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.

Almond ( Prunus dulcis ) canker diseases and decline syndromes have been reported during the past decade in the main almond‐growing regions worldwide. The notable expansion of almond cultivation in intensive farming systems to increase yields, particularly in countries across the Mediterranean Basin such as Italy and Spain, as well as in Australia, United States (California) and South Africa has led to their emergence. This review focuses on the different canker diseases and decline syndromes affecting almond crops and their symptoms, causal agents and life cycles. The influence of biotic and abiotic factors on the emergence and development of some of these diseases and syndromes under altered environmental conditions because of climate change is discussed. Finally, the challenges that the control of these complex diseases and syndromes poses for global food security are presented, including an overview of the integrated management strategies currently available. This comprehensive review aimed to elucidate the aetiology of almond cankers and decline syndromes as well as raise awareness within the scientific community of the need to deepen our knowledge of their aetiology and epidemiology. This should help in developing effective management strategies to reduce their high economic impact on almond (and other woody crops) worldwide, in the current era of climate change.

Botryosphaeriaceae fungi are the main pathogens associated with almond decline syndrome in southern Spain, with Botryosphaeria dothidea and Neofusicoccum parvum being the most frequent and most aggressive species, respectively. They can occur singly or coinfect trees, with the disease severity usually being higher in younger than in older trees. In this study, coinoculations of B. dothidea and N. parvum with adjusted conidial suspensions were conducted at different timings in detached shoots and potted plants, using green (1 year old) or lignified (2 to 3 years old) tissues of cultivar Avijor to determine the effect of coinfections and wood age on disease development. Cankers developed on lignified tissues were significantly smaller than on green tissues. There were no significant differences in aggressiveness among treatments in lignified tissues, whereas B. dothidea and N. parvum were significantly more aggressive in green tissues when inoculated individually, or when N. parvum was inoculated 4 days before B. dothidea. The biomass of each pathogen was quantified by multiplex qPCR in the margin and 2 cm beyond the margin of necrotic lesions in green tissues. The highest relative inoculum density was found in plants inoculated with B. dothidea, N. parvum, coinoculated simultaneously, or in those where B. dothidea was inoculated 4 days before N. parvum. Although at a much lower density, both pathogens were detected in asymptomatic tissues 2 cm beyond the necrotic lesions. These findings suggest that interactions during coinfections alter the relative abundance and aggressiveness of these pathogens, with disease development being strongly influenced by the age of the host tissue.

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. Copyright © 2026 The Author(s). This is an open access article distributed under the CC BY 4.0 International license .