ANATOMICAL-MORPHOLOGICAL AND ITS-BASED PHYLOGENETIC EVALUATION OF SELECTED LICHENIZED FUNGI FROM ROBERT ISLAND (ANTARCTIC PENINSULA, ANTARCTICA)

Objective To profile the intraspecific type of Trichophyton mentagrophytes clinically isolated from different anatomical sites of patients, and to compare the performance of different target sites for the identification of Trichophyton mentagrophytes complex strains. Methods A total of 48 Trichophyton mentagrophytes strains, which were clinically isolated from Department of Dermatology, Wuhan No. 1 Hospital in the latest 3 years, were included in this study. The phenotypes of these Trichophyton mentagrophytes isolates were primarily determined by morphological observation and the urease test. PCR was performed to amplify the nuclear ribosomal internal transcribed spacer (ITS) region and the D1-D2 domains of the large-subunit ribosomal DNA (28S rDNA) followed by DNA sequencing. Then, Clustal X2 software and MEGA 6.0 software were used to analyze the ITS and D1-D2 sequences and to build phylogenetic trees by the maximum-likelihood method (bootstrap= 2000) . Results As the ITS sequence-based phylogenetic tree showed, the probability that the 48 isolates were grouped into the Trichophyton interdigitale clade reached 92%. However, Trichophyton interdigitale could not be effectively differentiated from Trichophyton quinckeanum by the D1-D2 sequence-based phylogenetic tree. In addition, Trichophyton interdigitale showed various appearances, including woolen type, downy type, cream type, powdery type and granular type. Conclusions Trichophyton mentagrophytes can be identified to the species level based on the sequence of ITS region, which shows higher efficiency in identifying Trichophyton mentagrophytes complex than the D1-D2 domains. Morphological characteristics can not serve as the basis for intraspecific typing of Trichophyton mentagrophytes. Key words: Trichophyton; DNA barcoding, taxonomic; DNA, ribosomal spacer; Ribosome subunits, large; Mycological typing techniques

PDF HTML阅读 XML下载 导出引用 引用提醒 11种鲈形目鱼类ITS2特征及系统应用 DOI: 作者: 作者单位: 1. 中国科学院热带海洋生物资源与生态重点实验室, 广东 广州 510301;2. 中国科学院大学, 北京 100049;3. 中国科学院海洋研究所, 海洋生物分类与系统演化实验室, 山东 青岛 266071;4. 广东省应用海洋生物学重点实验室, 广东 广州 510301 作者简介: 武宝生(1991-),男,硕士,从事鱼类分类及系统进化研究.E-mail:wubaosheng15@mails.ucas.ac.cn 通讯作者: 中图分类号: S917;Q96 基金项目: 国家自然科学基金项目（31272273）. Study on feature of ITS2 in 11 Perciformes species and the application on phylogenetic relationship Author: Affiliation: 1. Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, China;2. University of Chinese Academy of Sciences, Beijing 100049, China;3. Laboratory of Marine Organism Taxonomy and Phylogeny, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China;4. Guangdong Provincial Key Laboratory of Applied Marine Biology, Guangzhou 510301, China Fund Project: 摘要 | 图/表 | 访问统计 | 参考文献 | 相似文献 | 引证文献 | 资源附件 | 文章评论 摘要:ITS2（Internal transcribed spacer 2）是位于核糖体5.8S和28S基因之间的非编码序列。为了探讨该片段的多样性特征以及进化模式，本研究选取了鲈形目（Perciformes）5科11种鱼类为研究对象，共获得了444条ITS2克隆序列，其长度范围为332~515 bp。比较种内不同序列的长度发现，金带细鲹（）在种内存在32 bp的差异，这2种鱼类的差异较为明显；其余9种鱼类的长度相对比较保守，长度差异小于14 bp。依据11种鱼类的保守位点数、变异位点数、简约信息位点数、单倍型数、保守位点比例、单倍型多样性指数、核苷酸多样性等特征分析发现，种内存在着不同程度差异，特别是金带细鲹的ITS2序列存在着Type A、Type B和Type C 3种类型，各类型间差异较大。根据序列的多样性特征推断，金带细鲹和剑鱼的进化方式为非协同进化；蓝圆鲹（Alepes djedaba）和日本竹鱼䇲（Scomberoides tala）、布氏鲳鲹（Toxotes chatareus）和军曹鱼（）5种鱼类为协同进化；另外，协同和非协同进化状态与分类系统没有相关性。序列比对发现，大甲鲹种内存在着由协同进化方式演变为非严格的协同进化方式的过度序列；在金带细鲹的3个不同个体中，序列间存在着从协同进化、非严格的协同进化演变为非协同进化的3种进化方式。基于ITS2序列构建的11种鱼类的邻接系统树显示，每种鱼类的克隆都分别按种单独聚为一支，鲹科7属鱼类各属也是单独聚支，表明ITS2不仅可以用在种类的分子鉴定，同时也可以作为分子标记应用于鲹科和属级水平的系统关系研究。 Abstract:The nuclear ribosomal RNA (nrRNA) genes of eukaryotes are organized in clusters of tandem repeat units, including three genes (18S, 5.8S, and 28S) and two internal transcribed spacers (ITS1 and ITS2). The ITS2 is located between the 5.8S and 28S genes. Because multiple copies of ITS2 have different intra-and inter-species evolution rates, ITS2 is often used as the molecular marker to identify species or to infer the phylogenetic relationship at the rank of species or genera. In this study, 11 species from five families of Perciformes were selected as the representatives to investigate the characteristics and the evolutionary pattern of the ITS2 in fishes. A total of 444 cloning sequences of ITS2 were obtained from 24 samples through PCR and cloning methods. The length of these sequences ranged between 332-515 bp. A comparison of sequences within species found that the length of (32 bp) were extremely varied, and that of others, less than 14 bp, were relatively conservative. The evolutionary pattern of ITS2 of 11 species within species were conjectured based on the difference in several polymorphism characteristics, including the difference in length, conserved and variable sites, parsimony-informative site, the number of haploid type, proportion of conserved and variable sites, haploid type diversity index, nucleotide diversity and genetic diversity. The two out of 11 species () had obvious differences, especially the three different sequence types (Type A, Type B and Type C) detected in three individuals, suggesting a non-concerted evolution. Although, to a certain extent, length and variable sites were observed in four other species (, and S. leptolepis and , therefore, the four species were not subjected to strict concerted evolution (no-strict concerted evolution). The five other species (Toxotes chatareus, and ) almost had no intraspecific variations, suggesting a concerted evolution process. Meanwhile, there was no correlation between the evolution pattern of 11 species and the taxonomic system. In addition, sequence comparison revealed that transitive sequences between concerted evolution and non-concerted evolution were observed in , all three evolutionary patterns (concerted evolution, no-strict concerted evolution and non-concerted evolution) were detected in three individuals. Based on 444 cloning sequences of ITS2 from 11 species with as the outgroup, two phylogenetic trees were constructed using the neighborjoining and maximum likelihood methods. The results showed that both trees were largely congruent with each other. The topology showed that sequences from the same species clustered together and sequences from each genus of seven genera in Carangidae claded together. These results supported that the ITS2 is applicable as a molecular marker for species identification, but also useful for phylogenetic relation analysis at the rank of genus in Carangidae. 参考文献 相似文献 引证文献

Dekopon citrus (Citrus reticulata Shiranui) is a three-way hybrid (Citrus unshiu Marcov. × C. sinensis Osbeck × C. reticulata Blanco) developed in Japan in 1972. This citrus is popular in China due to its sweet and tender taste (Lim 2012). In November of 2021, a brown spot disease on Dekopon fruits with about 20% disease incidence was observed in an orchard of the Institute of Citrus Research in Ganzhou, Jiangxi Province, China. Initially, the symptoms appeared as slightly sunken deep red to purple spots on the fruit surface, with the disease progression, lesions became brown to brown-black large necrotic regions covered with a fluffy layer of gray spores. Infected fruits were surface sterilized with 70% ethanol for 30 sec and rinsed three times with sterile distilled water. Diseased tissues from the edge of lesions were cut into small segments, placed onto potato dextrose agar and incubated at 25℃ for 7 days. Ten single-spore isolates were obtained in total. Fungal colonies were olive green to dark green, velvet-like in texture and sporulated abundantly, surrounded by grayish-white hyphae. Conidiophores were subcylindrical, straight, septate, solitary or in clusters of two to three, and ranged in size from 65 to 550 × 3.8 to 6.3 µm (x ̅= 261.7 ± 60.5 × 5.2 ± 0.4 µm, n=50). Ramoconidia were cylindrical，aseptate, and 10 to 22 × 2.8 to 4.5 µm (x ̅= 15.5 ± 1.4 × 4.0 ± 0.9 µm, n=50). Conidia were lemon-shaped to oval-shaped, smooth-walled, and 1.8 to 5.0 × 1.4 to 2.5 µm (x ̅= 3.9 ± 0.4 × 2.2 ± 0.2 µm, n=50). The morphological characteristics of the pathogen were consistent with those of Cladosporium tenuissimum Cooke (Li et al. 2021). For further identification, DNA was extracted from two representative isolates. The internal transcribed spacer (ITS) region, translation elongation factor (EF1-α), and actin (ACT) were amplified by using primers ITS1/ITS4, EF1-728F/EF1-986R, and ACT-512F/ACT-783R (Bensch et al. 2012), respectively. ITS (OM232067, OM232068), EF1-α (OM256525, OM256526) and ACT (OM256529, OM256530) sequences were deposited in GenBank. Multi-gene (combined data set of ITS, EF1-α and ACT) phylogenetic analysis was conducted using the Maximum Likelihood method (Nguyen et al. 2015). Based on the morphological characteristics and the molecular data, two fungal isolates were identified as C. tenuissimum. To evaluate pathogenicity, fifteen fruits were surface sterilized with 1% NaClO solution for 30 sec, rinsed twice with sterile distilled water and dried. Dekopon fruits (n=10) were wounded with a sterile needle and inoculated with a 10 µL drop of conidial suspension (1 × 106 conidia/mL) of isolate GZCJ-1, followed by incubation at 25℃ and 80% relative humidity. The controls (n=5) were treated with sterile water and maintained under the same conditions. Five days after inoculation, small brown sunken spots were observed on the wounded and inoculated fruits. After 7 days, lesions were coated by a layer of brown conidia that were similar to those described above, whereas control remained symptomless. Pathogenicity test was repeated twice. Cladosporium tenuissimum was consistently re-isolated from inoculated fruits and confirmed by morphological and molecular data, fulfilling the Koch's postulates. To our knowledge, this is the first report of C. tenuissimum causing the brown spot of dekopon fruit in China and perhaps the world. The disease may become the potential risk for fruit production, making fruits unfit for marketing purposes, and the appropriate management actions will be necessary.

Species identification via DNA barcodes is contributing greatly to current bioinventory efforts. The initial, and widely accepted, proposal was to use the protein-coding cytochrome c oxidase subunit I (COI) region as the standard barcode for animals, but recently non-coding internal transcribed spacer (ITS) genes have been proposed as candidate barcodes for both animals and plants. However, achieving a robust alignment for non-coding regions can be problematic. Here we propose two new methods (DV-RBF and FJ-RBF) to address this issue for species assignment by both coding and non-coding sequences that take advantage of the power of machine learning and bioinformatics. We demonstrate the value of the new methods with four empirical datasets, two representing typical protein-coding COI barcode datasets (neotropical bats and marine fish) and two representing non-coding ITS barcodes (rust fungi and brown algae). Using two random sub-sampling approaches, we demonstrate that the new methods significantly outperformed existing Neighbor-joining (NJ) and Maximum likelihood (ML) methods for both coding and non-coding barcodes when there was complete species coverage in the reference dataset. The new methods also out-performed NJ and ML methods for non-coding sequences in circumstances of potentially incomplete species coverage, although then the NJ and ML methods performed slightly better than the new methods for protein-coding barcodes. A 100% success rate of species identification was achieved with the two new methods for 4,122 bat queries and 5,134 fish queries using COI barcodes, with 95% confidence intervals (CI) of 99.75–100%. The new methods also obtained a 96.29% success rate (95%CI: 91.62–98.40%) for 484 rust fungi queries and a 98.50% success rate (95%CI: 96.60–99.37%) for 1094 brown algae queries, both using ITS barcodes.

Crotalaria juncea (Fabaceae), also known as sunn hemp or Indian hemp, is a warm-season legume grown as a cover crop that provides nitrogen and organic-matter to soils, prevents weed growth and suppresses nematode populations (Meagher et al., 2019). In September 2021, root rot symptoms were observed in sunn hemp fields distributed in Cocula, Guerrero, Mexico. Diseased plants showed reduced growth, root rot, chlorosis, and wilting (Fig. 1). The disease incidence was estimated to be up to 40%. For fungal isolation, diseased roots were surface sterilised with 2% sodium hypochlorite for two minutes, rinsed with sterilised distilled water twice and blotted dry on sterile filter paper. Small fragments of diseased roots were placed on potato dextrose agar (PDA) medium and incubated at 25°C in darkness for five days. Ceratobasidium-like colonies were obtained consistently and 10 isolates were purified by the hyphal-tip method. Fungal colonies on PDA were white initially (Fig. 2) and then turned brown, raised, and with entire or undulated edges. Septate hyphae were hyaline, 4.2-6.7 μm in width, smooth, and branched at right angles with a septum near the point of branching. Microscopic examination by lactophenol blue staining showed two nuclei per cell. The morphological features of the isolates resembled those of Ceratobasidium spp. (Gonzalez et al., 2016; Ferreira et al., 2021). Four representative isolates were selected for molecular analysis and pathogenicity tests. The isolates were deposited in the Culture Collection of Phytopathogenic Fungi at the Colegio Superior Agropecuario del Estado de Guerrero (Accession Nos. CSAEG28–CSAEG31). For molecular identification, genomic DNA from each of the five isolates was extracted from mycelia following the CTAB method, and the internal transcribed spacer (ITS) region and partial sequences of the second largest subunit of RNA polymerase II (rpb2) gene were amplified and sequenced with the primer pairs ITS5/ITS4 (White et al., 1990) and RBP2-980F/RPB2-7cR (Liu et al., 1999), respectively. The sequences were deposited in GenBank (Accession Nos. OM677842-OM677845 for ITS and OL677498-OL677501 for rpb2). Phylogenetic analyses were performed using the Maximum Likelihood method with ITS and rpb2 sequences for Ceratobasidium spp. (Gonzalez et al., 2016; Ferreira et al., 2021). Additionally, a phylogenetic analysis using ITS sequences data for anastomosis groups (AGs) of Ceratobasidium sp. was generated. The phylogenetic trees inferred with ITS and rpb2 sequences data grouped the four isolates CSAEG28–CSAEG31 within the Ceratobasidium sp. clade with high bootstrap support (Fig. 3). The tree for AGs of Ceratobasidium spp. grouped the four isolates into a distinct clade to the other AGs but closely related to AG-L (Fig. 4). All these results showed that the four isolates of this study belong to a new AG of Ceratobasidium sp. For pathogenicity tests, 5-mm-diameter plugs from the margin of seven-day-old colonies were placed near the roots of sunn hemp seedlings (21 days old) with five replicates per isolate. Control plants were inoculated with a sterile PDA plug. All plants were kept in a greenhouse where the temperature ranged from 25 to 30°C. Root rot and wilting symptoms appeared on inoculated seedlings after 12 days (Fig. 5), whereas control plants remained symptomless. The pathogenicity test was performed twice with similar results. Fungi were reisolated from the infected roots and found to be morphologically identical to the isolates used for inoculation, thus fulfilling Koch's postulates. Ceratobasidium sp. have been reported on other members of the Fabaceae, including Arachis hypogaea, Glycine max, Trifolium subterraneum, and Vigna sinensis (Farr & Rossman, 2023). This is the first report of a Ceratobasidium sp. causing root rot of Crotalaria juncea in Mexico and worldwide. According to our observations, this disease is a serious threat to the health of sunn hemp plants.

The lichen species Usnea aurantiaco-atra (Jacq.) Bory is the most dominant vegetation on the Fildes Peninsula, Antarctica. Most individuals grow on rocks, and some are found with mosses. During the 27th and 28th Chinese National Antarctic Research expeditions of the Great Wall Station, U. aurantiaco-atra was observed growing on the lichen thallus of Umbilicaria antarctica Frey & I.M. Lamb, or on wood, which indicated that Usnea aurantiaco-atra could grow on various substrates. The diversities of the symbionts in U. aurantiaco-atra collected in the Fildes Peninsula were investigated using ITS rDNA sequences. The results showed that the sequences from mycobionts of U. aurantiaco-atra growing on various substrates did not exhibit significant differences. All photobionts in this lichen species were the green algae Trebouxia jamesii (Hildreth & Ahmadjian) G鋜tner. The identical sequences from the photobionts of both Umbilicaria antarctica and Usnea aurantiaco-atra indicated there was an algae pool in this area and different mycobionts could obtain their algal partners from this pool. The variety of substrates for U. aurantiaco-atra suggested its photobiont could be obtained from a mature lichen thallus by vegetative propagation; from other lichen thalli (e.g. Umbilicaria antarctica ); or from the surroundings. This study will promote understanding of the distribution of photobionts and the process of lichenization. Citation: Cao S N, Zheng H Y, Liu C P, et al. The various substrates of Usnea aurantiaco-atra and its algal sources in the Fildes Peninsula, Antarctica. Adv Polar Sci, 2015, 26: 274-281, doi: 10.13679/j.advps.2015.4.00274

Inter- and intra-strain variation and PCR detection of the internal transcribed spacer 1 (ITS-1) sequences of Australian isolates of Eimeria species from chickens.

Eucalyptus urophylla × E. camaldulensis, named Chiwei eucalypt, is a hybrid species widely used in China. Many of its clones are cultivated for afforestation due to cold tolerance, high yield, high strength and disease resistance. Clone LH1 is planted extensively for its high stability and machinability in South China. In December 2021, severe powdery mildew signs were observed on clone LH1 in Zhanjiang, Guangdong (N28°8'29"; E110°17'5"). Whitish powder principally appeared on both abaxial and adaxial leaf surfaces. All plants were infected within about a week and above 90% leaves were diseased, which resulted in abnormal growth and shrinkage of leaves. Hyphae with single, lobed appressoria were hyaline, septate, branched, 3.3-6.8 µm (ave. 4.9 µm, n>50) wide. Conidiophores with a straight to flexuous foot-cell (14.7-46.1×5.4-9.7 µm, ave. 25.8×7.9 µm, n>30) were erect, hyaline, 2-septate, unbranched, 35.4-81.8 × 5.7-10.7 (ave. 56.7×8.7 µm, n>50). Conidia were solitary, hyaline, cylindrical to elliptical, 27.7-46.6 ×11.2-19.0 (ave. 35.7×16.6 µm, n>50). Chamothecia were not found on infected trees. The further identification was confirmed by partial sequences of internal transcribed spacer (ITS), large submit rRNA gene (LSU), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glutamine synthetase (GS), and RNA polymerase II second largest subunit (RPB2) gene. A very small amount of mycelia and spores from voucher specimens CCAS-ASBF-1 and CCAS-ASBF-2 were deposited in the herbarium of Guangdong Ocean University. Specimens were PCR amplified and sequenced with primer pairs ITS1/ITS4 (White et al 1990), LROR/LR7 (Moncalvo et al 1995), PMGAPDH1/PMGAPDH3R, GSPM2/GSPM3R and PmRpb2_4/ PMRpb2_6R (Bradshaw, et al. 2022), respectively. BLASTn results showed that ITS (OP270019 and OQ380937), LSU (OP270018 and OQ380938), GAPDH, GS and RPB2 (OQ414445- OQ414450) were above 99% identical with those of E. elevata on Catalpa bignonioides (ITS: AY587013) (Cook et al 2004), Plumeria rubra (ITS: MH985631) (Yeh et al 2019), Cerbera manghas (ITS: MZ379159; LSU: MZ379160) (Mukhtar et al 2022), and Eucalyptus camaldulensis (LSU: LC177375-6) (Meebon et al. 2017), and above 99% identical with those of Erysiphe vaccinii FH00941201 on Vaccinium corymbosum (ITS: ON073869; RPB2: ON119159; GS: ON075687) and FH00112205 on V. vacillans (ITS: ON073870; GAPDH: ON075646) (Bradshaw et al 2022). This is the first sequence data for non rDNA of E. elevata. In an ITS tree phylogenetic analysis with Maximum likelihood (ML) method showed the fungus clustered in a highly supported clade with E. elevata and E. vaccinii. In a multi-locus tree, E. elevata grouped in a sister position to E. vaccinii FH00941201. Thus, the pathogen was identified as E. elevata based on morphology, DNA BLASTn and phylogenetic analysis (Braun and Cook 2012). Pathogenicity tests were conducted on healthy leaves of 1-year-old potted plants. Ten leaves were cleaned with sterile water, inoculated by gently dusting conidia from single lesion on the naturally infected leaves, and then covered with plastic bags containing wet absorbent cotton. Non-inoculated leaves served as controls. Symptoms developed on all inoculated leaves 3 to 5 days after inoculation, and the fungus was identical to the original fungus on the infected leaves, whereas control plants remained symptomless. This is the first report of powdery mildew caused by E. elevata on Eucalyptus sp. from China. This finding is helpful for land managers to diagnose and control the disease.

Data processing can mask biology: towards better reporting of fungal barcoding data?

Antarctica is one of the last frontiers of the planet to be investigated for the environmental transport and accumulation of persistent organic pollutants. Perfluorinated contaminants (PFCs) are a group of widely used anthropogenic substances, representing a significant risk to wildlife and humans due to their high biomagnification potential and toxicity risks, especially in food webs of the northern hemisphere and Arctic. Because the assessment of PFCs in the Antarctic continent is scarce, questions linger about the long-range transport and bioaccumulation capacity of PFCs in Antarctic food webs. To better understand the global environmental fate of PFCs, sediment, lichen (Usnea aurantiaco-atra), and seabird samples (southern giant petrel, Macronectes giganteus; gentoo penguin, Pygoscelis papua) were collected around the Antarctic Peninsula in 2009. PFC analytes were analyzed by LC/MS/MS, revealing the detection of PFHpA in seabirds’ feather and fecal samples, and PFHxS in lichens. PFBA and PFPeA were detected in 80% and 60% of the lichens, and PFTA in 60% of sediment samples. While oceanic currents and atmospheric transport of PFCs may explain the ubiquitous nature of these contaminants in the Antarctic Peninsula, military bases and research stations established there may also be contributing as secondary sources of PFCs in the Antarctic ecosystem.

Objective To clarify the taxonomic status of anopheline mosquitoes in Hainan Island and reconstruct the molecular phylogenetic relationship among Anopheles vagus and its cryptic species.Methods Anopheline mosquitoes from 8 sites in Hainan Island were distinguished based on morphology and molecular markers,with ribosomal DNA second internal transcribed spacer (rDNA-ITS2)and the third domain of 28S (28S-D3) sequences.The phylogenetic tree,using maximum likelihood method,was reconstructed by rDNA-ITS2 and 28S-D3 dataset,after the sequences of mosquito species within Pyretophorus series were analyzed.Results Total 407 individuals were identified in this study.The agreement between results of molecular identification and morphology in adults was 84.9％.In 228 ITS2 sequences of An.vagus,some individuals at two positions exhibitd double-peaks (G/A),the frequencies were 57.9％,64.8％,respectively,both for 57％.Other positions were intraspecific conservation.There were no differences of D3 sequences within An.vagus individuals.The ML tree based on ITS2 sequences showed that An.vagus and An.subpictus cytotype A/C/D were close on the same clade,while far away with the other clades with An.subpictus cytotype B and An.sundiacus complex.Anopheles sp.NBG was An.subpictus cytotype B by ITS2 marker.However,it was also likely a new member of An.sundiacus complex.Conclusion Molecular marker is more important in distinguishing anopheline mosquitoes species while there are many intraspecific morphology variations.The relationship between An.vagus and An.subpictus cytotype A/C/D is closer than that between subpictus cytotype B and An.subpictus cytotype A/C/D. Key words: Anopheline mosquitoes; Ribosomal DNA; Morphology; Anopheles vagus; Pyretophorus series

Muskmelon(Cucumis meloL.) is one of the most widely cultivated and economically important fruit crops in the world.However, many pathogens can cause decay ofmuskmelon fruit, including Fusarium asiaticum, F. equiseti, F. incarnatum and F. lateritium (Hao et al. 2021; Wang et al. 2019). Fusarium spp. are the most important pathogens affecting muskmelon fruit yield and quality (Wang et al. 2011). In August 2020, fruitrot symptoms were observed on ripening muskmelons (cv. Tianbao) in several fields in Jiyang District, Jinan City of Shandong Province, China. The incidences of infected muskmelon ranged from 15% to 30% and caused an average 20% yield loss. Symptoms appeared as pale brown, water-soaked lesions that were irregular in shape, with the lesion sizes ranging from a small spot (1 to 2 cm) to decay of the entire fruit. The core and surface of infected fruit were colonized and covered with white mycelia. Two infected muskmelons were collected from two fields, 7 km apart. Tissues removed from inside the infected fruit were surface disinfected with 75% ethanol for 30 s, and cultured on potato dextrose agar (PDA) at 25°C in the dark for 5 days. Four purified cultures were obtained using the single spore method.On carnation leaf agar (CLA), macroconidia were 1 to 5 septate, falcate, with a pronounced dorsiventral curvature with blunt to papillate apical cell, and barely to distinctly notched basal cell, measuring 12 to 35 × 3.5 to 6 μm. Microconidia and chlamydospores were not observed. These morphological characteristics were consistent with the description of Fusarium sp. Because these isolates had similar morphology, two representative isolates (XP9 and XP10) were selected for multilocus phylogenetic analyses. DNA was extracted from the representative isolates using a CTAB method. Nucleotide sequences of the internal transcribed spacers (ITS) (White et al. 1990), calmodulin (CAM), RNA polymerase II second largest subunit (RPB2), translation elongation factor 1-α gene (TEF1) (Xia et al. 2019) were amplified using specific primers, sequenced, and deposited in GenBank (ITS: MW391507 and MW391508, CAM: MW392787 and MW392788, RPB2: MW392795 and MW392796, TEF1: MW392791 and MW392792). The Fusarium MLST database pairwise alignment of ITS (546 bp), CAM (628 bp), RPB2 (902 bp) and TEF1 (718 bp) sequences from isolate XP9 showed 99.63%, 99.33%, 100.00% and 99.71% similarity with the corresponding sequences (GQ505685, GQ505508, GQ505774 and GQ505596) of the reference strain of F. nanum (NRRL 22244), respectively. The overlap of ITS, CAM, RPB2 and TEF1 sequences from XP9 and NRRL 22244 were 100.00%, 95.06%, 97.45% and 94.99%, respectively. Alignments of a combined dataset of ITS, CAM, RPB2 and TEF1 were made using MAFFT v. 7, and phylogenetic analyses were conducted in MEGA v. 7.0 using the maximum likelihood method. The muskmelon isolates (XP9 and XP10) clustered together with theF. nanum reference strain CGMCC3.19498 and NRRL 22244 (100% bootstrap) (Wang et al., 2019). To perform a pathogenicity test, 10 μl of conidial suspensions (1 × 106 conidia/ml) were injected into each muskmelon fruit using a syringe, and the control fruit was inoculated with 10 μl of sterile distilled water. There were ten replicated fruits for each treatment. The test was repeated three times. After 7 days at 25°C, the interior of the inoculated muskmelons begun to rot, and the rot lesion expanded from the core towards the surface of the fruit, then white mycelia were produced on the surface. Ten isolations were re-isolated from the infected tissues and identified by morphological and phylogenetic analyses and confirmed to fulfill Koch's postulates. No symptoms were observed on the control muskmelons. To our knowledge, this is the first report of muskmelon fruit rot caused by F. nanum in China. Considering the economic value of the muskmelon crop, correct identification can help farmers select appropriate field management measures for control of this disease.

Sonchus oleraceus, common sow thistle, is native to Europe, Northern Africa, and Western Asia. This plant has become a common weed throughout the world. In Mexico, this weed has become widely naturalized by replacing indigenous plants and invading many agricultural areas. During the spring of 2018 and 2019, common sow thistle plants showing typical symptoms and signs of powdery mildew, were collected from agricultural fields in Ahome, Sinaloa, Mexico. As much as 30% of plants were diseased and 60 to 95% of the foliage was affected. Mycelium was conspicuous and white-gray, and on stems and both surfaces of leaves. Appressoria were nipple-shaped to crenulate. Conidiophores (n= 30) were hyaline, cylindrical, erect, and up to 150 μm long. Foot-cells (n= 30) were distinctly curved, 47 to 75 × 10 to 13 μm, slightly constricted, followed by 1-3 shorter cells and formed conidia in chains. Conidia (n= 100) were ellipsoid to doliiform to subcylindrical, 28 to 37 × 14 to 19 μm, lacked fibrosin bodies, and germinated from the apex. Chasmothecia were not observed. The morphological characters were consistent with those of the anamorphic state of Golovinomyces sonchicola (Braun and Cook 2012, Jakše et al. 2019). A voucher specimen (accession no. FAVF215) was deposited in the Herbarium of the Faculty of Agriculture of El Fuerte Valley at the Autonomous University of Sinaloa (Juan Jose Rios, Sinaloa, Mexico). To confirm the morphological identification, genomic DNA was extracted from mycelium and conidia, and the internal transcribed spacer (ITS) region and part of the 28S gene were amplified by PCR and sequenced. The ITS region of rDNA was amplified using the primers ITS5/ITS4 (White et al. 1990). For amplification of the 28S rRNA partial gene, a nested PCR was performed using the primer sets PM3 (Takamatsu and Kano 2001)/TW14 (Mori et al. 2000) and NL1/TW14 (Mori et al. 2000) for the first and second reactions, respectively. Phylogenetic analyses using the maximum parsimony and maximum likelihood methods (Braun et al. 2019), including ITS and 28S sequences of isolates of Golovinomyces spp. were performed and confirmed the results obtained from the morphological analysis. Isolate FAVF215 grouped in a clade with the other isolates of G. sonchicola. The ITS and 28S sequences were deposited in GenBank under accession numbers MW425872 and MW442972, respectively. Pathogenicity was demonstrated by gently dusting conidia from infected leaves onto leaves of 20 healthy plants and covered with plastic bags for 24 h. Ten non-inoculated plants served as controls. All plants were maintained in a greenhouse at 25 to 35ºC. All inoculated plants developed similar symptoms to those observed in the field from natural infections after 12 days, whereas powdery mildew symptoms and signs were not observed on control plants. The morphology asexual structures of fungus on inoculated plants were identical to those on naturally infected plants, fulfilling Koch's postulates. Inoculation tests were repeated twice with identical results. Based on the morphological data and phylogenetic analysis, the fungus was identified as G. sonchicola. This fungus has been reported causing powdery mildew on S. oleraceus in Germany, The Netherlands, Slovenia, and The United Kingdom (Farr and Rossman 2021). To the best of our knowledge, this is the first report of G. sonchicola causing powdery mildew on S. oleraceus in Mexico. This powdery mildew pathogen may represent an option for the biological control of common sow thistle.

From 2018 to 2020, powdery mildew-like signs and symptoms were observed on chayote (Sechium edule var. virens levis) in a commercial field located in Santa María del Río, San Luis Potosí, Mexico. Signs appeared as whitish powdery masses on both sides of leaves and stems. Disease incidence was about 30% and signs covered up to 70% of leaf surface. Ten samples were collected and analyzed. Mycelium was amphigenous, persistent, white, in dense patches. Hyphal appressoria were lobed and solitary. Conidiophores (n = 30) were hyaline, erect, straight, and 62 to 101 μm long. Foot cells were cylindrical and straight, followed by 1-3 shorter cells, and forming conidia in short chains. Conidia (n = 100) were hyaline, surface striate, cylindrical-ellipsoid, doliiform or ovoid, 25.7 to 37.6 × 11.9 to 18.4 μm, without fibrosin bodies, and with germ tubes terminal or subterminal. Conidial appressoria were lobed. Chasmothecia were not observed. The morphological characters were consistent with those of the anamorphic state of Neoerysiphe sechii (Gregorio-Cipriano et al. 2020). A voucher specimen was deposited in the Herbarium of the Department of Agricultural Parasitology at the Chapingo Autonomous University under accession number UACH192. To confirm the identification of the fungus, genomic DNA was extracted from conidia and mycelium, and the internal transcribed spacer (ITS) region and part of the 28S gene were amplified by PCR and sequenced. The ITS region of rDNA was amplified using the primers ITS5/ITS4 (White et al. 1990). For amplification of the 28S rRNA partial gene, a nested PCR was performed using the primer sets PM3 (Takamatsu and Kano 2001)/TW14 (Mori et al. 2000) and NL1/TW14 (Mori et al. 2000) for the first and second reactions, respectively. Phylogenetic analyses using the maximum parsimony and maximum likelihood methods, including ITS and 28S sequences of isolates of Neoerysiphe spp. were performed and confirmed the results obtained in the morphological analysis. The isolate UACH192 grouped in a clade with isolates of N. sechii. The ITS + 28S sequence was deposited in GenBank under accession number MZ468642. Pathogenicity was confirmed by gently dusting conidia from infected leaves onto ten leaves of healthy chayote plants. Five non-inoculated leaves served as controls. The plants were maintained in a greenhouse at 25 to 30 ºC, and relative humidity of 60 to 70%. All inoculated leaves developed similar symptoms to the original observation after 8 days, whereas control leaves remained disease free. Microscopic examination of the fungus on inoculated leaves showed that it was morphologically identical to that originally observed. The pathogenicity test was repeated twice with similar results. Based on morphological data and phylogenetic analysis, as well as pathogenicity test, the fungus was identified as N. sechii. This pathogen has been previously reported causing powdery mildew on S. edule and S. mexicanum in Veracruz, Mexico (Gregorio-Cipriano et al. 2020). However, to our knowledge, this is the first report of N. sechii causing powdery mildew on chayote in San Luis Potosí (Central Mexico). This pathogen represents a serious threat to chayote production and disease management strategies should be developed.

Symphyotrichum subulatum (Michx.) G. L. Nesom (syn. Aster subulatus), an annual herb in the Asteraceae family, is native to North America. Nowadays, it has become an invasive weed in several provinces of China, including Jiangsu, Zhejiang, and Sichuan (Li and Xie, 2002). Despite being invasive, this species holds significance in Chinese medicine, where it is used for the external treatment of eczema and swollen sore poison (Hu, 2020). In June 2023, symptoms of powdery mildew were observed in S. subulatum populations in Deyang and Nanchong, Sichuan Province, China. About 32.73% among 55 surveyed S. subulatum plants showed signs of infection. Symptoms initially appeared as small, scattered white powdery patches on the leaves, which enlarged and coalesced over time. Subsequently, hyphal growth forming extensive conidia covered up to 90% of the leaf area on both surfaces (Fig. S1A, B), and the infected leaves withered and fell off (Fig. S1A). A specimen was archived at China West Normal University (SsPM-ZL). Conidiophores were cylindrical and erect, 66.4 to 183.2 µm (avg. 108.2±40.8 μm) in length (n=30) (Fig. S1C). Conidia, produced singly, were ellipsoid-ovoid to nearly cylindrical, measuring 29.5 to 36.7 μm in length (avg. 32.9±2.6 μm) and 16.0 to 19.9 μm in width (avg. 17.4±1.3 μm), lacking distinct fibrosin bodies (n=30) (Fig. S1D). Under a scanning electron microscope, turgid conidia displayed reticulate wrinkles on the surface, with gentle contractions or bulges at both poles (Fig. S1E, F). Based on these characteristics, the powdery mildew fungus was consistent with the genus Golovinomyces (Bradshaw et al. 2022a). To confirm the identity of the causal fungus of specimen (SsPM-ZL), the calmodulin (CAM), RNA polymerase II subunit (RPB2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glutamine synthetase (GS), and the internal transcribed spacer (ITS) were amplified using PMCAMF/R, PMRPB2F/R, PMGAPDH1/3R, PMGSF/R, and PM5/ITS4 primers (Bradshaw et al. 2022b), and deposited in GenBank (CAM: OR761878; RPB2: OR761881; GAPDH: OR761879; GS: OR761880; ITS: OR758452). BLAST analysis showed 99 to 100% identity with the sequences of Golovinomyces ambrosiae (FH00941234) for CAM (ON101658, 99.65%), RPB2 (ON119165, 100%), GS (ON075690, 99.78%), and ITS (ON073876, 99.47%). Phylogenetic analysis was performed in MEGAX with maximum likelihood method (Kumar et al. 2016) and clustered SsPM-ZL into the G. ambrosiae clade with a 100% bootstrap support value based on the concatenated sequences of CAM, RPB2, GAPDH, GS and ITS (Fig. S2). Combining morphological and phylogenetic analyses, SsPM-ZL was identified as Golovinomyces ambrosiae. To evaluate pathogenicity, leaves of 3 healthy potted S. subulatum plants (3 leaves per plant) were inoculated by gently pressing them with diseased leaves, while 3 non-contact plants were used as control. Plants in two groups were incubated in separate greenhouses maintained at 27±1°C, with a photoperiod of 14 hours and a relative humidity of 80%. After 7 days, the inoculated plants exhibited symptoms of powdery mildew (Fig. S1H, J), while the control plants remained asymptomatic (Fig. S1G, I). Morphological characteristics of the artificially induced powdery mildew were consistent with those on naturally infected plants. Powdery mildew caused by G. ambrosiae has been reported affecting Helianthus tuberosus (Huang et al. 2017) and Bidens pilosa (Mukhtar et al. 2022) in China. To our knowledge, this is the first report of powdery mildew caused by G. ambrosiae on S. subulatum in China. Our finding will provide the fundamental knowledge for future powdery mildew diagnosis and the development of potential control strategies.