Maintenance of physiological races of P. phaseoli.

Isolates of P. phaseoli characterized as races E and F were maintained in pure cultures for long-term storage at 20 °C in screw-cap test tubes containing sterile water and corn (Zea mays L.) and/or cucumber (Cucumis sativus L.) seeds as substrates. Isolates of P. phaseoli races E and F were reactivated by transferring to the semiselective media lima bean agar (Calvert et al., 1960) or modified pea agar (Dance et al., 1975). After transfer, the cultures were maintained in an incubator set at 20 °C.

Inoculum production and hypocotyl screening of germplasm.

Inoculum of P. phaseoli was produced and maintained on hypocotyls of the susceptible lima bean cultivar Concentrated Fordhook. Untreated seeds were rinsed in sterile distilled water to remove any surface contaminants and to begin the imbibition process. The seeds were placed in 10-cm plastic pots with a commercial plug and seedling mix, five to six seeds per pot, placed close together. The pots were placed on heat mats set to 26 °C, on greenhouse benches. Four days after seeding when hypocotyls began to emerge, some potting mix was gently removed by hand from the pots to reveal more of the developing hypocotyls. Emerging seedlings with cotyledons were watered lightly to soften seedcoats and whenever possible, seedcoats were removed by hand. Inoculum was prepared by harvesting 7-d-old infected hypocotyls and stem material which were cut into small (3–6 mm) sections on a sterile surface. This material was then mixed with sterile distilled water to make a slurry. The slurry was placed onto expanding seedlings in direct contact with the new tissue. The pots were placed in a dew chamber for up to 7 d. Symptoms of stunting and distortion typical of the disease developed within 6 d with abundant mycelia and sporangia produced inside of the dew chamber. The two races of P. phaseoli were maintained in separate digital-controlled dew chambers (Percival Scientific, Inc., Perry, IA). The dew chambers were set at a continuous 20 °C, 94% ± 1% relative humidity, and continuous 50 μmol·m⁻²·s⁻¹ of photosynthetic active radiation. When seedlings exhibited symptoms of downy mildew, the hypocotyls were harvested to prepare inoculum. Lines may be screened for resistance at the seedling emergence stage or later, during early pod set. When screened at seedling emergence, susceptible plants develop symptoms on hypocotyl tissue and resistant plants do not. For screening at the seedling emergence stage, the inoculum was prepared as previously described. For larger studies, ≈200 g of infected tissue and 1000 mL of water filtered by reverse osmosis were placed in a 2-L beaker and mixed thoroughly with a glass stirring rod; the suspension was refrigerated at 5 °C for 30 min. Finally, plant tissue pieces were removed by passing the solution through a sieve (U.S. Standard test sieve No. 18). The concentration of sporangia was determined from a sample of this suspension using a hemocytometer (Bright-Line^®; American Optical Corporation, Buffalo, NY) and a compound microscope at ×200. The suspension was adjusted to a density of 1 × 10³ sporangia/mL.

Evaluation of P. lunatus accessions and commercial cultivars in the field for reaction to downy mildew incited by P. phaseoli races E and F.

A total of 43 P. lunatus genotypes (35 accessions and eight cultivars) were screened in the field for their reaction to races E and F of the downy mildew pathogen from 2003 to 2010. Nineteen accessions were from the USDA-National Plant Germplasm System and 16 from the International Center for Tropical Agriculture. The remaining eight were cultivars obtained from commercial seed suppliers. A species native to eastern North America, Phaseolus polystachios (L.) BSP, was also screened to determine its reaction to races E and F in the field (four plants for each race), and in a dew chamber (five plants for each race) during 2010. Seed used for screening was collected from a wild population in Maryland and subsequently submitted to the USDA-NPGS collection, accession number W636674. The species P. polystachios, which was not previously identified as a host of P. phaseoli, is closely related to lima bean (Delgado-Salinas et al., 1999).

Lima bean accessions were evaluated in the field for their reactions to two races of P. phaseoli separately at the University of Delaware’s Agricultural Experiment Stations in Newark (race E) and Georgetown (race F) in 2004, 2005, 2006, 2009, and 2010. Plots were established during the first week of July in each year and arranged in a randomized complete block design with three replications. Each plot consisted of 4.6-m row with rows spaced 0.76 m apart. The cultivars selected were planted by hand or hand-operated vegetable seeder, 12 seeds/m row. For some cultivars with limited seed, evaluation plots were half the length (2.44 m row). Susceptible cultivars (Eastland race E and 184-85 race F) were planted every fifth row as controls and to facilitate pathogen spread.

Each year, plots were inoculated in early September at the newly formed pod (pin) to flat pod stage. Inoculum was prepared as previously described and applied with a backpack low-pressure manual pump sprayer (SOLO backpack sprayer, model 425; SOLO Incorporated, Newport News, VA) in the early evening when temperatures were between 18 and 21 °C. Plants were sprayed until runoff with a sporangial suspension containing 1 × 10³ sporangia/mL of P. phaseoli, and the inoculation was repeated 7–10 d later. Beginning the first night after inoculation, the plants were misted intermittently each night using Micro-Bird misting nozzles (Rain Bird Corp., Glendora, CA) from 1800 to 0600 hr to increase humidity and leaf wetness duration. Irrigation was provided as needed throughout the season using trickle tape in Newark and overhead irrigation in Georgetown. The middle 2.3 m of each row was evaluated for susceptibility 10–15 d after the second inoculation. The plants were rated for signs of infection (white fluffy mycelium on shoot tips, pins, and flat pods) and symptoms of downy mildew (red line surrounding infected areas on pods). If necessary, confirmation of infection was made by evaluating samples morphometrically for sporangia of P. phaseoli using a compound microscope.

Inheritance of resistance of F₂ populations of P. lunatus to races E and F of P. phaseoli.

To facilitate breeding for resistance to downy mildew, a study of the inheritance of resistance to races E and F of P. phaseoli was started. The cultivars used in the study were Cypress, Dover Tucker, Maffei 15, Jackson Wonder, and germplasm release B2C. Parents for the crosses were chosen based on their disease reaction in previous disease screening: ‘Cypress’ and ‘Dover Tucker’ are resistant to race E but susceptible to race F, whereas ‘Maffei 15’ and B2C are susceptible to race E and resistant to race F (Table 1). ‘Jackson Wonder’ is susceptible to both races. Crosses were made between ‘Jackson Wonder’ and the resistant parents in the greenhouse. The F₁ plants from these crosses were planted in the greenhouse and the seed from each F₁ plant was harvested and maintained separately during inoculation to facilitate detection of accidental self-pollination. Five F₂ populations (Tables 3–5) were screened to observe the segregation pattern of the resistance reaction. A total of 254 F₂ plants from ‘Jackson Wonder’ (susceptible) × ‘Cypress’ (E resistant) and 315 F₂ plants from ‘Jackson Wonder’ × ‘Dover Tucker’ (E resistant) were screened. A total of 209 F₂ plants from ‘Jackson Wonder’ (susceptible) × ‘Maffei 15’ (F resistant) and 271 F₂ plants from ‘Jackson Wonder’ (susceptible) × B2C (F resistant) were screened. From the E-resistant by E-resistant test cross ‘Dover Tucker’ × ‘Cypress’, 223 F₂ plants were screened. The F₂ seeds were planted in 10-cm pots in seed starter substrate and grown until hypocotyl emergence.

Table 1.Results of screening for downy mildew races E and F resistance in small-seeded lima bean cultivars and germplasm accessions and Phaseolus polystachios germplasm in the field and greenhouse from 2003 to 2010.

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Table 2.Results of screening for downy mildew races E and F resistance in large-seeded lima bean cultivars and germplasm accessions in the field and greenhouse from 2004 to 2009.

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Table 3.Inheritance of resistance to downy mildew race E in lima bean populations derived from crosses of cultivars Cypress, Dover Tucker, Bridgeton, and Jackson Wonder.

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Table 4.Inheritance of resistance to downy mildew race F in lima bean populations derived from crosses of cultivars Maffei 15, B2C, and Jackson Wonder with genotypes categorized as resistant or susceptible.

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Table 5.Inheritance of resistance to downy mildew race F in lima bean populations derived from crosses of cultivars Maffei 15, B2C, and Jackson Wonder with genotypes categorized as resistant, moderately susceptible, or very susceptible.

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At the time of hypocotyl emergence from the substrate, the pots were transferred to two separate greenhouse humidity chambers with wick mats and timer-controlled misting system as described previously to provide optimal conditions for infection. The F₂ populations were inoculated with either race E or F by applying a 10-mL suspension of 1 × 10³ sporangia/mL of P. phaseoli and at 5 and 10 d postemergence by spray inoculation to ensure that there were no escapes from infection. The plants were visually evaluated for infection daily over a 7-d period and infection confirmed by making scrapings of hypocotyls on microscope slides and observing the presence or absence of sporangia of P. phaseoli.

Evaluation of a population RILs of P. lunatus for resistance of downy mildew incited by P. phaseoli race E in 2011.

A set of 121 F₉ RILs from the cross ‘Bridgeton’ × ‘Jackson Wonder’ and the two parental lines were screened in the field for their reaction to race E of P. phaseoli. Ten seeds of each line were planted in the field on 6 July 2011. The plants were inoculated at the pin pod to flat pod stage with a sporangial suspension as previously described. Individual plants were rated as susceptible or resistant on 21 Sept. 2011 3 months later. Ratings were based on the presence of signs and symptoms of downy mildew.

Statistical analysis.

Observed ratios of resistant to susceptible plants from resistant × susceptible test crosses were compared with the expected ratio of 3 resistant : 1 susceptible for F₂ populations and 1 resistant : 1 susceptible for the RIL population to test for monogenic inheritance of resistance using the chi-square goodness of fit test. Observed ratios for resistant × resistant test crosses were compared with a 15 resistant : 1 susceptible ratio to test for independence of the resistance loci.

Evaluation of germplasm for resistance to races E and F of downy mildew.

Of the seven baby lima bean cultivars obtained from commercial seed suppliers that were screened in inoculated field plots in 2003–05, four were resistant to race E and susceptible to race F: ‘Eastland Select’, ‘184-85’, ‘Cypress’, and ‘C-elite Select’ (Table 1). Three of the commercial cultivars were resistant to race F and susceptible to race E: Maffei 15, ‘Eastland’, and ‘8-78’. Of the 26 small-seeded accessions screened in inoculated field plots, three were resistant to race E and susceptible to race F, ‘Dover Tucker’ (PI 549456), PI 189403, and ‘Bridgeton’ (PI 549508). Two accessions, B2C (PI 549515) and PI 195342, were resistant to race F and susceptible to race E. None of the accessions were resistant to both races E and F.

Nine large-seeded genotypes were screened in the field for resistance to downy mildew races E and F (Table 2). The germplasm release F-169 (PI 549514) was resistant to race E but susceptible to race F and the germplasm release MRF-79 (PI 549524) was resistant to race F but susceptible to race E. Two other USDA Fordhook releases which had been bred for resistance to downy mildew races A, B, C, or D, ‘F-1072’ and ‘MRF 90-1’, were not resistant to either race E or F.

Two of four plants of P. polystachios that were inoculated in the field with race E and three of four plants inoculated with race F exhibited signs and symptoms of infection of P. phaseoli identical to the reaction produced on lima bean. Samples taken from these plants were confirmed morphometrically to be due to infection by P. phaseoli, indicating that P. polystachios is an alternate host for downy mildew. In later dew chamber evaluations, five of five plants inoculated at the hypocotyl stage were susceptible to race E and two of five plants were susceptible to race F. These preliminary results suggest that this particular wild population of P. polystachios has genes conferring resistance to downy mildew.

Determining the inheritance of resistance to races E and F of P. phaseoli.

The two F₂ populations from the crosses between downy mildew–susceptible ‘Jackson Wonder’ and the race E–resistant cultivars Cypress and Dover Tucker segregated in a 3:1 (resistant/susceptible) ratio with P > 0.05. The χ² values were 2.09 (P = 0.1484) for ‘Jackson Wonder’ × ‘Cypress’ and 1.37 (P = 0.2421) for ‘Jackson Wonder’ × ‘Dover Tucker’ (Table 3), indicating that in both cultivars, a single dominant gene conferred resistance to race E of P. phaseoli. In the R × R cross ‘Dover Tucker’ × ‘Cypress’, all 223 F₂ plants were resistant to race E. This result does not fit a 15:1 (resistant/susceptible) ratio to suggest the presence of two independent dominant resistance genes; the χ² value is 14.94 (P = 0.0001). The observed result suggests that these two cultivars carry the same resistance gene or two different resistance alleles at the same locus (Table 3).

Four to 10 individual plants were rated for their reaction to race E for each of the ‘Bridgeton’ × ‘Jackson Wonder’ RILs and parent lines. The cultivar Bridgeton is resistant to race E and Jackson Wonder is susceptible. All of the plants in each RIL either had signs and symptoms of downy mildew (rated susceptible) or were without signs and symptoms of the disease (rated resistant). Sixty-nine of the lines were rated resistant and 52 were susceptible (Table 3), which fits a 1:1 segregation ratio with a χ² value of 2.39 (P = 0.1222), indicating that resistance to race E is conferred by a single gene.

When inoculated, the F₂ populations from the crosses between ‘Jackson Wonder’ and the race F–resistant ‘Maffei 15’ and B2C resulted in plants with three different disease reactions. Some plants were completely resistant, some were very susceptible (i.e., exhibited obvious mycelial growth and died), whereas others were stunted, had some tissue death, but did not die. Mycelium and sporangia of P. phaseoli were detectable on these stunted plants microscopically. In Table 4, the plants are categorized as either resistant or susceptible, with plants exhibiting partial resistance classified as susceptible. When classified this way, both populations fit a 3:1 (resistant/susceptible) ratio with P > 0.05. The χ² value is 0.10 (P = 0.7490) for ‘Jackson Wonder’ × ‘Maffei 15’ and 1.59 (P = 0.2073) for ‘Jackson Wonder’ × B2C, indicating that both ‘Maffei 15’ and B2C carry a single dominant gene which confers complete resistance to downy mildew race F (Table 4).

However, in Table 5, the plants are categorized as resistant, partially resistant, or very susceptible. The segregation ratio is suggestive of the presence of recessive or partially dominant genes that confer partial resistance. These partial resistance genes are masked in ‘Maffei 15’, B2C, and their fully resistant progeny by the single dominant gene for resistance, which makes determining the segregation ratio of the genes for partial resistance in these particular populations difficult. It is likely that ‘Maffei 15’ and B2C carry the same downy mildew resistance genes because ‘Maffei 15’ was selected from the germplasm release B2C.