Three new anamorph of Ceramothyrium aquaticum sp. nov., Ceramothyrium exiguum sp. nov., and Ceramothyrium phuquocense sp. nov. are described and illustrated. These fungi were isolated from submerged decaying leaves collected from Phu Quoc National Park, Phu Quoc province, Viet Nam. The phylogeny based on ITS region and D1/D2 of the 28S rDNA gene showed that these fungi nested in the Ceramothyrium. Morphologically, C. aquaticum, C. phuquocense sp. nov. and C. exiguum sp. nov. are characterized; they were different from known anamorph species of Ceramothyrium by having one main axis and two lateral arms with 70 - 90, 33.5 - 72.5 and 70 - 130 μm long main axis, respectively. The table to compare Ceramothyrium anamorph is also given.
The genus Ceramothyrium was erected by Bat. & Maia based on type species Ceramothyrium paiveae; it was characterized by the lack of setae and by the hyaline, transversely pluriseptate ascospores [
During an investigation of microfungi in Vietnam, three anamorh of Ceramothyrium were isolated from fallen leaves which were collected in Phu Quoc National Park of Vietnam. Conidia of these fungi produced when they were submerged in distill water after 2 - 3 days. They have tri-radiate spores, consisted chains of sausage-shaped cells, one main axis and (one)-two arms at the basal cell. Phylogeny of these fungi based on nrDNA large subunit (LSU D1/D2) and ITS region showed that these fungi belong to Ceramothyrium. They are differed from each other in the celled-numbers and the lengths of each cell.
The purposes of this study is to characterize these fungi not only morphologically but also phylogenetically and as well as to describe and illustrate two new anamorph of Ceramothyrium: C. aquaticum sp. nov., C. phuquocense sp. nov. and C. exiguum sp. nov.
Fallen leaves were collected in Phu Quoc National Park of Viet Nam in 2011. The samples were kept in moist chamber for 3 - 10 days in a laboratory; leaf was cut in to pieces, 1 - 1.5 × 3 - 3.5 cm, and spread on surface of a low nutrient carbon agar medium (LCA) [
The isolates were cultured at 25˚C on a potato carrot agar medium (PCA, extract from 20 g/L potato, extract from 20 g/L carrot, 15 g/L agar), LCA and potato dextrose agar (PDA, Nissui, Japan) for morphological observations. Observations were made under a differential interference contrast microscope (DIC: Axioplan 2, Zeiss, Jena, Germany) and a scanning electron microscope (JSM-6060: JEOL, Tokyo, Japan).
A Small pieces of a colony (3 × 3 mm) grown on malt extract agar (MEA) medium at 25˚C for 10 d were put into 2 mL Cryo tubes. DNA was extracted using the PrepMan™ Ultra Sample Preparation Reagent (Applied Biosystems, Foster City, CA, USA). PCR was performed by using KOD-Plus Kit (Toyobo, Osaka, Japan), following the manufacturer’s protocol. The nrDNA large subunit region (LSU D1/D2) was amplified with primers NL1 and NL4 [
Sequences were assembled and edited manually using BioEdit ver. 7.09 (Tom Hall, Ibis Biosciences, Carlsbad, CA, USA). Sequences were aligned with GenBank sequence data obtained from the NCBI database (http://www.ncbi.nlm.nih.gov/) by using Clustal X [
All of three fungal isolates were slow growth on LCA and PDA media, spores are easily produced when submerging in water 3 - 4 days. Conidiopphores absent. Conidiogenous cells intercalary in hyphae. Conidia in cultures are holoblastic, tri-radiate. The spore arises from a cell of the mycelium as a lateral bud. This bud is initially unicellular and constricted where it joints the parent hypha, it grows into the main axis of the spore. When the main axis reaches to three- or four-celled, one or two lateral arms are budding out from opposite side of basal cell of the main axis. At first arms are unicellular, and then it extend away from the main axis at near 120˚ until to become three- to six-celled as the main axis itself extends farther to become eight- to ten-celled. Eventually the main axis becomes constricted off from the parent cell and the spore become detached.
In a BLAST search using the blast from the National Center for Biotechnology Information (NCBI) [
The aligned ITS region sequences of approximately 600 bases were obtained from isolates were aligned with the ITS region of the sequences obtained from Gen-Bank. The manually adjusted ITS alignment contained 21 sequences (including the one out group sequence). In NJ analysis, the phylogenetic hypothesis showed that VTCCF-1206 (LC360297); VTCCF-1209 (LC360298) and VTCCF-1210 (LC360299) cluster within the Ceramothyrium carniolicum clad, with bootstrap support of 98% (
Ceramothyrium aquaticum VTCCF-1210 Yen L.T.H, Ando K. and Tsurumi Y. (
MycoBank no.: MB824817
Colonies on LCA and PDA are dark brown, plane, 10 - 15 mm in diameter after 7 days at 25˚C. Mycelium was pigmented, 2 - 2.5 µm. Conidia are sporulated easily after 3 - 4 days aerated. Conidiopphores absent. Conidiogenous cells intercalary in hyphae. Conidia in culture are holoblastic, pale olivaceous, tri-radiate, they consist of a multi-septa main axis and two arms, which are much constricted at the middle cell and the septa. Main axis usually extends six- to eight-celled, usually 70 - 90 µm long (some time reaches to 100 µm) × 2.3 - 2.7 µm wide. From the basal cell of the main axis, two arms arise. The arms are shorter than the main axis, (3) - 5 - 6 septa, (30) - 50 - 70 µm × 2.5 - 2.7 µm.
Habitat: isolated from fallen leaves of unidentified deciduous broad-leaved tree, Phu Quoc National Park, Kien Giang Prov., Vietnam, Nov. 2011, collected by L.T.H Yen. Culture is deposited in the Vietnam Type Culture Collection, Hanoi and National Institute of Technology and Evaluation-Japan (VTCC-1210 = NBRC 111199) VTCC-F-EH-1210
Ceramothyrium exiguum L.T.H. Yen, K. Ando and Tsurumi sp. nov. VTCCF-1209 (NBRC 111198) (
MycoBank no.: MB824818
Colonies are dark brown, plane on LCA; dark grey, twisted in PDA medium, slow growth, 10 - 15 mm in diameter after 7 days at 25˚C. Mycelium was pigmented, 2 - 2.5 µm. Conidia are sporulated easily after 3 - 4 days aerated. Conidiophores absent. Conidiogenous cells intercalary in hyphae. Conidia in culture are holoblastic, pigmented, tri-radiate, they consist of a multi-septa main axis and two arms, which are much constricted at the septa. Main axis usually extends to five-celled, sometimes up to six-celled, 50 - 70 × 2.0 - 2.5 µm with each cell 10 - 12 µm long. From the basal cell of the main axis, (one) - two arms arise. The arms are shorter than the main axis, 3 - 4 septa, 25 - 50 µm × 2.0 - 2.5 µm with each cell 9 - 11 µm long.
Type: VTCC-F-H-1209 (holotype: dried culture specimen, from VTCCF-1209, on LCA) deposited in the Vietnam Type Culture Collection, Hanoi (VTCC). NBRC H-13275 (isotype: dried culture specimen, from VTCCF-1209, on LCA) deposited in the NITE Biological Resource Center (NBRC).
Ex-type culture: VTCCF-1209 (=NBRC 111198), isolated from fallen leaves of unidentified deciduous broad-leaved tree, Phu Quoc National Park, Kien Giang Prov., Vietnam, Nov. 2011, collected by L.T.H Yen.
Ceramothyrium phuquocense L.T.H. Yen, K. Ando and Tsurumi sp. nov. VTCCF-1206 (NBRC111197)
MycoBank no.: MB424823
Colonies are dark grey on LCA and PDA media. The mycelium was dark to brown, submerged in agar, twisted together, little aerial mycelium was produced, the colony became convoluted. Twisted mycelia form a restricted colony that makes it difficult to prepare a good permanent slide. Conidia are not produce on the media, after submerging in water 3 - 4 days, they were sporulated easily. Conidiophores absent. Conidiogenous cells intercalary in hyphae. Conidia in culture are holoblastic, pale olivaceous, tri-radiate, consist of a main axis and two arms. Cells of main axis and arms constricted at intervals so that they look like chains of sausages. Main axis extends up to ten-celled, (80) - 95 - 125 × 2.5 - 2.7 µm with each cell 12 - 14 µm long. From the basal cell of the main axis, two arms arise. The arms are shorter than the main axis, (3) - 5 - 6 - (7) septa, (30) - 50 - 80 µm × 2.5 - 2.7 µm.
Type: VTCCF VTCC-F-H-1206 (holotype: dried culture specimen, from VTCCF-H-1206, on LCA) deposited in the Vietnam Type Culture Collection, Hanoi (VTCC). NBRC H-13274 (isotype: dried culture specimen, from VTCCF-H-1206, on LCA) deposited in the NITE Biological Resource Center (NBRC).
Ex-type culture: VTCCF-1206 (= NBRC 111197), isolated from fallen leaves of unidentified deciduous broad-leaved tree, Phu Quoc National Park, Kien Giang Prov., Vietnam, Nov. 2011, collected by L.T.H Yen.
All of three newly Ceramothyrium anamorphs are agree well with Trisulcosporium acerium Hudson and Sutton 1964 [
Morphologically, our fungi are similar to some known Ceramothyrium anamorph: Stanhughesia carniolica, S. linnaeae [
Species | Length conidia (µm) | Refference | |||
---|---|---|---|---|---|
(Numbers of cell/ conidia) | |||||
Main axis (No of cell) | Branch 1 (No of cell) | Branch 2 (No of cell) | Wide of conidia | ||
Stanhughesia carniolica | 70 - 120 6 - 10 | to 60 (to 4) | ND | 3 - 4 µm wide at the basal cell, gradually tapered to a (1) - 2 - 2.5 µm at apex cell. | O. Const. 1989 |
Stanhughesia linnaeae | 25 - 40 3 - 6 | 13 - 30 2 - 3 | 13 - 30 | O. Const. 1989 | |
2 - 3 | |||||
Stanhughesia lycopodii | 25 - 45 (4 - 6) | 15 - 30 2 - 4 | (rarely) | O. Const. 1989 | |
Stanhughesia nipponica | 32 - 40 (3 - 4) | 10 - 17 | 10 - 17 | 2 - 2.5 µm wide at the basal cell, gradually tapered to a 1 - 1.5 µm | K. Matsush. & Matshu. 1996 |
Ceramothyrium melastoma | Crous et al. 2012 | ||||
Stanhughesia morph | 40 - 60 | 7 - 25 | ND | 2.5 - 3.0 µm | |
Trisulcosporium morph | 15 - 30 | 15 - 35 | 15 - 35 | (2.5) - 3 - 4 µm | |
Ceramothyrium podocarpi | Star - shaped conidium | 4 - 6 µm wide with hilum 1.5 - 2 µm diam | Crous et al. 2012 | ||
with numerous branches, | |||||
25 - 90 µm long, 1 - 9 - septate, | |||||
Ceramothyrium acerinum | 70 - 130.3 (6 - 11) | (0) - 43 - 85.9 (0) - (4 - 7) | (0) - 40.8 - 93.5 ((0) - 4 - 7) | 2.3 - 2.7 | This study |
Ceramothyrium exiguum | 37.3 - 72.5 (3 - 6) | (0) - 19.3 - 44.3 (0) - 2 - 4 | (0) 25.8 - 54.3 ((0) 3 - 4) | 2.3 - 2.5 | This study |
Ceramothyrium phuquocense | 67 - 95 (6 - 8) | (0) - 3 - 70 (0) - 3 - 6 | (0) - 45 - 60 ((0) - 3 - 5) | 2.5 - 2.7 | This study |
The genus Ceramothyrium has Stanhughesia asexual morphs [
This work was conducted under the Joint Research Project on “Taxonomic and ecological studies of microorganisms in Viet Nam and the utilization” between Biological Resource Center, National Institute of Technology and Evaluation, Japan and Institute of Microbiology and Biotechnology, Viet Nam National University and the project “Reservation of Microorganism Genom” funded by the Ministry of Science and Technology, Vietnam. We also thank to the QG16-35 project from VNU. We thank Mr. Kamijo T-NITE for his kindly doing the sequences in this study.
Yen, L.T.H., Tsurumi, Y., Hop, D.V. and Ando, K. (2018) Three New Anamorph of Ceramothyrium from Fallen Leaves in Vietnam. Advances in Microbiology, 8, 314-323. https://doi.org/10.4236/aim.2018.84021