Semi-High Throughput Screening for Potential Drought-tolerance in Lettuce (<em>Lactuca sativa</em>) Germplasm Collections

This protocol describes a method by which a large collection of the leafy green vegetable lettuce (Lactuca sativa L.) germplasm was screened for likely ...

United States Department of Agriculture

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.

A real-time PCR assay for detection and quantification of Verticillium dahliae in spinach seed.

Lettuce is a major vegetable in western countries. Mutations generated genetic variations and played an important role in the domestication of the crop. Many traits derived from natural and induced mutations, such as dwarfing, early flowering, male sterility, and chlorophyll deficiency, are useful in physiological and genetic studies. Mutants were also used to develop new lettuce products including miniature and herbicide-tolerant cultivars. Mutant analysis was critical in lettuce genomic studies including identification and cloning of disease-resistance genes. Mutagenesis combined with genomic technology may provide powerful tools for the discovery of novel gene alleles. In addition to radiation and chemical mutagens, unconventional approaches such as tissue or protoplast culture, transposable elements, and space flights have been utilized to generate mutants in lettuce. Since mutation breeding is considered nontransgenic, it is more acceptable to consumers and will be explored more in the future for lettuce improvement.

Mutations in lettuce improvement.

Verticillium wilt on spinach (Spinacia oleracea) is caused by the soilborne fungus Verticillium dahliae. The pathogen is seedborne and transmission through seed is a major concern because of the dispersal of the pathogen to areas where fresh and processing spinach crops are grown in rotation with susceptible crops. Reduction in seedborne inoculum minimizes pathogen spread; therefore, knowledge of pathogen localization in seed is critical to develop methods to reduce seedborne inoculum. Spinach seedlings were inoculated with conidial suspensions of a green fluorescent protein-tagged strain of V. dahliae and colonization events were followed through seed production by confocal laser-scanning microscopy. Between 24 to 96 h postinoculation (PI), conidia germinated and formed hyphal colonies on root tips and in root elongation zones. Hyphae colonized root cortical tissues both intra and intercellularly by 2 weeks, and colonized the taproot xylem with abundant mycelia and conidia that led to vascular discoloration coincident with foliar symptom expression by 8 weeks PI. At 10 weeks PI, the xylem of the upper stem, inflorescence, and spinach seed parts, including the pericarp, seed coat, cotyledons, and radicle, had been colonized by the pathogen but not the perisperm (the diploid maternal tissue). Maximum concentration of the fungus was in the seed coat, the outermost layer of the vasculature. Infection of V. dahliae in spinach seed was systemic and transmissible to developing seedlings. Additional analyses indicated that fungicide and steam seed treatments reduced detectable levels of the pathogen but did not eliminate the pathogen from the seed. This information will assist in the development of seed treatments that will reduce the seedborne inoculum transmission to crop production fields.

Beiquan Mou

Agricultural Research Service

United States Department of Agriculture

Semi-High Throughput Screening for Potential Drought-tolerance in Lettuce (Lactuca sativa) Germplasm Collections

Beiquan Mou

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Agricultural Research Service

United States Department of Agriculture

Semi-High Throughput Screening for Potential Drought-tolerance in Lettuce (Lactuca sativa) Germplasm Collections

Agricultural Research Service