Endophytic fungi have a symbiotic relationship with the plants . They live in healthy tissues of the plants and do not cause disease unless there is a stressful condition in the plant [2,3]. However, they can protect the hos plants from pathogens or predators through the production and secretion of secondary metabolites such as alkaloids [4,5]. In particular, since these metabolites can exhibit bioactive effects such as anti-proliferative or anti-microbial properties in plant as well as in human body [6,7], it is essential to confirm the diversity of endophytic fungi and secure the strains that can produce metabolites. In this study, we described the morphological characteristics and the results of molecular analysis of two unrecorded endophytic fungi isolated from the conifer leaves of Pinus densiflora S. et Z.
MATERIALS AND METHODS
The sampling was conducted in April 2016, around Gyehwasan mountain (N35°47'18.6", E126°37'56.1") in Buan-gun, Jeollabuk-do. We sampled healthy conifer leaves without any symptoms. Sampled leaves were placed in polyethylene bags and transported to the laboratory within 24 h. After washing the leaves with distilled water, they were sterilized in 1% NaClO solution for 1 min and 70% EtOH for 2 min, and then divided into appropriate sizes to place into potato dextrose agar (PDA) medium . The culture medium was incubated for 3 d or more in a dark place at 25℃ until the mycelium grew out, after which, it was subcultured in the fresh PDA medium using a scalpel to obtain pure culture. Morphological characteristics were observed on a dissecting microscope and an optical microscope after culturing for 7 d in PDA and malt extract agar (MEA) medium, respectively (Table 1). For the identification of fungal strains, genomic DNA was extracted from hyphae using the DNeasy plant mini kit (Qiagen, USA) according to the manufacturer’s instructions, followed by amplification of the internal transcribed spacer (ITS) containing 5.8S region by PCR using ITS1F and ITS4 primers . Then, the small subunit (SSU) region of rDNA, the large subunit (LSU) region, and the β-tubulin (TUB) region were amplified using NS1/NS4 , LR0R/LR16 , and Bt2a/Bt2b primer set , respectively. During PCR, the annealing temperature was set to 50, 40, 44, and 55℃ for ITS, SSU, LSU and TUB, respectively. After the PCR, agarose gel electrophoresis was performed on 1.5% agarose gel for 20 min to confirm the size of each DNA fragment, followed by DNA sequencing (SolGent, Daejeon, Korea). We used BLAST on the National Biological Information Center (NCBI) to confirm the similarity of the species and presence of the reference DNA sequences (Table 2). The phylogenetic tree was constructed by using neighbor-joining method by concatenating DNA sequences of two or three regions, using a MEGA7 program . The identified fungal strains were deposited to the National Institute of Biological Resources (NIBR, Incheon, Korea). The DNA sequences used for the identification were submitted to the NCBI by obtaining the accession number.
Table 1. Morphological characteristics of fungal strains isolated from conifer leaves of Pinus densiflora
RESULTS AND DISCUSSION
Lophiostoma macrostomum (Tode) Ces. & De Not., Commentario della Società Crittogamologica Italiana 1:219 (1863) MB#149287
The colonies cultured for 7 d in the PDA medium had the diameters of about 14 to 16 mm. The top side of colony was observed to be bright gray, and the bottom side was observed to be white in color. The colonies were slightly elevated the medium, and their margin was close to a crushed oval. Aerial mycelium was slightly covered at the center of the colony (Fig. 1A and 1E). Diameters of the colonies cultured for 7 d in the MEA medium were about 11 to 12 mm, indicating slower growth than in the PDA. The color of the colonies was observed to be white on both the obverse and the reverse. The colonies were slightly raised upward the medium, and their margin was irregular (Fig. 1B and 1F). Conidiophores were circular, formed at the end of the hyphal growth (Fig. 1I), which swelled up to develop into a chlamydospore (Fig. 1J). Chlamydospores were observed to be globose or cylindrical, hyaline, and colorless, with 0-1 septate, (12.65-) 13.90 (-15.02)×(9.14-) 12.16 (-14.24) μm in diameter (n=10).
Specimen examined. Mt. Gyehwasan, Buan-gun, Jeollabuk-do, Korea, N35°47'19.4", E126°37'54.0", April 8, 2016, Lophiostoma macrostomum, isolated from conifer leaf of Pinus densiflora, strain 16B060, NIBRFG0000503375, GenBank Nos. MT323102 (SSU), MT323120 (ITS), and MT323121 (LSU).
Notes. L. macrostomum was first reported as a Sphaeria macrostoma by Tode in 1791 and became a type species of the genus Lophiostoma in 1863 when the genus Lophiostoma was suggested by Cesati & De Notaris . Only the teleomorph forming the asci was recorded in the original literature and other references [14-16]. However, we confirmed anamorph and chlamydospores in this study. There is a record about this species isolated from the branches of mulberry (Morus bombycis Koidz.) as an endophytic fungus in Japan . The BLAST result showed that the SSU rDNA sequence of 16B060 was closely related to that of L. macrostomum AB521731.1 with 98.92% similarity, the ITS sequence was closely related to that of L. macrostomum MH389072.1 with 99.40% similarity, and the LSU sequence was closely related to that of L. macrostomum EU552140.1 with 99.52% similarity. The concatenated DNA sequences of 16B060 also formed a monophyletic group with those of L. macrostomum, strains KT635 and KT709 (Fig. 2).
Pestalotiopsis lespedezae (Syd.) Bilgrami, Proceedings of the National Academy of Sciences of India 33:441 (1963) MB#336017
The diameters of the colonies cultured for 7 d in PDA medium were about 38 to 40 mm, and the color was observed to be bright white on both the obverse and the reverse. The colonies were slightly raised upward the medium. The aerial mycelium was highly covered; therefore, the overall shape of the margin was close to the entire (Fig. 1C and 1G). The diameters of the colonies cultured for 7 d in the MEA medium were about 33 to 35 mm. The obverse of the colony was observed to be ivory at the center, and white at the margin. The reverse of the colony was observed to be dark yellowish-brown at the center, and yellow at the margin. The colonies were slightly raised upward the medium, and aerial mycelium spread irregularly toward the margin (Fig. 1D and 1H). At the distal end of the hyphal growth, the formation of cylindrical conidiophore was observed. Conidiophores were hyaline, with 1-2 septate (Fig. 1K), and developed into the fusiform conidia. Conidia were translucent, pale brown, with 3-4 septate, and the color of the middle cells appeared darker than the base (Fig. 1L), (11.22-) 13.34 (-17.63)×(5.31-) 6.11 (-7.24) μm in diameter (n=20).
Specimen examined. Mt. Gyehwasan, Buan-gun, Jeollabuk-do, Korea, N35°47'19.4", E126°37'54.0", April 8, 2016, Pestalotiopsis lespedezae, isolated from conifer leaf of Pinus densiflora, strain 16B115, NIBRFG0000503376, GenBank No. MT323122 (ITS), MT613344(TUB)
Notes. P. lespedezae was first reported as Pestalotia lespedezae in 1900 and then recombined into genus Pestalotiopsis by Bilgrami in 1963 . There is a report on P. lespedezae isolated as an endophytic fungus from the conifer leaves and bark of Pinus armandii in China . The length of the conidia was slightly longer than that isolated in the present study; however, the color of the middle cells was darker than the base, and the fusiform conidia that divided by the septa were consistent with that isolated in this study . It has been reported that P. lespedezae inhibits the growth of pathogenic fungi belonging to genus Alternaria, when cultured together in one medium . The BLAST result showed that the ITS sequence was closely related to that of P. lespedezae EF055203.1 with 99.01% similarity and the β-tubulin DNA sequence was closely related to that of P. lespedezae EF055243 with 99.33% similarity. The concatenated DNA sequences of 16B115 also formed a monophyletic group with the concatenated sequences of P. lespedezae, strains SY16E and YY12A (Fig. 3).