J. Bio. & Env. Sci

Mt. Makiling Forest Reserve (MFR), a well-known forest in the Philippines, functions as an instruction, research and training laboratory for the advancement of scientific and technical knowledge on the protection, conservation and development of forests and other natural resources. However, data are scarce on the diversity of fungi in MFR as previous studies relied on characteristics of spores to enumerate fungal population. Sterile fungi have been ignored or discarded. In this study, 37 and 22 sterile fungi were isolated during the wet season and dry season, respectively, from the leaf litter of MFR. The sequences of the internal transcribed spacer (ITS) regions of the ribosomal RNA were used to determine the diversity of sterile fungi along 100 meters above sea level (masl) to 1000 masl altitudinal gradients of the forest. Based on the similarity of sequences at the 97% criterion, the fungal isolates were classified into 28 molecular operational taxonomic units (MOTUs). However, fungal diversity and species richness seemed to decrease with increasing altitude during dry and wet season. While analysis of a bigger fungal population is warranted, the results of this study suggest that fungal communities in MFR are not significantly affected by vegetation and abiotic factors such as temperature and precipitation. *Corresponding Author: R.B. Opulencia  rbopulencia@up.edu.ph Journal of Biodiversity and Environmental Sciences (JBES) ISSN: 2220-6663 (Print) 2222-3045 (Online) Vol. 7, No. 4, p. 43-50, 2015 http://www.innspub.net J. Bio. & Env. Sci. 2015 44 | Aborot et al. Introduction Mount Makiling, located 65 km southeast of Manila, Philippines, covers a total area of 5,907 ha and is considered as a relatively well conserved small mountain ecosystem within a highly diversified and urbanizing environment. Eighty percent of the whole Mt. Makiling is a reservation area set aside primarily as an instruction, research, and training laboratory, and includes the Mt. Makiling Forest Reserve (MFR) (Sargento et al., 1997). MFR, one of the most popular forests in the Philippines, is about 42.44 km2 and about 4, 244 ha (Combalicer et al., 2010). It has an annual mean temperature of 27°C, and Macolod clayloam type soil derived from volcanic tuff and ash. The soil pH ranges from 4.3 to 6.5 (Combalicer et al., 2010; Sargento et al., 1997). The topography of MFR is generally rugged with elevation ranging from 40 to 1100 meters above sea level (masl). MFR is home to a large number of biological species, several of which are considered threatened by the International Union for the Conservation of Nature. On October 2013, ASEAN declares MFR as 33rd ASEAN Heritage Park. Plant litter represents a major source of organic carbon in forest soils. Fungi are considered the key players in litter decomposition because they can produce a wide range of extracellular enzymes that can efficiently attack the recalcitrant lignocellulose matrix in plants (Osono, 2007). Fungi present in decomposing forest litter are believed to be among the most diverse mycological collection (Zak and Visser, 1996). Previous studies on fungal communities of leaf litter of MFR included identification using morphological features, which depends heavily on characteristics of spores and spore-bearing structures (unpublished data). However, few fungal species do not sporulate in natural habitat and when cultured in the laboratory, and are referred to as sterile fungi. “Sterile fungi” is considered a form group, not a taxonomic division, and is used as a matter of convenience (Hall, 1987; Shivanna et al., 1994; Vinod et al., 2012). Fungi that fail to sporulate in vitro are difficult, if not impossible, to identify, hence, have usually been avoided and neglected (Currah and Tsuneda, 1993), and there is no specific species concept that is applicable to them (Vinnere, 2004). Therefore, past studies on biodiversity might have underestimated the fungal flora in leaf litter of MFR. In this study, the diversity of sterile fungi from leaf litter of MFR was determined based on analysis of the sequences of the internal transcribed spacer (ITS) regions of the ribosomal RNA (rRNA). The ITS regions (ITS 1 and ITS 2) are non-coding but are involved in rRNA processing by forming specific secondary structures needed for recognition of cleavage sites and by providing binding sites for nucleolar proteins and RNAs during ribosome maturation (Hausner and Wang, 2005). However, the ITS regions vary in size and sequence that have been useful in determining interand intraspecies variability in fungal isolates (Iwen et al., 2002). The ITS region is now the preferred DNA barcoding marker both for the identification of single taxa and mixed environmental templates ('environmental DNA barcoding') (Bellemain et al., 2010). It has recently been proposed as the official primary barcoding marker for fungi (Schoch et al., 2012). Materials and methods Sample collection Leaf litter samples were collected from 100 masl to 1000 masl altitudinal gradients of MFR on November 2010 and May 2011 to represent wet and dry seasons, respectively. Litter samples included any aboveground leaves. Samples were collected every 100 masl, starting from 100 masl up to 1000 masl altitudinal gradients of MFR; hence, there were 10 sample collection sites. Each specific collection site was 20 m away from the main road or hiking trail. Within each site, three samplings were performed. Three areas, 10 m away from each other, were measured and marked. In each of the three areas, litter samples were collected in 10 points that were 1 m apart in a zigzag manner. Leaf litter samples from the 10 points of each of the three sampling sites were put together in a pail then mixed using a trowel. Consequently, three bags of leaf litter were obtained from each collection site or altitudinal gradient. Samples were placed in autoclavable plastic bags, J. Bio. & Env. Sci. 2015 45 | Aborot et al. sealed and labeled properly. Isolation and purification of filamentous fungi Fifty grams of leaf litter samples were first homogenized with 450 ml of 1% peptone using a sterile blender and then serially diluted using 1% peptone diluent. Each dilution was plated onto Potato Dextrose Agar (PDA) supplemented with streptomycin (100 ppm). Cultures were incubated at 30°C for 5-7 days. After incubation, filamentous colonies were picked from the plates and transferred on PDA that were also incubated at 30°C for 5-7 days. Successive transfers to PDA were performed until colonies had been purified as determined by cultural characteristics. Collection of sterile fungal isolates Slide culture technique was used for the observation of reproductive structures of fungal isolates. The slide culture setup that used PDA for agar block was incubated up to one month at room temperature (~30°C) and observed periodically under the microscope until sporulation had occurred. Those isolates that failed to produce spores after four weeks of incubation were arbitrarily declared as sterile fungi and collected for this study. Preparation of isolates for DNA extraction Hyphae were picked from a fresh culture of each isolate with the aid of dissecting microscope, and then inoculated in Potato Dextrose Broth (PDB) containing streptomycin (100 ppm). Liquid cultures were incubated on a shaker at 125 rpm at room temperature for 3-7 days. After incubation, the spherical-shaped mycelia were collected from the liquid medium by filtration using sterile filter paper. Genomic DNA extraction Genomic DNA was extracted from the isolates using the ZR Fungal/Bacterial DNA KitTM (Zymo Research, California, USA) by following the manufacturer’s instructions. Polymerase Chain Reaction Universal primers, ITS1 (5’TCCGTAGGTGAACCTGCGG-3’) and ITS4 (5’ TCCTCCGCTTATTGATATGC-3’) (White et al., 1990), were used to amplify the ITS regions. Amplification was performed in a 50 μl reaction containing 25 μl 2X Taq Master Mix (Vivantis Technologies, California, USA), 1.5 mM MgCl2, 0.3 μM of each primer, and 3 μl of template DNA. The thermal cycling program was run on MyCyclerTM Thermal Cycler (Bio-Rad Laboratories, Inc., California, USA) as follows: 3 minutes initial denaturation at 95°C, followed by 35 cycles of 40 seconds denaturation at 94°C, 50 seconds primer annealing at 52°C, 1 minute extension at 72°C, and a final 10 minutes extension at 72°C (Lacap et al., 2003). DNA Sequencing The PCR products were sent to Macrogen, Inc., (Seoul, South Korea) for purification and sequencing. Both strands of the DNA molecule were sequenced using the universal primers ITS1 and ITS4. Sequence analysis Forward and reverse sequences from Macrogen, Inc. of each isolate were aligned using ClustalW2 (Larkin et al., 2007) to obtain overlapping sequences into consensus sequence. Overhang sequences and ambiguous nucleotides were excluded using ChromasPro software (ChromasPro ver. 1.3). The sequence of each isolate was used as query sequence to search for the most similar reference sequences in GenBank using Basic Local Alignment Tool (BLAST) program (Altschul et al., 1990). To reveal the general taxonomic placement of the unknown fungal isolates, reference sequences with 97-100% similarity were considered for multiple sequence alignment in ClustalW in MEGA 6 software. Test for the best-fit substitution model was performed to select for the most suitable model for phylogenetic tree analysis (Posada and Crandall, 2001). Thus, maximum likelihood was used to infer phylogenetic tree using Kimura 2-parameter with gamma distribution as the suitable substitution model. Genetic distance was considered to determine the molecular operational J. Bio. & Env. Sci. 2015 46 | Aborot et al. taxonomic unit (MOTU) of the sequences. The MOTUs were group of sequences that were most similar to each other; i.e, for which the genetic distance was the smallest (Van de Peer, 2009). The sequences with similar smallest genetic distance values were clustered tog