Hamatispora, a New Genus of Aquatic Fungi in Microthyriales Isolated From

Mycoscience 59 (2018) 467e472

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Alication of an Enironental Paease Assa Mycoscience Sale6 etect HL for te etection of journal homepage: www.elsevier.com/locate/myc s in Ice Crea Full paper Elba eronica Arias-Rios, Kristina Tenney, Tam Mai, Sam Anderson, Hamatispora, a new genus of aquatic fungi in Microthyriales isolated Ruth Marie Cantera, Jasmine M. Pando, Brandon Selover, Lourdes M. Nadala, from fallen leaves in Vietnam Seana K. Davidson, Mansour Samadpour Le Thi Hoang Yen a, *, Kaoru Yamaguchi b, Yasuhisa Tsurumi b, Duong Van Hop a, Katsuhiko Ando b

ackroun: Dairy products are common sources of Listeria outbreaks, and early a Institute of Microbiology and Biotechnology, Vietnam National University, E2, 144 Xuan Thuy, Cau Giay, Ha Noi, Viet nam b detection of the pathogen is critical to prevent outbreaks of illnesses and financial losses Biological Resource Center, National Institute of Technology and Evaluation, 2-5-8, Kazusakamatari, Kisarazu, Chiba, 292-0818, Japan for dairy producers. Objective: This study aimed to evaluate Sample6 Detect TL for article info abstract effective detection of Listeria monocytoenes and L. innocua in ice cream. etos: Performance of the Sample6 DETECT TL was compared with U.S. Food and Drug Article history: A new aquatic fungus was isolated from submerged, decaying leaves collected at Phu Quoc Island in Received 10 April 2017 Vietnam. The fungus produced hyaline, unique-shaped conidia consisting of a hook-shaped main axis Received in revised form and three arms at the helicoid part of the main axis. Based on its conidial development and morpho- Administration (FDA) Bacterioloical Analytical Manual (BAM) Chapter 10 method for 4 April 2018 logical characteristics, Hamatispora phuquocensis was newly introduced. Phylogenetic analyses based on Accepted 10 April 2018 LSU nrDNA sequences showed that it clusters with Microthyrium spp. and belongs to Microthyriales. detection of Listeria spp. in ice cream using an unpaired study design. Results: R2- Available online 11 June 2018 Furthermore, we generated ITS barcode for this hyphomycetous fungus. enriched samples tested with Sample6 Detect TL performed as well as the reference © 2018 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved. Keywords: method at all time points tested from 15 to 24 h. R2 is a proprietary blend for use with the Freshwater Helico-staurospore test kit that helps with early detection. All the dPODC values (Sample6 Detect TL Phylogeny Taxonomy presumptive and confirmed results) equaled zero, indicating 100 concordance between the methods. Both Sample6 Detect TL and FDA BAM results showed low dPODC values, with confidence intervals indicating no significant differences between Sample6 1. Introduction described and illustrated as belonging to a new genus and its Detect TL and reference method results. Conclusions: Sample6 Detect TL is suitable phylogenetic positon of the new fungus is also discussed. During a survey of micro-fungi in Vietnam, an undescribed to detect Listeria spp. in ice cream, even with a 12 h enrichment. Sample6 Detect TL species was detected and isolated from fallen leaves. The shape of 2. Materials and methods demonstrated equivalent detection of L. monocytoenes and L. innocua from R2-enriched the conidium was characterized by a main axis and three arms. The main axis was shaped like a question mark and the three arms 2.1. Sampling, isolation and preservation samples as expected with 15 and 18 h enrichment when compared with the 24 h FDA developed from three different cells of the helicoid part of the main axis. Matsushima (1975) reported a fungus from decayed leaves of Fallen broad leaves were collected from the Suoi Tranh stream at BAM method for L. monocytoenes. Hilits: These results indicate that Sample6 Podocarpusm acrophyllus (Thunb.) Sweet collected in Kobe city, Phu Quoc Island, Vietnam in Nov. 2011. Samples were put in clean Detect TL, primarily developed for environmental samples, can be used to detect Hyogo Pref., Japan which possess similar morphology to ours and plastic bags with a zip and transported to the laboratory. The leaves described the fungus as “Fungus Imperfectus nonnominates, MFC- were rinsed in tap water, and were incubated in moist chambers. Listeria spp. in ice cream with less incubation time, resulting in faster detection. 1585”. Subsequently, Nawawi (1985) reported the fungus from After 3e7 d, each leaf was stamped on low carbon agar (LCA) (Miura foam sample in Malaysia. Goncz€ ol€ and Revay� (2006) reported a & Kudo, 1970). A single spore on the LCA was isolated by a Sker- similarly shaped conidium from draining rainwater from intact man's micromanipulator under a light microscope to obtain the Abstract of an article in Journal o AOAC nternational 102: 1-6 (2019). leaves of Picea abies (L.) H. Karst in Romania and identiﬁed the pure culture. Cultures have been deposited to the Vietnam Type fungus as Tricladiella pluvialis (Ando & Tubaki, 1984). Hosoya and Culture Collection, Institute of Microbiology and Biotechnology, Lourdes M. Nadala: Participant of the 15th UM, 1987-1988. Tanaka (2007) surveyed freshwater hyphomycetes in Yakushima Vietnam National University, Ha Noi, Vietnam (VTCC) and to the Island, Kagoshima Pref., Japan and reported a conidium like Mat- National Institute of Technology and Evaluation, Biological Re- sushima's fungus. The conidial shape was unique and the fungus sources Center, Chiba, Japan (NBRC). appeared to be a hitherto undescribed species which could not be accommodated into any a genus. In this study, the fungus is 2.2. Morphological study

The isolates were cultured at 25 �C on a potato carrot agar me- * Corresponding author. dium (PCA, extract from 20 g/L potato, extract from 20 g/L carrot, 2% E-mail address: [email protected] (L.T.H. Yen). agar), LCA and potato dextrose agar (PDA, Nissui, Japan) for

Reproduced from Mycoscience 59: 467e472 (2018). Duong Van Hop: Participant of the 15th UM, 1987-1988.

362 363 468 L.T.H. Yen et al. / Mycoscience 59 (2018) 467e472

morphological observations. Observations were made under a dif- shaped with a long tail bearing lateral branches; lateral branches ferential interference contrast microscope (DIC: Axioplan 2, Zeiss, straight-shaped develop from different cells of the helicoids part of Jena, Germany) and a scanning electron microscope (JSM-6060: the main axis. JEOL, Tokyo, Japan). For SEM observation, a small piece (ca. 5 5 mm) of the colony was cut and fixed with 1% OsO4 aq. sol. at Hamatispora phuquocensis L.T.H. Yen, K. Yamag. & K. Ando, sp. � room temperature for 2 h, dehydrated in an ethanol series and nov. Figs. 1e3. finally substituted with isoamyl acetate. After critical point drying MycoBank no.: MB 822832. (HCP-2: Hitachi, Tokyo, Japan) and coating with platinum- Type: VIETNAM, Phu Quoc Island, Kien Giang province, from palladium (JUC-5000: JEOL), the specimens were observed under fallen leaves of an unidentified deciduous broad-leaved tree in the the SEM at 15 kV. Suoi Tranh stream, 5 Nov 2011, leg. L.T.H. Yen (Holotype, VTCCF-H- 1219; ex-holotype culture, VTCCF-1219 NBRC 111195; isotype, ¼ 2.3. Molecular phylogeny NBRC H-521261; ex-isotype culture, VTCCF-1219 NBRC 111195). ¼ Gene sequences ex-holotype: LC064073 (LSU) and LC064074 2.3.1. DNA extraction, PCR and sequencing (ITS). Small pieces of a colony (3 3 mm) grown on malt extract agar Etymology: The epithet phuquocensis refers to the location � (MEA) medium at 25 �C for 10 d were put into 2 mL Cryo tubes. DNA where the fungus was discovered. was extracted using the PrepMan™ Ultra Sample Preparation Re- Mycelium composed of branched, septate, hyaline to olivaceous agent (Applied Biosystems, Foster City, CA, USA). PCR was per- brown, smooth, 1.2e2 mm wide hyphae. Sexual morph: Undeter- formed by using KOD-Plus Kit (Toyobo, Osaka, Japan), following the mined. Asexual morph: Conidiophore reduced to conidiogenous cells. manufacturer's protocol. The nrDNA large subunit region (LSU D1/ Conidiogenous cells holoblastic, solitary, hyaline and simple, pro- D2) was amplified with primers NL1 and NL4 (O'Donnell, 1993). To duced on hyphae, 8e15 1.5e2 mm. Conidia multi-septate, consist- � amply the ITS region, the combination ITS1 and ITS4 (White, Bruns, ing of a main axis and 3(e5) arms; the main axis comprised of two Lee, & Taylor, 1990) were used. Amplification of the DNA fragments parts: a helicoid part and a tail part, constricted at the joint portion of was performed using the GeneAmp PCR System 9700 (Applied the helicoid and the tail part; the helicoid part 3(e5)-celled, 7e10 mm Biosystems) under the following thermal cycling programme: an in diam; the tail part 5e6-celled, 35e40 1.8e2.5 mm. The arms � initial denaturation at 94 �C for 2 min, 35 cycles of denaturation at developing from each cellof the helicoid partof the main axis; the first 94 C for 15 s, annealing at 56 C for 30 s, extension at 68 C for arm (4e)5(e6)-celled, 35e40 1.8e2.5 mm; the second arm 4e5- � � � � 1 min 30 s, a final extension at 68 C for 10 min, and a 16 C soak. celled, 25e35 1.8e2.5 mm; the third arm 3e4-celled, � � � PCR products were checked by agarose gel electrophoresis, and 20e30 1.8e2.5 mm; constricted at the base of the first and second � were purified by using AMPureKit (Agencourt Biosciences, Beverly, arms, not constricted at the base of the third arm. MA, USA). Sequencing reactions were performed by using the Big Cultural characteristics: The isolate (VTCCF-1219 NBRC ¼ Dye Terminator V.3.1 Cycle Sequencing Kit (Applied Biosystems) 111195) grows slowly on PCA, LCA and PDA, with a blight grey, black and the primers of the PCR. The newly generated sequence data colony which is dark grey in reverse. The colony reaches to were deposited in GenBank. 20e30 mm in diam after incubating for 3e4 wk at 25 �C. No sporulation was observed on those media. However, when the colony 2.3.2. Phylogenetic analysis slivers on PCA medium were submerged in sterile distilled water in Sequences were assembled and edited manually using BioEdit a Petri dish at 20 �C, many conidia were produced after 3e5 d. V.7.09 (Tom Hall, Ibis Biosciences, Carlsbad, CA, USA). Sequences Habitat and distribution: On fallen leaves of unidentified de- were aligned with GenBank sequence data obtained from the NCBI ciduous broad-leaved tree in the Suoi Tranh stream in Phu Quoc database (http://www.ncbi.nlm.nih.gov/) by using Clustal X Island, Vietnam. (Thompson, Gibson, Plewniak, Jeanmougin, & Higgins, 1997). The Notes: The conidia are of quite unique shape, with staurospores alignment was manually refined and gap positions were removed that are question mark shaped or hook-shaped with 3 arms (rarely using the programme Se-Al v2.0 a11 (http://tree.bio.ed.ac.uk/ 4e5 arms) developing from each cell of the helicoid part (Figs. 1e3). software/seal/) and the alignment was deposited in TreeBASE The conidiogenous cell develops from the vegetative mycelium. (http://www.treebase.org/) with the accession number S21330. A When it reaches a length of 8e15 mm, a septum is formed at the phylogenetic tree was inferred with neighbor-joining (NJ) method upper part. Then, the upper cell becomes a conidial initial cell. The (Saitou & Nei, 1987) and the Knuc value (Kimura, 1980) by using conidial initial cell elongates, and when it reaches a length of Clustal X. The topology of the tree was evaluated by the bootstrap 5e7 mm, it bends down one circle to make a helicoid shaped resampling method (Felsenstein, 1985) with 1000 replicates. The conidium (Fig. 1A and B, 2A, 3A). Three arms bud out from the first NJplot programme (Perriere & Gouy, 1996) was used for plotting cell nearest the conidiogenous cell (Figs. 1C and 2B), the second cell the phylogenetic tree. (Figs. 1D, 2C and 3B), and the third cell (Figs. 1E, 2D and 3C) of the helicoid part of the main axis, and three arms continue to elongate 3. Results (Figs. 1F, 2E and 3D). The main axis is constricted at the septum between the third and the fourth cells, following which the tail part 3.1. Taxonomy elongates to make a question mark-shaped main axis, along with the elongation of the main axis (Figs. 1GeI, 2F, G, 3E, F). Hamatispora L.T.H. Yen, K. Yamag. & K. Ando, gen. nov. MycoBank no.: MB 822829. 3.2. Molecular phylogeny Type species: Hamatispora phuquocensis L.T.H. Yen, K. Yamag. & K. Ando. To determine the relationship of Hamatispora to other ascomy- Etymology: Hamatispora, from the hook-shape with a long tail. cetous fungi, phylogenetic analysis of LSU region was performed. As Asexual fungus. Mycelium composed of branched, septate hy- a result, Hamatispora phuquocensis is nested in the Microthyriales in phae. Conidiophores unclear. Conidiogenous cells holoblastic, a clade, together with the Condylospora vietnamensis L.T.H Yen & K. produce from hyphae. Conidia solitary, acrogenous, multi-septate, Ando with 76% bootstrap value and Spirosphaera minuta Hennebert consisting of a main axis and lateral branches; main axis hook- (GenBank HQ696659) with 65% bootstrap support (Fig. 4). This

364 365 468 L.T.H. Yen et al. / Mycoscience 59 (2018) 467e472 L.T.H. Yen et al. / Mycoscience 59 (2018) 467e472 469 morphological observations. Observations were made under a dif- shaped with a long tail bearing lateral branches; lateral branches ferential interference contrast microscope (DIC: Axioplan 2, Zeiss, straight-shaped develop from different cells of the helicoids part of Jena, Germany) and a scanning electron microscope (JSM-6060: the main axis. JEOL, Tokyo, Japan). For SEM observation, a small piece (ca. 5 5 mm) of the colony was cut and fixed with 1% OsO4 aq. sol. at Hamatispora phuquocensis L.T.H. Yen, K. Yamag. & K. Ando, sp. � room temperature for 2 h, dehydrated in an ethanol series and nov. Figs. 1e3. finally substituted with isoamyl acetate. After critical point drying MycoBank no.: MB 822832. (HCP-2: Hitachi, Tokyo, Japan) and coating with platinum- Type: VIETNAM, Phu Quoc Island, Kien Giang province, from palladium (JUC-5000: JEOL), the specimens were observed under fallen leaves of an unidentified deciduous broad-leaved tree in the the SEM at 15 kV. Suoi Tranh stream, 5 Nov 2011, leg. L.T.H. Yen (Holotype, VTCCF-H- 1219; ex-holotype culture, VTCCF-1219 NBRC 111195; isotype, ¼ 2.3. Molecular phylogeny NBRC H-521261; ex-isotype culture, VTCCF-1219 NBRC 111195). ¼ Gene sequences ex-holotype: LC064073 (LSU) and LC064074 2.3.1. DNA extraction, PCR and sequencing (ITS). Small pieces of a colony (3 3 mm) grown on malt extract agar Etymology: The epithet phuquocensis refers to the location � (MEA) medium at 25 �C for 10 d were put into 2 mL Cryo tubes. DNA where the fungus was discovered. was extracted using the PrepMan™ Ultra Sample Preparation Re- Mycelium composed of branched, septate, hyaline to olivaceous agent (Applied Biosystems, Foster City, CA, USA). PCR was per- brown, smooth, 1.2e2 mm wide hyphae. Sexual morph: Undeter- formed by using KOD-Plus Kit (Toyobo, Osaka, Japan), following the mined. Asexual morph: Conidiophore reduced to conidiogenous cells. manufacturer's protocol. The nrDNA large subunit region (LSU D1/ Conidiogenous cells holoblastic, solitary, hyaline and simple, pro- D2) was amplified with primers NL1 and NL4 (O'Donnell, 1993). To duced on hyphae, 8e15 1.5e2 mm. Conidia multi-septate, consist- � amply the ITS region, the combination ITS1 and ITS4 (White, Bruns, ing of a main axis and 3(e5) arms; the main axis comprised of two Lee, & Taylor, 1990) were used. Amplification of the DNA fragments parts: a helicoid part and a tail part, constricted at the joint portion of was performed using the GeneAmp PCR System 9700 (Applied the helicoid and the tail part; the helicoid part 3(e5)-celled, 7e10 mm Biosystems) under the following thermal cycling programme: an in diam; the tail part 5e6-celled, 35e40 1.8e2.5 mm. The arms � initial denaturation at 94 �C for 2 min, 35 cycles of denaturation at developing from each cellof the helicoid partof the main axis; the first 94 C for 15 s, annealing at 56 C for 30 s, extension at 68 C for arm (4e)5(e6)-celled, 35e40 1.8e2.5 mm; the second arm 4e5- � � � � 1 min 30 s, a final extension at 68 C for 10 min, and a 16 C soak. celled, 25e35 1.8e2.5 mm; the third arm 3e4-celled, � � � PCR products were checked by agarose gel electrophoresis, and 20e30 1.8e2.5 mm; constricted at the base of the first and second � were purified by using AMPureKit (Agencourt Biosciences, Beverly, arms, not constricted at the base of the third arm. Fig. 1. Conidia of Hamatispora phuquocensis (VTCCF-1219 NBRC 111195) and the conidial development. A: A conidial initial and a conidiogenous cell developed from hyphae. B: fi ¼ fi fi fi MA, USA). Sequencing reactions were performed by using the Big Cultural characteristics: The isolate (VTCCF-1219 NBRC The rst and second cells of the main axis grow out and produce a curved main axis. C: A budding of the rst arm from the rst cell of the main axis. D: Development of the rst and ¼ the second arms from the first and the second cells of the main axis, respectively. E, F: Development of the first, the second and the third arms along with the elongation of the main Dye Terminator V.3.1 Cycle Sequencing Kit (Applied Biosystems) 111195) grows slowly on PCA, LCA and PDA, with a blight grey, black axis of conidia. GeI: Mature conidia. Bars: AeF5mm; GeI 10 mm. and the primers of the PCR. The newly generated sequence data colony which is dark grey in reverse. The colony reaches to were deposited in GenBank. 20e30 mm in diam after incubating for 3e4 wk at 25 �C. No sporulation was observed on those media. However, when the colony clade is also nested in Microthyrium spp. that are representative of to disperse and colonize on the litter patches (Dang, Gessner, & 2.3.2. Phylogenetic analysis slivers on PCA medium were submerged in sterile distilled water in Microthyriales with 99% bootstrap value (Fig. 4). Based on a Meg- Chauvet, 2007). This kind of spore has been detected in aquatic Sequences were assembled and edited manually using BioEdit a Petri dish at 20 �C, many conidia were produced after 3e5 d. aBLAST search of NCBI GenBank, nucleotide database, the closest environments in many country, such as Japan, Malaysia and V.7.09 (Tom Hall, Ibis Biosciences, Carlsbad, CA, USA). Sequences Habitat and distribution: On fallen leaves of unidentified de- hits using the ITS sequence are Unculture fungus [GenBank Romania (Goncz€ ol€ & Revay,� 2006; Hosoya & Tanaka, 2007; were aligned with GenBank sequence data obtained from the NCBI ciduous broad-leaved tree in the Suoi Tranh stream in Phu Quoc GU174368, Identities 189/204 (92.6%), Gaps 1/204 (0.5%)], Spi- Matsushima,1975; Nawawi, 1985). Unfortunately, like other aquatic ¼ ¼ database (http://www.ncbi.nlm.nih.gov/) by using Clustal X Island, Vietnam. rosphaera minuta [GenBank HQ696659, Identities 209/234 fungi, this fungus is difficult to culture on artificial media and it is ¼ (Thompson, Gibson, Plewniak, Jeanmougin, & Higgins, 1997). The Notes: The conidia are of quite unique shape, with staurospores (89.3%)], Condylospora vietnamensis [GenBank LC146723, Identi- also difficult to sporulate on the culture medium (Descals & alignment was manually refined and gap positions were removed that are question mark shaped or hook-shaped with 3 arms (rarely ties 158/163 (96.9%), Gaps 0/163 (0%)], Triscelophorus mono- Moralejo, 2001). In this study, we succeeded in culturing the fun- ¼ ¼ using the programme Se-Al v2.0 a11 (http://tree.bio.ed.ac.uk/ 4e5 arms) developing from each cell of the helicoid part (Figs. 1e3). sporus Ingold [GenBank KF730840, Identities 175/189 (92.6%), gus on artificial media and inducing its sporulation by submerging ¼ software/seal/) and the alignment was deposited in TreeBASE The conidiogenous cell develops from the vegetative mycelium. Gaps 3/189 (1.5%)], and several the other Unculture fungi. the colony pieces in water. ¼ (http://www.treebase.org/) with the accession number S21330. A When it reaches a length of 8e15 mm, a septum is formed at the phylogenetic tree was inferred with neighbor-joining (NJ) method upper part. Then, the upper cell becomes a conidial initial cell. The (Saitou & Nei, 1987) and the Knuc value (Kimura, 1980) by using conidial initial cell elongates, and when it reaches a length of 4. Discussion 4.2. Taxonomy and phylogeny of Hamatispora phuquocensis Clustal X. The topology of the tree was evaluated by the bootstrap 5e7 mm, it bends down one circle to make a helicoid shaped resampling method (Felsenstein, 1985) with 1000 replicates. The conidium (Fig. 1A and B, 2A, 3A). Three arms bud out from the first 4.1. Ecology of Hamatispora phuquocensis Morphological observations of H. phuquocensis showed that the NJplot programme (Perriere & Gouy, 1996) was used for plotting cell nearest the conidiogenous cell (Figs. 1C and 2B), the second cell spore shape of this fungus was similar to that of the fungus recor- the phylogenetic tree. (Figs. 1D, 2C and 3B), and the third cell (Figs. 1E, 2D and 3C) of the Hamatispora phuquocensis was isolated from submerged fallen ded by Matsushima (1975), Nawawi (1985) and Hosoya and Tanaka helicoid part of the main axis, and three arms continue to elongate leaves collected from the Suoi Tranh stream in Vietnam. There were (2007) as an unnamed fungus. Goncz€ ol€ and Revay� (2006) identified 3. Results (Figs. 1F, 2E and 3D). The main axis is constricted at the septum no sporulation in solid media but abundantly sporulated in water. the fungus as Tricladiella pluvialis K. Ando & Tubaki, but Hamatis- between the third and the fourth cells, following which the tail part This result suggests that H. phuquocensis belongs to the ecological pora is clearly distinct morphology from T. pluvialis. The conidia of 3.1. Taxonomy elongates to make a question mark-shaped main axis, along with group of aquatic hyphomycetes, also known as Ingoldian fungi our new fungus have one main axis and three arms which bud out the elongation of the main axis (Figs. 1GeI, 2F, G, 3E, F). (Descals & Moralejo, 2001). Aquatic hyphomycetes were first from the first, the second and the third cell of the helicoid part of Hamatispora L.T.H. Yen, K. Yamag. & K. Ando, gen. nov. detected by Ingold (1942) as an artificial group of non- the main axis, while Tricladiella has one main axis and two short MycoBank no.: MB 822829. 3.2. Molecular phylogeny taxonomically related organisms. Biomass of aquatic hyphomy- arms which develop from different levels of the main axis (Ando & Type species: Hamatispora phuquocensis L.T.H. Yen, K. Yamag. & cetes can reach up to 6.1e15% of the total detrital biomass (Gessner Tubaki, 1984). Furthermore, the main axis of Hamatispora is K. Ando. To determine the relationship of Hamatispora to other ascomy- & Chauvet, 1994) and 96% of the microbial biomass (Baldy, Gessner, helicoid-shaped at the base, but this character does not occur in Etymology: Hamatispora, from the hook-shape with a long tail. cetous fungi, phylogenetic analysis of LSU region was performed. As & Chauvet, 1995) making them important plant litter decomposers Tricladiella. Thus, we conclude that Hamatispora and Tricladiella are Asexual fungus. Mycelium composed of branched, septate hy- a result, Hamatispora phuquocensis is nested in the Microthyriales in in the nutrient recycling of forest ecosystems (Barlocher€ & Boddy, distinct genera. phae. Conidiophores unclear. Conidiogenous cells holoblastic, a clade, together with the Condylospora vietnamensis L.T.H Yen & K. 2016). Hyde et al. (2013) accepted the Microthyriales based on produce from hyphae. Conidia solitary, acrogenous, multi-septate, Ando with 76% bootstrap value and Spirosphaera minuta Hennebert Like the most other aquatic fungi, the H. phuquocensis produces morphological data and included the families Microthyriaceae and consisting of a main axis and lateral branches; main axis hook- (GenBank HQ696659) with 65% bootstrap support (Fig. 4). This big, tetraridiate and branched spores which could help them easily Micropeltidaceae. Members of this order are reported as epiphytes,

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Fig. 2. Hamatispora phuquocensis (VTCCF-1219 NBRC 111195) under a light microscope. A: A conidial initial and a conidiogenous cell developed from hyphae. B: A budding of the ¼ first arm from the first cell of the main axis. C: A budding of the second arm from the second cell of the main axis. D: A development of the third arm initial from the third cell of the main axis. E: Development of the first, the second, and the third arms along with the elongation of the main axis of conidia. F, G: Mature conidia. Bars: AeF 10 mm; G 20 mm.

Fig. 3. Hamatispora phuquocensis (VTCCF-1219 NBRC 111195) under a SEM. A: A helicoid-shaped conidial initial developed from a conidiogenous cell. BeD: Development of the ¼ ﬁrst, the second, and the third arms along with the elongation of the main axis of conidia. E, F: A matured conidium and the conidiogenous cell. Arrows: Separating parts between a conidiogenous cell and a conidium. Bars: AeE5mm; F 10 mm. pathogens or saprobes (Hongsanan et al., 2015; Hyde et al., 2013; Li, thyriothecia with radiating cells, a prominent central ostiole, fusi- Haixia, & Hyde, 2011; Schoch et al., 2009; Wu, Tian, Li, & Hyde, form to cylindrical asci, and 1-septate ascospores, often with ciliate 2014a). Microthyriaceae is the type family of Microthyriales and appendages (Hofmann, 2010; Hyde et al., 2013; Wu et al. b, 2014a; differs from Micropeltidaceae in the following ways: it has Hongsanan, Chomnunti, Crous, Chukeatirote, & Hyde, 2014). Kirk,

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Fig. 4. Neighbor joining phylogenetic tree inferred from LSU nrDNA sequence data. Bootstrap values above 50% are given for the corresponding branches. The scale is shown so that evolutionary distances can be inferred. Sequences downloaded from GenBank are given with accession numbers.

Cannon, Minter, and Stalpers (2008) estimated there are 54 genera The classiﬁcation of Mycrothyriales is almost exclusively based and 278 species of Microthyriaceae based on their morphological on morphological characters. There is little molecular data in the Fig. 3. Hamatispora phuquocensis (VTCCF-1219 NBRC 111195) under a SEM. A: A helicoid-shaped conidial initial developed from a conidiogenous cell. BeD: Development of the ¼ features. In Microthyriaceae, Hyde et al. (2013) accepted 13 genera, GenBank because phylogeny of Mycrothyriales has been poorly ﬁrst, the second, and the third arms along with the elongation of the main axis of conidia. E, F: A matured conidium and the conidiogenous cell. Arrows: Separating parts between a seven of which are sexual genera (Arnaudiella Petr., Calothyriopsis studied. The clade of Microthyriales contains Microthyrium micro- conidiogenous cell and a conidium. Bars: AeE5mm; F 10 mm. Hohn.,€ Caribaeomyces Cif., Microthyrium Desm., Palawania Syd., & P. scopicum Desm. (type species of Microthyrium), Microthyrium Syd., Paramicrothyrium H.X. Wu & K.D. Hyde, and Seynesiella G. propagulensis H.X & K.D. Hyde, Micropeltis zingiberacicola H.X. Wu pathogens or saprobes (Hongsanan et al., 2015; Hyde et al., 2013; Li, thyriothecia with radiating cells, a prominent central ostiole, fusi- Arnaud) and six asexual genera (Asterostomula Theiss., Isthmospora & K.D. Hyde, Paramicrothyrium chinensis H.X. Wu & K.D. Hyde, and Haixia, & Hyde, 2011; Schoch et al., 2009; Wu, Tian, Li, & Hyde, form to cylindrical asci, and 1-septate ascospores, often with ciliate F. Stevens, Hansfordiella S. Hughes, Leptothyrium Kunze, Xen- Stomiopeltis betulae I.P. Ellis (Wu et al., 2014b). 2014a). Microthyriaceae is the type family of Microthyriales and appendages (Hofmann, 2010; Hyde et al., 2013; Wu et al. b, 2014a; ogliocladiopsis Crous & W.B. Kendr., and Zalerion R.T. Moore & In this study, our fungus nested in the Microthyriales clade differs from Micropeltidaceae in the following ways: it has Hongsanan, Chomnunti, Crous, Chukeatirote, & Hyde, 2014). Kirk, Meyers). which contained Condylospora vietnamensis, Microthyrium buxicola

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Species diversity of rain borne hyphomycete conidia only clearly confirmed that Hamatispora phuquocensis belongs to from living trees. Fungal Diversity, 22, 37e54. Hofmann, T. A. (2010). Plant parasitic asterinaceae and Microthyriaceae from the the Microthyriaceae/Microthyriales but also suggested the Con- neotropics (Panama). PhD thesis. Germany: J.W. Goethe-University Frankfurt am dylospora is also a member of the Microthyriales. Condylospora is an Main. anamorphic aquatic genus comprising of four species with hyaline, Hongsanan, S., Chomnunti, P., Crous, P. W., Chukeatirote, E., & Hyde, K. D. (2014). Introducing Chaetothyriothecium, a new genus of Microthyriales. Phytotaxa, 161, multiseptate, wormlike conidia (Type species: Condylospora spu- 157e164. migena Nawawi) or L-shaped (C. gigatea Nawawi & Kuthub.), S- Hongsanan, S., Tian, Q., Bahkali, A. H., Yang, J. B., McKenzie, E. H. C., Chomnunti, P., & shaped conidia (C. flexuosa Nawawi & Kuthub.) or N/U-shaped Hyde, K. D. (2015). 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