INTRODUCTION

Apple (Malus pulmina) is a Rosaceae plant whose fruit is consumed worldwide fresh or processed. In Korea, apples are representative fruit trees. In 2018, the cultivation area of apples was 33,234 ha, accounting for 21.2% fruit tree cultivation area [1]. Recently, fire blight has damaged many apple trees in orchards across several provinces in Korea [2]. It is caused by Erwinia amylovora, a bacterium belonging to the Enterobacteriaceae [2]. According to the burial control regulations, fire-blighted apple trees are buried 4-5 m below the soil [3]. To ensure burial control safety, soil microorganisms in apple orchards must be explored to understand the environment of the burial soil.

Apple trees buried in the soil are mostly degraded and deteriorated by saprophytic soil fungi [4]. Thus, information on soil fungal diversity is important to understand the decay process of fire-blight-controlled apple trees. Saprophytic fungi produce enzymes that decompose cellulose, hemicellulose, and pectin present in plant materials. We investigated the fungal diversity in orchard soil where fire-blighted apple trees are buried. In this study, we report the description and morphological features of seven ascomycetes previously unreported in Korea.

MATERIALS AND METHODS

RESULTS AND DISCUSSION

The morphological characteristics and the results of the molecular phylogenetic analysis of the seven unrecorded soil fungi are described below. The nucleotide sequences of these seven DUCC fungal strains were registered in the GenBank database, and the accession numbers are listed in Table 1. The strains were deposited at the National Institute of Biological Resources of the Republic of Korea and received NIBRFGC numbers.

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].

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.