ABSTRACT Nearly 50 years has passed since brown spot of pear (BSP), caused by the fungus Stemphylium vesicarium, was detected in Europe. Despite significant progress in disease management and the identification of resistance targets in pear, BSP remains a major threat for European pear production. The extensive knowledge recently acquired on the biology, genomics and epidemiology of S. vesicarium , together with the increasing importance of this pathogen in Europe, prompted the preparation of this review to provide a comprehensive overview of its current geographic distribution, as well as newly discovered aspects of its biology, infection strategies and host specificity. This review also examines the symptoms and epidemiology of BSP, highlighting key factors influencing disease development, including spore dispersal and environmental conditions. The susceptibility of different pear cultivars is discussed, together with recent advances in genetic studies related to resistance breeding, providing guidance for future breeding programmes. In addition, publicly available genomic data for S. vesicarium are summarized to provide a foundation for identifying pathogenicity‐related characteristics. Finally, potential disease control strategies are outlined to offer new perspectives for the effective management of BSP.

Since the late 1970s, brown spot of pear (BSP), a fungal disease caused by Stemphylium vesicarium (Wallr.) Simmons, has been one of the most important pear fungal diseases in Italy. To protect orchards from BSP, frequent fungicide application is essential throughout the period spanning petal fall to the onset of fruit maturation. In Italy, boscalid was the first succinate dehydrogenase inhibitor (SDHIs) fungicide authorised against BSP; subsequently, penthiopyrad and fluxapyroxad were authorised against the disease. In 2016 and 2017, SDHI compounds were applied against BSP as solo products at the University of Bologna’s experimental farm, showing a reduction in efficacy. Stemphylium vesicarium strains were isolated from leaves and fruit, and sensitivity assays and molecular analyses were performed. In vitro tests confirmed resistance to SDHIs, and two specific single-nucleotide polymorphisms were discovered, SDHB P230L and SDHC H134R, both leading to amino acid substitutions in succinate dehydrogenase subunits and confirming the resistant phenotype.

Plasmopara viticola is controlled by fungicides with different modes of action, which include zoxamide. Zoxamide was developed in 1998 by Dow AgroSciences LIC (Indianapolis, IN) and has been commercialized since 2001 by Rohm and Haas Company. This fungicide is highly effective against oomycetes, used for foliar application in vine crops, potato, and other vegetables to control oomycete-induced diseases. Zoxamide acts by causing mitotic arrest by binding to β-tubulin, inhibiting tubulin polymerization and cell division of the pathogen. In the past three decades, the management of fungicide resistance has emerged as a significant concern for both growers and regulatory authorities; this is primarily due to the substantial increase in resistance, in terms of occurrence and spread, towards most fungicide groups. The monitoring of the effectiveness of fungicides is the only way to accurately identify as soon as possible the onset of resistance. In this study, we were interested in tracking the changes in the sensitivity of zoxamide to P. viticola populations collected during a 6-year monitoring (2017–2022) in two Italian locations: the autonomous province of Trento and the autonomous region of Friuli-Venezia Giulia. Bioassays on leaf discs were carried out, and EC50 and MIC values were elaborated. From our results, zoxamide showed for a long period of time high sensitivity, but over the last years we observed a change, as in 2022 most of the samples tested in these two regions showed MIC > 100 mg L−1.

Several oomycete species are pathogenic to plants or animals, including humans, and cause economic and environmental damage, nevertheless no effective molecules against these pathogens are available. Dimethomorph (DMM), is a fungicide with high activity against plant pathogenic oomycetes used to control downy mildew. The current study aims at developing a transversal approach and to do that, we evaluated: 1) the effect of two concentrations of DMM on in vitro mycelial growth on Saprolegnia and Pythium isolated from fish and aquatic environment; 2) the effect of DMM on hyphal morphology of S. parasitica and on the sporangia and hyphae of a P. viticola strain using Scanning Electron Microscopy. Results indicated that radial growth of Saprolegnia species was reduced at 50 and 100 mg/L of DMM after 24h of incubation. For P. viticola at 50 mg/L sporangia are rarely present with respect to the control and in 100 mg/L are not present at all. Moreover, a SEM protocol was developed, and allowed to observe how the fungicide induced changes at the morphological level in both oomycete species.

Plasmopara viticola is the causal agent of Grapevine Downy Mildew (GDM), which is a devastating disease of grapevines in humid temperate regions. The most employed method for protecting grapevines against GDM is the application of chemical fungicides. In Spain, Carboxylic Acid Amides (CAAs) are a fungicide group currently utilized in GDM control. In P. viticola, resistance to CAAs is conferred by G1105S and G1105V mutations in the CesA3 gene. Droplet digital polymerase chain reaction (ddPCR) is an innovative technique that combines PCR and droplet microfluidics to disperse the sample into thousands of water-in-oil droplets in which an amplification reaction is individually performed. In this study, we set up a ddPCR protocol to quantify S1105 and V1105 mutations conferring resistance to CAAs in P. viticola. The optimal PCR conditions were established, and the sensitivity and precision of the protocol were assessed. Four P. viticola populations coming from commercial vineyards in northern Spain were analyzed, and different allele frequencies were found in the analyzed samples corresponding to the different fungicide management strategies, ranging from 7.72% to 100%. Knowing the level of mutated alleles allows for designing resistance management strategies suited for each location. This suggests that similar ddPCR assays could be developed for studying mutations implicated in fungicide resistance in other fungicide groups and plant pathogens.

Fungi of genera Phiyctema and Neofabraea are the causal agents of bull's eye rot, a major postharvest disease of pome fruits. To investigate their morphological and genetic diversity, isolates obtained in Italy and Chile from decayed fruit and rainwater between 2014 and 2019 were grown on two agar media, inoculated onto four fruit cultivars and compared using four marker genes. Consistent intra- and interspecies phenotypic differences were recorded among isolates identified as P. vagabunda (two main morphotypes, PvM-I and PvM-II, were distinguished) and N. kienholzii. In particular, the Chilean isolates belonging to PvM-I showed low sporulation in vitro, while isolates belonging to PvM-II showed the most abundant sporulation and also formed conidiomata deep within fruit tissue. Host cultivar influenced the disease incidence in unwounded, inoculated fruit. Cripps Pink and Golden Delicious apples favoured the formation of P. vagabunda conidiomata and macroconidia, while Granny Smith apples and/or Kaiser pears restricted sporulation of some isolates of PvM-I. Mycelial cords of P. vagabunda and N. kienhoizii were consistently recorded in inoculated fruit, suggesting their possible involvement as a source of inoculum. Propagules of P. vagabunda were present in rainwater collected from apple plants from September to October in Italy. According to sequence analysis of ITS, EF-1 alpha, TUB2 and ACT1 regions of the fungi, 12 distinct sequence types were identified, three of which were characteristic of isolates from the Southern Hemisphere. The condensed maximum-likelihood phylogenetic tree separated the 50 P. vagabunda isolates into six phylogroups, suggesting a correlation with their geographical distribution.

Z. tritici first appeared in Italy later than in northern-central European countries. QoIs fungicides currently play a role in STB control, used in combination with Demethylation Inhibitors (DMIs) or Succinate dehydrogenase Inhibitors (SDHIs). In this study, we set up a fast, sensitive, and accurate ddPCR protocol in order to investigate the presence and frequency of G143A substitution, causing a reduction in strobilurins’ efficacy in Z. tritici. The best PCR conditions for the clear separation of positive and negative droplets were identified. The lowest wild-type and resistant alleles frequencies were accurately determined on samples consisting of mixed DNAs from monoconidial cultures of Z. tritici and were expressed as fractional abundance. The protocol was tested by determining the copy number and frequency of alleles on gDNA purified in three Italian Z. tritici field populations representative of different fungicide management strategies. For the first time, the determination of allele concentration and the frequency of a mutation involved in Z. tritici fungicide resistance was carried out by employing digital PCR. This new approach provides a diagnostic tool that is rapid and able to detect very low G143A substitution percentages, which is very useful for fungicide resistance detection at early stages, thus, informing field management strategies for contrasting STB disease.

Downy mildew caused by Plasmopara viticola is one of the most devastating diseases of grapevine, attacking all green parts of the plant. The damage is severe when the infection at flowering stage is left uncontrolled. P. viticola management consumes a significant amount of classical pesticides applied in vineyards, requiring efficient and environmentally safe disease management options. Spray-induced gene silencing (SIGS), through the application of exogenous double-stranded RNA (dsRNA), has shown promising results for the management of diseases in crops. Here, we developed and tested the potential of dsRNA targeting P. viticola Dicer-like (DCL) genes for SIGS-based crop protection strategy. The exogenous application of PvDCL1/2 dsRNA, a chimera of PvDCL1 and PvDCL2, highly affected the virulence of P. viticola. The reduced expression level of PvDCL1 and PvDCL2 transcripts in infected leaves, treated with PvDCL1/2 dsRNA, was an indication of an active RNA interference mechanism inside the pathogen to compromise its virulence. Besides the protective property, the PvDCL1/2 dsRNA also exhibited a curative role by reducing the disease progress rate of already established infection. Our data provide a promising future for PvDCL1/2 dsRNA as a new generation of RNA-based resistant plants or RNA-based agrochemical for the management of downy mildew disease in grapevine.

Apple scab caused by Venturia inaequalis (Cke.) Wint. is the most important disease of apple trees worldwide and requires a high number of fungicide applications. The G143A substitution in the inhibitor binding site of cytochrome b of V. inaequalis confers a high level of resistance to strobilurins targeting the bc1 complex. The aim of this work was to substitute the labor intensive in vitro assays, with the faster quantitative PCR. An allele-specific qPCR method with a newly designed primer set was successfully developed to quantitatively determine the frequency of QoI-resistant A143 allele in populations of V. inaequalis. To be able to suggest that the molecular method could be applied as unique and robust technique, we carried out in vitro sensitivity test to trifloxystrobin; first testing the relative germination and subsequently confirmed with the quantification of mutated allele frequencies by qPCR on forty-nine Italian V. inaequalis populations. qPCR gave a similar pattern to that obtained using in vitro conidial germination test in predominantly sensitive and resistant populations, the variability between these two tests was observed in some heterogeneous populations. The qPCR assay developed in this study efficiently quantifies the A143 allele and we can conclude that this method could be useful for the study of the fungicide resistance at population level in the fields, giving a quick response also with a large amount of samples.

Atypical scab symptoms were reported for the first time at the end of July in 2012 in Northern Italy (Cesena province, Emilia Romagna region) on fruit apple cultivar CIVG198 Modì® carrying the Rvi6 (= Vf ) major resistance gene to Venturia inaequalis ; it is a hybrid of ‘Gala’ x ‘Liberty’ with more than half the total world production area of cv ‘Modì’ located in Italy. The symptoms were also established in all the following years and Venturia asperata was identified as a causal agent of these atypical scab symptoms through morphological studies and molecular analysis of conidia from fruits and ascospores from overwintered leaves, using specific primers for V. asperata, V. inaequalis and V. pirina . Pathogenicity tests carried out on fruit at different phenological stages showed only light symptoms. Venturia asperata could represent an emergent disease for cv ‘Modì’ and disease control may be necessary.

Within the Research Project on the bacterial canker of kiwifruit caused by Pseudomonas syringae pv. actinidiae (PSA) sponsored by Emilia-Romagna Region during the period 2012-2015, the results of 4 trials in open field conditions - in presence of natural inoculum - to verify the efficacy of different active ingredients to control the disease are presented. The field trials confirmed previous studies on the efficacy of copper-based products and acibenzolar-S-methyl. A formulation of poliglucosammine and a formulation which combines the action of poliglucosammine with copper and boron contributed to reduce disease symptoms too. An inorganic compound based on aluminum potassium sulfate dodecahydrate confirmed in open field the good results showed in vitro and in greenhouse assays. These results give good indication for the PSA management with copper and acibenzolar-S-methyl, while further studies to confirm the activity of the new compounds presented in this study are needed.

Brown spot (Stemphylium vesicarium) is a fungal disease widespread in European pear industry. The high number of fungicide applications required to control the disease can promote strains with fungicide resistance. The aim of the present study was to assess the effectiveness of soil- applied water solution of calcium chloride in combination with rootstock to control the incidence of brown spot in Abbé Fétel pear. Soil salinity, stem water and osmotic potential, along with fruit fraction of calcium and defence-mechanism related-enzymes such as peroxidase, polyphenol oxidase, phenyl-alanine ammonia-lyase, and β-1,3-glucanase were investigate in tree grafted on Fox 11 (Pyrus communis L.) and Sydo®, a quince (Cydonia oblonga) rootstock. Both grafting combinations were fertigated or not with calcium chloride. Sydo® showed a positive effect in reducing brown spot severity on Abbé Fétel. Compared to Fox 11, fruits from trees grafted on Sydo® showed higher calcium concentrations, fraction of calcium pectate, fruit firmness, polyphenol oxidase, phenyl- alanine ammonia-lyase and β-1,3-glucanase levels. Fruit calcium concentration was positively correlated with disease fruit tolerance. However, soil calcium applications were ineffective in promoting fruit calcium partitioning. Calcium chloride applications decreased leaf osmotic and stem water potential in trees grafted onto Sydo®, but this wasn’t found in those grafted on Fox 11. In conclusion, Sydo® rootstock promoted Ca acquisition, osmotic adjustment at leaf and fruit level, fruit synthesis of defence-related enzymes that all together reduced brown spot severity in Abbé Fétel pear.

Stemphylium vesicarium is the causal agent of several plant diseases as well brown spot of pear (BSP), which is one of the most economically important fungal diseases in European pear-production areas. In addition to the relevance of the economic impact, conidia spread widely from plant material infected by the pathogen can trigger respiratory allergy. Here, we report the first genome of a S. vesicarium strain, 173-1a13FI1M3, isolated from pear and sensitive to the mostly used fungicide classes currently authorized in Europe against BSP. The availability of this draft genome could represent a first important step in understanding the physiology and the infection mechanism of the pathogen. Furthermore, this contribution could be fundamental in order to design more effective and sustainable strategies to control the disease.

Brown spot is one of the most serious fungal diseases that can affect pear fruits and leaves in the Po valley (Italy). Stemphylium vesicarium is the causal agent of this disease, and several antifungal treatments, repeated throughout the period between bloom and harvest, are needed to control its spread. Many of the most important pear cultivars (such as ‘Abbé Fétel’) are very susceptible to this fungus, while others (such as ‘Bartlett’ and its mutated sports) are known to be resistant. Our research aimed to develop molecular markers linked to this trait. To this end, 92 seedlings derived from an ‘Abbé Fétel’ × ‘Max Red Bartlett’ cross were evaluated for resistance to S. vesicarium for two consecutive years by artificial inoculation with conidia on detached leaves and fruits under controlled conditions in greenhouse. The extent of the lesions was recorded at different time points. A major QTL for susceptibility was located at the lower end of linkage group 15 of ‘Abbé Fétel’. This region was saturated with three SSR markers, and the putative position of a susceptibility gene was also estimated by the single gene mapping approach. This putative gene was located at 2 cM far from the lower end of the linkage group. Molecular markers tightly associated to this locus represent a first step towards the development of MAS (marker-assisted selection) to support the selection of new pear genotypes more resistant to brown spot.

Late blight caused by Phytophthora infestans is the most devastating disease of tomato ( Solanum lycopersicum L.) and causes important economically losses if not properly controlled. Control is achieved mainly by preventive fungicide applications. However, even if curative applications are discouraged because they increase the risk of resistance development in the target pathogens, in practice fungicides may be applied also when the disease is already present, a situation that commonly occurs in the field. The aim of this work was to study the curative activity of several fungicides toward P. infestans to determine their efficacy when applied after the infection process. Nine trials were performed in greenhouse using potted tomato plants that were treated 24 h after inoculation. Disease severity was assessed three times from the development of the symptoms on the untreated plants and data analysed using a linear mixed model. Differences in post-infection control between the different chemical classes were found. Metalaxyl-M and cymoxanil showed the best curative activity while among the CAA fungicides, a good efficacy was expressed by dimethomorph. Interestingly, evidence of synergy between active ingredients having different modes of action was observed such as in the mixtures containing dimethomorph + ametoctradin, dimethomorph + pyraclostrobin and fosetyl-Al + propamocarb. This study provided useful information on the post-infection activity of some fungicides used to control tomato late blight and should be taken into account to perform more in depth studies at the field level and to improve the management strategies of the disease.

Neofabraea vagabunda is the prevalent cause of bull's eye rot, one of the main postharvest diseases of apple, in many producing areas, but its biology has not been studied in detail. The molecular identification, by DNA sequencing of the β‐tubulin region, of 41 isolates collected from apples showing bull's eye rot in the Emilia‐Romagna region confirmed N. vagabunda as the main species in Italy. A biological and morphological characterization of N. vagabunda isolates was performed in vitro. Assays at temperatures ranging from 0 to 30 °C carried out on 10 isolates demonstrated: (i) a marked influence of temperature on colony morphology, conidial production, conidial size and mycelial growth, showing the cold‐tolerant character of N. vagabunda; and (ii) that culture at 15 °C on tomato agar (TA) for 14 days is a rapid and reliable method to favour pathogen conidial production. Trials performed on 38 isolates using these incubation conditions recorded the presence of two N. vagabunda morphotypes, differing for colony morphology, conidial size, conidiomata formation and temperature requirement. The alkalizing ability of the pathogen during growth on TA was also demonstrated for the first time. The pathogenicity of 25 N. vagabunda isolates was proved in vivo on artificially infected Cripps Pink apples. A pH increase was also recorded in apple tissue infected by N. vagabunda isolates (on average 0.2 and 0.3 units of pH after 60 and 120 days of incubation, respectively), suggesting that the N. vagabunda transition from quiescence to necrotrophic colonization in apples could involve the secretion of alkalizing compounds.

Phytoplasmas and mycoplasmas are bacteria belonging to the class Mollicutes. In this study, a fine tuning of quantitative polymerase chain reaction (qPCR) with a universal mycoplasma primer pair (GPO3F/MGSO) targeting the 16S rRNA gene was carried out on phytoplasmas. The dissociation curves of DNAs from Catharanthus roseus phytoplasma-infected micropropagated shoots and from phytoplasma field-infected plant samples showed a single peak at 82.5 °C (±0.5) specifically detecting phytoplasmas belonging to several ribosomal groups. Assay specificity was determined with DNA of selected bacteria: 'Candidatus Liberibacter solanacearum', Xylella fastidiosa, Ralstonia solanacearum and Clavibacter michiganensis. No amplification curves were observed with any of these tested bacteria except 'Ca. L. solanacearum' that was amplified with a melting temperature at 85 °C. Absolute quantification of phytoplasma titer was calculated using standard curves prepared from serial dilutions of plasmids containing the cloned fragment GPO3F/MGSO from European stone fruit yellows phytoplasma. Phytoplasma copy number ranged from 106 to 103 according with the sample. The sensitivity evaluated comparing plasmid serial dilutions resulted 10-6 for conventional PCR and 10-7 for qPCR. The latter method resulted therefore able to detect very low concentrations of phytoplasma in plant material.