Geographical location of the experiments – This work was carried out in the Plant Pathology and Plant Breeding Laboratories and in the greenhouses of the Embrapa Vegetable Crops (CNPH). The experimental farm is located at 996 meters above sea level and geographical coordinates of 15º56’00” South latitude and 48º08’00” longitude to the West, in the Administrative Region of Gama in Brasília-DF, Central Brazil, during the years 2021 and 2022.
Major characteristics of the contrasting parental cultivars – Controlled crosses between contrasting cultivars were carried out in order to investigate the mode of inheritance of resistance to different isolates of the two Berkeleyomyces species. The hybridization process was carried out between the cultivar ‘Elisa’ (used as a susceptible male parent) and ‘La Brillante’ (employed as the resistant female parent). ‘La Brillante’ is an heirloom cultivar developed in the 1930s in Europe; it has bright green leaves and multiple disease resistance traits (Sala et al. 2008; Hayes et al. 2011; 2014; Simko et al. 2015; Perdomo 2022). The cultivar ‘Elisa’ belongs to the ‘Butterhead’ morphotype with large plants, light green foliage and cubic shape. ‘Elisa’ is resistant to lettuce mosaic virus (LMV) pathotype II, but displays susceptibility to Septoria lactucae, B. basicola, and B. rouxiae (Sala et al. 2008; Perdomo 2022; Perdomo et al. 2023).
Berkeleyomyces isolates and inoculum production for the bioassays – The B. basicola and B. rouxiae isolates were obtained from symptomatic lettuce plants. These isolates were previously identified at species level via molecular analyses (Souza 2022). The isolates B. rouxiae (EH–2743 and EH–2763) were collected in Santa Maria de Jetibá, Espírito Santo–ES (20º 2’ 27” S; 40º 44’ 45” W) and Campinas, São Paulo–SP (47º 04’ 40” W; 22º 53’ 20” S), in the year 2021. The B. basicola isolates (EH–2783 and EH–2784) were collected in Teresópolis, Rio de Janeiro–RJ (22° 24’ 44’’ S; 42° 57’ 59’’ W) and Sumidouro, Rio de Janeiro–RJ (22º 02’ 46” S; 42º 41’ 22” W), in 2021 respectively. These isolates were grown in Petri dishes (9-cm-diameter), containing Potato Dextrose Agar (PDA-t) culture medium (200 g of potato, 20 g of dextrose, 20 g of agar, 1000 mL of distilled water and 30 mg of tetracycline/L). Fungal colonies were kept in a BOD incubator at a constant temperature of 23°C (12 hours light and 12 hours dark) for 15 days (Dhingra and Sinclair 1995). Conidia suspension was prepared by adding 10 mL of sterilized distilled water to each plate, and then the conidia were released with the aid of a soft brush. The spore suspension was subsequently filtered through a double-layer gauze. The spore concentration was estimated under an optical microscope by counting them with the assistance of a Neubauer’s chamber. In the final step, the suspension was adjusted to concentrations of either 7.5 x 105 or 2 x 106 conidia per mL.
Inoculation protocol – At 21 days after germination, the seedlings were gently removed from the cells and washed in running tap water to get rid of the substrate adhered to the roots. The root system of each seedling was then immersed into 3 mL of the spore suspension (2 x 106 conidia/mL) for three minutes. The residual suspension was placed near the crown area of each transplanted seedling with the aid of a micropipette. The seedlings were transplanted into trays (72 cells) containing 1/3 of the substrate (Plantmax®) infested 10 days before with Berkeleyomycesspores at a concentration of 7.5 x 105 conidia/gram of substrate. Control (mock-inoculated) seedlings were dipped into sterilized distilled water and then transplanted to trays containing non-colonized substrate (at least one meter away from the inoculated plants) under identical environmental conditions.
Controlled crossings for production of F1 hybrid plants, and development of F2 segregating populations – Seeds of the contrasting cultivars ‘Elisa’ and ‘La Brillante’ were sown (3 mm depth) in 6.2 cm deep polystyrene trays with 128 cells (containing previously sterilized substrate) at 7-day intervals during three consecutive weeks. With this scheme, a greater coincidence of flowering between the contrasting male and female parental plants was obtained. The flower buds of the cultivar ‘La Brillante’ (chosen to be the ♀ parent), before anthesis, were emasculated and washed, according to the pollen removal technique proposed by Nagata (1992), which combines two traditional lettuce breeding methods (Oliver 1910; Pearson 1962). Emasculation was carried out with a sharp blade by cutting the upper third of each bud, thus cutting the anthers of the flowers of the recipient female plant, at the intersection of the corolla with the floral envelope, before sunrise (5:00–6:00 am). Then, after exposition of the stigma in development of the female parent, we proceed with the elimination of pollen grains in the floral stigma, with the help of a jet of water via a manual sprayer. Once the stigma was bifid and fully receptive, cross-pollination procedure was carried out using freshly collected pollen grains from the cultivar ‘Elisa’ (chosen to be the male parent). Fully open flowers were collected from ‘Elisa’ plants and then rubbed into previously emasculated flower buds of the female parent. Subsequently, each pollinated flower bud was identified and covered with a paper bag. After the ripening of the seeds in the female parent, only previously emasculated and pollinated flowers were harvested. The seeds were then cleaned, packed, identified as having putative F1 origin and stored in a cold chamber.
Evaluation of the reaction to Berkeleyomyces isolates of putative F1 plants – These putative F1 seeds were subsequently sown in trays of 128 cells and 21-days. After seed germination, 300 F1 seedlings were inoculated with the isolates B. rouxiae EH–2763 (150 seedlings) and B. basicola EH–2783 (150 seedlings). Plants were individually evaluated for the reaction to both fungi. Then, leaf samples of plants identified as resistant to either B. rouxiae or B. basicola and displaying morphologically distinct features in comparison with the original parents were collected, aiming to confirm their hybrid origin. The selection of truly hybrid plants was carried out via genotyping with randomly amplified polymorphic DNA (RAPD) markers (see section below).
DNA extraction from the contrasting parents and individuals from the F1 populations and confirmation of their hybrid origin via RAPD marker genotyping – DNA was extracted individually from each plant of the parents and from putative F1 individuals, according to a slightly modified 2X CTAB methodology (Boiteux et al. 1999). DNA samples were quantified using 1.5% agarose gel in 0.5X TBE buffer (Tris-borate-EDTA), stained with GelRed® Nucleic Acid Gel Stain (Biotium, USA) and visualized in a photodocumentator L-Pix ST (Loccus Biotechnology). The quantified DNA samples were diluted with TE + RNAse (75 ng/μL) and used as template in the RAPD assays. The final volume of the reaction was 12.5 μL, consisting of 2 μL of DNA, 5.95 μL of Milli-Q water; 1.25 μL of 10X buffer (100 mM Tris-HCl, 500 mM KCl, pH 8.3), 0.6 μL of MgCl2, 0.5 μL of dNTPs, 0.2 μL of Taq DNA polymerase and 2 μL of primer. Forty-eight primers (Operon random primer kit; Operon Technologies, Alameda, CA) were used to search for polymorphisms between the contrasting cultivars ‘Elisa’ and ‘La Brillante’. Template DNA of cultivars ‘BRS Mediterrânea’, ‘PI 342444’, and ‘Salinas 88’ were used as controls for primer selection in the RAPD assays. The primer OP-H7 was selected due to its ability to generate a sharp band consistently present in the male parent and absent in the female parent. This primer was then used for genotyping putative F1 individuals. Polymerase chain reaction (PCR) assays were performed using a Veriti® thermocycler, Applied Biosystems (ABI). The PCR program consisted of an initial cycle of 94°C for 2 minutes, followed by 35 cycles of 94°C for 30 seconds (denaturation), 36°C for 1 minute (primer annealing), and 72°C for 1 minute (extension), followed by a final cycle of 68ºC for 10 minutes and 4ºC for an indefinite period of time (∞). The PCR products (amplicons) were analyzed in agarose gels (1.5%) stained with ethidium bromide and visualized under ultraviolet light, using the 1 Kb Plus DNALadder® marker (Invitrogen, Carlsbad-CA, USA) for analysis of the products obtained.
Study of the segregation patterns of resistance to two species of Berkeleyomyces in the contrasting parents, F1 hybrids and in F2 populations –The hybrid origin of individual F1 plants was assessed via RAPD marker genotyping. Two bona-fide F1 plants were then self-fertilized to obtain two independent F2 generations. Altogether, 413 F2 seedlings (21-day-old) were inoculated separately with B. rouxiae ‘EH–2743’ (247 seedlings) and B. basicola ‘EH–2784’ (166 seedlings). Two groups of 30 plants of the contrasting parents were also inoculated with each pathogen.
Disease severity assessment and statistical analysis – The disease assessment was performed 30 days after inoculation (DAI), using the disease severity scale based upon the degree of visual severity of the roots evaluated according to the scale proposed by O’Brien and Davis (1994). This 1-5 grade scale is as follows: grade score (1) = absence of symptoms; grade (2) = traces of necrosis in the radicles; grade (3) = less than 50 % of necrotic roots; grade (4) = more than 50% and less than 90% of necrotic roots; and grade (5) = more than 90% of the roots severely affected. To determine the mode of inheritance, an arbitrary evaluation criterion was adopted, where: plants with scores between (1) and (2) were arbitrarily classified as resistant and plants with scores between (3) and (5) as susceptible. The data obtained for genetic analysis were evaluated by the chi-square (χ2) significance test at 5% probability the check the goodness of fit to predicted Mendelian ratios. The expression for the chi-square significance test was χ2 = Σ [(O-E) % E], where: O = observed frequency in the segregating population and E = expected frequency in the segregating population. Plants classified as resistant were transplanted into plastic pots (5L), containing a mixture of 110 g of ammonium sulfate, 510 g of super simple, 200 g of limestone, 20 liters of raw rice straw, 20 liters of rice straw and 40 liters of cattle manure for every 200 liters of soil. The pots with the transplanted plants were placed in a greenhouse to advance the F3 progenies via controlled self-fertilization of individual F2 plants.