Marina Collina

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 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.

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.

Fungi of genera Phlyctema 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. kienholzii 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‐1α , 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.

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.

Bull’s eye rot is an important postharvest disease affecting apples (Malus domestica) worldwide. Fruit infection occurs in the field, but rot lesions develop only after a period in cold storage (about 90 days). The disease is caused by fungi belonging to genus Neofabraea (N. vagabunda, N. malicorticis, N. perennans and N. kienholzii) (Spotts et al. 2009). However, no data are available on the presence of species other than N. vagabunda in Italy. To verify the current causal agents of apple bull’s eye rot in Emilia-Romagna Region, a pathogen survey of apples stored in several local packinghouses showing bull’s eye rot symptoms was carried out in 2014. Small sections from the edges of necrotic tissue of one hundred fruit were placed on tomato agar (TA) and kept at 15°C. Sporulation was observed after 14 days of incubation. Mycelia originated from the single spore of twenty isolates were used for DNA extraction. Polymerase chain reaction (PCR) was used to amplify the β-tubulin (tub2) gene using primers F 5’CTTTCTCCGTTGTCCCATCC-3’ and R 5’-GAACATTGCGCATCTGGTCC-3’ (Cao et al. 2013). After sequencing and sequence assembly, BLAST analysis showed that one isolate (M1) had 99% identity to N. malicorticis, while other isolates belonged to N. vagabunda. The sequence of N. malicorticis isolate was submitted to Genbank (accession number KP263368). Morphological characteristics of N. malicorticis isolate were examined after 14 days of incubation on TA at 15°C. Colonies were circular, centrally umbonate, with abundant white floccose mycelium. Average growth rate was 22mm ± 2 in 14 days. Conidia were one-celled, cylindrical, rounded at apex, aseptate, hyaline and measured 6 to 16 μm (mean 10μm ± 2.4) x 2 to 5 μm (mean 3 μm ± 0.8), similarly to micro and intermediate conidia formed on oatmeal (Chen et al. 2015). To prove pathogenicity, Koch’s postulates were conducted on ten ‘Cripps Pink’ apples, surface-sterilized, wounded and inoculated with 20 μl conidial suspension (104 conidia ml-1). After 90 days of storage at 0°C, artificially infected apples showed lesions of 21 mm 29 ± 1 diameter without conidiomata on peel surface (symptoms identical to those observed on apples that were originally obtained from cold storage). Water-only controls were symptomless. The pathogen was re-isolated from decayed tissue. To our knowledge, this is the first report of N. malicorticis causing bull’s eye rot of apples in Italy. This finding suggests that, beside N. vagabunda, other species of Neofabraea may occur in Italy and this could represent an emerging problem on apples. Epidemiological studies performed on a wider scale could improve the knowledge of current Neofabraea species occurring in Italian orchards, leading to the adoption of specific control strategies to reduce fruit postharvest losses.