INTRODUCTION

Since long time, apples and pears have been in the spotlight as internationally important crops to the extent that more than 100 million tons of apples and pears are produced worldwide annually [1-3]. In 2020, Korea produced approximately 550, 000 tons of apples and pears. Soil is indispensable for growing apple and pear trees. Soil supports the roots of trees and stores moisture; various interactions, such as helping or hindering the growth of trees by various rhizosphere microorganisms, occur in the soil [4,5].

Diverse fungi infect apple and pear trees [6]. Orchard soil serves as a reservoir for these pathogenic fungi, as they can overwinter in soil. We investigated fungi from apple and pear trees and orchard soils in diverse places to monitor diseases. Diverse fungi, including pathogenic fungi, were identified. Among the identified fungi, seven species belonging to the phylum Ascomycota have not been recorded in Korea. In this paper, we report the full descriptions and provide illustrations of their morphological characteristics and molecular phylogenetic positions.

MATERIALS AND METHODS

Collecting rhizosphere soil samples: The rhizosphere soils of apple and pear trees were collected from orchards in Cheongju, Anseong, and Cheonan, Korea. After excavating approximately 30 cm from the surface, the soil (approximately 100 g) around the roots of apple and pear trees was collected using a spade and collected in a 50 mL conical tube. Soil-containing tubes were stored in a cooler and transported to the laboratory for fungal isolation.

Isolation of fungi: Further, 3 g of the collected soil was placed in a 50 mL conical tube containing 10 mL of sterile distilled water and vortexed for 30 min. The mixture was then diluted up to 1,000 times through serial dilution; 1 mL of muddy water was mixed with 9 mL of sterile distilled water. Diluted muddy water (100 µL) was dispensed onto Dichloran-Glycerol 18% (DG18) agar medium and spread using a glass spreader. DG18 agar plates spread with soil dilution were incubated for at least three weeks in an incubator at 25℃. Among the growing fungal mycelia, mycelia that appeared different in morphology were collected and separated in pure form on potato dextrose agar (PDA). Purely isolated strains were cultured on PDA, malt extract agar (MEA), czapek yeast extract agar (CYA), and oatmeal agar (OA) to observe colony morphology.

Microstructure observation: The microstructures of the isolated fungi were observed using an optical microscope (BX53; Olympus, Tokyo, Japan). The size of each microstructure was measured 50 times, with the range shown.

DNA extraction and phylogenetic analysis: Purely isolated fungi were cultured on PDA lined with sterilized cellophane for another week and approximately 50 mg of mycelia was scraped with a surgical blade and collected in a 2 mL tube. DNA was extracted from the collected mycelia using a Plant/Fungi DNA Isolation Kit (Norgen Biotek Corp., Thorold, Canada). Using the extracted DNA as a template, PCR primers in Table 1 were used to amplify the internal transcribed spacer (ITS) region (ITS1-5.8S-ITS2), the large subunit of nuclear ribosomal RNA (28S rDNA), and partial β-tubulin gene (BenA) sequences. PCR was performed under the conditions listed in Table 1 using a Bio-Rad T100 Thermal cycler. After confirming PCR product amplification by electrophoresis on a 0.8% agarose gel, the PCR amplicon was purified and sequenced by Macrogen Corp. (Seoul, Korea).

The determined nucleotide sequences were analyzed for homology using the National Center for Biotechnology Information (NCBI, https://www.ncbi.nlm.nih.gov/) search engine GenBank BLASTN. Fungal species closely related to the taxa isolated in this study and belonging to the same or different genera, were included in the analysis. Reference sequences were obtained from GenBank and are listed in Tables 2-8. Individual sequence datasets of the ITS region, 28S rDNA, and the β-tubulin gene were aligned using the MUSCLE (multiple Sequence Comparison by Log- Expectation) alignment tool of MEGA XI [11] and improved manually where necessary using BioEdit [12]. Based on the aligned sequences, a maximum likelihood (ML) phylogenetic tree was constructed and the reliability of each node in the phylogenetic tree was evaluated using 1,000 bootstraps [13].

Table

RESULTS AND DISCUSSION

The morphological characteristics of each species of unrecorded soil fungi isolated and identified in this study and the results of phylogenetic analysis based on nucleotide sequence analysis of ITS and 28S rDNA are presented below. The seven DUCC strains were deposited at the National Institute of Biological Resources of the Republic of Korea (https://www.nibr.go.kr/cmn/main/enMain.do) and received the NIBRFGC numbers. The analyzed nucleotide sequences were registered in GenBank of the NCBI database and the registration numbers of the seven DUCC strains are provided in Tables 2-8.