Use of a Root Tumorigenesis Assay to Detect Genotypic Variation in Susceptibility of Thirty-four Cultivars of Pisum sativum to Crown Gall

Abstract

We developed a quantitative assay to measure tumorigenesis on roots and root crowns, the natural sites of Agrobacterium tumefaciens infection. Efficiency of tumor formation and tumor weight on seedlings of Pisum sativum 'Little Marvel' were directly proportional to the logarithm of inoculum concentration. Depth of wounding prior to inoculation also significantly influenced tumor weight but not efficiency. Mean weight of tumors that developed in response to inoculation with strain B6 varied significantly among 34 different commercial cultivars. Tumors on the most susceptible cultivar, Target, were more than tenfold heavier than those formed on the least susceptible cultivar, Sweet Snap. Efficiency of tumorigenesis on 'Sweet Snap' was also relatively low: only 64% of inoculated seedlings developed tumors compared with 89 to 100% efficiencies for all other cultivars. evaluate resistance and susceptibility in Pisum sativum L., a well characterized diploid species that is a known host of A. tumefaciens (16). To initiate studies of crown gall resistance in pea, we established a reproducible quantitative assay for tumorigenesis on roots of intact plants and used it to survey genotypic variation among 34 commercial cultivars of pea. MATERIALS AND METHODS Bacterial Strains and CultureSingle colony cultures of bacterial strains were maintained at -70C in 50:50 (v/v) glycerol:yeast extract-mannitol (YEM) medium. Strain B6 was a gift from J. A. Lippincott (Northwestern University). For assays, cultures were grown overnight on YEM solidified with 0.7% agar and were suspended in water. Inoculum concentrations were estimated turbidimetrically and confirmed by dilution plating.Agrobacterium tumefaciens is a soil-borne bacterial pathogen that causes crown gall on most dicots and some gymnosperms and monocots (5). Although most parts ofmany plants are susceptible to experimental inoculation, A. tumefaciens normally infects through wounds in roots and at the rootshoot interface, the crown (18). Crown gall is among the major disease problems in fruit tree and ornamental nurseries and vineyards (25,26 Plant Inoculation and AssayPisum sativum L. seeds were surface-sterilized by consecutive 5-min immersions in 95% ethanol and 50% commercial bleach and then washed 4x in at least 50 mL of sterile water. Seeds were germinated on water agar overlaid with filter paper for 2 to 3 d or until the radicles were approximately 1 to 3 cm in length. Each seedling was wounded either by stabbing the root with a marked scalpel blade to a depth of 1 mm or by pushing the blade through the root to a depth of approximately 3 mm. Three wounds were made on each plant at approximately 5-mm intervals from the crown to the root tip. Seedlings were immersed into 5 mL of inoculum for 5 min and were then placed into moistened growth pouches (Northrup King, Minneapolis, MN), with 4 or 5 seedlings per pouch. Pouches were moistened daily with approximately 5 mL of water and were incubated at 250C day/230C night for 2 weeks.

Inheritance of Resistance to Crown Gall in Pisum sativum

“…Three of the lines-P1272185, B586-105(l), and B786-287( 1)-developed unusually large tumors (more than 9 mm in diameter), and this 'supersusceptible' phenotype was transmitted to Fl and F2 progeny from selfed parents. Tumor response phenotypes were also compared with those of 34 commercial cultivars whose responses were documented in a previous study (4). Tumors on the three supersusceptible lines (for example, Fig.…”

“…4,8,15,18), studies on inheritance have been limited to grapevine (12,20). We recently developed an assay that results in a 95 to 100% infection frequency on susceptible pea cultivars and used it to demonstrate the occurrence of quantitative variation in susceptibility of 34 cultivars (4). The objectives of the current study were to identify variation in responses of more than 1300 pea genotypes.…”

Requirement for chemotaxis in pathogenicity of Agrobacterium tumefaciens on roots of soil-grown pea plants

“…Roots were immersed for 5 min in inoculum at different concentrations and then were planted in growth pouches. Infection was rated after 2 weeks by the percentage of inoculation sites that developed tumors (17). Soil Mutants that exhibited wild-type virulence in the growth pouch assay were selected for studies of behavior in soil and in sand (Table 2).…”

“…A growth pouch assay was used to measure virulence of mutants in vitro (17). Seedlings with root lengths of 10 to 20 cm were wounded at two sites:…”

A semiquantitative bioassay for relative virulence of Agrobacterium tumefaciens strains on Bryophyllum daigremontiana

“…Various assays have been used to study tumor formation and to determine the relative virulence of mutant strains with respect to that of the wild-type bacterium. These assays include Anand and Heberlein's potato disc assay (1), Lippincott and Heberlein's pinto bean leaf assay (4), the pea root assay described by Hawes et al (2), and a carrot root assay originally described by Klein and We found that the best way to quantitate bacterial virulence in this assay was to determine the number of bacteria needed to form tumors at 50% of the inoculated sites. Other parameters, such as tumor weight, diameter, and length of time to form tumors, were explored but were not found to give a linear response as the number of bacteria inoculated increased.…”