Vladimiro Guarnaccia

Apple bitter rot (ABR) and Glomerella leaf spot (GLS), caused by Colletotrichum spp., have recently become an increasing concern in most Italian apple-growing areas. Surveys were conducted between 2023 and 2025 in 28 commercial apple orchards in Piedmont, Northwestern Italy, to investigate the occurrence and diversity of Colletotrichum species associated with symptomatic fruits and leaves. A total of 155 isolates were obtained and initially screened using gapdh, while 49 representative isolates were selected for species-level identification through multilocus phylogenetic analyses. Five species belonging to the C. gloeosporioides and C. acutatum species complexes were identified: C. chrysophilum, C. alienum, C. fioriniae, C. godetiae, and C. nymphaeae. Eight representative isolates were evaluated for mycelial growth at different temperatures and pathogenicity on apple fruits and leaves, revealing differences in growth and virulence. All tested isolates were pathogenic and fulfilled Koch’s postulates. Isolates were characterized for their in vitro sensitivity to fludioxonil, mefentrifluconazole, and pyraclostrobin. EC₅₀ values were generally low, although greater variability was observed, particularly within the C. gloeosporioides species complex for pyraclostrobin. This study expands current knowledge of the Colletotrichum species associated with ABR and GLS in apple production and provides relevant biological and fungicide sensitivity data as a reference for future epidemiological investigations and disease management strategies.

‘Mal dello stacco’, a destructive fungal trunk disease of hazelnut, is caused by Anthostoma decipiens. This disease manifests as wedge-shaped cankers, vascular necroses, branch cracking, and reddish masses on bark surfaces. The disease has rapidly increased in incidence and spread, resulting in significant qualitative and quantitative yield reductions. Disease management strategies remain insufficient, without reliable tools for early pathogen detection in hazelnut nurseries or field plantations. This study developed and validated an SYBR Green quantitative real-time PCR (qPCR) assay for the detection and quantification of A. decipiens in hazelnut wood. Primers were designed on the nuclear ribosomal internal transcribed spacer (ITS) region. The assay was optimized and validated according to EPPO standard PM 7/98 (6), for analytical specificity and sensitivity, selectivity, repeatability, and reproducibility. The primer pair AD8_F/AD16_R successfully amplified a 154 bp fragment in all of 32 A. decipiens isolates, without amplification for 42 off-target species up to 35 cycles. The assay detected up to 100 fg of pathogen DNA in water and hazelnut DNA, with reaction efficiencies from 97% to 100%, and high reproducibility across operators and sessions. The assay was validated on artificially inoculated and naturally infected hazelnut plants from Italy, Spain, and Serbia, and from ten hazelnut cultivars. The qPCR assay developed is a reliable, rapid, and cost-effective diagnostic tool for early detection of A. decipiens, with potential applications in nursery certification, orchard monitoring and epidemiological studies.

BACKGROUND: Downy mildew of grapevine, caused by Plasmopara viticola , is primarily managed with copper‐based fungicides in organic farming, otherwise by preventive treatments with chemical fungicide. However, regulatory restrictions and environmental concerns require strategies that reduce copper inputs, without compromising disease control. Essential oil (EO)‐based formulations are emerging as promising alternatives, due to their antimicrobial and antifungal activities. RESULTS: In vitro assays showed dose‐dependent inhibition of P. viticola sporulation by the EO‐based formulation. The concentration of 500 ppm provided efficacy comparable to the copper standard (67%) with no phytotoxicity. Two‐year vineyard trials demonstrated that integrating the EO formulation with reduced copper rates consistently maintained low disease incidence and severity. The copper‐EO combination strategy achieved 85% protection on both foliage and clusters, while reducing copper input by 45%. CONCLUSION: Integration of EO‐based formulation within reduced‐copper spray programs can effectively control grapevine downy mildew under field conditions, without compromising disease control. These findings support the use of EO‐based products as viable components of sustainable disease management strategies aligned with current European sustainability policies. © 2026 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Valorizing agro-industrial by-products within a circular bioeconomy provides sustainable alternatives to synthetic pesticides. This study evaluated a coffee production waste (CPW) aqueous extract, obtained via subcritical water extraction, as a multifunctional biopesticide. We assessed its chemical profile, antifungal and insecticidal efficacy, ecotoxicology, allelopathy, and biochemical phytotoxicity. HPLC-DAD-MS revealed high levels of caffeine (12.9 mg/g DM), trigonelline (10.8 mg/g DM), and chlorogenic acids (~8.5 mg/g DM). It demonstrated broad in vitro antifungal activity, inhibiting Botrytis cinerea mycelial growth (84.0%) and conidial germination (98.1%). In vivo, CPW reduced apple gray mold severity to 34.9%, though in planta protection on tomato was partial. The extract reduced Halyomorpha halys egg hatching to 33.0% and increased nymphal mortality to 49.0% via symbiotic disruption, without harming its parasitoid Trissolcus japonicus. However, high acute mortality in the pollinator Osmia bicornis highlights the need for risk mitigation. Crucially, CPW showed negligible phytotoxicity on crops but selectively reduced weed germination by up to 25%, supported biochemically by its non-inhibition of plant defense CYP74 enzymes. In conclusion, CPW extract is a promising bioactive candidate for sustainable crop protection, requiring formulation optimization to enhance field safety and performance.

Several fungal species in the Botryosphaeriaceae family are associated with trunk diseases of major agricultural crops, such as almond and grapevine. These diseases can lead to considerable yield losses in vineyards and orchards. Botryosphaeriaceae can also infect plant material and seedlings during nursery operations. Latent infections in contaminated propagation materials play an important role in the spread of these fungal pathogens. Accurate identification and early detection of these pathogens are crucial for improving disease management. Molecular tools, such as real-time PCR, are widely used as rapid and sensitive approaches to improve disease diagnostics, allowing early detection and quantification of target pathogens. In this study, we developed two SYBR Green qPCR assays for the specific detection and quantification of Botryosphaeria dothidea and species within the Neofusicoccum parvum-ribis complex. Assays were validated using artificially inoculated and naturally infected grapevine and almond plants. We assessed the specificity of the assays on 79 isolates, including various Botryosphaeriaceae and off-target species, such as other pathogens associated with grapevine trunk diseases. Target pathogens were successfully detected in all artificially inoculated plants, as well as in most naturally infected field samples tested. These results demonstrate that both SYBR Green qPCR assays are specific, selective, and sensitive, validating their use for detecting B. dothidea and N. parvum-ribis species in wood samples. This cost-effective tool can be applied for accurate detection of these pathogens from both symptomatic and asymptomatic plants, enabling large-scale analyses and early screening to promote the production of healthy planting material.

In the last decade, avocado production has increased in Italy due to the fruit’s high nutritional quality and economic value. During 2024, stem lesions, wood discoloration and dieback, often starting at the grafting point, were observed in young plants in a nursery in Sicily (Italy). Colletotrichum-like colonies were frequently isolated from symptomatic tissues. Multi-locus phylogenetic analysis (gapdh, chs-1, act, tub2, cal, gs and ApMat) was conducted on 11 representative isolates, identifying 6 as C. perseae and 5 as C. gloeosporioides sensu stricto (s.s.). Two representative isolates were selected for pathogenicity tests performed on 2-year-old avocado plants cultivated in a greenhouse. After two months, necrotic lesions, wood discoloration and reddish-brown streaking at the inoculation point were induced in both species. Additional inoculations of avocado fruit confirmed the ability of both species to cause fruit rot. All inoculated fungi were successfully re-isolated and identified, fulfilling Koch’s postulates. This is the first report of stem lesions and dieback caused by Colletotrichum species and the first occurrence of C. perseae in avocado plants in Europe. The results highlight the importance of early monitoring in nurseries during the propagation process and contribute to a better understanding of fungal diseases in avocado crops in Italy.

Trichoderma afroharzianum, the causal agent of Trichoderma ear rot, is an emerging pathogen of maize (Zea mays L.). It was recently reported as a maize pathogen in Germany, France, and in Italy. In 2023, nine seed lots from three farms in Northern Italy were tested for their phytosanitary conditions, revealing infection rates of up to 71% with Trichoderma spp. All seed lots showed symptoms of Trichoderma ear rot infection, and 26 out of 50 isolates were identified as T. afroharzianum. The study confirmed that T. afroharzianum infects maize seeds causing severe disease. Thirteen isolates from infected seeds were used to design species-specific primers on the translation elongation factor 1α gene and to develop a SYBR Green quantitative PCR to detect and quantify T. afroharzianum in maize seeds. The assay was validated following EPPO standard PM 7/98 guidelines, assessing analytical sensitivity, specificity, selectivity, repeatability, and reproducibility. The specificity of the method was validated using 19 T. afroharzianum strains and 16 nontarget species, including Trichoderma species belonging to the T. harzianum species complex. Only target DNA produced positive amplifications. Analytical sensitivity was tested using serial dilutions of T. afroharzianum DNA, revealing a detection limit of 50 fg, even in the presence of maize seed DNA. The assay enables specific and sensitive detection of target DNA in asymptomatic samples, providing a valuable tool for early target detection and quantification during seed certification.

Citrus is one of the most economically important crops in Florida. In 2022 surveys of 46 citrus groves in southwest Florida, twig dieback and anthracnose on fruit, leaves and twigs were frequently observed. The occurrence, diversity, pathogenicity and sensitivity to chemical compounds of Colletotrichum strains isolated from symptomatic Citrus sinensis cv. Valencia were investigated. One hundred representative monoconidial isolates were identified as Colletotrichum gloeosporioides by phylogenetic analysis based on five genomic loci (GAPDH, ACT, TUB2, GS and ApMat). Fruit pathogenicity tests performed on seven representative isolates confirmed the pathogenicity of the identified species. This study is the first report of C. gloeosporioides associated with twig dieback and preharvest anthracnose on Ci. sinensis cv. Valencia in Florida. The sensitivity of 100 isolates of C. gloeosporioides to fenbuconazole (DMI), fluopyram (SDHI) and pyraclostrobin (QoI) was assessed by inhibition of conidial germination. Over 50% of tested isolates showed reduced sensitivity to fenbuconazole, fluopyram and pyraclostrobin, providing the first evaluation of the in vitro sensitivity of C. gloeosporioides strains isolated from Ci. sinensis cv. Valencia in Florida. These molecules appear unable to control the spread and development of C. gloeosporioides on Ci. sinensis cv. Valencia in Florida. Due to several citrus diseases that have severely affected Florida's citrus industry over the past 20 years, fungal diseases such as anthracnose have been neglected, but are increasing in importance in recent years worldwide. Our study represents a starting point for the development of new solutions to manage this problem for citrus growers.