Grapevine Resistance to Botrytis Cinerea

Two strains of P. ampelicida isolated by FEM in Trentino (Italy), and one isolated by the Julius Kühn Institute (JKI)–Institute for Grapevine Breeding Geilweilerhof (Siebeldingen, Germany), were previously genetically characterized and propagated on oatmeal agar (0.5% w/v) [22 ]. These strains were combined and used for artificial infection, following the protocol developed by Bettinelli et al. [22 ]. Briefly, fresh leaf tissues with mature BR lesions were used as the inoculum source. In the greenhouse, the cuttings of the mapping population were cultivated in a climatic chamber at 24 °C, young growing shoots with at least five fully expanded leaves were sprayed with conidia suspension adjusted to 10⁴ conidia/mL in the late afternoon and kept at 100% relative humidity overnight. In the field, clusters at susceptible phenological stage [26 ] BBCH 77 (berries beginning to touch) [89 ] and growing shoots were sprayed after the sunset to avoid direct UV irradiation. Plastic bags were used to wrap inoculated organs, ensuring a high humidity treatment overnight. Bags were removed the following early morning before sunrise. Overall, two experiments were conducted on clusters in the two growing seasons 2020 (FC1) and 2021 (FC2), while in 2022, disease progression did not occur due to unusually high temperatures during the night (>30 °C). In each field inoculation trial, the entire population was screened simultaneously, and two clusters were evaluated per genotype, generating four sub-experiments (FC1a, FC1b, FC2a, FC2b). Shoots were evaluated once in the field in 2021, distinguishing between leaves (FL) and shoot internodes (FS). A total of eight greenhouse inoculation trials were performed between 2020 and 2021 on subgroups of the population to obtain three experiments (GL1, GL2, GL3). To produce an overall dataset of the resistance trait, the replicated data of GL and FC trials were also combined by (i) retaining only the minimum resistance value per each genotype and experiment type (GL min and FC min), and (ii) calculating the median (GL median, FC median). While the minimum should exclude possible false positive resistance evaluations, the median is the most robust statistical parameter for non-normally distributed data. Resistance evaluation of leaves in the greenhouse was conducted following the 5-step scale proposed by Rex et al. [21 (link)], which resembles OIV descriptors [90 ], i.e., (1) very low, (3) low, (5) medium, (7) high, and (9) very high resistance. Contrariwise, leaves and shoot internodes in the field were evaluated with a binomial scale as susceptible (S that is 1) or resistant (R that is 9), respectively, in the presence or absence of lesions, because the occurrence of other lesions caused by multiple abiotic and biotic stresses did not permit using a more detailed scale. To screen cluster resistance in the field, a 5-step rating scheme was developed based on the percentage of infected berries (Table 3), following the example of OIV₄₅₉ descriptor for degree of cluster resistance to Botrytis bunch rot [90 ]. Berry color (white or black, coded as a 0/1 valued binary variable) was also recorded to be used as a positive control in QTL analyses, thanks to the well-known location of the responsible QTL on chromosome 2 [33 (link)]. The normality of trait distribution was determined by means of the Shapiro–Wilk normality test (p < 0.05). All statistical analyses were executed with the software PAST 3.26 [91 ].