Comparison of different methods to assess downy mildew symptoms on either grapevine leaf discs or plants A. Vecchione, S. Vezzulli, E. Peressotti, S. Micheli, M. Stefanini, G. Malacarne, C. Moser and L. Zulini Department of Genomics and Biology of Fruit Crops, Research and Innovation Centre - Fondazione Edmund Mach (FEM) Via Mach 1, I-38010 San Michele all’Adige (TN), Italy - antonella.vecchione@fmach.it Introduction Downy mildew, caused by the oomycete Plasmopara viticola (Berk. & Curt.) Berl. & de Toni, is one of the major threats to grapevine particularly in warm and humid climate (Agrios G. N., 1997). All traditional grapevine cultivars (Vitis vinifera L.) are susceptible to downy mildew, the control of which requires regular application of fungicides. One alternative to the use of pesticides is the development of new varieties with innate resistance to this disease. An early characterization of the resistance level of new genotypes allows for elimination of highly susceptible plants during breeding program. Several laboratory screening procedures have been developed for grapevine, using artificial inoculations with a suspension of P. viticola sporangia. These methods provide controlled environmental conditions favourable to infection and development of the parasite, thus leading to the optimal expression of the disease. Materials and Methods An interspecific population derived from Merzling (M) (complex hybrid of V. vinifera descending from V. rupestris and V. lincecumii) × V. vinifera cv Teroldego (T) was characterized for resistance to P. viticola. The cross was developed at the Fondazione Edmund Mach and consisted of 131 F1 individuals, replicated annually by grafting wood cuttings onto rootstock Kober 5BB and grown in pots. Artificial infections on plants: 10 week old grafted plants were inoculated by spraying a conidial suspension of about 105 spores/ml onto the abaxial leaf surface and were kept overnight in the dark in a growth chamber at 21°C with 100% RH. The extent of sporulation was assessed by visually estimating the percentage area of sporulation (% Sp) at 6 dpi (days post infection) on the lower leaf surface on all infected leaves of 3 replicates according to OEPP/EPPO (1997). Plants were also classified using the OIV 452 descriptor, recommended by the Office International de la Vigne et du Vin (OIV, 2013). Classes were named 1, 3, 5, 7 and 9 from the most susceptible to the totally resistant, identified by absence of sporulation. Leaf disc bioassay: each progeny individual was grown in the greenhouse in three replicates. For each replicate the fourth and fifth leaves beneath the shoot apex were detached and rinsed with water. Eight leaf discs of 2.5 cm diameter were excised from each genotype with a cork borer and plated onto wet paper in Petri dishes with the abaxial side up. Discs were sprayed with P. viticola inoculum suspension at about 105 spores/ml. Petri dishes were incubated in a growth chamber at 21 °C and dark, for 6 days. Drops were removed with sterile filter paper after 24 h. At 6 dpi the sporulation was assessed by visually estimating the percentage area of sporulation (% Sp) and scoring the OIV 452-1 descriptor on all infected discs and by image analysis. Pictures of leaf discs were taken at 6 dpi using a digital camera (Nikon D80) under natural light conditions and at constant focal length. Images were stored in jpeg format and processed using the open source software ImageJ as described in Peressotti et al. (2011). The main goal of this work is to compare different methods of downy mildew symptoms assessment both on plants and leaf discs upon artificial infection. Comparisons were made between: i) visual estimation of sporulation and OIV code on plants, ii) visual estimation and image analysis on leaf discs, and iii) OIV code and image analysis on leaf discs. The first comparison was performed on all progeny genotypes for two artificial infections (during 2012), while the second and the third ones were done on a pool of 13 genotypes for four artificial infections (during 2012 and 2013). Results For each comparison, the R2 value of the linear correlation and the corresponding significance level are indicated. Visual estimation vs Image analysis Fig. 2 - Correlation between quantification of downy mildew sporulation on leaf discs using visual estimation and image analysis. A) and B) two different artificial infections carried out in year 2012. C) and D) Two different artificial infections carried out in year 2013. Each data point corresponds to the average of 8 discs belonging to the same genotype. *** = significant at P ≤ 0.001 Visual estimation vs OIV Code Fig. 1 - Correlation between quantification of downy mildew sporulation on potted plants using visual estimation and OIV code. A) and B) two different artificial infections carried out in year 2012. Each data point corresponds to the average of 8 plants belonging to the same genotype. *** = significant at P ≤ 0.001 A B A B C D Image analysis vs OIV Code Fig. 3 - Correlation between quantification of downy mildew sporulation on leaf discs using image analysis and OIV code. A) Two different artificial infections carried out in year 2012. B) Two different artificial infections carried out in year 2013. Each data point corresponds to the average of 8 discs belonging to the same genotype. *** = significant at P ≤ 0.001 A B Conclusions In general image analysis showed a very good linear correlation with the other methods as revealed by the high R2 values. In particular, on leaf discs the comparison between this method and visual estimation revealed a higher values of R2 respect to the comparison with OIV code. This is likely due to the fact that the first two methods are based on quantitative evaluations (% of infected disc area), while the OIV code is a qualitative screening based on the assignment to fixed resistance classes. We observed a slightly lower R2 (but highly significant) also in the correlation between visual estimation and OIV code assessed on plants, confirming a good correspondence among the different methods of evaluation. Literature Agrios G. N. 1997. Plant Pathology. Academic Press, San Diego, CA, USA. OEPP/EPPO. 1997. Guidelines for the efficacy evaluation of plant protection products. In EPPO Bulletin. Volume 27: 385-387. OIV. 2013. Descriptor list for grapevine varieties and Vitis species. Office International de la Vigne et du Vin (OIV). http://www.oiv.int Peressotti E., Duchene E., Merdinoglu D., Mestre P. 2011. A semi-automatic non-destructive method to quantify grapevine downy mildew sporulation. Journal of Microbiological Methods. 84: 265-271.