Morphological and Physiological Features of Tremella yokohamensis: A First Report from Eucalyptus Trees in Southern China

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The isolates exhibited typical cryptococcal traits: mucoid colonies, polysaccharide capsule, positive urease, and growth only at 28 °C. ITS rDNA sequencing revealed two sequence types with 100% identity to type strains JCM 16990 and JCM 16989; phylogeny confirmed placement in Tremella. Antifungal testing showed lowest MICs for amphotericin B and itraconazole, highest for fluconazole. Exclusive leaf recovery indicates a phyllosphere niche, and the antifungal profile suggests ecologically driven adaptation within Eucalyptus habitats, providing critical strain resources and baseline data for understanding this rare yeast’s ecology and public health relevance. Biological sciences/Ecology Earth and environmental sciences/Ecology Biological sciences/Microbiology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction Eucalyptus spp. are widely planted worldwide, with China and Brazil among the largest producers; in China, Guangxi accounts for roughly 40% of national Eucalyptus area and timber output, and extensive monocultures in Nanning, Liuzhou and Guigang[1]may alter phyllosphere microbial communities. The Eucalyptus phyllosphere hosts diverse yeasts (e.g., Candida, Debaryomyces)[2,3] and can include known zoonotic cryptococci such as Cryptococcus neoformans and Cryptococcus gattii , indicating that plantations may act as environmental reservoirs for some pathogens[4-5] Cryptococcus yokohamensis (now treated in Tremella ) was first described from trees and a koala in Japan (2011)[6] and has had few confirmed records outside Japan and the Russian Far East [7,8]. We report the first environmental isolation of T. yokohamensis from Eucalyptus in Guangxi, China, expanding its known geographic range and providing strain material for follow‑up studies of ecology, antifungal susceptibility and host interactions. No human infections have been reported for this species to date, but targeted antifungal, virulence and One‑Health surveillance studies are recommended to assess potential risk. 2. Materials and Methods 2.1 Specimen Collection Samples were collected from three regions in Guangxi between April and August 2025: Binyang County in Nanning City, Sanmenjiang Forest Park and surrounding areas in Liuzhou City, and Qintang District in Guigang City. The collected materials included senescent leaves, bark, twigs, and rhizosphere soil from forest stands of Eucalyptus urophylla , Eucalyptus grandis , and hybrid clones (e.g., DH32-28, DH32-26, etc.). A total of 2,000 samples were obtained. 2.2 Isolation and Culture of Fungi The collected leaf, bark, twig, and soil samples were each mixed with an appropriate volume of sterile physiological saline and thoroughly shaken to prepare a homogeneous suspension. After allowing the suspension to settle, the supernatant was collected and centrifuged at 3,500 rpm for 5 minutes. The supernatant was discarded, and the pellet was concentrated to approximately 0.5 mL, then evenly spread onto CHROMagar™ Candida chromogenic medium plates. The plates were incubated at 28°C in a constant-temperature incubator, observed periodically for up to 14 days, and suspected yeast-like colonies were recorded and isolated. 2.3 Species identification by Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) Routine fungal pretreatment method Colonies were directly spotted onto the MALDI target plate and air-dried. Each sample was overlaid with 1 μL of formic acid, air-dried, and then mixed with 1 μL of α-cyano-4-hydroxycinnamic acid (HCCA) matrix solution. Mass spectra were acquired using the Autof ms1000 MALDI-TOF MS system (Autobio Bio-Technology Co., Ltd., China). Identification scores were interpreted as follows: ≥9.0, reliable species-level identification; 6.0–9.0, genus-level identification; <6.0, unreliable. Special pretreatment procedure for T. yokohamensis Fresh colonies were transferred with a cotton swab into 1 mL of 75% ethanol, mixed thoroughly, and centrifuged for 2 min. The supernatant was discarded, and residual ethanol was removed with a paper towel. The pellet was resuspended in 40 μL of formic acid and ground for 30 s using a handheld tissue grinder (LC-MY-10, Lichen, China). After centrifugation for 2 min, 1 μL of the supernatant was spotted onto the target plate, air-dried, and overlaid with 1 μL of HCCA matrix solution. Mass spectral acquisition and result interpretation were performed as described above. 2.4 Amplification and DNA Sequence Analysis Amplification The internal transcribed spacer1 (ITS1)-5.8S-ITS2 of the ribosomal DNA region was chosen as the target for amplification by ITS1 (5’-TCCGTAGGTGAACCTGCGG-3’) and ITS4 (5’-TCCTCCGCTTATTGATATGC-3’) primers [9]. Sequencing primers were commercially synthesized, and nucleic acid extraction services were provided by Shanghai Saiyin Biotechnology Co., Ltd. The isolates were then identified by sequencing the ITS1/ITS4 region. 2.5 Biochemical Identification of T. yokohamensis After growth on Sabouraud Dextrose Agar (SDA) for five days at 28°C, colonies were suspended in sterile saline and adjusted to a 2.0 McFarland standard. Biochemical identification was performed using the VITEK 2 system with YST cards (bioMérieux) according to the manufacturer’s instructions; the YST panel covers multiple assimilation/enzymatic substrates and results were recorded substrate-by-substrate as positive/negative, and the system identification and confidence score were reported. 2.6 Scanning electron microscopy (SEM) T . yokohamensis and a clinical isolate of C . gattii were cultured on SDA at 28°C for 5 days. Cells were harvested and subjected to freeze‑drying preparation. Briefly, the samples were fixed, dehydrated, and freeze‑dried, followed by sputter‑coating with gold‑palladium. Observations were performed using a scanning electron microscope (Apreo 2C HiVac, Thermo Fisher Scientific, USA) at an accelerating voltage of 5 kV. 2.7 Phylogenetic tree construction and analysis Complete ITS rDNA sequences were obtained by assembling forward and reverse sequencing reads using MEGA version 12. The newly assembled sequences were deposited in the GenBank database under accession numbers PX915820 and PX915821. Phylogenetic analysis was performed using the Neighbor‑Joining method implemented in MEGA 12. The analysis was based on the sequences obtained in this study together with reference sequences of relevant type strains retrieved from GenBank. Evolutionary distances were calculated using the Kimura 2‑parameter model. Gaps and missing data were treated by pairwise deletion. Bootstrap support values were calculated from 1,000 replicates, and values ≥75% are shown at branch nodes. The sequence of C.marinus CBS 5235 T (KF36593.1)was used as the outgroup to root the tree. 2.8 Antibiotic Susceptibility Antifungal susceptibility testing was conducted following the Clinical and Laboratory Standards Institute (CLSI, 2022, M27/A5) guidelines [10], using the ATB FUNGUS 3 broth microdilution system (bioMérieux, USA). The test strips contained predefined gradients of five antifungal agents: amphotericin B (0.5–16 μg/mL), itraconazole (0.125–4 μg/mL), voriconazole (0.06–8 μg/mL), fluconazole (1–64 μg/mL), and 5-fluorocytosine (4–16 μg/mL). After culturing T. yokohamensis on Sabouraud Dextrose Agar (SDA) for 4 days, the turbidity of the fungal suspension was adjusted to a 0.5 McFarland standard using a densitometer, A 20 μL aliquot of the suspension was transferred into the accompanying broth medium, yielding a final inoculum density of 0.5–1.5 × 10 3 CFU/mL. Then, 135 μL of the inoculated broth was dispensed into each well of the strip. Incubation was carried out at 28 °C for 48 hours. To ensure test reliability, the quality control strain Candida parapsilosis ATCC 22019 was included in the experiment. 3. Results 3.1 Sample sources and isolation rate of T. yokohamensis Samples were collected from three geographic regions in the Guangxi Zhuang Autonomous Region: Binyang County (Nanning City), Sanmenjiang Forest Park and surrounding areas (Liuzhou City), and Qintang District (Guigang City) (Figure 1), covering both plains and mountainous areas. Eucalyptus sampling sites are shown in Figure 2. A total of 2,000 Eucalyptus-related samples (bark, leaves, twigs, and soil beneath the trees) were collected, and 30 isolates of T. yokohamensis were recovered. Within the scope of our sampling, T. yokohamensis was recovered exclusively from Eucalyptus leaves and not from bark, twigs, or soil. The overall isolation rate was 1.5% (Table 1). Table 1. Isolation of T. yokohamensis exclusively from leaf samples in Eucalyptus plantations of Southern China Total samples Nanning Liuzhou Guigang Total Recovery rate （ % ） Number of T. yokohamensis Leaves 1550 10 20 0 30 2.0 Bark 190 0 0 0 0 0 Twigs 130 0 0 0 0 0 Soil 130 0 0 0 0 0 Total 2000 10 20 0 30 1.5 3.2 MALDI‑TOF MS identification of yeast‑like fungi and ITS rDNA sequencing The remaining yeast-like isolates (excluding the 30 T. yokohamensis strains) were identified by MALDI-TOF MS and ITS sequencing, and classified into 28 species across 20 genera (Fig. 3). MALDI-TOF MS enables accurate species-level identification of clinically common fungi, such as Candida albicans , C. neoformans , and C. gattii , typically with scores above 9.0. However, for several other fungal species, including T. yokohamensis , Syzygospora sp. , Cryptotrichosporon brontis , Tremella basidiomaticola voucher , and Colacogloea terpenoidalis , this method fails to achieve reliable genus-level identification, with scores often below 6.0, necessitating the use of ITS sequencing for accurate identification. After the specialized pretreatment involving mechanical grinding, T. yokohamensis exhibited six characteristic ion peaks in the mass spectrum, located at m/z 3142.524, 3402.751, 6283.049, 6803.279, 7370.305, and 8563.406. In comparison, C. gattii showed peaks at m/z 3374.345, 3659.501, 6076.732, 6747.308, 7316.443, and 8685.785. Although the spectral profiles of the two species were similar, distinct differences were observed (Fig. 4). 3.3 Morphological characteristics and optimal growth temperature T. yokohamensis grew slowly under aerobic conditions at 28°C, with colonies becoming visible after 4–5 days. On Sabouraud Dextrose Agar (SDA) plates, colonies were milky white and mucoid; on CHROMagar™ Candida, colonies were light pink and mucoid after 8 days (Figure 5). The isolate failed to grow on Columbia blood agar, chocolate agar, or routine nutrient agar. All isolates grew at 28°C but did not grow at 37°C or 40°C, indicating an optimal growth temperature near 28°C. Microscopic examination showed budding cells without pseudohyphae. India ink staining revealed a thick capsule surrounding the cells; the cryptococcal capsular antigen test was positive, and the urease test was also positive. Biochemical testing with the VITEK 2 YST card showed positive reactions for sucrose fermentation (SAC), gluconate fermentation (DGLU), tyrosine arylamidase (TYRA), ornithine decarboxylase (ODC), β‑galactosidase (BGALI), leucine arylamidase (LEUA), alkaline phosphatase (PHOS), and pyruvate utilization (PVATE). Negative reactions were recorded for arginine dihydrolase (ArgA), γ‑glutamyl aminopeptidase (GGT), lysine decarboxylase (LlsA), phenylalanine deaminase (PHEA), proline arylamidase (PROA), pyrrolidonyl arylamidase (PYRA), tryptophan deaminase (APPA), L‑malate utilization (ELLM), and esculin hydrolysis (PHC). The isolate did not ferment glycogen (GLYG), D‑mannitol (DMNE), D‑melezitose (DMAL), N‑acetylglucosamine (NAG), D‑galacturonic acid (dGAL), D‑xylose (DXYL), L‑arabinose, D‑maltose (DMLT), or D‑ribose (DRIB2), nor did it utilize malonate (MTE) or D‑lactate (LGLM) as carbon sources. Note that the VITEK 2 YST card did not accurately identify this isolate to the genus level, probably because the card’s database is optimized for commonly encountered clinical yeasts. Therefore, the biochemical profile above is presented only as a phenotypic description; the final taxonomic assignment was based on ITS (internal transcribed spacer) sequence phylogenetic analysis. 3.4 Morphological and staining characteristics of five mucoid yeast-like fungi confirmed by ITS sequencing After 8 days of incubation on CHROMagar Candida, the five ITS‑confirmed mucoid fungi showed growth rates and colony morphologies similar to those of C. gattii . Members of the genus Syzygospora produced the most abundant mucoid growth, whereas Cryptotrichosporon brontis produced the least. In terms of pigmentation, C. brontis formed pale‑orange colonies; T. yokohamensis , Tremella basidiomaticola and Colacoglaea terpenoidalis produced pink, mucoid colonies resembling those of C. gattii . India ink staining revealed polysaccharide capsules in all five fungi, with T. yokohamensis and T. basidiomaticola showing the widest and thickest capsules. The capsule morphology of T. yokohamensis closely resembled that of pathogenic cryptococci. All strains reproduced by budding and lacked pseudohyphal structures. Gram staining was positive for all isolates, and oval to spherical yeast‑like cells were seen microscopically. Compared with clinically pathogenic cryptococci, capsule expression was influenced by culture conditions. Clinical isolates of novel cryptococcal species exhibit wide, thick capsules in cerebrospinal fluid but often show reduced or absent capsules and generally do not form mucoid colonies after growth on CHROMagar Candida; by contrast, C. gattii maintains a wide, thick capsule and produces abundant mucoid colonies both in CSF and on CHROMagar. Unlike some clinical cryptococcal isolates that lose their capsules on CHROMagar Candida, this fungus retained its capsule and produced mucoid colonies on this medium, a phenotype more closely 3 .5 Ultrastructural Comparison between T. yokohamensis and C. gattii Scanning electron microscopy preliminarily revealed that the capsule of T. yokohamensis exhibits a loose, porous, fibrous network‑like ultrastructure, contrasting with the more compact capsule morphology observed in a C. gattii complex strain. However, given that freeze‑drying preparation may introduce artifacts such as shrinkage or deformation of surface structures, these observations should be considered preliminary and do not constitute definitive evidence for distinguishing the two species at the ultrastructural level. Future studies employing cryo‑electron microscopy or chemical fixation combined with critical point drying would be better suited for a reliable characterization of the capsule ultrastructure in this species (Figure 7) 3.6 Phylogenetic analysis A total of 30 isolates of T . yokohamensis were obtained. ITS rDNA sequencing revealed that the sequences of these 30 isolates could be divided into two distinct sequence types. Two representative isolates (one for each sequence type) were selected and submitted to the NCBI database, with the assembled complete sequence accession numbers being PX915820 and PX915821, respectively. BLAST analysis showed that these two representative sequences exhibited 100% identity with the type strains of T. yokohamensis , JCM 16990 and JCM 16989. Phylogenetic analysis indicated that all isolates obtained in this study belong to the genus Tremella (Figure 8). 3.7 Antifungal Susceptibility Testing Antifungal susceptibility of 30 T. yokohamensis strains was tested using the ATB FUNGUS 3 panel after 48 hours of incubation. MIC ranges were as follows: amphotericin B <0.5 µg/mL, flucytosine ≤4 µg/mL, itraconazole 0.125–1 µg/mL, voriconazole 0.25–2 µg/mL, and fluconazole 2–16 µg/mL. The lowest MICs were observed for amphotericin B and itraconazole, while fluconazole showed the highest MICs. This susceptibility profile closely aligned with the epidemiological cutoff values established for the C . gattii species complex ( Table 2 ） . Table 2 Antifungal susceptibility of 30 T. yokohamensis isolates Antifungal drugs Number of isolates with MIC (μg/mL) 0.125 0.25 0.5 1 2 ≤4 8 16 32 MIC 50 /（μg/mL） MIC 90 /（μg/mL） Amphotericin B 30 0.5 0.5 Itraconazole 8 12 7 3 0.25 0.5 Voriconazole 1 5 17 6 1 1 2 Fluconazole 1 1 27 1 16 16 5-Fluorocytosine 30 4 4 4. Discussion 4.1 Geographic distribution and niche specialization The original description reported isolation of this species from a tree trunk ( Eucalyptus spp. ) and the nostril of a koala kept in a Japanese zoological park[6]., but no detailed ecological association was established. Within the scope of our sampling, T. yokohamensis was recovered exclusively from Eucalyptus leaves, suggesting that the phyllosphere may be a natural habitat for this species. The absence of isolates from bark, twigs, or soil in our sample set is consistent with a possible preference for leaf surfaces, but it does not prove strict niche specialization. Several factors could contribute to this pattern, including sampling bias, seasonal variation, or the culture conditions employed. Specifically, before concluding that T. yokohamensis is strictly confined to Eucalyptus leaves, several limitations should be considered. First, although we collected 2,000 samples from three regions, the sampling window was limited to the period from April to August 2025. Seasonal or phenological changes in the phyllosphere microbiome may affect the detectability of this yeast on different plant parts[11].Second, the culture conditions used in this study (CHROMagar™ Candida and Sabouraud Dextrose Agar incubated at 28°C) may favor the growth of leaf-associated yeasts but may be suboptimal for recovering the species from bark or soil, where different microbial communities or physiological states (e.g., dormancy, biofilm formation) might require alternative isolation protocols[12]. Therefore, while our data demonstrate a clear association between T. yokohamensis and Eucalyptus leaves within the sampled regions, the possibility of occasional occurrence on other plant parts or in soil under different conditions cannot be ruled out. Future studies employing more diverse isolation methods and year-round sampling are needed to clarify the true ecological breadth of this species. This phyllosphere association is distinct from that of C. gattii , which is frequently isolated from bark, tree hollows, and soil[13-16]. Thus, T. yokohamensis may occupy a unique ecological niche within the Eucalyptus ecosystem, offering a new model for studying phyllosphere-specific adaptation in basidiomycetous yeasts—but this conclusion requires confirmation through more comprehensive sampling and alternative isolation methods. 4.2 MALDI‑TOF MS with a novel pretreatment Routine MALDI‑TOF MS failed to identify T. yokohamensis (scores <6.0). A mechanical grinding step combined with formic acid extraction produced six characteristic ion peaks, distinct from those of C. gattii . However, because these peaks are absent from commercial databases, ITS sequencing remained necessary for definitive identification. The documented peaks provide reference data for future database expansion. 4.3 Phenotypic consistency with the original description All isolates exhibited typical cryptococcal traits: mucoid colonies, polysaccharide capsules, positive urease, and inability to grow at 37°C. These features match the original description of T. yokohamensis [6]. ITS phylogeny showed 100% identity with type strains JCM 16990 and JCM 16989. The presence of two ITS sequence types suggests previously unreported intraspecific variation[7,8]. 4.4 Antifungal susceptibility profile All 30 isolates showed low MICs for amphotericin B and itraconazole, while fluconazole MICs were higher. This profile is similar to the epidemiological cutoff values(EVCs) for the C. gattii complex[10]. Should this species ever become opportunistic, these drugs would likely remain effective. Future studies should examine whether Eucalyptus metabolites influence azole susceptibility. 4.5 Methodological note: susceptibility testing at 28°C Because T. yokohamensis does not grow at 35°C, we performed antifungal testing at 28°C, its optimal growth temperature. This deviates from the CLSI M27 recommendation (35°C), but the deviation was unavoidable for this thermosensitive species. MICs obtained at 28°C may not be directly comparable to those generated at 35°C for pathogenic yeasts, as temperature can affect drug activity. Therefore, our MIC values should be interpreted as ecological susceptibility data rather than clinical breakpoints. They provide a baseline for environmental comparisons but should not be used to predict clinical outcomes. 4.6 Thermal intolerance and pathogenic potential The inability of T. yokohamensis to grow at 37°C strongly suggests that it is not adapted to the mammalian host environment and likely has very low pathogenic potential in humans. In contrast, C. gattii grows readily at 37°C, correlating with its ability to cause systemic infections. Although thermosensitivity does not absolutely preclude opportunistic infections, it is a strong indicator that T. yokohamensis is unlikely to cause invasive disease in immunocompetent individuals. Nevertheless, given its phenotypic similarities to C. gattii , formal virulence studies (e.g., macrophage or invertebrate models) are warranted. 4.7 Diagnostic pitfalls due to phenotypic resemblance The striking similarity between T. yokohamensis and C. gattii —mucoid pink colonies, India-ink-positive capsules, urease activity, and yeast-like morphology—raises concerns for clinical laboratories. Without molecular identification, T. yokohamensis could be misidentified as C. gattii using routine phenotypic tests, potentially leading to unnecessary clinical concern. Commercial systems such as VITEK 2 failed to identify this species, and MALDI‑TOF databases lack its reference spectra. We therefore recommend that laboratories exercise caution when encountering cryptococcus-like isolates and use ITS sequencing for definitive identification. Expanding MALDI‑TOF databases to include emerging environmental yeasts is strongly encouraged. 4.8 One Health implications and conclusions Although no human infections have been reported, the exclusive association of T. yokohamensis with Eucalyptus leaves and its phenotypic resemblance to C. gattii argue for including this species in environmental surveillance programs within a One Health framework. This study provides the first comprehensive report of T. yokohamensis from mainland China, offering new data on its ecology, identification, and antifungal susceptibility. Our findings highlight the importance of exploring under-sampled phyllosphere habitats and lay a foundation for future studies on this rare basidiomycetous yeast. Declarations Acknowledgments We would like to acknowledge Antu I1000 Bio-Technology Co., Ltd., for technical Support. Ethics Statement As this study did not involve human or animal subjects, informed consent was not required Funding Acknowledgements:None Data Availability Statement:Not applicable. Consent for publication:Not applicable. Competing interests Chunlan Huang, Beilei Hou, Yueyuan He, Xiaojing Huang, Chunfeng Wu declare that they have no conflicts of interest. References Liang C, Chen ZJ, You GH, Wu XH, Huang HM, He GK, Yang SJ. Effect of Eucalyptus dieback on soil bacterial diversity in plantations. J Trop Biol. 2025;16:218–26. Chen QL, Hu HW, Yan ZZ, Li CY, Nguyen BT, Zhu YG, He JZ. 2021. Precipitation increases the abundance of fungal plant pathogens in Eucalyptus phyllosphere. Environmental Microbiology 23:7688–7700.DOI:10.1111/1462-2920.15728 Nouraei H, Gharechahi F, Zareshahrabadi Z, Zomorodian K, Gharavi A, Khodadadi H, et al. 2024. Molecular characterization of non-Cryptococcus yeast communities isolated from Eucalyptus trees. Current Medical Mycology 10:e2024.345184.1500. 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International Journal of Environmental Research and Public Health 19:4603. DOI:10.3390/ijerph19084603 Elhariri M, Hamza D, Elhelw R, et al. 2016. Eucalyptus tree: a potential source of Cryptococcus neoformans in Egyptian environment. International Journal of Microbiology 2016:4080725. DOI:10.1155/2016/4080725 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Reviewers invited by journal 23 Apr, 2026 Editor assigned by journal 16 Apr, 2026 Submission checks completed at journal 16 Apr, 2026 First submitted to journal 14 Apr, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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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-9421352","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":633578663,"identity":"c8845c7f-9c7e-4703-876f-d620bb16aa07","order_by":0,"name":"Chunlan Huang","email":"","orcid":"","institution":"Liuzhou P eople's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Chunlan","middleName":"","lastName":"Huang","suffix":""},{"id":633578665,"identity":"04bb04a2-171f-4343-ba89-b2c385af5ff0","order_by":1,"name":"Beilei Hou","email":"","orcid":"","institution":"Liuzhou P eople's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Beilei","middleName":"","lastName":"Hou","suffix":""},{"id":633578668,"identity":"03026465-2525-4f97-b254-cea6787b218d","order_by":2,"name":"Yueyuan He","email":"","orcid":"","institution":"Liuzhou P eople's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yueyuan","middleName":"","lastName":"He","suffix":""},{"id":633578670,"identity":"7e648d85-5703-4507-9213-d53217bfe686","order_by":3,"name":"Xiaojing Huang","email":"","orcid":"","institution":"Liuzhou P eople's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xiaojing","middleName":"","lastName":"Huang","suffix":""},{"id":633578672,"identity":"89de9b59-1da4-48f9-adbf-975d28ac7f92","order_by":4,"name":"Chunfeng Wu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyElEQVRIiWNgGAWjYBACNvbGBoMPPyTq+dkbiNTCx3O4oXBmj02CZM8BIrXISaQ3fOZgS0swuJFArMMYEhs3M/AczjO4+XjjDYYam2gitBxsNi6wOFwseTut2ILhWFpuA0EtjI1txjN4DjP23c4xk2BsOEyEFmbG9t88bIcZG26eIVYLG2ODMQ9bWuKEGzzEauFhbDAEBrKxZA/QLwnE+EV+/vMHoKiU42c/vPHGhxobwlqQgYFEAinKIVpI1TEKRsEoGAUjAwAAGnBBPVrZYM0AAAAASUVORK5CYII=","orcid":"","institution":"Liuzhou P eople's Hospital","correspondingAuthor":true,"prefix":"","firstName":"Chunfeng","middleName":"","lastName":"Wu","suffix":""}],"badges":[],"createdAt":"2026-04-15 03:54:29","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9421352/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9421352/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108390764,"identity":"c9afdd83-6abc-430e-8ce2-a444da102145","added_by":"auto","created_at":"2026-05-04 06:57:52","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":254237,"visible":true,"origin":"","legend":"\u003cp\u003eGeographic map of the sampling sites\u003c/p\u003e","description":"","filename":"Fig.1Geographicmapofthesamplingsites.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9421352/v1/c1d0f4e3df11eb22d12ae238.jpg"},{"id":108390754,"identity":"09620f81-be97-44e4-81b2-bbbc7910e4e7","added_by":"auto","created_at":"2026-05-04 06:57:50","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":7947262,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eEucalyptus spp.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Fig.2Eucalyptusspp..jpg","url":"https://assets-eu.researchsquare.com/files/rs-9421352/v1/73ebde260cf008c62bf5f345.jpg"},{"id":108390760,"identity":"a97c0a39-cd3a-4fd7-8b7b-543a1bd2dade","added_by":"auto","created_at":"2026-05-04 06:57:51","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":75154,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparison of mass spectral peaks between \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eT.\u003c/strong\u003e\u003c/em\u003eyokohamensis\u003cstrong\u003e and \u003c/strong\u003eC.gattii\u003c/p\u003e","description":"","filename":"Fig.3DistributionProportionofMajorYeasts.png","url":"https://assets-eu.researchsquare.com/files/rs-9421352/v1/15519b96749a18630e7d908e.png"},{"id":108390763,"identity":"8b89806d-4a0d-4f20-8741-7a287d6ff6ff","added_by":"auto","created_at":"2026-05-04 06:57:51","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":6929896,"visible":true,"origin":"","legend":"\u003cp\u003eDistribution Proportion of Major Yeasts\u003c/p\u003e","description":"","filename":"Fig.4ComparisonofmassspectralpeaksbetweenT.yokohamensisandC.gattii.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9421352/v1/ae798a4bf75699118048ab0f.jpg"},{"id":108390753,"identity":"1b495e22-31c8-40be-826b-4cee138dbf6a","added_by":"auto","created_at":"2026-05-04 06:57:50","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":325268,"visible":true,"origin":"","legend":"\u003cp\u003eGrowth rate of \u003cem\u003eT.yokohamensis\u003c/em\u003e under aerobic culture at 28 °C. (A) Sabouraud Dextrose Agar (SDA); (B) CHROMagar™ Candida (CA)\u003c/p\u003e","description":"","filename":"Fig.5GrowthrateofT.yokohamensisunderaerobiccultureat28C.ASabouraudDextroseAgarSDABCHROMagarCandidaCA.png","url":"https://assets-eu.researchsquare.com/files/rs-9421352/v1/e2befbab4d479af8ee3a9697.png"},{"id":108390761,"identity":"fc02d490-a930-4c15-a297-0b3553690d91","added_by":"auto","created_at":"2026-05-04 06:57:51","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":790657,"visible":true,"origin":"","legend":"\u003cp\u003eColony morphology and microscopic characteristics of the seven yeast-like fungi:\u003cem\u003eC.neoformans\u003c/em\u003e, \u003cem\u003eC. gattii\u003c/em\u003e,\u003cem\u003e Syzygospora sp\u003c/em\u003e., \u003cem\u003eCryptotrichosporon brontis\u003c/em\u003e, \u003cem\u003eTremella basidiomaticola,\u003c/em\u003eand \u003cem\u003eColacogloea terpenoidalis\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eA. Colony morphology on CHROMagar™ Candida (5 days, 28 °C)\u003c/p\u003e\n\u003cp\u003eB. India‑ink staining (×1000)\u003cbr\u003e\nC. Gram staining(×1000)\u003c/p\u003e","description":"","filename":"Fig.6ColonymorphologyandmicroscopiccharacteristicsofthefivemucoidyeastlikefungiandtworeferencecryptococcalspeciesC.neoformansC.gattiiSyzygosporasp.Cryptotrichosporonbron.png","url":"https://assets-eu.researchsquare.com/files/rs-9421352/v1/50ce39ecb44c1292ab1a7b6d.png"},{"id":108390758,"identity":"33996262-07b2-447d-aa1e-e60dbc235ceb","added_by":"auto","created_at":"2026-05-04 06:57:51","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":4435775,"visible":true,"origin":"","legend":"\u003cp\u003eMorphological comparison between \u003cem\u003eT.yokohamensis \u003c/em\u003eand \u003cem\u003eC. gattii( from a clinical strain)\u003c/em\u003e by scanning electron microscopy (SEM)（×20000）\u003c/p\u003e","description":"","filename":"Fig.7MorphologicalcomparisonbetweenT.yokohamensisandC.gattiifromaclinicalstrainbyscanningelectronmicroscopySEM20000.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9421352/v1/3dca094503b200adce88574f.jpg"},{"id":108390797,"identity":"9e0beb45-2d4b-4ddb-a491-076ef8c70b2b","added_by":"auto","created_at":"2026-05-04 06:57:57","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":9691757,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree drawn from neighbor-joining analysis of the ITS1-5.8S-ITS2 region of\u0026nbsp;\u003cem\u003eTremella yokohamensis\u003c/em\u003e\u0026nbsp;and closely related species. \u003cem\u003eC.marinus \u003c/em\u003eCBS 5235\u003csup\u003eT\u003c/sup\u003e(KF36593.1)\u003c/p\u003e","description":"","filename":"Fig.8PhylogenetictreedrawnfromneighborjoininganalysisoftheITS15.8SITS2regionofTremellayokohamensisandcloselyrelatedspecies.Cryptococcus.marinusCBS5235TKF36593.1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9421352/v1/ed7668a2d90940fc1b91cfb4.jpg"},{"id":108493398,"identity":"dd7a2914-2d23-4425-b4de-95a3186f4d08","added_by":"auto","created_at":"2026-05-05 10:00:15","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":30730472,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9421352/v1/3630edad-bd41-419f-aacb-831d00b0ef87.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Morphological and Physiological Features of Tremella yokohamensis: A First Report from Eucalyptus Trees in Southern China","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003e\u003cem\u003eEucalyptus\u003c/em\u003e\u003cem\u003e\u0026nbsp;spp.\u003c/em\u003e are\u0026nbsp;widely planted worldwide, with China and Brazil among the largest producers; in China, Guangxi accounts for roughly 40% of national Eucalyptus area and timber output, and extensive monocultures in Nanning, Liuzhou and Guigang[1]may alter phyllosphere microbial communities. The \u003cem\u003eEucalyptus\u0026nbsp;\u003c/em\u003ephyllosphere hosts diverse yeasts (e.g., Candida, Debaryomyces)[2,3] and can include known zoonotic cryptococci such as \u003cem\u003eCryptococcus neoformans\u003c/em\u003e and \u003cem\u003eCryptococcus gattii\u003c/em\u003e, indicating that plantations may act as environmental reservoirs for some pathogens[4-5] \u003cem\u003eCryptococcus yokohamensis\u003c/em\u003e (now treated in \u003cem\u003eTremella\u003c/em\u003e) was first described from trees and a koala in Japan (2011)[6] and has had few confirmed records outside Japan and the Russian Far East [7,8]. We report the first environmental isolation of \u003cem\u003eT. yokohamensis\u003c/em\u003e from \u003cem\u003eEucalyptus\u003c/em\u003e in Guangxi, China, expanding its known geographic range and providing strain material for follow‑up studies of ecology, antifungal susceptibility and host interactions. No human infections have been reported for this species to date, but targeted antifungal, virulence and One‑Health surveillance studies are recommended to assess potential risk.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003e2.1\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eSpecimen Collection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSamples were collected from three regions in Guangxi between April and August 2025: Binyang County in Nanning City, Sanmenjiang Forest Park and surrounding areas in Liuzhou City, and Qintang District in Guigang City. The collected materials included senescent leaves, bark, twigs, and rhizosphere soil from forest stands of \u003cem\u003eEucalyptus\u003c/em\u003e\u003cem\u003e\u0026nbsp;urophylla\u003c/em\u003e,\u0026nbsp;\u003cem\u003eEucalyptus\u003c/em\u003e\u003cem\u003e\u0026nbsp;grandis\u003c/em\u003e, and hybrid clones (e.g., DH32-28, DH32-26, etc.). A total of 2,000 samples were obtained.\u003c/p\u003e\n\u003cp\u003e2.2 Isolation and Culture of Fungi\u003c/p\u003e\n\u003cp\u003eThe collected leaf, bark, twig, and soil samples were each mixed with an appropriate volume of sterile physiological saline and thoroughly shaken to prepare a homogeneous suspension. After allowing the suspension to settle, the supernatant was collected and centrifuged at 3,500 rpm for 5 minutes. The supernatant was discarded, and the pellet was concentrated to approximately 0.5 mL, then evenly spread onto CHROMagar\u0026trade; Candida chromogenic medium plates. The plates were incubated at 28\u0026deg;C in a constant-temperature incubator, observed periodically for up to 14 days, and suspected yeast-like colonies were recorded and isolated.\u003c/p\u003e\n\u003cp\u003e2.3 Species identification by Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)\u003c/p\u003e\n\u003cp\u003eRoutine fungal pretreatment method\u003cbr\u003e\u0026nbsp;Colonies were directly spotted onto the MALDI target plate and air-dried. Each sample was overlaid with 1 \u0026mu;L of formic acid, air-dried, and then mixed with 1 \u0026mu;L of\u0026nbsp;\u0026alpha;-cyano-4-hydroxycinnamic acid (HCCA) matrix solution. Mass spectra were acquired using the Autof ms1000 MALDI-TOF MS system (Autobio Bio-Technology Co., Ltd., China). Identification scores were interpreted as follows:\u0026nbsp;\u0026ge;9.0, reliable species-level identification; 6.0\u0026ndash;9.0, genus-level identification; \u0026lt;6.0, unreliable.\u003c/p\u003e\n\u003cp\u003eSpecial pretreatment procedure for \u003cem\u003eT. yokohamensis\u003cbr\u003e\u0026nbsp;\u003c/em\u003eFresh colonies were transferred with a cotton swab into 1 mL of 75% ethanol, mixed thoroughly, and centrifuged for 2 min. The supernatant was discarded, and residual ethanol was removed with a paper towel. The pellet was resuspended in 40 \u0026mu;L of formic acid and ground for 30 s using a handheld tissue grinder (LC-MY-10, Lichen, China). After centrifugation for 2 min, 1 \u0026mu;L of the supernatant was spotted onto the target plate, air-dried, and overlaid with 1 \u0026mu;L of HCCA matrix solution. Mass spectral acquisition and result interpretation were performed as described above.\u003c/p\u003e\n\u003cp\u003e2.4 Amplification and DNA Sequence Analysis Amplification\u003c/p\u003e\n\u003cp\u003eThe internal transcribed spacer1 (ITS1)-5.8S-ITS2 of the ribosomal DNA \u0026nbsp;region was chosen as the target for amplification by ITS1 (5\u0026rsquo;-TCCGTAGGTGAACCTGCGG-3\u0026rsquo;) and ITS4 (5\u0026rsquo;-TCCTCCGCTTATTGATATGC-3\u0026rsquo;) primers [9]. Sequencing primers were commercially synthesized, and nucleic acid extraction services were provided by Shanghai Saiyin Biotechnology Co., Ltd. The isolates were then identified by sequencing the ITS1/ITS4 region.\u003c/p\u003e\n\u003cp\u003e2.5 Biochemical Identification of \u003cem\u003eT. yokohamensis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAfter growth on Sabouraud Dextrose Agar (SDA) for five days at 28\u0026deg;C, colonies were suspended in sterile saline and adjusted to a 2.0 McFarland standard. Biochemical identification was performed using the VITEK 2 system with YST cards (bioM\u0026eacute;rieux) according to the manufacturer\u0026rsquo;s instructions; the YST panel covers multiple assimilation/enzymatic substrates and results were recorded substrate-by-substrate as positive/negative, and the system identification and confidence score were reported.\u003c/p\u003e\n\u003cp\u003e2.6 Scanning electron microscopy (SEM)\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eT\u003c/em\u003e\u003cem\u003e.\u0026nbsp;\u003c/em\u003e\u003cem\u003eyokohamensis\u003c/em\u003e and a clinical isolate of \u003cem\u003eC\u003c/em\u003e\u003cem\u003e.\u003c/em\u003e\u003cem\u003e\u0026nbsp;gattii\u003c/em\u003e \u0026nbsp;were cultured on SDA at 28\u0026deg;C for 5 days. Cells were harvested and subjected to freeze‑drying preparation. Briefly, the samples were fixed, dehydrated, and freeze‑dried, followed by sputter‑coating with gold‑palladium. Observations were performed using a scanning electron microscope\u0026nbsp;\u0026nbsp;(Apreo 2C HiVac, Thermo Fisher Scientific, USA)\u0026nbsp;\u0026nbsp;at an accelerating voltage of\u0026nbsp;5\u0026nbsp;kV.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e2.7 Phylogenetic tree construction and analysis\u003c/p\u003e\n\u003cp\u003eComplete ITS rDNA sequences were obtained by assembling forward and reverse sequencing reads using MEGA version 12. The newly assembled sequences were deposited in the GenBank database under accession numbers PX915820 and PX915821. Phylogenetic analysis was performed using the Neighbor‑Joining method implemented in MEGA 12. The analysis was based on the sequences obtained in this study together with reference sequences of relevant type strains retrieved from GenBank. Evolutionary distances were calculated using the Kimura 2‑parameter model. Gaps and missing data were treated by pairwise deletion. Bootstrap support values were calculated from 1,000 replicates, and values \u0026ge;75% are shown at branch nodes. The sequence of \u003cem\u003eC.marinus\u003c/em\u003e CBS 5235\u003csup\u003eT\u003c/sup\u003e(KF36593.1)was used as the\u0026nbsp;outgroup\u0026nbsp;to root the tree.\u003c/p\u003e\n\u003cp\u003e2.8 Antibiotic Susceptibility\u003c/p\u003e\n\u003cp\u003eAntifungal susceptibility testing was conducted following the Clinical and Laboratory Standards Institute (CLSI, 2022, M27/A5) guidelines [10], using the ATB FUNGUS 3 broth microdilution system (bioM\u0026eacute;rieux, USA). The test strips contained predefined gradients of five antifungal agents: amphotericin B (0.5\u0026ndash;16 \u0026mu;g/mL), itraconazole (0.125\u0026ndash;4 \u0026mu;g/mL), voriconazole (0.06\u0026ndash;8 \u0026mu;g/mL), fluconazole (1\u0026ndash;64 \u0026mu;g/mL), and 5-fluorocytosine (4\u0026ndash;16 \u0026mu;g/mL).\u003c/p\u003e\n\u003cp\u003eAfter culturing \u003cem\u003eT. yokohamensis\u003c/em\u003e on Sabouraud Dextrose Agar (SDA) for 4 days, the turbidity of the fungal suspension was adjusted to a 0.5 McFarland standard using a densitometer, A 20 \u0026mu;L aliquot of the suspension was transferred into the accompanying broth medium, yielding a final inoculum density of 0.5\u0026ndash;1.5 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e CFU/mL. Then, 135 \u0026mu;L of the inoculated broth was dispensed into each well of the strip. Incubation was carried out at 28 \u0026deg;C for 48 hours. To ensure test reliability, the quality control strain \u003cem\u003eCandida parapsilosis\u0026nbsp;\u003c/em\u003eATCC 22019 was included in the experiment.\u003c/p\u003e"},{"header":"3. Results","content":"\u003cp\u003e3.1 Sample sources and isolation rate of \u003cem\u003eT. yokohamensis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eSamples were collected from three geographic regions in the Guangxi Zhuang Autonomous Region: Binyang County (Nanning City), Sanmenjiang Forest Park and surrounding areas (Liuzhou City), and Qintang District (Guigang City) (Figure 1), covering both plains and mountainous areas. Eucalyptus sampling sites are shown in Figure 2. A total of 2,000 Eucalyptus-related samples (bark, leaves, twigs, and soil beneath the trees) were collected, and 30 isolates of \u003cem\u003eT. yokohamensis\u0026nbsp;\u003c/em\u003ewere recovered. Within the scope of our sampling, \u003cem\u003eT. yokohamensis\u003c/em\u003e was recovered exclusively from \u003cem\u003eEucalyptus\u003c/em\u003e leaves and not from bark, twigs, or soil. The overall isolation rate was 1.5% (Table 1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1. Isolation of\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eT. yokohamensis\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;exclusively from leaf samples in\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eEucalyptus\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;plantations of Southern China\u003c/strong\u003e\u003c/p\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eTotal samples\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eNanning\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eLiuzhou\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eGuigang\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eTotal\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eRecovery rate\u003c/strong\u003e\u003cstrong\u003e（\u003c/strong\u003e\u003cstrong\u003e%\u003c/strong\u003e\u003cstrong\u003e）\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\"\u003e\n \u003cp\u003eNumber of \u003cstrong\u003e\u003cem\u003eT. yokohamensis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eLeaves\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1550\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e10\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e20\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e30\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e2.0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eBark\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e190\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eTwigs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e130\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eSoil\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e130\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e2000\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e10\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e20\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e30\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e1.5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e3.2 MALDI‑TOF MS identification of yeast‑like fungi and ITS rDNA sequencing\u003c/p\u003e\n\u003cp\u003eThe remaining yeast-like isolates (excluding the 30 \u003cem\u003eT. yokohamensis\u003c/em\u003e strains) were identified by MALDI-TOF MS and ITS sequencing, and classified into 28 species across 20 genera (Fig. 3).\u003c/p\u003e\n\u003cp\u003eMALDI-TOF MS enables accurate species-level identification of clinically common fungi, such as \u003cem\u003eCandida albicans\u003c/em\u003e, \u003cem\u003eC. neoformans\u003c/em\u003e, and \u003cem\u003eC. gattii\u003c/em\u003e, typically with scores above 9.0. However, for several other fungal species, including \u003cem\u003eT. yokohamensis\u003c/em\u003e, \u003cem\u003eSyzygospora\u0026nbsp;sp.\u003c/em\u003e, \u003cem\u003eCryptotrichosporon brontis\u003c/em\u003e,\u003cem\u003e\u0026nbsp;Tremella basidiomaticola voucher\u003c/em\u003e, and \u003cem\u003eColacogloea terpenoidalis\u003c/em\u003e, this method fails to achieve reliable genus-level identification, with scores often below 6.0, necessitating the use of ITS sequencing for accurate identification.\u003c/p\u003e\n\u003cp\u003eAfter the specialized pretreatment involving mechanical grinding, \u003cem\u003eT. yokohamensis\u003c/em\u003e exhibited six characteristic ion peaks in the mass spectrum, located at \u003cem\u003em/z\u003c/em\u003e 3142.524, 3402.751, 6283.049, 6803.279, 7370.305, and 8563.406. In comparison, \u003cem\u003eC. gattii\u003c/em\u003e showed peaks at \u003cem\u003em/z\u003c/em\u003e 3374.345, 3659.501, 6076.732, 6747.308, 7316.443, and 8685.785. Although the spectral profiles of the two species were similar, distinct differences were observed (Fig. 4).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e3.3 Morphological characteristics and optimal growth temperature\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eT. yokohamensis\u003c/em\u003e grew slowly under aerobic conditions at 28\u0026deg;C, with colonies becoming visible after 4\u0026ndash;5 days. On Sabouraud Dextrose Agar (SDA) plates, colonies were milky white and mucoid; on CHROMagar\u0026trade; Candida, colonies were light pink and mucoid after 8 days (Figure 5). The isolate failed to grow on Columbia blood agar, chocolate agar, or routine nutrient agar.\u003c/p\u003e\n\u003cp\u003eAll isolates grew at 28\u0026deg;C but did not grow at 37\u0026deg;C or 40\u0026deg;C, indicating an optimal growth temperature near 28\u0026deg;C. Microscopic examination showed budding cells without pseudohyphae. India ink staining revealed a thick capsule surrounding the cells; the cryptococcal capsular antigen test was positive, and the urease test was also positive.\u003c/p\u003e\n\u003cp\u003eBiochemical testing with the VITEK 2 YST card showed positive reactions for sucrose fermentation (SAC), gluconate fermentation (DGLU), tyrosine arylamidase (TYRA), ornithine decarboxylase (ODC), \u0026beta;‑galactosidase (BGALI), leucine arylamidase (LEUA), alkaline phosphatase (PHOS), and pyruvate utilization (PVATE). Negative reactions were recorded for arginine dihydrolase (ArgA), \u0026gamma;‑glutamyl aminopeptidase (GGT), lysine decarboxylase (LlsA), phenylalanine deaminase (PHEA), proline arylamidase (PROA), pyrrolidonyl arylamidase (PYRA), tryptophan deaminase (APPA), L‑malate utilization (ELLM), and esculin hydrolysis (PHC). The isolate did not ferment glycogen (GLYG), D‑mannitol (DMNE), D‑melezitose (DMAL), N‑acetylglucosamine (NAG), D‑galacturonic acid (dGAL), D‑xylose (DXYL), L‑arabinose, D‑maltose (DMLT), or D‑ribose (DRIB2), nor did it utilize malonate (MTE) or D‑lactate (LGLM) as carbon sources.\u003c/p\u003e\n\u003cp\u003eNote that the VITEK 2 YST card did not accurately identify this isolate to the genus level, probably because the card\u0026rsquo;s database is optimized for commonly encountered clinical yeasts. Therefore, the biochemical profile above is presented only as a phenotypic description; the final taxonomic assignment was based on ITS (internal transcribed spacer) sequence phylogenetic analysis.\u003c/p\u003e\n\u003cp\u003e3.4\u0026nbsp;Morphological and staining characteristics of five mucoid yeast-like fungi confirmed by ITS sequencing\u003c/p\u003e\n\u003cp\u003eAfter 8 days of incubation on CHROMagar Candida, the five ITS‑confirmed mucoid fungi showed growth rates and colony morphologies similar to those of \u003cem\u003eC. gattii\u003c/em\u003e. Members of the genus \u003cem\u003eSyzygospora\u003c/em\u003e produced the most abundant mucoid growth, whereas \u003cem\u003eCryptotrichosporon brontis\u003c/em\u003e produced the least. In terms of pigmentation, \u003cem\u003eC. brontis\u003c/em\u003e formed pale‑orange colonies; \u003cem\u003eT. yokohamensis\u003c/em\u003e, \u003cem\u003eTremella basidiomaticola\u003c/em\u003e and \u003cem\u003eColacoglaea terpenoidalis\u003c/em\u003e produced pink, mucoid colonies resembling those of \u003cem\u003eC. gattii\u003c/em\u003e. India ink staining revealed polysaccharide capsules in all five fungi, with \u003cem\u003eT. yokohamensis\u003c/em\u003e and \u003cem\u003eT. basidiomaticola\u003c/em\u003e showing the widest and thickest capsules. The capsule morphology of \u003cem\u003eT. yokohamensis\u003c/em\u003e closely resembled that of pathogenic cryptococci. All strains reproduced by budding and lacked pseudohyphal structures. Gram staining was positive for all isolates, and oval to spherical yeast‑like cells were seen microscopically. Compared with clinically pathogenic cryptococci, capsule expression was influenced by culture conditions. Clinical isolates of novel cryptococcal species exhibit wide, thick capsules in cerebrospinal fluid but often show reduced or absent capsules and generally do not form mucoid colonies after growth on CHROMagar Candida; by contrast, \u003cem\u003eC. gattii\u003c/em\u003e maintains a wide, thick capsule and produces abundant mucoid colonies both in CSF and on CHROMagar. Unlike some clinical cryptococcal isolates that lose their capsules on CHROMagar Candida, this fungus retained its capsule and produced mucoid colonies on this medium, a phenotype more closely\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003cstrong\u003e.5 Ultrastructural Comparison between\u003c/strong\u003e\u003cem\u003eT. yokohamensis\u003c/em\u003e\u003cstrong\u003e\u0026nbsp;and\u0026nbsp;\u003c/strong\u003e\u003cem\u003eC. gattii\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eScanning electron microscopy preliminarily revealed that the capsule of \u003cem\u003eT. yokohamensis\u003c/em\u003e exhibits a loose, porous, fibrous network‑like ultrastructure, contrasting with the more compact capsule morphology observed in a \u003cem\u003eC. gattii\u003c/em\u003e complex strain. However, given that freeze‑drying preparation may introduce artifacts such as shrinkage or deformation of surface structures, these observations should be considered preliminary and do not constitute definitive evidence for distinguishing the two species at the ultrastructural level. Future studies employing cryo‑electron microscopy or chemical fixation combined with critical point drying would be better suited for a reliable characterization of the capsule ultrastructure in this species (Figure 7)\u003c/p\u003e\n\u003cp\u003e3.6 Phylogenetic analysis\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;A total of 30 isolates of \u003cem\u003eT\u003c/em\u003e\u003cem\u003e.\u003c/em\u003e\u003cem\u003e\u0026nbsp;yokohamensis\u003c/em\u003e were obtained. ITS rDNA sequencing revealed that the sequences of these 30 isolates could be divided into two distinct sequence types. Two representative isolates (one for each sequence type) were selected and submitted to the NCBI database, with the assembled complete sequence accession numbers being PX915820 and PX915821, respectively. BLAST analysis showed that these two representative sequences exhibited 100% identity with the type strains of \u003cem\u003eT. yokohamensis\u003c/em\u003e, JCM 16990 and JCM 16989. Phylogenetic analysis indicated that all isolates obtained in this study belong to the genus \u003cem\u003eTremella\u003c/em\u003e (Figure 8).\u003c/p\u003e\n\u003cp\u003e3.7 Antifungal Susceptibility Testing\u003c/p\u003e\n\u003cp\u003eAntifungal susceptibility of 30 \u003cem\u003eT. yokohamensis\u003c/em\u003estrains was tested using the ATB FUNGUS 3 panel after 48 hours of incubation. MIC ranges were as follows: amphotericin B \u0026lt;0.5 \u0026micro;g/mL, flucytosine \u0026le;4 \u0026micro;g/mL, itraconazole 0.125\u0026ndash;1 \u0026micro;g/mL, voriconazole 0.25\u0026ndash;2 \u0026micro;g/mL, and fluconazole 2\u0026ndash;16 \u0026micro;g/mL. The lowest MICs were observed for amphotericin B and itraconazole, while fluconazole showed the highest MICs. This susceptibility profile closely aligned with the epidemiological cutoff values established for the \u003cem\u003eC\u003c/em\u003e\u003cem\u003e.\u003c/em\u003e\u003cem\u003e\u0026nbsp;gattii\u0026nbsp;\u003c/em\u003especies complex (\u003cstrong\u003eTable\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003cstrong\u003e）\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e2\u003c/strong\u003e Antifungal susceptibility of 30 \u003cem\u003eT. yokohamensis\u0026nbsp;\u003c/em\u003eisolates\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\"\u003e\n \u003cp\u003eAntifungal drugs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"11\"\u003e\n \u003cp\u003eNumber of isolates with MIC (\u0026mu;g/mL) \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e0.125\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026le;4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eMIC\u003csub\u003e50\u003c/sub\u003e/（\u0026mu;g/mL）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eMIC\u003csub\u003e90\u003c/sub\u003e/（\u0026mu;g/mL）\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eAmphotericin B\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eItraconazole\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eVoriconazole\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eFluconazole\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e5-Fluorocytosine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"4. Discussion","content":"\u003cp\u003e4.1 Geographic distribution and niche specialization\u003c/p\u003e\n\u003cp\u003eThe original description reported isolation of this species from a tree trunk (\u003cem\u003eEucalyptus\u003c/em\u003e\u003cem\u003e\u0026nbsp;spp.\u003c/em\u003e) and the nostril of a koala kept in a Japanese zoological park[6]., but no detailed ecological association was established. Within the scope of our sampling, \u003cem\u003eT. yokohamensis\u003c/em\u003e was recovered exclusively from\u0026nbsp;\u003cem\u003eEucalyptus\u003c/em\u003eleaves, suggesting that the phyllosphere may be a natural habitat for this species. The absence of isolates from bark, twigs, or soil in our sample set is consistent with a possible preference for leaf surfaces, but it does not prove strict niche specialization. Several factors could contribute to this pattern, including sampling bias, seasonal variation, or the culture conditions employed.\u003c/p\u003e\n\u003cp\u003eSpecifically, before concluding that \u003cem\u003eT. yokohamensis\u003c/em\u003e is strictly confined to\u0026nbsp;\u003cem\u003eEucalyptus\u003c/em\u003eleaves, several limitations should be considered. First, although we collected 2,000 samples from three regions, the sampling window was limited to the period from April to August 2025. Seasonal or phenological changes in the phyllosphere microbiome may affect the detectability of this yeast on different plant parts[11].Second, the culture conditions used in this study (CHROMagar™ Candida and Sabouraud Dextrose Agar incubated at 28°C) may favor the growth of leaf-associated yeasts but may be suboptimal for recovering the species from bark or soil, where different microbial communities or physiological states (e.g., dormancy, biofilm formation) might require alternative isolation protocols[12].\u003c/p\u003e\n\u003cp\u003eTherefore, while our data demonstrate a clear association between \u003cem\u003eT. yokohamensis\u003c/em\u003e and\u0026nbsp;\u003cem\u003eEucalyptus\u003c/em\u003e leaves within the sampled regions, the possibility of occasional occurrence on other plant parts or in soil under different conditions cannot be ruled out. Future studies employing more diverse isolation methods and year-round sampling are needed to clarify the true ecological breadth of this species.\u003c/p\u003e\n\u003cp\u003eThis phyllosphere association is distinct from that of \u003cem\u003eC. gattii\u003c/em\u003e, which is frequently isolated from bark, tree hollows, and soil[13-16]. Thus, \u003cem\u003eT. yokohamensis\u003c/em\u003e may occupy a unique ecological niche within the\u0026nbsp;\u003cem\u003eEucalyptus\u003c/em\u003e ecosystem, offering a new model for studying phyllosphere-specific adaptation in basidiomycetous yeasts—but this conclusion requires confirmation through more comprehensive sampling and alternative isolation methods.\u003c/p\u003e\n\u003cp\u003e4.2 MALDI‑TOF MS with a novel pretreatment\u003c/p\u003e\n\u003cp\u003eRoutine MALDI‑TOF MS failed to identify \u003cem\u003eT. yokohamensis\u003c/em\u003e (scores \u0026lt;6.0). A mechanical grinding step combined with formic acid extraction produced six characteristic ion peaks, distinct from those of \u003cem\u003eC. gattii\u003c/em\u003e. However, because these peaks are absent from commercial databases, ITS sequencing remained necessary for definitive identification. The documented peaks provide reference data for future database expansion.\u003c/p\u003e\n\u003cp\u003e4.3 Phenotypic consistency with the original description\u003c/p\u003e\n\u003cp\u003eAll isolates exhibited typical cryptococcal traits: mucoid colonies, polysaccharide capsules, positive urease, and inability to grow at 37°C. These features match the original description of \u003cem\u003eT. yokohamensis\u003c/em\u003e[6]. ITS phylogeny showed 100% identity with type strains JCM 16990 and JCM 16989. The presence of two ITS sequence types suggests previously unreported intraspecific variation[7,8].\u003c/p\u003e\n\u003cp\u003e4.4 Antifungal susceptibility profile\u003c/p\u003e\n\u003cp\u003eAll 30 isolates showed low MICs for amphotericin B and itraconazole, while fluconazole MICs were higher. This profile is similar to the epidemiological cutoff values(EVCs) for the \u003cem\u003eC. gattii\u0026nbsp;\u003c/em\u003ecomplex[10]. Should this species ever become opportunistic, these drugs would likely remain effective. Future studies should examine whether\u0026nbsp;\u003cem\u003eEucalyptus\u003c/em\u003e metabolites influence azole susceptibility.\u003c/p\u003e\n\u003cp\u003e4.5 Methodological note: susceptibility testing at 28°C\u003c/p\u003e\n\u003cp\u003eBecause \u003cem\u003eT. yokohamensis\u003c/em\u003e does not grow at 35°C, we performed antifungal testing at 28°C, its optimal growth temperature. This deviates from the CLSI M27 recommendation (35°C), but the deviation was unavoidable for this thermosensitive species. MICs obtained at 28°C may not be directly comparable to those generated at 35°C for pathogenic yeasts, as temperature can affect drug activity. Therefore, our MIC values should be interpreted as ecological susceptibility data rather than clinical breakpoints. They provide a baseline for environmental comparisons but should not be used to predict clinical outcomes.\u003c/p\u003e\n\u003cp\u003e4.6 Thermal intolerance and pathogenic potential\u003c/p\u003e\n\u003cp\u003eThe inability of \u003cem\u003eT. yokohamensis\u003c/em\u003e to grow at 37°C strongly suggests that it is not adapted to the mammalian host environment and likely has very low pathogenic potential in humans. In contrast, \u003cem\u003eC. gattii\u003c/em\u003e grows readily at 37°C, correlating with its ability to cause systemic infections. Although thermosensitivity does not absolutely preclude opportunistic infections, it is a strong indicator that \u003cem\u003eT. yokohamensis\u003c/em\u003e is unlikely to cause invasive disease in immunocompetent individuals. Nevertheless, given its phenotypic similarities to \u003cem\u003eC. gattii\u003c/em\u003e, formal virulence studies (e.g., macrophage or invertebrate models) are warranted.\u003c/p\u003e\n\u003cp\u003e4.7 Diagnostic pitfalls due to phenotypic resemblance\u003c/p\u003e\n\u003cp\u003eThe striking similarity between \u003cem\u003eT. yokohamensis\u0026nbsp;\u003c/em\u003eand \u003cem\u003eC. gattii\u003c/em\u003e—mucoid pink colonies, India-ink-positive capsules, urease activity, and yeast-like morphology—raises concerns for clinical laboratories. Without molecular identification, \u003cem\u003eT. yokohamensis\u0026nbsp;\u003c/em\u003ecould be misidentified as \u003cem\u003eC. gattii\u0026nbsp;\u003c/em\u003eusing routine phenotypic tests, potentially leading to unnecessary clinical concern. Commercial systems such as VITEK 2 failed to identify this species, and MALDI‑TOF databases lack its reference spectra. We therefore recommend that laboratories exercise caution when encountering cryptococcus-like isolates and use ITS sequencing for definitive identification. Expanding MALDI‑TOF databases to include emerging environmental yeasts is strongly encouraged.\u003c/p\u003e\n\u003cp\u003e4.8 One Health implications and conclusions\u003c/p\u003e\n\u003cp\u003eAlthough no human infections have been reported, the exclusive association of \u003cem\u003eT. yokohamensis\u0026nbsp;\u003c/em\u003ewith\u0026nbsp;\u003cem\u003eEucalyptus\u003c/em\u003e leaves and its phenotypic resemblance to \u003cem\u003eC. gattii\u0026nbsp;\u003c/em\u003eargue for including this species in environmental surveillance programs within a One Health framework. This study provides the first comprehensive report of \u003cem\u003eT. yokohamensis\u003c/em\u003e from mainland China, offering new data on its ecology, identification, and antifungal susceptibility. Our findings highlight the importance of exploring under-sampled phyllosphere habitats and lay a foundation for future studies on this rare basidiomycetous yeast.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAcknowledgments\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe would like to acknowledge Antu I1000 Bio-Technology Co., Ltd., for technical Support. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eEthics Statement\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAs this study did not involve human or animal subjects, informed consent was not required\u003c/p\u003e\n\u003cp\u003eFunding Acknowledgements:None\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eData Availability Statement:Not applicable.\u003c/p\u003e\n\u003cp\u003eConsent for publication:Not applicable.\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eChunlan Huang, Beilei Hou, Yueyuan He, Xiaojing Huang, Chunfeng Wu declare that they have no conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eLiang C, Chen ZJ, You GH, Wu XH, Huang HM, He GK, Yang SJ. Effect of Eucalyptus dieback on soil bacterial diversity in plantations. J Trop Biol. 2025;16:218–26.\u003c/li\u003e\n \u003cli\u003eChen QL, Hu HW, Yan ZZ, Li CY, Nguyen BT, Zhu YG, He JZ. 2021. Precipitation increases the abundance of fungal plant pathogens in \u003cem\u003eEucalyptus\u003c/em\u003e phyllosphere. \u003cem\u003eEnvironmental Microbiology\u003c/em\u003e 23:7688–7700.DOI:10.1111/1462-2920.15728\u003c/li\u003e\n \u003cli\u003eNouraei H, Gharechahi F, Zareshahrabadi Z, Zomorodian K, Gharavi A, Khodadadi H, et al. 2024. Molecular characterization of non-Cryptococcus yeast communities isolated from \u003cem\u003eEucalyptus\u003c/em\u003e trees. \u003cem\u003eCurrent Medical Mycology\u003c/em\u003e 10:e2024.345184.1500. DOI:10.22034/CMM.2024.345184.1500\u003c/li\u003e\n \u003cli\u003eChowdhary A, Rhandhawa HS, Prakash A, Meis JF. 2012. Environmental prevalence of \u003cem\u003eCryptococcus neoformans\u003c/em\u003e and \u003cem\u003eCryptococcus gattii\u003c/em\u003e in India: an update. \u003cem\u003eCritical Reviews in Microbiology\u0026nbsp;\u003c/em\u003e38:1–16.\u0026nbsp;DOI:10.3109/1040841X.2011.606426\u003c/li\u003e\n \u003cli\u003eGugnani HC, Mitchell TG, Litvintseva AP, Lengeler KB, Heitman J, Kumar A, et al. 2005. Isolation of \u003cem\u003eCryptococcus gattii\u003c/em\u003e and \u003cem\u003eCryptococcus neoformans\u003c/em\u003e var. \u003cem\u003egrubii\u003c/em\u003e from the flowers and bark of \u003cem\u003eEucalyptus\u003c/em\u003e trees in India. \u003cem\u003eMedical Mycology\u003c/em\u003e 43:565–569. DOI:10.1080/13693780500160785\u003c/li\u003e\n \u003cli\u003eAlshahni MM, Makimura K, Satoh K, Nishiyama Y, Kido N, Sawada T. 2011. \u003cem\u003eTremella yokohamensis\u003c/em\u003e sp. nov., a basidiomycetous yeast isolated from trees and a Queensland koala kept in a Japanese zoological park. \u003cem\u003eInternational Journal of Systematic and Evolutionary Microbiology\u003c/em\u003e 61:3068–3071. DOI:10.1099/ijs.0.027144-0\u003c/li\u003e\n \u003cli\u003eChiku K, Sugiyama S, Akimoto K, Yoshida M. 2022. Draft genome sequences of two basidiomycetous yeasts, \u003cem\u003eTremella yokohamensis\u003c/em\u003e and \u003cem\u003eTremella fuciformis\u003c/em\u003e. \u003cem\u003eMicrobiology Resource Announcements\u0026nbsp;\u003c/em\u003e11:e0057322.\u0026nbsp;DOI:10.1128/mra.00573-22\u003c/li\u003e\n \u003cli\u003eKaeoniwong N, Sotome K, Ichiyanagi T, Shimomura N, Aimi T. 2024. Life cycle and mating compatibility in the Japanese white jelly mushroom, \u003cem\u003eTremella yokohamensis\u003c/em\u003e. \u003cem\u003eMycoscience\u003c/em\u003e 65:208–215. DOI:10.47371/mycosci.2024.05.003\u003c/li\u003e\n \u003cli\u003eFonseca RSK, Lotas KM, Cortez ACA, Fernandes FDS, de Souza ÉS, Dufossé L, et al. 2025. Exploration of carotenoid-producing Rhodotorula yeasts from Amazonian substrates for sustainable biotechnology applications. \u003cem\u003eCurrent Research in Microbial Sciences\u003c/em\u003e 8:100373. DOI:10.1016/j.crmicr.2025.100373\u003c/li\u003e\n \u003cli\u003eClinical and Laboratory Standards Institute. 2022. Performance standards for antifungal susceptibility testing of yeasts. CLSI supplement M27M44S. Wayne (PA): CLSI.\u003c/li\u003e\n \u003cli\u003eChen QL, Hu HW, Yan ZZ, Li CY, Nguyen BT, Zhu YG, He JZ. 2021. Precipitation increases the abundance of fungal plant pathogens in\u0026nbsp;Eucalyptus\u0026nbsp;phyllosphere.\u003cem\u003e\u0026nbsp;Mycosphere\u003c/em\u003e 23(12): 7688–7700. doi: 10.1111/1462-2920.15728\u003c/li\u003e\n \u003cli\u003eTadesse T, Dese D, Dabassa A, Bacha K. 2025. Diversity, distribution, and phenotypic characterization of cultivable wild yeasts isolated from natural forest.\u003cem\u003e\u0026nbsp;Mycosphere\u003c/em\u003e 14: 105. doi: 10.12688/f1000research.160250.3\u003c/li\u003e\n \u003cli\u003eSerna-Espinosa BN, Guzmán-Sanabria D, Forero-Castro M, Escandón P, Sánchez-Quitian ZA. 2021. Environmental status of \u003cem\u003eCryptococcus neoformans\u003c/em\u003e and \u003cem\u003eCryptococcus gatti\u003c/em\u003ei in Colombia. \u003cem\u003eJournal of Fungi\u003c/em\u003e 7:410. DOI:10.3390/jof7060410\u003c/li\u003e\n \u003cli\u003eKidd SE, Bach PJ, Hingston AO, Mak S, Chow Y, MacDougall L, et al. 2007. \u003cem\u003eCryptococcus gattii\u0026nbsp;\u003c/em\u003edispersal mechanisms, British Columbia, Canada. \u003cem\u003eEmerging Infectious Diseases\u003c/em\u003e 13:51–57. DOI:10.3201/eid1301.060823\u003c/li\u003e\n \u003cli\u003eKan A, Schmertmann LJ, McArthur C, Mella VSA, Crowther MS, Miranda L, et al. 2022. A possible link between the environment and \u003cem\u003eCryptococcus gattii\u003c/em\u003enasal colonisation in koalas (Phascolarctos cinereus) in the Liverpool Plains, New South Wales. \u003cem\u003eInternational Journal of Environmental Research and Public Health\u003c/em\u003e 19:4603. DOI:10.3390/ijerph19084603\u003c/li\u003e\n \u003cli\u003eElhariri M, Hamza D, Elhelw R, et al. 2016. \u003cem\u003eEucalyptus\u003c/em\u003e tree: a potential source of \u003cem\u003eCryptococcus neoformans\u003c/em\u003e in Egyptian environment. \u003cem\u003eInternational Journal of Microbiology\u003c/em\u003e 2016:4080725. DOI:10.1155/2016/4080725\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"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":"npj-fungal-science","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [npj Fungal Science](https://www.nature.com/npjfungalsci/)","snPcode":"44512","submissionUrl":"https://submission.springernature.com/new-submission/44512/3?","title":"npj Fungal Science","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-9421352/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9421352/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"This first large-scale isolation of Tremella yokohamensis (formerly Cryptococcus yokohamensis) from Eucalyptus plantations in southern China recovered 30 isolates exclusively from leaves, marking the first mainland China record and extending its known range beyond Japan and the Russian Far East. The isolates exhibited typical cryptococcal traits: mucoid colonies, polysaccharide capsule, positive urease, and growth only at 28 °C. ITS rDNA sequencing revealed two sequence types with 100% identity to type strains JCM 16990 and JCM 16989; phylogeny confirmed placement in Tremella. Antifungal testing showed lowest MICs for amphotericin B and itraconazole, highest for fluconazole. Exclusive leaf recovery indicates a phyllosphere niche, and the antifungal profile suggests ecologically driven adaptation within Eucalyptus habitats, providing critical strain resources and baseline data for understanding this rare yeast’s ecology and public health relevance.","manuscriptTitle":"Morphological and Physiological Features of Tremella yokohamensis: A First Report from Eucalyptus Trees in Southern China","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-04 06:57:33","doi":"10.21203/rs.3.rs-9421352/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewersInvited","content":"","date":"2026-04-23T10:01:30+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-16T13:34:33+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-16T10:56:39+00:00","index":"","fulltext":""},{"type":"submitted","content":"npj Fungal Science","date":"2026-04-15T03:47:29+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"npj-fungal-science","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [npj Fungal Science](https://www.nature.com/npjfungalsci/)","snPcode":"44512","submissionUrl":"https://submission.springernature.com/new-submission/44512/3?","title":"npj Fungal Science","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6ccd07a1-8674-44c0-8931-9f4069f67993","owner":[],"postedDate":"May 4th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":67434146,"name":"Biological sciences/Ecology"},{"id":67434147,"name":"Earth and environmental sciences/Ecology"},{"id":67434148,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2026-05-04T06:57:33+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-04 06:57:33","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9421352","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9421352","identity":"rs-9421352","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}