View of New Report of Endophytic Sordariomycetes from the Seagrasses of Cebu, Central Philippines

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http://dx.doi.org/10.11594/jtls.13.03.14 Research Article

New Report of Endophytic Sordariomycetes from the Seagrasses of Cebu, Central Philippines

Venus Bantoto-Kinamot ¹*, Alvin Monotilla ²

1 Biology Department, Negros Oriental State University, 6200 Dumaguete City, Philippines

2 Biology Department, University of San Carlos, 6000 Cebu, Philippines

Article history:

Submission October 2022 Revised November 2022 Accepted December 2022

ABSTRACT

Sordariomycete is one of the dominant components of the endophytic community in seagrasses. However, there are still many species under this group that still need to be described. Among the tropical seagrasses, the Philippines had the least number of endophytic Sordariomycete reported. This study reports the isolation of endophytic Sordariomycete from Enhalus acoroides, Cymodocea serrulata, and Thalassia hemprichii. Seagrass samples were collected from the coastal areas of Hilutungan Channel, Cebu, Central Philippines using a sterilized razor. Explants from seagrass samples were surface-sterilized, inoculated in culture plates, and incubated for 14 days at 27±2°C. Fungi that grew from the edge of explants were purified and identified through morphological and molecular methods using ITS sequences. Molecular phylogeny was inferred by neighbor-joining with 1000 bootstrap replications. Three species of Sordariomycete were identified in this study. Fungal isolates from the rhizomes and roots of Cymodocea serrulata had 99.84% nucleotide similarity with Eutypella sp. Isolates from the rhizomes and roots of Enhalus acoroides had a 100% match identity with Beauveria bassiana. While the isolate from the leaf of Thalassia hemprichii had a 99% nucleotide affinity to Xylaria apoda. These isolates formed a well-supported clade with their closest match taxa at 100% bootstrap support. The estimate of evolutionary divergence between the isolates’ sequences and their reference taxa was 0.0, suggesting no nucleotide base substitutions per site.

The identification of seagrass isolates as Eutypella sp., Beauveria bassi- ana, and Xylaria apoda is hereby supported. Phylogenetic analysis revealed that endophytic fungi from the Philippine seagrass did not cluster with any fungal species previously reported from the Philippines, suggesting different species. To our knowledge, Eutypella sp., Beauveria bassiana, and Xylaria apoda were the first reports of endophytic Sordariomycete in Philippine seagrasses. The isolation of these endophytes is promising because these genera are a rich source of novel compounds and biocontrol agents.

Keywords: Endophytic fungi, Philippines, Seagrass, Sordariomycete

*Corresponding author:

E-mail: [email protected]

Introduction

Fungi are one of the most diverse groups of organisms on earth, with a total species estimate ranging from 2.2-3.8 million based on host associ- ation [1]. Among the class with significant diversity is Sordariomycete, with 32 orders, 105 families, and 1331 genera [2]. Members of Sordari- omycete are found in different niches, including inside the plant tissues as endophytes. It was estimated that every plant host has 4-5 fungal taxa [3]

in which Sordariomycete is dominant. In Enhalus

acoroides of Thailand, 85% of the endophytic fungal isolates were Sordariomycete. In Cymodocea serrulata and Thalassia hemprichii, 67% and 54%

were Sordariomycete, respectively [4]. They are prolific producers of bioactive secondary metabolites, which could be potential sources of medicine like xylariphilone, a new azaphilone derivative from seagrass-derived Xylariales [5].

Despite the diversity of Sordariomycete, many taxa of this group are underrepresented and refer-

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ence sequences in databases need to be included [6, 7, 8]. For instance, Xylaria comprises at least 1340 species, but most were unrecognized [9]. As a result, some proposed orders were referred to as insertae sedis, like Xylariomycetidae [10], Co- niochaetales, and Cordanales [11], because the classification was not supported by phylogenetic analyses [2]. Other fungal isolates cannot be identified at the species level because no taxa matched the sequence in the database. For instance, in seagrasses, the fungal isolates PSU-ES106, PSU- ES116, and PSU_ES147 from E. acoroides were classified at the family level as Xylariaceae [6].

In Zostera marina, three fungal isolates (CLE105, CLE153, CLE61) were classified at the ordinal level as Hyprocreales sp. [8]. Greater sampling, especially in tropical regions, is recommended to provide a stable classification [7].

Philippines is considered a hotspot of biodiversity. However, only Trichoderma was reported as an endophyte of seagrasses under Sordariomy- cete [12]. In comparison, E. Acoroides in Thailand had 16, T. hemprichii had 15, and C. serrulata had four fungal strains classified as Sordariomy- cete [4]. This means many fungal taxa must still be discovered from the Philippine seagrasses. In this study, three more species that belong to Eutypella, Beauveria, and Xylaria were identified from E. acoroides, T. hemprichii, and C.

serrulata. These genera still needed to be reported in Philippine seagrasses. The previous reports of Eutypella sp. were from Garcinia species [13], lotus, Nelumbo nucifera [14]), Camellia sinensis [15], and Centella asiatica [16]. Xylaria was isolated from terrestrial plants like Moringa oleifera [9] Casuarina equisetifolia, Pinus kesiya [17], and Usnea spp. [18, 19], and mangroves [12, 20, 21, 22]. On the other hand, B.

bassiana was isolated from Tedania species, a mangrove associated sponge [23]. The species of Sordariomycete identified in this study serve as a new update to Philippine marine mycoflora.

Material and Methods Seagrass sample collection

Seagrass samples were collected from the coastal areas of Hilutungan Channel, Central Phil- ippines (10°16’20” N, 124° 0’ 50” E) using a sterilized razor. A total of 15 sampling stations were set up perpendicular to the shore, where each station was 100 m long. Five quadrats having an area of 30 × 30 cm were laid in every station. Live and

intact seagrasses identified as E. acoroides, C. ser- rulata, and T. hemprichii inside the quadrat were collected. The whole plant was cut using a sterilized razor, placed in a plastic container, and brought to the University of San Carlos-Marine Research Station. In the laboratory, the samples were washed with filtered seawater until the epi- phytes were removed. The whole seagrass plant was then divided into 3 parts, leaves, roots, and rhizomes. Each part was used to prepare 3-5mm explants.

Isolation of endophytic fungi associated with seagrass

All explants were surface-sterilized using 10%

ethanol (EtOH) for 3 mins, 3% sodium hypo- chlorite (NaClO) for 10s, 10% EtOH for 3 mins, and finally washed twice with sterile distilled water and blotted dry with a sterile tissue paper [6].

Sterilized explants from the three parts of each seagrass species were inoculated in a culture plate with potato dextrose agar (PDA) containing chlo- ramphenicol (150 mg/L). Inoculated plates were then incubated at 25 ± 2°C for 14 days.

For the effectivity of surface sterilization and sterility check, tissue printing and exposure of un- inoculated plates to air were performed, respectively. Fungal colonies were sub-cultured on a new culture medium and purified by the hyphal tip method. In this method, the hyphal tips of each fungal isolate were transferred onto a new culture medium using a sterile loop. The purified strains were then transferred to PDA slants for further study.

Morphological identification of the fungal endo- phytes

Seventy-nine (79) pure fungal isolates were identified based on macroscopic and microscopic characteristics. The macroscopic characteristics include colony color, form, texture, and margin of the colony. Microscopic characteristics of each purified strain were examined using a light micro- scope and characterized based on the types of veg- etative hyphae, diameter of the hyphae, conidia, and conidiophore. Measurement of the microscopic structure was done using ImageJ software.

Scientific articles were used as references in the morphological identification [24, 25, 26]. Each isolate identified by morphological features was then processed for molecular identification.

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DNA extraction, sequencing, and bioinformatics analyses

Using a sterile loop, the mycelia of a 1-week pure culture was transferred to a 1.5 µl microcen- trifuge with nuclease-free water and sent to Mac- rogen (Korea) for DNA extraction, amplification, and sequencing. The genomic DNA of each endophytic fungal isolate was extracted using In- staGene Matrix (Bio-Rad). The DNA fragments were amplified in DNA Engine Tetrad 2 Peltier Thermal Cycler using Internal Transcribed Region (ITS). ITS5 (5’-GGAAGTAAAAGTCG- TAACAAGG-3’) was the forward primer, while ITS4 (5’- TCCTCCGCTTATTGATATGC-3’) was the reverse primer [27]. The PCR cycle started with an initial denaturation at 95°C for 5 mins, then 35 cycles of denaturation at 95°C for 30 secs, primer annealing at 55°C for 30 secs, extension at 72°C for 1 min, and final extension at 72°C. PCR products were purified using a multiscreen filter plate and sequenced using BigDye (R) Terminator v3.1 Cycle Sequencing Kit in an ABI PRISM 3730XL Analyzer following the manufacturer’s protocol.

Sequences were edited using BioEdit Se- quence Alignment Editor 7.2, and sequences' contig assembly was done using the cap contig assembly program. The sequences were then analyzed for the most probable closely related taxa using BLAST search. The subsequent phylogenetic analysis used the most similar sequences (99- 100%) and sequence data from publications.

Phylogenetic tree construction and analysis A total of 59 ITS sequences of closely- matched Xylaria, Beauveria, and Eutypella from GenBank were aligned using MUSCLE in Molec- ular Evolutionary Genetic Analysis (Mega) ver- sion X software [28]. To check if our sequences

are phylogenetically related to the previously reported endophytic Sordariomycete from the Phil- ippines, species of Xylaria, Beauveria, and Eu- typella published in scientific journals were also included as reference taxa [12, 17, 18, 19, 20, 21, 22, 32]. The phylogenetic tree was constructed using neighbor-joining with 1000 bootstrap replications using the Kimura-2 parameter model. Dis- crete gamma distribution (+G) with 5 categories was used to model the non-uniformity of evolutionary rates among sites. The estimate of evolutionary divergence between sequences was deter- mined by the number of base substitutions per site based on pairwise distance. The between-group distance was also calculated. The site coverage cut-off was set at 80% and ambiguous bases, miss- ing data, and less than 20% alignment gaps was partially deleted.

Results and Discussion

Philippines is one of the countries in Southeast Asia with the largest seagrass meadows having as many as eight species [29]. Aside from the ecological importance of seagrasses, it was also reported to host endophytic fungi [12]. In this study, four endophytic fungal isolates were recorded; CRz1, CRt2, ERt2, and TL2. CRz1 and CRt2 were isolated from the rhizomes and roots of C. serrulata, respectively. Their colony color was white at the surface, cream with black center reverse, flat, cir- cular, cottony, and plumose. Growth was 40 - 60mm in 7 days and not sporulating in culture (Figure 1).

Isolate ERt2 had white to cream colonies with irregular edges and a powdery appearance. The hyphae were septate and measured 1.152 µm in diameter. The conidiophores were densely clustered in a whorl. The conidiogenous cells had a mean diameter of 5.197 µm (SD= 1.241 µm) with zigzag

Figure 1. Colony and microscopic characteristics of CRz1 and CRt2 isolates in PDA at 25°C for 14 days. (A) Surface view of the colony, (B) Reverse view, (C) Hyphae viewed with high power objective (400×).

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rachis in which a chain of conidia emerged. The conidia were globose measuring 0.533 µm in diameter (SD= 0.219 µm). Sporulation was high and growth was dispersed (Figure 2). The characteristics of this isolate were typical of the genus Bassi- ana as described in terrestrial plants [25, 26].

Isolate TL2 had a white colony that turned black starting from the center when it matured. It had conspicuous radial lines and plumose margins.

It had septate hyphae with a mean diameter of 0.711 µm. The conidia were ovoid to clavate, measuring 2.93 µm (SD = 0.46 µm). Sporulation was very low (Figure 3). This isolate is morphologically similar to Xylaria apoda from Den- drobium spp. [24].

BLAST search revealed that isolates CRz1, CRt2, ERt2, and TL2 had >99% nucleotide similarity with Eutypella sp., Beauveria bassiana, and Xylaria apoda, respectively (Table 1). The molecular phylogeny of our isolates formed a well-supported clade with their closest-matched taxa at 100% bootstrap support. CRz1 and CRt2 clustered with the clade of Eutypella sp. ERt2 shared the same clade with B. bassiana and TL2 was within the clade of Xylaria apoda (Figure 4). The estimate of evolutionary distance was 0.00 suggesting

no nucleotide base substitution per site between our isolates’ sequences and their closest matched taxa. Thus, CRz1 and CRt2 are identified as Eu- typella sp., ERt2 as B. bassiana, and TL2 as Xylaria apoda.

Eutypella sp. is less reported as an endophyte.

But interestingly, in this study, a well-supported clade of Eutypella sp. included the endophytes of Garcinia sp. (Accession number: DQ480354), Ne- lumbo nucifera (Acc. No. KT868952) and Camel- lia sinensis (Acc. No. KP743030) (Figure 4). This suggests that this species may colonize several hosts as an endophyte. Previous report of endophytic Eutypella sp. was on terrestrial plants [13, 14, 16]. It is the first time that Eutypella sp. was isolated as an endophyte of seagrasses. The inclu- sion of Eutypella sp. as marine-derived fungi was reported by Jones et al. [30]. The clustering of CRz1 and CRt2 with Eutypella sp. from marine sources like a marine sponge (Acc. No.

MH729005, MH729003, JQ922165) and coral (Acc. No HM486432) supported that this species colonized and adapted the marine habitat.

B. bassiana was reported as an endophyte of Posidonia oceanica [31] and Zostera spp. [32, 33], however, it was identified morphologically. Thus, Figure 2. Colony and microscopic characteristics of ERt2 isolate in PDA at 25°C for 14 days. (A) Surface view

of the colony, (B) hyphae viewed with high power objective (400×) (C) Reverse view of the colony, (D) Conidiophore viewed with high power objective (400×).

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it was difficult to track the phylogenetic relation- ship of this species with our isolates from Philip- pine seagrasses. Phylogenetic analysis using sequences of Hypocreales from E. acoroides (Acc.

No. JN116711) in Thailand [6] and Zostera ma- rina (Acc. No. MN543958, MN543932) in the USA [21] showed low nucleotide similarity (Fig- ure 4). The p-distance between these sequences was quite high ranging from 0.3 - 0.4 and the between-group distance was 0.238. Moreover, our

isolate did not cluster with any Sordariomycete previously reported from the Philippines and the pairwise distance between sequences was 0.24.

Thus, B. bassiana from E. acoroides in the Philip- pines is a different strain from the previously reported Hypocrealean species.

Xylaria has been reported in several plants as well as lichen hosts in the Philippines. Two species of Xylaria were reported from the leaves of terrestrial plants [17, 34], 14 from lichens [18, 19]

Figure 3. Colony and microscopic characteristics of TL2 isolate. (A) Surface view of the colony in PDA at 25°C for 14 days, (B) Reverse view, (C) Hyphae at 400x, (D) 7-day old colony, (E) 28-day old colony with characteristic black coloration, (F) Spore at 400x.

Table 1. Isolation source and identification of the fungal isolates from the three seagrass species Isolate

Code

GenBank Accession

ID Source Morphological

Identification

Molecular Identification

by BLAST search

GenBank Accession No.

of the Closest Matched taxa

Match Identity

(%) CRz2 ON627708 Cymodocea

serrulata

rhizome Sterile mycelia Eutypella sp. MK775825 99.84

CRt2 ON627708

Cymodocea serrulata rhizome and roots

Sterile mycelia Eutypella sp. MK775825 99.84

ERt2 ON507746

Enhalus acoroides roots

and rhizomes Beauveria Beauveria

bassiana MT530083 100.00

TL2 ON644448 Thalassia hemprichii leaves

Xylaria Xylaria apoda MZ423071 99.29

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and three from the leaves, stems, and roots of mangroves [20, 21, 22]. In this study, our Xylaria isolate formed a separate clade. The bootstrap support with any of the Xylaria sequences previously reported from the Philippines was 22% (Figure 4).

Further, our isolate did not cluster with any of the Xylariaceae species (Acc. No. JN116641, JN116712, JN116632) from the seagrasses of

Thailand [6]. This suggests that our isolate which is identified as Xylaria apoda is a different species of endophytic Xylariaceous fungi colonizing the seagrasses. This observation was similar to Pidoplitchkoviella terricola in that it formed a separate sub-clade from the reference strain indicat- ing a difference in phylogenetic strains [35].

The distribution of the three species of endo- Figure 4. A molecular phylogeny of ERt2, CRz1, CRt2 and TL2 based on neighbor-joining inferred from 59

ITS sequences. Fungal isolates from seagrasses are in black box. The phylogenetic tree was rooted with the Pleurocatena brevior as outgroup.

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phytes in the tissues of its host implied their preference in the particular microenvironment. Eu- typella sp. was isolated in the roots and rhizomes of C. serrulata. This is parallel with the findings of Zhou et al. [36] in which Eutypella sp. was dis- tributed only in the roots of mangroves citing its preference in this microenvironment. Likewise, B.

bassiana was isolated from the roots and rhizomes of E. acoroides. In P. oceanica, this species also colonized the roots of the seagrass [37]. Bayesian source tracking classifier showed that root micro- biomes of seagrasses were primarily estimated to be sourced from the sediments [38]. A high colony forming unit (CFU) of B. bassiana was recorded in the soil which may be made entry to the seagrass tissues through the roots [39]. On the other hand, Xylaria apoda was isolated from the leaves of T.

hemprichii. It seemed common for Xylaria to colonize the leaves of its hosts since it was also isolated from the leaves of mangroves [21, 22], and terrestrial plants, Casuarina equisetifolia, Pinus kesiya [17], and Moringa oleifera in the Philip- pines [34]. In Thailand, Xylaria was isolated from the leaves of E. acoroides [6]. The tissue-specific differences like leaf area, root structure, and types of antifungal compounds present in every seagrass host may be the reasons for the distribution of Eu- typella, Beauveria, and Xylaria as observed in other seagrass species. For instance, larger leaf area and dry matter content in Thalassia testudi- num than Halodule wrightii accounted for its high endophytic fungal diversity [39]. Also, the pres- ence of thick-walled hypodermis and the absence of root hairs are considered factors for the colonization of dark septate endophytes in the roots of P.

oceanica not in Cymodocea nodusa even though these two species were often associated with each other in the Mediterranean Sea [40].

The three Sordariomycete isolated in this study were known as prolific producers of bioactive secondary metabolites. Eutypella sp. isolated from marine organisms was discovered to produce novel compounds like sesquiterpenes and cyto- chalasins [41, 42] while Xylaria produced like xylariphilone [5], cytochalasin D [43], and phe- nolics compounds [18]. These compounds were reported to have antimicrobial [18, 20, 44], cytotoxic [22], antifungal [17], and antioxidant activities [18]. On the other hand, B. bassiana produced iron siderophores and solubilize phosphate, pro- moting plant growth [39, 45]. It is also an active biocontrol agent against agricultural pests [46,

47]. It produces bassianolide, beauvericin, and bassiacridin metabolites which are toxic to insects [45]. These three species might also produce bioactive substances in seagrasses which may protect their hosts against microbial infections and pro- mote growth.

Conclusion

Many fungal species that are still undescribed.

In this study, three more species of endophytic fungi in seagrasses under Sordariomycete were identified by morphological and molecular methods: Beauveria bassiana, Eutypella sp. and Xylaria apoda. Based on phylogenetic analysis, these species were different from the previously isolated endophytic Sordariomycete in the Philip- pines. To our knowledge, this is the first report of these species in seagrasses. Exploration of the biotechnological potential of Beauveria bassiana, Eutypella sp., and Xylaria apoda is promising.

Acknowledgment

The authors would like to thank the USC Med- ical Biophysics Laboratory and the Marine Biol- ogy Laboratory for the facilities and assistance.

This study was supported by the Department of Science and Technology-Science Education Insti- tute (DOST-SEI) through the Accelerated Science and Technology Human Resource Development Program (ASTHRDP).

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