Host resistance offers the most cost-effective method of Verticillium wilt control in lettuce (Lactuca sativa). In 2004 and 2005, 107 and 22 lettuce cultivars, respectively, were screened for resistance in a field infested with Verticillium dahliae, and disease progress on resistant and susceptible cultivars was determined. Greenhouse experiments were conducted to evaluate 16 cultivars for resistance to a race 1 and a race 2 isolate. Significant differences for resistance were observed within cultivated lettuce. In susceptible cultivars, disease levels increased through the season, whereas disease in resistant cultivars remained constant. Resistance in greenhouse tests was dependant upon the race used. Seven cultivars were resistant to race 1, whereas all were susceptible to race 2. Cultivar reactions to race 1 in greenhouse and field experiments were correlated, indicating the utility of greenhouse evaluations. The identification of resistance in diverse lettuce types is beneficial to the breeding process. However, because of the existence of resistance-breaking race 2 isolates, this resistance may not be durable. Alternatively, targeted releases of race-1-resistant cultivars to fields with only race 1 pathogen genotypes may extend the life of these cultivars.

Verticillium wilt, caused by Verticillium dahliae, poses a major threat to lettuce (Lactuca sativa) production in California. Incorporation of resistance into commercial lettuce cultivars offers the least expensive technique of sustaining production in infested areas. To test the breadth of the resistance identified in field experiments, a pair of susceptible (‘Salinas’ and ‘Sniper’) and resistant (‘La Brillante’ and ‘Little Gem’) lettuce cultivars were used as differentials and individually inoculated with 29 isolates of V. dahliae and two isolates of V. albo-atrum from several hosts, including lettuce, in replicated greenhouse experiments. The reactions of the four cultivars were determined based on the disease severity at maturity. None of the V. albo-atrum isolates or V. dahliae isolates from cruciferous hosts caused significant disease on lettuce. Both Salinas and Sniper were susceptible to many isolates of V. dahliae (21 of 23) from noncruciferous hosts, and the isolates varied in their overall virulence. However, of these, only three isolates caused significant disease on the resistant cvs. La Brillante and Little Gem. These three isolates also were distinct from the other V. dahliae isolates based on sequence data from the intergenic spacer (IGS) region of the nuclear ribosomal RNA gene, suggesting that they form a phylogenetically distinct subgroup that differs in virulence toward specific lettuce genotypes. Accordingly, isolates of V. dahliae virulent on all tested cultivars, including the resistant La Brillante and Little Gem, were designated as race 2, whereas those virulent only on the susceptible Salinas and Sniper were designated as race 1. Although a range of virulence among isolates has been described in other hosts, this is the first description of distinct virulence phenotypes in V. dahliae since a similar race structure was described in tomato in the 1960s.

Verticillium isolates collected from lettuce and artichoke were characterized for morphology, growth and pathogenicity. Several isolates were identified as Verticillium tricorpus based on morphological and cultural characteristics, including the production of dark resting mycelia, chlamydospores, microsclerotia, and yellow to orange pigmentation in culture. Compared with isolates of V. dahliae, these isolates also produced microsclerotia and conidia that were significantly larger and exhibited a distinct growth pattern at varying temperatures. Using database sequence information, primers were developed from the internal transcribed spacer region to produce a diagnostic 337-bp product specific to V. tricorpus and used to confirm the identification of isolates. Pathogenicity tests indicated that isolates of V. tricorpus were weak pathogens, causing a median disease severity (DS) of 3.5 on lettuce and 5.0 on artichoke. Although lettuce and artichoke inoculated with isolates of V. tricorpus exhibited reduced height and fresh foliar and root weight, the reductions were not statistically significant, unlike in plants inoculated with isolates of V. dahliae. Lettuce co-inoculated with isolates of V. tricorpus and V. dahliae exhibited reduced symptoms of Verticillium wilt and improved growth relative to those inoculated with V. dahliae alone. The early introduction of V. tricorpus in soil-drench inoculations appeared to provide better relief from subsequent V. dahliae inoculation than when the two species were co-inoculated simultaneously using the root-dip method, suggesting competitive exclusion as a plausible mechanism. A spore-polymerase chain reaction assay developed using cultured spores directly as template and primers specific to V. tricorpus confirmed the presence of V. tricorpus on inoculated roots. This work demonstrates the potential use of V. tricorpus to directly reduce the effect of V. dahliae on lettuce and artichoke and, to our knowledge, is the first reported characterization of V. tricorpus isolates collected from lettuce and artichoke.

Field experiments were conducted over 2 years in Yuma County, AZ, and Imperial County, CA, to determine the efficacy of several biocontrol agents for the management of lettuce drop caused by Sclerotinia spp. Commercial formulations of Trichoderma harzianum (Plantshield, Supersivit), Gliocladium virens (Soilgard), Coniothyrium minitans (Contans), and Bacillus subtilis (Companion) were evaluated and compared with the chemical fungicide iprodione (Rovral) against Sclerotinia sclerotiorum and S. minor. A single application of biocontrol products or of Rovral did not reduce lettuce drop caused by either Sclerotinia species. However, two applications of Contans, one at planting and one at post-thinning, significantly reduced the incidence of lettuce drop caused by S. sclerotiorum and increased yield but had no effect on S. minor at both locations in both years. Two applications of other biocontrol products did not significantly reduce disease incidence despite medium to high recovery following application. In contrast, Contans was only sporadically recovered following application. In vitro fungicide sensitivity evaluation revealed that both Trichoderma and Gliocladium species were tolerant to iprodione, dicloran (Botran), and vinclozolin (Ronilan) up to 1,000 ppm a.i., whereas both Sclerotinia spp. and C. minitans were sensitive to all three fungicides above 1 ppm. In summary, Contans was the most effective treatment for the control of lettuce drop caused by S. sclerotiorum, but no treatment was effective against S. minor in the desert lettuce production systems.

Survival of sclerotia of Sclerotinia minor and S. sclerotiorum was compared in irrigated fields during the summer in two major lettuce production areas in California. More than 50% sclerotia of S. sclerotiorum compared with 4 and 35% of S. minor remained viable after 24 weeks of burial at 15 and 5 cm depths, respectively, in the San Joaquin Valley while >80% of sclerotia survived in the Salinas Valley for both species. The results explain in part the lower infections from S. minor in the San Joaquin Valley. To identify factors that contribute to the rapid decline in the viability of sclerotia, the effects of soil moisture, temperature, and oxygen levels were studied in laboratory. More than 90% of sclerotia of both species survived for at least 3 months in sterilized dry soils at temperatures between 15 and 40°C. Soil moisture did not affect survival at 15 and 25°C. At 35°C, however, survival rates were significantly lower at high (–0.3 to –0.01 MPa) water potential than at low (<–1.0 MPa) water potential. Incubation under ultralow oxygen concentration (0.01%) significantly reduced survival of sclerotia in nonautoclaved moist soils at 25°C, with less than 2% sclerotia surviving over 4 weeks compared with about 45% sclerotia surviving at the ambient oxygen level (21%). The combination of high temperature, high soil moisture, and reduced oxygen in irrigated fields contribute to the lower survival of both Sclerotinia species and the responses of the two species to these conditions shape their relative geographical distribution.

Interactions between lettuce and a green fluorescent protein (GFP)- expressing, race 1 isolate of Verticillium dahliae, were studied to determine infection and colonization of lettuce cultivars resistant and susceptible to Verticillium wilt. The roots of lettuce seedlings were inoculated with a conidial suspension of the GFP-expressing isolate. Colonization was studied with the aid of laser scanning confocal and epi-fluorescence microscopes. Few differences in the initial infection and colonization of lateral roots were observed between resistant and susceptible cultivars. Hyphal colonies formed on root tips and within the root elongation zones by 5 days, leading to the colonization of cortical tissues and penetration of vascular elements regardless of the lettuce cultivar by 2 weeks. By 8 to 10 weeks after inoculation, vascular discoloration developed within the taproot and crown regions of susceptible cultivars well in advance of V. dahliae colonization. Actual foliar wilt coincided with the colonization of the taproot and crown areas and the eruption of mycelia into surrounding cortical tissues. Advance colonization of stems, pedicels, and inflorescence, including developing capitula and mature achenes was observed. Seedborne infection was limited to the maternal tissues of the achene, including the pappus, pericarp, integument, and endosperm; but the embryo was never compromised. Resistant lettuce cultivars remained free of disease symptoms. Furthermore, V. dahliae colonization never progressed beyond infected lateral roots of resistant cultivars. Results indicated that resistance in lettuce may lie with the plant’s ability to shed infected lateral roots or to inhibit the systemic progress of the fungus through vascular tissues into the taproot.

Extensive studies have been conducted on the carpogenic germination of Sclerotinia sclerotiorum, but carpogenic germination in S. minor has not been studied adequately. It remains unclear why apothecia of this pathogen have seldom been observed in nature. In this study, a new method was developed to produce apothecia in the absence of soil or sand, and carpogenic germination without preconditioning was recorded for 95 of the 96 S. sclerotiorum isolates tested. Carpogenic germination of the two species was compared under a variety of temperature, soil moisture, burial depths, and short periods of high temperature and low soil moisture. The optimal temperatures for rapid germination and for maximum germination rates were both lower for S. minor than for S. sclerotiorum. The temperature range for carpogenic germination was also narrower for S. minor than for S. sclerotiorum. A 5-day period at 30°C, either starting on the 10th or 20th day of incubation, did not significantly affect carpogenic germination of S. sclerotiorum. For both S. minor and S. sclerotiorum, the percentage of carpogenically germinated sclerotia increased as soil water potential increased from –0.3 to –0.01 MPa. In the greenhouse, a 10- or 20-day dry period completely arrested carpogenic germination of S. sclerotiorum, and new apothecia appeared after an interval of 35 days following rewetting, similar to the initial carpogenic germination regardless of when the dry period was imposed. In naturally infested fields, the number of sclerotia in 100 cc of soil decreased as depth increased from 0 to 10 cm before tillage, but became uniform between 0 and 10 cm after conventional tillage for both species. Most apothecia of S. minor were, however, produced from sclerotia located at a depth shallower than 0.5 cm while some apothecia of S. sclerotiorum were produced from sclerotia located as deep as 4 to 5 cm. These results provide the much needed information to assess the epidemiological roles of inoculum from sexual reproduction in diseases caused by the two Sclerotinia species in different geographical regions. However, more studies on effects of shorter and incompletely dry periods are still needed to predict production of apothecia of S. sclerotiorum in commercial fields under fluctuating soil temperature and moisture.

Fungal colonies initially extend exponentially and reach a constant linear extension rate determined solely by their growth in the peripheral zone. However the radial extension rates of Sclerotinia sclerotiorum and S. minor accelerate over time on PDA. Experiments were conducted to analyze the variable extension rates of the two Sclerotinia species and compare than with those of Verticillium dahliae and Cladosporium sp. In addition, the effects of starter disk size, disk position in the parent colony, the age of the parent colony, the concentration of potato dextrose broth and of incubation temperature also were determined. While the growth of Cladosporium sp. and V. dahliae followed established linear trends, the radial extension of S. sclerotiorum and S. minor colonies continuously accelerated over time until they reached the edge of the (150 mm diam) Petri dish. A polynomial model fitted the radial extension of colonies of Sclerotinia spp. Furthermore the accelerating colony extension rate was partly due to increasing colony radius. The rates of extension from mycelial disks transferred from the parental colony were positively correlated with the radius of the mycelial disks transferred. The rates of extension also were dependent on where the transferred disks were taken from parent colonies and the age and radius of the parent colony. On potato dextrose agar medium the extension rates of colonies of S. sclerotiorum and S. minor also were affected by broth concentration and temperature. With increasing nutrient concentration colony extension rates increased and were highest at 25 C. This study revealed a novel pattern of radial growth for Sclerotinia spp. that diverged from the established growth patterns of fungal colonies. Knowledge of the differences in growth behavior may be exploited in the laboratory studies on fungal competition and hyperparasitism and potentially in disease control strategies.

The influence of crop rotation on soilborne diseases and yield of strawberry (Fragaria × ananassa) was determined at a site infested with Verticillium dahliae microsclerotia and at another with no known history of V. dahliae infestation during 1997 to 2000. The rotations studied at the V. dahliae-infested site were (i) broccoli-broccoli-strawberry, (ii) Brussels sproutsstrawberry, and (iii) lettuce-lettuce-strawberry; the treatments at the site with no history of V. dahliae were (i) broccoli-broccoli-strawberry, (ii) cauliflower-cauliflower-strawberry, and (iii) lettuce-lettuce-strawberry. The effects of rotation on V. dahliae and Pythium populations, strawberry vigor, Verticillium wilt severity, and strawberry fruit yield were compared with a standard methyl bromide + chloropicrin fumigated control treatment at both sites. Rotations did not alter total population levels of Pythium spp. at either study site. However, V. dahliae microsclerotia were significantly reduced with broccoli and Brussels sprouts rotations compared with lettuce rotations at the V. dahliae-infested site. Reduced propagules led to lower Verticillium wilt severity on strawberry plants in the broccoli and Brussels sprouts rotations than in lettuce-rotated plots. Strawberry vigor and fruit yield were significantly lower in lettuce-rotated plots than in broccoli- and Brussels sprouts-rotated plots. Despite no detectable microsclerotia at the other site, strawberry vigor and fruit yield were greatest in plots rotated with broccoli, intermediate with cauliflower, and lowest with lettuce. None of the rotation treatments were better than the fumigated control for all variables measured. In the absence of fumigation, rotation with broccoli and Brussels sprouts is an effective cultural practice for managing Verticillium wilt in strawberry production; whereas, in fields with no detectable V. dahliae, broccoli is also a feasible rotational crop that enhances strawberry growth and yield. According to a cost-benefit analysis, the broccoli- strawberry rotation system could be an economically viable option provided growers are able to alternate years for strawberry cultivation.

The effects of broccoli and lettuce rotations on population densities of Verticillium dahliae and Pythium spp. in soil and on strawberry (Fragaria × ananassa) growth, yield, and Verticillium wilt were evaluated in conventional and organic production systems in California for 2 years. Under both management systems, strawberry was planted after two successive crops of broccoli or lettuce. The control treatment in the conventional field was strawberry planted in soils fumigated with methyl bromide + chloropicrin. Preplant densities of V. dahliae and Pythium sp. did not differ in these fields. At the end of the second broccoli crop, V. dahliae densities in conventional plots had declined by 44% in both years. In contrast, after the second broccoli crop, densities in organic fields decreased 47% in 2000 and 25% in 2001. In general, there were no differences in V. dahliae inoculum densities in organic and conventional plots following lettuce rotations. After the second vegetable production cycle, population densities of V. dahliae in broccoli rotated organic (24 CFU/g of soil in 2000 and 27 CFU/g of soil in 2001) or conventional (23 CFU/g of soil in 2000 and 19 CFU/g of soil in 2001) fields were significantly lower than those in lettuce rotated organic (40 CFU/g of soil in 2000 and 42 CFU/g of soil in 2001) or conventional (39 CFU/g of soil in 2000 and 35 CFU/g of soil in 2001) fields. However, crop rotation treatments had no consistent effect on the inoculum densities of Pythium spp. Canopy diameters of strawberry plants grown in rotation with broccoli were not different from those in fumigated control plots, whereas those from lettuce plots were 10% smaller. Strawberry plant nutrient analysis showed that fertilizer inputs into organic or conventional production were not responsible for the observed differences in plant size. Increases in strawberry yields were not consistent between years. Verticillium wilt incidence on strawberry was 12 to 24% lower in fields rotated with broccoli compared with fields rotated with lettuce. Wilt severity on strawberry was 22 to 36% lower in fields rotated with broccoli compared with those rotated with lettuce. The strategy of using broccoli rotation coupled with postharvest incorporation of broccoli residue continues to show promise as a tool in the management of Verticillium wilt in both conventional and organic strawberry production systems.