Ryan J. Hayes

Population analyses of the vascular plant pathogen Verticillium dahliae detect recombination and transcontinental gene flow.

The fungal pathogen Verticillium dahliae has resulted in significant losses in numerous crops in coastal California, but lettuce remained unaffected until the mid-1990s. Since then outbreaks have decimated entire fields, but the causes of this sudden susceptibility of lettuce remain elusive. The population structure of V. dahliae isolated from coastal California (n=123) was investigated with 22 microsatellite markers, and compared with strains from tomato in central California (n=60), spinach seed imported from Washington State and Northern Europe (n=43), and ornamentals from Wisconsin (n=17). No significant differentiation was measured among hosts in coastal California or with the spinach and Wisconsin ornamental sampling groups. In contrast, the tomato sampling group was significantly differentiated. Significant gene flow was measured among the various geographic and host sampling groups, with the exception of tomato. Evidence of recombination in V. dahliae was identified through gametic disequilibrium and an exceedingly high genotypic diversity. The high incidence of V. dahliae in spinach seed and high planting density of the crop are sources of recurrent gene flow into coastal California, and may be associated with the recent outbreaks in lettuce.

Selenium accumulation in lettuce germplasm

Selenium (Se) is an essential micronutrient for animals and humans. Increasing Se content in food crops offers an effective approach to reduce the widespread selenium deficiency problem in many parts of the world. In this study, we evaluated 30 diverse accessions of lettuce (Lactuca sativa L.) for their capacity to accumulate Se and their responses to different forms of Se in terms of plant growth, nutritional characteristics, and gene expression. Lettuce accessions responded differently to selenate and selenite treatment, and selenate is superior to selenite in inducing total Se accumulation. At least over twofold change in total Se levels between cultivars with high and low Se content was found. Synergistic relationship between Se and sulfur accumulation was observed in nearly all accessions at the selenate dosage applied. The change in shoot biomass varied between lettuce accessions and the forms of Se used. The growth-stimulated effect by selenate and the growth-inhibited effect by selenite were found to be correlated with the alteration of antioxidant enzyme activities. The different ability of lettuce accessions to accumulate Se following selenate treatment appeared to be associated with an altered expression of genes involved in Se/S uptake and assimilation. Our results provide important information for the effects of different forms of Se on plant growth and metabolism. They will also be of help in selecting and developing better cultivars for Se biofortification in lettuce.

Identification of Pathogenicity-Related Genes in the Vascular Wilt Fungus Verticillium dahliae by Agrobacterium tumefaciens-Mediated T-DNA Insertional Mutagenesis

Verticillium dahliae is the causal agent of vascular wilt in many economically important crops worldwide. Identification of genes that control pathogenicity or virulence may suggest targets for alternative control methods for this fungus. In this study, Agrobacteriumtumefaciens-mediated transformation (ATMT) was applied for insertional mutagenesis of V. dahliae conidia. Southern blot analysis indicated that T-DNAs were inserted randomly into the V. dahliae genome and that 69% of the transformants were the result of single copy T-DNA insertion. DNA sequences flanking T-DNA insertion were isolated through inverse PCR (iPCR), and these sequences were aligned to the genome sequence to identify the genomic position of insertion. V. dahliae mutants of particular interest selected based on culture phenotypes included those that had lost the ability to form microsclerotia and subsequently used for virulence assay. Based on the virulence assay of 181 transformants, we identified several mutant strains of V. dahliae that did not cause symptoms on lettuce plants. Among these mutants, T-DNA was inserted in genes encoding an endoglucanase 1 (VdEg-1), a hydroxyl-methyl glutaryl-CoA synthase (VdHMGS), a major facilitator superfamily 1 (VdMFS1), and a glycosylphosphatidylinositol (GPI) mannosyltransferase 3 (VdGPIM3). These results suggest that ATMT can effectively be used to identify genes associated with pathogenicity and other functions in V. dahliae.

Variation for Resistance to Verticillium Wilt in Lettuce (Lactuca sativa L.)

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.

A Real-Time PCR Assay for Detection and Quantification of Verticillium dahliae in Spinach Seed

Verticillium dahliae is a soilborne fungus that causes Verticillium wilt on multiple crops in central coastal California. Although spinach crops grown in this region for fresh and processing commercial production do not display Verticillium wilt symptoms, spinach seeds produced in the United States or Europe are commonly infected with V. dahliae. Planting of the infected seed increases the soil inoculum density and may introduce exotic strains that contribute to Verticillium wilt epidemics on lettuce and other crops grown in rotation with spinach. A sensitive, rapid, and reliable method for quantification of V. dahliae in spinach seed may help identify highly infected lots, curtail their planting, and minimize the spread of exotic strains via spinach seed. In this study, a quantitative real-time polymerase chain reaction (qPCR) assay was optimized and employed for detection and quantification of V. dahliae in spinach germplasm and 15 commercial spinach seed lots. The assay used a previously reported V. dahliae-specific primer pair (VertBt-F and VertBt-R) and an analytical mill for grinding tough spinach seed for DNA extraction. The assay enabled reliable quantification of V. dahliae in spinach seed, with a sensitivity limit of ≈1 infected seed per 100 (1.3% infection in a seed lot). The quantification was highly reproducible between replicate samples of a seed lot and in different real-time PCR instruments. When tested on commercial seed lots, a pathogen DNA content corresponding to a quantification cycle value of ≥31 corresponded with a percent seed infection of ≤1.3%. The assay is useful in qualitatively assessing seed lots for V. dahliae infection levels, and the results of the assay can be helpful to guide decisions on whether to apply seed treatments.

Molecular Variation Among Isolates of Verticillium dahliae and Polymerase Chain Reaction-Based Differentiation of Races

Verticillium dahliae is a soilborne fungal pathogen that causes vascular wilt in a variety of economically important crops worldwide. There are two races of V. dahliae that infect tomato and lettuce. Although race-1-specific resistance has been identified in both tomato and lettuce, no resistant sources are available for race 2. Molecular analyses were employed to characterize the genetic variability and race structure of 101 isolates of V. dahliae from a variety of hosts, mainly from central and coastal California, and 10 isolates exotic to this area. Analyses of the 16 simple sequence repeat (SSR) markers illustrated that tomato subpopulations from central California were distinct relative to the marigold subpopulations. In contrast, cotton and olive isolates showed admixture with tomato isolates. Analyses of both the ribosomal DNA intergenic spacer regions and SSR markers revealed high genetic variability among isolates but were unable to delineate races of V. dahliae. However, a polymerase chain reaction (PCR) assay was applied to amplify a race-1-specific amplicon from the isolates in many hosts from different geographic areas, and was coupled with virulence assays for validation of the data. Results of the PCR assay showed 100% concordance with the virulence assay to differentiate race 1 from race 2 of 48 isolates from tomato. The results indicate that the PCR assay can be applied to differentiate the two races to support our related aim of breeding host resistance, and further reveal insights into the distribution of races in tomato and lettuce cropping systems in California.

Host Resistance to Mirafiori lettuce big-vein virus and Lettuce big-vein associated virus and Virus Sequence Diversity and Frequency in California

Big vein is an economically damaging disease of lettuce (Lactuca sativa) caused by the Olpidium brassicae-vectored Mirafiori lettuce big-vein virus (MLBVV). Lettuce big-vein associated virus (LBVaV) is also frequently identified in symptomatic plants, but no causal relationship has been demonstrated. Although big vein is a perennial problem in the United States, the extent of MLBVV and LBVaV infection and diversity is unknown. Lettuce cultivars partially resistant to big vein reduce losses, but do not eliminate disease. While Lactuca virosa does not develop big vein symptoms, it has not been tested for infection with MLBVV or LBVaV. Lettuce cultivars Great Lakes 65, Pavane, Margarita, and L. virosa accession IVT280 were evaluated for big vein incidence and virus infection in inoculated greenhouse trials. Additional lettuce samples were collected from field sites in California, classified for symptom severity, and evaluated for virus infection. Reverse transcription-polymerase chain reaction and nucleotide sequencing were used to determine infection with MLBVV and LBVaV, and sequence diversity among viral isolates, respectively. Infections with MLBVV and MLBVV/LBVaV were dependent on big vein symptom expression in California production areas, and isolates were closely related to those found in Europe and Japan. Partial big vein resistance was identified in Margarita and Pavane; however, MLBVV infection was found in asymptomatic plants. L. virosa IVT280 remained symptomless and virus free, suggesting that it is immune to MLBVV and LBVaV.

A soilless Verticillium wilt assay using an early flowering lettuce line.

A soilless growth chamber assay was evaluated for rapid assessment of Verticillium wilt on lettuce (Lactuca sativa). Seedlings of the early flowering Plant Introduction (PI) 251246 were inoculated in tubes with conidial suspensions of isolates of Verticillium dahliae from lettuce or cauliflower. PI 251246 developed significant leaf and root symptoms by 21 days following inoculation with isolates of race 1 and race 2 from lettuce, but not following inoculation with an isolate from cauliflower. In contrast, leaf symptoms on the cultivar La Brillante and root symptoms on the cultivar Salinas were not easily differentiated from the symptoms observed on the noninoculated control treatments in the soilless assay, even at the highest inoculum concentration of 1 × 107 conidia/ml. Comparison of the soilless growth chamber assay and a soil-based greenhouse assay revealed a significantly higher proportion of PI 251246 with root vascular discoloration in the soilless assay compared with the soil-based greenhouse assay (χ2, 1 df = 8.9; P = 0.003). There is thus an advantage to using the soilless growth chamber assay with PI 251246. Compared with the soil-based greenhouse assay, the soilless assay reduced the time required for evaluation of Verticillium wilt symptoms on lettuce from about 90 days to 42 days.

The Three Lineages of the Diploid Hybrid Verticillium longisporum Differ in Virulence and Pathogenicity

Verticillium longisporum is an economically important vascular pathogen of Brassicaceae crops in different parts of the world. V. longisporum is a diploid hybrid that consists of three different lineages, each of which originated from a separate hybridization event between two different sets of parental species. We used 20 isolates representing the three V. longisporum lineages and the relative V. dahliae, and performed pathogenicity tests on 11 different hosts, including artichoke, cabbage, cauliflower, cotton, eggplant, horseradish, lettuce, linseed, oilseed rape (canola), tomato, and watermelon. V. longisporum was overall more virulent on the Brassicaceae crops than V. dahliae, which was more virulent than V. longisporum across the non-Brassicaceae crops. There were differences in virulence between the three V. longisporum lineages. V. longisporum lineage A1/D1 was the most virulent lineage on oilseed rape, and V. longisporum lineage A1/D2 was the most virulent lineage on cabbage and horseradish. We also found that on the non-Brassicaceae hosts eggplant, tomato, lettuce, and watermelon, V. longisporum was more or equally virulent than V. dahliae. This suggests that V. longisporum may have a wider potential host range than currently appreciated.

Dynamics of Verticillium Species Microsclerotia in Field Soils in Response to Fumigation, Cropping Patterns, and Flooding

Verticillium dahliae is a soilborne, economically significant fungal plant pathogen that persists in the soil for up to 14 years as melanized microsclerotia (ms). Similarly, V. longisporum is a very significant production constraint on members of the family Brassicaceae. Management of Verticillium wilt has relied on methods that reduce ms below crop-specific thresholds at which little or no disease develops. Methyl bromide, a broad-spectrum biocide, has been used as a preplant soil fumigant for over 50 years to reduce V. dahliae ms. However, reductions in the number of ms in the vertical and horizontal soil profiles and the rate at which soil recolonization occurs has not been studied. The dynamics of ms in soil before and after methyl bromide+chloropicrin fumigation were followed over 3 years in six 8-by-8-m sites in two fields. In separate fields, the dynamics of ms in the 60-cm-deep vertical soil profile pre- and postfumigation with methyl bromide+chloropicrin followed by various cropping patterns were studied over 4 years. Finally, ms densities were assessed in six 8-by-8-m sites in a separate field prior to and following a natural 6-week flood. Methyl bromide+chloripicrin significantly reduced but did not eliminate V. dahliae ms in either the vertical or horizontal soil profiles. In field studies, increases in ms were highly dependent upon the crop rotation pattern followed postfumigation. In the vertical soil profile, densities of ms were highest in the top 5 to 20 cm of soil but were consistently detected at 60-cm depths. Six weeks of natural flooding significantly reduced (on average, approximately 65% in the total viable counts of ms) but did not eliminate viable ms of V. longisporum.