Front. Microbiol. Frontiers in Microbiology Front. Microbiol. 1664-302X Frontiers Media S.A. 10.3389/fmicb.2022.887880 Microbiology Original Research Diversity of endophytic fungi isolated from different plant parts of Acacia mangium, and antagonistic activity against Ceratocystis fimbriata, a causal agent of Ceratocystis wilt disease of A. mangium in Malaysia Ahmad Mohd Farid 1 Zahari Rozihawati 2 Mohtar Mastura 3 Wan-Muhammad-Azrul Wan Azhar 4 Hishamuddin Muhammad Syahmi 2 Samsudin Nik Iskandar Putra 5 6 Hassan Affendy 7 Terhem Razak 2 * 1Mycology and Pathology Unit, Forest Research Institute Malaysia, Kepong, Malaysia 2Laboratory of Forest Pathology and Tree Health, Department of Forestry Science and Biodiversity, Faculty of Forestry and Environment, Universiti Putra Malaysia (UPM), Serdang, Malaysia 3Bio Activity Programme, Natural Products Division, Forest Research Institute Malaysia, Kepong, Malaysia 4Pest and Disease Management Programme, Horticulture Research Centre, Malaysian Agriculture Research and Development (MARDI), Persiaran Mardi-UPM, Serdang, Malaysia 5Department of Food Science, Faculty of Food Science and Technology, Universiti Putra Malaysia (UPM), Serdang, Malaysia 6Laboratory of Food Safety and Food Integrity, Institute of Tropical Agriculture and Food Security, Universiti Putra Malaysia (UPM), Serdang, Selangor, Malaysia 7Faculty of Tropical Forestry, Universiti Malaysia Sabah, Kota Kinabalu, Sabah, Malaysia

Edited by: Giuseppe Ianiri, University of Molise, Italy

Reviewed by: Laith Khalil Tawfeeq Al-Ani, Universiti Sains Malaysia, Malaysia; Samantha Chandranath Karunarathna, Qujing Normal University, China

 *Correspondence: Razak Terhem, razakterhem@upm.edu.my

This article was submitted to Microbe and Virus Interactions with Plants, a section of the journal Frontiers in Microbiology

 08 11 2022 2022 13 887880 02 03 2022 04 10 2022 Copyright © 2022 Ahmad, Zahari, Mohtar, Wan-Muhammad-Azrul, Hishamuddin, Samsudin, Hassan and Terhem. 2022 Ahmad, Zahari, Mohtar, Wan-Muhammad-Azrul, Hishamuddin, Samsudin, Hassan and Terhem

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Acacia mangium is an important wood for commercial products especially pulp and medium-density fibreboard. However, it is susceptible to Ceratocystis fimbriata infection, leading to Ceratocystis wilt. Therefore, the present work aimed to (i) establish the diversity of endophytic fungi in different plant parts of A. mangium,and (ii) evaluate the antifungal potentials of the isolated and identified endophytic fungi against C. fimbriata. Endophytic fungal identification was conducted by PCR amplification and sequencing of the internal transcribed spacer 1 (ITS1) and ITS4 regions of nuclear ribosomal DNA. A total of 66 endophytic fungi were successfully isolated from different parts of A. mangium; leaf (21), stem (13), petiole (12), root (9), flower (6), and fruit (5). The endophytic fungal isolates belonged to Ascomycota (95.5%) and Zygomycota (4.5%). For Ascomycota 13 genera were identified: Trichoderma (28.6%), Nigrospora (28.6%), Pestalotiopsis (12.7%), Lasiodiplodia (9.5%), Aspergillus (6.3%), Sordariomycetes (3%), and Neopestalotiopsis, Pseudopestalotiopsis, Eutiarosporella, Curvularia, Fusarium, Penicillium, and Hypoxylon each with a single isolate. For Zygomycota, only Blakeslea sp. (5%) was isolated. Against C. fimbriata, Trichoderma koningiopsis (AC 1S) from stem, Nigrospora oryzae (AC 7L) from leaf, Nigrospora sphaerica (AC 3F) from the flower, Lasiodiplodia sp. (AC 2 U) from fruit, Nigrospora sphaerica (AC 4P) from petiole, and Trichoderma sp. (AC 9R) from root exhibited strong inhibition for C. fimbriata between 58.33 to 69.23%. Thus, it can be concluded that certain endophytic fungi of A. mangium have the potential to be harnessed as anti-Ceratocystis agent in future biotechnological applications.

 Acacia mangium endophytic fungi Ceratocystis fimbriata Ceratocystis wilt antagonism GP-IPM/2017/9565600 Universiti Putra Malaysia10.13039/501100004530 Introduction

Acacia mangium Willd., a fast-growing and flowering leguminous tree native to Indonesia, Papua New Guinea, and Australia, has been introduced and cultivated into humid tropical lowland regions of Asia, South America, and Africa (Pinyopusarerk et al., 1993). In 1966, forest plantation of A. mangium began in Sabah, Malaysia, pioneered by D.I. Nicholson, an Australian forester. Commercial cultivation of A. mangium began in 1976 (Udarbe and Hepburn, 1986; Pinyopusarerk et al., 1993). The species was considered promising due to its stellar performance, superior growth, and multiple uses especially for pulp and medium-density fibreboard (Potter et al., 2006). Furthermore, pharmacological studies have also shown that the leaves of A. mangium exhibit antibacterial (Sarah Shafiei et al., 2017), antifungal (Mihara et al., 2005), antifilarial, and antihelmintic (Chaki et al., 2015) properties.

Despite its various commercial applications, A. mangium is susceptible to the infection of the ascomycetous pathogen, Ceratocystis fimbriata, which infects the wounds of A. mangium trees in plantations, and causes the Ceratocystis wilt disease (Kile, 1993; Roux and Wingfield, 2009; Tarigan et al., 2011; Brawner et al., 2015). Wounded tree caused by humans, other mammals including monkeys, elephants, squirrels or boring insects, and others factor such as wind, are likely to increase the disease spreading and tree mortality as the wound become the entrance for this Ceratocystis species to invade (Nasution et al., 2019). In Malaysia at the year of 2011, a severe case which was the first report of this disease infected approximately 40% of A. mangium trees in plantation at Tawau, Sabah. Later, this disease spreads to other regions on A. mangium plantation in Sabah such as Pitas, Kota Belud and Sipitang, where the incidence of this disease were about in range of 6–60% (Mandy and Wickneswari, 2014; Farid et al., 2018). Johor, Pahang and Sarawak were also reported faced the same disease problems to the A. mangium plantation in respective state. 50% out of 1,500 trees that were accessed in a 2-year-old Acacia mangium plantation in Johor have been infected by this disease (Farid et al., 2018). The main reason of this disease spreading and uncontrollable was due to lack of knowledge, researches and studies on how to overcome or prevent this disease to happen towards Acacia mangium trees (Lee, 2018).

There are no specific methods or guidelines established on how to handle this disease in Malaysia yet up to now. But there were several actions that commonly are used by the plantation managers to prevent the infection of this disease. As Ceratocystis species penetrate and invade the trees by wounds, this problems can be prevent by avoid the occurance of wound itself (Kile, 1993; Harrington, 2013; Nasution et al., 2019). Silviculture practice should be done in correct way and cautions. The timing of doing work for silviculture is also important to reduce the risk of disease development (Pilotti et al., 2016; Farid et al., 2018). Problems involved with wildlife in plantation areas also are count on in management such as establishment of wildlife management plan to overcome the conflicts occurred (Farid et al., 2018). Chemical control is one of application they used to delay the symptoms of the disease development and help the infected trees to live longer for at least 2 years (Blaedow, 2009; Nasution et al., 2019). Although the use of chemical fungicides are more preferred due to their rapid action, they are often associated with high production and application costs, human health hazards, restriction by domestic and international regulatory limits, trade bans, residual effects, environmental pollution, resistance development in pests, and potential elimination of beneficial natural enemies of the targeted pests (Yazid et al., 2020). Therefore, biological control is seen as a safer and cheaper alternative. Biological control is the use of living organisms (including microorganisms) to eliminate or reduce the density of pests / pathogens to safe levels (Wyckhuys et al., 2013). Often, indigenous organisms or microorganisms are utilised as biological control agent to minimise the risk of introducing foreign species that might grow uncontrollably and in turn become invasive. One such example of indigenous organisms or microorganisms is endophyte. The research is about using a microorganism (endophyte) to fight the pathogen (Ceratocystis fimbriata) which is one of biological control.

Like many other plant species, A. mangium is also associated with endophytes. Endophytes are usually bacteria or fungi that endosynbiotically live within a plant host without causing disease. These endophytes function to enhance the plant host growth and nutrient acquisition improve the plant host’s ability to tolerate abiotic stresses or decrease biotic stresses by enhancing the plant host’s resistance to infections (Farahat, 2020). Recently, an endophytic actinomycete of the genus Fodinicola was isolated from the roots of A. mangium, and has shown potential activity as a beneficial plant-growth promoter and specialised secondary metabolite producer (Phạm et al., 2020).

Despite endophytic fungi being regarded as new sources of novel bioactive compounds (Daouk et al., 1995; Cui et al., 2015), biological activities, and biotechnological developments, their true potential in controlling A. mangium diseases caused by C. fimbriata remains underexplored and underreported. Moreover, the leaf and root parts of A. mangium have been found to provide the habitats for various endophytic fungi (Mihara et al., 2005; Sarah Shafiei et al., 2017; Phạm et al., 2020). Nevertheless, besides leaf and root, other plant parts of the species should also be explored for endophytic fungi which might offer novel species or strains that possess valuable bioactive compounds useful in controlling the Ceratocystis wilt disease. Therefore, the objectives of the present work were (i) to establish the diversity of endophytic fungi in different plant parts of A. mangium, and (ii) to evaluate the antifungal potentials of the isolated and identified endophytic fungi against C. fimbriata.

 Materials and methods Plant materials

Ten seedlings of Acacia mangium (≈30–50 cm in height) and 2 A. mangium trees (≈30 cm in diameter at breast height) free from disease and insect infestation were randomly sampled, and identified at Serdang, Selangor (coordinate E 101○ 42.6333 N 2○ 59.1833). The root, stem, petiole, and leaf from healthy A. mangium seedlings were sampled in three replicates, respectively. In addition, three replicates of flower and fruit were also sampled from mature trees, respectively. Each plant part was cut into five 0.5 cm2 segments using a blade. These plant parts were washed thoroughly under running tap water to remove adherent debris on the surface.

 Isolation of endophytic fungi

Plant part segments were surface-sterilised following the protocol suggested by Nuangmek et al. (2021). Briefly, the plant part segments were washed thoroughly under running tap water, immersed in 70% ethanol (Cerilliant Corporation, United States) for 1 min, soaked in 4% NaOCl (Malay-Sino Chemical, Malaysia) for 1 min, rinsed thrice in sterile distilled water, and blot-dried using a sterile filter paper. Next, the surface-sterilised plant part segments were excised 1–2 mm from the edge, and explant-plated onto a Potato Dextrose Agar (PDA; Merck Milipore, Germany). The PDA plates were incubated at 27°C for 7 d. Single hyphae growing out from the cultivated plant part segments were sub-cultured onto fresh PDA. Pure cultures were grouped according to the six types of plant parts (root, stem, petiole, leaf, flower, and fruit). Isolates were group based on colour and morphology on PDA (Yoo and Eom, 2012). Cultures were maintained on PDA for 5 d before sub-cultured into Potato Dextrose Broth (PDB; Neogen®, United States) while shaken at 150 rpm at 26°C for 3–6 d. Following incubation, the culture supernatant was filtered through Whatman filter paper (Cytiva™ Sigma-Aldrich Chemie GmbH, Germany) before being used for genomic DNA extraction.

 DNA extraction and PCR amplification

A total of 100 mg of fungal mycelia harvested from PDB was used for fungal genomic DNA extraction. Fungal genomic DNA was extracted as previously described by Landum et al. (2016), in accordance with the manufacturer’s instructions, using the FAVORGEN Fungi/ Yeast Genomic DNA Extraction Mini Kit (Taiwan). The nuclear ribosomal DNA internal transcribed spacer (ITS) of the fungal isolates were amplified using the forward primer, ITS-F (5’-CTT GGT CAT TTA GAG GAA GTA A-3′) and the reverse primer, ITS4 (5’-TCC TCC GCT TAT TGA TAT GC-3′; White et al., 1990). The final reaction volume was 25 μl, containing 12.5 μl of 2X PCRBio Tag Mix Red (PCR Biosystems, UK), 0.4 μM of forward and reverse primers, and 10 mg of genomic DNA template. For negative control, the DNA was replaced with distilled water to verify the absence of contamination. The PCR was carried out using MyCycler™ (Bio-Rad, USA), programmed for 5 min at 95°C; 30 cycles for 30 s at 95°C, 30 s at 54.8°C, and 1 min at 72°C; and a final 10 min extension at 72°C. The PCR products were separated using 1% agarose gel in 1X TAE buffer (90 mM Tris-acetate and 2 nM EDTA, pH 8.0), stained with ethidium bromide (0.5 μg/ml), and visualised using FluorChem TM (Alpha Innotech, USA). The PCR products were sequenced by Apical Scientific Sdn. Bhd. (Malaysia). The sequences were deposited in NCBI GenBank, and compared with those already deposited in there via BLAST searches.

 Sequence and phylogenetic analyses

The resulting DNA sequences were aligned using MUSCLE software embedded in MEGA software version 10.0.5 (Kumar et al., 2018), and manually trimmed and edited to obtain the complete sequences. Homology searches were carried out using the BLAST program against the NCBI GenBank database.1 The Maximum Likelihood tree was constructed using MEGA software version 10.0.5 with all positions containing gaps and missing data were included for analysis. Clade supports were calculated based on 1,000 bootstrap replications. A total of 64 sequences of close relatives were downloaded from the NCBI GenBank, and combined with sequences of the 66 endophytic fungi isolated in the present work for phylogenetic tree construction. Two wood decay macrofungi namely Schizophyllum commune (phylum Basidiomycota, family Schizophyllaceae) and Phellinus gabonensis (phylum Basidiomycota, family Hymenochaetaceae) were included as out-group.

 Antagonism assay

Endophytic fungal isolates were cultivated on PDA plates at 26°C for 7 days. The antagonistic activity was evaluated through the dual culture assay against C. fimbriata. The pathogenic C. fimbriata (FRIM1162) isolate used in this study was isolated from a infected Acacia mangium (Syazwan et al., 2021) and maintained at 27°C on PDA media at the Mycology & Pathology Unit, Forest Research Institute Malaysia (FRIM). Briefly, a fungal disc of 5 mm in diameter was taken from C. fimbriata, and placed 3 cm from the margin of the PDA plate (9 cm in diameter). Next, a 5 mm disc of the endophytic fungus was placed 3 cm from the margin of the PDA plate, and directly opposite of the C. fimbriata disc. Inoculated PDA plates were incubated at room temperature for 7 days. PDA plates inoculated with C. fimbriata in the absence of endophytic fungus served as negative controls. The assay was performed in triplicates. Observations were carried out for 6 days, after which the mycelial radial growth of test pathogen (C. fimbriata) on a control plate (rl) and in the presence of the antagonistic fungus (r2) were measured, and the percentage inhibition (I%) in mycelial growth was calculated as: I% = [(r1 – r2) / r1] × 100 (Hajieghrari et al., 2008). The I% data were analysed statistically with ANOVA using the SAS statistical software. To examine the significance between endophytic fungal isolates, Fisher’s LSD was performed at p ≤ 0.05.

 Results Identification of endophytic fungi

A total of 66 endophytic fungal isolates were successfully isolated from different parts of healthy A. mangium (Table 1); 21 from leaf, 12 from petiole, 13 from stem, nine from root, six from flower, and five from fruit. Correspondingly, 66 isolates were successfully amplified using primers ITS1 and ITS4. The endophytic fungal isolates mostly belonged to Ascomycota (95.5%) followed by Zygomycota (4.5%) based on the BLAST searches analysis (Table 2). For Ascomycota, 13 genera were identified; Trichoderma (28.6%), Nigrospora (28.6%), Pestalotiopsis (12.7%), Lasiodiplodia (9.5%), Aspergillus (6.3%), Sordariomycetes (3%), and genera that were represented by a single isolate were Neopestalotiopsis, Pseudopestalotiopsis, Eutiarosporella, Curvularia, Fusarium, Penicillium, and Hypoxylon. Only Blakeslea sp. (4.5%) of Zygomycota was identified in the present work (Table 1). All the fungal ITS rDNA sequences exhibited high similarity with existing sequences in the NCBI database (Table 1).

Endophytic fungi isolated from different plant part of healthy Acacia mangium.

Plant part Individual nnumber Total
Fruit 1 2 1 1 5
Flower 1 2 1 1 1 6
Leaf 4 2 1 4 1 1 8 21
Petiole 1 1 1 1 4 4 12
Stem 2 1 1 1 4 3 1 13
Root 2 3 2 1 1 9
Total 8 1 1 6 1 4 1 18 1 1 18 2 1 3 66
Pestalotiopsis Pseudopestalotiopsis Neopestalotiopsis Lasiodiplodia Eutiarosporella Aspergillus Penicillium Trichoderma Fusarium Curvularia Nigrospora Sordariomycetes Hypoxylon Blakeslea

Percentage of identity matches of 66 fungal isolates from different plant parts of Acacia mangium based on ITS sequences using BLAST analyses, and their percentage of inhibition against Ceratocystis fimbriata.

No. Endophytic isolate ID Plant part Inhibition activities (%) (mean ± standard error) GenBank Accession number ITS region
Match identity (%) E-value Identification in GenBank BLAST match in GenBank Phylum, Class, Family
1 AC 1R Root 55 ± 0.58 MW254902 99.28 0 Blakeslea trispora HQ248186 Zygomycota, Zygomycetes, Choanephoraceae
2 AC 2R Root 0 ± 0.00 MW254903 99.63 0 Trichoderma gamsii KX009501 Ascomycota, Sordariomycetes, Hypocreaceae
3 AC 3R Root 0 ± 0.00 MW254904 100 0 Aspergillus aculeatinus MK281555 Ascomycota, Eurotiomycetes, Trichocomaceae
4 AC 4R Root 44 ± 2.08 MW254905 99.38 0 Nigrospora sphaerica MH368102 Ascomycota, Sordariomycetes, Trichosphaeriales
5 AC 5R Root 20 ± 0.00 MW254913 99.21 0 Aspergillus niger MN474007 Ascomycota, Eurotiomycetes, Trichocomaceae
6 AC 6R Root 8.88 ± 0.66 MW254916 99.63 0 Trichoderma spirale MN227543 Ascomycota, Sordariomycetes, Hypocreaceae
7 AC 7R Root 14.28 ± 0.43 MW254942 99.17 0 Sordariomycetes sp. JQ759985 Ascomycota, Sordariomycetes,
8 AC 8R Root 25 ± 2.89 MW254956 99.58 0 Nigrospora oryzae MN382281 Ascomycota, Sordariomycetes, Trichosphaeriales
9 AC 9R Root 58.33 ± 5.02×1015 MW254964 99.81 0 Trichoderma sp. MK870905 Ascomycota, Sordariomycetes, Hypocreaceae
10 AC 1S Stem 58.33 ± 5.02 ×1015 MW254907 99.81 0 Trichoderma koningiopsis KY807125 Ascomycota, Sordariomycetes, Hypocreaceae
11 AC 2S Stem 33.33 ± 0.29 MW254909 99.79 0 Nigrospora sphaerica KJ572188 Ascomycota, Sordariomycetes, Trichosphaeriales
12 AC 3S Stem 0 ± 0.00 MW254914 99.63 0 Pestalotiopsis vismiae KP747709 Ascomycota, Sordariomycetes, Sporocadaceae
13 AC 4S Stem 0 ± 0.00 MW254920 99.81 0 Pestalotiopsis sp. KY413701 Ascomycota, Sordariomycetes, Sporocadaceae
14 AC 5S Stem 45.45 ± 5.02 ×1015 MW254924 99.45 0 Trichoderma sp. MK870688 Ascomycota, Sordariomycetes, Hypocreaceae
15 AC 6S Stem 20 ± 3.61 MW254925 99.15 0 Lasiodiplodia theobromae GQ502461 Ascomycota, Dothideomycetes, Botryosphaeriaceae
16 AC 7S Stem 45 ± 0.00 MW254931 99.25 0 Trichoderma gamsii KX009501 Ascomycota, Sordariomycetes, Hypocreaceae
17 AC 8S Stem 40 ± 0.00 MW254937 99.59 0 Nigrospora oryzae MN38228 Ascomycota, Sordariomycetes, Trichosphaeriales
18 AC 9S Stem 0 ± 0.00 MW254940 99.63 0 Trichoderma ovalisporum FJ442652 Ascomycota, Sordariomycetes, Hypocreaceae
19 AC 10S Stem 0 ± 0.00 MW254944 99.8 0 Aspergillus niger MN559950 Ascomycota, Eurotiomycetes, Trichocomaceae
20 AC 11S Stem 45.45 ± 2.60 MW254951 100 0 Sordariomycetes sp. KC178665 Ascomycota, Sordariomycetes,
21 AC 12S Stem 14.28 ± 0.30 MW254954 97.98 0 Eutiarosporella sp. KX464132 Ascomycota, Dothideomycetes, Botryosphaeriales
22 AC 13S Stem 0 ± 0.00 MW254959 100 0 Nigrospora sp. MT556677 Ascomycota, Sordariomycetes, Trichosphaeriales
23 AC 1 l Leaf 55.55 ± 5.02 ×1015 MW254906 99.63 0 Trichoderma gamsii KM103313 Ascomycota, Sordariomycetes, Hypocreaceae
24 AC 2 l Leaf 37.5 ± 1.44 MW254908 99.38 0 Nigrospora sphaerica MN625838 Ascomycota, Sordariomycetes, Trichosphaeriales
25 AC 3 l Leaf 45.45 ± 0.75 MW254910 99.81 0 Trichoderma gamsii KX009501 Ascomycota, Sordariomycetes, Hypocreaceae
26 AC 4 l Leaf 16.67 ± 9.53 MW254918 100 0 Curvularia pandanicola MH275056 Ascomycota, Dothideomycetes, Pleosporaceae
27 AC 5 l Leaf 16.67 ± 0.00 MW254919 99.63 0 Pestalotiopsis microspora MT597837 Ascomycota, Sordariomycetes, Sporocadaceae
28 AC 6 l Leaf 45.45 ± 1.16 MW254921 99.81 0 Pestalotiopsis microspora EU137910 Ascomycota, Sordariomycetes, Sporocadaceae
29 AC 7 l Leaf 58.3 ± 5.02 ×1015 MW254922 98.77 0 Nigrospora oryzae MN382281 Ascomycota, Sordariomycetes, Trichosphaeriales
30 AC 8 l Leaf 28.57 ± 2.51 ×1015 MW254923 99.58 0 Fusarium chlamydosporum MT448890 Ascomycota, Sordariomycetes, Nectriaceae
31 AC 9 l Leaf 0 ± 0.00 MW254926 99.38 0 Nigrospora sphaerica MN566004 Ascomycota, Sordariomycetes, Trichosphaeriales
32 AC 10 l Leaf 30 ± 5.77 MW254934 99.57 0 Lasiodiplodia theobromae KF293981 Ascomycota, Dothideomycetes, Botryosphaeriaceae
33 AC 11 l Leaf 12.5 ± 6.93 MW254936 99.63 0 Trichoderma koningiopsis JQ617301 Ascomycota, Sordariomycetes, Hypocreaceae
34 AC 12 l Leaf 0 ± 0.00 MW254938 99.44 0 Pestalotiopsis neglecta MN006391 Ascomycota, Sordariomycetes, Sporocadaceae
35 AC 13 l Leaf 22.22 ± 0.00 MW254939 99.62 0 Trichoderma gamsii KX009501 Ascomycota, Sordariomycetes, Hypocreaceae
36 AC 14 l Leaf 0 ± 0.00 MW254943 99.58 0 Nigrospora oryzae JX966549 Ascomycota, Sordariomycetes, Trichosphaeriales
37 AC 15 l Leaf 33.33 ± 1.59 MW254945 99.79 0 Nigrospora sp. MT561433 Ascomycota, Sordariomycetes, Trichosphaeriales
38 AC 16 l Leaf 45.45 ± 5.02 ×1015 MW254946 99.58 0 Lasiodiplodia theobromae MK696043 Ascomycota, Dothideomycetes, Botryosphaeriaceae
39 AC 17 l Leaf 40 ± 5.77 MW254948 99.43 0 Pestalotiopsis vismiae KP747709 Ascomycota, Sordariomycetes, Sporocadaceae
40 AC 18 l Leaf 25 ± 0.00 MW254949 99.59 0 Nigrospora sphaerica MT043797 Ascomycota, Sordariomycetes, Trichosphaeriales
41 AC 19 l Leaf 0 ± 0.00 MW254950 99.8 0 Aspergillus aculeatus KJ605160 Ascomycota, Eurotiomycetes, Trichocomaceae
42 AC 20 l Leaf 40 ± 5.77 MW254962 99.59 0 Nigrospora sphaerica MH368102 Ascomycota, Sordariomycetes, Trichosphaeriales
43 AC 21 l Leaf 0 ± 0.00 MW254963 99.58 0 Nigrospora sphaerica MT561433 Ascomycota, Sordariomycetes, Trichosphaeriales
44 AC 1P Petiole 0 ± 0.00 MW254917 99.81 0 Trichoderma crissum MK911703 Ascomycota, Sordariomycetes, Hypocreaceae
45 AC 2P Petiole 0 ± 0.00 MW254932 99.79 0 Nigrospora sphaerica MT561433 Ascomycota, Sordariomycetes, Trichosphaeriales
46 AC 3P Petiole 50 ± 4.91 MW254933 97.68 0 Nigrospora sphaerica MN795570 Ascomycota, Sordariomycetes, Trichosphaeriales
47 AC 4P Petiole 58.33 ± 5.02 ×1015 MW254935 99.38 0 Nigrospora sphaerica KJ572188 Ascomycota, Sordariomycetes, Trichosphaeriales
48 AC 5P Petiole 45.45 ± 5.02 ×1015 MW254947 99.79 0 Nigrospora sphaerica MT561433 Ascomycota, Sordariomycetes, Trichosphaeriales
49 AC 6P Petiole 45 ± 5.77 MW254952 99.8 0 Penicillium rolfsii MK120600 Ascomycota, Eurotiomycetes, Trichocomaceae
50 AC 7P Petiole 20 ± 5.77 MW254957 100 0 Trichoderma longibrachiatum FJ462745 Ascomycota, Sordariomycetes, Hypocreaceae
51 AC 8P Petiole 30 ± 5.77 MW254958 99.37 0 Neopestalotiopsis cubana LC521857 Ascomycota, Sordariomycetes, Pestalotiopsidaceae
52 AC 9P Petiole 45.45 ± 5.02 ×1015 MW254961 99.79 0 Pestalotiopsis sp. JN116590 Ascomycota, Sordariomycetes, Sporocadaceae
53 AC 10P Petiole 45.45 ± 5.02 ×1015 MW254965 99.57 0 Lasiodiplodia theobromae MT075441 Ascomycota, Dothideomycetes, Botryosphaeriaceae
54 AC 11P Petiole 14.28 ± 0 MW254966 70.1 4.00E-20 Trichoderma sp. GU973813 Ascomycota, Sordariomycetes, Hypocreaceae
55 AC 12P Petiole 20 ± 5.77 MW254967 99.44 0 Trichoderma koningiopsis MT102395 Ascomycota, Sordariomycetes, Hypocreaceae
56 AC 1F Flower 8.88 ± 0.00 MW254911 99.28 0 Blakeslea trispora HQ248186 Zygomycota, Zygomycetes, Choanephoraceae
57 AC 2F Flower 8.88 ± 5.14 MW254915 94.87 0 Hypoxylon monticulosum KY610404 Ascomycota, Sordariomycetes, Hypoxylaceae
58 AC 3F Flower 58.33 ± 5.02 ×1015 MW254927 100 0 Nigrospora sphaerica MT561433 Ascomycota, Sordariomycetes, Trichosphaeriales
59 AC 4F Flower 40 ± 0.00 MW254941 99.62 0 Trichoderma longibrachiatum MH745146 Ascomycota, Sordariomycetes, Hypocreaceae
60 AC 5F Flower 45.45 ± 2.60 MW254953 99.59 0 Pseudopestalotiopsis theae KX401429 Ascomycota, Sordariomycetes, Pestalotiopsidaceae
61 AC 6F Flower 45.45 ± 5.02 ×1015 MW254955 100 0 Trichoderma koningiopsis JQ278013 Ascomycota, Sordariomycetes, Hypocreaceae
62 AC 1 U Fruit 8.88 ± 0.00 MW254912 100 0 Pestalotiopsis microspore EU137910 Ascomycota, Sordariomycetes, Sporocadaceae
63 AC 2 U Fruit 69.23 ± 0.00 MW254928 99.79 0 Lasiodiplodia theobromae MK696044 Ascomycota, Dothideomycetes, Botryosphaeriaceae
64 AC 3 U Fruit 25 ± 2.89 MW254929 98.39 0 Blakeslea trispora HQ248186 Zygomycota, Zygomycetes, Choanephoraceae
65 AC 4 U Fruit 45 ± 2.89 MW254930 99.79 0 Lasiodiplodia venezuelensis MH865369 Ascomycota, Dothideomycetes, Botryosphaeriaceae
66 AC 5 U Fruit 16.16 ± 0.00 MW254960 100 0 Trichoderma harzianum MF537642 Ascomycota, Sordariomycetes, Hypocreaceae

The ITS sequences obtained in the present work were deposited in the NCBI GenBank (MW254902 - MW254967) for future reference. A total of 66 sequences of close relatives were downloaded from the NCBI GenBank, and combined with sequences of the 66 endophytic fungi for phylogenetic tree construction (Figure 1). Nine different orders were observed, of which six belonged to Ascomycota (Amphisphaeriales, Brotryosphaerialase, Eurotiales, Hypocreales, Pleosporales and Trichosphaeriales), one belonged to Zygomycota, and two belonged to Basidiomycota (out-group). Most of the endophytic fungal isolates clustered under the order Trichosphaeriales (20 isolates) belonged to genus Nigrospora, and under the order Hypocreales (19 isolates) belonged to genera Fusarium and Trichoderma. Tables 3 and 4 summarises these results.

Maximum likelihood (ML) phylogenetic tree based on rDNA ITS sequences of endophytic fungal isolates and fungal ITS sequences from the GenBank. ML tree was constructed using the Kimura 2-parameter (K2) model and gamma distributed (+G) model. All positions containing gaps and missing data were included for analysis. Clade supports were calculated based on 1,000 bootstrap.

Endophytic fungal orders from the phylum Ascomycota.

No. ID GenBank Accession no. Plant part Amphisphaeriales
1 AC 3S MW254914 Stem Pestalotiopsis vismiae Pestalotiopsis clade (94% bootstrap)
2 AC 4S MW254920 Stem Pestalotiopsis sp.
3 AC 9P MW254961 Petiole Pestalotiopsis sp.
4 AC 5L MW254919 Leaf Pestalotiopsis microspora
5 AC 6L MW254921 Leaf Pestalotiopsis microspora
6 AC 12L MW254938 Leaf Pestalotiopsis neglecta
7 AC 1U MW254912 Fruit Pestalotiopsis microspora
8 AC 5F MW254953 Flower Pseudopestalotiopsis theae
9 AC 17L MW254948 Leaf Pestalotiopsis vismiae Pseudopestalotiopsis clade (77% bootstrap)
10 AC 8P MW254958 Petiole Neopestalotiopsis cubana Brotryosphaerialase Neopestalotiopsis clade (77% bootstrap)
11 AC 6S MW254925 Stem Lasiodiplodia theobromae Lasiodiplodia clade (97% bootstrap)
12 AC 10P MW254965 Petiole Lasiodiplodia theobromae
13 AC 10L MW254934 Leaf Lasiodiplodia theobromae
14 AC 16L MW254946 Leaf Lasiodiplodia theobromae
15 AC 2U MW254928 Fruit Lasiodiplodia theobromae
16 AC 4U MW254930 Fruit Lasiodiplodia venezuelensis
17 AC 12S MW254954 Stem Eutiarosporella sp. Eurotiales Eutiarosporella clade (97% bootstrap)
18 AC 10S MW254944 Stem Aspergillus niger Aspergillus clade (97% bootstrap)
19 AC 3R MW254904 Root Aspergillus aculeatinus
20 AC 5R MW254913 Root Aspergillus niger
21 AC 19L MW254950 Leaf Aspergillus aculeatus
22 AC 6P MW254952 Petiole Penicillium rolfsii Hypocreales Penicillium clade (97% bootstrap)
23 AC 2R MW254903 Root Trichoderma gamsii Trichoderma clade (95% bootstrap)
24 AC 6R MW254916 Root Trichoderma spirale
25 AC 9R MW254964 Root Trichoderma sp.
26 AC 1S MW254907 Stem Trichoderma koningiopsis
27 AC 5S MW254924 Stem Trichoderma sp.
28 AC 7S MW254931 Stem Trichoderma gamsii
29 AC 9S MW254940 Stem Trichoderma ovalisporum
30 AC 1P MW254917 Petiole Trichoderma crissum
31 AC 7P MW254957 Petiole Trichoderma longibrachiatum
32 AC 11P MW254966 Petiole Trichoderma sp.
33 AC 12P MW254967 Petiole Trichoderma koningiopsis
34 AC 1L MW254906 Leaf Trichoderma gamsii
35 AC 3L MW254910 Leaf Trichoderma gamsii
36 AC 11L MW254936 Leaf Trichoderma koningiopsis
37 AC 13L MW254939 Leaf Trichoderma gamsii
38 AC 4F MW254941 Flower Trichoderma longibrachiatum
39 AC 5U MW254960 Fruit Trichoderma harzianum
40 AC 6F MW254955 Fruit Trichoderma koningiopsis
41 AC 8L MW254923 Leaf Fusarium chlamydosporum Pleosporales Fusarium clade (95% bootstrap)
42 AC 4L MW254918 Leaf Curvularia pandanicola Curvularia clade (95% bootstrap)
Hypocreales
43 AC 4R MW254905 Root Nigrospora sphaerica Nigrospora and Sordariomycete polytomy clade (95% bootstrap)
44 AC 8R MW254956 Root Nigrospora oryzae
45 AC 2S MW254909 Stem Nigrospora sphaerica
46 AC 8S MW254937 Stem Nigrospora oryzae
47 AC 13S MW254959 Stem Nigrospora sp.
48 AC 2P MW254932 Petiole Nigrospora sphaerica
49 AC 3P MW254933 Petiole Nigrospora sphaerica
50 AC 4P MW254935 Petiole Nigrospora sphaerica
51 AC 5P MW254947 Petiole Nigrospora sphaerica
52 AC 2L MW254908 Leaf Nigrospora sphaerica
53 AC 7L MW254922 Leaf Nigrospora oryzae
54 AC 9L MW254926 Leaf Nigrospora sphaerica
55 AC 14L MW254943 Leaf Nigrospora oryzae
56 AC 15L MW254945 Leaf Nigrospora sp.
57 AC 18L MW254949 Leaf Nigrospora sphaerica
58 AC 20L MW254962 Leaf Nigrospora sphaerica
59 AC 21L MW254963 Leaf Nigrospora sphaerica
60 AC 3F MW254927 Flower Nigrospora sphaerica
61 AC 7R MW254942 Root Sordariomycetes sp.
62 AC 11S MW254951 Stem Sordariomycetes sp. Hypocreales
63 AC 2F MW254915 Flower Hypoxylon monticulosum Hypoxylon clade (99% bootstrap)

Endophytic fungal order from the phylum Zygomycota.

No. ID GenBank Accession number Plant part Mucorales
1 AC 1R MW254902 Root Blakeslea trispora Blakeslea clade (99% bootstrap)
2 AC 1F MW254911 Flower Blakeslea trispora
3 AC 3U MW254929 Fruit Blakeslea trispora
Antagonism assay

All 66 endophytic fungal isolates were tested in the antagonism assay against C. fimbriata. After 5 days of incubation, six fungal isolates namely Trichoderma koningiopsis (AC 1S) stem, Nigrospora oryzae (AC 7L) leaf, Nigrospora sphaerica (AC 3F) flower, Lasiodiplodia sp. (AC 2 U) fruit, Nigrospora sphaerica (AC 4P) petiole, and Trichoderma sp. (AC 9R) root were observed to exhibit stronger inhibition where the mycelia of the antagonists had breached into C. fimbriata colony (Figure 2). Of these, four fungal isolates namely T. koningiopsis (AC 1S) stem, Lasiodiplodia sp. (AC 2 U) fruit, N. sphaerica (AC 4P) petiole, and Trichoderma sp. (AC 9R) root colonised almost 99% of the culture plate. Although N. sphaerica (AC 7 l) leaf and N. sphaerica (AC 3F) flower did not colonise the entire culture plate, there was no growth of C. fimbriata observed.

Dual culture plate assay between six endophytic fungal isolates against the pathogen C. fimbriata (A). (B) Trichoderma koningiopsis AC1S – stem; (C) Nigoshora spharenica AC7L – leaf; (D) Nigoshora spharenica AC3F - flower; (E) Lasiodiplodia sp. AC2U – fruit; (F) Nigoshora spharenica AC4P-petiole; (G) Trichoderma sp. AC9R-root. The plates were cultivated for 5 days at 27°C. Radial growths were measured and interaction were observed.

The inhibition percentages (I%) of endophytic fungi against the pathogen C. fimbriata in dual culture assay are shown in Figure 3. Lasiodiplodia sp. (AC 2 U) isolated from fruit recorded the highest I% (69.23%), followed by Trichoderma sp. (AC 9R) isolated from root, Nigrospora sphaerica (AC 4P) isolated from petiole, Nigrospora sphaerica (AC 3F) isolated from flower, Trichoderma koningiopsis (AC 1S) isolated from stem, and Nigrospora oryzae (AC 7L) isolated from leaf with value 58.33%, respectively.

Inhibition percentages (I%) of endophytic fungi against the pathogen Ceratocystis fimbriata in dual culture assay. Data are mean ± standard error (SE) of triplicates.

Thirteen endophytic fungi from various plant parts of A. mangium showed no inhibition against C. fimbriata (Figure 4) namely A. aculeatinus (AC 3R) isolated from root, A. aculeatus (AC 19L) isolated from leaf, A. niger (AC 10S) isolated from stem, N. oryzae (AC 14L) isolated from leaf, Nigrospora sp. (AC 13S) isolated from stem, N. sphaerica (AC 9L) isolated from leaf, N. sphaerica (AC 2P) isolated from petiole, P. neglecta (AC 12L) isolated from leaf, Pestalotiopsis sp. (AC 4S) isolated from stem, P. vismiae (AC 3S) isolated from stem, T. crissum (AC 1P) isolated from petiole, T. gamsii (AC 2R) isolated from root, and T. ovalisporum (AC 9S) isolated from stem.

The inhibition percentages (I%) of endophytic fungi isolated from root (A), stem (B), petiole (C), leaf (D), flower (E), and fruit (F) against the pathogen Ceratocystis fimbriata. Data are mean ± standard error (SE) of triplicates. Means followed by the same letter in each group are not significantly different at α = 0.05 according to DuncanLSD.

 Diversity of endophytic fungi

Endophytic fungi are ubiquitous, and every plant species examined to date have been found colonised by them (Arnold et al., 2001). A single plant species may harbour hundreds of endophytes which may inhabit all available tissues, including leaves, petioles, stems, twigs, barks, xylems, roots, fruits, flowers, and seeds (Chapela and Boddy, 1988; Fisher et al., 1993; Saikkonen et al., 1998; Jena and Tayung, 2013). In the present work, endophytic fungi were isolated from different plant parts of A. mangium with the highest number of isolates found in leaf and dominated by the genera Trichoderma and Nigrospora. Trichoderma spp. were present in all plant parts, while Nigrospora spp. were present in all but fruit. In total, 66 endophytic fungal isolates were obtained from different plant parts of A. mangium.

Trichoderma and Nigrospora have also been reported as endophytes in other plants such as Rauvolfia serpentine, Prosopis cineraria, and Piper nigrum (Gehlot et al., 2008; Dutta et al., 2014; Sopialena et al., 2018). Trichoderma is also found in many ecosystems, and can reduce the severity of plant diseases by inhibiting the plant pathogens in the soil through their highly potent antagonistic and mycoparasitic activities (Hermosa et al., 2012). Moreover, as revealed by research in recent decades, some Trichoderma strains can interact directly with roots, thus increasing plant growth potential, resistance to disease, and tolerance to abiotic stresses (Mastouri et al., 2010; Hermosa et al., 2012; Brotman et al., 2013). Nigrospora is also a beneficial member of the foliar endophytic community due to its mutualistic existence with their host plants, and having a potential for biological control strategies (Zakaria et al., 2016). Other than Nigrospora, Pestalotiopsis also is a beneficial member of the foliar endophytic community due to its ability to switch its nutritional mode, thus able to stay as an endophyte or switch to saprophyte when necessary (Douanla-Meli et al., 2013; Hamzah et al., 2018). Besides Trichoderma, Nigrospora, and Pestalotiopsis, other fungal genera such as Lasiodiplodia, Sordariomycetes, and Aspergillus have also been reported as predominant endophytic fungi in other plants species (Li et al., 2012; del Castillo et al., 2016), and have an antagonism ability (Chen et al., 2010). Fusarium too is a common endophytic fungal genus found in trees (Zakaria et al., 2010). Although it is widely available in most tropical plants investigated in past studies (Warman and Aitken, 2018), we recorded a low isolation frequency of Fusarium. Our finding also revealed lesser-known fungal genera, namely Eutiarosporella, Curvularia, Glomerella, and Hypoxylon in A. mangium.

In the present work, ITS sequences identified 63 endophytic fungal isolates from the phylum Ascomycota, and three from Zygomycota. The phylum Ascomycota has been reported to be the most common endophytic fungal phylum when isolated using standard isolation protocols (Koukol et al., 2012; Hamzah et al., 2018). Fungi from the phylum Zygomycota have been reported to be culture-method dependent (Crozier et al., 2006; Hamzah et al., 2018), which might explain the small isolate number reported in the present work. Comparative studies also show that only a small fraction of microorganisms in nature can be cultured using conventional microbiological techniques (Amann et al., 1995). There are many factors that can affect the microbial viability under laboratory conditions, for example the lack of knowledge about their nutritional requirements.

 Antagonism activities against <italic>Ceratocystis fimbriata</italic>

Fungal antagonism can manifest in many ways such as nutrition competition, niche exclusion, mycoparasitism, and the production of extracellular metabolites (Siameto et al., 2010). These metabolites, especially antibiotics and lytic enzymes, have been widely applied in various fields like crop-pathogen controls. Endophytic microorganisms isolated from plants can produce various novel bioactive metabolites (Ramasamy et al., 2010). The bioactive metabolites produced by plants, microorganisms, and organisms are useful for the discovery and development of new drugs.

In the present work, Lasiodiplodia sp., T. koningiopsis, N. sphaerica and Trichoderma sp. successfully inhibited the pathogen C. fimbriata in the dual culture assay. The ability to out-grow the pathogen in vitro suggested that these fungi competed for the space and nutrient with the pathogen. In theory, biological agents with antifungal properties are known to secrete certain enzymes which break down their competitors’ cell wall, thus restricting their growth (Sharon et al., 2001). The antagonism displayed by Lasiodiplodia sp. was more aggressive as compared to other endophytic fungi (Figure 3). This could be attributed to the production of lytic enzymes by Lasiodiplodia sp. (Anitha and Rabeeth, 2010). The antagonism displayed by Lasiodiplodia sp., T. koningiopsis, N. sphaerica and Trichoderma sp. could also be explained by their secretion of secondary metabolites into the growth medium, as well as nutrient depletion in the growth medium (Robinson et al., 2014). The antagonism displayed might also be influenced by the antibiotics or hydrolytic enzymes they produced (Kamala and Indira, 2011). The difference in antagonism magnitude observed in the present work could also be dependent on specific fungal species (Kai et al., 2007). Previously, Lasiodiplodia sp. from the flower of Viscum coloratum also exhibited antimicrobial activity which could be due to the presence of cyclo-(Trp-Ala), ICA, indole-3-carbaldehyde, mullein, and 2-phenylethano in their extract (Qian et al., 2014). Lasiodiplodia sp. isolated from the twig of Aegle marmelos has also been shown to have in vitro fibrinolytic activities (Meshram and Saxena, 2016). Another plant parts such as bark and leaf of Terminalia sp. has also been isolated with Lasiodiplodia sp. which not only exhibited antimicrobial and antioxidant activities, but also aided the plant to withstand stressful environmental conditions (Patil et al., 2014).

 Conclusion

Diversity of endophytic fungi were successfully isolated from different parts of A. mangium, with Trichoderma spp. being the most prevalent, and were isolated from all six plant parts. Against C. fimbriata, the crude extracts from Trichoderma spp., N. sphaerica, and Lasiodiplodia sp. exhibited strong inhibition in the dual culture assay. Thus, it can be concluded that certain endophytic fungi of A. mangium have the potential to be harnessed as anti-Ceratocystis agent in future biotechnological applications.

 Data availability statement

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/supplementary material.

 Author contributions

RT designed the study, collected, identified plant materials, and edited the manuscript. RT and RZ conducted the experiments, drafted, and revised the manuscript. Data analysis performed by RT, MA, MM, NS, WAW-M-A, and AH. MH assisted in DNA extraction. RT, MA, MM, NS, and AH supervised. RT acquired funding. All authors contributed to the article and approved the submitted version.

 Funding

The present work was financially supported by Universiti Putra Malaysia under the Putra Grant Scheme (GP-IPM/2017/9565600).

 Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

 Publisher’s note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

The authors gratefully acknowledge the Laboratory of Wood Deterioration and Protection, Department of Natural Resource Industry, Faculty of Forestry and Environment, Universiti Putra Malaysia for the research facilities.

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1https://blast.ncbi.nlm.nih.gov/Blast.cgi