Location of the field and greenhouse assays
All experimental activities were carried out in the field, greenhouses, and laboratories of Embrapa Vegetables (CNPH), located at an altitude of 996 meters above sea level and geographical coordinates of 15º56’00” South latitude and 48º08’00” West longitude, in the Administrative Region of Gama in Brasília-DF, Brazil.
Screening germplasm accessions from distinct lettuce morphotypes to Septoria lactucae with natural inoculum under open-field conditions
The field experiment was carried out from January to March 2014. Forty-two (42) lettuce accessions were evaluated. The seedlings were produced in styrofoam trays with 128 cells filled with sterilized substrate (Plantmax®) and transplanted (after 21 days) into beds fertilized with simple superphosphate. Over time, three topdressing fertilizations with urea (10 g/m2) were carried out at ten, 20, and 30 days. Infestation with the inoculum of S. lactucae occurred naturally from old lettuce fields located in the vicinity of the experiment. Evaluations were performed by visual analysis of the leaf damage (= severity) induced by the pathogen at 50, 57, 64, and 71 days after sowing. The progress and degree of disease severity were evaluated according to the following scale (Sousa et al., 2003): grade 1 (= 1 to 25%), grade 2 (= 26 to 50%), grade 3 (= 51 to 75%), grade 4 (= 76 to 100% of leaf tissue injured). From the seedling transplant period until the last severity assessment, the average temperature was 21.7°C (range from 19.5 to 24.3°C); the average accumulated precipitation was 86.0 mm (range from 40.0 to 148.0 mm) and the average relative humidity was 73.43% (range from 54.2 to 82.9%). In this study, an experimental design of randomized blocks was used with 42 treatments (= lettuce accessions) x three replications (= plots). The plots consisted of 16 plants in a spacing of 0.30 x 0.30 m.
Field-selection of a subset of lettuce accessions for subsequent greenhouse assays aiming to assess the Septoria lactucae resistance stability
From the initial screening in the field, a subset of accessions classified as promising sources of resistance/tolerance was selected. The cultivars ‘BRS Mediterrânea’ and ‘Romana LNS’ (identified as being tolerant accessions under commercial field conditions) were also included in these subsequent assays. Seeds of the nine field-selected accessions/cultivars (‘Vera’, ‘Vanda’, ‘Romana Lente New Selection’, ‘Rubi’, ‘Isabela’, ‘Elisa’, ‘Banchu New Red Fire’, ‘BRS Mediterrânea’, and ‘Veneranda’) were sowed in styrofoam trays of 126 cells filled with sterilized substrate (Plantmax®) and kept in a greenhouse. Seedlings (15 days after sowing) were removed from the cells and then transplanted into plastic pots (1.0 L) containing a previously sterilized mixture. The mixture was composed of 110 g of ammonium sulfate, 510 g of super simple fertilizer, 200 g of limestone, 20 liters of raw rice straw, 20 liters of carbonized rice straw and 40 liters of cattle manure for every 200 liters of soil.
Fungal isolates and inoculum production for the subsequent greenhouse bioassays
Four S. lactucae isolates were employed in the bioassays viz. Sep13 (063.482 TRA), Sep26 (063.490 TRA), Sep34 (063.496 TRA) and Sep39 (063.501 TRA). All these isolates were classified as S. lactucae via molecular analyses (Cabral et al., 2022). The isolates were cultivated in 9 cm diameter Petri dishes containing oatmeal and agar culture medium (60 grams of oat flour + 18 grams of agar + 1000 mL of distilled water) in an incubator type BOD at constant temperature of 17 oC (12 hours light and 12 hours dark) for 13 days until sporulation (Dhingra and Sinclair 1995) for each isolate. For inoculum production (= conidial suspension for each isolate), 10 mL of sterile distilled water was added to each plate. The conidia were released into the suspension using a soft bristle brush and the spore suspension was subsequently filtered through a double layer of gauze. The spore concentration was estimated by counting under an optical microscope using a Neubauer chamber. The suspension was adjusted to a concentration of 2 x 105 conidia/mL. In the final step, 1 mL of Tween 20® per liter of water, was added to the suspension aiming to increase spore adhesion to inoculated leaves.
Greenhouse assay for Septoria lactucae reaction of field-selected lettuce accessions at the early vegetative phenological stage
The experiment was carried out during the months of October and November 2021. One hour before inoculation, the 1L-pots containing the lettuce plants were irrigated up to maximum soil saturation. Then, the plants (27 days of sowing) were inoculated by spraying the leaves until the beginning of the suspension run-off. After inoculation, the plants were kept in a humid chamber (23ºC ± 2ºC) for 48 hours. Control plants were sprayed only with sterile distilled water plus Tween 20®. In order to avoid cross-contamination between isolates, all plants treated with different isolates were kept in different benches (at least one meter apart). Disease incidence assessments were performed by counting the number of leaves with symptoms at 11 days after inoculation and the severity at 11, 13, 19 and 23 days after inoculation, using a scale of scores as a function of the degree of visual leaf damage (= severity). The experiment was carried out in a completely randomized design in a 9 x 4 factorial arrangement (nine accessions x four isolates) with four replications and each replication constituted a pot with two plants each. The cultivar ‘Rubi’ served as a susceptible control.
Greenhouse assay for Septoria lactucae reaction of field-selected lettuce accessions at the vegetative-reproductive phenological stage
In May-June, 2022, a third bioassay was carried out with older lettuce plants of the same subset of accessions that were inoculated in order to verify the reaction to fungus S. lactucae in the transition of the vegetative to the early reproductive phenological stage. This assay was carried out with identical methodological approaches of the first greenhouse assay with exception that the plants were inoculated at the age of 50-days-old. Evaluations were performed by visual analysis of the severity of leaf damage induced by the pathogen at 57, 64, and 71 days after sowing. The progress and degree of disease severity were evaluated according to the following scale of Sousa et al. (2003).
Statistical analyses for all assays
The Area Under the Disease Progress Curve (AUDPC) was calculated from the pathogen reaction data (= average severity grades) of each lettuce accession expressed by the arithmetic mean of the scores of all evaluated plants. AUDPC was calculated using the following formula: AUDPC = Σ [((y1 + y2) /2) *(t2 - t1)], where y1 and y2 are two consecutive evaluations performed at times t1 and t2, respectively. After obtaining the severity grade values for each lettuce accession, the data were submitted to analysis of variance and the means were grouped by the Scott-Knott test (P ≤ 0.05) with the assistance of the SISVAR statistical program (Ferreira 2011).