Different Pestalotiopsis-like species have been reported in strawberry worldwide, as agents of leaf spot, root rot, and crown rot. The identification of Pestalotiopsis-like fungi is based on both molecular and morphological analyses to discriminate between species and clarify phylogenetic relationships. Recent studies have provided robust multi-locus analyses, which reclassified most Pestalotiopsis-like isolates associated with strawberry root and crown rot diseases as Neopestalotiopsis spp. Numerous disease outbreaks have been observed in strawberry fields in Italy in recent years, showing that Neopestalotiopsis is an emerging pathogen. A survey was conducted in Northern Italy during 2022–2023 to investigate the diversity and distribution of Neopestalotiopsis species. Morphological and phylogenetic characterization, based on ITS, tef1 and tub2, led to the identification of four species: Neopestalotiopsis rosae, N. iranensis, N. hispanica (syn. vaccinii) and N. scalabiensis. Based on our results from multi-locus phylogenetic analysis, N. hispanica and N. vaccinii were grouped in the same cluster; thus, they were confirmed to be the same species. Pathogenicity tests with representative isolates of each species were conducted on strawberry ‘Portola’ transplants. All isolates were shown to be wound pathogens in strawberry, causing crown rot, and were successfully re-isolated. Neopestalotiopsis rosae was confirmed to be the most dominant and virulent species associated with these symptoms, as well as the most dominant among the obtained isolates. To the best of our knowledge, this work represents the first report of N. scalabiensis being associated with the crown rot of strawberry in Italy and the first report of N. iranensis in association with the crown rot of strawberry worldwide.

Grapevine Trunk Diseases (GTDs) are major threats in Mediterranean countries, causing economic losses due to reduced grape yields and long-term vine productivity, as well as death of grapevines. A survey was conducted in Piedmont (Northern Italy) during 2021-2022 to investigate the species diversity and distribution of GTD pathogens in this important Italian wine region. Morphological and multi-locus phylogenetic analyses (based on ITS, tef1, tub2, act and rpb2) identified species of Botryosphaeriaceae at high frequency, including Botryosphaeria dothidea, Diplodia mutila, Diplodia seriata and Neofusicoccum parvum. Other pathogens commonly associated with GTDs, including Eutypa lata, Fomitiporia mediterranea and Phaeomoniella chlamydospora, were also isolated. Less commonly isolated species included Neocucurbitaria juglandicola, Paraconiothyrium brasiliense, Seimatosporium vitis-viniferae and Truncatella angustata. Pathogenicity tests with two representative isolates of each species were carried out using one-year-old potted grapevine cuttings (‘Barbera’). All isolates (except N. juglandicola) caused brown wood necrotic vascular discolourations on inoculated plants and were successfully re-isolated. Effects of temperature on colony growth were also assessed. For all tested isolates there was no growth at 5°C, only four isolates (Botryosphaeriaceae) grew at 35°C, and optimum growth temperatures were between 20 and 25°C. This is the first record of Paraconiothyrium brasiliense and Neocucurbitaria juglandicola associated with symptomatic grapevines in Italy.

Florida ranks among the most important citrus growing regions in the USA. The present study investigates the occurrence, diversity, and pathogenicity of fungal species associated with symptomatic sweet orange (Citrus sinensis) cv. Valencia plants and fruit. The survey was conducted on twigs and fruit collected in Southwest Florida during 2022. Based on morphological and molecular characteristics, the identified isolates belonged to the species Lasiodiplodia iraniensis, Diaporthe pseudomangiferae, and Diaporthe ueckerae. The pathogenicity of representative isolates was evaluated on citrus fruit and plants. Lasiodiplodia iraniensis was the most virulent on fruit and plants, followed by Diaporthe pseudomangiferae. Diaporthe ueckerae had the lowest virulence on fruit, and it was not pathogenic to plants. In vitro tests were performed to assess the effect of temperature on mycelial radial growth. The optimum temperature of growth ranged from 26.0 to 28.4 °C for all the evaluated species, and L. iraniensis showed the fastest mycelial growth. This study represents the first report of L. iraniensis as a causal agent of tree dieback and fruit stem-end rot on C. sinensis worldwide. Moreover, D. pseudomangiferae and D. ueckerae are reported here for the first time in association with citrus diseases worldwide.

Monilinia spp. cause brown rot and blossom blight of stone fruit. This study characterized the diversity of Monilinia spp. associated with stone fruit rots in the Cuneo province, the major fruit production area in Piedmont, and assessed their sensitivity to azoxystrobin, cyprodinil, fenhexamid, fludioxonil and tebuconazole. Species diversity was determined by PCR amplification and sequencing of isolate internal transcribed spacer (ITS) regions. Sensitivity to fungicides was determined by measuring in vitro mycelium growth on fungicide-amended media. Fifty isolates were obtained from apricot, cherry, or peach fruits with typical brown rot symptoms. Thirteen isolates were identified as M. fructicola, and 37 as M. laxa. Nine isolates of Monilinia laxa and two of M. fructicola had resistance factor (RF) values greater than 10 for different fungicides. The greatest (RF) value (48.96) was measured for azoxystrobin against the M. fructicola isolate CVG 1514. Among the M. laxa isolates, isolate CVG 1547 had the greatest RF value to cyprodinil, while isolate CVG 1709 had RF values greater than 10 for cyprodinil and tebuconazole. A systematic and wider sampling should be carried out in the Piedmont region to determine the distribution of fungicide resistant Monilinia spp. in stone fruit crops. The use of site-specific fungicides remains the most effective strategy for control brown rot, and continued monitoring for fungicide resistance within Monilinia spp. populations is recommended.

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.

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.