Corresponding author: Saisamorn Lumyong (
Academic editor: Maarja Öpik
A new species of soil fungi, described herein as
Khuna S, Suwannarach N, Kumla J, Meerak J, Nuangmek W, Kiatsiriroat T, Lumyong S (2019)
The genus
During the isolation of non-soluble mineral solubilizing fungi from agricultural soil in northern Thailand, we found a particular population of
Soil samples were collected from agricultural areas of Mae Wang District, Chiang Mai Province, Thailand. The samples were air-dried at room temperature for 3 d, sieved and mixed through a 2 mm mesh prior to isolation of fungi by serial dilution. The dilution spread plate method was used with three serial dilutions in 0.5% NaCl solution. After dilution, 0.1 ml of suspension was spread on modified Aleksandrov agar (5.0 g glucose, 0.5 g MgSO4•7H2O, 0.1 g CaCO3, 0.005 g FeCl3, 2.0 g Ca3PO4, 3.0 g K2HPO4, and 15.0 g agar, pH 7.0, in 1 L of deionized water) for detection of non-soluble mineral solubilizing fungi. The plates were incubated at 30 °C in darkness for 5 d. Colonies which produced clear zones were considered mineral solubilizing strains and were selected for further studies.
The colonies’ morphology on potato dextrose agar (
Carbon source assimilation profiles were determined with the API 50CH commercial kit (bioMérieux, France), following the methods described by
For nitrogen source assimilation we prepared inoculum as described above, but the yeast nitrogen base broth was replaced by carbon nitrogen base broth, and testing was performed in sterile, disposable, multiwell microplates. The medium with the nitrogen sources was dispensed into the wells in 150 µl, and each well was inoculated with 50 µl of the spore containing medium. The microplates were incubated at 37 °C in darkness for 48–72 h. Growth on NaCl (2%, 5%, 7%, and 10%), 2% MgCl2 and 0.1% cycloheximide was determined. All tests were performed in three replicates.
Genomic DNA of five day-old fungal mycelia on
Details of the sequences used for phylogenetic analysis obtained from this study and from previous studies are provided in Table
Sequences used for phylogenetic analysis. Type species of
Taxa | Strain/isolate | GenBank accession number | References | ||
---|---|---|---|---|---|
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D1/D2 domain |
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CBS 476.78 |
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CBS 477.78 |
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|
|
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FMR 12015 |
|
– | – |
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CBS 658.93 |
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UTHSC 06-4222 |
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|
|
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|
UTHSC 03-3644 |
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GMCH 480/07 |
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IMI 338332 |
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IMI 338333 |
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GMCH 211/09 |
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FMR 13881 |
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|
– | Unpublished |
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FMR 13217 |
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– | Unpublished |
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FMR 12016 |
|
– | – |
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|
GMCH M333/05 |
|
– | – |
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GMCH M52/05 |
|
– | – |
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UTHSC 08-1425 |
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UTHSC 08-2146 |
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UTHSC 06-2356 |
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UTHSC 04-891 |
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UTHSC R-3841 |
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UTHSC 04-838 |
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UTHSC 07-204 |
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CBS 136361 |
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SDBR-CMUS24 |
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|
This study |
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SDBR-CMUS26 |
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This study |
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SDBR-CMUS219 |
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This study |
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ATCC 60625 |
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– |
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UTHSC 08-3606 |
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– |
|
This experiment was carried out using basal medium (10.0 g glucose, 0.5 g (NH)4SO4, 0.2 g NaCl, 0.1 g MgSO4•7H2O, 0.2 g KCl, 0.5 g yeast extract, 0.002g MnSO4•H2O, and 15.0 g agar per liter of deionized water, pH 7.0) with addition of non-soluble metal minerals including Ca3(PO4)2, CaCO3, CuCO3•Cu(OH)2, CuO, CoCO3, FePO4, MgCO3, MnO, ZnCO3, ZnO, feldspar (KAlSi3O8), and kaolin (Al2Si2O5(OH)4) to the desired final concentration of 0.5% according to the method described by
The data were analyzed by one-way analysis of variance (ANOVA) by SPSS program version 16.0 (SPSS Inc., USA) for Windows, and Tukey’s range test was used for significant differences (
Mycelial growth of the three
Growth rate of
Medium | Temperature (°C) | Isolate/growth rate (mm/day) | ||
---|---|---|---|---|
SDBR-CMUS24 | SDBR-CMUS26 (Holotype) | SDBR-CMUS219 | ||
|
4 | – | – | – |
20 | 5.78 ± 0.51 i | 5.78 ± 0.19 jk | 5.67 ± 0.67 i | |
25 | 8.58 ± 0.76 g | 8.67 ± 0.76 g | 8.83 ± 0.88 f | |
30 | 28.33 ± 0.00 b | 28.33 ± 0.00 b | 28.33 ± 0.00 b | |
37 | 40.64 ± 0.00 a | 45.04 ± 0.00 a | 42.64 ± 0.00 a | |
42 | 16.73 ± 0.47 d | 17.00 ± 0.00 d | 16.89 ± 0.19 d | |
45 | – | – | – | |
50 | – | – | – | |
|
4 | – | – | – |
20 | 3.64 ± 0.62 k | 3.55 ± 0.16 l | 3.69 ± 0.36 k | |
25 | 5.89 ± 019 i | 6.11 ± 0.19 ij | 6.00 ± 0.33 hi | |
30 | 7.00 ± 0.71 h | 7.57 ± 0.74 h | 6.95 ± 0.70 gh | |
37 | 9.80 ± 1.00 f | 9.93 ± 1.10 f | 9.07 ± 0.99 f | |
42 | 6.13 ± 0.63 i | 6.38 ± 0.57 i | 6.08 ± 0.62 hi | |
45 | – | – | – | |
50 | – | – | – | |
|
4 | – | – | – |
20 | 4.60 ± 0.20 j | 4.93 ± 0.76 k | 4.67 ± 0.99 j | |
25 | 7.89 ± 0.35 g | 8.33 ± 0.76 gh | 7.28 ± 0.19 g | |
30 | 17.00 ± 0.00 d | 17.00 ± 0.00 d | 17.00 ± 0.00 d | |
37 | 21.25 ± 0.00 c | 21.25 ± 0.00 c | 21.25 ± 0.00 c | |
42 | 13.79 ± 0.46 e | 14.09 ± 0.13 e | 13.94 ± 0.39 e | |
45 | – | – | – | |
50 | – | – | – |
Carbon assimilation profiles of the three strains of
Carbon assimilation profiles for
Carbon source |
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||
---|---|---|---|---|---|---|---|---|
SDBR-CMUS24 | SDBR-CMUS26T | SDBR-CMUS219 | CBS 476.78 T | CBS 136361 T | UTHSC 04-838 T | UTHSC 08-1425 T | CBS 658.93 T | |
GLY (glycerol) | + | + | + | + | + | + | + | + |
ERY (erythritol) | – | – | – | – | – | – | – | – |
DARA (D-arabinose) | – | – | – | – | – | – | – | – |
LARA (L-arabinose) | + | + | + | + | – | + | + | + |
RIB (D-ribose) | + | + | + | + | + | + | + | + |
DXYL (D-xylose) | + | + | + | + | + | + | + | + |
LXYL (L-xylose) | – | – | – | – | – | – | – | – |
ADO (D-adonitol) | + | + | + | + | + | + | + | + |
MDX ( |
– | – | – | – | – | – | – | – |
GAL (D-galactose) | – | – | – | – | – | – | – | – |
GLU (D-glucose) | + | + | + | + | + | + | + | + |
FRU (D-fructose) | + | + | + | + | + | + | + | + |
MNE (D-mannose) | + | + | + | + | + | + | + | + |
SBE (L-sorbose) | – | – | – | – | – | – | – | – |
RHA (L-rhamnose) | – | – | – | – | – | – | – | – |
DUL (dulcitol) | – | – | – | – | – | – | – | – |
INO (inositol) | – | – | – | – | – | – | – | – |
MAN (D-mannitol) | + | + | + | + | + | + | + | + |
SOR (D-sorbitol) | + | + | + | + | + | + | + | + |
MDM ( |
– | – | – | – | – | – | – | – |
MDG ( |
– | – | – | – | – | – | – | – |
NAG ( |
+ | + | + | + | + | + | + | + |
AMY (amygdalin) | – | – | – | – | – | – | – | – |
ARB (arbutin) | – | – | – | – | – | – | – | – |
ESC (esculin) | – | – | – | + | – | – | – | – |
SAL (salicin) | – | – | – | – | – | – | – | – |
CEL (D-cellobiose) | – | – | – | + | – | + | + | + |
MAL (D-maltose) | + | + | + | + | + | + | + | + |
LAC (D-lactose) | – | – | – | – | – | – | – | – |
MEL (D-melibiose) | – | – | – | – | – | – | – | – |
SAC (D-saccharose) | – | – | – | – | – | – | – | – |
TRE (D-trehalose) | + | + | + | + | + | + | + | + |
INU (inulin) | – | – | – | – | – | – | – | – |
MLZ (D-melezitose) | + | + | + | + | – | + | + | + |
RAF (D-raffinose) | – | – | – | – | – | – | – | – |
AMD (amidon) | + | + | + | + | – | + | + | + |
GLYG (glycogen) | + | + | + | + | + | + | + | + |
XLT (xylitol) | + | + | + | + | + | + | + | + |
GEN (gentiobiose) | – | – | – | – | – | – | – | – |
TUR (D-turanose) | + | + | + | – | – | – | – | – |
LYX (D-lyxose) | + | + | + | – | + | + | – | – |
TAG (D-tagatose) | + | + | + | – | – | – | – | – |
DFUC (D-fucose) | + | + | + | – | – | – | – | – |
LFUC (L-fucose) | + | + | + | – | – | – | – | – |
DARL (D-arabitol) | + | + | + | + | + | + | + | + |
LARL (L-arabitol) | – | – | – | + | + | + | + | + |
GNT (potassium gluconate) | – | – | – | – | + | – | – | – |
2KG (potassium 2-keto- gluconate) | – | – | – | – | – | – | – | – |
5KG (potassium 5-keto- gluconate) | – | – | – | – | – | – | – | – |
aThis study, b
Nitrogen assimilation and tolerance to chemical compounds for
Nitrogen source and other tests |
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---|---|---|---|---|---|---|---|---|
SDBR-CMUS24 | SDBR-CMUS26 T | SDBR-CMUS219 | CBS 476.78 T | CBS 136361 T | UTHSC 04-838 T | UTHSC 08-1425 T | CBS 658.93 T | |
Creatine | + | + | + | + | + | + | + | + |
L-lysine | + | + | + | + | + | + | + | + |
Nitrate | + | + | + | + | + | + | + | + |
Nitrite | + | + | + | – | – | – | – | – |
L-tryptophan | + | + | + | + | + | + | + | + |
L-proline | + | + | + | + | + | + | + | + |
L-leucine | + | + | + | + | + | + | + | + |
L-ornithine | + | + | + | + | + | + | + | + |
L-cysteine | + | + | + | + | + | + | + | + |
Arginine | + | + | + | + | + | + | + | + |
2% NaCl | – | – | – | + | + | + | + | + |
5% NaCl | – | – | – | – | – | – | – | – |
7% NaCl | – | – | – | – | – | – | – | – |
10% NaCl | – | – | – | – | – | – | – | – |
2% MgCl2 | + | + | + | + | + | + | + | + |
Cycloheximide 0.1% | – | – | – | – | – | – | – | – |
aThis study, b
The topologies of each single-gene and the multi-gene (
Phylogenetic tree derived from maximum likelihood analysis of a combined
The percentage of nucleotide distances of
Mean percentage nucleotide
Number | Within species | 1 | 2 | 3 | 4 | 5 | |
---|---|---|---|---|---|---|---|
1 | 0.0 ± 0.00 | ||||||
2 | 1.15±0.31 | 4.53±0.43 | |||||
3 | 0.10±0.00 | 5.25±0.07 | 4.70±0.28 | ||||
4 | 0.55±0.26 | 4.96±0.05 | 5.85±0.24 | 5.95±0.13 | |||
5 | – | 15.60±0.00 | 16.30±0.00 | 15.30±0.00 | 16.10±0.00 | ||
6 | 0.10±0.00 | 4.56±0.26 | 6.18±0.17 | 5.75±0.21 | 3.00±0.10 | 16.75±0.38 |
The ability of
Solubilization of non-soluble minerals in agar media by
Solubilization index of the ability to solve non-soluble mineral by
For ‘
THAILAND. Chiang Mai Province: Mae Wang District, (
Distinguished from other
Colonies on
THAILAND. Chiang Mai Province: Mae Wang District, (
1 | Sporangiospores trapezoid, ellipsoid, subtriangular or claviform in shape |
|
– | Sporangiospores less variable in shape |
|
2 | Sporangiospores slightly trapezoidal to trapezoidal in shape |
|
– | Sporangiospores other shapes |
|
3 | Sporangiospores 2–3 µm wide |
|
– | Sporangiospores 3–5 µm wide |
|
4 | Apophyses cup-funnel shape, 8–15 µm long |
|
– | Apophyses funnel-shaped, 15–20 µm long |
|
5 | Sporangiospores bone-like in shape |
|
– | Sporangiospores ovoid, broadly ellipsoidal to barrel-shaped |
|
The present study identifies a new species of
Origin, isolation source and microscopic observation of
Origin | Isolation source | Microscopic observation | |||||
---|---|---|---|---|---|---|---|
Hyphae width (µm) | Sporangiophores (µm) | Sporangia (µm) | Apophyses shape / size (µm) | Sporangiospore shape / size (µm) | |||
|
India | Soil | 3.4–8 | 400–540 × 3.4–7.5 | 20–60 | Funnel to bell / 10–46 × 11–46 | Ovoid, broadly ellipsoidal to barrel-shaped / 5.4–12 × 3–8 |
|
Mexico | Human necrotic lesion | 3–5.5 | 100–700 × 3.5–7.0 | 25–30 | Cub-funnel / 12–20 × 8–15 | Slightly trapezoidal / 5–10 × 3–4 |
|
USA | Cellulitis of human leg wound | 3–5.5 | 100–400 × 2–3.5 | 15–50 | Funnel / 15–20 × 15–20 | Bone-like / 6–8 × 3–5.5 |
|
USA | Abdominal abscess of human | 3–5.5 | 400 × 2–3.5 | 15–50 | Funnel / 15–20 × 15–20 | Trapezoid / 5–8.5 × 3–5 |
|
Thailand | Soil | 5–15 | 60–890 × 3.75–7.5 | 25–58 | Funnel to bell / 21–52 × 19–46 | Slightly trapezoidal / 5–9 × 2–3 |
|
Netherlands | Osteomyelitis of human | 3–5.5 | 100–400 × 2–3.5 | 15–50 | Funnel / 15–20 × 15–20 | Trapezoid, ellipsoid, subtriangular or claviform / 5–14 × 3–6 |
a
Carbon assimilation profiles have been shown to be useful for differentiation of mucoralean genera (
In the phylogenetic analysis based on multi-gene sequences of combined
In the terrestrial environment, fungi play important roles in the biogeochemical cycling of elements (
In conclusion, the combination of morphological and physiological characteristics, and the molecular analysis strongly support our claim of a new fungus species. This discovery is considered important in terms of stimulating the investigations of soil fungi in Thailand and will help researchers to better understand the distribution and ecology of the genus
This work was supported by grants from Center of Excellence on Biodiversity (BDC), Office of Higher Education Commission (BDC-PG4-161008), Center of Excellence for Renewable Energy, and Center of Excellence in Microbial Diversity and Sustainable Utilization, Chiang Mai University, Chiang Mai, Thailand. We are grateful to Dr Eric McKenzie for proofreading the English.
Sporangiospores and apophyses observed on Czapek agar in culture.