There were four fungal species in the genus Grovesiella M. Morelet. But two fungal species - G. abieticola and G. grantii - are remained in this genus since G. ericae and G. ledi have been reclassified as Erissonopsis ericae and Godronia ledi, respectively [1, 2]. Until now, there has been no description of Grovesiella spp. in Korea.
The genus Grovesiella is defined as a solid black apothecium and club-shaped ascus with eight ascospores. In the asexual stage, it will characteristically have cylindrical macroconidia with septate and oval microconidia without septate [3].
Alpine conifers are vulnerable to climate change and are directly affected by global warming [4]. Therefore, the Ministry of Environment (National Institute of Ecology) is conducting an effective conservation process for alpine conifers through basic ecological studies on vulnerable species including Abies koreana.
Based on this context, this study focuses on the biodiversity of endophytic fungi in alpine conifers. So we try to elucidate the classification and ecological matters of endophytic fungi on fir. Through this, we have discovered an unrecorded endophytic fungus G. abieticola in A. koreana and reported it.
Needle leaves of A. koreana were harvested from Mt. Halla (33° 20' N, 126° 31' E, 1,952 m) in the Jeju Special Self-governing Province of Korea in 2018. The samples were transported in a zipper bags, and fungi were isolated within 24 hours.
All samples were washed with tap water and cut into 1-cm pieces. Surface sterilization was performed; they were immersed in 96% ethyl alcohol for 1 minute, sodium hypochlorite for 3 min, and then 96% ethyl alcohol for 30 seconds; finally, they were washed twice with sterilized water.
Each sample was placed on potato dextrose agar (PDA, MBcell, Seoul, Korea) and incubated in the dark for 4 weeks at 25℃ to isolate endophytic fungi [5].
PDA and maltose extract agar (MEA, MBcell, Seoul, Korea) media were used for the pure culture of endophytic fungi.
The fungi’s macroscopic and microscopic features were measured by light microscopy (DM2500, Leica Microsystems, Wetzlar, Germany). Finally, this unrecorded endophytic fungus was deposited in the Korean Collection for Type Cultures (KCTC).
Genomic DNA was extracted from the fungus using a plant tissues genomic DNA extraction kit (Xi’an Tianlong Science & Technology, Shaanxi, Taiwan) following the manufacturer’s instructions.
Polymerase Chain Reaction (PCR) was performed using primers ITS1 (internal transcribed spacer 1) and LR3, which can selectively amplify from the ITS1 region to the D2 region of 28S ribosomal DNA [6, 7].
The conditions of PCR were as follows: Pre-denaturing for 5 minute at 94℃ with 1 cycle, denaturing for 30 seconds at 94℃, annealing for 30 sec at 50℃, extending for 1 min at 72℃ in 30 cycles, and then finally stabilizing for 10 min at 72℃ in 1 cycle. The PCR product was confirmed by electrophoresis using 1.5% agarose gel.
DNA sequencing was applied to Macrogen (Seoul, Korea), and the analyzed DNA sequence was then identified based on similarity with the National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov/) using the Basic Local Alignment Search Tool (BLAST). A maximum-likelihood tree was generated by MEGA 10.0.5 based on the Kimura-2 parameter distance model with the 1,000-times bootstrap method [8].
Grovesiella abieticola (Zeller & Goodd.) M. Morelet & Gremmen
[MB#314737]
The colony diameter was <1.0 mm on PDA and MEA after 7 days. Mycelium hardly grew in either media. However, the colony diameter was 49.5-51.2 mm on PDA after 300 days and the mycelium was dense. The surface color is brownish black (Munsell color notation: 5YR 3/1) to brownish gray (Munsell color notation: 7.5GY 6/2) [9] with undulate margin, velvety texture at the center, whitish aerial hyphae at the margin, and no exudates. The reverse color is bluish black (Munsell color notation: 10PB 3/2) to dark grayish blue (Munsell color notation: 5PB 3/2) [9]. Macroconidia are 31.5-42.5×1.6-2.8 μm (n=20), cylindrical with rounded-to-pointed ends, hyaline and dyed well with lactophenol cotton blue and have 2-3 septa inside. Microconidia are 5.0-7.3×2.6-3.1 μm (n=20), ovoid, hyaline, and dyed well with lactophenol cotton blue without a septum inside (Fig. 1 and 2).
Specimen examined: Mt. Halla, Jeju special self-governing province, Korea, 2018.5.14., isolated from leaves of Abies koreana, strain NIE7113, KCTC no. 56675, GenBank no. MT157258.
Note: According to the Index of Fungi (1971-1980), three species-G. abieticola, G. ericae, and G. ledihave been reported in the genus Grovesiella [1]. After that, Funk discovered G. grantii on Abies grandis (Douglas ex D. Don) Lindl. in 1977 [2], however in 1971 Morelet reclassified G. ericae as Erikssonopsis ericae [10] and in 1885 Krasten also reclassified G. ledi as Godronia ledi [11]. Therefore, a total of two species have been remained in this genus to date. This genus has few molecular studies compared to other taxa outside the ITS barcode region. The ITS barcode region in this species showed 96.3% similarity with G. abieticola (KX358852.1). In this study, large subunit ribosomal nucleic acid (LSU rDNA) were aligned and loaded into NCBI but there are no reference sequences to compare with this study. The distribution region of G. abieticola has been reported in the United States, Canada, France, and the United Kingdom (Scotland). In addition, host plants in which G. abieticola has been found include Abies alba Mill., A. amabilis Douglas ex J. Forbes, A. balsamea (L.) Mill., A. concolor (Gordon) Lindley ex. Hildebrand, A. grandis (Douglas ex D. Don) Lindley, A. lasiocarpa (Hooker) Nuttall, Picea abies (L.) H. Krast., and A. procera Rehder. Host plants are all woody plants [3, 12]. The present study confirmed for the first time that this fungus is distributed in Korea (Asia) and can be isolated from A. koreana in the genus Abies. Further observation of the fungus presence is also needed in other Korean fir plants such aAs. holophylla Maxim. and A. nephrolepis (Truatv.) Maxim. Finally, Sieber and Kowalski commented on Agyriellopsis caeruleoatra Höhn isolated from A. alba in Switzerland as the same species as G. abieticola [12] but there were no morphological characteristics or DNA barcode regions for reference, so further studies are needed on the identity of A. caeruleoatra.
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Table 1.Morphological characteristics of Grovesiella abieticola NIE7113 isolated from needle leaves of Abies korenana 
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Specimen examined: Mt. Halla, Jeju special self-governing province, Korea, 2018.5.14., isolated from leaves of Abies koreana, strain NIE7113, KCTC no. 56675, GenBank no. MT157258.
