토양에서 분리된 곰팡이의 마이코바이러스 유병률 조사와 3종 dsRNA 바이러스의 분자적 특성 = Prevalence of mycoviruses in fungi isolated from soil and molecular characterization of three dsRNA viruses

다국어 초록 (Multilingual Abstract)

Mycoviruses infect a wide range of fungi and influence host growth, pathogenicity, and stress tolerance, thereby drawing attention as potential biological control agents. However, information on the distribution and molecular characteristics of mycoviruses in South Korea remains highly limited. Therefore, this study investigated the prevalence of mycoviruses in 64 ascomycetous fungi isolated from soil in Korea and examined the molecular and phylogenetic characteristics of three detected viruses. As a result of screening, dsRNA viruses were found in three strains, corresponding to a prevalence of 4.7%. These viruses were classified as Tolypocladium polymycovirus 1 (TPmV1), Cladosporium cladosporioides partitivirus 1 (CcPV1-IPBL11), and Marquandomyces marquandii polymycovirus 1 (MmPmV1), representing one Gammapartitivirus and two Polymycovirus spp.. Among them, CcPV1-IPBL11 was confirmed as a new isolate of the previously reported CcPV1, while TPmV1 and MmPmV1 were identified as novel species. Transmission electron microscopy revealed filamentous virus-like particles, several hundred nanometers in length, in TPmV1 and MmPmV1, consistent with recent research on polymycovirus capsid architecture. To analyze host responses to mycovirus infection, a virus-free isogenic strain of MmPmV1 was obtained through single-spore isolation. MmPmV1 exhibited asymptomatic infection, with no detectable effects on colony morphology, growth rate, pigmentation, conidiation, or conidial germination. Furthermore, qPCR analysis of viral ORF expression showed that all six ORFs of MmPmV1 decreased in expression during early culture stages and increased during later stages, suggesting replication linked to the host growth phase. This study characterizes Gammapartitivirus and Polymycovirus from ascomycetous fungi isolated in Korea, expanding the known diversity and molecular features of mycoviruses. In particular, the findings broaden the host range of Polymycoviridae and provide understanding of host-virus interactions, structural characteristics, and replication patterns. These results provide foundational knowledge for future research exploring the biocontrol potential and applied uses of mycoviruses.

목차 (Table of Contents)

• INTRODUCTION 1
• MATERIALS AND METHODS 5
• 1. Fungal isolates and culture conditions 5
• 2. Total nucleic acid extraction 5
• 3. Isolation of double-stranded RNA (dsRNA) 7
• 4. Identification of fungal host strain 8
• 5. dsRNA sequencing using SPAT and RLM-RACE 12
• 6. Cloning 15
• 7. Sequence and phylogenetic analysis 16
• 8. Purification of virus-like particle from mycelia and TEM 19
• 9. Generation of virus-free isogenic strain 20
• 10. Comparison of fungal traits between virus-infected and virus-free strains 22
• 10-1. Morphology and colony diameter 22
• 10-2. Stress tolerance 22
• 10-3. Conidia productivity 22
• 10-4. Thermotolerance 22
• 11. Quantitative analysis of viral ORF expression by RT-qPCR 23
• 12. Statistical analysis 25
• RESULTS 26
• 1. Prevalence of dsRNA in ascomycetes 26
• 2. Identification of fungal host strains 28
• 2-1. Tolypocladium sp. IPBL-4 28
• 2-2. Cladosporium anthropophilum IPBL-11 28
• 2-3. Marquandomyces marquandii IPBL-196 29
• 3. Identification of three mycoviruses 33
• 3-1. Cladosporium cladosporioides partitivirus 1 (CcPV1-IPBL11) 33
• 3-2. Tolypocladium polymycovirus 1 (TPmV1) 36
• 3-3. Marquandomyces marquandii polymycovirus 1 (MmPmV1) 39
• 4. Phylogenetic analysis 42
• 4-1. CcPV1-IPBL11 42
• 4-2. TPmV1 and MmPmV1 44
• 5. Virus-like particle 46
• 6. Generation of virus-free isogenic strain 48
• 7. Phenotypic characterization of virus-infected and virus-free isogenic lines 50
• 8. Relative expression profiles of viral ORF 54
• DISCUSSION 56
• LITERATURE CITED 62
• ABSTRACT IN KOREAN 79