Evaluation location: New Hampshire, United States
Plant and fungal materials. Information about the studied plant accessions, their geographic origins (wild-collected materials) or breeding program sources, and their identification numbers (local and/or Plant Introduction) is provided for diploids and polyploids, respectively, in Tables 1 and 2. Subspecies designations are not provided for California accessions of F. vesca due to uncertainty in differentiating the subspecies Fragaria vesca ssp. bracteata from F. vesca ssp. californica and their possible hybrids. Among diploid accessions (Table 1); CFRA364.002, CFRA333.001, and CFRA520.001 were obtained from the NCGR, accessions from Hokkaido, Japan were collected by Thomas M. Davis and Kim Hummer [32], and GS1J was collected by Gunter Staudt, while all other UNH-numbered accessions were collected by Davis.
The polyploid accessions of octoploids Fragaria virginiana and F. chiloensis, and decaploid F. cascadensis belong-ing to the Fragaria supercore collection were obtained from the NCGR. The studied F.×ananassa accessions were of interest to our breeding programs. Plants of ‘Sparkle’ and ‘Tristar’ were purchased from Nourse Farms, Whately MA, while the other cultivars and breeding lines were provided by Andrew R. Jamieson. F.×ananassa cultivars ‘Annapolis’, ‘Cavendish’, ‘Evangeline’, ‘Mira’, ‘Wendy’ (aka ‘AC Wendy’), and ‘Laurel’ (tested as K93-20), and numbered breeding clones K05-9 and M903 were developed at the Agri-Food Canada Kentville Research Station, Nova Scotia, Canada. Cultivar ‘Sparkle’ is a 1942 release from the New Jersey State University breeding program [18], while ‘Tristar’ is a 1981 release from the USDA Beltsville program.
Screening methods. A series of trials was conducted, in which from five to 20 genotypes were screened. In each trial, each genotype was represented by three orfour inoculated and two uninoculated (control) plants. Pots containing inoculated or uninoculated plants were maintained within separate containment trays, and plants were randomly distributed within the trays. Some genotypes were included in multiple trials.
Verticillium dahliae isolate V1 was obtained from Mansun Kong at Driscoll Strawberry Associates, Watsonville, CA, USA, and was originally isolated from an infected strawberry plant (M. Kong, personal communication). Plants were maintained in the UNH MacFarlane Greenhouse facility in Pro-Mix Mycorrhizae™ (Premier Tech Horticulture LTD, Canada), and were propagated by rooting stolons that were still attached to mother plants to produce runner plantlets. Runner nodes were pinned onto the surface of Metro-mix 360 (Hummert™ International, Missouri) medium in 4” standard round polypropylene pots (Dillen Products Company, Ltd., Middlefield, Ohio) using staples made from plastic-coated wire. Plantlets were allowed to root for two weeks prior to separation from the mother plant and inoculation. Ten plantlets were rooted from each mother plant, and eight were ultimately used in each trial (four inoculated plantlets and four uninoculated controls). Concurrent with plant propagation, fungal inoculum was cultured in an appropriate volume (∼10 ml for each plant to be inoculated) of autoclaved Difco™ Czapek-Dox broth (BD Biosciences). The broth, contained in 1L flasks, was inoculated in a laminar flow hood with two to four ∼1cm2 pieces of fungus-covered Czapek-Dox agar from fungal culture plates. The inoculated broth was then incubated for two weeks at room temperature on an orbital shaker at 200 RPM.
On the day of inoculation, the fungal culture was strained through two layers of cheesecloth and one layer of Miracloth (EMD Millipore, Billerica, Massachusetts), and then centrifuged at 10,000× g for 5 min. The culture medium was decanted and the conidial pellet was resuspended in a volume of sterile distilled water equivalent to that of the decanted culture medium. Conidia were quantified using a hemacytometer (Bright Light Counting Chamber Improved Neubauer, Hausser Scientific, Horsham Pa), and the suspension was diluted, if necessary, with sterile distilled water to ∼2 × 107 conidia/ml. Immediately prior to root dipping, rooted plantlets were separated from mother plants and trimmed to remove runners. Soil was shaken from roots prior to dipping. Root dipping was performed in 50-ml polyvinyl chloride pipet basins (Fisher Scientific, Pittsburgh, PA). Root systems were immersed, two at a time per basin, for 5 min in either 20 ml fungal spore suspension or 20 ml sterile distilled water (uninoculated control). After dipping, the plants were replanted in new 4” pots of sterile (autoclaved) Metromix medium, then moved to a greenhouse under ambient light and temperature conditions or to a 22◦ C temperature-controlled growth room under broad-spectrum (140 moles/m2/sec) fluorescent lights, then maintained in containment trays with minimal watering for a period of at least eight weeks.
Plant verticillium wilt disease ratings. At the end of the observational period, each individual plant was rated relative to controls according to the following rating scale, as exemplified by plants shown in Fig. 1. A rating of 1 (healthy) was given to plants closely resembling uninoculated controls. A rating of 1.5 (slightly symptomatic) indicated mild stunting and/or mild outer leaf necrosis and/or browning. A rating of 2.0 (mildly symptomatic) indicated distinct stunting and/or distorted growth, more leaf necrosis and browning, and perhaps one or two dead leaves. A rating of 2.5 (very symptomatic) was given to plants with severe stunting, leaf necrosis and browning, yet still having one to a few green leaves. A 3.0 (dead) rating indicated that all of the leaves were necrotic and the plant was considered to be nearly or completely dead.
A mean disease rating was then calculated for each germplasm accession or cultigen on the basis of the respective total number of rated plants. As guided by previous literature [9, 31, 33] and to facilitate comparison with prior studies, we then assigned a qualitative classification to each accession or cultigen based upon its mean disease rat-ing. For this purpose, the scale of mean disease ratings was partitioned into five ordered categories. The category ranges and corresponding classifications (in parentheses) were: 1.0 to 1.3 (very resistant = VR), 1.4 to 1.7 (moder-ately resistant = MR), 1.8 to 2.2 (intermediate = I), 2.3 to 2.6 (moderately susceptible = MS), and 2.7 to 3.0 (very susceptible = VS). For example, an accession with a mean disease rating of 1.5 would have been classified as MR.