Selection for Resistance to Verticillium Wilt Caused by ... - HortScience

HORTSCIENCE 46(2):201–206. 2011.

Selection for Resistance to Verticillium Wilt Caused by Race 2 Isolates of Verticillium dahliae in Accessions of Lettuce (Lactuca sativa L.) Ryan J. Hayes1 U.S. Department of Agriculture, Agricultural Research Service, Crop Improvement and Protection Unit, 1636 E. Alisal Street, Salinas, CA 93905 Karunakaran Maruthachalam Department of Plant Pathology, University of California, Davis, c/o U.S. Agricultural Research Station, 1636 E. Alisal Street, Salinas, CA 93905 Gary E. Vallad Gulf Coast Research and Education Center, University of Florida, 14625 CR 672, Wimauma, FL 33598 Steven J. Klosterman U.S. Department of Agriculture, Agricultural Research Service, Crop Improvement and Protection Unit, 1636 E. Alisal Street, Salinas, CA 93905 Krishna V. Subbarao Department of Plant Pathology, University of California, Davis, c/o U.S. Agricultural Research Station, 1636 E. Alisal Street, Salinas, CA 93905 Additional index words. genetic variation, breeding, cultivars, disease resistance Abstract. Verticillium wilt of lettuce caused by Verticillium dahliae can cause severe economic damage to lettuce producers. The pathogen exists as two races (Races 1 and 2) in lettuce, and complete resistance to Race 1 is known. Resistance to Race 2 isolates has not been reported, and production of Race 1-resistant cultivars will likely increase the frequency of Race 2 strains. The objective of this research was to select lettuce accessions for resistance to Race 2 isolates of V. dahliae. Two independent populations totaling 314 randomly sampled PIs were evaluated for Verticillium wilt disease incidence (DI) caused by V. dahliae isolate VdLs17 in one unreplicated and two replicated greenhouse experiments. Selection for PIs with reduced DI was conducted between each experiment and plant stems were plated on semiselective media to identify colonized plants that remained non-symptomatic. No accession with complete resistance was identified, although accessions with partial resistance were selected. Genetic variation for the frequency of V. dahliae-colonized plants that remain symptomless was detected. Four PIs (169511, 171674, 204707, and 226641) were selected for further testing in three replicated greenhouse experiments and demonstrated significantly lower disease incidence than the susceptible control cultivars. The results indicate that lettuce has genetic variation for partial resistance to a Race 2 isolate of V. dahliae. The resistant PIs selected in this research are morphologically diverse, and no dependence between rate of bolting and resistance was found. PIs with partial resistance may be useful for breeding lettuce cultivars with resistance to Race 2 isolates of V. dahliae. Lettuce (Lactuca sativa L.) is a globally important horticultural crop, and many preexisting and emerging diseases constrain pro-

Received for publication 17 Nov. 2010. Accepted for publication 23 Nov. 2010. This research was supported by the California Leafy Greens Research Program, the Leafy Greens Crop Germplasm Committee, the California Department of Food and Agriculture under the ‘‘Buy California Initiative,’’ the California Department of Food and Agriculture Specialty Crop Block Grant Program, and the U.S. Department of Agriculture, National Research Initiative, Crops at Risk. 1 To whom reprint requests should be addressed; e-mail [email protected].

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duction throughout the world. Host resistance is often the most sustainable and economically viable control method, if available (Lebeda et al., 2009). To achieve this, continual screening for new/additional resistances is necessary because new diseases emerge or change. Verticillium wilt caused by Verticillium dahliae Kleb. is a serious disease of lettuce that was first identified in the central coast of California in 1995 (Subbarao et al., 1997). The disease has subsequently spread within coastal California (Atallah et al., 2010) and has been identified in the Mediterranean basin (Garibaldi et al., 2007; Ligoxigakis et al., 2002). The pathogen is seed-transmitted in lettuce and other crops commonly grown in rotation with lettuce,

raising concerns regarding its spread to other lettuce production areas (Atallah et al., 2010; Vallad et al., 2005). The disease is particularly destructive on lettuce, and all market types are susceptible. Plants often remain symptomless until they near harvest maturity, at which time the symptoms develop quickly. Host resistance is the best long-term control method in lettuce, because current cultural control methods are cost-prohibitive, potentially damaging to the environment, or of limited feasibility (Subbarao et al., 1997). Verticillium dahliae of lettuce exists as two pathogenic races (Race 1 and Race 2). The Batavian cultivar La Brillante and several other heirloom cultivars are resistant to Race 1 isolates, whereas no source of resistance against Race 2 isolates is known (Hayes et al., 2007; Vallad et al., 2006). The system is similar to that described in tomato (Alexander, 1962; Vallad et al., 2006), and the pathogenicity of isolates (Race 1 or Race 2) from lettuce and tomato is strongly correlated (Maruthachalam et al., 2010). Race 1 resistance in ‘La Brillante’ is complete (no symptom development) (Vallad and Subbarao, 2008) and continues to be effective in grower fields for the time being (Hayes et al., 2007). In tomato, widespread use of cultivars carrying the Race 1 resistance gene Ve+ led to the discovery of resistance-breaking Race 2 isolates that now predominate in many tomato production regions (Alexander, 1962; Pegg and Brady, 2002). Although the existence of Race 2 lettuce isolates in California production districts has been established (Maruthachalam et al., 2010), no fields that are predominantly or exclusively infested with Race 2 isolates have been reported. Regardless, it is highly likely that production of Race 1-resistant lettuce cultivars will increase the frequency of Race 2. Therefore, there is a need to rapidly identify sources of resistance to Race 2 isolates of V. dahliae in lettuce. Substantial diversity exists within cultivated lettuce and wild relatives for resistance to biotic and abiotic stresses and for horticultural traits (Ryder, 1999). The primary gene pool of lettuce comprises germplasm that is fully interfertile with L. sativa, including the wild species L. serriola and several L. serriolalike species. Lactuca saligna and L. virosa represent the secondary and tertiary gene pools, respectively (Lebeda et al., 2009; Ryder, 1999). All are 2n = 2x = 18 and are autogamous, resulting in cultivars, landraces, accessions, and wild populations of highly homozygous plants. Within cultivated lettuce, numerous market types are known, including crisphead, romaine, butterhead, Latin, and red or green leaf and the less widely grown stem and oil seed types (Ryder, 1999). Cultivated lettuce may be derived from multiple gene pools (Kesseli et al., 1991), and each lettuce type may represent an important and unique resource for the identification of useful new genes. Screening diverse Lactuca for disease resistance is often complicated by correlations with morphology, maturity, or rate of bolting. In the V. dahliae–lettuce pathosystem, disease severity and incidence may be related to market or harvest maturity as well as to

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reproductive maturity (Hayes et al., 2007; Vallad and Subbarao, 2008). Disease evaluations must be delayed past market maturity or until flowering to reduce the number of lines incorrectly categorized as resistant. Furthermore, foliar symptoms can be similar to other diseases or disorders. Examination of root and foliar symptoms in conjunction with plating plant samples on semiselective media may be required for accurate evaluations. Taking these factors into account, successful strategies were used to identify resistance to Race 1 isolates (Hayes et al., 2007) and are expected to successfully identify resistance to Race 2 isolates. The objective of this research was to identify accessions with resistance to Race 2 isolates of V. dahliae. Materials and Methods Greenhouse experiments. Verticillium wilt resistance was assessed using greenhousegrown plants inoculated with V. dahliae isolate VdLs17, a highly aggressive Race 2 isolate. The isolate was prepared and maintained according to Vallad et al. (2006). Seeds of each cultivar were sown in 200-well plug trays, incubated at 10 C in the dark for 48 h in a growth chamber, and then germinated at 20 C with a 16-h photoperiod. Seedlings were inoculated at 2, 3, and 4 weeks after sowing by saturating the soil in each plug tray well with a 3-mL suspension containing 2 · 106 conidia/ mL in sterile, distilled water. Seedlings were incubated for another 1 to 2 weeks after the third inoculation and transplanted into 0.5-L foam-insulated cups filled with a pasteurized sand:potting soil mixture (3:1 v/v). All replicated experiments used a randomized complete block design with three blocks and five plants per block per accession. Unreplicated experiments used 10 plants per accessions and were transplanted in alpha-numerical order based on the accession number. Inoculated and noninoculated plants of the cultivar Salinas 88

were included in each replicated and unreplicated experiment for each population as a susceptible control; inoculated plants of the cultivar La Brillante were included in replicated experiments for each population as a Race 1-resistant control. Plants were maintained until flowering and then evaluated for DI (proportion of symptomatic plants). Plants were uprooted, roots were cleaned of soil, and cut longitudinally to evaluate for the presence of root discoloration and foliar symptoms typical of Verticillium wilt. Crown and stem sections (1 to 2 inches long) of non-symptomatic plants were sampled and plated on semiselective NP-10 medium (Kabir et al., 2004) to determine the presence or absence of the pathogen. This was conducted only with accessions having less than 20% DI within each experiment. Stems from symptomatic plants as well as non-symptomatic uninoculated plants were collected and plated as positive and negative controls. Up to six cross-sections of each plant stem sample were plated, and identification of V. dahliae from at least one section was interpreted as a positive result (infected) for that plant. Accessions evaluated. Selection for Verticillium wilt resistance was conducted in two independent populations of accessions (Populations A and B) (Table 1). Each population was comprised of randomly selected PIs, which are part of the Western Regional Plant Introduction Station (WRPIS) in Pullman, WA. The source of the seeds used in these experiments came from a working collection of the WRPIS genebank maintained at the U.S. Department of Agriculture (USDA), U.S. Agriculture Research Station in Salinas, CA. A total of 314 accessions were tested from 29 countries in Europe, Africa, and Asia (Table S1). The species were primarily L. sativa and included a diversity of market types based on historical passport data located at the USDA in Salinas, CA (Table 1 and Table S1). However, market type classification for L. sativa acces-

Table 1. The number of wild Lactuca and cultivated L. sativa of each market type tested for resistance to Verticillium wilt caused by Race 2 V. dahliae isolate VdLs17. Number of y Number and type of cultivated L. sativax accessions Number of wild species z ser sal sat-ser BAT BUT COS LAT LEF PRI STM MX ND Population tested Population A 155 2 1 6 69 3 48 3 8 14 1 Population B 159 6 1 1 11 60 26 2 30 3 1 14 4 Total 314 8 1 1 12 66 95 5 78 6 9 28 5 z Populations A and B are independent populations of randomly sampled accessions (Table S1). y Wild Lactuca species: ser = L. serriola; sal = L. saligna; sat-ser = probable L. sativa · L. serriola hybrids. x Cultivated lettuce types BAT = Batavia; BUT = butterhead; COS = cos or romaine; LAT = Latin; LEF = leaf; PRI = primitive and transitional types, including oil seed lettuce; STM = Stem or Balady; MX = population of diverse cultivated types; ND = no data.

sions were not field-grown to confirm. Ten accessions of L. serriola, L. saligna, and apparent L. sativa · L. serriola hybrids were also tested. Evaluation procedure. All accessions were initially tested in an unreplicated greenhouse experiment followed by two replicated experiments (replicated greenhouse Expts. 1 and 2, GH1 and GH2). During these experiments, accessions were selected for low DI and low occurrence of infected plants that were nonsymptomatic. Selection within each population was conducted separately in independent experiments. The number of accessions tested in each experiment was as follows. Population A, unreplicated: 155 accessions, GH1: 37 accessions, GH2: 16 accessions. Population B, unreplicated: 159 accessions, GH1: 58 accessions, GH2: 21 accessions. Finally, four accessions from Population A (PI169511, PI171674, PI204709, and PI226641) were tested in three additional replicated greenhouse experiments. Data analysis. All analyses were conducted separately within each population using DI data. To determine if selection from unreplicated greenhouse experiments was successful at improving resistance, the DI of accessions was compared with the susceptible control, ‘Salinas 88’, for the unreplicated and replicated greenhouse Expts. 1 and 2. Additionally, the proportion of accessions with DI numerically lower than ‘Salinas 88’ in unreplicated and the subsequent replicated experiment was compared to determine if selection increased the frequency of accessions with better resistance than ‘Salinas 88’. In all comparisons, exact binomial confidence intervals (95%) were calculated for each proportion, and confidence intervals that did not overlap were considered significantly different. Disease incidence data from replicated greenhouse experiments were analyzed using analysis of variance-type statistics of ranked data using the PROC Mixed procedure in SAS (Version 9.1; SAS Institute Inc., Cary, NC) and the LD_CI macro to generate relative marginal effects (RME) for each treatment and 95% confidence intervals for detection of statistical differences between treatments (Brunner et al., 2002; Shah and Madden, 2004). This analysis method was used because it has no assumptions regarding normality and constant variances. Confidence intervals that did not overlap were considered a significant difference with a lower RME corresponding to a lower DI. Only comparisons between susceptible control cultivars and accessions were considered. The median and maximum DI was also calculated for each accession and cultivar. The non-inoculated

Table 2. Mean Verticillium wilt disease incidence (DI) caused by Race 2 Verticillium dahliae in Salinas 88 and two populationsz of Lactuca accessions in one unreplicated and two replicated greenhouse experiments (GH1, GH2). Population A DIy Unreplicated Replicated GH1 Replicated GH2 Unreplicated Treatment Salinas 88 0.21 0.45 0.45 0.25 0.20* 0.20* 0.27 NS Accessions 0.27 NS z Populations A and B are independent populations of randomly sampled accessions (Table 1). y DI is the proportion of symptomatic plants. NS = non-significant; *significant at P < 0.05; comparisons are between Salinas 88 and accessions within columns

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Population B DI Replicated GH1 0.58 0.27*

Replicated GH2 0.67 0.31 NS


controls in the greenhouse experiments remained free of Verticillium wilt and were not included in the analysis. The date of disease evaluation was recorded for each accession and used to determine the number of weeks from planting to flowering. The correlation between DI and weeks to flowering was calculated to determine the dependence between these variables. The proportion of V. dahliae-positive lettuce stems from non-symptomatic plants was calculated, and exact binomial confidence intervals (99%) were calculated to compare accessions. Non-overlapping confidence intervals were considered significantly different. As a result of an incubator failure, some data were discarded, and the remaining data were unbalanced. Consequently, data from select accessions were pooled across experiments and analyzed only if the accession’s proportion of the total number of plated stems was consistent across all experiments. This was done to achieve a balanced data set and eliminate any bias resulting from differences among experiments. This criterion was only satisfied by comparing PI169511, PI204707, and PI226641 from Population A across two replicated experiments and PI273582, PI274366, PI278074, and PI342450 across two replicated experiments from Population B. Results Foliar and root vascular symptoms of Verticillium wilt observed in cultivated lettuce were typical of those previously reported (Hayes et al., 2007; Subbarao et al., 1997; Vallad et al., 2006). In many early-bolting L. sativa, L. serriola, and L. saligna, wilting was negligible or absent, because leaves rapidly developed necrosis followed by defoliation (Fig. S1). Plants also exhibited extensive auxiliary branching in some cases (Fig. S1). In unreplicated experiments, the correlations between the number of weeks to flowering and DI was –0.12 (Population A) and –0.09 (Population B). Neither of these correlations was significant (P > 0.08). In unreplicated greenhouse experiments using Populations A and B, the Verticillium wilt DI ranged from 0 to 1.0 (data not shown). Although numerous accessions had zero symptomatic plants in the unreplicated experiment, complete resistance was not reproducible in any accession in subsequent experiments (data not shown). However, the results indicate that selection using data from unreplicated experiments was effective at eliminating highly susceptible accessions. The mean DI of all accessions in unreplicated experiments was 0.27 in both populations, which was not significantly different from the DI of the susceptible control cultivar Salinas 88 (Population A DI = 0.21, Population B DI = 0.25) (Table 2). In subsequent replicated experiments using selected accessions from Population A, the DI for accessions was 0.20 in GH1 and GH2. This was significantly lower than the DI for ‘Salinas 88’ (DI = 0.45 in GH1 and GH2). A similar result was found in Population B, although the difference between ‘Salinas 88’ and accessions HORTSCIENCE VOL. 46(2) FEBRUARY 2011

in Population B was not significant in GH2 (Table 2). The frequency of accessions with lower DI than ‘Salinas 88’ was increased in replicated experiments by discarding highly susceptible accessions based on the results of the unreplicated greenhouse experiments. The proportion of accessions in unreplicated experiments with numerically lower DI than ‘Salinas 88’ was 0.53 in Populations A and B (Table 3). In Population A, 37 and 16 accessions were selected for retesting in GH1 and GH2, respectively, and resulted in 0.97 (GH1) and 0.94 (GH2) proportion of accessions with numerically lower DI than ‘Salinas 88’ (Table 3). The proportions for GH1 and GH2 are significantly higher than the unreplicated experiments. A significantly higher proportion of accessions with lower DI than ‘Salinas 88’ in GH1 and GH2 compared with the unreplicated experiment was also observed in Population B (Table 3). Further selection after replicated greenhouse Expt. 1 was not effective at improving the level of resistance in either population. Comparing replicated greenhouse Expts. 1 and 2, the mean DI of Population A (GH1 = 0.20; GH2 = 0.20), Population B (GH1 = 0.27; GH2 = 0.31), and the cultivar Salinas 88 (tested with Population A: GH1 = 0.45, GH2 = 0.45; tested with Population B: GH1 = 0.58, GH2 = 0.67) was relatively unchanged (Table 2). Furthermore, the DI of individual accessions selected for testing in GH1 and GH2 were inconsistent, and few lines had a level of resistance that was repeatedly significantly lower than ‘Salinas 88’ or ‘La Brillante’ (Table 4). In a rare but extreme example of inconsistent performance, two accessions in Population B (PIs 250428 and 358014) had no disease (median and maximum DI = 0) in GH1 but had median and maximum DI of 1.0 in GH2 (Table 4). In Population A, only four accessions had significantly lower RME than either ‘Salinas 88’ or ‘La Brillante’ in both GH1 and GH2 (Table 4). In Population B, only one accession had significantly lower RME than either ‘Salinas 88’ or ‘La Brillante’ in both experiments (Table 4). Four accessions from Population A were tested in three independent greenhouse experiments with no selection between experiments. The differences among lines was significant

(P = 0.009), whereas the experiment · accession interaction was not (P = 0.11). The median DI was zero for all accessions compared with 0.45 and 0.60 for ‘La Brillante’ and ‘Salinas 88’, respectively (Table 5). The maximum DI (the plot with the highest DI) was relatively similar for all accessions and cultivars, ranging from 0.67 to 1.0. The RME of all four accessions was significantly lower than ‘Salinas 88’, and the RME of PI 204707 was significantly lower than ‘La Brillante’ and ‘Salinas 88’. The frequency of non-symptomatic plants that were nonetheless colonized with V. dahliae was compared among select accessions and indicated that genetic differences exist in Lactuca for the frequency of non-symptomatic-infected plants. Recovery of V. dahliae from non-symptomatic plants in accessions from Population A ranged from a proportion of 0.29 (PI 204707) to 0.95 (PI 169511), and the 99% confidence interval for PI 204707 and PI 169511 did not overlap indicating a significant difference (Table 6). Among the four accessions evaluated from Population B, the proportion of recovery ranged from 0.30 (PI 274366) to 1.0 (PI 273582). The 99% confidence interval for PI 274366 and PI 273582 did not overlap, indicating a significant difference (Table 6). Discussion Identification of resistance to Race 2 isolates of V. dahliae is critically important to sustaining the lettuce industry, and we have screened 314 accessions for DI using isolate VdLs17. No complete resistance was found in this collection of germplasm. However, genetic variation for reduced DI does appear to exist in Lactuca. Partial resistance has been described for tomato and other crops (Fradin and Thomma, 2006; Okie and Gardner, 1982a, 1982b), and partial resistance is currently the best description of the disease reactions reported here. However, we cannot exclude the possibility that some accessions are mixtures of resistant and susceptible genotypes, and sub-selection within accessions could result in lines with complete resistance. Four accessions (PIs 169511, 171674, 204707, and 226641) were identified with a repeatable

Table 3. Mean Verticillium wilt disease incidence (DI) caused by Race 2 Verticillium dahliae in ‘Salinas 88’ and the proportion of accessions in two populationsz with lower disease incidence (DI) than Salinas 88 in one unreplicated and two replicated experiments. Accessions Accessions with lower DI than Salinas 88 99% confidence interval Proportion Lower Upper

Number tested Expt.y Population A Unreplicated 155 0.53 0.46 Replicated GH1 37 0.97 0.87 Replicated GH2 16 0.94 0.74 Population B Unreplicated 159 0.53 0.46 Replicated GH1 58 0.97 0.90 Replicated GH2 21 0.83 0.67 z Populations A and B are independent populations of randomly sampled accessions (Table 1). y GH1 = greenhouse Expt. 1; GH2 = greenhouse Expt. 2.

0.60 1.00 1.00 0.60 1.00 0.93

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Table 4. Median and maximum Verticillium wilt disease incidence (DI) caused by Race 2 Verticillium dahliae and relative marginal effect (RME) of DI in replicated greenhouse experiments of selected accessions from two populations.z Accession Population A PI 206964 PI 226641 PI 204707 PI 171674 PI 179295 PI 206965 PI 226513 PI 141680 PI 142871 PI 204584 PI 198733 PI 220665 PI 169511 PI 164939 PI 169508 PI 169512 PI 164937 PI 167140 PI 169494 PI 184113 PI 171675 PI 206963 PI 221936 PI 172914 PI 181882 PI 179297 PI 146078 PI 169514 PI 121935 PI 181947 PI 207490 PI 181883 PI 140398 PI 171666 PI 140395 PI 140392 PI 162787 Salinas 88 La Brillante Population B PI 250428 PI 254368 PI 273582 PI 324242 PI 342492 PI 358014 PI 358018 PI 269500 PI 271940 PI 274358 PI 278092 PI 342442 PI 278074 PI 358024 PI 271476 PI 278102 PI 342450 PI 344448 PI 342508 PI 274366 PI 278080 PI 342054 PI 358002 PI 342464 PI 278070 PI 342540 PI 342486 PI 274364 PI 278072 PI 358008 PI 342474

x

RME

Replicated greenhouse Expt. 1 DIy Median Maximum

0.29 L, S 0.29 L, S 0.34 L, S 0.35 L, S 0.36 L, S 0.37 L, S 0.37 L, S 0.39 L, S 0.41 L, S 0.41 L, S 0.43 L, S 0.43 L, S 0.43 L, S 0.44 L, S 0.45 L 0.45 L 0.46 L 0.46 L 0.46 L 0.49 L 0.54 L 0.55 L 0.54 0.56 0.56 0.57 0.57 0.58 0.59 0.60 0.64 0.67 0.67 0.70 0.71 0.72 0.74 0.78 0.89

0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.10 0.10 0.10 0.10 0.00 0.20 0.00 0.20 0.20 0.25 0.17 0.13 0.25 0.20 0.23 0.38 0.20 0.50 0.50 0.40 0.60 0.40 0.70

0.00 0.00 0.20 0.25 1.00 0.20 0.20 0.50 0.40 0.40 0.60 0.60 1.00 0.40 0.50 0.80 0.20 0.20 0.20 0.40 0.67 0.60 0.75 0.40 0.40 0.60 1.00 1.00 1.00 0.60 1.00 0.60 0.50 0.60 0.75 0.75 0.67 0.80 0.80

0.18 L, S 0.18 L, S 0.18 L, S 0.18 L, S 0.18 L, S 0.18 L, S 0.18 L, S 0.27 S 0.27 S 0.27 S 0.27 S 0.27 S 0.35 0.35 0.37 0.37 0.37 0.37 0.40 0.41 0.44 0.44 0.44 0.46 0.50 0.50 0.51 0.52 0.52 0.52 0.52

0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.20 0.20 0.20 0.20 0.00 0.20 0.20 0.20 0.20 0.20 0.20 0.20 0.20 0.40 0.40 0.40 0.33

0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.20 0.20 0.20 0.20 0.20 0.40 0.40 0.20 0.20 0.20 0.20 0.60 0.25 0.40 0.40 0.40 0.20 0.60 0.60 1.00 0.40 0.40 0.40 0.50

RME

Replicated greenhouse Expt. 2 DI Median Maximum

0.67 0.62 0.53 0.38 L 0.58 0.46 L 0.52 L 0.55 0.70 0.60 0.80 0.62 0.44 L 0.61 0.63 0.51

0.25 0.37 0.13 0.00 0.20 0.20 0.20 0.13 0.33 0.40 0.40 0.25 0.00 0.33 0.20 0.25

0.75 0.67 0.75 0.25 0.75 0.40 0.40 1.00 0.67 0.60 0.75 0.75 0.80 0.60 0.60 0.40

0.72 0.81

0.20 0.40

0.20 0.60

0.76

100.00

100.00

0.29 S 0.38 0.57 0.83 0.45 0.36

0.00 20.00 33.00 100.00 20.00 0.00

20.00 20.00 100.00 100.00 40.00 40.00

0.75 0.62 0.39 0.21 S 0.41 0.45 0.53 0.29 S 0.44 0.33 0.21 S 0.29 S

67.00 50.00 0.00 0.00 20.00 20.00 40.00 0.00 0.00 0.00 0.00 0.00

100.00 100.00 60.00 0.00 25.00 40.00 50.00 20.00 100.00 33.00 0.00 20.00

0.78 0.79

60.00 70.00

100.00 100.00

(Continued on next page)

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Table 4. (Continued) Median and maximum Verticillium wilt disease incidence (DI) caused by Race 2 Verticillium dahliae and relative marginal effect (RME) of DI in replicated greenhouse experiments of selected accessions from two populations.z Replicated greenhouse Expt. 2 DI Replicated greenhouse Expt. 1 DIy RMEx Accession Median Maximum RME Median Maximum PI 278078 0.53 0.20 0.80 PI 278098 0.55 0.40 0.50 PI 342458 0.57 0.33 0.67 PI 358004 0.58 0.25 0.40 PI 278096 0.59 0.20 0.60 PI 289060 0.59 0.20 0.60 PI 278110 0.60 0.40 0.75 PI 257288 0.63 0.60 0.60 PI 342516 0.63 0.60 0.60 PI 278090 0.63 0.25 0.60 PI 289096 0.64 0.40 0.50 La Brillante 0.64 0.35 0.60 0.40 0.00 67.00 PI 278068 0.66 0.25 0.75 PI 289026 0.67 0.40 0.60 PI 289056 0.67 0.40 0.60 PI 321012 0.67 0.40 0.60 PI 278064 0.67 0.60 1.00 PI 342448 0.69 0.40 0.40 PI 358012 0.70 0.40 0.80 PI 339262 0.71 0.40 1.00 PI 278112 0.73 0.60 0.60 PI 342468 0.73 0.60 0.60 PI 274416 0.75 0.40 0.60 PI 342460 0.75 0.40 0.60 PI 278106 0.76 0.60 0.80 Salinas 88 0.78 0.50 1.00 0.76 60.00 100.00 PI 342494 0.85 0.60 1.00 PI 342476 0.86 0.60 0.60 PI 342456 0.87 0.80 0.80 z Populations A and B are independent populations of randomly sampled accessions (Table 1). y DI is the proportion of symptomatic plants. x Relative marginal effect calculated from rank analysis of DI data. S = significantly less disease than Salinas 88 at P < 0.05; L = significantly less disease than La Brillante at P < 0.05.

Table 5. Median and maximum Verticillium wilt disease incidence (DI) caused by Race 2 Verticillium dahliae and relative marginal effect of DI in three replicated greenhouse experiments. Disease incidence (proportion symptomatic plants) RMEy Median Maximum Accessionz PI 204707 0.35 L, S 0 0.75 PI 171674 0.41 S 0 0.75 PI 226641 0.42 S 0 0.67 PI 169511 0.44 S 0 1.00 La Brillante 0.66 0.45 0.80 Salinas 88 0.73 0.60 1.00 z Accessions previously selected from Population A using unreplicated and replicated greenhouse experiments (Tables 1 and S1). y Relative marginal effect calculated from rank analysis of DI data. S = significantly less disease than Salinas 88 at P < 0.05; L = significantly less disease than La Brillante at P < 0.05.

Table 6. Recovery of Race 2 Verticillium dahliae colonies on semiselective NP-10 media from select accessions of two populationsz grown in two independent greenhouse experiments. No. of plants No. of positivey plants (proportion positive) Accession tested Population A PI 204707 21 6 (0.29) PI 226641 28 18 (0.64) PI 169511 20 19 (0.95) Population B PI 274366 27 8 (0.30) PI 278074 11 5 (0.45) PI 342450 24 16 (0.67) PI 273582 36 36 (1.0) z Independent populations of randomly sampled PIs; see Table 1. y At least one V. dahliae colony identified.


99% confidence interval Lower bound Upper bound 0.07 0.36 0.66

0.61 0.87 1.00

0.09 0.10 0.36 0.85

0.58 0.85 0.89 1.00

level of partial resistance relative to the iceberg cultivar Salinas 88. These accessions are considerably different morphologically (Table S2), and this diversity will likely be useful for their future use in lettuce breeding (Hayes et al., 2007). Furthermore, there is no evidence that reduced Verticillium wilt DI in these populations is associated with plant morphology or rate of bolting. The evaluation strategy of initial unreplicated experiments followed by replicated experiments ultimately identified partial resistance to Race 2 isolates of V. dahliae in Lactuca. Although it appears initial unreplicated experiments were useful at discarding extremely susceptible accessions, the overall selection strategy using greenhouse experiments does not seem to be highly efficient. This was particularly apparent in replicated experiments using accessions previously selected for resistance based on data from an unreplicated trial. It is likely that our greenhouse assay lacks sufficient precision in a single experiment to reliably detect genetic differences in populations of selected accessions, which likely have reduced genetic variation. This is supported by the lack of consistent performance among selected accessions over multiple experiments. This may also result from a genotype · environment interaction. Regardless, confirmation of resistance in selected accessions will require extensive testing over multiple experiments. Furthermore, it is possible that discarded

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accessions may have potential as sources of resistance. It is not known whether the partial resistance reported here is effective in field environments, because infested fields containing a majority of Race 2 isolates are not known at this time. However, greenhouse and field experiments evaluating resistance to Race 1 isolates were strongly correlated (Hayes et al., 2007). Development and commercial production of partially resistant cultivars may allow the pathogen to propagate and increase field inoculum levels. The relationship between DI and field inoculum density is not known for partially resistant lines of lettuce. Therefore, it is conceivable that repeated production of partially resistant cultivars may increase the level of V. dahliae microsclerotia in soil rendering even partial resistance ineffective. Furthermore, plating of stem sections from symptomless plants on NP-10 media identified plants that exhibited vascular colonization of V. dahliae. High frequencies of infected but symptomless plants were found in two specific accessions (PI 169511 and PI 273582), indicating a genetic basis for this character. Notably, both PIs had previously undergone selection in unreplicated experiments against the occurrence of symptomless infection. It is likely that the environment and genotype · environment interactions have a large effect on this character. Additionally, quantitative differences in stem colonization among PIs, which were not evaluated in this research, could explain why some colonized plants had low or no visible symptoms in some experiments. Regardless, accessions that are colonized by V. dahliae but do not show symptoms have been reported in other crops (Fradin and Thomma, 2006) and can be useful tools for studying host–pathogen interactions. Although we have identified useful germplasm for Verticillium wilt resistance breeding, genetics, and pathology research in lettuce, continued screening for high levels of resistance to Race 2 isolates is needed. The International Lactuca Database (http://documents. plant.wur.nl/cgn/pgr/ildb/), the most comprehensive catalog of Lactuca genetic resources,

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lists more than 11,000 accessions from all major international genebanks (Lebeda et al., 2007). Additionally, wild Lactuca from the center of diversity in Southwest Asia and the Eastern Mediterranean remains poorly collected (Lebeda et al., 2009). Consequently, large amounts of diverse germplasm have not been tested for resistance to Race 2 isolates and could harbor resistance that is superior to what is reported here. Literature Cited Alexander, L.J. 1962. Susceptibility of certain Verticillium-resistant tomato varieties to an Ohio isolate of the pathogen. Phytopathology 52:998–1000. Atallah, Z.K., K. Maruthachalam, L. du Toit, S.T. Koike, R.M. Davis, S.J. Klosterman, R.J. Hayes, and K.V. Subbarao. 2010. Population analyses of the vascular plant pathogen Verticillium dahliae detect recombination and transcontinental gene flow. Fungal Genet. Biol. 47:416–422. Brunner, E., S. Domhof, and F. Langer. 2002. Nonparametric analysis of longitudinal data in factorial experiments. John Wiley & Sons, New York, NY. Fradin, E.F. and B.P.H.J. Thomma. 2006. Physiology and molecular aspects of Verticillium wilt diseases caused by V. dahliae and V. alboatrum. Mol. Plant Path. 7:71–86. Garibaldi, A., G. Gilardi, and M.L. Gullino. 2007. First report of Verticillium wilt caused by Verticillium dahliae on lettuce in Italy. Plant Dis. 91:770. Hayes, R.J., G.E. Vallad, Q.-M. Qin, R.C. Grube, and K.V. Subbarao. 2007. Variation for resistance to Verticillium wilt in lettuce (Lactuca sativa L.). Plant Dis. 91:439–445. Kabir, Z., R.G. Bhat, and K.V. Subbarao. 2004. Comparison of media for recovery of Verticillium dahliae from soil. Plant Dis. 88:49–55. Kesseli, R., O. Oswaldo, and R.W. Michelmore. 1991. Variation at RFLP loci in Lactuca spp. and origin of cultivated lettuce (L. sativa). Genome 34:430–436. Lebeda, A., I. Dolezalova, E. Kristkova, M. Kitner, I. Petrzelova, B. Mieslerova, and A. Novotna. 2009. Wild Lactuca germplasm for breeding: Current status, gaps, and challenges. Euphytica 170:15–34.

Lebeda, A., E.J. Ryder, R. Grube, I. Dolezalova, and E. Kristkova. 2007. Lettuce (Asteraceae; Lactuca spp.), p. 377–472. In: Singh, R. (ed.). Genetic resources, chromosome engineering, and crop improvement series. Vol 3—Vegetable crops. CRC Press, Boca Raton, FL. Ligoxigakis, E.K., D.J. Vakalounakis, and C.C. Thanassoulopoulos. 2002. Weed hosts of Verticillium dahliae in Crete: Susceptibility, symptomatology and significance. Phytoparasitica 30:511–518. Maruthachalam, K., Z. Atallah, G.E. Vallad, S.J. Klosterman, R.J. Hayes, R.M. Davis and K.V. Subbarao. 2010. Molecular variation among isolates of Verticillium dahliae and PCR-based differentiation of races. Phytopathology 100: 1222–1230. Okie, W.R. and R.G. Gardner. 1982a. Screening tomato seedlings for resistance to Verticillium dahliae races 1 and 2. Plant Dis. 60:34–37. Okie, W.R. and R.G. Gardner. 1982b. Breeding for resistance to Verticillium dahliae race 2 of tomato in North Carolina. J. Amer. Soc. Hort. Sci. 107:552–555. Pegg, G.F. and B.L. Brady. 2002. Verticillium wilts. CABI Publishing, New York, NY. Ryder, E.J. 1999. Lettuce, endive and chicory. Crop production science in horticulture series. CABI Publishing, New York, NY. Shah, D.A. and L.V. Madden. 2004. Nonparametric analysis of ordinal data in designed factorial experiments. Phytopathology 94:33–43. Subbarao, K.V., J.C. Hubbard, A.S. Greathead, and G.A. Spencer. 1997. Verticillium wilt, p. 26– 27. In: Davis, R.M., K.V. Subbarao, R.N. Raid, and E.A. Kurtz (eds.). Compendium of lettuce diseases. The American Phytopathological Society, St. Paul, MN. Vallad, G.E., R.G. Bhat, S.T. Koike, E.J. Ryder, and K.V. Subbarao. 2005. Weedborne reservoirs and seed transmission of Verticillium dahliae in lettuce. Plant Dis. 89:317–324. Vallad, G.E., Q.-M. Qin, R.C. Grube, R.J. Hayes, and K.V. Subbarao. 2006. Characterization of race-specific interaction among isolates of Verticillium dahliae pathogenic on lettuce. Phytopathology 96:1380–1387. Vallad, G.E. and K.V. Subbarao. 2008. Colonization of resistant and susceptible lettuce cultivars by a green fluorescent protein-tagged isolate of Verticillium dahliae. Phytopathology 98:871–885.
