First Report of a New Species Hyalopsora erlangensis Causing Rust Disease on Cystopteris chinensis in China

First Report of a New Species Hyalopsora erlangensis Causing Rust Disease on Cystopteris chinensis in China | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article First Report of a New Species Hyalopsora erlangensis Causing Rust Disease on Cystopteris chinensis in China An Yu, Xia Zhao, Xiaohong Chen This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4226118/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract A newly discovered rust fungus poses a significant threat to the survival and reproduction of the endangered plant Cystopteris chinensis in a forest in western Sichuan province, China. This study utilized both morphological analysis and molecular phylogenetic approaches to classify this fungal taxon. Artificial inoculation experiments, which involved sampling uredia from the field, were conducted to fulfill Koch’s postulates. Our results designate this rust fungus as a novel species within the Pucciniastraceae family, named Hyalopsora erlangensis A. Yu & X.H. Chen. Morphological feature, including hyaline peridia cells, yellowish urediospores, dark brown amphidspores with three germ pores, and pigmental cytoplasma, differentiate this species from others in the genera Milesina, Uredinopsis, and Pucciniastrum. Phylogenetic analysis, based on internal transcribed spacer (ITS) sequences and 28S rDNA gene fragments, further confirms its distinctiveness from other Hyalopsora species, supported by high Maximum Parsimony (MP), Maximum Likelihood (ML), and Bayesian Inference (BI) bootstrap values. Artificial inoculation of both field-collected and tissue-cultured seedlings verified this fungus as the causing agent of C. chinensis rust disease, indicating a broad host range within the Cystoperis genus. Our research not only identifies the pathogen but also offers fundamental insights for the future management and conservation of this endangered fern, aiming to mitigate the impact of this devastating rust. Cystoperis chinensis Hyalopsora erlangensis A. Yu & X.H. Chen rust fungi taxonomy Figures Figure 1 Figure 2 Figure 3 Introduction Rust fungi affecting ferns, such as species within the genera Hyalopsora, Milesina and Uredinopsis, comprise a significant portion of fern pathogens (Arthur 1924 );To date,a total of 26 species of Hyalopsora have been reported globally (Arthur 1906 ; Kern 1914; Chen et al. 1980 ; Hiratsuka et al. 1992 ; Saba et al. 2012 ), with eleven of these species documented in China, including H. aspidiotus, H. cryptogrammes, H. hakodatensis, H. japonica, H. polypodii, H. pseudocys-topteridis, H. taiwaniana, H. yunnanensis, H. tibetica, H. minispora, H . neocheilanthis (Chen 1982 , 1989 ; Jian-yun 1983 ; Hiratsuka and Chen 1991 ; Yu et al. 1997 ; Cao et al. 2000 ; Kirschner et al. 2019 ; Wang et al. 2021 ). Rust fungi are known to infect a wide range of plant species, including crops like wheat, barley, corn, and soybeans, as well as ornamental plants, trees, and shrubs (Blythe et al. 2014 ; Savary et al. 2019 ). Despite the known diversity, it is likely that additional fern rust species await discovery, given the extensive habitats suitable for fern growth. Rust fungi are characterized by complex life cycles often involving different hosts and spore stages producing abundant spores that facilitate widespread transmission via wind, water, or insects (McTaggart et al. 2022 ). The lifecycle of Hyalopsora rust on ferns remain elusive, with many species lacking reported alternative hosts. The subtle morphological distinctions between their urediospores, amphispores, and teliospores pose challenges for species delineation within the genus Hyalopsora. Some species exhibit telia parasitizing the epidermal cells of ferns, producing dark brown teliospores that are nearly hyaline, thin-walled, and frequently septate (Hiratsuka et al. 1992 ), complicating the task of accurately identifying Hyalopsora species. Ferns play a pivotal role among vascular plants, representing the second largest group after flowering plants, with approximately 12,240 species globally (Ranker and Haufler 2008 ). They occupies an intermediate phylogenetic position between mosses and angiosperms. China boasts a rich diversity of ferns, comprising 63 families, 230 genera, and around 2,600 species, which accounts for 21.7–26.0% of the global fern species total. Southwestern China, in particular, is a hotspot for fern biodiversity in Asia, with Sichuan Province's fern species count second only to Yunnan Province. Mount Emei alone is home to about 400 species of lycophytes and ferns (Zhang 2012 ). Most ferns are naturally found in their native habitats, typically growing in lithophytic or epiphytic conditions. Some species may also be found in moist or aquatic environments. They thrive in warm, humid forest settings and are particularly abundant in tropical and subtropical regions ( http://flora.huh.harvard.edu/China/mss/volume13/index.htm ). Fungi belonging to the genera Hyalopsora , Milesina , and Uredinopsis (Pucciniastraceae) exhibit a unique ecological specialization, exclusively parasitizing fern hosts during their sporophytic phase (uredinia and telia). The gametophytic phase of these fungi predominantly occurs on conifers in the Northern Hemisphere, especially within the Pinaceae family, including species of Abies. Rust fungi outside the Pucciniastraceae family that parasitize ferns are rare, with only a single known species of Desmella and Uncol, the former likely belonging to the Pucciniaceae and the latter to the Phakopsoraceae family (Berndt 2008 ). Cystopteris chinensis (Ching) X.C. Zhang&R. Wei, synonymously known as Cystopathyrium chinensis Ching, is a distinctive fern indigenous to China, classified under the family Cystopteridaceae and genus Cystopteris (Wei and Zhang 2014 ). It has been designated as an endangered species in the first volume of the "Red Book of Chinese Plants", recognized as a national level I endangered protected plant. Currently, its habitat is limited to Erlang Mountain in Tianquan County and Emei Mountain in Leshan City, Sichuan Province. C. chinensis has a limited reproductive capacity, which makes it particularly vulnerable to population decline. Moreover, it has specific habitat requirements that challenge its continued survival and reproductive success. Consequently, C. chinensis 's population is critically endangered and faces the threat of extinction. Conservation measures are imperative to safeguard this species and avert its loss from the ecosystem. The prevalence of rust disease significantly impacts the stability of C. chinensis populations, with an 80% infection rate and over 30% mortality observed in natural habitats. This study is the first to report the causative agent of this fern rust disease as the new species Hyalopsora erlangensis , identified through morphological and molecular phylogenetic methods. Materials and Methods Morphological study Rust leaves were collected at Erlang Mt. in Tianquan county, Sichuan China every July from 2021–2023, and the holotype specimens were kept in the herbarium of Sichuan Agriculture University under the accession numbers HAMS AY-2003a, HAMS AY-2003b. For morphological analysis, fresh leaf sections displaying uredia prepared manually and examined under a microscope. Initial observations of uredia size, shape, and development location were conducted using a stereomicroscope. To further investigate spore morphology and dimensions, spores from the sori were mounted in a drop of lactophenol solution on glass slides and observed with a light microscope (Olympus 51). Approximately 30 spores were randomly selected and measured for each specimen using an ocular micrometer at 40× magnification. The number and placement of germ pores were examined by mounting spores in lactic acid, briefly heating to boiling, and adding a drop of lactophenol solution with aniline blue as needed. DNA Extraction, PCR Amplification, and DNA Sequencing Genomic DNA was extracted from the urediniospres with BioRad Chelex-100 following published procedures (Scholin et al. 1999 ). Approximately six uredia were carefully scraped with a sterilized scalpel under a dissecting microscope and gently placed on microslides. The spores were softly crushed under a coverslip, which, along with the slide, was then rinsed with 50 µL of 5% Chelex-100 in sterilized water into a 1.5 mL centrifuge tube. This tube was incubated at 56℃ for 20 minutes, centrifuged at 5000 rpm for 1 minute, subsequently heated at 100℃ for 8 minutes, vortexed vigorously, and centrifuged again at 15000 rpm for 1 minute. The supernatant served as the template DNA for the ITS amplification. PCR amplification was performed on a Bioer GeneAmp PCR TC-96 system, starting with a 4-minute initial denaturation at 95℃, followed by 35 cycles (1 minute at 94℃, 1 minute at 51℃, 1 minute at 72℃), and ending with a 10-minute final extension at 72℃. The LSU/28S rDNA region was amplified under identical conditions, except for an annealing temperature of 60℃, using primers LSU1 and LSU2. PCR products were separated by electrophoresis on a 1% agarose gel in TAE buffer and purified using the Bioteke ZymocleanTM Gel DNA Recovery Kit according to the manufacturer’s instructions. The purified DNA products as substrate were amplified again as depicted above, and the PCR products were then sequenced by Shanghai Sangon Biotech Co., Ltd. Phylogenetic analysis Sequences were edited using BioEdit 7.2.5, verified against NCBI with BLAST, and submitted to GenBank (accession numbers: OR365513.1, OR372494.1, OR372639.1). Other homologous sequences from the GenBank database were downloaded for comparative analysis (supplemental Table 1). DNA sequences were aligned using ClustalX vers. 1.8. ( http://www-igbmc.u-strasbg.fr/BioInfo/ ); gaps or missing data were eliminated from the datasets. Maximum-parsimony (MP) and maximum-likelihood (ML) were generated with PAUP 4.0b10 ( http://phylosolutions.com/paup-test/ ) as described in the reference and RAxMLGUI 2.0 under a GTR + GAMMA model (Cummins and Hiratsuka 2004 ; Edler et al. 2021 ). Bootstrap values, obtained from 1000 replicates, are shown at tree nodes. Bayesian Inference (BI) analysis was conducted in MrBayes 3.1.2, using a partitioned mixed model for LSU and ITS sequences with the GTR + I + G substitution model. Markov chain Monte Carlo (MCMC) simulations ran for 1,000,000 generations until ASDSF values were below 0.01, with trees sampled every 100 generations post-burn-in. Consensus trees were built using 50% majority rule, and clades were considered significantly supported with bootstrap values ≥ 70% and Bayesian posterior probabilities ≥ 0.95. Phylogenetic trees were visualized with FigTree v1.4.3 and manually annotated using Adobe Illustrator 2019 software. Inoculation tests Urediniospores were carefully collected from infected ferns using a camel hair brush, part of which was transported to the lab in a cooler while another portion was suspended in sterilized water at a concentration of 1.0x10 5 spores/mL. This suspension was sprayed onto healthy C. chinensis and Adiantum capillus-veneris plants located approximately 3 km from the collection sites until runoff occurred. For indoor tests, two groups of three artificially cultured ferns each were sprayed with the urediniospore suspension (1.0x10 5 spores/mL) and sterilized water (1 mL), respectively, These were initially kept in a dark chamber at 20℃ and 100% relative humidity for 24 h, and then moved into the greenhouse at 25℃, 70–80% relative humidity, and 16 h photoperiod (2500 Lux at leaf level) for in planta experiments. When the uredia appeared at abaxial leaf, scrap the urediniospora and check again under the microscope.. Results Morphology Uredia are subepidermal, forming long, rectangle blister-shaped structures predominantly on the abaxial (underside) leaf surfaces; they measure 0.2–0.4 mm in width and 0.6–0.8 mm in length, appearing yellowish to orange and becoming pulverulent upon maturity. The peridia are hemispherical, bursting through with an apical crack, and composed of peridial cells that are smaller, irregularly polygonal, hyaline, and finely echinulate compared to the urediniospores. Fresh urediniospores and amphispores with yellow contents fade over time. Urediniospores are oblong, ovoid, or obovate-elliptic, measuring 18.5–26.0 × 15.0–23.5 µm (average 23.0 × 18.5 µm, n = 30), with colorless walls 1.0–2.0 µm thick, featuring a hyaline sporophore at the base and finely echinulate, equipped with 3 scattered germ pores. Amphispores are similar to urediniospores but have thicker walls (1.5-3.0µm), are darker in color, and lack a sporophore at the base, Fig. 1 . They predominate in mature uredia. These characteristics led to the preliminary identification of the samples as a new species, Hyalopsora erlangensis A. Yu & X.H Chen, in the family Pucciniastraceae. Phylogenetic Analyses The combined ITS + 28S sequence alignment consisted of 1620 characters, with 1281 constant and 69 parsimony-uninformative variable characters. MP analysis with the remaining 270 parsimony-informative characters resulted in six equally parsimonious trees with the following values: tree length (TL) = 520; consistency index (CI) = 0.762; retention index (RI) = 0.662; and rescaled consistency index (RC) = 0.238. Both MP analysis and BI analysis resulted in the same topology as the presented ML phylogram. Hyalopsora was found to be monophyletic, forming a separate lineage from Milesina and Uredinopsis which are also parasitic on ferns, with strong bootstrap support (ML/BI = 97%/0.97) (Fig. 2 ). Our research formed 8 separate species level lineages within the Hyalospora clade, H. erlangensis is a monophyletic clade with MPBS/MLBS/BI 88/100/1.0, supported a novel species in genus Hyalospora, causing the endangered fern rust, C. chinensis .. Note Parsimony bootstrap, likelihood bootstrap greater than 50% and Bayes inference are shown (MP% /ML%/BI). The new species are indicated in colorful background. Pucciniastrum agrimoniae was selected as the outgroup. Bars: 0.02 like hood of nucleotide substitutions. Pathogenic tests In all three instances where urediniospore suspensions were applied, uredia development was observed 7 days post-inoculation, similarly affecting Adiantum capillus-veneris in the field. In contrast, the controls remained healthy (see Fig. 3 ). The symptoms on artificially tissue-cultured seedlings mirrored those observed in the field, albeit with uredia that were smaller and less dense. Only one of the three seedlings developed uredia 2 weeks post-inoculation. These pathogenicity tests suggest that Hyalopsora erlangensis likely has a broad host range within the genera Adiantum and Cystopteris, including the endangered fern C. chinensis . Discussion Rust fungi on ferns include species in genus Hyalopsora, Milesina and Uredinopsis. Hyalopsora is distinguished from Milesina and Uredinopsis by its hyaline peridial cells and pigmented cytoplasm. Phylogenetic analysis based on ITS + 28S sequences further supports their classification into separate genera within the family Pucciniastraceae (Gardes and Bruns 1993 ; Aime and McTaggart 2021 ). Globally, 26 Hyalopsora species have been identified on ferns, with 11 species reported in China on various hosts, Cheilanthes chusana, C. pellucida, Athyrium spinulosum, Blechnopsis orientalis ( http://www.sp2000.org.cn/browse/browse_taxa ). The presence of urediniospora and amphispores, alongside the morphology of sori on the host plant, are essential factors in distinguishing rust species (Ishaq et al. 2011 ). The color, shape, and arrangement of sori provide crucial information for identification. Additionally, the size, shape, and presence of septal teliospores play a vital role in separating between species in this genus; however, telia are seldom found on ferns. It is imperative for researchers and scientists to meticulously examine these distinguishing features in order to accurately classify and study rust fungi, for example, H. minispora also formed a distinct lineage in phylogenetic analyses, with a close sister relationship to H. neocheilanthis , but H. neocheilanthis lacks amphispores (Wang et al. 2021 ), H. aspidiotus , H. hakodatensis , and H. polypodii are distinguished by the presence of these two types of spores (Hiratsuka et al. 1992 ). Amphispores are a specialized type of spore with unique characteristics such as increased darkness and thicker walls. These features are believed to provide enhanced protection and support for the organism in times of adversity. The dark pigmentation of the amphispores may act as a defense mechanism against harmful UV radiation and excessive light exposure. Furthermore, the thicker walls of the amphispores can help prevent desiccation and damage from external stresses. In conclusion, these adaptations in amphispores likely confer a competitive advantage to the rust fungus in changing or challenging environments (Cummins and Hiratsuka 2004 ). Hyalopsora is distinguishable from the other two genera(Milesina and Uredinopsis)primarily by the presence of hyaline peridial walls and pigmented cytoplasm in its urediniospores, along with its predominant production of amphispores (Cummins and Hiratsuka 2004 ). The unique spore morphology, size, wall thickening, and pore arrangement of H. erlangensis set it apart from other Hyalopsora species. For instance, H. polypodii , previously reported on C. pellicida , shares similar urediniospore and amphispore morphology with H. erlangensis , except for the smoother peridial cell walls, germ pores located in the equatorial zone of the urediniospores, and the absence of a stem at the base. Phylogenetic analyses separate these two rusts into different clades, supporting the classification of H. erlangensis as a novel species (Cao et al. 2000 ; Liu et al. 2006 ). This study underscores the importance of integrating morphological and molecular data for the accurate identification of such cryptic species. The phylogenetic analysis affirms the unique position of H. erlangensis within the Hyalopsora genus, illustrating its close relationships with other species within a distinct clade. This insight contributes to our understanding of the evolutionary history of the genus and the interconnections between H. erlangensis and other species (Jones and Dangl 2006 ). Hyalopsora erlangensis ' presence on the endangered Cystopteris chinensis carries profound implications for conservation. Gaining a thorough understanding of the rust fungus's biology, ecology, and distribution is pivotal for devising effective management strategies and conservation efforts for both the fungus and its host plant. This study sheds light on fungal diversity, evolution, and plant-fungus interactions, highlighting the critical need for continued research in this domain.. Declarations Competing Interests: The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. Funding: This research was funded by Natural Science Foundation of Sichuan Province: Study on the Reproductive Characteristics and Formation Mechanism of Cystoperis chinensis , a National-level Protected Plant, 22NSFSC0230 and Project of Sichuan Forestry and Grassland Bureau: Developmental Biology Research and Artificial Breeding Technique of Cystoperis chinensis. Author Contribution Xiaohong Chen and Xia Zhao contributed to the conception of the study; An Yu and Xia Zhao performed the experiment; An Yu contributed significantly to analysis and manuscript preparation; An Yu performed the data analyses and wrote the manuscript. Data Availability: The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. References Aime MC, McTaggart AR (2021) A higher-rank classification for rust fungi, with notes on genera. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4226118","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":289284911,"identity":"d8466615-e9e2-4e28-bbab-1b70aec382ac","order_by":0,"name":"An Yu","email":"","orcid":"","institution":"Sichuan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"An","middleName":"","lastName":"Yu","suffix":""},{"id":289284912,"identity":"a5f89922-bffb-4aaa-9b57-8a3eed278ed1","order_by":1,"name":"Xia Zhao","email":"","orcid":"","institution":"Sichuan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Xia","middleName":"","lastName":"Zhao","suffix":""},{"id":289284913,"identity":"35ceaaff-0492-4b3b-91dd-52fd9f429114","order_by":2,"name":"Xiaohong Chen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABDUlEQVRIiWNgGAWjYDACZjACAR4grgCTQMBGtJYzxGhhQNbC2AYTw6PF4Djzw8cFFXfsGtjPHnxcOK9Ohr/9jAHDh7LDDPyzG7BqkWxmMzaeceZZcgNPXrLxzG2HeSTO5Bgwzjh3mEHizgGsWviZGcykedsOJzNI8AAZ2w7wGDDkGDADRRgMJBKwamFjZv8G02L+m3dOHY8B/xsD5r94tPAz84BtsQPZwszbwMxjIAG0hRGPFslmnmJjnjOHExh4coyleY4B/XLjWcHBnnPpQAZ2LQbnj298zFNx2J6B/YzhZ56aOnv+/uSND36UWcvxz8CuBQYS9x9A4oHYPHjVA4E9IQWjYBSMglEwggEA3ilP3JSuEZ0AAAAASUVORK5CYII=","orcid":"","institution":"Sichuan Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Xiaohong","middleName":"","lastName":"Chen","suffix":""}],"badges":[],"createdAt":"2024-04-06 06:59:31","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4226118/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4226118/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":54526748,"identity":"a75dbd2e-13a6-4782-82e0-b694b391b253","added_by":"auto","created_at":"2024-04-11 21:07:55","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":447147,"visible":true,"origin":"","legend":"\u003cp\u003eSymptoms and Morphologies: (A) Uredia; (B) Matured uredia cracked at the apical; (C) Dominated amphispora (blue arrow), urediniospora (yellow arrow),hyaline peridia cell (dark arrow), germ pores (red arrow); (D) Urediniospora with germ pores\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4226118/v1/dca9052cee54cf48015783d3.jpg"},{"id":54526746,"identity":"c2dd9bef-26f7-4767-8a6b-f8105207ca02","added_by":"auto","created_at":"2024-04-11 21:07:55","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":77799,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogram based on ITS2+28S sequences.\u003c/p\u003e\n\u003cp\u003eNote: Parsimony bootstrap, likelihood bootstrap greater than 50% and Bayes inference are shown (MP% /ML%/BI). The new species are indicated in colorful background. \u003cem\u003ePucciniastrum agrimoniae\u003c/em\u003e was selected as the outgroup. Bars: 0.02 like hood of nucleotide substitutions.\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4226118/v1/4bb1906938ba1d63a99ebff4.jpg"},{"id":54526747,"identity":"92920485-7bad-4bef-8411-7e32cbd0f584","added_by":"auto","created_at":"2024-04-11 21:07:55","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":197785,"visible":true,"origin":"","legend":"\u003cp\u003eInoculated leaves by spraying urediniospora suspension on the two sides of leaves: A. the treatment with uredia occurred on the adaxia and abaxia leave of \u003cem\u003eC. chinensis\u003c/em\u003e; B. the control in the field without typical symptoms on both sides of the leaves.\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4226118/v1/52a46a3ed2449e8c401abab6.jpg"},{"id":54686747,"identity":"526564ef-f20c-4160-9f94-4691625dee32","added_by":"auto","created_at":"2024-04-15 09:17:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":491078,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4226118/v1/d70b6d4a-7379-4761-8560-ea5a8efa7a37.pdf"},{"id":54526749,"identity":"7f53d6de-8084-4fea-9225-f6dbb353cda2","added_by":"auto","created_at":"2024-04-11 21:07:55","extension":"zip","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":9524,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalTable11.zip","url":"https://assets-eu.researchsquare.com/files/rs-4226118/v1/3b578293bad2e90ea0fd06d2.zip"}],"financialInterests":"No competing interests reported.","formattedTitle":"First Report of a New Species Hyalopsora erlangensis Causing Rust Disease on Cystopteris chinensis in China","fulltext":[{"header":"Introduction","content":"\u003cp\u003eRust fungi affecting ferns, such as species within the genera Hyalopsora, Milesina and Uredinopsis, comprise a significant portion of fern pathogens (Arthur \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1924\u003c/span\u003e);To date,a total of 26 species of Hyalopsora have been reported globally (Arthur \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1906\u003c/span\u003e; Kern 1914; Chen et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1980\u003c/span\u003e; Hiratsuka et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1992\u003c/span\u003e; Saba et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), with eleven of these species documented in China, including \u003cem\u003eH. aspidiotus, H. cryptogrammes, H. hakodatensis, H. japonica, H. polypodii, H. pseudocys-topteridis, H. taiwaniana, H. yunnanensis, H. tibetica, H. minispora, H\u003c/em\u003e. \u003cem\u003eneocheilanthis\u003c/em\u003e (Chen \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1982\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1989\u003c/span\u003e; Jian-yun \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1983\u003c/span\u003e; Hiratsuka and Chen \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1991\u003c/span\u003e; Yu et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Cao et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Kirschner et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Rust fungi are known to infect a wide range of plant species, including crops like wheat, barley, corn, and soybeans, as well as ornamental plants, trees, and shrubs (Blythe et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Savary et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Despite the known diversity, it is likely that additional fern rust species await discovery, given the extensive habitats suitable for fern growth. Rust fungi are characterized by complex life cycles often involving different hosts and spore stages producing abundant spores that facilitate widespread transmission via wind, water, or insects (McTaggart et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The lifecycle of Hyalopsora rust on ferns remain elusive, with many species lacking reported alternative hosts. The subtle morphological distinctions between their urediospores, amphispores, and teliospores pose challenges for species delineation within the genus Hyalopsora. Some species exhibit telia parasitizing the epidermal cells of ferns, producing dark brown teliospores that are nearly hyaline, thin-walled, and frequently septate (Hiratsuka et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1992\u003c/span\u003e), complicating the task of accurately identifying Hyalopsora species.\u003c/p\u003e \u003cp\u003eFerns play a pivotal role among vascular plants, representing the second largest group after flowering plants, with approximately 12,240 species globally (Ranker and Haufler \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). They occupies an intermediate phylogenetic position between mosses and angiosperms. China boasts a rich diversity of ferns, comprising 63 families, 230 genera, and around 2,600 species, which accounts for 21.7\u0026ndash;26.0% of the global fern species total. Southwestern China, in particular, is a hotspot for fern biodiversity in Asia, with Sichuan Province's fern species count second only to Yunnan Province. Mount Emei alone is home to about 400 species of lycophytes and ferns (Zhang \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Most ferns are naturally found in their native habitats, typically growing in lithophytic or epiphytic conditions. Some species may also be found in moist or aquatic environments. They thrive in warm, humid forest settings and are particularly abundant in tropical and subtropical regions (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://flora.huh.harvard.edu/China/mss/volume13/index.htm\u003c/span\u003e\u003cspan address=\"http://flora.huh.harvard.edu/China/mss/volume13/index.htm\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Fungi belonging to the genera \u003cem\u003eHyalopsora\u003c/em\u003e, \u003cem\u003eMilesina\u003c/em\u003e, and \u003cem\u003eUredinopsis\u003c/em\u003e (Pucciniastraceae) exhibit a unique ecological specialization, exclusively parasitizing fern hosts during their sporophytic phase (uredinia and telia). The gametophytic phase of these fungi predominantly occurs on conifers in the Northern Hemisphere, especially within the Pinaceae family, including species of Abies. Rust fungi outside the Pucciniastraceae family that parasitize ferns are rare, with only a single known species of Desmella and Uncol, the former likely belonging to the Pucciniaceae and the latter to the Phakopsoraceae family (Berndt \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eCystopteris chinensis\u003c/em\u003e (Ching) X.C. Zhang\u0026amp;R. Wei, synonymously known as \u003cem\u003eCystopathyrium chinensis\u003c/em\u003e Ching, is a distinctive fern indigenous to China, classified under the family Cystopteridaceae and genus Cystopteris (Wei and Zhang \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). It has been designated as an endangered species in the first volume of the \"Red Book of Chinese Plants\", recognized as a national level I endangered protected plant. Currently, its habitat is limited to Erlang Mountain in Tianquan County and Emei Mountain in Leshan City, Sichuan Province. \u003cem\u003eC. chinensis\u003c/em\u003e has a limited reproductive capacity, which makes it particularly vulnerable to population decline. Moreover, it has specific habitat requirements that challenge its continued survival and reproductive success. Consequently, \u003cem\u003eC. chinensis\u003c/em\u003e's population is critically endangered and faces the threat of extinction. Conservation measures are imperative to safeguard this species and avert its loss from the ecosystem. The prevalence of rust disease significantly impacts the stability of \u003cem\u003eC. chinensis\u003c/em\u003e populations, with an 80% infection rate and over 30% mortality observed in natural habitats. This study is the first to report the causative agent of this fern rust disease as the new species \u003cem\u003eHyalopsora erlangensis\u003c/em\u003e, identified through morphological and molecular phylogenetic methods.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eMorphological study\u003c/p\u003e \u003cp\u003eRust leaves were collected at Erlang Mt. in Tianquan county, Sichuan China every July from 2021\u0026ndash;2023, and the holotype specimens were kept in the herbarium of Sichuan Agriculture University under the accession numbers HAMS AY-2003a, HAMS AY-2003b. For morphological analysis, fresh leaf sections displaying uredia prepared manually and examined under a microscope. Initial observations of uredia size, shape, and development location were conducted using a stereomicroscope. To further investigate spore morphology and dimensions, spores from the sori were mounted in a drop of lactophenol solution on glass slides and observed with a light microscope (Olympus 51). Approximately 30 spores were randomly selected and measured for each specimen using an ocular micrometer at 40\u0026times; magnification. The number and placement of germ pores were examined by mounting spores in lactic acid, briefly heating to boiling, and adding a drop of lactophenol solution with aniline blue as needed.\u003c/p\u003e \u003cp\u003eDNA Extraction, PCR Amplification, and DNA Sequencing\u003c/p\u003e \u003cp\u003eGenomic DNA was extracted from the urediniospres with BioRad Chelex-100 following published procedures (Scholin et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). Approximately six uredia were carefully scraped with a sterilized scalpel under a dissecting microscope and gently placed on microslides. The spores were softly crushed under a coverslip, which, along with the slide, was then rinsed with 50 \u0026micro;L of 5% Chelex-100 in sterilized water into a 1.5 mL centrifuge tube. This tube was incubated at 56℃ for 20 minutes, centrifuged at 5000 rpm for 1 minute, subsequently heated at 100℃ for 8 minutes, vortexed vigorously, and centrifuged again at 15000 rpm for 1 minute. The supernatant served as the template DNA for the ITS amplification. PCR amplification was performed on a Bioer GeneAmp PCR TC-96 system, starting with a 4-minute initial denaturation at 95℃, followed by 35 cycles (1 minute at 94℃, 1 minute at 51℃, 1 minute at 72℃), and ending with a 10-minute final extension at 72℃.\u003c/p\u003e \u003cp\u003eThe LSU/28S rDNA region was amplified under identical conditions, except for an annealing temperature of 60℃, using primers LSU1 and LSU2. PCR products were separated by electrophoresis on a 1% agarose gel in TAE buffer and purified using the Bioteke ZymocleanTM Gel DNA Recovery Kit according to the manufacturer\u0026rsquo;s instructions. The purified DNA products as substrate were amplified again as depicted above, and the PCR products were then sequenced by Shanghai Sangon Biotech Co., Ltd.\u003c/p\u003e \u003cp\u003ePhylogenetic analysis\u003c/p\u003e \u003cp\u003eSequences were edited using BioEdit 7.2.5, verified against NCBI with BLAST, and submitted to GenBank (accession numbers: OR365513.1, OR372494.1, OR372639.1). Other homologous sequences from the GenBank database were downloaded for comparative analysis (supplemental Table\u0026nbsp;1). DNA sequences were aligned using ClustalX vers. 1.8. (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www-igbmc.u-strasbg.fr/BioInfo/\u003c/span\u003e\u003cspan address=\"http://www-igbmc.u-strasbg.fr/BioInfo/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e); gaps or missing data were eliminated from the datasets. Maximum-parsimony (MP) and maximum-likelihood (ML) were generated with PAUP 4.0b10 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://phylosolutions.com/paup-test/\u003c/span\u003e\u003cspan address=\"http://phylosolutions.com/paup-test/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) as described in the reference and RAxMLGUI 2.0 under a GTR\u0026thinsp;+\u0026thinsp;GAMMA model (Cummins and Hiratsuka \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Edler et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Bootstrap values, obtained from 1000 replicates, are shown at tree nodes. Bayesian Inference (BI) analysis was conducted in MrBayes 3.1.2, using a partitioned mixed model for LSU and ITS sequences with the GTR\u0026thinsp;+\u0026thinsp;I\u0026thinsp;+\u0026thinsp;G substitution model. Markov chain Monte Carlo (MCMC) simulations ran for 1,000,000 generations until ASDSF values were below 0.01, with trees sampled every 100 generations post-burn-in. Consensus trees were built using 50% majority rule, and clades were considered significantly supported with bootstrap values\u0026thinsp;\u0026ge;\u0026thinsp;70% and Bayesian posterior probabilities\u0026thinsp;\u0026ge;\u0026thinsp;0.95. Phylogenetic trees were visualized with FigTree v1.4.3 and manually annotated using Adobe Illustrator 2019 software.\u003c/p\u003e \u003cp\u003eInoculation tests\u003c/p\u003e \u003cp\u003eUrediniospores were carefully collected from infected ferns using a camel hair brush, part of which was transported to the lab in a cooler while another portion was suspended in sterilized water at a concentration of 1.0x10\u003csup\u003e5\u003c/sup\u003e spores/mL. This suspension was sprayed onto healthy \u003cem\u003eC. chinensis\u003c/em\u003e and \u003cem\u003eAdiantum capillus-veneris\u003c/em\u003e plants located approximately 3 km from the collection sites until runoff occurred. For indoor tests, two groups of three artificially cultured ferns each were sprayed with the urediniospore suspension (1.0x10\u003csup\u003e5\u003c/sup\u003e spores/mL) and sterilized water (1 mL), respectively, These were initially kept in a dark chamber at 20℃ and 100% relative humidity for 24 h, and then moved into the greenhouse at 25℃, 70\u0026ndash;80% relative humidity, and 16 h photoperiod (2500 Lux at leaf level) for in planta experiments. When the uredia appeared at abaxial leaf, scrap the urediniospora and check again under the microscope..\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eMorphology\u003c/p\u003e \u003cp\u003eUredia are subepidermal, forming long, rectangle blister-shaped structures predominantly on the abaxial (underside) leaf surfaces; they measure 0.2\u0026ndash;0.4 mm in width and 0.6\u0026ndash;0.8 mm in length, appearing yellowish to orange and becoming pulverulent upon maturity. The peridia are hemispherical, bursting through with an apical crack, and composed of peridial cells that are smaller, irregularly polygonal, hyaline, and finely echinulate compared to the urediniospores. Fresh urediniospores and amphispores with yellow contents fade over time. Urediniospores are oblong, ovoid, or obovate-elliptic, measuring 18.5\u0026ndash;26.0 \u0026times; 15.0\u0026ndash;23.5 \u0026micro;m (average 23.0 \u0026times; 18.5 \u0026micro;m, n\u0026thinsp;=\u0026thinsp;30), with colorless walls 1.0\u0026ndash;2.0 \u0026micro;m thick, featuring a hyaline sporophore at the base and finely echinulate, equipped with 3 scattered germ pores. Amphispores are similar to urediniospores but have thicker walls (1.5-3.0\u0026micro;m), are darker in color, and lack a sporophore at the base, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. They predominate in mature uredia. These characteristics led to the preliminary identification of the samples as a new species, \u003cem\u003eHyalopsora erlangensis\u003c/em\u003e A. Yu \u0026amp; X.H Chen, in the family Pucciniastraceae.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePhylogenetic Analyses\u003c/p\u003e \u003cp\u003eThe combined ITS\u0026thinsp;+\u0026thinsp;28S sequence alignment consisted of 1620 characters, with 1281 constant and 69 parsimony-uninformative variable characters. MP analysis with the remaining 270 parsimony-informative characters resulted in six equally parsimonious trees with the following values: tree length (TL)\u0026thinsp;=\u0026thinsp;520; consistency index (CI)\u0026thinsp;=\u0026thinsp;0.762; retention index (RI)\u0026thinsp;=\u0026thinsp;0.662; and rescaled consistency index (RC)\u0026thinsp;=\u0026thinsp;0.238. Both MP analysis and BI analysis resulted in the same topology as the presented ML phylogram. Hyalopsora was found to be monophyletic, forming a separate lineage from Milesina and Uredinopsis which are also parasitic on ferns, with strong bootstrap support (ML/BI\u0026thinsp;=\u0026thinsp;97%/0.97) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Our research formed 8 separate species level lineages within the Hyalospora clade, \u003cem\u003eH. erlangensis\u003c/em\u003e is a monophyletic clade with MPBS/MLBS/BI 88/100/1.0, supported a novel species in genus Hyalospora, causing the endangered fern rust, \u003cem\u003eC. chinensis\u003c/em\u003e..\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eNote\u003c/strong\u003e \u003cp\u003eParsimony bootstrap, likelihood bootstrap greater than 50% and Bayes inference are shown (MP% /ML%/BI). The new species are indicated in colorful background. \u003cem\u003ePucciniastrum agrimoniae\u003c/em\u003e was selected as the outgroup. Bars: 0.02 like hood of nucleotide substitutions.\u003c/p\u003e \u003c/p\u003e \u003cp\u003ePathogenic tests\u003c/p\u003e \u003cp\u003eIn all three instances where urediniospore suspensions were applied, uredia development was observed 7 days post-inoculation, similarly affecting \u003cem\u003eAdiantum capillus-veneris\u003c/em\u003e in the field. In contrast, the controls remained healthy (see Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The symptoms on artificially tissue-cultured seedlings mirrored those observed in the field, albeit with uredia that were smaller and less dense. Only one of the three seedlings developed uredia 2 weeks post-inoculation. These pathogenicity tests suggest that \u003cem\u003eHyalopsora erlangensis\u003c/em\u003e likely has a broad host range within the genera Adiantum and Cystopteris, including the endangered fern \u003cem\u003eC. chinensis\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eRust fungi on ferns include species in genus Hyalopsora, Milesina and Uredinopsis. Hyalopsora is distinguished from Milesina and Uredinopsis by its hyaline peridial cells and pigmented cytoplasm. Phylogenetic analysis based on ITS\u0026thinsp;+\u0026thinsp;28S sequences further supports their classification into separate genera within the family Pucciniastraceae (Gardes and Bruns \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Aime and McTaggart \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Globally, 26 Hyalopsora species have been identified on ferns, with 11 species reported in China on various hosts,\u003cem\u003eCheilanthes chusana, C. pellucida, Athyrium spinulosum, Blechnopsis orientalis\u003c/em\u003e (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.sp2000.org.cn/browse/browse_taxa\u003c/span\u003e\u003cspan address=\"http://www.sp2000.org.cn/browse/browse_taxa\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The presence of urediniospora and amphispores, alongside the morphology of sori on the host plant, are essential factors in distinguishing rust species (Ishaq et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The color, shape, and arrangement of sori provide crucial information for identification. Additionally, the size, shape, and presence of septal teliospores play a vital role in separating between species in this genus; however, telia are seldom found on ferns. It is imperative for researchers and scientists to meticulously examine these distinguishing features in order to accurately classify and study rust fungi, for example, \u003cem\u003eH. minispora\u003c/em\u003e also formed a distinct lineage in phylogenetic analyses, with a close sister relationship to \u003cem\u003eH. neocheilanthis\u003c/em\u003e, but \u003cem\u003eH. neocheilanthis\u003c/em\u003e lacks amphispores (Wang et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), H. \u003cem\u003easpidiotus\u003c/em\u003e, \u003cem\u003eH. hakodatensis\u003c/em\u003e, and \u003cem\u003eH. polypodii\u003c/em\u003e are distinguished by the presence of these two types of spores (Hiratsuka et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1992\u003c/span\u003e). Amphispores are a specialized type of spore with unique characteristics such as increased darkness and thicker walls. These features are believed to provide enhanced protection and support for the organism in times of adversity. The dark pigmentation of the amphispores may act as a defense mechanism against harmful UV radiation and excessive light exposure. Furthermore, the thicker walls of the amphispores can help prevent desiccation and damage from external stresses. In conclusion, these adaptations in amphispores likely confer a competitive advantage to the rust fungus in changing or challenging environments (Cummins and Hiratsuka \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2004\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eHyalopsora\u003c/em\u003e is distinguishable from the other two genera(Milesina and Uredinopsis)primarily by the presence of hyaline peridial walls and pigmented cytoplasm in its urediniospores, along with its predominant production of amphispores (Cummins and Hiratsuka \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). The unique spore morphology, size, wall thickening, and pore arrangement of \u003cem\u003eH. erlangensis\u003c/em\u003e set it apart from other Hyalopsora species. For instance, \u003cem\u003eH. polypodii\u003c/em\u003e, previously reported on \u003cem\u003eC. pellicida\u003c/em\u003e, shares similar urediniospore and amphispore morphology with \u003cem\u003eH. erlangensis\u003c/em\u003e, except for the smoother peridial cell walls, germ pores located in the equatorial zone of the urediniospores, and the absence of a stem at the base. Phylogenetic analyses separate these two rusts into different clades, supporting the classification of \u003cem\u003eH. erlangensis\u003c/em\u003e as a novel species (Cao et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Liu et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). This study underscores the importance of integrating morphological and molecular data for the accurate identification of such cryptic species. The phylogenetic analysis affirms the unique position of \u003cem\u003eH. erlangensis\u003c/em\u003e within the Hyalopsora genus, illustrating its close relationships with other species within a distinct clade. This insight contributes to our understanding of the evolutionary history of the genus and the interconnections between \u003cem\u003eH. erlangensis\u003c/em\u003e and other species (Jones and Dangl \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). \u003cem\u003eHyalopsora erlangensis\u003c/em\u003e' presence on the endangered \u003cem\u003eCystopteris chinensis\u003c/em\u003e carries profound implications for conservation. Gaining a thorough understanding of the rust fungus's biology, ecology, and distribution is pivotal for devising effective management strategies and conservation efforts for both the fungus and its host plant. This study sheds light on fungal diversity, evolution, and plant-fungus interactions, highlighting the critical need for continued research in this domain..\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting Interests:\u003c/h2\u003e \u003cp\u003eThe authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e \u003cp\u003eThis research was funded by Natural Science Foundation of Sichuan Province: Study on the Reproductive Characteristics and Formation Mechanism of \u003cem\u003eCystoperis chinensis\u003c/em\u003e, a National-level Protected Plant, 22NSFSC0230 and Project of Sichuan Forestry and Grassland Bureau: Developmental Biology Research and Artificial Breeding Technique of \u003cem\u003eCystoperis chinensis.\u003c/em\u003e\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eXiaohong Chen and Xia Zhao contributed to the conception of the study; An Yu and Xia Zhao performed the experiment; An Yu contributed significantly to analysis and manuscript preparation; An Yu performed the data analyses and wrote the manuscript.\u003c/p\u003e\u003ch2\u003eData Availability:\u003c/h2\u003e \u003cp\u003eThe datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAime MC, McTaggart AR (2021) A higher-rank classification for rust fungi, with notes on genera. 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Beijing:Peking University Press\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Cystoperis chinensis, Hyalopsora erlangensis A. Yu \u0026 X.H. Chen, rust fungi, taxonomy","lastPublishedDoi":"10.21203/rs.3.rs-4226118/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4226118/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eA newly discovered rust fungus poses a significant threat to the survival and reproduction of the endangered plant \u003cem\u003eCystopteris chinensis\u003c/em\u003e in a forest in western Sichuan province, China. This study utilized both morphological analysis and molecular phylogenetic approaches to classify this fungal taxon. Artificial inoculation experiments, which involved sampling uredia from the field, were conducted to fulfill Koch\u0026rsquo;s postulates. Our results designate this rust fungus as a novel species within the Pucciniastraceae family, named \u003cem\u003eHyalopsora erlangensis\u003c/em\u003e A. Yu \u0026amp; X.H. Chen. Morphological feature, including hyaline peridia cells, yellowish urediospores, dark brown amphidspores with three germ pores, and pigmental cytoplasma, differentiate this species from others in the genera Milesina, Uredinopsis, and Pucciniastrum. Phylogenetic analysis, based on internal transcribed spacer (ITS) sequences and 28S rDNA gene fragments, further confirms its distinctiveness from other Hyalopsora species, supported by high Maximum Parsimony (MP), Maximum Likelihood (ML), and Bayesian Inference (BI) bootstrap values. Artificial inoculation of both field-collected and tissue-cultured seedlings verified this fungus as the causing agent of \u003cem\u003eC. chinensis\u003c/em\u003e rust disease, indicating a broad host range within the Cystoperis genus. Our research not only identifies the pathogen but also offers fundamental insights for the future management and conservation of this endangered fern, aiming to mitigate the impact of this devastating rust.\u003c/p\u003e","manuscriptTitle":"First Report of a New Species Hyalopsora erlangensis Causing Rust Disease on Cystopteris chinensis in China","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-11 21:07:50","doi":"10.21203/rs.3.rs-4226118/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"cdf5b74b-b28d-4b1a-aae4-4af0a3d1205f","owner":[],"postedDate":"April 11th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-04-15T09:16:40+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-11 21:07:50","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4226118","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4226118","identity":"rs-4226118","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}