Strawberry (Fragaria x ananassa Duch.) is one of the most important crops in Huelva (southwestern Spain) with 7,300 ha cultivated in 2012 yielding 275,300 tonnes (98% of the total Spanish production). At the beginning of the 2013-14 season, a low incidence of mortality (~1%) in strawberry plants cultivated at Huelva province was observed. Symptoms consisted in discoloration or brown necrotic areas in crown and root (Fig. 1.A and 1.B, respectively). To isolate the causal agent, crowns and roots were surface disinfected for 2 min in 1% sodium hypochlorite, rinsed twice in sterile distilled water, air dried in a laminar flow-cabinet, and plated on potato dextrose agar (PDA). Pure cultures of the fungus were grown on PDA at 25ºC with a 12-h photoperiod for 7-10 days. Fungal colonies were white and cottony, edge undulate, circular growth appearance (upper side) and pale luteous (reverse side) (Fig. 1.C and 1.D). The acervuli (average 280 μm) were black, concentric and appeared throughout the entire plate after...

Woody canker diseases caused by fungi of the Botryosphaeriaceae family are producing increasing losses in many economically important woody crops, including almond. To develop a molecular tool for the detection and quantification of the most aggressive and threatening species is of main importance. This will help to prevent the introduction of these pathogens in new orchards and to conveniently apply the appropriate control measures. Three reliable, sensitive and specific duplex qPCR assays using TaqMan probes have been designed for the detection and quantification of (a) Neofusicoccum parvum and the Neofusicoccum genus, (b) N. parvum and the Botryosphaeriaceae family and (c) Botryosphaeria dothidea and the Botryosphaeriaceae family. The multiplex qPCR protocols have been validated on artificially and naturally infected plants. Direct systems to process plant materials, without DNA purification, allowed high-throughput detection of Botryosphaeriaceae targets even in asymptomatic tissues. These results validate the qPCR using the direct sample preparation method as a valuable tool for Botryosphaeria dieback diagnosis allowing a large-scale analysis and the preventive detection of latent infection.

ISHS VII International Strawberry Symposium EFFICIENCY OF NATURAL PRODUCTS IN THE CONTROL OF COLLETOTRICHUM ACUTATUM MONITORED BY REAL-TIME PCR

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.