Diversity and bioactivities of mangrove fungal endophytes ...

Vol. 12 (Supplement) | 2019 Philippine Science Letters

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Diversity and bioactivities of mangrove fungal endophytes from Leyte and Samar, Philippines Carlo Chris S. Apurillo1,3,4, Lei Cai5, and Thomas Edison E. dela Cruz*1,2,3 1The Graduate School 2Department of Biological Sciences, College of Science, and 3Fungal Biodiversity, Ecogenomics and Systematics Group, Research Center for

Natural and Applied Sciences, University of Santo Tomas, Manila, Philippines 4Philippine Science High School-Eastern Visayas Campus, Leyte, Philippines 5State Key Laboratory of Mycology, Institute of Microbiology,

Chinese Academy of Sciences, Beijing, China

angrove fungal endophytes (MFEs) are known to be viable sources of metabolites with potential antibacterial and cytotoxic properties. In this study, 73 fungal endophytes were isolated from 4 mangrove hosts, namely, Sonneratia alba J.

Smith., Rhizophora mucronata Lamk., Aegiceras floridum Roemer & Schultes, and Avecinnia marina (Forssk.) Vierh., collected in Leyte and Samar, Eastern Philippines. Among these mangrove hosts, R. mucronata from Samar had the highest MFE diversity and species richness. Differences in fungal species composition were also noted between the same host mangroves collected from different sites. Although fungal endophytes are known to exhibit host specificity, in this study, similar species of mangroves are actually hosts to different fungal endophytes. The MFEs were characterized as belonging to 24 morphospecies. Identities of the representative strains of the 24 morphospecies were then confirmed by multigene analysis, i.e., ITS (internal transcribed spacer region), CAL (partial calmodulin gene), HIS (partial histone H3 gene), GAPDH (partial glyceraldehyde-3-phosphate dehydrogenase gene), TEF (partial translation elongation factor 1-alpha gene), TUB (partial beta-tubulin gene), ApMAT (Apn2/MAT locus), and ACT (partial actin gene). Of these, 16 MFEs were mass-produced to extract secondary metabolites for antibacterial and cytotoxic tests. Culture extracts of Pestalotiopsis adusta were most

effective against Pseudomonas aeruginosa with a MIC (minimum inhibitory concentration) of 80 µg/ml. Twelve MFEs grown under stationary conditions and 15 MFEs grown under agitated conditions showed an inhibitory concentration (IC50) of less than 500 µg/ml against the K562 myelogenous leukemia cell line. Of these, Xylaria cubensis grown under stationary conditions exhibited the lowest IC50 of 99.35±19.5 µg/ml. This study paves the way for the first report of the MFEs from the central part of the Philippines and the discovery of potential metabolites which can be explored for pharmaceutical applications. KEYWORDS antibacterial, bioprospecting, cytotoxicity, diversity, Philippine fungi, natural products INTRODUCTION Mangrove species diversity is extremely high in the Philippines, with 40 of the 65 species of mangroves known worldwide (Primavera 2000). About 45% of the remaining mangrove forests in the Philippines are located in Eastern Visayas (Primavera 2000). These mangrove ecosystems are “hot spots” of biodiversity for fungi (Shearer et al. 2007). Thus, mangrove forests in Eastern Visayas may contain many host plants that harbor unique and novel species of fungi with special metabolic capabilities. Our particular interest are the fungal endophytes associated with the leaves of mangrove plants. Endophytes are

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*Corresponding author Email Address: [email protected] Date received: February 20, 2019 Date revised: July 1, 2019 Date accepted: July 2, 2019

Philippine Science Letters Vol. 12 (Supplemental) | 2019 34

Table 1: Mangrove fungal endophytes used in this study. The GenBank accession numbers for each gene markers are also listed. fscxx Taxa ITS TUB TEF-1 HIS3 CAL GAPDH ApMAT ACT M141 Guignardia mangiferae KR056282 M111 Guignardia mangiferae KR056283 M113 Guignardia mangiferae KR056284 M161 Guignardia mangiferae KR056285 B141 Valsa brevispora KR056286 M151 Aspergillus nidulans KR056287 B261 Aspergillus oryzae KR056288 M133 Marasmiellus palmivorus KR056289 M121 Marasmiellus palmivorus KR056290 M261 Phaeosphaeriopsis musae KR056291 M225 Cytospora rhizophorae KR056292 M264 Pestalotiopsis adusta KR056293 KR262520 KR262530 M263 Xylaria cubensis KR056294 M221 Diaporthe siamensis KR056295 KR262523 KR262533 KR262538 M231 Phomopsis pittospori KR056296 KR262522 KR262532 KR262537 B443 Diaporthe sp. KR056297 KR262524 KR262534 KR262539 B423 Diaporthe sp. KR056298 KR262525 KR262535 KR262540 B251 Phomopsis pittospori KR056299 KR262521 KR262531 KR262536 M331 Verticillium nigrescens KR056300 B431 Colletotrichum queenslandicum KR056301 KR262527 KR262542 KR262546 KR262550 KR262554 B462 Colletotrichum fructicola KR056302 KR262529 KR262544 KR262548 KR262552 KR262556 B461 Colletotrichum fructicola KR056303 KR262528 KR262543 KR262547 KR262551 KR262555 B464 Colletotrichum tropicale KR056304 KR262526 KR262541 KR262545 KR262549 KR262553

a ITS = nuclear ribosomal internal transcribed spacer region, TUB = partial beta-tubulin gene, TEF = partial translation elongation factor 1-alpha gene, HIS = partial histone H3 gene, CAL = partial calmodulin gene, GAPDH = partial glyceraldehyde-3-phosphate dehydrogenase gene, ApMAT = Apn2/MAT locus, ACT = partial actin gene

microbes that reside inside host plants without causing any harm to the host (Petrini 1991). The relationship between an endophyte and its host is described as a gradient from a beneficial or harmless relationship such as mutualism and commensalism to a harmful parasitism (Aly et al. 2011).

Studies in past decades have reported mangrove fungal endophytes (MFEs) in Asia and Southeast Asia. In the Philippines, the following MFEs from Luzon Island were reported (Moron et al. 2018): Arthirinium phaeosmermum, Colletotrichum siamense, Colletotrichum tropicale, Fusariusm oxysporum, Fusarium chladysporum, Fusarium proliferatum, Fusarium solani, Lasiodiplodia theobromae, Nodulisporium sp., Paecilomyes formosus, Penicillium citrinum, and Pestalotiopsis microspora. These were isolated from roots and stems of 12 healthy host mangroves. Species of Pestalotiopsis, Alternaria, and Cladosporum from Rhizophora mucronata collected in Malaysia were also reported (Hamzah et al. 2018). Phomopsis, Aspergillus, and Pestalotiopsis were among the most common MFEs from Rhizophoraceae collected in Hainan, China (Xing and Guo 2011). Mangroves belonging to the genus Sonneratia were also found to be the hosts of Diaporthe, Fusarium, and Phomopsis (Xing et al. 2011). While marine-derived fungi from macroalgae, seagrasses, and other marine substrata (dela Cruz et al. 2006; Schulz et al. 2008; Yao et al. 2009; Lavadia et al. 2017; Notarte et al. 2017, 2018) and fungal endophytes from terrestrial plants [e.g., Musa spp. (Dagamac et al. 2008), Pandanus (Bungihan et al. 2011, 2013a), Canarium ovatum (Torres and dela Cruz 2015), and medicinal plants (Eskandarighadikolaii et al. 2015)] were reported for their bioactivities, with some species producing novel metabolites (Bungihan et al. 2010, 2013b) and xylanolytic and dye-degrading enzymes (Torres et al. 2011; Torres and dela Cruz 2013), MFEs can also be tapped for their antimicrobial and cytotoxic activities. For example, Pestalotiopsis microspora showed antibacterial activity against gram-positive bacteria (Moron et al. 2018). Extracts from an MFE, Phyllosticta sp., also contained Tyrosol C and Cytosporone B, which have antimicrobial and antioxidant activities, respectively (Tan et al. 2015). Hypocrea lixii, a mangrove-derived fungus, including those belonging to the genus Xylaria and Cladosporum, had potent antibacterial activities against Escherichia, Pseudomonas, Enterococcus, Staphylococcus, and Bacillus, with the highest activity seen against Pseudomonas (Chaeprasert et al. 2010; Bhimba et al. 2012). Species of Cladosporum, Colletotrichum, and Xylaria

showed cytotoxic activities against malignant melanoma A375, colorectal adenocarcinoma SW620, gastric carcinoma Kato III, liver hepatoblastoma HepG2, and acute T cell leukemia Jurkat (Chaeprasert et al. 2010). A taxol-producing fungal endophyte was also isolated from Rhizophora annamalayana (Elavarasi et al. 2012). MFEs certainly merit a closer look for bioprospecting and drug discovery.

Despite the high diversity of mangrove species in the Philippines, there is limited knowledge on MFEs in the country. With the mangrove ecosystems continually being threatened by human activities resulting in habitat loss, we may lose many of these interesting fungal endophytes. Thus, we aimed in this study to isolate and identify MFEs from mangrove hosts collected in Leyte and Samar, Eastern Visayas, Philippines. These areas in the central Philippines were chosen for this study because no records of MFEs have been reported despite the large number of mangroves in those areas. The crude culture extracts of the isolated fungal endophytes were then explored for bioactivities, including tests for antibacterial and cytotoxic activities. MATERIALS AND METHODS Sampling sites and host plants Two sites were selected for the study: an island mangrove site in Babatngon, Leyte (11o25’45.3”N, 124o51’39.8”E) and a coastal mangrove site in Marabut, Samar (11o7’15.9”N, 125o12’34.5”E). Here healthy, mature leaves were collected from four mangrove species: Sonneratia alba, Rhizophora mucronata, Aegiceras floridum, and Avicennia alba. Healthy and mature leaves were collected at random from different branches and twigs of the host mangroves. The first three hosts were present in both collecting sites, while the last host was present only in Babatngon, Leyte. Collected leaf samples were then placed in ice and processed in the laboratory within 24 to 48 hours.

Isolation of mangrove fungal endophytes Mangrove leaves were initially washed in tap water to remove dirt and any attached debris. Leaf explants with a diameter of 6 mm. were surface-sterilized by sequential washing of 75% EtOH (1 minute), commercial bleach (5% NaOCl, 3 minutes), and 75% EtOH (30 seconds) and then washed with distilled water three times (Ariffin et al. 2011). For each host plant, five surface-sterilized leaf explants were plated on each of the six


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Figure 1: (A) Phylogram for Xylaria obtained from maximum likelihood based on ITS (internal transcribed spacer) alignment. Values above the branches represent maximum likelihood bootstrap values and posterior probability from Bayesian inference. This isolate was identified as Xylaria cubensis, with a bootstrap support of 1.00 and Bayesian posterior probability of 1.00. Sequences of mangrove fungal endophyte (MFE) isolates obtained in this study and the closely related taxa are highlighted in yellow. (B) Colony of the MFEs identified as Xylaria cubensis.

potato dextrose agar (PDA) plates supplemented with 500 mg/L streptomycin sulfate, i.e., 30 total number of explants per mangrove host. Streptomycin sulfate was added to inhibit growth of leaf-associated bacteria. Tissue prints were prepared by touching leaf fragments on agar for 10 seconds in order to test the efficacy of the surface sterilization method. The absence of any fungal or bacterial growth on the tissue prints indicates an effective surface sterilization. The plates were then incubated at room temperature and checked for fungal growth daily up to one week, and then observation was extended up to a month to isolate fungi which grew even after one week. All fungi growing out of the leaf explants were subcultured for isolation. To compute for percent colonization rate, the number of explants with a particular species of fungi was divided by the total number of explants multiplied by 100. The isolates were then characterized morphologically and initially classified into morphospecies based on characteristics of their colonies and spores for isolates that produced spores. DNA extraction and sequencing of target genes Representative strains of each of the MFE morphospecies were initially grown on PDA for at least seven days. Genomic DNA was then extracted by placing fungal mycelia and spores in a cell

lysis solution consisting of 1M Tris-HCl, 0.5M EDTA, 10% SDS, and H2O. These were tissue-lysed by using MP FastPrep-24 (USA) for three 50s cycles and centrifuged at high speed for 10 minutes to pellet the cell tissue. The supernatants were then transferred to a fresh tube and mixed with 100 µl of protein precipitation solution (ammonium acetate). These were vortexed for 30s and incubated at 4oC for 1 hour. After incubation, the tubes were centrifuged at high speed for 10 minutes to pellet the protein. The supernatants were then transferred to a fresh tube with 400 µl isopropanol and then incubated for 10 minutes at room temperature. Subsequently the tubes were centrifuged at high speed for 10 minutes to pellet the DNA. The supernatants were discarded, and 600 µl of 70% ethanol was added and centrifuged at high speed for 5 minutes to wash the DNA. The supernatants were again discarded, and the DNA pellets were allowed to dry for 10 minutes, resuspended in 50 µl of DNAse free water, and stored at -20oC until used for polymerase chain reaction (PCR).

To identify the MFEs, fragments of the nuclear ribosomal internal transcribed spacer (ITS) region were amplified for all morphospecies with ITS1 (5’-CTTGGTCATTTAGAGGAAGTAA-3’, Gardes and Bruns

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Figure 2: (A) Phylogram for Colletotrichum obtained from maximum likelihood analysis based on the ITS (internal transcribed spacer region), CAL (partial calmodulin gene), TUB (partial beta-tubulin gene), GAPDH (partial glyceraldehyde-3-phosphate dehydrogenase gene), ApMAT (Apn2/MAT locus), and ACT (partial actin gene) alignments. Values above the branches represent maximum likelihood bootstrap values and posterior probability from Bayesian inference. Sequences of mangrove fungal endophyte (MFE) isolates obtained in this study and the closely related taxa are highlighted in yellow. (B) Colonies of the MFEs identified as Colletotrichum queenslandicum and (C) Colletotrichum fructicola. (D) Colony, (E) conidiomata, and (F) conidia of MFEs identified as Collectotrichum tropicale.

1993) and ITS4 (5’- TCCTCCGCTTATTGATATGC-3’, White et al. 1990) primer pairs. PCR was performed in a 25 µl reaction containing 19 µl distilled water, 2.5 µl buffer, 0.3 µl dNTP, 0.2 µlTaq, 1 µl of each primer, and 1 µl of fungal DNA. The quality and quantity of amplified genes were checked visually by staining with ethidium bromide after 1% agar electrophoresis. For other MFEs whose identification could not be confirmed by the ITS sequences only, other target gene fragments were amplified by using the following primer pairs: EF1-728F (5’-CATCGAGAAGTTCGAGAAGG-3’) / EF1-986R (5’-ACTTGAAGGAACCCTTACC-3’) for the partial translation elongation factor 1-alpha gene (TEF, Carbone and Kohn 1999),

ACT-512F (5’-ATGTGCAAGGCCGGTTTCGC-3’) / ACT-783R (5’-TACGAGTCCTTCTGGCCCAT-3’) for the partial actin gene (ACT, Carbone and Kohn 1999), T1 (5’-AACATGCGTGAGATTGTAAGT-3’, O’Donell and Cigelnik 1997) / Bt-2b (5’-ACCCTCAGTGTAGTGACCCTTGGC-3’, Glass and Donaldson 1995) for the partial beta-tubulin (TUB) gene, CYLH3F (5’-AGGTCCACTGGTGGCAAG-3’) / CYLH3R (5’-AGCTGGATGTCCTTGGACTG-3’) for the partial histone H3 gene (HIS, Crous et al 2004), CL1C (5’-GAATTCAAGGAGGCCTTCTC-3’) / CL2C (5’-CTTCTGCATCATGAGCTGGAC-3’) for the partial calmodulin gene (CAL, Weir et al 2012), GDF1 (5’-

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Figure 3: (A) Phylogram for Guignardia obtained from maximum likelihood analysis based on the ITS (internal transcribed spacer region) sequence alignment. Values above the branches represent maximum likelihood bootstrap values and posterior probability from Bayesian inference. Sequences of mangrove fungal endophyte (MFE) isolates obtained in this study and the closely related taxa are highlighted in yellow. (B) Colony and (C) conidia of the MFEs identified as Guignardia mangiferae.

GCCGTCAACGACCCCTTCATTGA-3’) / GDR1 (5’-GGGTGGAGTCGTACTTGAGCATGT-3’) for the partial glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH, Guerber et al 2003), and AM-F (5’-TCATTCTACGTATGTGCCCG-3’) / AM-R (5’-CCAGAAATACACCGAACTTGC-3’) for the Apn2/MAT locus (ApMAT, Silva et al 2012). The PCR was performed with initial denaturation of 94oC, and 37 cycles of 94 oC for 45s, 52

oC for 45s, and 72 oC for 1 minute. The PCR products were then sent for sequencing. Sequence analysis Basic Local Alignment Search Tool (BLAST) search was conducted for related sequences. The MFE sequences were then aligned with these reference sequences by using MAFFT (Multiple Sequence Alignment using Fast Fourier Transform, https://mafft.cbrc.jp/alignment/server/) (Katoh and Standley 2013). Maximum likelihood trees were then generated by using PhyML (Phylogenetic inferences using Maximum Likelihood,

http://www.atgc-montpellier.fr/phyml/) (Guindon 2010) with 1,000 bootstrap replicates. For Bayesian analyses, the model of evolution was determined by MrModeltest 2.2. Posterior probalities (PP) (Zhaxybayeva and Gogarten 2002) were determined by Markov chain Monte Carlo sampling (MCMC) in MrBayes 3.2 (Ronquist et al. 2012) under the estimated model. Six simultaneous Markov chains were run for 1,000,000 generations, sampled every 100th generation, thus generating 10,000 trees. The first 2,000 trees were discarded as part of the burn-in phase, and the remaining 8,000 trees were used to calculate the PP in the majority rule consensus tree. Generated phylogenetic trees were viewed by using FigTree (http://tree.bio.ed.ac.uk/software/figtree/). Gene sequences generated from this study were deposited in GenBank (table 1). Diversity assessment The colonization frequency was initially determined as the number of explants with a particular species of MFEs. Diversity indices were then computed per host mangrove in each of the

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Figure 4: Phylogram for Verticillium obtained from maximum likelihood analysis based on ITS (internal transcribed spacer region) alignment. Values above the branches represent maximum likelihood bootstrap values and posterior probability from Bayesian inference. Sequences of mangrove fungal endophyte (MFE) isolates obtained in this study and the closely related taxa are highlighted in yellow. (B) Colony of the MFEs identified as Verticillium nigrescens.

two sites. Gleason’s species richness was computed by using the formula d = (S-1)/ln N, where S is the number of species and N is the number of individuals. Other indices such as species evenness, Shannon-Wiener index, and Simpson Index (1-D) were computed by using the biodiversity calculator (https://alyoung.com/labs/biodiversity_calculator.html). Mass production To extract the secondary metabolites, 16 different fungal species were initially selected and then mass-produced in potato dextrose broth (PDB) under submerged liquid fermentation at room temperature for four weeks. For the extracts used for antibacterial activity testing, mass production was done only under stationary conditions. The mass production for cytotoxic tests was done at the State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences. To determine any effects of incubation conditions on the bioactivities, mass production was done under both stationary and agitated conditions. The culture broths were extracted with ethyl acetate, and the culture extracts were concentrated in vacuo at 30oC. These were then allowed to dry and reconstituted with 10% DMSO (dimethyl sulfoxide) to a final concentration of 10 mg/ml (for antibacterial tests) and 1 mg/ml (for cytotoxic tests).

Antibacterial tests Microbroth dilution was used to test the antibacterial activity of the crude culture extracts against S. aureus ATCC 25923, E. coli ATCC 25922, and P. aeruginosa ATCC 27853. The Clinical Laboratory Standards Institute (CLSI) recommends these three as the initial test bacteria when testing compounds with potential antibacterial activity. The crude culture extracts were reconstituted to an initial concentration of 10 mg/ml. For the microbroth dilution method, 50 µl of the reconstituted crude culture extract was serially diluted (twofold) in eight wells, and then 40 µl of Mueller-Hinton Broth (MHB) was added. Ten µl of bacterial inoculum standardized to 5 x 105 CFU/ml was added to each well to achieve a final volume of 100 µl in each well. The microtiter plates were incubated at 37oC for 24 hours. Growth was determined in the wells by observing for turbidity by using a microplate optical density reader. The lowest concentration showing no growth was reported as the minimum inhibitory concentration (MIC) and was presented as mean values. All wells with no growth were then subcultured in nutrient agar plates to determine the minimum bactericidal concentration (MBC). The lowest concentration which showed no growth on the NA (Nutrient Agar) plate was reported as the

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Table 2: Mangrove fungal endophytes per mangrove host and sampling locality

Hosts Sites

Babatngon, Leyte Marabut, Samar

Sonneratia alba Valsa brevispora Guignardia mangiferae

Marasmiellus palmivorus

Aspergillus nidulans

Rhizophora

mucronata

Aspergillus oryzae

Phomopsis pittospori

Phaeosphaeriopsis musae

Pestalotiopsis adusta

Xylaria cubensis

Diaporthe siamensis

Cytospora rhizophorae

Phomopsis pittospori

Aegiceras floridum No MFE isolated Verticillium nigrescens

Avicennia marina Colletotrichum fructicola

Colletotrichum

queenslandicum

Colletotrichum tropicale

Diaporthe sp.

Host not found in this site

MBC. Growth control (10% DMSO + inoculum) and sterility control (MHB only) were run together with the crude culture extracts. All tests were run in triplicates. Cytotoxicity test The cytotoxicity of the crude culture extracts was tested on K562 myelogenous leukemia cells by using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay. The crude culture extracts were reconstituted to an initial concentration of 1 mg/ml. Here 100 µl of the crude culture extracts at different concentrations were added to each well containing K562 myelogenous leukemia cells. These were then incubated for 48 hours at 37oC in a humidified atmosphere of 5% CO2 air. Afterward 20 µl of MTT/medium (5 mg/ml) was added to each well and then incubated for 4 hours. The MTT/medium was then removed, and 100 µl of DMSO was added to each well. This was agitated at 6,000 rpm for 5 minutes to dissolve the precipitate. The microtiter plates were read at 540 nm by using a microplate reader. The inhibition rate was calculated and plotted versus the culture extract concentrations to determine the inhibitory concentration (IC50) by using linear interpolation of inhibition curves from the triplicates. Linear interpolation was done with the aid of a computer software. RESULTS Fungal endophytes were isolated from four mangrove species collected in two sampling sites: Leyte and Samar. Seventy-three MFEs were then isolated from these mangrove hosts. These were then classified into 24 morphospecies based on their morphological characteristics. ITS genes were used initially to confirm the identities of all MFEs. Additional gene markers were also amplified for some taxa whose identification could not be resolved by the ITS genes (table 1). Following gene sequence analysis, the 24 morphospecies were identified as 16 species belonging to 10 genera with high bootstrap support and Bayesian posterior probabilities. Phylogenetic trees for representative taxa are shown in figures 1–10. Identification of

Table 3: Colonization frequency of fungal endophytes in relation to host mangroves.

Mangrove fungal endophytes

Mangrove hosts per site Leyte Samar

SAa RM AM SA RM AF Aspergillus nidulans - - - 10 - - Aspergillus oryzae - 17 - - - - Colletotrichum fructicola - - 13 - - - Colletotrichum queenslandicum

- - 30 - - -

Colletotrichum tropicale - - 33 - - - Cytospora rhizophorae - - - - 10 - Diaporthe siamensis - - - - 7 - Diaporthe sp. - - 13 - - - Guignardia mangiferae - - - 27 - - Marasmiellus palmivorus - - - 13 - - Pestalotiopsis adusta - - - - 10 - Phaeosphaeriopsis musae - - - - 13 - Phomopsis pittospori - 13 - - 10 - Valsa brevispora 7 - - - - -

Verticillium nigrescens - - - - - 10 Xylaria cubensis - - - - 7 -

a host mangroves: Sonneratia alba (SA), Rhizophora mucronata (RM), Avicennia marina (AM), Aegiceras floridum (AF)

Table 4: Diversity indices of fungal endophytes from mangrove hosts collected in Leyte and Samar, Eastern Philippines.

Host Site Gleason’s species richness

Species evenness

Shannon (H’)

Simpson (1/D)

S. alba Leyte 0 - 0 1.00 Samar 0.74 0.92 1.01 1.42

R. mucronata Leyte 0.46 0.99 0.69 1.10 Samar 1.76 0.98 1.76 4.00

A. floridum Leyte nia ni ni ni Samar 0 - 0 1.00

A. marina Leyte 0.91 0.94 1.3 1.89 Samar ncb nc nc nc

ani = no MFE isolated from host; bnc = not collected (host not present in site)

the MFEs based on phylogenic analysis was supported by morphological data.

The percent colonization rate of the MFEs was determined per host mangrove (fig. 11). The highest colonization rate was observed in Avicennia marina, found only in Leyte. When the three species of mangroves present in the two sites were compared, MFEs had higher colonization rates in the host mangroves from Samar. In fact, Aegiceras floridum from Leyte did not yield any MFEs. For the host mangroves where MFEs were isolated, Sonerratia alba from Leyte had the lowest total colonization rate, with only one species of fungal endophytes isolated. The absence or presence of a small number of fungal isolates from these mangroves could be due to the stringent surface-sterilization protocol used in the study. Table 2 shows the distribution of the different MFEs among the mangrove hosts from the two sites. Only Phomopsis pittospori was isolated from the same mangrove species found in the two sites. Colletotrichum tropicale had the highest colonization frequency in Avicennia marina from Leyte (table 3). This was followed by C. queenslandicum on the same host. Guignardia mangiferae also had high colonization frequency on Sonneratia alba collected in Samar. Note, however, that different mangrove species hosted different MFE species. Among these mangroves, diversity indices in table 4 show that R. mucronata from Marabut, Samar, had the highest MFE species richness with six species. The same host had also the highest diversity as shown by the Shannon and Simpson indices. In terms of species evenness, the computed values for all hosts were all higher than 0.90, which means that there were no dominant fungal species living within the mangrove hosts.

MIC and MBC assays showed Pestalotiopsis adusta crude culture extract as the most potent against P. aeruginosa with an MIC of 80 µg/ml (table 5). Against S. aureus, an MIC of 630 µg/ml was observed with three MFEs: P. adusta, Colletotrichum


Table 5: Antibacterial activity of mangrove fungal endophytes against P. aeruginosa, E. coli and S. aureus.

MFE

MIC (mg/ml) MBC (mg/ml)

P. aeruginosa

ATCC 27853

E. coli

ATCC 25922

S. aureus

ATCC 25923

P. aeruginosa

ATCC 27853

E. coli

ATCC 25922

S. aureus

ATCC 25923

P. adusta 0.08 1.25 0.63 0.63 5.00 0.63

C. queenslandicum 0.63 1.25 0.63 1.25 2.50 1.25

C. tropicale 0.63 1.25 0.63 5.00 5.00 2.50

M. palmivorus 0.63 1.25 2.50 1.25 5.00 2.50

D. siamensis 1.25 1.25 1.25 2.50 2.50 2.50

G. mangiferae 1.25 2.50 1.25 >5.00 >5.00 2.50

V. brevispora 1.25 1.25 1.25 >5.00 5.00 2.50

V. nigrescens 1.25 1.25 1.25 2.50 2.50 2.50

X. cubensis 1.25 1.25 2.50 2.50 5.00 5.00

Diaporthe sp. 3.13 6.25 6.25 6.25 25.00 25.00

a Other fungal extracts which showed MIC >5.0 mg/ml are not shown here. Values presented are mean of three technical replicates (n = 3). bMIC = minimum inhibitory concentration; MBC = minimum bactericidal concentration.

Table 6: Cytotoxic activity of mangrove fungal endophyte extracts.

MFE IC50 (ug/ml) ± SD

Stationary Agitated

X. cubensis 99.4 ± 19.5 306.2 ± 42.8

Diaporthe sp. 111.5 ± 15.4 157.9 ± 43.3

P. adusta 195.7 ± 13.7 212.4 ± 32.2

C. tropicale 210.5 ± 24.6 226.0 ± 40.6

A. nidulans 213.6 ± 46.7 134.7 ± 31.8

V. nigrescens 216.4 ± 25.6 245.7 ± 23.6

P. pittospori 228.2 ± 26.7 195.2 ± 46.2

C. fructicola 231.7 ± 38.7 218.4 ± 28.2

M. palmivorus 296.8 ± 39.3 >500

A. oryzae 341.3 ± 31.1 223.6 ± 28.7

D. siamensis 483.5 ± 36.5 146.7 ± 26.1

V. brevispora 487.1 ± 49.0 213.6 ± 46.0

C. queenslandicum >500 185.2 ± 41.5

G. mangiferae >500 199.9 ± 29.3

C. rhizophorae >500 319.68 ± 49.68

P. musae >500 426.1 ± 48.52

Etoposide 4.0

queenslandicum, and C. tropicale. Most of the MFEs exhibited an MIC value of 1,250 µg/ml against E. coli. As expected, the MBC values against any of the test bacteria was greater than their MIC values for a majority of the MFEs. Table 6 also shows the activities of the crude culture extracts against K562 myelogenous leukemia cells. Inhibitory activities differed between the incubation conditions. Some MFEs performed

better when they were mass-produced under static conditions while others did well when mass-produced under agitated conditions in a rotary shaker. For example, X. cubensis, grown under stationary conditions, exhibited the lowest IC50 of 99.4 ± 19.5 µg/ml. By contrast, among the MFEs grown under agitated conditions, Aspergillus nidulans had the most potent cytotoxic effect with an IC50 of 134.7 ± 31.8 µg/ml. All other extracts


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Figure 5: (A) Phylogram for Phaeosphaeriopsis obtained from maximum likelihood analysis based on the ITS (internal transcribed spacer region) alignment. Values above the branches represent maximum likelihood bootstrap values and posterior probability from Bayesian inference. Sequences of mangrove fungal endophyte (MFE) isolates obtained in this study and the closely related taxa are highlighted in yellow. (B) Colony of the MFEs identified as Phaeosphaeriopsis musae.

showed IC50 values less than 500 ug/ml in either stationary or agitated conditions or both. Although these values are higher than the standard IC50< 30 ug/ml set by the National Cancer Institute, note that these represent only the crude ethyl acetate extracts from these fungal endophytes. Possibly these extracts, if fractionated with other solvents, may yield better results. DISCUSSION Plant leaves and other plant parts have been traditionally explored for their bioactivities (Laluces et al. 2015; Tan et al. 2014) and may even be used as functional colorants in food (Aril-dela Cruz et al. 2018). Lichens are also tapped for bioactivities against pathogenic microorganisms (Santiago et al. 2010, 2013; de Jesus et al. 2016) and biocontrol of weeds (Gazo et al. 2019). With the rise in cases of cancer and antibacterial resistance, the search for novel drugs from other sources is urgent. Fungal endophytes constitute an interesting group of fungi ideal for the study of their bioactivities. Fungal endophytes

such as Pestalotiopsis were initially reported to produce the potent cytotoxic compound taxol (Strobel et al. 1996). Other fungal endophytes produce antibacterial compounds (Buatong et al. 2011; Bhimba et al. 2012). In our study, we focused on fungal endophytes isolated from mangroves collected in Leyte and Samar in Eastern Philippines. Our results showed that MFEs varied from one host mangrove to another and exhibited varying degrees of bioactivities.

Mangroves are host to more than 200 species of fungal endophytes, mainly belonging to the genera Alternaria, Aspergillus, Cladosporium, Colletotrichum, Fusarium, Paecilomyces, Penicillium, Pestalotiopsis, Phoma, Phomopsis, Phyllosticta, and Trichoderma (Liu et al. 2007). Xylaria and Guignardia were also isolated from mangroves collected in Asia (Pang et al. 2008). These genera including Arthrinium, Lasiodiplodia, and Nodulisporium were also isolated from mangroves collected in Luzon Island (Moron et al. 2018). Not surprisingly, some of these species could also be isolated from mangroves collected in Eastern Philippines (tables 1–3),

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Figure 6: (A) Phylogram for Valsa/Cytospora obtained from maximum likelihood analysis based on the ITS (internal transcribed spacer region) alignment. Values above the branches represent maximum likelihood bootstrap values and posterior probability from Bayesian inference. Sequences of mangrove fungal endophyte (MFE) isolates obtained in this study and the closely related taxa are highlighted in yellow. (B) Colony of the MFEs identified as Valsa brevispora and (C) Cytospora rhizophorae.

Figure 7: (A) Phylogram for Pestalotiopsis obtained from maximum likelihood analysis based on the ITS (internal transcribed spacer region) alignment. Values above the branches represent maximum likelihood bootstrap values and posterior probability from Bayesian inference. Sequences of mangrove fungal endophyte (MFE) isolates obtained in this study and the closely related taxa are highlighted in yellow. (B) Colony and (C) conidia of the MFEs identified as Pestalotiopsis adusta.

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Figure 8: (A) Phylogram for Diaporthe/Phomopsis obtained from maximum likelihood analysis based on the ITS (internal transcribed spacer region) alignment. Values above the branches represent maximum likelihood bootstrap values and posterior probability from Bayesian inference. Sequences of mangrove fungal endophyte (MFE) isolates obtained in this study and the closely related taxa are highlighted in yellow. (B) Colony of the MFEs identified as Diaporthe siamensis. (C) Colony and (D) conidia of MFEs identified as Phomopsis pittospori. (E) Colony and (F) conidia of the MFEs described as a potentially novel species of Diaporthe.

Figure 9: (A) Phylogram for Marasmiellus obtained from maximum likelihood analysis based on the ITS (internal transcribed spacer region) alignment. Values above the branches represent maximum likelihood bootstrap values and posterior probability from Bayesian inference. Sequences of mangrove fungal endophyte (MFE) isolates obtained in this study and the closely related taxa are highlighted in yellow. (B) Colony of the MFEs identified as Marasmiellus palmivorus.

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Figure 10: (A) Phylogram for Aspergillus obtained from maximum likelihood analysis based on the ITS (internal transcribed spacer region) alignment. Values above the branches represent maximum likelihood bootstrap values and posterior probability from Bayesian inference. Sequences of mangrove fungal endophyte (MFE) isolates obtained in this study and the closely related taxa are highlighted in yellow. (B) Colony of the MFEs identified as Aspergillus nidulans and (C) Aspergillus oryzae.

Figure 11: Percent colonization rate of fungal endophytes per mangrove hosts.

although diversity varied between host mangroves and geographic locations (table 4). We also isolated Colletotrichum queenslandicum. This species was first reported from Carica papaya in Queensland, Australia (Weir et al. 2012) and is now reported to be associated with mangroves. Another isolate we identified as Phaeosphaeriopsis musae was previously reported as an endophyte of seagrass (Sakayaroj et al. 2010), but not of mangroves. Some of our MFE isolates, i.e., Verticillium nigrescens and Marasmiellus palmivorus, were also known as pathogens in other plants. Verticillium nigrescens causes Verticillium wilt in alfalfa (Hu et al. 2011), while Marasmiellus palmivorus causes disease in coconuts (Almaliky et al. 2013).

Perhaps these taxa, in spite of their pathogenicity to other plants, may have existed as asymptomatic endophytes in mangroves. The host-endophyte relationship is usually of a balanced antagonism (Schulz et al. 1999). Here a fungal endophyte usually just overcomes the host defense in order to live inside the plant tissues without causing any damage. The host plant may in turn produce metabolites that keep the endophytes in check. Interestingly, fungal endophytes also produce antifungals and antibacterials to reduce competing microorganisms that may colonize their host. This could explain why fungal endophytes are promising sources of bioactive secondary metabolites targeting other pathogenic microorganisms.

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Sonneratia alba Rhizophoramucronata

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MFEs produced bioactive metabolites. Species of Colletotrichum, Penicillium, Aspergillus, and Xylaria, isolated as fungal endophytes, were reported for their antibacterial activities (Chaeprasert et al. 2010). An MFE, Phyllosticta sp., isolated from a Philippine mangrove, also produced cytosporone B, which has antibacterial activity (Tan et al. 2015). In our study, Pestalotiopsis adusta had the most potent antibacterial activity against P. aeruginosa (table 5). P. aeruginosa is a major cause of infection with a high intrinsic resistance to antibiotics (Hancock and Speert 2000). Colletotrichum queenslandicum and Colletotrichum tropicale also showed potent antibacterial activity against S. aureus (table 5). Colletotrichum, Xylaria, and Guignardia have been previously reported to have antibacterial activities (Chaeprasert et al. 2010; Tan et al. 2015). The fungal endophytes under the genera Valsa, Verticillium, and Marasmiellus are reported for their antibacterial activities for the first time in our study. The Aspergillus isolates, though known for their bioactivities, failed to show any antibacterial activity. This shows that fungal endophytes isolated from different mangrove hosts behave differently, for example, in terms of their antimicrobial activity. In terms of cytotoxicity, Xylaria cubensis had the lowest IC50 among all the MFEs (table 6) - a good indication of its potential for further therapeutic studies. Colletotrichum and Xylaria species as shown in our study have also been previously reported to be effective against cancer cell lines (Chaeprasert et al. 2010). Phyllosticta sp. from a Philippine mangrove produced tyrosol C, which is also known for its cytotoxic effects (Tan et al. 2015). Thus a promising future endeavor is to isolate, purify, and identify the bioactive metabolites from our study and determine the exact mode of action by these compounds. In summary our study highlighted the potential of MFEs for drug discovery programs. The differences in the isolated taxa in our study and their varying degrees of bioactivity against test bacteria and cancer cells show the importance of developing a strategy that will look into the different host mangroves in the country. The Philippines, as already mentioned, is home to numerous mangrove taxa. More than 65% of its plants are endemic. These all represent many unique potential hosts for fungal endophytes and an opportunity to explore secondary metabolites for novel and structural diversity. ACKNOWLEDGMENTS Carlo Chris S. Apurillo acknowledges the Philippines’ Department of Science and Technology–Human Resource Development Program (HRDP) for partially funding this study through a graduate scholarship grant. We would like to express our gratitude to Prof. Dr. Hongwei Liu, Institute of Microbiology, Chinese Academy of Sciences, for his technical assistance on the cytotoxicity assay. We also thank the Institute of Microbiology, Chinese Academy of Sciences (IMCAS), and the Research Center for Natural and Applied Sciences, University of Santo Tomas, for using their laboratory facilities. CONFLICT OF INTEREST The authors declare no conflict of interest. CONTRIBUTIONS OF INDIVIDUAL AUTHORS Carlo Chris S. Apurillo and Thomas Edison dela Cruz performed the planning, experimentation, data analysis, and manuscript writing. Lei Cai assisted in the molecular identification of the isolates.

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