Descriptive Assessment of Colletotrichum Overwintering in Walnut Tissues: Complementary Insights from Traditional Isolation and High-Throughput Sequencing

Understanding the overwintering patterns of Colletotrichum spp. is important for managing walnut anthracnose. This exploratory study integrated traditional tissue isolation with high-throughput sequencing (HTS) to describe the pathogen’s overwintering distribution across four orchards in Hubei Province. Buds, twigs, fallen leaves, and mummified fruits were sampled in January (late dormancy) and March (pre-budbreak), with five trees pooled into composite samples per tissue–orchard–month combination. Traditional isolation identified buds and fallen leaves as tissues with higher recovery rates, frequently exceeding 70% in March. HTS revealed a more complex community composition, with Ascomycota dominance and Fusarium, Alternaria, and Colletotrichum as co-dominant genera. Notably, the HTS-detected relative abundance of Colletotrichum reached 40.19% in mummified fruits at one orchard; however, because HTS cannot distinguish viable from non-viable organisms, this should be interpreted as a potential DNA signal rather than a confirmed epidemiological reservoir. The two methods showed qualitatively different patterns across tissues, underscoring that traditional isolation detects the culturable fraction whereas HTS captures total DNA, which may include DNA from non-viable or non-culturable sources. These descriptive findings elucidate candidate overwintering niches and temporal differences between January and March, providing a preliminary basis for future hypothesis-driven studies. We suggest that integrated analysis of the culturable and DNA-detected fractions may improve disease risk assessment, pending experimental validation.