Abstract
Fusarium wilt of lettuce is caused by the pathogen Fusarium oxysporum f. sp. lactucae (Fol) and is a growing threat to global lettuce production. Fol was first detected in Florida in 2017 and was subsequently confirmed as race 1. Management strategies for this long-persisting soil pathogen are limited, time-consuming and expensive, and they may lack efficacy. Identifying diverse sources of genetic resistance is imperative for breeding adapted cultivars with durable resistance. The objectives of this study were to identify sources of resistance against a race 1 isolate of Fol in Florida, delineate the relationship between foliar and taproot symptoms, and investigate the inheritance of resistance and partial resistance in two F2 populations. Thirteen experiments were conducted in greenhouse and field locations to characterize the diversity of genetic resistance in the genus Lactuca. Leaf cultivars Dark Lollo Rossa and Galactic; romaine breeding lines 43007, 60182, and C1145; and iceberg breeding line 47083 consistently exhibited low foliar and taproot disease symptoms. Resistance was not identified among the wildtype Lactuca or primitive plant introductions (PI) in this study based on taproot symptoms. An additional test was conducted to study the segregation pattern of Fol resistance between one resistant and one susceptible accession (R × S) and one partial resistant and one susceptible accession (PR × S). The F2 population from ‘60182 × PI 358001-1’ fit the expected segregation ratio for a single recessive locus model, whereas the ratio for ‘Dark Lollo Rossa × PI 358001-1’ did not fit either recessive or dominant single locus models. These sources of resistance are potential candidates for developing commercial cultivars with multiple resistance loci against Fol race 1, especially for the Florida lettuce production system.
Fusarium wilt of lettuce (Lactuca sativa L.) is caused by the soilborne fungus Fusarium oxysporum f. sp. lactucae (Fol) and was first described as a root rot around 1955 in Japan (Matuo and Motohashi, 1967). The fungus infects susceptible lettuce roots by colonizing and congesting the central taproot, causing vascular discoloration, as well as foliar symptoms of wilting, stunting, and chlorosis due to constricted water transport (Gordon, 2017). Disease symptoms caused by Fol appear to be host-specific; however, the pathogen has been detected in asymptomatic taproots of broccoli, cauliflower, and spinach (Scott et al., 2014). Severely infested fields can result in significant crop losses that persist for years due to the production of pathogen survival structures called chlamydospores (Gordon and Koike, 2015). Since its discovery in Japan, race 1 of Fol has been detected in San Joaquin Valley in California; Yuma, AZ; several European countries; and in other worldwide locations including Iran, Egypt, Brazil, and Argentina (El-Sayed et al., 2018; Garibaldi et al., 2002, 2017b, 2019; Guerrero et al., 2020; Hubbard and Gerik, 1993; Malbrán et al., 2014; Matheron and Koike, 2003; Millani, 1999; Pasquali et al., 2007; Ventura and Costa, 2008). Three other Fol races have been reported, with race 2 occurring only in Japan (Aruga et al., 2012; Seki et al., 2021); race 3 in Japan and Taiwan; and the most recently discovered race 4 has been reported in The Netherlands, Belgium, Italy, England, and Ireland (Claerbout et al., 2018; Gilardi et al., 2017a, 2019; Taylor et al., 2019). It is believed that the pathogen has spread over these large distances through contaminated seed (Garibaldi et al., 2004).
Florida is the third largest lettuce producing state in the United States, and Fol symptoms were first detected in its primary lettuce producing region of the Everglades Agricultural Area (EAA) in 2017 (Murray et al., 2020). Although the pathogen has been confined to a localized region within the EAA, it has the potential to spread to nearby fields through contaminated equipment and will likely remain problematic for years to come, as has occurred in other lettuce production areas (Gordon and Koike, 2015). In fact, long-term fusarium wilt infestations have already occurred in other crops planted Florida, including watermelon (Citrullus lanatus), basil (Ocimum basilicum), Senegal date palm (Phoenix reclinata), cucumber (Cucumis sativus), tomato (Solanum lycopersicum), banana (Musa spp.), and roselle (Hibiscus sabdariffa var. sabdariffa) (Amaradasa et al., 2018; Datnoff et al., 1997; Elliott, 2015; Owen, 1955; Ploetz and Haynes, 2000; Ploetz et al., 1999, 2007). This makes it imperative to derive long-term solutions for mitigating the threat fusarium wilt may pose to the lettuce industry.
Although fallowing fields can reduce the number of viable chlamydospores by roughly half every 6 months, it can take many years before inoculum densities are low enough in the soil to safely replant lettuce (Gordon and Koike, 2015). Effective soil treatments including solarization may be cost-prohibitive, whereas others like methyl bromide may have toxic effects and have been phased out (Desaeger et al., 2017). As a result, breeding lettuce for Fol resistance offers one of the most effective solutions to reduce producer costs relative to other management strategies (Gordon et al., 2019).
Sources of resistance to fusarium wilt have been identified in cultivated lettuce, and research has shown that romaine cultivars tend to be more resistant than iceberg cultivars, particularly when exposed to warmer temperatures toward the beginning and end of the lettuce growing season in warmer climates (Matheron et al., 2005; Scott et al., 2010b). Previous resistance tests have found a high degree of Fol race 1 resistance in the romaine cultivars Costa Rica #4, Caesar, King Henry, Slugger (PI 632267), King Louie, and Valmaine, as well as leaf cultivars Lolla Rossa (PI 617943) and Red Rossa (Matheron and Porchas, 2010; Scott et al., 2012, 2010b). Iceberg cultivars Shinano Hope, Salinas, Salinas 88 (PI 667817), and Calmar show at least moderate resistance (McCreight et al., 2005; Ryder, 1970; Tsuchiya et al., 2004). These cultivars are not adapted to Florida growing conditions; thus, this work includes extensive screening of Florida-adapted germplasm.
There may potentially be new sources of resistance among Lactuca wildtypes, and screening wildtype accessions for Fol resistance has not yet been reported. Although L. sativa is the only cultivated species, there are ≈100 other species in the genus Lactuca, with the greatest genetic diversity present in the Mediterranean and Southwest Asia regions (Lebeda et al., 2014). Wildtype accessions are valuable sources of resistance against several lettuce pathogens, including the oomycete Bremia lactucae that causes lettuce downy mildew, the Lettuce mosaic virus, and the fungus Verticillium dahliae that causes verticillium wilt (Lebeda et al., 2014; Sandoya et al., 2021; Sequiera, 1978). Recently, several sources of resistance have been identified among accessions of L. georgica, L. saligna, L. serriola, and L. virosa for the pathogens Golvinomyces bolayi causing lettuce powdery mildew, B. lactucae, and Lettuce mosaic virus (Beharav, 2021; Giesbers et al., 2018; Maisonneuve et al., 2018; Pelgrom et al., 2019). Hundreds of wildtype accessions are deposited in the U.S. Department of Agriculture Germplasm Information Network (USDA-GRIN) and remain unexplored for novel sources of Fol resistance.
Characterizing the inheritance of resistance in identified sources provides essential information for cultivar development. McCreight et al. (2005) performed segregation analyses in the F1 generation of six cross combinations and found evidence that ‘Costa Rica #4’, ‘Salinas’, and ‘Salinas 88’ each contributed a single recessive allele controlling Fol resistance. In contrast, evidence for a single dominant allele controlling resistance in ‘Vanda’ was found in a segregating population from a cross of ‘Vanda × Gisele’ (Cabral et al., 2019). In support of these findings, quantitative trait loci (QTL) analyses conducted in a recombinant inbred lines (RILs) population and in several F2 mapping populations has demonstrated distinct QTLs involved in resistance to lettuce Fusarium wilt race 1. These QTLs are found in several linkage groups (LG1, LG2, LG4, LG7, and LG8) of the lettuce genome in ‘Valmaine’, ‘Green Towers’, ‘King Louie’, ‘Salinas’, and ‘Lolla Rossa’ (Michelmore et al., 2017; Scott et al., 2012; Seki et al., 2020). Together this indicates that Fol race 1 resistance may be controlled by one to few loci, although several other minor loci may influence the effectiveness of the resistance response (McCreight et al., 2005; Michelmore et al., 2017; Scott et al., 2012; Seki et al., 2020). These results suggest that discovering new accessions with different genetics for resistance may be useful for combining complementary resistance loci into novel cultivars.
The objectives of this study were to identify sources of resistance against Fol race 1 isolates in Florida, delineate the relationship between foliar and taproot symptoms, and investigate the inheritance of resistance and partial resistance.
Materials and Methods
Experiments and germplasm
Field initial screenings.
Three replicated field trials (Field 1, Field 2.1, and Field 2.2) were conducted in 2017, 2018, and 2019, respectively, in a plot with confirmed Fol infestation near Belle Glade, FL (Table 1). Each experiment was planted as a randomized complete block design (RCBD) with three blocks to initially test 32 lettuce accessions including commercial cultivars available to lettuce producers in Florida, Fol differential cultivars, lettuce breeding lines from the University of Florida Institute of Food and Agricultural Sciences (UF/IFAS) germplasm, and the susceptible control ‘Chosen’ (Supplemental Table 4). The experiments were hand-planted in double seed lines in raised beds of high organic matter “muck” soil that were 10 m long. Lettuce seedlings were thinned after 3 weeks to a distance of 0.30 m between plants. Plants were grown until maturity using the recommended farming practices for the EAA lettuce production system.
Summary of Fol race 1 screening experiments conducted on diverse germplasm of Lactuca spp. accessions in field and greenhouse.z
Initial greenhouse screening of wildtypes.
Two unreplicated experiments (GH-4 and GH-5) were conducted in a greenhouse at the Everglades Research and Education Center (EREC) of UF/IFAS near Belle Glade, FL (Table 1). GH-4 included 35 PIs of L. sativa and 13 wildtype of L. serriola accessions obtained from the USDA-GRIN that represented a global distribution concentrated in the Lactuca Caucasus center of origin (Supplemental Table 1). Seeds for GH-5 were received from the University of California–Davis and consisted of L. serriola (98), L. saligna (5), and Lactuca spp. (3) from countries within the Caucasus center of origin for Lactuca (Supplemental Table 2). Both experiments were planted in an augmented block design (ABD) with five seedlings of the 48 (GH-4 distributed in five blocks) and 106 (GH-5 distributed in 15 blocks) accessions planted once but the controls ‘Dark Lollo Rossa’ (PI 667690), ‘Lolla Rossa’ (PI 617943), 60182, and ‘Chosen’ were replicated five times in GH-4 and 15 times in GH-5, respectively (Supplemental Table 4). The cultivar Lolla Rossa was described as resistant (R) (Michelmore et al., 2017); Dark Lollo Rossa was found to be resistant in our initial screenings, romaine breeding line 60182 was found to be partially resistant (PR), and the iceberg commercial cultivar Chosen was found to be susceptible (S) (Supplemental Table 4).
UF/IFAS germplasm initial screening.
Two additional ABD experiments (GH-6 and Field 7) were planted to screen 80 cultivars and breeding lines from the UF/IFAS germplasm (Table 1; Supplemental Table 3). GH-6 was planted in a greenhouse at EREC using ‘Salinas’ (PR), 60182 (PR), and ‘Chosen’ (S) (replicated nine times) as controls (Supplemental Table 4). Field 7 was conducted in the same Fol-infested plot previously described and following the same planting and maintenance protocols. Breeding line 60182 (PR) and ‘Chosen’ (S) were replicated six times, whereas ‘Dark Lollo Rossa’ (R) was replicated four times and ‘Lolla Rossa’ (R) twice as controls (Supplemental Table 4).
Resistant confirmation.
All germplasms that showed the highest levels of resistance in experiments 1 through 7 were retested in one greenhouse and two field experiments: Expt. GH-8 in the EREC greenhouse and Expts. Field 9.1 and 9.2 in the same Fol-infested field plot used in prior experiments. Fourteen wildtypes and PIs, 11 UF/IFAS breeding lines, and five commercial cultivars were tested in an RCBD with three replications alongside the controls ‘Chosen’ (S) and ‘Dark Lolla Rossa’ (R) (Supplemental Table 4). GH-8 contained five plants per replicate, whereas the plots in Field 9.1 and 9.2 typically contained ≈20 plants per replicate.
Segregation analysis.
Before these experiments, crosses between resistant × susceptible and partial resistant × susceptible accessions were made in greenhouse following a well-established protocol for crossing lettuce (Ryder, 1999). The F1 hybrid was then planted to self-pollinate and produce the F2 seeds used in this study. Three hundred F2 disease-free seeds from population ‘Dark Lollo Rossa (R) × PI 358001-1 (S)’ and ‘60182 (PR) × PI 358001-1 (S)’ were planted in 200-cell seeding trays, as well as 30 seeds of each of the parents from these populations and grown for 30 d before inoculation.
Seedling preparation and inoculation protocol for greenhouse experiments
The same inoculation protocol was used for greenhouse Expts. GH-4, GH-5, GH-6, and GH-8. Two lettuce seeds per cell were planted in 200-cell seeding trays containing sterilized potting soil (Sungro Horticulture, Agawam, MD). Emerging seedlings were thinned to one plant per cell, watered daily, and fertilized weekly for 30 d before inoculation. Plants were inoculated with the monoconidic EAA isolate Fol #51 previously verified for high virulence (Murray et al., 2020). Twelve days before inoculation, isolate Fol #51 was grown on plates of potato dextrose agar (Difco Laboratories, Detroit, MI) for 1 week to obtain mycelia. Five days before inoculation, a plug of Fol #51 mycelia was placed in sterile 100-ml potato dextrose broth (PDB; Difco Laboratories) and maintained at room temperature on a G10 Gyrotory Shaker (New Brunswick Scientific Co., Edison, NJ) set to 130 rpm for 5 d. On the day of inoculation, the PDB cultures were filtered through four layers of sterilized cheese cloth and the suspensions were standardized to 1 × 106 mL−1 conidia using a Petroff-Hauser E60512-20 counting chamber (Hausser Scientific, Horscham, PA).
During inoculation, 30-d-old seedlings at approximately the four-leaf stage were removed from the trays without disrupting the roots. Untrimmed roots of all five seedlings for each accession were submerged and agitated in 200 mL of the conidial suspension for ≈15 s and then individually transplanted in separate 10-cm plastic pots containing a 1:1 potting soil/sand mixture (Sungro Horticulture). The substrate was previously sterilized in an SST-15 soil sterilizer (All-American Associates, Mason City, IL) at 93 °C for 48 h and maintained in a closed container before inoculations (Gilardi et al., 2017a). Each accession was submerged in a new batch of conidial suspension to avoid contamination. Five seedlings of control plants were mock inoculated by submerging their roots for 15 s in sterile deionized water and transplanted in separate plots as previously described (Supplemental Table 4). Plants were kept in a greenhouse dedicated to Fol screening, monitored, and watered daily until evaluation.
Segregation analysis.
Fol conidia suspensions from Fol #51 were grown as previously described 5 d before inoculation and used to inoculate 300 F2 seedlings from each population. Twenty plants of each parent were inoculated with Fol #51 as a positive control, and 10 were mock inoculated for negative control. All plants were grown for 30 d in a greenhouse until evaluation.
Disease ratings
Field screenings.
Disease ratings for Expts. Field 1 and Field 2.1 and 2.2 were taken at 30 d postplanting and at market maturity 60 d postplanting. Foliar disease severity (FDS) was measured on a 0 to 5 scale, where 0 = no foliar symptoms; 1 = slight stunting and/or chlorosis; 2 = moderate stunting and/or chlorosis; 3 = wilting, moderate stunting/chlorosis; 4 = severe wilting, stunting, and chlorosis; 5 = plant dead (Fang et al., 2012). Foliar disease incidence (FDI) was calculated as the percentage of plants showing foliar symptoms per plot.
In Expts. Field 7 and Field 9.1 and 9.2, plants were rated for foliar symptoms at 30 d postplanting using the previously described scale. At 60 d postplanting, taproots were excavated from the soil, dissected longitudinally to reveal vascular tissue, and rated for root discoloration severity (RDS) using a 0 to 5 scale, where 0 = no discoloration, 1 = slight vascular discoloration, 2 = reddish-brown discoloration, 3 = severe discoloration and beginning necrosis, 4 = severe discoloration and necrosis, and 5 = plant dead (Fang et al., 2012). The root discoloration incidence (RDI) was calculated as the percentage of taproots displaying disease symptoms in each plot.
Greenhouse screenings.
Asymptomatic plants dying within 1 to 5 d after inoculation were discarded as transplant errors in Expts. GH-4, GH-5, GH-6, and GH-8. Ratings for taproot discoloration occurred at weekly intervals as the accessions started to develop moderate foliar wilting symptoms, except for occasions when accessions were evaluated when bolted. The taproots of all five plants within each plot visually displaying foliar symptoms or bolting were dissected to record RDI and RDS using the same scale described earlier. The remaining plots without foliar symptoms or bolting were evaluated for RDI and RDS at 30 d postinoculation.
In Expts. GH-4, GH-5, and GH-6, asymptomatic taproots were surface disinfected in 10% sodium hypochlorite in a laminar hood for 10 s, rinsed twice in sterile deionized water, allowed to partially dry on autoclaved paper towels, and implanted directly in Komada’s semiselective medium to determine whether the pathogen was present in taproot vascular tissue (Komada, 1975). The percentage of taproots with positive Fol identification from the semiselective medium was used to calculate a corrected root discoloration incidence (RDIC), so that the final RDIC was the percentage of taproots displaying discoloration plus the percentage of asymptomatic taproots with positive Fol identification on Komada’s Medium.
Segregation analysis.
Plants showing moderate foliar symptoms of fusarium wilt were rated for disease incidence and severity using the 0 to 5 scale of taproot vascular discoloration previously described, and all remaining plants were rated for taproot discoloration at 30 d post–Fol inoculation.
Data analysis
The foliar and root DI, DIC, and DS were analyzed as an RCBD in Expts. Field 1, Fields 2.1 and 2.2, GH-8, Fields 9.1 and 9.2, Field 11, and Field 12 (Table 1). The analysis of variance (ANOVA) was obtained using nonparametric statistic with ranked methods. The analysis was conducted in PROC MIXED of SAS Institute Version 9.4. (2019; Cary, NC). In the ANOVA, replicates were nested to the experiment and accessions were used as the repeated statement. Relative marginal effects (RMEs) and confidence intervals (CIs) at 95% probability were calculated using the macro LD_CI generated in PROC IML of SAS Institute Version 9.4. (Shah and Madden, 2004).
The foliar and root DI, DIC, and DS were analyzed as an augmented design in Expts. GH-4, GH-5, GH-6, and Field 7. An ANOVA using PROC MIXED of SAS software Version 9.4. was computed. In the ANOVA, blocks were considered as random effects, and the newly tested accessions and controls were considered as fixed effects. To test differences between the newly tested accessions and each of the controls, a Dunnett two-tailed t test was performed between the tested accessions and resistant and susceptible controls.
The number of observed and expected resistant and susceptible plants for each segregating F2 population were used to calculate a chi-square test (P = 0.05) to determine whether the observed results fit the expected ratios based on the inheritance of either one dominant gene (3 Resistant: 1 Susceptible) or one recessive gene (3 Susceptible: 1 Resistant).
Results and Discussion
Initial screening of Florida-adapted germplasm
Fusarium wilt of lettuce was first detected in Florida in 2017 and has been confirmed as race 1 within a small, localized region (Murray et al., 2020). The pathogen has persisted in initial fields, and newer detections have occurred in surrounding plots when planted with Fol susceptible cultivars. Due to the long-term persistence of Fol, it was imperative to first identify genetic resistance in cultivars adapted to growing conditions in South Florida. An initial field experiment (Field 1) from Winter 2017 indicated significant differences (P = 0.0004) for FDI and FDS among the tested accessions (Table 2). None of the commercial cultivars currently planted by growers in Florida or previously released cultivars from UF/IFAS had complete resistance to Fol race 1 in this experiment. The commercial cultivars Manatee, Homestead, and RSX743; the UF/IFAS cultivars Floribibb, Floricos 83, Floricrisp 1265, and Terrapin; and UF/IFAS breeding lines 60176 and 60182 did have low levels of FDI and FDS in this first assessment (Table 3). Similar to previous studies, iceberg cultivars had greater overall FDI and FDS compared with romaine cultivars (Table 3) (Matheron et al., 2005; Scott et al., 2010b). Butterhead cultivars had the lowest overall FDI and FDS and Fol resistance has not yet been extensively reported for this lettuce type.
ANOVA of the foliar disease incidence (FDI) and disease severity (FDS) in field experiments initially conducted in a naturally infested Everglades Agricultural Area field to identify resistance to Fol race 1.
Relative marginal effects (RME), lower and upper confidence intervals for foliar disease incidence (FDI) and disease severity (FDS) in an initial screening Expt. Field 1 of lettuce cultivars and lines in an Everglades Agricultural Area field naturally infested with Fol race 1.
Cultivars Floribibb and Floricos 83, as well as breeding line 60182, have the romaine cultivar Valmaine as a parent in their pedigree. ‘Valmaine’ has previously been described as resistant to Fol race 1 (Michelmore et al., 2017) and may contribute resistance alleles to these genotypes. Additionally, ‘Floribibb’ has ‘Calmar’ in its pedigree, which McCreight et al. (2005) found to be resistant to Fol race 1 in Arizona. Future segregation or QTL analyses could ascertain whether ‘Valmaine’ or ‘Calmar’ have donated resistance alleles to cultivars and lines in this study or any of their other contemporary progeny. Crossing accessions with ‘Valmaine’ and ‘Calmar’ in their pedigree could be useful in developing Florida-adapted germplasm with transgressive resistance loci.
Two additional experiments were planted in the same field, one in Winter 2018 and the second in Spring 2019 to verify results on Florida-adapted accessions and identify resistance in additional tested accessions. Significant differences in FDI and FDS (P = 0.0128 and P < 0.0001, respectively) were found among the accessions (Table 2). No Florida-adapted germplasm was resistant to the pathogen; however, breeding line 60182 and cultivar Floricrisp 1265 continued to exhibit low FDI and FDS (Table 4). The UF/IFAS cultivar Tall Guzmaine and cultivars not adapted to Florida Green Towers, Valmaine, Salinas, and Lolla Rossa had low levels of FDI and FDS (Table 4). The resistance displayed by cultivars Green Towers, Valmaine, Salinas, and Lolla Rossa agree with previous screening results for Fol race 1 (Matheron et al., 2005; Scott et al., 2010b, 2012, 2014). Romaine and leaf cultivars continued to show lower FDI and FDS than iceberg cultivars; however, butterhead cultivars had greater FDI and FDS during these trials (Table 4).
Relative marginal effects (RME), lower and upper confidence intervals for foliar disease incidence (FDI) and severity (FDS) in an initial screening Expt. Field 2.1 and 2.2 of lettuce cultivars and lines in an Everglades Agricultural Area field naturally infested with Fol race 1.
Relationship between foliar wilting symptoms and taproot colonization
Identifying genotypes resistant to Fol race 1 based on the absence of foliar symptoms helps fast-track breeding solutions for reducing damage caused by this pathogen, but evaluating taproot colonization should be considered for long-term protection. Fol has been documented to extensively colonize the taproots of lettuce that does not display foliar symptoms, allowing the pathogen to continue proliferating in infested soils (Gordon and Koike, 2015). Identifying genotypes with no foliar symptoms and the least taproot colonization may help avoid increasing Fol inoculum in infested soils.
Significant differences in RDI and RDS (P < 0.0001, respectively) were observed among the tested accessions for Expts. GH-4 and GH-5 in plants inoculated with the isolate Fol #51 (Table 5). RDIC was measured by observing Fol colony formation of taproot sections plated on Komada’s medium. In GH-4, only PI 165063 had RDI and RDIC of 0% (Supplemental Table 5). Additionally, PI 220665 and PI 615055 showed no taproot discoloration and had RDIC of 20%, which was significantly less colonized than susceptible check ‘Chosen’ (Supplemental Table 5). The abovementioned PIs and PI 164940 were selected for future resistance confirmation experiments. Five L. serriola accessions in Expt. GH-5 had RDI and RDIC of 0% (Supplemental Table 6). An additional five L. serriola accessions had no taproot discoloration and RDIC of 20% when plated on Komada’s medium, which was significantly less colonized than ‘Chosen’ (Supplemental Table 6). Similarly, these 10 L. serriola accessions were selected for future experiments.
ANOVA for the root disease incidence (RDI) and severity (RDS) of accessions tested in augmented design in greenhousez and fieldy experiments.
Expts. GH-6 and Field 7 in Spring 2020 (Table 1) were conducted to evaluate additional Florida-adapted germplasm for taproot vascular discoloration. Significant differences in RDI were detected among tested accessions for both experiments (P = 0.0002 in GH-6 and P < 0.0001 in Field 7; Table 5). No root discoloration was observed in breeding line 60182, ‘Costa Rica #4’, ‘Floribibb’, ‘Floricrisp 1265’, and ‘Terrapin’ when inoculated with Fol #51 in the greenhouse experiment; however, only ‘Costa Rica #4’ and ‘Floribibb’ had RDIC of 0% (Supplemental Table 7). The breeding lines 47083, 49530, 49889, 50100, and 60168, as well as cultivar Galactic, had RDI and RDIC of 0% (Supplemental Table 7). In a similar study, cultivars King Henry, Caesar, Red Rossa, Lolla Rossa, and Green Forest were asymptomatic when rated for Fol foliar symptoms but Fol isolation rates from taproots remained high when whole sections of individual taproot tissues were homogenized and plated by serial dilution on Komada’s medium (Scott et al., 2014).
In the Field 7 experiment conducted in a naturally Fol-infested field, the nonadapted cultivars Dark Lollo Rossa, Costa Rica #4, Galactic, and Red Rage exhibited no taproot vascular discoloration (Supplemental Table 8). The Florida-adapted cultivar Floribibb displayed a relatively low RDI of 17% (Supplemental Table 8). Florida breeding lines 43007, C1139, C1144, and C1145 had an RDI of 0%. Additionally, each of these lines have Fol-resistant ‘Valmaine’ in their pedigree. On the basis of the results from these two experiments, 11 UF breeding lines and five nonadapted commercial cultivars to the Florida environment were selected for future confirmation experiments (Supplemental Tables 7 and 8).
Initially, minimal taproot colonization and foliar symptoms were identified in 30 accessions consisting of two butterheads, three icebergs, four leaf types, one leaf/romaine type, 10 romaines and 10 wildtypes. To maintain lettuce marketability and mitigate proliferation and spread of Fol, it is recommended that Fol resistance selections be based on both plants with no above-ground symptoms and little to no root colonization (Scott et al., 2014). Incorporating these two measurements may assist with combining resistance alleles in new cultivars and improve resistance durability by preventing an increase in Fol inoculum in infested fields. Avoiding further increases of the pathogen is paramount for the Florida lettuce production system because Fol is still localized within a few plots in the EAA.
Confirmation of taproot resistance in Florida
Thirty Florida-adapted genotypes, PIs, and Lactuca wildtypes demonstrating highest resistance in the initial screening experiments were further evaluated in replicated RCBD greenhouse and infested field experiments (Table 1). The ANOVA for Expt. GH-8 showed significant differences for RDI and RDS among accessions (P < 0.0001; Table 6). In greenhouse experiments, only cultivar Dark Lollo Rossa was asymptomatic for root discoloration; however, its RMEs for RDI and RDS were not significantly different from 50100 and 60182 (Fig. 1). Breeding lines 43007, 47083, 49530, C1139, C1144, and C1145 had similarly low RMEs (Fig. 1) and the same median of RDI values (20%) as partially resistant 60182 (Table 7).
Relative marginal effects (RME) for the root discoloration incidence (RDI) and severity (RDS) of accessions tested in GH-8 with inoculation of isolate Fol #51. Error bars represent confidence intervals at 95%.
Citation: HortScience 56, 12; 10.21273/HORTSCI16186-21
ANOVA for the root disease incidence (RDI) and severity (RDS) of accessions tested in randomized complete block design greenhousez and fieldy experiments.
Root discoloration incidence (RDI) and discoloration severity (RDS) dispersion measurements of 28 Lactuca accessions tested in Expt. GH-8 at the Everglades Research and Education Center, Belle Glade, FL.
Field experiments conducted in the same Fol-infested field showed significant differences for RDI and RDS among accessions (P = 0.0149 and P < 0.0001, respectively) between the two experiments (P < 0.0001) and for the Experiment × Accession interaction for RDS (P < 0.0001) (Table 6). In general, disease incidences and severities were higher in Expt. Field 9.1 conducted during the first planting, in which higher temperatures may have exacerbated Fol symptoms as previously reported (Table 1) (Matheron et al., 2005; Scott et al., 2010a). The cultivar Galactic was the only genotype that was nearly asymptomatic (Table 8). Romaine breeding lines 43007, 50100, and 60182 had median RDI of 0% and RDS of 0, and C1144 had a RME that was not significantly different from 60182 (Table 8; Fig. 2). Iceberg breeding line 47083 continued to show low disease symptoms in Field 9.1 and 9.2. Across these resistance confirmation experiments, leaf and romaine cultivars performed similarly overall and exhibited the lowest RDI and RDS as in previous studies (Tables 7 and 8) (Matheron et al., 2005; Scott et al., 2010b). Butterhead and iceberg cultivars had slightly higher RDI and RDS overall, but the two types performed similar to each other as a group (Tables 7 and 8). Each of the wildtypes and PIs selected for these resistance confirmation experiments displayed much greater root discoloration incidence than in the initial experiments.
Relative marginal effects (RME) for the root discoloration incidence (RDI) and severity (RDS) of accessions in Expt. Fields 9.1 and 9.2 tested in a field near Belle Glade, FL, naturally infested with Fol race 1. Error bars represent confidence intervals at 95%.
Citation: HortScience 56, 12; 10.21273/HORTSCI16186-21
Root discoloration incidence (RDI) and discoloration severity (RDS) dispersion measurements of 28 Lactuca accessions tested in Expts. Field 9.1 and Field 9.2 near Belle Glade, FL.
Testing resistant Florida selections in highly Fol-infested Arizona fields
Fusarium wilt has existed in the United States for decades since its discovery in the San Joaquin Valley of California in 1992 (Hubbard and Gerik, 1993) and Yuma, AZ, in 2002 (Matheron and Koike, 2003). Fol has been more difficult to avoid in Arizona due to its proliferation and because warmer temperatures increase the susceptibility of lettuce to this pathogen, especially during the first planting slot in September (Matheron et al., 2005; Scott et al., 2010a). Test plots with natural Fol infestation and high disease pressure have been used for screening lettuce in Yuma County since 2015. Other crops are planted in these test sites between lettuce screening experiments, yet high pathogen inoculum persists (S. Slinski, personal communication). These sites in Wellton and Yuma, AZ, were used in experiments Field 11 and Field 12, respectively, to further validate resistance in the initial Florida experiments (Table 1).
Foliar disease incidences were significantly different among accessions in Field 11 and Field 12 (P = 0.0075 and P = 0.0107, respectively; Table 9). In both experiments, the susceptible check El Guapo showed consistently high FDS and FDI, whereas the resistant control Meridian showed less FDI and FDS (Fig. 3; Tables 10 and 11). Romaine breeding lines 43007, 50100, 60182, C1144, and C1145; iceberg breeding line 47083; and leaf cultivars Dark Lollo Rossa and Galactic showed low FDI and FDS in both Field 11 and Field 12 (Fig. 3; Tables 10 and 11). This result was consistent with their low RDI and RDS in the Florida screening experiments. Additionally, romaine breeding line 49530, romaine cultivars Red Rage and Terrapin, and bibb cultivar Floribibb showed low FDI and FDS in the Arizona trials. Similar to the taproot confirmation experiments, leaf and romaine types as a group had the lowest FDI and FDS (Tables 10 and 11). This was followed by butterhead genotypes, which had lower foliar symptoms than iceberg genotypes (Tables 10 and 11). Poor germination among wildtypes and some PIs made it difficult to draw conclusions regarding foliar symptoms in these experiments; however, the leaf type PI 615055 displayed low foliar symptoms in both Fields 11 and 12. Although some of these latter genotypes may not possess the same level of taproot resistance to Fol, they may still have resistance alleles that permit relatively few foliar symptoms and may be considered for future breeding programs or genetic studies.
Relative marginal effects (RME) for the foliar disease incidence (FDI) and severity (FDS) of accessions tested in a field naturally infested with Fol race 1 near Wellton (A) and Yuma (B). Error bars represent confidence intervals at 95%.
Citation: HortScience 56, 12; 10.21273/HORTSCI16186-21
ANOVA of the foliar disease incidence (FDI) and severity (FDS) in a randomized complete block design in Field 10 and Field 11 Expts. conducted in fields in Wellton and Yuma, Arizona, infested with Fusarium oxysporum f. sp. lactucae race 1.
Foliar disease incidence (FDI) and disease severity (FDS)z dispersion measurements of 18 Lactuca accessions tested in a field naturally infested with Fol race 1 near Wellton, AZ (Field 11).
Foliar disease incidence (FDI) and disease severity (FDS)z dispersion measurements of 17 Lactuca accessions tested in a field infested with Fol race 1 near Yuma, AZ (Field 12).
Segregation analysis
Two F2 populations segregating for resistance to Fol race 1 ‘Dark Lollo Rossa × PI 358001-1’ and ‘60182 × PI 358001-1’ were screened for taproot discoloration with virulent isolate Fol #51 in greenhouse experiments (GH-13 and GH-14; Table 1). The parent ‘Dark Lollo Rossa’ had 18 plants with no root discoloration and four plants with mild symptoms (ratings of ≤2), and PI 358001-1 had three plants with no root discoloration and 18 plants with severe symptoms (Table 12). The observed resistant-to-susceptible ratio for the F2 population from ‘Dark Lollo Rossa × PI 358001-1’ was 132:199, which deviated from the expected ratio (P < 0.0001) for both single recessive and single dominant models (Table 12). This suggests that multiple alleles may confer resistance in ‘Dark Lollo Rossa’ (Table 12). The parent 60182 had 15 plants without root discoloration and eight plants with mild symptoms, and PI 358001-1 had four plants without root discoloration and 20 with severe symptoms (Table 12). The ratio of resistant: susceptible F2 plants from ‘60182 × PI 358001-1’ was 61:235, which did not deviate from the expected model of inheritance of a single recessive allele (P = 0.0812; Table 12).
Root discoloration severity (RDS) of two F2 populations from crosses ‘Dark Lollo Rossa × PI 358001-1’ and ‘60182 × PI 358001-1’ inoculated with Fol #51 in greenhouse. Chi-square and P values are provided for an analysis of the expected segregation ratios based on the model of inheritance for a single resistant locus (χ2 df = 1, α = 0.05 = 3.841).
McCreight et al. (2005) found evidence for the simple inheritance of a recessive allele from ‘Salinas’ in their segregation analysis of an F2 population from ‘Vanguard × Salinas’. ‘Salinas’ is considered partially resistant to Fol race 1 and 60182 can be considered as partially resistant (Table 12). Although the exact number of loci involved in the resistance response for ‘Dark Lollo Rosso’ and 60182 remains undetermined, the proportion of resistant F2 plants in ‘Dark Lollo Rossa × PI 358001-1’ population was roughly twice that of ‘60182 × PI 358001-1’ population. This may signify the potential for combining different sources of vertical and horizontal resistance in one genotype by crossing ‘Dark Lollo Rossa × 60182’. This cross combination could result in greater taproot resistance to Fol, in addition to longer durability of resistance in highly infested regions.
Conclusions
In this study, a combination of foliar and taproot disease ratings was used to help identify germplasm that is resistant to Fol. The genotypes displaying consistently low foliar and taproot symptoms to Fol race 1 across all experiments and locations in this study were UF/IFAS romaine breeding lines 43007, 50100, 60182, and C1144; iceberg breeding line 47083; and leaf cultivars Dark Lollo Rossa and Galactic. UF/IFAS cultivars that have demonstrated low foliar symptoms, but more substantial taproot colonization, included the romaine type Terrapin, the bibb Floribibb, and the iceberg Floricrisp 1265. The same resistance profile has been demonstrated by UF/IFAS breeding lines 49530 and C1145, as well as PI 615055. Leaf and romaine genotypes demonstrated lowest foliar and taproot symptoms overall. Butterhead and iceberg genotypes had higher, but similar, taproot symptoms; however, the butterhead genotypes displayed lower foliar symptoms in Arizona. Consistent resistance to root symptoms of Fol race 1 was not identified among the wildtype Lactuca tested in this study. Finally, the F2 segregation analysis conducted in this research suggests that using ‘Dark Lollo Rossa’ and 60182 as Fol race 1 resistance donors in breeding programs could provide complementary resistance alleles. Further genetic studies are necessary to understand the number of loci controlling resistance in these accessions and whether these alleles will confer greater inhibition of taproot colonization or increased durability in Fol infested fields over time.
Literature Cited
Amaradasa, B.S., Beckham, K., Dufault, N., Sanchez, T., Ertek, T.S., Iriarte, F., Paret, M. & Ji, P. 2018 First report of Fusarium oxysporum f. sp. niveum race 3 causing wilt of watermelon in Florida, USA Plant Dis. 102 5 1029 https://doi.org/10.1094/PDIS-10-17-1649-PDN
Aruga, D., Tsuchiya, N., Matsumura, H., Matsumoto, E. & Hayashida, N. 2012 Analysis of RAPD and AFLP markers linked to resistance to Fusarium oxysporum f. sp. lactucae race 2 in lettuce (Lactuca sativa L.) Euphytica 187 1 1 9 https://doi.org/10.1007/s10681-012-0665-5
Beharav, A 2021 Lactuca georgica, a new wild source of resistance to downy mildew: Comparative study to other wild lettuce relatives Eur. J. Plant Pathol. 160 127 136 https://doi.org/10.1007/s10658-021-02228-5
Cabral, C.S., Fonseca, M.E.N., Oliveira, V.R., Boiteux, L.S. & Reis, A. 2019 A single dominant gene/locus model for control of Fusarium oxysporum f. sp. lactucae race 1 resistance in lettuce (Lactuca sativa) Euphytica 215 114 https://doi.org/10.1007/s10681-019-2441-2
Claerbout, J., Venneman, S., Vandevelde, I., Decombel, A., Bleyaert, P., Volckaert, A., Neukermans, J. & Höfte, M. 2018 First report of Fusarium oxysporum f. sp. lactucae race 4 on lettuce in Belgium Plant Dis. 102 5 1037 https://doi.org/10.1094/pdis-10-17-1627-pdn
Datnoff, L., Liang, L. & Wick, R. 1997 Recent outbreak of Fusarium wilt of basil in Florida Plant Dis. 81 10 1214 https://doi.org/10.1094/PDIS.1997.81.10.1214C
Desaeger, J., Dickson, D.W. & Locascio, S.J. 2017 Methyl bromide alternatives for control of root-knot nematode (Meloidogyne spp.) in tomato production in Florida J. Nematol. 49 2 140 149 https://doi.org/10.21307/jofnem-2017-058
Elliott, M.L 2015 First report of Fusarium wilt caused by Fusarium oxysporum f. sp. canariensis on Phoenix reclinata in Florida Plant Dis. 99 6 887 https://doi.org/10.1094/PDIS-09-14-0897-PDN
El-Sayed, S.F., Reyad, N.E.-H.A., Abdel-Wahab, A. & Hanafy, A.M. 2018 Occurrence of lettuce Fusarium wilt caused by Fusarium oxysporum f. sp. lactucae in Egypt and its management by using solarization, metam sodium and certain of bioproducts Bioscience Research 15 2 602 609
Fang, X., You, M.P. & Barbetti, M.J. 2012 Reduced severity and impact of Fusarium wilt on strawberry by manipulation of soil pH, soil organic amendments and crop rotation Eur. J. Plant Pathol. 134 619 629 https://doi.org/10.1007/s10658-012-0042-1
Garibaldi, A., Gilardi, G. & Gullino, M.L. 2002 First report of Fusarium oxysporum on lettuce in Europe Plant Dis. 86 9 1052 https://doi.org/10.1094/PDIS.2002.86.9.1052B
Garibaldi, A., Gilardi, G. & Gullino, M.L. 2004 Seed transmission of Fusarium oxysporum f. sp. lactucae Phytoparasitica 32 1 61 65 https://doi.org/10.1007/BF02980861
Giesbers, A.K.J., den Boer, E., Braspenning, D.N.J., Bouten, T.P.H., Specken, J.W., van Kaauwen, M.P.W., Visser, R.G.F., Niks, R.E. & Jeuken, M.J.W. 2018 Bidirectional backcrosses between wild and cultivated lettuce identify loci involved in nonhost resistance to downy mildew Theor. Appl. Genet. 131 1761 1776 https://doi.org/10.1007/s00122-018-3112-8
Gilardi, G., Franco Ortega, S., van Rijswick, P.C.J., Ortu, G., Gullino, M.L. & Garibaldi, A. 2017a A new race of Fusarium oxysporum f. sp. lactucae of lettuce Plant Pathol. 66 4 677 688 https://doi.org/10.1111/ppa.12616
Gilardi, G., Garibaldi, A., Matic, S., Senatore, M.T., Pipponzi, S., Prodi, A. & Gullino, M.L. 2019 First report of Fusarium oxysporum f. sp. lactucae race 4 on lettuce in Italy Plant Dis. 103 10 2680 https://doi.org/10.1094/PDIS-05-19-0902-PDN
Gilardi, G., Pons, C., Gard, B., Franco-Ortega, S. & Gullino, M. 2017b Presence of Fusarium wilt, incited by Fusarium oxysporum f. sp. lactucae, on lettuce in France Plant Dis. 101 1053 https://doi.org/10.1094/PDIS-12-16-1815-PDN
Gordon, T.R 2017 Fusarium oxysporum and the Fusarium wilt syndrome Annu. Rev. Phytopathol. 55 23 39 https://doi.org/10.1146/annurev- phyto-080615-095919
Gordon, T.R. & Koike, S.T. 2015 Management of Fusarium wilt of lettuce Crop Prot. 73 45 49 https://doi.org/10.1016/j.cropro.2015.01.011
Gordon, T.R., Stueven, M., Pastrana, A.M., Henry, P.M., Dennehy, C.M., Kirkpatrick, S.C. & Daugovish, O. 2019 The effect of pH on spore germination, growth, and infection of strawberry roots by Fusarium oxysporum f. sp. fragariae, cause of Fusarium wilt of strawberry Plant Dis. 103 697 704 https://doi.org/10.1094/PDIS-08-18-1296-RE
Guerrero, M.M., Martínez, M.C., León, M., Armengol, J. & Monserrat, A. 2020 First report of Fusarium wilt of lettuce caused by Fusarium oxysporum f. sp. lactucae race 1 in Spain Plant Dis. 104 1858 https://doi.org/10.1094/PDIS-10-19-2143-PDN
Hubbard, J. & Gerik, J. 1993 A new wilt disease of lettuce incited by Fusarium oxysporum f. sp. lactucum forma specialis nov Plant Dis. 77 7 750 754
Komada, H 1975 Development of a selective medium for quantitative isolation of Fusarium oxysporum from natural soil Rev. Plant Protection Res. 8 114 124
Lebeda, A., Křístková, E., Kitner, M., Mieslerová, B., Jemelková, M. & Pink, D.A.C. 2014 Wild Lactuca species, their genetic diversity, resistance to diseases and pests, and exploitation in lettuce breeding Eur. J. Plant Pathol. 138 597 640 https://doi.org/10.1007/s10658-013-0254-z
Maisonneuve, B., Pitrat, M., Gognalons, P. & Moury, B. 2018 Growth stage-dependent resistance to the potyviruses lettuce Italian necrotic virus and Lettuce mosaic virus displayed by Lactuca sativa introgression lines carrying the Mo3 locus from L. virosa Plant Pathol. 67 9 2013 2018 https://doi.org/10.1111/ppa.12909
Malbrán, I., Mourelos, C.A., Mitidieri, M.S., Ronco, B.L. & Lori, G.A. 2014 Fusarium wilt of lettuce caused by Fusarium oxysporum f. sp. lactucae in Argentina Plant Dis. 98 9 1281 https://doi.org/10.1094/PDIS-04- 14-0372-PDN
Matheron, M.E. & Koike, S.T. 2003 First report of Fusarium wilt of lettuce caused by Fusarium oxysporum f. sp. lactucae in Arizona Plant Dis. 87 10 1265 https://doi.org/10.1094/PDIS.2003.87.10.1265C
Matheron, M.E. & Porchas, M. 2010 Evaluation of soil solarization and flooding as management tools for Fusarium wilt of lettuce Plant Dis. 94 11 1323 1328 https://doi.org/10.1094/PDIS-04-10-0296
Matheron, M.E., McCreight, J.D. & Tickes, B.R. 2005 Effect of planting date, cultivar, and stage of plant development on incidence of Fusarium wilt of lettuce in desert production fields Plant Dis. 89 6 565 570 https://doi.org/10.1094/PD-89-0565
Matuo, T. & Motohashi, S. 1967 On Fusarium oxysporum f. sp. lactucae n.f. causing root rot of lettuce Trans. Mycol. Soc. Japan 8 13 15
McCreight, J.D., Matheron, M.E., Tickes, B.R. & Platts, B. 2005 Fusarium wilt race 1 on lettuce HortScience 40 3 529 531 https://doi.org/10.21273/HORTSCI.40.3.529
Michelmore, R.W., Truco, M.J. & Ochoa, O.E. 2017 Breeding crisphead and leafy lettuce California Leafy Greens Research Program. 14 Mar. 2021. <https://calgreens.org/wp-content/uploads/2017/10/UCD-Lettuce-Breeding-Michelmore.pdf>
Millani, M 1999 Occurrence of Fusarium wilt of lettuce in Shahre-Ray, Varamin and Karaj areas Iranian Journal of Plant Pathology 35 44 45
Murray, J., Raid, R.N., Miller, C.F. & Sandoya-Miranda, G. 2020 First report of Fusarium oxysporum f. sp. lactucae causing vascular wilt of lettuce in Florida Plant Dis. 104 11 3069 https://doi.org/10.1094/PDIS-12-19-2625-PDN
Owen, J.H 1955 Fusarium wilt of cucumber Phytopathology 45 8 435 439
Pasquali, M., Dematheis, F., Gullino, M.L. & Garibaldi, A. 2007 Identification of race 1 of Fusarium oxysporum f. sp. lactucae on lettuce by inter-retrotransposon sequence-characterized amplified region technique Phytopathology 97 8 987 996 https://doi.org/10.1094/PHYTO-97-8-0987
Pelgrom, A.J.E., Eikelhof, J., Elberse, J., Meisrimler, C.N., Raedts, R., Klein, J. & Van den Ackerveken, G. 2019 Recognition of lettuce downy mildew effector BLR38 in Lactuca serriola LS102 requires two unlinked loci Mol. Plant Pathol. 20 2 240 253 https://doi.org/10.1111/mpp.12751
Ploetz, R.C. & Haynes, J.L. 2000 First report of race 3 of Fusarium oxysporum f. sp. lycopersici in Southeastern Florida Plant Dis. 84 2 199 https://doi.org/10.1094/PDIS.2000.84.2.199B
Ploetz, R.C., Palmateer, A.J., Geiser, D.M. & Juba, J.H. 2007 First report of Fusarium wilt caused by Fusarium oxysporum on roselle in the United States Plant Dis. 91 5 639 https://doi.org/10.1094/PDIS-91-5-0639A
Ploetz, R.C., Haynes, J.L. & Vázquez, A. 1999 Responses of new banana accessions in South Florida to Panama disease Crop Prot. 18 7 445 449 https://doi.org/10.1016/S0261-2194(99)00043-5
Ryder, E.J 1970 Inheritance of resistance to common lettuce mosaic J. Amer. Soc. Hort. Sci. 95 378 379
Ryder, E 1999 Lettuce, Endive and Chicory CABI Publishing New York, NY
Sandoya, G.V., Truco, M.J., Bertier, L.D., Subbarao, K.V., Simko, I., Hayes, R.J. & Michelmore, R. 2021 Genetics of partial resistance against Verticillium dahliae race 2 in wild and cultivated lettuce Phytopathology PHYTO-09 https://doi.org/10.1094/PHYTO-09-20-0396-R
Scott, J.C., Gordon, T., Kirkpatrick, S.C., Koike, S.T., Matheron, M.E., Ochoa, O.E., Truco, M.J. & Michelmore, R.W. 2012 Crop rotation and genetic resistance reduce risk of damage from Fusarium wilt in lettuce Calif. Agr. 66 1 20 24 https://doi.org/10.3733/ca.v066n01p20
Scott, J.C., Gordon, T.R., Shaw, D.V. & Koike, S.T. 2010a Effect of temperature on severity of Fusarium wilt of lettuce caused by Fusarium oxysporum f. sp. lactucae Plant Dis. 94 1 13 17 https://doi.org/10.1094/PDIS-94-1-0013
Scott, J.C., Kirkpatrick, S.C. & Gordon, T.R. 2010b Variation in susceptibility of lettuce cultivars to Fusarium wilt caused by Fusarium oxysporum f. sp. lactucae Plant Pathol. 59 1 139 146 https://doi.org/10.1111/j.1365-3059.2009.02179.x
Scott, J.C., McRoberts, D.N. & Gordon, T.R. 2014 Colonization of lettuce cultivars and rotation crops by Fusarium oxysporum f. sp. lactucae, the cause of Fusarium wilt of lettuce Plant Pathol. 63 3 548 553 https://doi.org/10.1111/ppa.12135
Seki, K., Komatsu, K., Hiraga, M., Tanaka, K., Uno, Y. & Matsumura, H. 2020 Identification of two QTLs for resistance to Fusarium wilt race 1 in lettuce (Lactuca sativa L.) Euphytica 216 174 https://doi.org/10.1007/s10681-020-02713-8
Seki, K., Komatsu, K., Hiraga, M., Tanaka, K., Uno, Y. & Matsumura, H. 2021 Development of PCR-based marker for resistance to Fusarium wilt race 2 in lettuce (Lactuca sativa L.) Euphytica 217 6 1 11 https://doi.org/10.1007/s10681-021-02857-1
Sequiera, L 1978 Two root rot resistant varieties of head lettuce Ohio Agricultural Experimental Research Station Bulletin 359 197 214
Shah, D.A. & Madden, L.V. 2004 Nonparametric analysis of ordinal data in designed factorial experiments Phytopathology 94 1 33 43 https://doi.org/10.1094/PHYTO.2004.94.1.33
Slinski, S.L., Pochas, M.A., Zima, H.V. & Correll, J.C. 2021 Evaluation of iceberg lettuce cultivars for resistance to Fusarium wilt, 2020 Plant Dis. Manag. Rep. V15 https://doi.org/10.1094/PDMR13
Taylor, A., Jackson, A. & Clarkson, J. 2019 First report of Fusarium oxysporum f. sp. lactucae Race 4 causing lettuce wilt in England and Ireland Plant Dis. 103 5 1033 https://doi.org/10.1094/PDIS-10-18-1858-PDN
Tsuchiya, N., Fujinaga, M., Ogiso, H., Usui, T. & Tsukada, M. 2004 Resistance tests and genetic resources for breeding Fusarium root rot resistant lettuce J. Jpn. Soc. Hort. Sci. 73 2 105 113 https://doi.org/10.2503/jjshs.73.105
University of Arizona 2021 AZMET: The Arizona Meteorological Network 8 Sept. 2021. <https://cals.arizona.edu/azmet/index.html>
University of Florida 2021 FAWN: Florida Automated Weather Network 8 Sept. 2021. <https://fawn.ifas.ufl.edu/data/>
Ventura, J.A. & Costa, H. 2008 Fusarium wilt caused by Fusarium oxysporum on lettuce in Espirito Santo, Brazil Plant Dis. 92 6 976 https://doi.org/10.1094/PDIS-92-6-0976C
