Internal Transcribed Spacer rDNA Sequences Research Articles

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.

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

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

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.

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.

Macrofungi as Medicinal Resources in Uzbekistan: Biodiversity, Ethnomycology, and Ethnomedicinal Practices.

Interest in edible and medicinal macrofungi is millennial in terms of their uses in health and food products in Central Asia, while interest in inedible and medicinal macrofungi has grown in popularity in recent years. Edible and inedible medicinal basidiomycetes were collected during field surveys from different regions of Uzbekistan. The morphological characters and similarity assessment of rDNA-Internal Transcribed Spacer sequence data were used to measure diversity and habitat associations. A number of 17 species of medicinal macrofungi of ethnomycological and medicinal interest was found associated with 23 species of trees and shrubs belonging to 11 families and 14 genera. Polyporaceae and Hymenochaetaceae were represented by the highest number of species followed by Ganodermataceae, Fomitopsidaceae, Auriculariaceae, Cerrenaceae, Grifolaceae, Phanerochaetaceae, Laetiporaceae, Schizophyllaceae, and Stereaceae. The highest number of medicinal basidiomycete species was reported in the following host genera: Acer, Betula, Celtis, Crataegus, Juglans, Juniperus, Lonicera, Malus, Morus, Platanus, Populus, Prunus, Quercus, and Salix. An updated list of edible and inedible medicinal mushrooms identified in Uzbekistan, their morphological characteristics, and phylogenetic placement are given for the first time. Information is provided on their uses in traditional and modern medicine. Their bioactive compounds and extracts can be applied as medicines, as well as food and cosmetic ingredients.

First Report of Alternaria alternata Causing Leaf Spot on Broccoli in Ecuador.

In Ecuador, broccoli (Brassica oleracea var. italica) production is located in the Andean region, specifically Cotopaxi-Ecuador (INEC, 2019). A leaf pathogen has been constantly observed in this area, showing brown circular necrosis surrounded by yellowish halo-like spots causing leaf death (Fig. 1a). This pathogen was believed to be Alternaria sp.; however, the species was not determined either using classical or molecular tools. In 2021, ten leaves showing similar symptoms were collected in Cotopaxi and sent for pathogen identification. Here, leaf explants (0.25 cm2) showing disease symptoms were surface sterilized with 2% sodium hypochlorite (NaClO) and 70% ethanol (C2H6O), rinsed with sterile water, and transferred to Potato Dextrose Agar (PDA) media. Petri dishes were incubated in darkness at 25°C for five days. The single hyphal tip method was used to purify the cultures on PDA. Fifteen pure isolates were obtained after incubating for 14 days. Isolates were incubated under blacklight for two days to induce fungal sporulation. All isolates presented early white cotton-like mycelium that later became dark green (Fig 1b). Under the microscope, we observed straight primary conidia in simple or branched chains. Also, the conidia were obclavate, long ellipsoids, moderate in size (19.5-43.9 μm in length, 7.1-17.2 μm in width), and septate with few longitudinal septa. Lastly, the conidium body can narrow itself into a secondary conidia (Fig 1c) (Woudenberg et al., 2013). According to colony and conidia morphology, isolates were identified as Alternaria sp. (Woudenberg et al., 2013). Five isolates were randomly selected for DNA extraction and sequencing of ITS (internal transcribed spacer; Chou, H.H. and Wu, W.S. 2002), TEF (translation elongation factor; O'Donnell et al., 1998), and RPB2 (RNA polymerase II second largest subunit; Liu et al., 1999) gene regions. DNA sequences obtained from each marker were identical for all isolates. Consensus sequences and alignment were built using ClustalX in MEGA X (Kumar et al., 2018). Consensus sequences were deposited in GenBank with the following accession numbers: ITS, ON982232; TEF, ON983964; RPB2, ON983963. A multilocus Bayesian inference phylogenetic tree was constructed in Beast software (version 1.8.4) using the concatenated sequences (Drummond et al., 2012; Maharachchikumbura et al., 2014). The isolates in our study clustered with isolates of Alternaria alternata, confirming their identity (Figure 2). For Koch's postulates, healthy broccoli plants were grown in sterile soil for six weeks. The fungal conidia were suspended in sterile distilled water (1×106 conidia/ml), and the leaves were inoculated by spraying the spore solution. The control treatment was sprayed with sterile distilled water alone. Plants were maintained at 28°C and had more than 85% relative humidity (Sigillo et al., 2020). Seven days after inoculation, plants showed chlorosis and necrosis. Ten days later, 100% of the treated leaves presented brown circular necrosis (Fig. 1d). Control plants showed no disease symptoms. Re-isolation of the pathogen from the diseased leaf tissue was performed as previously described. The isolates presented the exact morphology of pure cultures obtained from field-diseased leaves. The pathogenicity test was performed twice. To our knowledge, this is the first report on A. alternata being the causal agent of leaf spot on broccoli in Ecuador. Disease diagnosis contributes to providing strategies against this pathogen. Further investigations are needed to find biological/chemical techniques or cultivar resistance to control this pathogen in broccoli.

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.

Different Techniques reveal the Difference of Community Structure and Function of Fungi from Root and Rhizosphere of Salvia miltiorrhiza Bunge.

•Fungi have essential functions in plant health and performance. However, the plant-associated functions of many cultured fungi have not been established in detail. • Here, the fungal species diversity of the Salvia miltiorrhiza root and rhizosphere were first obtained by culturomics and high-throughput sequencing. In addition, we present a comprehensive functional metagenomic analysis of the cultured fungi, and agar plates verified the activities of cellulase and chitinase predicted by metagenomic. • We first present a collection of cultured fungi from the root and rhizosphere of Salvia miltiorrhiza, including 92 species across 37 families and 5 phyla, Ascomycota was the dominant species. Many rDNA internal transcribed spacer sequences were unassigned at lower taxonomic levels. 19 genera of endophytic fungi and 37 genera of rhizosphere fungi are first reported. The cultivation-dependent approach demonstrated a lower taxonomic diversity than high throughput sequencing, but some fungi existed only in cultures. Structural analyses indicated that the dominant species differed in cultured and noncultured samples at other levels except for the phylum level. Functional analysis showed that 223 carbohydrate enzyme families and 393 pathway maps were identified in the CAZy and KEGG databases, respectively. The most abundant families were glycoside hydrolases and carbohydrate metabolism. As predicted by metagenomics, we experimentally verified cellulase and chitinase activities for 29 and 74 fungi, respectively. •We provide the first evidence for biomass recycling by fungi associated with plants. Culturing is necessary to decipher the hidden microbial community and the critical functions in plant-microbe interactions.

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.

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.

Endophytic fungi associated with Brucea mollis Wall. ex Kurz.: a hidden source of antimicrobial and antioxidant metabolites

ABSTRACT Geosmithia pallida (KU693285) was isolated from Brucea mollis an endangered medicinal plant of North-East India. The secondary metabolites, produced by the endophytic fungi, extracted by ethyl acetate were screened for antimicrobial activity. G. pallida extract displayed the highest antimicrobial activity against Candida albicans with a minimum inhibitory concentration of 80.5 ± 1.25 µg/mL. G. pallida also showed the highest antioxidant activity which differed insignificantly from Penicillium sp. (P > 0.05). The G. pallida extract also exhibited the highest cellulase activity and also amylase and protease activities. The cytotoxicity assay of the ethyl acetate extract of this endophyte showed negligible effect (1.93 ± 0.42%) on chromosomal aberration as compared to the control (cyclophosphamide monohydrate) (7.20 ± 1.51%). The internal transcribed spacer rDNA sequence of G. pallida was submitted to the NCBI (Accession number KU693285) from India for the first time. The FT-IR spectrophotometry of the bioactive metabolite of G. pallida showed the presence of different functional groups such as alcohol, carboxylic acids, amines, aromatics, alkyl halides, aliphatic amines and alkynes. The GC-MS analysis revealed the presence of acetic acid, 2-phenylethyl ester; tetracosane; cyclooctasiloxane hexadecamethyl; cyclononasiloxane octadecamethyl; octadecanoic acid; phthalic acid, di(2-propylpentyl) ester and nonadecane, 2,6,10,14,18-pentamethyl as the major compounds in the metabolite. The findings of the present work indicated G. pallida as a potential source of important biomolecules without mammalian cytotoxic effects, which can be utilized for pharmaceutical purposes.

Diaporthe and Diplodia Species Associated with Walnut (Juglans regia L.) in Hungarian Orchards

Walnut (Juglans regia L.) production is a developing sector in Hungarian horticulture, where preharvest fruit rot increasingly causes economic losses. Previously, fungi from the Diaporthaceae and Botryosphaeriaceae families were isolated with a high incidence from rotten fruits. Here, we identify these pathogens from different plant parts (twigs, buds, and shoots) in orchards in the north-east of Hungary, and test their pathogenicity on immature nuts. Diaporthe eres Nitschke and Diplodia seriata De Notaris were identified in isolates from different symptomatic and asymptomatic plant parts based on their ITS (nuclear ribosomal internal transcribed spacer) and tef1 (translation elongation factor EF-1-alpha gene) DNA sequences. Purified monocultures caused rot of immature nuts following in vitro inoculation. Our results suggest that D. eres Nitschke and D. seriata De Notaris lingering on buds and overwintering woody parts may affect the seasonal nuts through wound infection. Infection by Diaporthe and Botryosphaeriaceae species present on woody plant parts, as well as on/in buds has been reported in Mediterranean countries. This is the first report of such stepwise aetiology from a region with continental weather. Climate change, associated weather patterns and the limitations of fungicide use in the European Union, among other factors, could be responsible for the increasing number of infections and economic damage caused by these pathogens.

Efficient control of the fungal pathogens Colletotrichum gloeosporioides and Penicillium digitatum infecting citrus fruits by native soilborne Bacillus velezensis strains

Destruction of citrus fruits by fungal pathogens during preharvest and postharvest stages can result in severe losses for the citrus industry. Antagonistic microorganisms used as biological agents to control citrus pathogens are considered alternatives to synthetic fungicides. In this study, we aimed to identify fungal pathogens causing dominant diseases on citrus fruits in a specialized citrus cultivation region of Vietnam and inspect soilborne Bacillus isolates with antifungal activity against these pathogens. Two fungal pathogens were characterized as Colletotrichum gloeosporioides and Penicillium digitatum based on morphological characteristics and ribosomal DNA internal transcribed spacer sequence analyses. Reinfection assays of orange fruits confirmed that C. gloeosporioides causes stem-end rot, and P. digitatum triggers green mold disease. By the heterologous expression of the green fluorescent protein (GFP) in C. gloeosporioides using Agrobacterium tumefaciens-mediated transformation, we could observe the fungal infection process of the citrus fruit stem-end rot caused by C. gloeosporioides for the first time. Furthermore, we isolated and selected two soilborne Bacillus strains with strong antagonistic activity for preventing the decay of citrus fruits by these pathogens. Molecular analyses of 16 S rRNA and gyrB genes showed that both isolates belong to B. velezensis. Antifungal activity assays indicated that bacterial culture suspensions could strongly inhibit C. gloeosporioides and P. digitatum, and shield orange fruits from the invasion of the pathogens. Our work provides a highly effective Bacillus-based preservative solution for combating the fungal pathogens C. gloeosporioides and P. digitatum to protect citrus fruits at the postharvest stages.

Gyrodactylus sprostonae Ling, 1962 infects an indigenous cyprinid in southern Africa: An expanded description

Gyrodactylus sprostonae Ling, 1962 is a highly invasive parasite reported across freshwater environments of the northern hemisphere. The taxon was originally described from Carassius auratus (Linnaeus, 1758) and Cyprinus carpio Linnaeus, 1758 in China. This parasite has never been reported in Africa or the southern hemisphere. Recently, this taxon was collected from an indigenous yellowfish, Labeobarbus aeneus (Burchell, 1822), in the Vaal River, South Africa. The present study includes the conclusive identification of the gyrodactylid parasites collected from L. aeneus, including additional taxonomic data, using microscopy and molecular techniques. Microscopy included light microscopy (LM) of whole worms and scanning electron microscopy (SEM) of isolated haptoral sclerites. Additionally, morphometric data were obtained from SEM and compared to that generated using LM. For molecular analysis, the internal transcribed spacer (ITS) region of rDNA was amplified and phylogenetic topologies constructed. The specimens were morphometrically and genetically highly similar to other data for G. sprostonae. Additional point-to-point measurements and ITS rDNA sequences were generated for the taxon, contributing to the morphometric and molecular data for G. sprostonae. The study also includes the first study of the isolated haptoral sclerites of the taxon using SEM, with similar morphometric results to LM. This is the first record of G. sprostonae in the southern hemisphere and from a new, indigenous African host, L. aeneus, indicating host switching to smallmouth yellowfish. Furthermore, these results expand on the knowledge of the distribution of invasive parasites in South Africa, as well as Gyrodactylus species diversity in Africa.

Occurence of Powdery Mildew Caused by Podosphaera xanthii on Vigna nakashimae in Korea

In October 2022, common Asian cowpea (<i>Vigna nakashimae</i>) powdery mildew disease was investigated at Yesan-gun, Chungcheongnam-do, Korea (36°40’12.7"N,126°51’36.4"E). The disease mainly occurred on the front of the leaves, and weak powdery mildew disease occurred on the back side of leaf. The chasmothecia was identified on the back side of the leaf. Analysis of the internal transcribed spacer rDNA sequence and 28S large subunit sequence, along with the morphological characteristics of the anamorph and teleomorph of the causal fungus identified as <i>Podosphaera xanthii</i>. This is the first time in Korea to report common Asian cowpea (<i>Vigna nakashimae</i>) powdery mildew disease caused by <i>Podospaera xanthii</i>.

Diversity of Fungi and Bacteria in Bronchoalveolar Lavage Fluid during Development of Chronic Obstructive Pulmonary Disease.

Dynamic changes in the microbiome during chronic obstructive pulmonary disease (COPD) exacerbations remain unclear. Using 16S ribosomal DNA and fungal internal transcribed spacer DNA sequencing, we described the composition and changes in the bacterial and fungal microbiota of bronchoalveolar lavage fluid samples from 15 COPD patients and seven non-COPD patients. In patients with COPD, the dominant bacterial phyla were Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria. The most abundant fungal phyla were the Ascomycota and Basidiomycota. In terms of the genera of bacteria and fungi, the numbers of Veillonella, Prevotella-7, Haemophilus, and Aspergillus were significantly higher in patients with COPD than in the non-COPD patients. In addition, after the progression of COPD, the relative abundances of the fungal genera Aspergillus, Mortierella, Grifola, Thermoascus, Russula, and Thermomyces and the bacterial genus Proteobacteria increased significantly. Existing analyses have demonstrated changes in the diversity of bacterial and fungal communities, which appear to be related to COPD. Our results demonstrate the potential utility of microbiota as a possible biomarker for disease progression and provide therapeutic targets for COPD.

Identification of Malassezia globosa as a Gastric Fungus Associated with PD-L1 Expression and Overall Survival of Patients with Gastric Cancer.

Microbiotas affected the prognosis of cancer patients by regulating programmed death ligand-1 (PD-L1) expression. However, the relationship between gastric fungi and PD-L1 expression is still unclear in gastric cancer (GC). We aimed at exploring the association of gastric fungi with PD-L1 expression and overall survival in GC. A total of 61 GC patients were divided into the two groups based on the PD-L1 combined positive scores (CPS). Fungal profiling was performed by internal transcribed spacer rDNA sequencing, and the survival analyses were performed by Kaplan-Meier curves. We observed a taxonomic difference of fungi between the PD-L1-High (CPS ≥ 10) and PD-L1-Low group (CPS < 10) by principal coordinates analysis (PCoA) (P = 0.014 for Bray-Curtis and P = 0.042 for Jaccard). Malassezia had a higher abundance in the PD-L1-High group compared to the PD-L1-Low group (P = 0.045). Malassezia globosa elevated significantly in the PD-L1-High group. GC patients with PD-L1 low expression and low abundance of Malassezia globosa had a longer overall survival (OS) than others (P = 0.047). Malassezia globosa was associated with PD-L1 expression (Odds Ratio = 3.509, 95% Confidence Interval: 1.056-11.656, P = 0.040). Malassezia globosa was associated with the tumor size (P = 0.031) and PD-L1 status (P = 0.024). GC patients with a high abundance of Malassezia globosa had shorter OS than others (P = 0.028). Malassezia globosa was an independent factor (Hazard Ratio = 3.080, 95% Confidence Interval: 1.140-8.323, P = 0.027) for OS after adjusting for tumor stage. Malassezia globosa was figured out to be associated with- fatty acid and lipid biosynthesis and degradation via LIPASYN pathway. Conclusions. Malassezia globosa was identified as a PD-L1 expression-associated gastric fungus and associated with OS of GC patients, which calls for more studies to further explore its potential in PD-L1/PD-1 targeted immunotherapy.

First Report of Root and Hypocotyl Rot of Dry Bean Caused by Aphanomyces euteiches in Alberta, Canada.

Dry bean (Phaseolus vulgaris L.) is a high-value crop grown under irrigation on 45,000 ha in southern Alberta, Canada. In 2019, one field of red beans showed premature yellowing and stunting of shoots in mid-July near Bow Island, AB. Roots had brown lesions along the length of hypocotyl and tap root, and decay and sloughing of the root cortex of lateral roots (Fig. 1). Roots were washed for 10 min under running tap water, and portions of the lesions were excised for DNA extractions using the Plant DNeasy kit (Qiagen) and multiplex PCR assays for an array of pathogens, which included Aphanomyces euteiches (Chatterton et al. 2019). Lesion pieces were also surface sterilized and plated onto PDA or cornmeal agar amended with metalaxyl, benomyl and vancomycin (CMBV) and visualized microscopically for oospores. The roots were PCR positive for A. euteiches, Rhizoctonia solani, and Pythium ultimum. Isolations yielded R. solani, and Fusarium spp. on PDA, but cultures on CMBV were over-grown with Rhizopus. Oospores measuring 24.7 +/- 1.08 m, consistent with the expected size of A. euteiches oospores of 18 - 25 m (Papavizas and Ayers, 1974), were visible in some of the root pieces. To obtain an A. euteiches isolate, soil was collected from three areas of the red bean field and used in a bait assay. Tests were performed using pea (cv. CDC Meadow) and dry bean (cv. Redbond) seeds treated with metalaxyl. Five seeds per pot were planted into field soils in 10-cm pots with 4 replicate pots/field. Soils were watered as needed until the 2nd node stage and then kept at saturation for 14 days, at which time roots were washed. Roots from the dry bean, but not the pea, plants showed severe root rot, including honey-brown discolouration of the lateral roots and extending to the hypocotyl, degradation of the root cortex, and presence of oospores. Diseased root pieces were plated onto CMBV without surface sterilization. Cultures with fast growing, white, aerial mycelia characteristic of A. euteiches on CMBV were recovered from dry bean roots from the three soil samples, considered as three isolates, and were transferred to PDATo confirm identity, total DNA was extracted from 7-day old cultures of the three isolates growing on PDA using the Qiagen DNeasy Plant Kit. The ribosomal DNA internal transcribed spacer (ITS) region was amplified using the primer pair ITS1 and ITS4, and sequenced (White et al. 1990). The sequences, deposited in GenBank with accession numbers OM976770, OM976771, and OM976772, were 100% identical to the ITS rDNA sequence of several isolates of A. euteiches (e.g. KM486066.1; 669/669 bp; 670/670 bp; 670/670 bp) using a BLASTn query and 99.7% identical to the CBS15773 voucher specimen (HQ643116.1, 661/663 bp; 662/664 bp; 662/664 bp). To confirm pathogenicity of these three isolates, cultures were used to produce zoospores for inoculating 14-day old red bean and pea seedlings (Chatterton et al. 2015). Control plants received minimal salt media solution and did not develop symptoms. Dry bean, but not pea, seedlings displayed root rot symptoms (Fig. 2). A. euteiches was re-isolated from dry bean seedlings by plating onto CMBV, and identity confirmed as described above. Although a previous survey detected A. euteiches in a small number of dry bean fields in southern Alberta (Chatterton et al. 2019), tests to confirm Koch's postulates were not performed. Aphanomyces root rot can be a devastating pathogen of pulse crops. Precautionary measures should be taken to prevent spread of this pathogen in the small dry bean growing area of southern Alberta by encouraging producers to use rotation intervals, monitoring movement of the pathogen, and evaluating cultivars for resistance to this pathogen.