High-throughput sequencing analysis of fungal and Botryosphaeriaceae communities in symptomatic and asymptomatic almond, avocado, and blueberry plants

The Botryosphaeriaceae family comprises a large and diverse group of fungi that can behave as endophytes, saprophytes, or pathogens, causing dieback and canker diseases worldwide. These fungi can persist in asymptomatic plants, with symptoms often triggered under host stress. This study employed a high-throughput sequencing (HTS) metabarcoding approach to investigate Botryosphaeriaceae populations in symptomatic and asymptomatic almond, avocado and blueberry plants, and to assess other fungi that may act as disease triggers, symptom enhancers or potential biocontrol agents. Botryosphaeriaceae species were detected in 100%, 93%, and 80% of symptomatic almond, avocado, and blueberry plants, respectively, and in 60%, 73% and 66% of asymptomatic plants. The predominant genera were Neofusicoccum, Botryosphaeria, Diplodia and Lasiodiplodia, with significant host-dependent variation in relative abundance. Multiple Botryosphaeriaceae infections predominated over single infections in symptomatic and asymptomatic plants, particularly in avocado. High diversity of total fungal and Botryosphaeriaceae communities was observed across all crops, with greater richness and evenness in avocado. Beta diversity analyses revealed highly significant differences among the fungal microbiomes of the three crops. Several non-Botryosphaeriaceae wood-colonizing fungi, including Cylindrocarpon, Cytospora, Diaporthe, Fusarium and Phaeoacremonium were also detected in symptomatic almond samples, suggesting possible synergistic interactions, whereas potential biocontrol genera such as Trichoderma and Clonostachys were detected exclusively in some symptomatic almond plants. This work contributes to a better understanding of the fungal etiology of canker and dieback diseases in almond, avocado, and blueberry. Furthermore, the high concordance between HTS and multiplex qPCR supports their complementary use to improve plant disease diagnosis, particularly for early preventive detection.