular Verification of Trichophyton in the Brazilian URM Culture Collection.

Trichophyton species cause dermatophytosis in humans, with a high, worldwide frequency of reports and important public health relevance. We evaluated 61 Trichophyton strains from different sources deposited in the University Recife Mycology (URM) culture collection of the Universidade Federal de Pernambuco, Brazil. Strains were phenotypically identified and confirmed by sequencing Internal Transcribed Spacers rDNA and partial beta-tubulin 2-exon. Additionally, we evaluated their susceptibility to terbinafine and itraconazole. Physiological analyses included urease activity and growth in casein medium. Phenotypic methods allowed the reliable identification of T. rubrum only, whereas, for other species, molecular methods were mandatory. All Trichophyton species exhibited susceptibility profiles to itraconazole (0.04-5.33μg/mL) and terbinafine (0.17-3.33μg/mL). Our analyses revealed a heterogeneous distribution of T. mentagrophytes, which does not support the current distribution within the species complex of T. mentagrophytes and its genotypes.

Isolation, identification, and antibacterial activity of endophytic fungus from Acanthus ilicifolius

Abstract Antimicrobial-resistant bacteria pose serious public health risks, necessitating bioprospecting for novel antimicrobial drugs. The endophytic fungi of the mangrove ecosystem are hotspots for discovering new bioactive chemical compounds. In this context, an investigation was designed to determine the isolation of the major endophytic fungi inhabiting the leaves of Acanthus ilicifolius, a mangrove plant with a long history of traditional use in the Chinese and Indian medical systems. Based on the morphological characterizations and molecular analysis of internal transcribed spacer rDNA sequence data, the study identified three unique endophytic fungal species, namely, Periconia macrospinosa, Coprinopsis cinerea, and Alternaria sp. Of these, P. macrospinosa was identified as the most dominant one, with the highest relative frequency (35.22%). The antibacterial activity of P. macrospinosa isolate (CMFRI/fPM-01) was evaluated by the well and diffusion method against six human pathogens, viz., Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus. The results demonstrated a high and wide spectrum of antimicrobial action of the isolate against all the tested human pathogens, with no significant difference (P > 0.05) in the activity between the pathogens. The antibacterial activity was further confirmed by determining the fungal culture supernatant's minimum inhibitory concentration and minimum bactericidal concentration. Although the studied fungi are known from other sources, this is the first report of P. macrospinosa and C. cinerea as endophytes in A. ilicifolius leaves. The outcomes also showed that the P. macrospinosa isolate could be used to discover effective antibacterial drugs against various human diseases.

Deteksi dan Identifikasi Molekuler Kapang Berpotensi sebagai Penghasil Patulin pada Buah Tropis

Production and export of tropical fruits in Indonesia have an increasing trend every year. The important factor in the production and export of fruits is food safety. One of the contaminants that may be found in fruits is mycotoxin, especially patulin. Patulin mainly found in fruits such as apple, orange, grape, and pear. This study aimed to detect molds from tropical fruits and to identify potentially patulin-producing molds. Detection of potentially patulin-producing molds obtained from tropical fruits was carried out using the isoepoxydon dehydrogenase (idh) gene. Species identification was carried out using internal transcribed spacer (ITS) region. The mold isolate sequences of ITS rDNA region were analyzed for their homology using both BLAST search and phylogenetic tree. A total of 26 molds were isolated from four types of fruit (malang apple, ambon banana, medan orange, and avocado) obtained from five different places in Bogor including fruit stalls, traditional markets, and supermarkets. The positive results of idh gene were mold isolates that have an amplicon at 620 bp. The result showed that 5 of 26 (19,2%) isolates were positive for idh gene, namely A11, A34, A43, A51 and B23. The positive isolates for idh gene were identified as Aspergillus aculeatus, Aspergillus niger, Cladosporium anthropophilum, Cladosporium tenuissimum, and Talaromyces verruculosus.

Morphology and phylogenetic relationships of two Antarctic strains within the genera Carolibrandtia and Chlorella (Chlorellaceae, Trebouxiophyceae)

The genera Carolibrandtia and Chlorella have been described as small green algae with spherical cell shapes that inhabit various environments. Species of these genera are often difficult to identify because of their simple morphology and high phenotypic plasticity. We investigated two small coccoid strains from Antarctica based on morphology, molecular phylogeny by two alignment methods which have been applied to previous phylogenetic studies of the genus Chlorella, and comparison of the secondary structures of nuclear small subunit (SSU) and internal transcribed spacer (ITS) rDNA sequences. Light microscopy of two strains revealed spherical cells containing chloroplasts with pyrenoids, and the morphological characteristics of the strains were nearly identical to those of other Chlorella species. However, based on the phylogenetic analyses of nuclear SSU and ITS rDNA sequences, it was determined that the Antarctic microalgal strains belonged to two genera, as the Chlorella and Carolibrandtia. In addition, the secondary structures of the SSU and ITS2 sequences were analyzed to detect compensatory base changes (CBCs) that were used to identify and describe the two strains. A unique CBC in the SSU rDNA gene was decisive for distinguishing strain CCAP 211/45. The ITS2 rDNA sequences for each strain were compared to those obtained previously from other closely related species. Following the comparison of morphological and molecular characteristics, we propose KSF0092 as a new species, Chlorella terrestris sp. nov., and the reassignment of the strain Chlorella antarctica CCAP 211/45 into Carolibrandtia antarctica comb. nov.

Development and Validation of a Multiplex TaqMan Probe qPCR Assay Specific for Three Rust Fungi Infecting Meliosma myriantha

Rust fungi are the largest group of obligate plant pathogens and cause severe damage to global forests and agricultural security. Meliosma myriantha, a tree species native to East Asia (China, Japan, and Korea), is vulnerable to three rust species: Neophysopella meliosmae, N. meliosmae-myrianthae, and N. vitis. The early symptoms of infection are indistinguishable between these species, making accurate and rapid diagnosis challenging. The urediniospores of N. meliosmae-myrianthae and N. vitis are also known to infect economically relevant grapevines (Vitis spp.) and ivies (Parthenocissus spp.), respectively, rendering early detection and identification even more important. To address this issue, we developed a multiplex quantitative PCR (qPCR) assay equipped with TaqMan probes targeting the internal transcribed spacer rDNA sequences specific to the three rust pathogens. This assay successfully detected minute quantities (5 fg for N. meliosmae-myrianthae and 50 fg for N. meliosmae and N. vitis) of DNA from the three Neophysopella species and demonstrated consistent reliability when applied to fresh and herbarium samples collected from M. myriantha, grapevines, and ivies. In conclusion, this novel assay is a rapid and robust diagnostic tool for the three rust pathogens, N. meliosmae, N. meliosmae-myrianthae, and N. vitis, and offers the potential to identify and detect their global movement and spread to grapevines, ivies, and trees.

Morphology, Molecular Phylogeny, and Fungicide Sensitivity of Phytophthora nagaii and P. tentaculata in Korea

Phytophthora species, classified under Oomycota, cause significant damage to various crops and trees. The present study introduced Phytophthora species, P. nagaii and P. tentaculata, new to Korea, which pose notable risks to their respective host plants. Our research provided a comprehensive description of these species taking into account their cultural features, morphological characteristics, and molecular phylogenetic analysis using the internal transcribed spacer rDNA region and cytochrome c oxidase subunit mtDNA genes (cox1 and cox2) sequences. In addition, this study first evaluated the sensitivity of P. nagaii and P. tentaculata to five anti-oomycete fungicides, finding both species most responsive to picarbutrazox and P. tentaculata resistant to fluazinam. The data can guide targeted treatment strategies and offer insights into effective control methods. The findings expand our understanding of the diversity, distribution, and management of Phytophthora species in Korea.

First report of powdery mildew caused by Erysiphe cruciferarum on spider flower (Tarenaya hassleriana) in China.

Spider flower (Tarenaya (Cleome) hassleriana (Chodat) Iltis, Cleomaceae) is an excellent ornamental landscape plant and has an extensive flowering period, and therefore, plays an important role in horticulture (Parma et al. 2022). In May 2020 and April 2021, severe powdery mildew symptoms were observed on spider flower plants in a public garden (22.35°N and 113.56°E) in Shenzhen, China. Approximately 60 % of the plants were infected, and the adaxial surface of diseased leaves were covered with irregular white patches, which developed on tender to old leaves. In severe infections, drying and premature defoliation of infected leaves were observed. Microscopic examinations of mycelia showed irregularly lobed hyphal appressoria. Conidiophores (n = 30) were straight, unbranched, 65.65-92.11 μm long, and consisted of two to three cells. Conidia were formed singly on the top of conidiophores, cylindrical to oblong, 32.15-42.60 × 14.88-18.43 μm (mean 38.26 × 16.89, n = 50), and without distinct fibrosin bodies. Chasmothecia were not observed. The internal transcribed spacer (ITS) region and 28S rDNA was amplified using the primer sets ITS1/ITS5 and NL1/NL4, respectively. The representative sequences of ITS and 28S rDNA (GenBank accession nos.: MW879365 for ITS and MW879435 for 28S rDNA) analyzed by BLASTN search and showed 100 % identity with the sequences from Erysiphe cruciferarum found in GenBank (accession nos.: LC009943 for ITS and MF192846 for 28S rDNA). Phylogenetic analyses were conducted for further confirmation by using the combined sequences of ITS and 28S rDNA and indicated that the isolate ZDH046 grouped in a clade with isolates of E. cruciferarum (Figure S2). Based on morphological and molecular characteristics, this fungus was identified as E. cruciferarum (Braun and Cook, 2012). Koch's postulates were confirmed by gently pressing conidia from diseased leaves onto 30 leaves of healthy spider flower plants. After incubating for 10 d in a greenhouse (25 ℃ and 75 % relative humidity), similar symptoms to the diseased plants appeared on all inoculated leaves, whereas control leaves remained symptomless. Powdery mildew caused by E. cruciferarum on T. hassleriana has so far only been reported from France (Ale-Agha et al. 2008), Germany (Jage et al. 2010), Italy (Garibaldi et al. 2009), and New Zealand (Pennycook 1989, E. polygoni). To our knowledge, this is the first report of E. cruciferarum causing powdery mildew on T. hassleriana in China. This finding expands the known host range of E. cruciferarum in China and indicates a potential threat to plantations of T. hassleriana in China.

Intracellular presence and genetic relationship of Helicobacter pylori within neonates' fecal yeasts and their mothers' vaginal yeasts.

Helicobacter pylori are transmissible from person to person and among family members. Mother-to-child transmission is the main intrafamilial route of H. pylori transmission. However, how it transmits from mother to child is still being determined. Vaginal yeast often transmits to neonates during delivery. Therefore, H. pylori hosted in yeast might follow the same transmission route. This study aimed to detect intracellular H. pylori in vaginal and fecal yeasts isolates and explore the role of yeast in H. pylori transmission. Yeast was isolated from the mothers' vaginal discharge and neonates' feces and identified by internal transcribed spacer (ITS) sequencing. H. pylori 16S rRNA and antigen were detected in yeast isolates by polymerase chain reaction and direct immunofluorescence assay. Genetic relationships of Candida strains isolated from seven mothers and their corresponding neonates were determined by random amplified polymorphic DNA (RAPD) fingerprinting and ITS alignment. The Candida isolates from four mother-neonate pairs had identical RAPD patterns and highly homologous ITS sequences. The current study showed H. pylori could be sheltered within yeast colonizing the vagina, and fecal yeast from neonates is genetically related to the vaginal yeast from their mothers. Thus, vaginal yeast presents a potential reservoir of H. pylori and plays a vital role in the transmission from mother to neonate.

Molecular prevalence of Enterocytozoon bieneusi in stray cats of İzmir, Türkiye

The phylum Microsporidia contains obligate single celled parasites that can infect many vertebrate hosts including humans. Enterocytozoon bieneusi is considered as the most diagnosed species in humans. E. bieneusi has also been detected in many animals such as cats, dogs and cattle. Among these animals, cats are carriers of type D and IV which are the most common human pathogenic genotypes of E. bieneusi. In Türkiye, the prevalence of E. bieneusi in stray cats is not well known. Therefore, in this study, the molecular prevalence of E. bieneusi in stray cats (n = 339) was determined by Real-Time PCR targeting ribosomal DNA ITS (internal transcribed spacer) region of E. bieneusi. Initially, the analytical sensitivity of Real-Time PCR was determined by a plasmid control and then E. bieneusi DNA was investigated in fecal samples of stray cats. The results showed that the analytical sensitivity of Real-Time PCR targeting ITS region of E. bieneusi was ≤1 copy plasmid/reaction. Analysis of fecal samples revealed that the molecular prevalence of E. bieneusi was 50.15% (170/339). Overall, these results showed that the Real-Time PCR successfully detected E. bieneusi in cat’s fecal samples and stray cats can be an important source for transmission of E. bieneusi to humans and other animals.

First Report of Heterodera zeae Koshy, Swarup & Sethi, 1971 (corn cyst Nematode) Infecting Corn (Zea mays) in Spain.

Heterodera zeae Koshy, Swarup & Sethi, 1971 (corn cyst nematode) is an important disease of corn in several areas of the world, including India, Nepal, Pakistan, Egypt, USA, Greece and Portugal (Subbotin et al., 2010). It is a sedentary semi-endoparasite feeding on corn roots and other Poaceae plants and has been associated with significant yield losses in corn (Subbotin et al., 2010). During autumn 2022 a plant-parasitic nematode survey performed in corn at central-western area of Spain (Talavera de la Reina, Toledo), revealed a commercial field with stunted plants. Nematodes were extracted from soil by centrifugal-flotation method (Coolen, 1979). Corn roots inspection detected infections by immature and mature cysts, and soil revealed also mature live cysts and second-stage juveniles (J2s) with a population density of 1010 eggs and J2s/500 cm3 soil (including eggs from cysts). J2s and cysts were processed to pure glycerine using De Grisse's (1969) method. DNA was isolated from single live fresh J2s specimens for amplifying and sequencing of cytochrome c oxidase subunit II (COII) mitochondrial region using the primer pair species-specific H.Gly-COIIF_inFOR/P116F-1R (Riepsamen et al., 2011); D2 and D3 expansion domains of the 28S rRNA were amplified using the D2A/D3B primers (De Ley et al. 1999); internal transcribed spacer (ITS) region using primers TW81/AB28 (Subbotin et al., 2001); and cytochrome c oxidase subunit 1 (COI) gene was amplified using the primers JB3/JB5 (Bowles et al., 1992). Brown cysts were lemon-shaped with a protruding vulval cone with fenestra ambifenestrate, bullae prominent below underbridge and characteristically arranged in finger-like bullae (Fig. 1). J2 with slightly offset lip region (3-5 annuli), stylet strong with rounded stylet knobs, lateral field with four lines, and tail short and tapering conically. Measurements of cysts (n=10) included body length 559 (432-688) µm, body width 450 (340-522) µm, fenestral length 40 (36-43) µm, semifenestral width 19 (17-21) µm, and vulval slit 40 (35-44) µm. J2 measurements (n=10) included body length 477 (420-536) µm, stylet length 21 (20-22) µm, tail length 51 (47-56) µm, and tail hyaline region 23 (20-26) µm. Morphology and morphometrics of cysts and J2, fit with original description and others from several countries (Subbotin et al., 2010). Two J2s individuals were sequenced for COII region (OQ509010-OQ509011) showing 97.1-98.1% similarity with H. zeae from USA (HM462012). Six almost identical 28S rRNA sequences from J2s (OQ449649-OQ449654) were 99.2-99.4% similar to 28S rRNA sequences of H. zeae from Greece, Afghanistan and USA (GU145612, JN583885, DQ328695). Four identical ITS DNA fragments from J2s (OQ449655-OQ449658) were 97.0-97.8% similar to ITS sequences of H. zeae from Greece, and China (GU145616, MW785771, OP692770). Finally, six COI sequences of 400 bp obtained for J2s (OQ449699-OQ449704) were under 87% similarity to several COI sequences of Heterodera spp. in NCBI, being a new molecular barcoding for identifying this species. On the basis of these results, the cyst nematodes isolated from the corn plants from the central-western area of Spain (Talavera de la Reina, Toledo) were confirmed as H. zeae and up to our knowledge it is the first report in Spain. This is a well-known pest of corn, causing important losses in this crop (Subbotin et al., 2010) and it was previously regulated as a quarantine nematode in the Mediterranean region (EPPO).

DNA‐based fungal diversity in Madagascar and arrival of the ectomycorrhizal fungi to the island

AbstractMadagascar is known for its high diversity and endemism of fauna and flora. Fungi, however, have been largely overlooked in diversity and evolution studies on the island, and whether fungi exhibit the same patterns as animals and plants has yet to be further examined. We collected fungal sporocarps and ectomycorrhizal (EcM) roots during three opportunistic surveys in five forests in Madagascar and generated a dataset of fungal Internal Transcribed Spacer (ITS) DNA sequences. We analyzed them together with all publicly available fungal ITS DNA sequences and identified 620 Operational Taxonomic Units (OTUs) from Madagascar, 10% of which contained only sequences from our surveys. Two hundred and ninety‐two OTUs belonged to EcM species with /russula‐lactarius, /boletus, /tomentella‐telephora, /cortinarius, and /amanita being the most abundant EcM lineages. Overall, 60% of all fungi and 81% of the EcM species from Madagascar appear to be endemic. We conducted a phylogenetic analysis using all the OTUs in Amanitaceae, Boletaceae, and Russulaceae families to elucidate their relative timing of arrival in Madagascar. We found that most EcM species from Madagascar in the three families diverged less than 22 million years ago (mya), long after the separation of India and Madagascar (88 mya), which is consistent with a dispersal mediated process of arrival to the island. Our study provides the first comprehensive view of the overall DNA‐based fungal diversity in Madagascar and the current state of knowledge of EcM fungi based on DNA sequences, useful for further ecological and evolutionary studies.Abstract in Malagasy is available with online material.

Diversity of fungi from marine inundated wood from the Bay of Fundy, Nova Scotia, Canada

Abstract Marine fungi play an integral role in the decomposition of intertidal organic substrata but remain understudied in cold-water habitats including Atlantic Canada. Marine inundated wood from the intertidal zone was sampled from 30 sites along the Bay of Fundy coastline in Nova Scotia, Canada. Wood types studied included attached and loose intertidal wood, and driftwood. Emergent fungi were cultured and identified using ITS (internal transcribed spacers) rDNA barcoding. Two hundred and twenty cultures representing 86 fungi are reported. Sixty-one fungi were new records for the Bay of Fundy, 41 are first records from the marine environment, and 19 fungi are potentially new to science. Fungi identified included eight obligate marine fungi, with the remaining fungi being facultatively marine. Eight ascomycetes were soft rot fungi; this ecological strategy for decaying woody material in cold-water marine environments is discussed. Historical records and roles of wood type and site on fungal colonization are discussed.

First Report of Powdery Mildew Caused by Podosphaera macularis on Humulus scandens in China.

Humulus scandens, (Lour.) Merr., is a climbing herb which are used as traditional Chinese medicine, a raw material for papermaking, making soap, and replacing hops H. lupulus. This herb is distributed in many provinces of China, including Sichuan province. During March and June 2022, powdery mildew was found on leaves of H. scandens in the modern agricultural high-tech demonstration garden of Sichuan Academy of Agricultural Science, Chengdu, Sichuan Province, China. Abundant white or grayish powdery colonies could be seen on the surface of leaves, and 30%-100% of leaf areas were affected. Some of the infected leaves were either chlorotic or senescent. About 90% of the observed plants showed powdery mildew symptoms. Conidiophores (n = 25) were 74.0 to 160.1 μm × 8.7 to 12.7 μm (on average 120.9 × 10.4 μm) and composed of cylindrical foot cells (length 31.9-72.9 μm, average 50.1 µm) and conidia (mostly 10 conidia) in chains. Barrel-shaped conidia with fibrosin bodies (n = 30) were 12.8 to 21.0 μm × 7.9 to 15 μm, on average 16.7 × 11.3 μm, with a length/width ratio of 1.5. Chasmothecia were not found. Based on these morphologic characteristics, the pathogen was initially identified as Podosphaera macularis (Braun and Cook 2012; Mahaffee et al. 2009). To confirm the identification, two isolates (PDLC0315 and PDLC0412) of P. macularis mycelia and conidia were collected, and mycelia and conidia were combined for a single DNA extraction from each isolate. With the total genomic DNA, the sequences of the internal transcribed spacer (ITS), 5.8S rRNA, the 18S and 28S large subunit ribosomal DNA (LSU) (Bradshaw and Tobin 2020; White et al. 1990), were bi-directionally sequenced and deposited in GenBank (ON862625.1 and ON862630.1). The ON862625.1 and ON862630.1 showed 100% similarity with sequences of P. macularis isolate CT1 (MH687414.1). Phylogenetic analyses based on the combined ITS and 28S rDNA sequences indicated that the two specimens, PDLC0315 and PDLC0412 formed a monophyletic clade together with sequences retrieved from Podosphaera macularis CT1 and Head quarter 31 (KX842348.1). The pathogenicity test with the fungus was confirmed by gently pressing the infected leaves onto three healthy wild plants from the same geographical location. Three uninoculated wild plants served as controls. Six inoculated and non-inoculated plants were placed in different growth chambers with a 16-h photoperiod at 22±2°C and 70% of relative humidity. After 10 to 14 days, powdery mildew colonies developed on inoculated plants. Non-inoculated control plants did not show any symptoms. The fungus on inoculated leaves was morphologically identical to that first observed in the garden. As far as we know, this study is the first report of powdery mildew disease in Humulus scandens caused by Podosphaera macularis in China. Rapid expansion and wild distribution of H. scandens could lead to increased powdery mildew risk in outdoor cultivation. Due to the invasive potential of the powdery mildew fungi, this record is important in the context of the range extension of Podosphaera macularis.

Morphological and Physiological Development of Pyricularia oryzae Isolates from North-western Region of Sarawak on Different Media under Laboratory Conditions

Rice blast (causal agent: Pyricularia oryzae) is an important disease of rice in Sarawak. Understanding the pathogen’s morphological characteristics, genetic diversity and pathogenicity is important. Having a suitable medium for culturing and maintaining P. oryzae is important to ensure the availability of inoculum or materials under laboratory conditions. Oatmeal agar (OMA) and potato dextrose agar (PDA) are common media used for growing P. oryzae. OMA allows better mycelial growth and better sporulation as compared to PDA. There are also other alternatives such as fresh rice leaf agar and rice straw agar. Although OMA seems to be the best medium, unfortunately the opaqueness of the medium causes difficulty in observing the morphology and growth of mycelia. In addition, it is known that different isolates of P. oryzae will respond differently to different media. This study aims to identify the best media for culturing and maintaining P. oryzae isolates from Sarawak. A total of 14 P. oryzae isolates were characterised for their morphological characteristics, growth rate and sporulation rate using seven growing media. These 14 isolates included seven newly identified isolates in this study and seven isolates from a previous study, which were verified using internal transcribed spacer DNA sequence. The colony surface of the 14 P. oryzae isolates varied on different growing media. The pigmentation of colony surface varied from different shades of grey, translucent light brown, white and colourless. Pyricularia oryzae isolates grew better on OMA and PDA, while OMA was the best for sporulation. These two media can be recommended for culturing and maintaining different P. oryzae isolates under laboratory conditions.

(2952) Proposal to conserve the name Coccidioides posadasii against Posadasia esferiformis, Coccidium posadasi, Pseudococcidioides mazzae, Geotrichum louisianoideum, Glenospora meteuropea, Glenospora metamericana and Trichosporon proteolyticum (Ascomycota)

(2952) Coccidioides posadasii M.C. Fisher & al. in Mycologia 94: 78. 2002, nom. cons. prop. Lectotypus (hic designatus): [cryopreserved culture] “Pappagianis isolate ‘Silveira’” from “severe primary coccidioidal infection [human] with erythema nodosum”, U.S.A., California, San Joaquin Valley, 1951 (ATCC No. 28868). MBT 10011881 (=) Posadasia esferiformis Cantón ex Posadas, Ensayo Nueva Neoplasia Hombre: Psorospermiosis Infect. Generaliz. 2: [3, footnote]–94. 1898 (‘esferiforme’), nom. rej. prop. Lectotypus (hic designatus): preserved infected human male, left foot, Pieza No. 779 (Mus. Patol., Dept. Patol., Fac. Med. Univ. Buenos Aires). MBT 10011896 (=) Coccidium posadasi Cantón, Tratado Zooparásitos Cuerpo Humano: 108. 1898 (‘posadas’), nom. rej. prop. Lectotypus (hic designatus): [icon in] Cantón, Tratado Zooparásitos Cuerpo Humano: 119, fig. 6. 1898. MBT 10011898 (=) Pseudococcidioides mazzae Fonseca in Bol. Inst. Clín. Quir. Fac. Ci. Méd. Univ. Nac. 4: 495. Apr–Mai 1928, nom. rej. prop. Lectotypus (hic designatus): [icon in] Bol. Inst. Clín. Quir. Fac. Ci. Méd. Univ. Nac. 4: 499, fig. 19. Apr–Mai 1928. MBT 10011882 (=) Geotrichum louisianoideum Castell. in Med. Press Circ. 136: 439. 31 Mai 1933, nom. rej. prop. Neotypus (hic designatus): [cryopreserved culture] Castellani, No. C. 8 (CBS No. 145.34). MBT 10011899 (=) Glenospora meteuropea Castell. in Med. Press Circ. 136: 440. 31 Mai 1933, nom. rej. prop. Lectotypus (hic designatus): [icon in] Med. Press Circ. 136: 440, fig. 4. 31 Mai 1933. MBT 10011900. Epitypus (hic designatus): [cryopreserved culture] isolated by A. Castellani, deposited by R. Ciferri (CBS No. 146.34). MBT 10011885 (=) Glenospora metamericana Castell. in Med. Press Circ. 136: 440. 31 Mai 1933, nom. rej. prop. Lectotypus (hic designatus): [icon in] Med. Press Circ. 136: 440, fig. 5. 31 Mai 1933. MBT 10011901. Epitypus (hic designatus): [cryopreserved culture] isolated by A. Castellani, deposited by R. Ciferri (CBS No. 196.34). MBT 10011886 (=) Trichosporon proteolyticum Negroni & Villafañe in Mycopathologia 2: 57. 1939, nom. rej. prop. Lectotypus (hic designatus): [icon in] Mycopathologia 2(1): t. VIII, fig. 2. 1939. MBT 10011902 Coccidioides immitis Rixford & Gilchrist (in Johns Hopkins Hosp. Rep. 1: 243. 1896) was designated by Clements & Shear (Gen. Fung., ed. 2: 410. 1931) as type of the fungal generic name Coccidioides Stiles in Rixford & Gilchrist (l.c.) (Ascomycota, Onygenales). This species name is occasionally incorrectly cited as “C. immitis C.W. Stiles in Rixford & Gilchrist”, due to a misreading of a statement by Rixford & Gilchrist in the original description “[…] we propose for it, in accordance with the suggestion of Dr. Stiles, the generic name Coccidioides, and we designate the species as Coccidioides immitis”. A quoted letter from Stiles republished by Rixford & Gilchrist (l.c.: 244) supplies the generic diagnosis differentiating Coccidioides from other protozoan genera, Eimeria Schneider and Klossia Schneider, so that the generic name should be attributed to Stiles (Art. 46.2 of the ICN; Turland & al. in Regnum Veg. 159. 2018). Stiles agreed with the suggested specific epithet “immitis” that was made by Rixford & Gilchrist during a face-to-face meeting (see quoted letter) for the one species but disagreed with a second species epithet, “superficialis” for a second species that Rixford & Gilchrist subsequently formally named C. pyogenes Rixford & Gilchrist (l.c.: 261). A sister species was molecularly recognized by M.C. Fisher, G.I. Koenig, T.J. White & J.W. Taylor (in Mycologia 94: 73–84. 2002), who described, identified, and separated it using an operational species concept, Genealogical Concordance Phylogenetic Species Recognition (GCPSR; Avise & Ball in Oxford Surv. Evol. Biol. 7: 45–67. 1990; Taylor & al. in Fungal Genet. Biol. 31: 21–32. 2000). The species was named Coccidioides posadasii M.C. Fisher & al. (l.c. 2002: 78). Sequences of the rDNA internal transcribed spacer (ITS) region provide diagnostic polymorphisms for the identification and differentiation of this species from C. immitis (Tintelnot & al. in Med. Mycol. 45: 385–393. 2007). Both species are etiologic agents of one of the classical systemic diseases, coccidioidomycosis, an infection known since the late 19th century (Posadas [as Posada] in Anales Circulo Méd. Argent. 15: 585–597, t. I–III. 1892; Wernicke in Centralbl. Bakteriol. Parasitenk. 12: 859–861. 1892). While both species exhibit a preference for arid desert regions of North, Central and South America, C. posadasii is found throughout these climatic zones, whereas C. immitis is primarily restricted in its distribution to the San Joaquin Valley area in California (Fisher & al., l.c. 2002: 73–98; Hamm & al. in J. Fungi 5(74): 1–11. 2019; Kollath & al. in Virulence 10: 222–233. 2019; Crum in Infect. Dis. Therapy 11: 713–742. 2022; Kirkland & al. in J. Fungi 8(859): 1–36. 2022). The purported “holotype” of Coccidioides posadasii is the frozen Californian strain RMSCC “Pappagianis isolate ‘Silveira’” (Fisher & al., l.c. 2002: 79), which, in the appendix to the publication (p. 82), is also listed as “Silveira4” from “D. Pappagianis”. Demosthenes Pappagianis is a well-known Coccidioides researcher who among other things was an author of a publication (Zimmermann & al. in J. Clin. Microbiol. 32: 3040–3042. 1994) that indicated that “Silveira” was isolated in 1951 from a human with severe primary coccidioidal infection with erythema nodosum and from the San Joaquin Valley, California. There were three other Silveira clones of the same isolate routed via the laboratory of Rebecca Cox, referred to as Silveira1, 2 and 3 by R. Cox used as controls (Fisher & al., l.c. 2002: 73–98). The history of these clonal isolates is further elucidated in a genome study of C. posadasii using the “Silveira” isolate in yet another lab (Teixeira & al. in G3 12(4): jkac031. 2022). RMSCC is an acronym for Roche Molecular Systems Culture Collection in Alameda, California. Article 40.7 of the ICN stipulates that beginning in 1990, “the single herbarium, collection, or institution in which the type is conserved must be specified” otherwise the name is not validly published. The type of C. posadasii is consequently interpreted as the RMSCC isolate stored in RMSCC because this was the only location and designation specified in the first sentence following “HOLOTYPE:”. The authors went on to say that it was a widely used isolate maintained in the American Type Culture Collection (ATCC) # 28868. Additionally, they indicated that a “killed sample of RMSCC Silveira has been lodged in the Jepson Herbarium, University of California at Berkeley, Berkeley, California 94720, USA” and that frozen samples were also in the “Centers for Disease Control and Prevention, Atlanta, Georgia”, thus listing three other locations ATCC (Manassas), JEPS (Berkeley) and CDC (Atlanta) later in the paragraph. Consequently, we interpret the holotype designation to be complete at the end of the first sentence, and the next three mentioned institutes to be indications of isotypes. Given the great importance of the organism involved, it is convenient to make this reasonable assumption of a single holotype with three isotypes. If it were not so interpreted, the name would not be validly published. Notably, the name Coccidioides posadasii was published in the first issue of Mycologia for 2002 and the paper had been in press when the 9/11 attacks occurred on September 11, 2001 in New York City, NY and the Pentagon, Arlington, VA. The species in the genus Coccidioides had become select agents in the U.S.A. (Dixon in J. Appl. Microbiol. 91: 602–605. 2001) and tightened rules followed (Federal Register 70: 13294–13325. 2005) for security reasons. Consequently the isolates, including the holotype, at Roche were destroyed (Matthew Fisher, pers. comm., 2 May 2012). The “killed” specimen was probably intended for UC (J. Taylor, pers. comm., 1 May 2012) rather than JEPS (both herbaria are in Berkeley but only UC held fungi), but the specimen never made it to either and was destroyed before deposit (J. Taylor, pers. comm., 1 May 2012). The number “94720” published by Fisher & al. (l.c. 2002: 79), is a U.S.A. postal code number for the herbaria rather than a specimen number. Given that the holotype was destroyed at RMSCC, and there are no illustrations, we hereby designated the frozen isotype at ATCC as lectotype in accordance with Art. 9.11–9.12; specifically ATCC No. 28868 (ex-RMSCC Silveira from San Joaquin, California, U.S.A.). We note that this isolate is also deposited elsewhere as CBS No. 113859. Ironically and confusingly, the name Coccidioides posadasii was proposed (Fisher & al., l.c. 2002) for the predominantly so called “non-California population” (Zimmermann & al., l.c.; Koufopanou & al. in Proc. Natl. Acad. Sci. U.S.A. 94: 5478–5482. 1997) but typified by an aberrant outlier, a Californian isolate. Should the Committee for Fungi and/or the General Committee decide that the name Coccidioides posadasii was not validly published in 2002 with regard to Art. 40.7 by not following our interpretation as explained above, then we request that the registered, validly published homonym (or isonym) Coccidioides posadasii M.C. Fisher & al. in de Hoog & al. (Index Fungorum No. 532. 2023) holotypified by the same lectotype (ATCC No. 28868) chosen here for the 2002 name, be substituted for conservation. Several older names long forgotten as supposed synonyms of Coccidioides immitis (see, e.g., Dodge, Med. Mycol.: 149. 1935) were not considered by Fisher & al. (l.c. 2002) and some of those names threaten the name C. posadasii: C. pyogenes Rixford & Gilchrist (l.c.: 261) was described simultaneously with C. immitis but, because we conclude that it is referable to that species, not C. posadasii, it is discussed in the Appendix to this proposal (below). Other names older than C. posadasii that threaten or potentially threaten the name include Posadasia esferiformis (‘esferiforme’) Cantón ex Posadas (Ensayo Nueva Neoplasia Hombre: Psorospermiosis Infect. Generaliz. 2: [3]. 1898), type of the generic name Posadasia Cantón ex Posadas, which was named after the same person, Dr. Alejandro Posadas. Posadas's famous patient was 33-year-old Mr. Domingo Escurra from Argentina. Posadas studied Mr. Escurra's disease for 7 years, publishing photographs of him depicting his face, full head, torso, all limbs, front and back throughout the years and conducted an autopsy on him following his death. Posadas believed the causal agent was a protozoan and published numerous illustrations and photographs. Although the organism was unnamed in his thesis and some of his publications, Dr. Elios Cantón, a famous Argentinian medical doctor, proposed a generico-specifica name to honour Posadas, “Posadasia esferiforme”, in a footnote in volume 2 of Posadas's Ensayo … for the organism infecting Mr. Escurra. In 1948 the infected head of Mr. Escurra was discovered preserved in a jar in formalin by Dr. Flavio Niño (in Bol. Inst. Clin. Quir. 26: 3–14. 1950), recognizable because the patient had been photographed while living. This was preceded by the discovery of other autopsy specimens (feet, hand) by Dr. R. Sammartino, as documented by Deresinski & Mirels (in Med. Mycol. 57: S3–S15. 2019). Escurra's infected left foot (specimen No. 779) was re-examined by Canteros & al. (in Medicina (Buenos Aires) 69: 215–220. 2009) using microscopy and molecular techniques to identify the fungus specifically as Coccidioides posadasii. Given a choice of original materials including numerous illustrations or Mr. Escurra's head (Exhibit No. 1, Museo del Instituto de Parasitología de la Facultad de Ciencias Médicas de Buenos Aires), versus the molecularly analysed left foot fungus (No. 779, Museo de Patología, Departamento de Patología, Facultad de Medicina de la Universidad de Buenos Aires., Pieza) we opt to select No. 779 as lectotype specimen of Posadasia esferiformis. Photographs of the lectotype were published by Niño (l.c.), Canteros & al. (l.c.) and Deresinski & Mirels (l.c.). This confirms that this earlier species name applies to C. posadasii. Authentic materials of some species names introduced by Aldo Castellani and co-workers (in Med. Press Circ. 136: 439–440. 31 Mai 1933; all often incorrectly attributed to Castellani & Jacono in J. Trop. Med. Hyg. 36: 297–321. 16 Oct 1933) are still available as living isolates in the CBS culture collection. Geotrichum louisianoideum Castell. (CBS No. 145.34, from a well-travelled patient living in Louisiana, U.S.A.), Glenospora meteuropea Castell. (CBS No. 146.34, from an English patient reported to be infected in the Balkans) and Glenospora metamericana Castell. (CBS No. 196.34, origin unknown) all proved to be identical to Coccidioides posadasii on the basis of morphological characteristics and sequences of the rDNA ITS region (Tintelnot & al., l.c.). As these names fulfil the requirements for novel species descriptions under the Code, they are older synonyms of C. posadasii, having priority by more than 60 years (Tintelnot & al., l.c.). In his May 1933 publication, Castellani (l.c.) published two hanging drop illustrations of mostly nondistinctive mycelium, one each of Glenospora metamericana and G. meteuropea that are here selected as their respective lectotypes. No illustration was supplied for Geotrichum louisianoideum in May 1933 (Med. Press Circ. 136), but a nondescript illustration was published in October 1933 (Castellani & Jacono, l.c.) where the earlier publication in May is attributed to G. louisianoideum. The cryopreserved epitypes selected here for the Glenospora names are based upon authentic identified isolates deposited by R. Ciferri many years ago in CBS and their metadata has been lost. It is logical and reasonable to assume that Ciferri, a contemporary of the older Castellani who was a fellow Italian medical mycology investigator researching the same diseases (see obituary by Baldacci in Mycologia 57: 198–201. 1965), would have acquired Castellani's isolates directly. Castellani & Jacono (l.c.: 320) stated: “Cultures of the fungi described in this paper will be gladly supplied to workers interested in the subject.” Additional names for which there are neither types nor authentic cultures have been listed (Baker & al. in Farlowia 1: 220. 1943; Dodge, l.c.) as synonyms of Coccidioides immitis but because they are from outside of California they were probably the more widespread C. posadasii. These are: “Coccidium neoplasicum” Cantón (Tratado Zooparasítica Cuerpo Humano: 123. 1898) that Cantón replaced by Coccidium posadas before publication (see below); Pseudococcidioides mazzae Fonseca (in Bol. Inst. Clín. Quir. Fac. Ci. Méd. Univ. Nac. 4: 495. Apr/Mai 1928; in Prensa Méd. Argent. 12: 532. post Aug 1928) from laryngeal abscess of male patient, Chaco, Argentina, 1927; Oidium protozoides Ophüls (in J. Exp. Med. 6: 459. 1905) isolated from disseminated infection of 19-year-old male cattle farmer, originating from the Azores, presenting in San Francisco, 1900 but now considered to be synonymous with C. immitis (see Appendix); and Trichosporon proteolyticum Negroni & Villafañe (l.c.) isolated from a pulmonary and disseminated fatal infection of 41-year-old male originating from Córdoba, Argentina, 1938. This last species name was based upon a culture forming arthrospores rather than on the characteristic spherules in tissues normally linked to Coccidioides. Negroni later synonymized Trichosporon proteolyticum with Pseudococcidioides mazzae following experiments with cultures of T. proteolyticum injected into guinea pigs that induced production of endospores characteristic of Pseudococcidioides (Negroni & Radice in Rev. Arg. Dermatosifilografia 30(3/4): 219–223. 1946), but this was followed by Artagaveytia-Allende, who studied the isolate and concluded P. mazzae was conspecific with Coccidioides immitis (in Mycopathologia 4: 375–378. 1949). As these originated in South America, they are here considered to be C. posadasii. The name Pseudococcidioides mazzae was published at least twice in 1928, with internal evidence suggesting that the article in Bol. Inst. Clín. Quir. Fac. Ci. Méd. Univ. Nac. volume 4 was published in the spring (April or May) because of articles dated April 1928 bracketing Fonseca's publication on pages 265 and 522. Whereas the publication in Prensa Méd. Argent. volume 12 begins on page 513 with a footnote saying the article was presented at a meeting in May, 1928 and it ends on page 536 where reference is made to a meeting on August 8, 1928. In both 1928 articles there is the same figure 19, here selected as lectotype. The introduction of Coccidioides posadasii as a species distinct from C. immitis has stimulated much debate (Millar & al. in J. Clin. Microbiol. 41: 5778–5780. 2003), as this was the first time that a novel species had been described through the use of purely molecular methods. The name is now widely accepted by mycologists and physicians (Umeyama & al. in J. Clin. Microbiol. 44: 1859–1862. 2006; Ramani & al. in Mycopathologia 163: 315–319. 2007; Maxwell & al. in Evolution 73: 42–58. 2018; Crum, l.c.; Teixeira & al., l.c.). Furthermore, the species is the etiologic agent of one of the major fungal diseases in endemic areas in the Americas, with thousands of infections each year (Laniado-Laborin in Ann. New York Acad. Sci. 1111: 19–34. 2007). Under the names Coccidioides immitis and C. posadasii, the two species have been the focus of genome studies (Sharpton & al. in Genome Res. 19: 1722–1731. 2009; Neafsey & al. in Genome Res. 20: 938–946. 2010; Maxwell & al., l.c; Teixeira & al., l.c.) and many medical studies (>215 listed in PubMed as of Jan 2022; e.g., Tortorano & al. in Mycopathologia 180: 229–235. 2015; Loudin & al. in Case Rep. Infect. Dis. 2016: No. 8715405. 2016) including whole-genome studies (Maxwell & al., l.c.; Teixeira & al., l.c.). Therefore, to resurrect an earlier name would be counterproductive as it would contribute to nomenclatural instability and confusion. In contrast, the names Coccidium posadasi, Geotrichum louisianoideum, Glenospora meteuropea, G. metamericana, Posadasia esferiformis, Pseudococcidioides mazzae, and Trichosporon proteolyticum have been lost and forgotten except in lists of synonyms as has Coccidioides pyogenes, and Oidium protozoides, recognized as applicable to Coccidioides immitis. Given the wide usage of the name C. posadasii and the significance of this species as the infectious agent of a major clinical entity, it is judged inappropriate to replace the rapidly entrenched name for the species. We therefore propose to conserve the name Coccidioides posadasii against its earlier synonyms. Finally, we note that many of the early reports on pathogenic fungi of humans in medical literature over a century ago often confused fungi for protozoans, and were often reported by investigators in several languages nearly simultaneously, which makes tracing of literature nearly impossible. For example, C.W. Dodge (l.c.) lists “Megalocitosporides” Wernicke 1892 as a synonym of Coccidioides which would make it an earlier generic name, possibly with earlier specific epithets. But, the one citation given by Dodge and often subsequently referenced by many, the landmark publication by Wernicke (l.c.), does not mention “Megalocitosporides”. We saw that Posadas (Ensayo Nueva Neoplasia Hombre: Psorospermiosis Infect. Generaliz. 1: 97. 1897) noted that Dr. Wernicke thought that Posadas's term, “megalosporideo” was not sufficient and that he recommended the term “megalocitospordeo” be used for the morphological structure. Dodge's copied synonymy was repeated many times in the Dictionary of the Fungi up to edition 10 (Kirk & al., Dict. Fung., ed. 10: 412. 2008) and in other literature (e.g., Seifert & al., Gen. Hyphomycetes: 150 & 284. 2011). Another long-cited synonymous “name”, “Coccidium neoplasicum” attributed to Cantón was traced to his now exceedingly rare book (Cantón, l.c. 1898), where on page 123 he noted that following Posadas's thesis presentation, they tentatively were going to name Posadas's parasitic protozoan “Coccidium neoplasicum” [not validly published], but he settled on the name “Coccidium posadas” instead. Coccidium posadasi (as posadas) Cantón was based upon descriptions supplied by Posadas and quoted text and illustrations by Posadas. Cantón (l.c. 1898) redrew Posadas's illustrations, as is evident from Cantón's fig. 6, no. 5 (p. 119) which is identical to Posadas's second drawing on page 79 of his published thesis (Posadas, Contribuciónal Studio de la Etologia de los Tumores Psorospermiosi Infectante Generalizada [thesis]. 1894). Cantón's fig. 6, no. 5 (p. 119) is here selected as lectotype. Therefore, Coccidium posadasi is synonymous with Coccidioides posadasii. Coccidium Leuckart, typified by C. oviforme Leuckart (Parasiten Menschen Krankheiten, ed. 2, 1: 254–255. 1879) remains a protozoan genus. We therefore, do not “correct” the epithet further than adding “i”. The generic name Coccidium Leuckart should not be confused with Agardh's earlier use of “Coccidium” or “Coccidia”, with or without a capitalized first letter, for a suprageneric grouping among Floridean algae. Failure to conserve the exact binomial, Coccidioides posadasii, would require publication of a new combination destabilizing medical mycology and governments’ listings causing considerable harm to the reputation of mycological taxonomy among user communities. Appendix: Application and typification of the names of the related species, Coccidioides immitis and C. pyogenes. Differentiation of Coccidioides posadasii from C. immitis not only involves differentiating two taxa using molecular analyses of sequence data, but is also dependent upon fixing the concept and application of the name, C. immitis, via its type (Principle II). When C. posadasii was described in 2002 the authors assumed the name C. immitis was applied to a different commonly recognized fungus. A second species, C. pyogenes, was simultaneously described with C. immitis in 1896 and could have been either C. immitis itself or C. posadasii. Neither the name C. immitis nor C. pyogenes have extant types and therefore both require typification and discussion to fix their application and to decide if C. pyogenes should be rejected against C. posadasii or listed as a synonym of C. immitis or if indeed C. posadasii should be named C. immitis! Effective typifications are required (Art. 7.10, 29.1) as explained below. Rixford & Gilchrist (l.c.: 209–268) introduced the name Coccidioides immitis, the type of Coccidioides, based upon observations of a specimen from a fatal case of disseminated coccidioidomycosis in a 40-year-old agricultural labourer who had been admitted to the municipal hospital of San Francisco in 1893. Authentic material of C. immitis is not known to be preserved at the Johns Hopkins Medical Center, Baltimore, Maryland (E. Hornsby and W.G. Merz, pers. comm., 2008) and the type specimen(s) reportedly deposited by Rixford & Gilchrist (l.c.: 243) at the “U.S. National Museum” (Smithsonian Institution, Washington, D.C.), consisting presumably of microscope slides prepared via alcohol, celloidin, haematoxylin and eosin, oil of cloves, and Canada balsam from tissue of diseased Homo sapiens [Mr. Joas Fertado Silverra, U.S.A., California], has been lost (Rusty Russell, pers. comm. 2008). Additional specimens purportedly held by Rixford, Gilchrist, Welch, Stiles, and Councilman have apparently been lost too. Tintelnot & al. (l.c.) therefore designated a dried culture of strain RMSCC No. 2394 preserved in the Centraalbureau voor Schimmelcultures (CBS No. H-19784), matching the original approximate geographical location, as the neotype of C. immitis. CBS is now the Westerdijk Fungal Biodiversity Institute. The ex-“neotype” culture is preserved as CBS No. 120936. Designation of the neotype was not in accordance with Art. 9.8 because other original materials, as photographs, were published in the protologue. Therefore, we hereby designate a photograph, plate XXV, fig. 1 (Rixford & Gilchrist, l.c.) as lectotype of Coccidioides immitis (MBT 10011883). Additionally, we hereby designate CBS No. H-19784 as epitype to the lectotype (MBT 10011884), thus maintaining Tintelnot & al.'s (l.c.) application of the name, C. immitis, as a species distinct from C. posadasii. We further note that the lectotype was from “Case 1” as reported by Rixford & Gilchrist (l.c.), a labourer in California who had worked in the interior, but which they suspected may or may not have been infected prior to emigrating from the Azores. Coccidioides pyogenes Rixford & Gilchrist (l.c.: 261) described simultaneously with C. immitis was isolated from a cutaneous infection of a 33-year-old male originating from the Azores, having worked in San Joaquin Valley, California, presenting in San Francisco, 1938. As with C. immitis, the name's authors purportedly deposited what are presumed to be microscope slides in the U.S. National Museum that are presumed lost as were the other materials with other researchers. For both protologues, C. immitis and C. pyogenes, Rixford and Gilchrist (l.c.: 243, 261) cited “(Rixford and Gilchrist, 1895)” following the binomials which suggests earlier publication of the names. A footnote on page 209 indicates that references were listed following another article on varicella. No reference to Rixford and Gilchrist 1895 is included there, but there are references by L.A. Duhring (Cutan. Med. 1: 156–157. 1895) to Gilchrist and Rixford 1894, 1895 and again to 1895 in another publication. The two former citations to “Rixford and Gilchrist, 1895” were public readings by the authors on the disease before scientific societies, whilst in the book by Duhring, both authors’ study of the disease is discussed, but no generic name or binomials are included. Rixford & Gilchrist (l.c.) differentiated the two species that they recognized based upon disease symptoms and morphological development but could not exclude the possibility that the same taxon was involved in both cases. We note that because the two species have a sympatric distribution and pathologically cannot be differentiated we still cannot exclude the possibility that the original material of C. pyogenes could have been C. immitis itself as both were described from California from labourers who had been in the San Joaquin Valley. However, to avoid continued ambiguity in name application in current literature, we hereby designate the illustration of Coccidioides pyogenes from “Case 2”, Plate XL Fig. 1 by Rixford & Gilchrist (l.c.) as lectotype of C. pyogenes (MBT 10011903). Additionally, we hereby designate as epitype the same specimen selected as epitype for C. immitis, viz. CBS No. H-19784 (MBT 10011887). This establishes that C. pyogenes is a synonym of C. immitis and not of C. posadasii. The epitype selected for both was molecularly characterized and differentiated from that for C. posadasii by Tintelnot & al. (l.c.) and maintains current usage. There was always a greater likelihood that C. pyogenes was C. immitis than C. posadasii because of the prevalence of C. immitis in California. Two other listed synonyms coined for Coccidioides pyogenes are superfluous and illegitimate. Ophüls (l.c.) considered that C. immitis Rixford & Gilchrist and C. pyogenes Rixford & Gilchrist represented a single species that should be included in Oidium and stated “[…] Its name then would be Oidium coccidioides, or, if there should be objections to the adoption of coccidioides, Oidium protozoides.” Being published prior to 1953 these are validly published alternative names (Art. 36.2), both are illegitimate as the epithets immitis or pyogenes were available for use in Oidium. It is also unclear which name is being replaced. Earlier C. pyogenes had been synonymized with C. immitis (Blanchard in Caus. Sci. Soc. Zool. France 1: 171. 1900), hence the alternative Oidium names should be considered as applying to C. immitis. SH, https://orcid.org/0000-0002-5344-257x KT, https://orcid.org/0000-0002-5788-1502 MCF, https://orcid.org/0000-0002-1862-6402 JWT, https://orcid.org/0000-0002-5794-7700 SAR, https://orcid.org/0000-0001-9715-3705 We thank Dr. Liliana Semenas, Laboratorio de Parasitología, Universidad Nacional del Comahue-CONICET, San Carlos de Bariloche, Argentina for supplying references including the rare Cantón (l.c. 1898) from her personal library and also Emilie Montreuil (Canadian Agriculture Library, Ottawa) for intensive interlibrary loan searches along with Fernanda da Silva Santos (Coleção de Culturas de Fungos Filamentosos, Instituto Oswaldo Cruz, Rio de Janeiro, Brasil). We also acknowledge that the late Drs. Walter Gams (1934–2017) and Ira F. Salkin (1941–2016) were collaborators and co-authors on early drafts.