Pertanika J. Trop. Agric. Sc. 42 (2): 735 - 743 (2019) TROPICAL AGRICULTURAL SCIENCE Journal homepage: http://www.pertanika.upm.edu.my/ Short Communication Identification of Microfungi Isolated from Belian Fruits Siti Fatimah Md-Isa, Nur Ain Izzati Mohd-Zainuddin, Christina Seok Yien Yong and Rusea Go* Department of Biology, Faculty of Science, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia ABSTRACT Belian tree (Eusideroxylon zwageri Teijsm. & Binn.) is one of the highly demanded commercial timber tree indigenous to the Southeast Asian region. The tree is threatened with over exploitation; habitat destruction and slow regeneration. While vascular plants are known as major reservoirs of fungi species, there have been no studies to identify the microfungi isolated from fruits of this endemic tree. By using internal transcribed spacers (ITS) sequence analysis, five genera were isolated and identified as Annulohypoxylon, Daldinia, Hypoxylon, Lasiodiplodia and Trichoderma. This result will be primarily used as baseline data for further investigations on microfungi diversity associated with Belian tree. Keywords: Annulohypoxylon, Borneo ironwood, Daldinia, Eusideroxylon zwageri, Hypoxylon, Lauraceae, Lasiodiplodia, Trichoderma INTRODUCTION of individual fungi in nature is unknown Fungi plays an important role in the optimal (Mueller & Bill, 2004). Apart from being functioning of the ecosystem. In general, an important contributors to the diversity they involved in the breakdown of nutrients of organisms in the forest, microfungi on the forest floor, which for ready uptake have been consistently utilized in the drug by the plants but in most cases the role production, food processing, bio-control agents as well as in enzyme biotechnolgy (Kumar et al., 2013; Snaddon et al., 2012). ARTICLE INFO A study on microfungi associated Article history: Received: 29 October 2018 with endemic plants in Mauritius reported Accepted: 04 January 2019 more than 200 taxa of microfungi with Published: 30 May 2019 E-mail addresses: approximately 90% of being new genus [email protected] (Siti Fatimah Md-Isa) (Dulymamode et al., 2001) while study in [email protected] (Nur Ain Izzati Mohd-Zainuddin) [email protected] (Christina Seok Yien Yong) Kenya reported four fungal taxa were new to [email protected] (Rusea Go) * Corresponding author science detected from their endemic and rare ISSN: 1511-3701 e-ISSN: 2231-8542 © Universiti Putra Malaysia Press Siti Fatimah Md-Isa, Nur Ain Izzati Mohd-Zainuddin, Christina Seok Yien Yong and Rusea Go plants (Siboe et al., 2000). This is shown 2017 from Semenggoh Reserved Forest that, many unknown microfungi are likely in Sarawak, Malaysia. The fruit samples to be rare and threatened to the same degree were collected on the forest floor near to as their plant host. the mother tree and were kept in brown However, very little is known and paper bags, labeled and transported to the reported on the microfungi diversity isolated laboratory for fungi isolation. from the endemic and endangered trees in Malaysia. Belian tree, scientifically known Isolation of Microfungi as Eusideroxylon zwageri Teijsm. & Binn. The fruits were washed under tap water is one of the most important commercial and were surface sterilized with 70% timber trees indigenous to Southeast Asian ethanol solution for 5 minutes, and then, the forests. It is native to Malaysia (Sarawak fruits were rinsed with sterilized distilled and Sabah), Brunei, Indonesia and The water for three times and blot-dried using Philippines. The tree is taxonomically sterilized filter paper (Kouame et al., 2010). classified in the Lauraceae family and The pericarp layer of the fruit samples listed as a vulnerable species by the IUCN was divided into three parts: tip, middle Global Red List of Threatened Species and bottom of the fruits to get an overall (version 2017.3) (International Union for estimation of the fungi reside in the pericarp Conservation of Nature [IUCN], 2017) part of the fruits. Then it was aseptically cut resulting from the over exploitation and into 1 cm2 using sterile surgical blades and habitat destruction. To date, only one study carefully transferred onto potato dextrose of the microfungal diversity on the green agar (PDA) plate supplemented with and leaf litters of Belian tree from Kubah Streptomycin (20 mL/L of medium). National Park, Sarawak has been reported The plates were sealed and labeled by (Lateef et al., 2015). fruits sample part, initial culturing date This study therefore, is aimed to identify and incubated at 28±2oC. Observation and microfungi community isolated from isolation of the growing microfungi on Belian fruits which will add value to the the surface of the media plates, colonies latest knowledge on Belian tree especially that differ in time of appearance, size, related to the diversity of microfungal. This color, and elevation were recorded. A loop information will aid further study on their of each original pure fungi colony was numerous roles with respect to Belian tree. picked up using plating technique, and re- plated onto a solid pure culture agar plates MATERIALS AND METHODS containing the same culture media but Materials without antibiotics. The plates were sealed, Ten available matured fruits were collected labeled and incubated until the fungi grew randomly under the adult Belian tree in May to half of the plate. 736 Pertanika J. Trop. Agric. Sc. 42 (2): 735 - 743 (2019) Microfungi Isolated from Belian Fruits The hyphae tip technique was performed were separated by electrophoresis in 1.5% to obtain pure culture isolate (Leslie & agarose gel in 1.0x Tris-boric acid-EDTA Summerell, 2006). Then the pure fungi (TBE) buffer with Etb “Out” Staining isolates were transferred to PDA slants for Solution (YEASTERN BIOTECH Co. short-term storage or in complete media Ltd) and photographed under UV light. xylose (CMX) 25% glycerol at -80oC for The images were captured with DOC long-term preservation. PRINT system (Vilber Lourmat, USA). A good quality of amplified PCR products DNA Extraction and PCR Amplification was sequenced in both directions using an Total genomic DNA were extracted from Applied Biosystems 3730xl DNA Analyzer ten successful pure cultured isolates by MyTACG Bioscience Company, MY. using an Ultra Clean ® Microbial DNA isolation kit (MO-BIO, Carlsbad, CA, Data Analysis USA) according to manufacturer’s ClustalW in Molecular Evolutionary Genetic instructions. Oligonucleotide primers ITS1 Analysis (MEGA 7) was used to generate (5’-TCCGTAGGTGAACCTGCGG- 3’) and aligned the consensus sequences. The and ITS4 (5’-TCCTCCGCTTATTGATA consensus sequences were search against TGC-3’) (White et al., 1990) were used to sequences in GenBank using Basic Local amplify internal transcribed spacer (ITS) Alignment Search Tool (BLAST) (http:// region of the nuclear ribosomal DNA. Each www.ncbi.nlm.gov) to determine the closest polymerase chain reaction (PCR) contained matched ITS sequence from the database. 5 uL of 5X PCR buffer, 4 uL of 25 mM All the assembled DNA sequences were MgCl2, 0.5 uL of 10 mM dNTPs (Promega), deposited in GenBank. 0.5 uL of each 10uM primer, 0.1uL of 5 A Maximum Likelihood analysis units/uL GoTaq® DNA polymerase, 0.5 was conducted using MEGA 7 with 1000 uL of 10X BSA and 2.0 uL of template bootstrap replication value based on Tamura- DNA adjusted with nuclease free water Nei Model (Kumar et al., 2016). Branches to a final volume of 50uL. Amplification corresponding to partitions reproduced in reactions were carried out in a thermal less than 50% bootstrap replicated were cycler (Eppendorf AG 22331, Hamburg). collapsed. The representative sequences of PCR cycling protocol was as follows: an each species were obtained from GenBank initial pre-denaturation for 2 min at 95ºC, and were included in the tree. The tree was followed by 35 cycles of denaturation at rooted with Puccinia graminis (HM131358) 95ºC for 20 s, annealing at 50ºC for 30 as an out-group for this phylogenetic tree. s, and extension at 72ºC for 30 s; with a The tree was constructed to comprehend the single cycle of final extension at 72ºC for placement of each isolates and the reference 5 min. The PCR amplification products sequences in the lineage. Pertanika J. Trop. Agric. Sc. 42 (2): 735 - 743 (2019) 737 Siti Fatimah Md-Isa, Nur Ain Izzati Mohd-Zainuddin, Christina Seok Yien Yong and Rusea Go RESULTS The phylogenetic tree was divided The phylogentic tree was constructed into two main clades; I and II (Figure 1). to understand the placement among the Main clade I clustered all species under isolates and the reference sequence (Figure Class Sordariomycetes while main clade 1) and colony features for each isolates II grouped Class Dothideomycetes. Main were shown in Figure 2. All fungi isolates clade I was further divided into subclades were identified belong to the phylum A and B which clustered the species into Ascomycota, which comprised nine taxa their specific order. Subclade A nested all from five genera; Annulohypoxylon (2 species under Xylariales order and subclade species); Daldinia (1 species); Hypoxylon B grouped Hypocreales species. (1 species); Lasiodiplodia (1 species); and Subclade A contained three genera from Trichoderma (4 species) (Table 1). The Xylariaceae family; Annulohypoxylon, molecular fragment size of ITS region Daldinia and Hypoxylon. All the isolates are amplified was between 500 – 600 bp except highly similar to their respective reference for ITS fragment of Annulohypoxylon sequences from GenBank and well supported species which was > 800 bp. Based on with 100% bootstrap value. Isolate Q2687 BLAST search, the ITS sequences showed was identified as Annulohypoxylon nitens high percentages similarities (99% and (KU684021) while isolate Q2688 was 100%) with sequences in the GenBank identified asAnnulohypoxylon viridistratum database (http://www.ncbi.nlm.gov). All (KX376325) both with 99% similarity. ITS sequences of the isolates were deposited Colony of A. nitens was observed with into GenBank and the accession number are filamentous white mycelium. The reverse listed in Table 1.