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 (PDA; CONDA, Spain), Czapek agar (CZA; Difco, France), and malt extract agar (MEA; Difco, France) was observed after 5 d of incubation in darkness at 37 °C. Three replicates were made in each medium. The colony diameter was measured. Micromorphological features were examined using a light microscope (Olympus CX51, Japan) following the methods described by Alvarez et al. (2010). The anatomical features were from at least 50 measurements of each structure.

Carbon source assimilation profiles were determined with the API 50CH commercial kit (bioMérieux, France), following the methods described by Schwarz et al. (2007). To obtain sufficient sporulation, all isolates were cultured for 1 week on CZA at 37 °C. A final concentration of 5 × 10⁵ spores/ml was prepared in 20 ml of yeast nitrogen base containing 0.5 g/l of chloramphenicol and 0.1% Bacto agar, and each well of the strips was inoculated with 300 µl of the spore containing medium. The inoculated API 50CH strips were incubated for 48–72 h at 37 °C in darkness. After incubation, the strips were read visually and growth or lack of growth was noted. Weak growth was considered as a positive result.

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 CZA was extracted using the fungal Genomic DNA Extraction Mini Kit (FAVOGEN, Taiwan). The ITS region of DNA was amplified by polymerase chain reactions (PCR) using ITS4 and ITS5 primers (White et al. 1990), the LSU of rDNA gene were amplified with NL1 and NL4 primers (Kurtzman and Robnett 1998), and histone 3 (H3) gene was amplified with the H3-1a and H3-1b primers (Glass and Donaldson 1995). The amplification program for these three domains were performed in separated PCR reaction and consisted of an initial denaturation at 95 °C for 5 min, followed by 35 cycles of denaturation at 95 °C for 30 s, annealing at 52 °C for 30 s (ITS); 52 °C for 45 s (LSU), and 54 °C for 1 min (H3), and extension at 72 °C for 1 min. Negative controls lacking fungal DNA were run for each experiment to check for any contamination of the reagents. PCR products were checked on 1% agarose gels stained with ethidium bromide under UV light and purified using NucleoSpin Gel and PCR Clean-up Kit (Macherey-Nagel, Germany). The purified PCR products were directly sequenced. Sequencing reactions were performed and sequences were automatically determined in a genetic analyzer at 1^st Base Company (Kembangan, Malaysia) using the same PCR primers mentioned above. Sequences were used to query GenBank via BLAST (http://blast.ncbi.nlm.nih.gov).

Details of the sequences used for phylogenetic analysis obtained from this study and from previous studies are provided in Table 1. The multiple sequence alignment was carried out using MUSCLE (Edgar 2004), and a combined ITS, LSU, and H3 alignments were deposited in TreeBASE under the study ID 23168. The combined ITS, LSU and H3 sequences dataset consisted of 28 taxa and the aligned dataset comprised 1991 characters including gaps (ITS: 1–942, LSU: 943–1620, and H3: 1621–1991). A maximum likelihood (ML) phylogenetic tree was constructed using RAxML v. 7.0.3 (Stamatakis 2006), applying the rapid bootstrapping algorithm for 1000 replications. Saksenaea vasiformis ATCC 60625 and S. erythrospora UTHSC 08-3606 were used as the outgroup. The ML trees were plotted with TreeView32 (Page 2001). Clades with bootstrap values (BS) ≥ 70% were considered as significantly supported (Hillis and Bull 1993). The best-fit substitution model for Bayesian inference algorithm was estimated by jModeltest v. 2.1.10 (Darriba et al. 2012) using Akaike information criterion. Bayesian phylogenetic analyses were carried out using the Metropolis-coupled Markov chain Monte Carlo (MCMCMC) method in MrBayes v. 3.2 (Ronquist et al. 2012), under a GRT+I+G model. Markov chains were run for one million generations, with six chains and random starting trees. The chains were sampled every 100 generations. Among these, the first 2000 trees were discarded as the burn-in phase of each analysis and the resulting trees were used to calculate Bayesian posterior probabilities. Bayesian posterior probabilities (PP) ≥ 0.95 were considered as a significant support (Alfaro et al. 2003). Pairwise genetic distances (proportions of variable sites) within and between five Apophysomyces species were computed using MEGA v. 6 (Tamura et al. 2013), with pairwise deletion of gaps and missing data.

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 Fomina et al. (2005). The medium was autoclaved at 121 °C for 15 min. After autoclaving, for each experiment, 25 ml of test media was poured into Petri dishes. Mycelial inocula were prepared by growing the fungus on CZA at 30 °C in darkness for 7 d. Mycelial plugs (5 mm in diameter) from the periphery of the growing colony were then used to inoculate the center of the tested media. All plates were incubated at 30 °C in darkness for 4 d. Colony diameter and solubilization zone (halo zone) were measured. Solubilization index (SI) was calculated as the halo zone diameter divided by the fungal colony diameter (Vitorino et al. 2012, Kumla et al. 2014). SI values of less than 1.0, between 1.0 and 2.0, and more than 2.0 were regarded as low, medium, and high solubilization activities, respectively. Three replications were made in each treatment.

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 (P <0.05) between treatments.

Mycelial growth of the three A. thailandensis isolates on three different agar media and at different temperatures is presented in Table 2. PDA promoted the best mycelial growth followed by CZA, and MEA. All isolates grew at temperatures ranging from 20–42 °C. The highest growth rate was observed on PDA at 37 °C.

Carbon assimilation profiles of the three strains of A. thailandensis are shown in Table 3. Assimilation patterns of all strains were positive for 23 carbon sources (amidon, D-adonitol, D-arabitol, D-fructose, D-fucose, D-glucose, D-lyxose, D-maltose, D-mannitol, D-mannose, D-melezitose, D-ribose, D-sorbitol, D-tagatose, D-trehalose, D-turanose, D-xylose, glycerol, glycogen, L-arabinose, L-fucose, N-acetyl-glucosamine and xylitol). Variability in nitrogen assimilation and tolerance to NaCl, MgCl₂, and cycloheximide of the three strains of A. thailandensis are presented in Table 4. All strains were positive for 10 nitrogen sources (arginine, creatine, L-cysteine, L-leucine, L-lysine, L-ornithine, L-proline, L-tryptophan, nitrate and nitrite). All strains were able to grow on 2% MgCl₂, but could not grow on 2%, 5%, 7%, and 10% NaCl, and on 0.1% cycloheximide.

The topologies of each single-gene and the multi-gene (ITS, LSU, and H3 genes) trees were similar. Therefore, we show only the multi-gene tree (Fig. 1). Our phylogenetic analysis separated Apophysomyces into three main clades. Clade I contained two species (A. variabilis and A. elegans). Apophysomyces trapeziformis, A. mexicanus, and A. ossiformis were assigned to clade II. Apophysomyces thailandensis was clearly separated from the other Apophysomyces species and formed a separate monophyletic clade (clade III) with high BS (100%) and PP (1.0) support.

Figure 1. 

Phylogenetic tree derived from maximum likelihood analysis of a combined ITS, LSU, and H3 genes of 28 sequences. Saksenaea vasiformis and S. erythrospora were used as outgroup. Numbers above branches are the bootstrap statistics percentages (left) and Bayesian posterior probabilities (right). Branches with bootstrap values ≥ 50% are shown at each branch and the bar represents 0.1 substitutions per nucleotide position. The fungal isolates from this study are in bold. Superscript T = type species.

The percentage of nucleotide distances of ITS (ITS1+5.8S+ITS2) sequence between A. thailandensis and other Apophysomyces species is shown in Table 5. The percentage nucleotide distance of A. thailandensis ranged from 4.53–15.60% from other Apophysomyces species.

The ability of A. thailandensis to solubilize metal minerals depended on the type of minerals and strain. In some cases, A. thailandensis produced a solubilization zone in agar that was larger than the fungal colonies (Fig. 2A–D), while in other cases the solubilization zones were found beneath the fungal colonies (Fig. 2E–H). The solubilization activities were expressed in terms of a solubilization index (SI) and are shown in Figure 3. The solubilization activity of all A. thailandensis strains in the presence of CaCO₃, Ca₃(PO₄)₂, CuCO₃•Cu(OH)₂, CuO, ZnCO₃, and ZnO was characterized as medium (SI value between 1.0 and 2.0) activity. All strains showed a low solubilization activity (SI value less than 1.0) for CoCO₃, FePO₄, MnO, feldspar, and kaolin.

Figure 2. 

Solubilization of non-soluble minerals in agar media by Apophysomyces thailandensis SDBR-CMUS26 (holotype). A Ca₃(PO₄)₂B CuCO₃•Cu(OH)₂C CuO D ZnCO₃E FePO₄F MnO G Feldspar H Kaolin. Scale bars: 10 mm. Fungal colonies in E and F were cut for the solubilization area (halo zone) observation.

Figure 3. 

Solubilization index of the ability to solve non-soluble mineral by Apophysomyces thailandensis. Data are means of three replicates. Error bar at each point indicates ± SD. Different letters above each graph indicate that the means are significantly different by Tukey’s test (P < 0.05)