Genetic Analysis of Resistance to Lettuce Drop Caused by Sclerotinia minor

Despite extensive germplasm screening, no lettuce accessions have been identified as possessing immunity to infection by Sclerotinia species. As previously reported, several genotypes have consistently shown a significant reduction in disease incidence compared with susceptible varieties following inoculation with S. minor. Many of these genotypes exhibit architectural features that may promote avoidance or escape from infection, such as upright growth and early bolting. To date, the genetic basis and mechanisms of resistance identified in lettuce remain unknown. Transfer of resistance that is due solely to avoidance into commercial cultivars without simultaneous transfer of unacceptable plant morphology may be difficult or impossible. In contrast, physiological resistance is likely to be more easily incorporated into acceptable cultivars. To facilitate the development of lettuce cultivars with S. minor resistance, we sought to ascertain the genetic basis of resistance from the primitive L. sativa accession PI 251246. Recombinant-inbred lines (RILs) were developed from a ‘Salinas’ x PI 251246 F2 population to determine the heritability and action of genes involved in resistance derived from PI 251246 and for mapping of quantitative resistance loci. Results and implications of preliminary evaluation of F2:4 RILs in a replicated field trial will be discussed. INTRODUCTION The disease lettuce drop is caused by two fungal species, Sclerotinia minor and S. sclerotiorum, and causes the complete collapse and soft rot of infected plants. Crop losses are routinely low to moderate in all production areas, and sporadically are very severe. Complete control is not acheived through cultural practices and fungicide applications, making resistant cultivars a top priority for the lettuce industry (Subbarao, 1998). Several lettuce (Lactuca sativa) cultivars and accessions have been described as partially resistant to either S. minor or S. sclerotiorum (Chupp and Sherf, 1960; Elia and Piglionica, 1964; Newton and Sequeira 1972; Madjid et al., 1983; Subbarao, 1998; Whipps et al., 2002). Since that time, partially resistant crisphead breeding lines have been developed (E.J. Ryder, pers. commun.), but commercial cultivars with an adequate level of resistance to lettuce drop are not available. This limited progress has been attributed to both the genetic complexity of and difficulties in assessing resistance. S. minor usually infects the plant at the crown via mycelia following germination of soilborne resting structures (sclerotia). Resistance to S. minor has been identified and confirmed among a diverse array of lettuce genotypes, using both field and greenhouse evaluation methods (Grube and Ryder, unpublished results). Although no lines have shown immunity when using these procedures, several genotypes showed lower incidence of disease (DI) than susceptible controls. Germination of sclerotia, and therefore DI, is highly influenced by environment (Imolehin et al., 1980; Pennypacker and Risius, 1999). As a result, variability in DI observed in different experiments is common and can obscure differences between partially resistant and susceptible genotypes. Accurate selection of resistance therefore requires time-consuming and laborious replication within and repetition of tests. Proc. XXVI IHC – Advances in Vegetable Breeding Eds. J.D. McCreight and E.J. Ryder Acta Hort. 637, ISHS 2004 Publication supported by Can. Int. Dev. Agency (CIDA) 50 To date, neither the inheritance nor the mechanism (s) of S. minor resistance identified in lettuce are known. Studies have suggested that resistance to S. sclerotiorum is under genetic control, but the specific genetic basis has not been examined (Newton and Sequiera, 1972). Our objective was to determine the feasibility of conducting genetic analyses of S. minor resistance using a RIL population developed by crossing the highly tolerant primitive L. sativa accession PI 251246 with the susceptible crisphead cultivar Salinas. We also sought to obtain preliminary information about possible mechanisms of resistance of PI 251246 by determining whether certain morphological traits showed an absolute association with resistance in the population examined. In this manuscript, we report results of field evaluation of F2:4 RILs from the population described above, and discuss implications for future experiments. MATERIALS AND METHODS Plant Material A recombinant-inbred line (RIL) population was created by crossing the S. minor – tolerant primitive L. sativa accession PI 251246 with the susceptible crisphead cultivar Salinas. Hybridity of F1 plants was confirmed by morphological markers. Generations were advanced from the F2 to F5 generation by single-seed descent in the greenhouse in Salinas, Calif. Forty-seven F4 RILs were evaluated for S. minor resistance and several morphological traits in a field plot at the USDA-ARS research station in Salinas, Calif. Pathogen High levels of S. minor were established in the experimental plot through a combination of continuous cropping with lettuce and the incorporation of additional S. minor-colonized rye seeds every 1 to 3 years for several years. Endemic inoculum was presumed to be a mixture of isolates. Supplemental inoculum was S. minor isolate ‘Sm18’, which was isolated from a lettuce field in Santa Maria, CA in 1993. To produce inoculum, rye seeds were mixed with water (1:1, v/v), autoclaved twice for 20 min, inoculated with mycelial plugs taken from the growing margin of 2-day-old potato dextrose agar (PDA) cultures, and incubated for 21 days at 20oC with a 14-hour photoperiod. One to two S. minor-colonized rye seeds were placed 1 to 2 cm from the base of each plant approximately four weeks after transplanting. Plot Layout Lettuce seeds were sown in the greenhouse in plug trays. Four-week old seedlings were transplanted into the field in two rows on 1 m wide beds with 30 cm spacing. Treatments were randomly assigned to experimental units in four replicates in a randomized complete block design. Each experimental unit consisted of a 3 m plot containing 20 plants. Trait Evaluation Total plant number was counted at the time of inoculation. Plants were monitored for the appearance of lettuce drop symptoms at regular intervals throughout the field season. Final disease incidence (DI = proportion of plants killed) was determined 28 days post-inoculation (dpi). Two of the four plots of each genotype were evaluated for one qualitative (leaf color) and several quantative traits. For all traits, the ratings obtained for the two plots evaluated were averaged. To permit timely evaluation, all traits except early bolting were evaluated categorically as follows: Leaf Surface – smooth = 0, intermediate = 1-2, blistered = 3; Heading Tendency – none = 0, slight = 1, strong = 2; Leaf Color – green = 0, segregating = 1, red = 2; Growth Habit – flat = 1, intermediate = 2, erect = 3; Axillary Branching – none = 0, intermediate = 1-4, strong = 5; Leaf Shape – narrow = 1, intermediate = 2-3, wide = 4; and Plant Diameter – small = 1, intermediate = 2-4, large = 5. For heterogeneous lines, the most extreme phenotypic values observed were averaged