First Report on Lophiostoma macrostomum and Pestalotiopsis lespedezae Isolated from Conifer Leaves of Pinus densiflora in Korea

Research Article
Hyeok Park1Ahn-Heum Eom1*

Abstract

In this study, we isolated endophytic fungi from the conifer leaves of Pinus densiflora inhabiting Mt. Gyehwasan in Korea. We identified the isolated fungal strains based on their morphological characteristics and phylogenetic analysis using DNA sequences of the internal transcribed spacer, small subunit rDNA, large subunit rDNA, and β-tubulin region. In the process of this study, we confirmed two endophytic fungi that have not been recorded previously in Korea, Lophiostoma macrostomum and Pestalotiopsis lespedezae. In this report, we described the morphological characteristics of these fungal strains and the results of their molecular analysis.

Keyword



INTRODUCTION

Endophytic fungi have a symbiotic relationship with the plants [1]. 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 [8]. 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 [9]. Then, the small subunit (SSU) region of rDNA, the large subunit (LSU) region, and the β-tubulin (TUB) region were amplified using NS1/NS4 [10], LR0R/LR16 [11], and Bt2a/Bt2b primer set [12], 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 [13]. 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

http://dam.zipot.com:8080/sites/ksom/images/N0320480205_image/Table_KJOM_48_02_05_T1.png

Table 2. DNA sequences used in phylogenetic analysis, including sequences used as the outgroups

http://dam.zipot.com:8080/sites/ksom/images/N0320480205_image/Table_KJOM_48_02_05_T2.png

*SSU: Small subunit rDNA; ITS: Internal transcribed spacer; LSU :Large subunit rDNA; TUB: β-tubulin DNA.

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).

http://dam.zipot.com:8080/sites/ksom/images/N0320480205_image/Figure_KJOM_48_02_05_F1.png

Fig. 1. Colonies of strain 16B060 (Lophiostoma macrostomum) grown for 7 days on potato dextrose agar (PDA) (A, E) and malt extract agar (MEA) (B, F); Conidiophore and chlamydospore (I, J). Colonies of strain 16B115 (Pestalotiopsis lespedezae) grown for 7 days on PDA (C, G) and MEA (D, H); conidiophore and conidia (K, L). (scale bars: I, J=20 μm; K, L=10 μm)

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 [14]. 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 [17]. 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).

http://dam.zipot.com:8080/sites/ksom/images/N0320480205_image/Figure_KJOM_48_02_05_F2.png

Fig. 2. Neighbor-joining phylogenetic tree based on a concatenated alignment of small subunit (SSU), internal transcribed spacer (ITS) and large subunit (LSU) rDNA sequences. Preussia lignicola was used as an outgroup. Numbers on branches indicate bootstrap values (1,000 replicates). Fungal strain isolated in this study is in bold.

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 [18]. There is a report on P. lespedezae isolated as an endophytic fungus from the conifer leaves and bark of Pinus armandii in China [19]. 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 [19]. It has been reported that P. lespedezae inhibits the growth of pathogenic fungi belonging to genus Alternaria, when cultured together in one medium [20]. 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).

http://dam.zipot.com:8080/sites/ksom/images/N0320480205_image/Figure_KJOM_48_02_05_F3.png

Fig. 3. Neighbor-joining phylogenetic tree based on a concatenated alignment of internal transcribed spacer (ITS) and beta-tubulin (TUB) sequences. Seiridium podocarpi was used as an outgroup. Numbers on branches indicate bootstrap values (1,000 replicates). Fungal strain isolated in this study is in bold.

Acknowledgements

This word was supported by a grant from the National Institate of Biologial Resources (NIBR), funded by the Ministry of Eavironment (MOE) of the Republic of Korea (NIBR 201902202)

References

1  1. Carroll G. Fungal endophytes in stems and leaves: from latent pathogen to mutualistic symbiont. Ecology 1988;69:2-9. 

2  2. Gao FK, Dai CC, Liu XZ. Mechanisms of fungal endophytes in plant protection against pathogens. Afr J Microbiol Res 2010;4:1346-51. 

3  3. Schulz B, Boyle C. The endophytic continuum. Mycol Res 2005;109:661-86. 

4  4. Bouton J, Gates R, Belesky D, Owsley M. Yield and persistence of tall fescue in the southeastern coastal plain after removal of its endophyte. Agron J 1993;85:52-5. 

5  5. Rowan DD, Latch GC, Bacon C, White J. Utilization of endophyte-infected perennial ryegrasses for increased insect resistance. Biotechnology of endophytic fungi of grasses 1994:169-83. 

6  6. Uzma F, Mohan CD, Hashem A, Konappa NM, Rangappa S, Kamath PV, Singh BP, Mudili V, Gupta VK, Siddaiah CN. Endophytic fungi-alternative sources of cytotoxic compounds: A review. Front Pharmacol 2018;9:309. 

7  7. Yu H, Zhang L, Li L, Zheng C, Guo L, Li W, Sun P, Qin L. Recent developments and future prospects of antimicrobial metabolites produced by endophytes. Microbiol Res 2010;165:437-49.  

8  8. Park H, Eom AH. Three unreported endophytic fungi isolated from conifer leaves of Pinus densiflora in Korea. Kor J Mycol 2019;47:35-42. 

9  9. Gardes M, Bruns TD. ITS primers with enhanced specificity for basidiomycetes-application to the identification of mycorrhizae and rusts. Mol Ecol 1993;2:113-8. 

10  10. White TJ, Bruns T, Lee S, Taylor J. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR protocols: a guide to methods and applications 1990;18:315-22. 

11  11. Moncalvo JM, Lutzoni FM, Rehner SA, Johnson J, Vilgalys R. Phylogenetic relationships of agaric fungi based on nuclear large subunit ribosomal DNA sequences. Syst b iol 2000;49:278-305. 12. Glass NL, Donaldson GC. Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous ascomycetes. Appl Environ Microbiol 1995;61:1323-30. 

12  12. Glass NL, Donaldson GC. Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous ascomycetes. Appl Environ Microbiol 1995;61:1323-30. 

13  13. Kumar S, Stecher G, Tamura K. MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 2016;33:1870-4. 

14  14. de Cesati V, de Notaris G. Schema di classificazione degli sferiacei italici aschigeri piu’o meno appartenenti al genere Sphaeria nell’antico significato attribuitoglide persoon. Erbario c rittogamico italiana 1863;1:177-240. 

15  15. Tanaka K, Harada Y. Pleosporales in Japan (1): The genus Lophiostoma . Mycoscience 2003;44:85-96. 

16  16. Thambugala KM, Hyde KD, Tanaka K, Tian Q, Wanasinghe DN, Ariyawansa HA, Jayasiri SC, Boonmee S, Camporesi E, Hashimoto A. Towards a natural classification and backbone tree for Lophiostomataceae, Floricolaceae, and Amorosiaceae fam. nov. Fungal Divers 2015;74:199-266. 

17  17. Hirayama K, Tanaka K. Taxonomic revision of Lophiostoma and Lophiotrema based on reevaluation of morphological characters and molecular analyses. Mycoscience 2011;52:401-12.  

18 18. Bilgrami K. Leaf spot diseases of some common ornamental plants. P Natl A Sci India A 1963;33:429-52.  

19 19. Hu H, Jeewon R, Zhou D, Zhou T, Hyde KD. Phylogenetic diversity of endophytic Pestalotiopsis species in Pinus armandii and Ribes spp.: Evidence from rDNA and β-tubulin gene phylogenies. Fungal Divers 2007;24:1-22 

20  20. Miao CP, Mi QL, Qiao XG, Zheng YK, Chen YW, Xu LH, Guan HL, Zhao LX. Rhizospheric fungi of Panax notoginseng: Diversity and antagonism to host phytopathogens. J Ginseng Res 2016;40:127-34.