Apophysomycesthailandensis (Mucorales, Mucoromycota), a new species isolated from soil in northern Thailand and its solubilization of non-soluble minerals

Abstract A new species of soil fungi, described herein as Apophysomycesthailandensis, was isolated from soil in Chiang Mai Province, Thailand. Morphologically, this species was distinguished from previously described Apophysomyces species by its narrower trapezoidal sporangiospores. A physiological determination showed that A.thailandensis differs from other Apophysomyces species by its assimilation of D-turanose, D-tagatose, D-fucose, L-fucose, and nitrite. A phylogenetic analysis, performed using combined internal transcribed spacers (ITS), the large subunit (LSU) of ribosomal DNA (rDNA) regions, and a part of the histone 3 (H3) gene, lends support to our the finding that A.thailandensis is distinct from other Apophysomyces species. The genetic distance analysis of the ITS sequence supports A.thailandensis as a new fungal species. A full description, illustrations, phylogenetic tree, and taxonomic key to the new species are provided. Its metal minerals solubilization ability is reported.

During the isolation of non-soluble mineral solubilizing fungi from agricultural soil in northern Thailand, we found a particular population of Apophysomyces which we describe here as a new species based on morphological, molecular, and physiological characteristics. To confirm its taxonomic status, the phylogenetic relationship was determined by analysis of the combined sequence dataset of the ITS and LSU of rDNA, and part of the histone 3 gene.

Fungal isolation
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 MgSO 4 •7H 2 O, 0.1 g CaCO 3 , 0.005 g FeCl 3 , 2.0 g Ca 3 PO 4 , 3.0 g K 2 HPO 4 , 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.

Morphological studies and growth observation
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.

Physiological studies
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 × 10 5 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% MgCl 2 and 0.1% cycloheximide was determined. All tests were performed in three replicates.

Molecular studies
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: 943-1620LSU: 943- , and H3: 1621LSU: 943- -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.

The non-soluble minerals solubilization ability
This experiment was carried out using basal medium (10.0 g glucose, 0.5 g (NH) 4 SO 4 , 0.2 g NaCl, 0.1 g MgSO 4 •7H 2 O, 0.2 g KCl, 0.5 g yeast extract, 0.002g MnSO 4 •H 2 O, and 15.0 g agar per liter of deionized water, pH 7.0) with addition of non-soluble metal minerals including Ca 3 (PO 4 ) 2 , CaCO 3 , CuCO 3 •Cu(OH) 2 , CuO, CoCO 3 , FePO 4 , MgCO 3 , MnO, ZnCO 3 , ZnO, feldspar (KAlSi 3 O 8 ), and kaolin (Al 2 Si 2 O 5 (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.

Statistical analysis
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.

Growth observation and physiological studies
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, Dmannitol, 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, MgCl 2 , 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,  Table 3. Carbon assimilation profiles for Apophysomyces species obtained with API 50 CH strips.

Phylogenetic results
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)

Metal minerals solubilization ability
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 CaCO 3 , Ca 3 (PO 4 ) 2 , CuCO 3 •Cu(OH) 2 , CuO, ZnCO 3 , 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 CoCO 3 , FePO 4 , MnO, feldspar, and kaolin. Etymology. For 'thailandensis', referring to Thailand, where soil containing the new fungus was collected.
In the phylogenetic analysis based on multi-gene sequences of combined ITS, LSU, and the histone 3 gene, A. thailandensis formed a monophyletic clade, separate from the other Apophysomyces species. The ITS (ITS1+5.8S+ITS2) genetic distance between A. thailandensis and other Apophysomyces species ranged from 4.53% to 15.60% (Table 5). This genetic distance of ITS was greater than 3%, which is sufficient to indicate a new fungal species (Leavitt et al. 2013;Nilsson et al. 2008).
In the terrestrial environment, fungi play important roles in the biogeochemical cycling of elements (Gadd 2017;Frąc et al. 2018). Soil fungi can mobilize and solubi-  Alvarez et al. 2010, b Misra et al. (1979, c Bonifaz et al. (2014) and d This study.
lize non-soluble minerals into forms available for cellular uptake and leaching from the system, e.g. complexation with organic acid, other metabolites and siderophores (Gadd 2010;Mapelli et al. 2012). In this study, pure cultures of A. thailandensis were able to solubilize different non-soluble minerals (Ca, Co, Cu, Fe, Mn, and Zn-containing minerals), and the solubilization demonstrated very different activities for the different minerals. This is similar to previous studies that reported other mucoralean genera (e.g. Absidia, Cunninghamella, Mucor, and Rhizopus) isolated from soils are able to solubilize non-soluble minerals (Ca, Fe, Mg and Zn-containing minerals) (Arrieta and Grez 1971;Kolo and Claeys 2005;Akintokun et al. 2007;Nenwani et al. 2010;Sharma et al. 2013;Patel et al. 2015;Alori et al. 2017;Ceci et al. 2018). This is the first report describing non-soluble mineral solubilization ability by the genus Apophysomyces.
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 Apophysomyces.