Euphytica (2017) 213:20 DOI 10.1007/s10681-016-1813-0

Genetics of resistance in lettuce to races 1 and 2 of dahliae from different host species

German V. Sandoya . Suraj Gurung . Dylan P. Short . Krishna V. Subbarao . Richard W. Michelmore . Ryan J. Hayes

Received: 23 June 2016 / Accepted: 2 December 2016 / Published online: 23 December 2016 Ó Springer Science+Business Media Dordrecht (outside the USA) 2017

Abstract Race 1 resistance against Verticillium originating from hosts other than lettuce. This indi- dahliae in lettuce was originally shown in the cultivar cated that cultivars resistant to race 1 and the breeding La Brillante to be conditioned by a single dominant lines derived from them would also be resistant to race gene (Verticillium resistance 1, Vr1). Multiple, mor- 1 isolates from other hosts; similarly, the partial phologically diverse sources of germplasm have been resistance would be effective against race 1 and 2 identified as resistant to race 1. In this study, allelism isolates from hosts other than lettuce. Nevertheless, tests indicated that resistance in these different lettuce there were specific interactions that warrant further cultivars is closely linked or allelic to the Vr1 gene. study. Although race 1 currently predominates in the The Vr1 gene is defeated by race 2 isolates of V. major lettuce production area of the Salinas Valley, dahliae. Only partial resistance to race 2 isolates is CA, breeding lettuce for resistance to V. dahliae available in a few plant introductions (PIs). Green- should take both races into account. house and field experiments conducted with these PIs demonstrated partial resistance to V. dahliae race 1 as Keywords Breeding Á Disease Á Disease resistance Á well as race 2 isolates from lettuce. Cultivars resistant Diversity Á Genetic Á Host-pathogen interaction Á to race 1 and PIs with partial resistance to race 2 were Lactuca sativa Á Vegetable Á challenged with several race 1 and 2 isolates

G. V. Sandoya Á R. W. Michelmore The Genome Center and Department of Plant Sciences, S. Gurung University of California, Davis, One Shields Avenue, Sakata Seed of America, Salinas, CA 93907, USA Davis, CA 95616, USA D. P. Short Present Address: Division of Plant and Soil Sciences, West Virginia G. V. Sandoya University, Morgantown, WV 26506, USA Everglades Research Education Center, Horticultural Sciences Department – IFAS, University of Florida, Belle Glade, R. J. Hayes (&) FL 33430, USA National Forage Seed Production Research Center, United States Department of Agriculture, USDA, 3450 SW S. Gurung Á D. P. Short Á K. V. Subbarao Campus Way, Corvallis, OR 97331, USA Department of , University of California, e-mail: [email protected] Davis, 1636 E Alisal St., Salinas, CA 93905, USA

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Introduction single aggressive isolate each of race 1 (VdLs16) and race 2 (VdLs17), Hayes et al. (2007) identified seven Fifty percent of the lettuce consumed in the USA is cultivars from a diversity of lettuce types that were grown in the Salinas Valley of California and is worth completely or highly resistant to race 1 but susceptible more than $1 billion annually (Anonymus 2014). to race 2 (Hayes et al. 2007). Only partial resistance to Verticillium wilt, caused by the soil borne race 2 of Verticillium wilt is known in lettuce (Hayes Verticillium dahliae Kleb., is a destructive disease of et al. 2011b). Plant introductions (PIs) 169511, lettuce in coastal California with the potential to 171674, 204707 and 226641 were shown to express severely reduce lettuce production in the region a lower incidence of Verticillium wilt compared to cv. (Subbarao et al. 1997). Symptoms of the disease Salinas when challenged with either the race 1 isolate include wilting of outer leaves, discoloration of the VdLs16 or race 2 isolate VdLs17 (Gurung et al. 2015; root vascular system, and ultimately plant death. Even Hayes et al. 2011b). though all lettuce types are susceptible, wilting is more The majority of V. dahliae isolates recovered from pronounced and economically damaging in iceberg the Salinas Valley are race 1 (Gurung et al. 2014); lettuce (Vallad et al. 2004). however, extensive crop production with Vr1 carrying Feasible methods for control of Verticillium wilt of cultivars, reduced fumigation, and inadvertent impor- lettuce are limited. Crop rotation is generally not tation of novel isolates could cause shifts in the effective since the pathogen is able to colonize a predominant populations in the future (Atallah et al. diverse range of dicotyledonous crops and weeds 2011; Short et al. 2014). Identifying new resistance (Atallah et al. 2011). This includes crops commonly genes, including among race 1 resistant cultivars (strawberry, artichoke and spinach) or occasionally would broaden the genetic base of resistance (Ye and (tomato, potato, eggplant and pepper) grown in Smith 2008). Additionally, the efficacy of resistance in rotation with lettuce (Bhat and Subbarao 1999). lettuce germplasm when challenged with V. dahliae Fumigation can reduce pathogen levels; however, isolates from diverse crops, particularly those com- the availability of fumigants is decreasing and monly rotated with lettuce, was unknown. As high- pathogen populations may return to pre-fumigation levels of resistance and race non-specific resistance is levels in as few as three lettuce crops (Short et al. generally more valuable, additional information on 2015). isolate specificity would be useful for prioritizing The most sustainable control method is to develop germplasm within current breeding efforts. Therefore and deploy resistant lettuce cultivars (Hayes et al. the objectives of this research were to determine: (1) 2007). There are two characterized races of Verticil- the allelic relationships of resistance in germplasm lium dahliae of lettuce (Vallad et al. 2006). The resistant to race 1 and (2) the effectiveness of resistant heirloom Batavian cultivar (cv.) La Brillante pos- lettuce germplasm against V. dahliae isolates origi- sesses the single dominant gene Verticillium resis- nating from diverse hosts other than lettuce. tance 1(Vr1), which confers complete resistance to race 1 isolates (Hayes et al. 2011c). Isolates that cause disease on Vr1 carrying germplasm are known and are Materials and methods designated as race 2 (Hayes et al. 2011c; Maruthacha- lam et al. 2010). Tomato also has two races of V. Description of experiments dahliae, where the Ve1 resistance gene interacts with the avr1 V. dahliae effector (de Jonge et al. 2012). In a Several trials were conducted in either the greenhouse handful of other crop species a single major gene or field using a diverse range of V. dahliae isolates confers high level resistance to Verticillium wilt (Mert (Table 1). The lettuce cultivars used in testing or as et al. 2005). However in other crops such as potato, parents were previously reported as resistant to race 1 multiple major Verticillium wilt resistance genes have (Hayes et al. 2007) or partially resistant to race 2 been reported (Jansky et al. 2004). (Hayes et al. 2011b). The field trials were grown in a Multiple lettuce accessions and cultivars possess race 1 artificially infested field site at the USDA-ARS resistance to race 1 isolates, though the genetic basis of Station in Salinas, CA using standard procedures for this resistance was unknown in most cases. Using a lettuce production on the Central Coast of California 123 Euphytica (2017) 213:20 Page 3 of 12 20

Table 1 Isolates of Isolate Originating host Geographical origin Racea Verticillium dahliae used in this study along with their VdLs16 Lettuce (Lactuca sativa L.) CA Race 1 hosts and geographic origin CS1616 Artichoke (Cynara scolymus L.) CA Race 1 and race classification based on Short et al. (2014) LE1087 Tomato (Solanum lycopersicum L.) CA Race 1 CL1 Watermelon (Citrullus lanatus L.) CA Race 1 VdLs17 Lettuce (L. sativa L.) CA Race 2 SM1 Eggplant (Solanum melongena L.) CA Race 2 VD57 Mint (Mentha longifolia L.) WA Race 2 CB86.7.2 Potato (Solanum tuberosum L.) WA Race 2 CF40 Pepper (Capsicum frutescens L.) CA Race 2 CF156 Pepper (C. frutescens L.) CA Race 2 303 Spinach (Spinacia oleracea L.) WA Race 2b G2D5 Spinach (S. oleracea L.) WA Race 2 a Short et al. (2014) PD494 Strawberry (Fragaria ananassa L.) CA Race 2 b Determined in this PD410 Strawberry (F. ananassa L.) CA Race 2b research

(Ryder 1999; Hayes et al. 2007). In greenhouse and &5 kg of potting mix. The second method of inoc- field trials, the first set were aimed at determining the ulation involved pipetting a 2 9 106 conidia/ml allelism of resistance in cv. La Brillante and five other suspension into each plug-tray cell. The conidia and race 1-resistant cultivars. The second set tested PIs suspension were produced using the methods of that were partially resistant to race 2 and two lettuce Vallad et al. (2006). Control plants for each lettuce isolates (VdLs16 and VdLs17, race 1 and 2 respec- cultivars were mock inoculated with sterile water and tively) under greenhouse conditions and against race 1 grown in a non-replicated block. in field conditions. The third set of were conducted with nine race 1-resistant cultivars plus four race 2 Disease assessment partially resistant PIs against 14 race 1 or race 2 isolates of V. dahliae isolated from diverse hosts. Disease symptoms depend on plant development, and unless otherwise stated, assessments of greenhouse Greenhouse experiments experiments were done when plants began to flower. Disease severity (DS) of each plant was evaluated by Seed germination longitudinally cutting the taproot and examining the leaves and root vascular tissue for symptoms typical of Seeds were germinated in Sunshine No. 4 plug mix V. dahliae. Each plant was rated on a 0 to 5 rating scale, (Sun Gro Horticulture, Canada) in 341 or 200 cell plug where 0 = roots with no discoloration; 1 = 1–25% of trays (each cell was 19 9 19 9 60 mm) and incu- the taproot with vascular discoloration; 2 = 26–50% bated in the dark at 10 °C for approximately 48 h. with discoloration; 3 = 51–75% with discoloration, Seedlings were transferred to a growth room for 4 = 76–100% with discoloration and no foliar symp- approximately 3 weeks at 20 °C and 16 h light before toms and 5 = 100% of taproot with discoloration and transplanting. leaves with wilting symptoms (Hayes et al. 2007). For each plot, disease incidence (DI) was calculated as the Inoculations proportion of plants with DS C 1. the DS was averaged across all plants within a plot before analysis. Two methods were used to inoculate plants. The first method involved mixing V. dahliae into potting mix NP10 and PDA media plating prior to seed sowing. Potato dextrose agar (PDA) plates with 2 week-old V. dahliae colonies were Putative symptomatic and non-symptomatic stems of mixed with potting mix in a ratio of ten plates: F2 plants and parents from the allelism test in the 123 20 Page 4 of 12 Euphytica (2017) 213:20 greenhouse experiments were plated on a modified conidia two and three weeks after sowing. Three

Sorenson’s NP10 semi-selective medium to confirm hundred plants of each F2 population were then the presence of V. dahliae using established protocols transplanted to a steam sterilized mix of 3 sand: 1 (Kabir et al. 2004). Stems from non-symptomatic potting soil in 500 ml foam-insulated cups. Each plant parents and the susceptible cv. Salinas were also was inoculated using 6 ml of a 2 9 106 conidia/ml plated on NP10. In field experiments, only putative suspension one week after transplanting. Plants were symptomatic plants were plated to confirm the pres- grown in the greenhouse and watered and fertilized as ence of V. dahliae. The putative V. dahliae positive needed until flowering at which time they were samples were then subcultured on PDA plates and assessed for disease severity. grown for approximately 3 days. A single spore was In a field trial F2 seed from crosses between cv. La transferred to a PDA plate, grown for 5 days, then Brillante to cvs. Annapolis, Eruption, Merlot, Little visually examined to determine if the fungus was V. Gem, Pavane and Sentry were planted in a race 1 dahliae based on morphology (Pegg and Brady 2002). infested field located at the USDA-ARS Salinas, California research station on 1 April 2015. After

Race determination of V. dahliae isolates thinning, approximately 400 F2 plants from each cross were grown for assessment. The race 1-resistant parent Each isolate of V. dahliae recovered above was cvs. (La Brillante, Annapolis, Eruption, Merlot, Little analyzed to determine which race it belonged to using Gem, Pavane and Sentry) and the susceptible cv. previously described methods (Short et al. 2014). Salinas were included and arranged as an RCD with

Briefly, mycelium was collected, drained and frozen in six replicates interspersed within the planting of F2 liquid nitrogen (N2), and freeze-dried before DNA progeny. Disease was assessed on all F2 plants when extraction (Gurung et al. 2014; Short et al. 2014). the susceptible cv. Salinas reached market maturity at Samples were ground with liquid nitrogen and DNA 85 days after sowing (DAS). Parental and check cvs. extraction was made using the Fast DNA extraction kit were assessed using a random sample of 10 plants per (Qiagen, Valencia CA). The PCR assay was followed plot. as described previously (de Jonge et al. 2012; Short et al. 2014). Challenge of plant introductions partially resistant to race 2 with single V. dahliae race 1 and 2 isolates Experiments from lettuce in greenhouse tests

Allelism tests The race specificity of germplasm with partial resis- tance (PR) to race 2 was unknown. We conducted two

F1 seed was produced through artificial hand cross greenhouse trials with cv. La Brillante (race 1 pollination and F2 seed was produced by allowing F1 resistant), three accessions with partial resistance plants to naturally self-pollinate. The F2 populations (PR) to race 2 (PIs 169511, 171674, and 204707), from crosses of cv. La Brillante to cvs. Merlot, and cv. Salinas against race 1 (VdLs16) and race 2 Annapolis and Eruption were planted in a greenhouse (VdLs17) lettuce isolates. The trials were conducted trial in February of 2012. F2 populations from cv. La twice and arranged as a six-block split-plot design Brillante crossed to cvs. Pavane and Little Gem were with isolates as whole plots and cultivars as split plots. planted in a second trial in May of 2012. Both trials Inoculum production, inoculum preparation and inoc- included the parents and susceptible cv. Salinas as ulation were done as previously described (Vallad controls. For analysis purposes, parents and Salinas et al. 2006). Disease incidence and severity were were considered as a randomized complete design assessed as previously described. (RCD) with four replicates and five plants per each cultivar. Additional seed of cv. Salinas 88 was planted Evaluation of race 2 partially resistant partially and used as a non-inoculated control. resistant in a race-1 field environment Plants were inoculated by mixing race 1 V. dahliae isolate VdLs16 into the potting mix prior to seed PIs that had previously expressed PR in the green- sowing and inoculating with a 3 ml suspension of house to a race 2 lettuce isolate were evaluated for 123 Euphytica (2017) 213:20 Page 5 of 12 20 resistance in a race 1-infested field at the USDA-ARS expected segregation of 15 non-symptomatic: 1 in Salinas, California. Trials were conducted twice and symptomatic plants using Chi square (v2). were arranged as a randomized complete block design The disease incidence (DI) and severity (DS) of the (RCBD) sown on May of 2012 and 2013. Seeds were parents included in the allelism test and the susceptible sown in three different beds that were 6.1 m long and cv. Salinas were analyzed as a separate RCD exper- 30 cm wide; each bed consisted of two seed lines iment using the ranked data in PROC MIXED of SAS 30 cm apart. The resistant cv. La Brillante and the to obtain an analysis of variance F (ANOVA-F). The susceptible cv. Salinas were included as controls in the LD_CI macro was used to calculate the relative planting. marginal effects (RME) for each treatment and 95% confidence intervals (CI); two treatments were con- Evaluation of V. dahliae race 1-resistant and race 2 sidered different when two CI did not overlap partially resistant lettuce cultivars against V. dahliae (Brunner et al. 2002; Shah and Madden 2004). A collected from diverse hosts similar analysis was conducted with the experiment with the race 2 partial resistant PIs in a race 1 field Nine lettuce cultivars (Annapolis, Defender, Eruption, environment. Infantry, Little Gem, Merlot, Pavane, and Sentry) with The race 1 resistant and the race 2 partial resistant complete resistance to V. dahliae race 1 isolates, and cultivars challenged with a variety of isolates of V. four PIs (169511, 171674, 204707, and 226641) with dahliae were analyzed individually as separate exper- PR against race 2 were tested against 12 V. dahliae iments, one analysis considered the race 1 resistant isolates. The pathogen isolates were obtained from cultivars and the other the race 2 partial resistant PIs. diverse crops that are grown in rotation with lettuce as Data was analyzed using the same non-parametric well as VdLs16 and VdLs17 as representative races 1 method with ranked data (Shah and Madden 2004)asa and 2 isolates, respectively. All isolates were previ- split-plot design. In this case the F1_LD_F1 and ously determined to be either race 1 or race 2 using a LD_CI macros were used to generate relative marginal molecular marker based on the Avr1 gene and a effects (RME) for each treatment and 95% CI. separate race 2 specific marker (de Jonge et al. 2012; Orthogonal contrasts were used to compare two Short et al. 2014) (Table 1). Seedlings were inoculated known lettuce strains of V. dahliae (race 1: VdLs16 at four weeks after sowing by saturating the soil in and race 2: VdLs17) to the strains isolated from each well with a 10-ml suspension containing 2 9 106 different hosts. A similar approach was used for the conidia/ml in sterile distilled water. Two additional experiment with the race 2 partial resistant PIs inoculations were performed on all lettuce cultivars at challenged with two isolates of V. dahliae in green- four and eight days after the first inoculation. Control house conditions. plants for each lettuce cultivar were mock inoculated with sterile water and grown in a non-replicated block. The trials were arranged as a three-block split-plot Results design, isolates were considered as whole plots and cultivars as the split plot, and the trial was conducted Allelism tests of race 1 resistant cultivars twice. Plants were grown on greenhouse benches for approximately eight weeks after the first inoculation Cultivar Salinas, the susceptible control, had fewer and were evaluated for Verticillium wilt disease symptomatic plants (53%) in the greenhouse com- severity. Ten plants per cultivar–isolate combination pared to field experiments (90%). Cultivar La Brillante were evaluated for DS in each plot as previously had no symptomatic plants in any greenhouse or field described. experiment. A few plants of cv. Merlot in the greenhouse experiments and a few plants of cvs. Little Statistical analysis Gem, Merlot, Pavane and Sentry in the field experi- ment exhibited an internal root discoloration. Plating To test the hypothesis that race-1 resistance is the root samples from resistant cvs. on NP10 media controlled by two independent dominant genes, the yielded no V. dahliae colonies and these plants were observed ratios in F2 families were compared to the deemed not infected by V. dahliae. 123 20 Page 6 of 12 Euphytica (2017) 213:20

Table 2 Total number of F2 plants tested, number with root discoloration, and number confirmed infected with race 1 Verticillium dahliae in F2 lettuce populations in greenhouse and in a race 1 field experiment in Salinas, California 2 Experiment Parent cv. La Brillante No. of F2 No. of F2 plants Expected no. race v Mean DI 9 plants tested 1 infectionsz Putative Plating experiments symptomsv NP10w PDAx PCRy

Greenhouse 0 Annapolis 283 5 7 2 0 18 – Field 0 383 2 0 0 0 25 – Greenhouse 0 Eruption 288 3 0 0 0 19 – Field 0 383 3 1 1 1 25 23.45 Greenhouse 0 Merlot 284 3 3 3 0 19 – Field 0 399 1 0 0 0 27 – Greenhouse 0 Pavane 297 10 12 1 1 20 19.35 Field 0 402 6 2 2 2 27 22.70 Greenhouse 0 Little Gem 291 8 0 0 0 19 – Field 0 385 7 1 1 1 26 23.58 Greenhouse – Sentry – – – – – – Field 0 369 0 0 0 0 25 – DI Median of disease incidence (% symptomatic plants) of race 1 resistant lettuce cultivars as the average of two greenhouse experiments, each greenhouse experiment had three replicates. In the field all cultivars were replicated six times v Putative symptomatic plants showing some type of root discoloration w Adjusted number of putative symptomatic plants after results from plating stems in NP10 semi-selective medium x Adjusted number of putative symptomatic plants after visual observation of mycelium and conidiophore growing of Verticillium dahliae in PDA y Adjusted number of putative symptomatic after running PCR with race 1 and 2 primers z Calculations were done considering 15 resistant to 1 susceptible segregation v2 values based on a 15:1 asymptomatic: symptomatic ratio. P values 0.05 = 3.841; 0.01 = 6.635. Therefore data does not fit the expected ratio 15:1

Large percentages ([97%) of F2 plants were Brillante 9 Pavane (two plants) and La Bril- initially rated as non-symptomatic in greenhouse and lante 9 Little Gem (one plant). None of the above

field experiments; only a few F2 plants exhibited root mentioned samples amplified with race 2 primers. The discoloration (Table 2). The subsequent isolation on two positive controls (fungal DNA) amplified with NP10 and PDA plates helped discriminate V. dahliae their respective primers indicating that the PCR and other Verticillium-like fungi. The number of conditions were suitable for amplification (data not symptomatic plants was then adjusted to determine the shown). number of V. dahliae infected plants. The crosses La Brillante 9 Merlot, La Brillante 9 Annapolis and La Partial resistant lettuce cultivars to race 2 and their Brillante 9 Pavane in greenhouse experiments all had interaction with two lettuce isolates of Verticillium less than three infected plants. These frequencies of dahliae in greenhouse conditions infected plants were too low to be an acceptable fit to a 15:1 (asymptomatic: symptomatic) segregation ratio Significant differences were found in DI and DS (Table 2). PCR testing of the isolates from these plants between trials (P = 0.0061, \0.0001), isolates demonstrated that only a single sample from La (P = 0.0128, \0.0001), while cultivars (P \ 0.0001 Brillante 9 Pavane was race 1 in greenhouse tests for DS) and the interaction (isolate 9 trial (Table 2). Four putative V. dahliae samples from the P \ 0.0001) was only significant for DS. The other field were concluded to be race 1. These samples came interactions were not significant in the analysis. from La Brillante 9 Eruption (one plant), La VdLs17 caused significantly more disease than

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1 1 80% L 0.9 0.9 70% 60% 0.8 50% 0.8 55% L 30% 0.7 20%40% 40% 30% S,L 0.7 40% S,L 0.6 0% 0.6 0.5

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0.4 RME 0.4 0.3 0% 0.3 0% S 0.2 0.2 0.1 0 0.1 171674 169511 204707 Salinas La Brillante 0 VdLs16 VdLs17 La Brillante 169511 204707 171674 226641 Salinas

Fig. 1 RME and medians (above the bars) of DI of different Fig. 2 RME and 95% CI (bars) of the disease incidence of six lettuce cultivars challenged with isolates VdLs16 and VdLs17 lettuce cultivar and PIs grown in a field predominantly infested of Verticillium dahliae in two greenhouse experiments with race 1 of V. dahliae. The data is the result of two experiments during 2012 and 2013. The data above bars represent the median DI of two experiments. S = DI signifi- cantly \ than Salinas, L = DI significantly [ than La Brillante VdLs16 on cv. La Brillante. In Fig. 1, only the RME and median of the DI are presented because DS was (Fig. 2). Among the partial resistant PIs tested in this highly correlated with DI. Differences between research, PI 169511 exhibited the lowest DI. The VdLs16 and VdLs17 within a PI were not significant, previously reported race 2 partial resistant PI 226641 though VdLs17 generally caused slightly more disease exhibited nearly as much root discoloration as cv. in 171674, 204707 and cv. Salinas (Fig. 1). Salinas. PI 171674 exhibited significantly less root discol- oration with isolate VdLs16 of race 1 compared to PIs Race 1 resistant cultivars tested with isolates 169511, 204707 and the susceptible cv. Salinas. of race 1 and 2 from hosts different than lettuce However, cv. Salinas had only 30% of plants with root discoloration. The resistant lettuce cv. La Bril- Cultivar Salinas exhibited a DS of 2 to 4.7 with all lante did not exhibit any sign of root discoloration and isolates causing different levels of root discoloration, no significant difference between this cultivar and the indicating that the environment was conducive for the partial resistant PI 171674 was detected (Fig. 1). PI disease. Cultivar Salinas was excluded from the subse- 171674 had significantly less root discoloration when quent analysis. Highly significant differenceswere found challenged with isolate VdLs17 compared to cvs. among nine race 1 resistant cultivars (P \ 0.0001), Salinas and La Brillante, but it was not different from isolates (P \ 0.0001) and the interaction isolate 9 cul- 1695511 and 204707 (Fig. 1). tivar (P \ 0.0001); interactions such as isolate 9 trial (P = 0.0548), cultivar 9 trial (P = 0.8822), and iso- Evaluation of race 2 partial resistant PIs in a race-1 late 9 cultivar 9 trial (P = 0.5656) were not field environment significant. In general race 1 isolates from different hosts did The ANOVA-F indicated significant differences not cause disease in race 1-resistant lettuce (Fig. 3). among cv. La Brillante, PIs 169511, 171674, Most of the remaining race 2 isolates produced 204707, 226641 and cv. Salinas for DI (P = 0.0007) substantial amounts of root discoloration, except for and DS (P = 0.0007). Slight differences were isolate CF40 from pepper. Isolates Vd57, (mint), observed in the interaction of cultivar 9 trial for DI CB86.7.2 (potato) CF156 (pepper) and 303 (spinach) (P = 0.0471). No diseased plants were found in cv. La caused intermediate levels of disease. Race 2 isolates Brillante. The RME values for three of the four partial G2D5 (spinach), SM1 (eggplant) and PD494 and resistant PIs were significantly lower than cv. Salinas, PD410 (strawberry) caused high levels of root discol- though all of them exhibited some diseased plants oration (Fig. 3). Race 2 isolate VdLs17 was the most

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Artichoke- Tomato- Lettuce- Watermelon Pepper - Mint - Potato -Pepper- Spinach - Strawberry- Strawberry - Eggplant - Spinach - Lettuce

Fig. 3 The RME of disease severity with 95% CI for four race 1 and ten race 2 V. dahliae isolates from diverse crop hosts averaged across nine Lactuca sativa race 1 resistant cultivars tested in two replicated greenhouse experiments aggressive of all isolates tested across the race exceptions as both isolates did not cause substantial 1-resistant cultivars. disease in the race 1 resistant cultivars. Race 1 resistant cultivars demonstrated diverse Orthogonal contrasts between race 2 lettuce isolate reactions to race 2 isolates. Figure 4 shows the RME VdLs17 and the race 1 isolates from hosts different of the DS of each of the race 1 resistant cultivars when than lettuce were significant (P \ 0.05). The contrasts challenged with different isolates of race 1 and 2. Race between VdLs17 and the other race 2 isolates were 2 isolates CF40, PD494, SM1, G2D5, and VdLs17 significant in most cases, as VdLs17 was generally exposed limited differences among these cultivars. more aggressive than other race 2 isolates. Isolate Conversely, some isolates demonstrated strong inter- G2D5 from spinach was not significantly different actions with specific cultivars. For example, race 2 than VdLs17 and caused similar levels of disease in isolates 303 (spinach), CF156 (pepper) and CB86.7.2 cvs. Annapolis, Eruption, Infantry, La Brillante, (potato) caused no root discoloration in cultivar Sentry Merlot and Pavane. while causing high levels of root discoloration in cv. La Brillante (Fig. 4). Evaluation of V. dahliae partially race 2-resistant The orthogonal contrasts between race 1 lettuce lettuce plant introductions against V. dahliae isolate VdLs16 and the race 1 isolates from different collected from different hosts hosts were not significant (P [ 0.05). Comparisons between VdLs16 and some race 1 isolates on Eruption, The ANOVA F detected significant differences among Infantry, La Brillante, Sentry and Pavane could not be isolates (P \ 0.0001), cultivars (P \ 0.0001) and the determined because all inoculations resulted in zero interactions of isolate 9 cultivar (P \ 0.0001); inter- disease and were considered as non-significant. These actions such as isolate 9 trial (P = 0.1325), culti- results provide additional evidence that isolates var 9 trial (P = 0.1215) and isolate 9 cultivar 9 trial CS1616, LE1087, and CL1 belong to race 1 of V. (P = 0.3880) were not significant for DS. dahliae. Contrasts between VdLs16 and the race 2 Overall race 1 isolates did not cause extensive root isolates from other hosts were mostly significant discoloration across these partially resistant PIs, (P \ 0.05). Pepper isolates CF40 and CF156 were the except for isolate CL1 (watermelon) (Fig. 5). The 123 Euphytica (2017) 213:20 Page 9 of 12 20

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Annapolis Defender Eruption Infantry La Brillante Little Gem Merlot Pavane Sentry

Fig. 4 RME of disease incidence with 95% CI for nine L. sativa race 1 resistant cultivars challenged with four race 1 and ten race 2 isolates of V. dahliae in two replicated greenhouse experiments partially resistant PIs also had similar levels of root SM1 (eggplant) and PD410 (strawberry) caused discoloration with isolates PD410 (strawberry), SM1 higher levels of root discoloration in the race 2 (eggplant), G2D5 and 303 (spinach) of race 2 as the partially resistant PIs. However, isolate 303 did not lettuce isolate VdLs17. The rest of race 2 isolates cause root discoloration in PI 2266641 (Fig. 6). caused little to no root discoloration (Fig. 5). The orthogonal contrasts showed that race 1 The individual RME of each race 2 partially isolates were similar to isolate VdLs16 (P [ 0.05) resistant PI challenged with 14 isolates of races 1 except for CL1 in PIs 171674 and PI 226641 and and 2 of V. dahliae from different hosts is presented in CS1616 in PI 226641. Several other race 2 isolates Fig. 6. CL1, a race 1 isolate from watermelon, caused were as aggressive as VdLs17 according to the slight root discoloration in PI 169511 and more severe orthogonal contrasts (P [ 0.05); such isolates were disease in PI 171674. However it caused no disease in 303, PD410, SM1, G2D5 in PI 169511; PD410, SM1 PIs 204707 and 226641. Isolate CS1616 from arti- and G2D5 in PI 171674 and 303, PD410 and G2D5 in choke caused little root discoloration in PIs 204707, PI 204707. 169511 and 171674 and none in 226641. The tomato isolate LE1087 and the race 1 lettuce isolate VdLs16 caused root discoloration only in PI 226641 (Fig. 6). Discussion Among race 2 isolates, all the PIs generally exhibited limited root discoloration with isolates The Vr1 gene confers resistance to race 1 of V. dahliae CF40 and CF516 from pepper, VD57 from mint and in the lettuce cv. La Brillante (Hayes et al. 2011c). We CB86.7.2 from potato (Fig. 6). CB86.7.2 was able to crossed several race 1 resistant lettuce cvs. to La cause extensive disease in PI 204707. PD494 from Brillante to determine if additional genes condition strawberry only produced intermediate root discol- resistance to race 1. Only a single confirmed race 1 oration on all PIs. Several race 2 isolates from hosts infected plant was discovered in greenhouse allelism different than lettuce such as 303 and G2D5 (spinach) tests and four in field experiments. The few plants

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0 VdLs16 CS1616 LE1087 CL1 CF40 CF156 VD57 CB86.7.2 PD494 303 G2D5 SM1 PD410 VdLs17

Lettuce-Artichoke-Tomato- Watermelon Pepper-Pepper-Mint-Potato-Strawberry-Spinach-Spinach-Eggplant-Strawberry-Lettuce

Fig. 5 The RME of disease severity with 95% CI for four race 1 and ten race 2 Verticillium dahliae isolates from diverse crop hosts averaged across four L. sativa PIs tested in two replicated greenhouse experiments

1 Race 1 Race 2 0.9

0.8

0.7

0.6

0.5 RME

0.4

0.3

0.2

0.1

0 VdLs16 CS1616 LE1087 CL1 CF40 CF156 VD57 CB86.7.2 PD494 303 G2D5 SM1 PD410 VdLs17

169511 171674 204707 226641

Fig. 6 RME of disease incidence with 95% CI for four L. sativa PIs challenged with four race 1 and ten race 2 isolates of V. dahliae in two replicated greenhouse experiments discovered with root discoloration may have resulted as saprophytes (Pegg and Brady 2002) and several from infection with a different Verticillium species species of Verticillium have similar morphology (Gurung et al. 2015). Organisms in this genus can live (Inderbitzin et al. 2011) and can cause root 123 Euphytica (2017) 213:20 Page 11 of 12 20 discoloration in susceptible lettuce (Gurung et al. Specifically, PI 226641 performed poorly against race 2015). The lack of segregation in these families 1 and 2 lettuce isolates but proved to have good levels indicates that only Vr1, or a gene closely linked to Vr1, of resistance against artichoke, watermelon, and likely confers resistance against race 1 of V. dahliae in spinach isolates in greenhouse experiments. Cultivar the ten diverse cultivars of lettuce analyzed. Sentry, which likely carries Vr1, had very little signs of Resistance against race 1 of V. dahliae in cv. La root discoloration with race 2 isolates 303 of spinach Brillante was successfully bred into iceberg germplasm and CB86.7.2 of potato. The genetic basis for this predominantly using isolate VdLs16 to test for resis- interaction is unknown, but may be distinct because cv. tance (Hayes et al. 2011a). However, V. dahliae can La Brillante was highly susceptible to both of these colonize several crops cultivated in the Salinas Valley isolates. The diversity of interaction phenotypes indi- and other areas in the western USA. None of the race cates that breeding lines should be tested for resistance 1-resistant cultivars (that presumably carry Vr1)tested against isolates from multiple sources as breeding for against the race 1 V. dahliae isolates from pepper, resistance progresses. tomato, strawberry, artichoke, eggplant, showed root New cultivars with high level resistance against discoloration. Plant stems plated on NP10 semi-selec- diverse V. dahliae isolates are likely to be needed for tive medium to determine whether the non-symp- reliable and sustained disease control. Among the tomatic plants had V. dahliae colonization failed to partially resistant accessions tested, PI 171674 most recover V. dahliae. Therefore, resistance identified with consistently expressed significantly less disease than race 1 isolates of V. dahliae from lettuce is also cv. Salinas in multiple greenhouse and field experi- effective against race 1 isolates from other hosts. This is ments and against multiple race 1 and 2 strains. Hence, consistent with previous reports showing that resistance PI 171674 is useful as a parent in breeding for in cvs. La Brillante and Little Gem is effective against a resistance against diverse V. dahliae isolates and broad range of race 1 isolates from diverse hosts races. Race 1 isolates of artichoke (CS1616) and (Gurung et al. 2015;Valladetal.2006). watermelon (CL1) caused significantly more disease The distribution of race 2 V. dahliae is largely than the lettuce isolate (VdLs16) in this PI. Therefore, limited to the Pajaro Valley but it is a concern that the resistance from PI 171674 should be combined with race 1 resistant breeding lines could become ineffec- Vr1 to achieve resistance against diverse V. dahliae tive through time if race 2 increases on the California isolates. Central Coast. Pre-emptive research is therefore being conducted to develop resistance before race 2 becomes Acknowledgements We extend special thanks to Crystal widespread and several sources of partial resistance Marchebout and Rosa Marchebout, laboratory technicians from the Department of Plant Pathology at UC Davis and Jose Orozco are known. The level of partial resistance to Verticil- and Dawn Avery of the USDA-ARS, Salinas, CA. This research lium wilt in lettuce appears to vary across environ- was funded by USDA NIFA Grant # 59-5305-4-002 and USDA ments, and we do not know if partial resistance is NIFA Specialty Crop Research Initiative (SCRI) Grant # expressed in race 2 infested field sites because no 2015-51181-24283 and by The California Leafy Greens Research Board. known sites are available. Three PIs with partial resistant to race 2 (169511, 171674, and 204707) (Hayes et al. 2011b) expressed partial resistance to race 1 in a field environment. Therefore, this research References suggests that partial resistance would also be effective against race 2 in field conditions. Anonymus (2014) Monterey County Crop Report. Vol. 2016. Accessions initially identified as partially resistance County of Monterey Agricultural Commissioner Atallah ZK, Hayes RJ, Subbarao KV (2011) Fifteen years of to VdLs17 generally expressed equivalent levels of verticillium wilt Of lettuce in America’s salad bowl: a tale resistance to lettuce isolates VdLs16 (race 1) and of immigration, subjugation, and abatement. Plant Dis VdLs17 (race 2) or may have had more resistance to the 95:784–792 race 1 lettuce isolate. However, testing diverse resis- Bhat RG, Subbarao KV (1999) Host range specificity in Verti- cillium dahliae. Phytopathology 89:1218–1225 tant lettuce germplasm against diverse V. dahliae Brunner E, Domhof S, Langer F (2002) Nonparametric analysis isolates demonstrated that the host-pathogen interac- of longitudinal data in factorial experiments. 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