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NOTES

An indirect test using oxalate to determine physiological resistance to white mold in common bean

Department of Crop and Soil Sciences, Michigan State University, East Lansing, MI 48824 USA

kellyj{at}msu.edu

	ABSTRACT

TOP ABSTRACT INTRODUCTION Materials and Methods Results and Discussion REFERENCES

 
In common bean (Phaseolus vulgaris L.), the detection of physiological resistance to white mold [Sclerotinia sclerotiorum (Lib.) de Bary] in the field is confounded by environmental factors and plant avoidance mechanisms. Development of a reliable screening procedure is needed to identify resistant bean germplasm and to develop resistant cultivars. The objective of this study was to determine if oxalate, a primary pathogenicity factor of S. sclerotiorum, could be used to indirectly screen for physiological resistance to white mold in common bean. Cut bean seedlings were placed in a 20 mM oxalate solution in the greenhouse. Genotypes were rated based on differences in wilting response to oxalate. Oxalate ratings of the 27 genotypes were correlated with field ratings of a white mold disease severity index and incidence , and negatively correlated with yield . The oxalate test is an efficient method to indirectly test for physiological resistance to white mold in common bean.

Abbreviations: DI, disease incidence • DSI, disease severity index • RCBD, randomized complete block design

	INTRODUCTION

TOP ABSTRACT INTRODUCTION Materials and Methods Results and Discussion REFERENCES

 
WHITE MOLD is a destructive fungal disease that can infect more than 400 plant species, including many important crop species, such as common bean, sunflower (Helianthus annuus L.), alfalfa (Medicago sativa L.), soybean [Glycine max (L.) Merr.], rape (Brassica napus L.), and peanut (Arachis hypogaea L.) (Boland and Hall, 1994). In common bean, total seed and pod yield are reduced due to lower number of seeds produced per plant, reduced number of pods per plant, and smaller seed size (Kerr et al., 1978). Progress in breeding for resistance is hindered by environmental conditions and plant avoidance mechanisms that confound the expression and detection of physiological resistance mechanisms in the field. Methods to detect physiological resistance to white mold in common bean include the limited-term inoculation method (Hunter et al., 1981), the excised-stem inoculation technique (Miklas et al., 1992a), the leaf-agar plug assay (Steadman et al., 1997), the straw test (Petzoldt and Dickson, 1996), and growing callus on medium containing pathogen filtrate (Miklas et al., 1992b). Most of these tests use a limited number of genotypes and depend on fungal mycelium in screening procedures. Variability in virulence among isolates (Maxwell and Lumsden, 1970; Morrall et al., 1971; Miklas et al., 1992a; Pratt and Rowe, 1995) and pathogen sensitivity to high temperatures (Abawi and Grogan, 1975; Boland and Hall, 1987) limit greenhouse screening methods using the pathogen.

White mold mycelium exude copious amounts of oxalate during infection of plant tissue (Maxwell and Lumsden, 1970). Using nonoxalate producing mutants, oxalate was identified as a primary mode of pathogenesis for S. sclerotiorum (Godoy et al., 1990). A low pH environment (pH 4.0) created by exuded oxalate is optimal for function of the polygalacturonase and pectolytic enzymes produced by the pathogen (Marciano et al., 1983). Differentiation for resistance to oxalate has been identified in a leaf test in sunflower (Noyes and Hancock, 1981), a germination test in alfalfa and crimson clover (Trifolium incarnatum L.) (Rowe, 1993), an excised stem test in soybean (Wegulo et al., 1998), and a leaf test in transgenic rape (Thompson et al., 1995). Common bean has also shown genotypic differentiation in response to oxalate. The uptake of oxalic acid by petioles of excised primary leaves of the resistant cultivar Bunsi (also known as Ex Rico 23) was shown to be slower than in two susceptible cultivars, Kentwood and Seafarer (Tu, 1985). Cultivars that were susceptible to white mold exhibited more severe structural damage to the plasma membranes and chloroplasts than resistant cultivars when exposed to an oxalate solution (Tu, 1989).

Breeding for resistance to white mold in common bean is limited by the lack of a simple, consistent screening method to quickly evaluate a broad array of genotypes for physiological resistance to white mold. An indirect screening method that bypasses the need for the plant to flower would be valuable in screening unadapted, photoperiod-sensitive germplasm for new sources of resistance to white mold. An indirect screening method that eliminates the use of the pathogen would also eliminate variability often associated with greenhouse tests. The objective of this study was to develop an indirect greenhouse test, using oxalate, a primary pathogenicity factor of S. sclerotiorum, to identify physiological resistance to white mold in common bean.

	Materials and Methods

TOP ABSTRACT INTRODUCTION Materials and Methods Results and Discussion REFERENCES

 
High-yielding common bean genotypes were evaluated for resistance to oxalate in three greenhouse tests. Thirty (Test 1) or 36 (Tests 2 and 3) genotypes were evaluated — including cultivars and breeding lines from the navy, black, pink, pinto, great northern, cranberry, and kidney commercial classes; new sources of resistance from breeding programs across North America and the Caribbean; and genotypes entered in the National Sclerotinia White Mold Nursery. Twenty-seven genotypes were common across all three tests. In the greenhouse oxalate test, each entry (genotype) was planted in three 15-cm-diameter pots containing Baccto High Porosity Professional Planting Mix (Michigan Peat Company, Houston, TX), with nine seeds per pot, and grown under greenhouse conditions with ambient temperature and a 16-h day length. Twenty-day-old seedlings (second trifoliate emerging) were cut at the base of the stem at night to avoid wilting due to the potential of high transpiration rates during daylight hours. A foam stopper was placed around the base of the seedling, and placed in a perforated foam board in a 78-L plastic container (67 cm long, 47 cm wide, 21 cm deep). Each container held 11 L of a 20 mM oxalic acid solution that had been adjusted to a pH of 4.0 with NaOH. The perforated foam board was positioned above the solution and kept the seedlings upright while the cut stem was immersed in the solution. Four (Tests 2 and 3) or five (Test 1) seedlings (samples) were used for each genotype in each of the three containers (replications) of the Randomized Complete Block Design (RCBD). In each experiment, a separate control replication (single container) consisted of two (Tests 2 and 3) or three (Test 1) seedlings per genotype being placed in an 11-L solution of distilled water that had been adjusted to a pH of 4.0 with HCl. The three replications and one control replication were placed in an enclosed greenhouse chamber to minimize wilting as a result of exposure to direct light.

The seedlings were rated for wilting symptoms after 12 to 15 h of exposure to the oxalate solution (6–9 h of daylight). A 1 to 6 scale was used to measure wilting, where 1 = no wilting symptoms visible, 2 = one leaf with wilting symptoms (the two unifoliate leaves were rated together as one leaf and the three leaflets of a trifoliate leaf were rated together as one leaf), 3 = two leaves with wilting symptoms, 4 = three or more leaves with wilting symptoms, 5 = petioles collapsing, 6 = main stem (total plant) collapsing. Wilting symptoms ranged from curled leaf tip to total loss of turgidity in the entire leaf.

Genotypes in the three greenhouse oxalate tests were evaluated for comparison with reaction to white mold resistance in the field. The field experiments were grown at the Montcalm Research Farm in Entrican, MI in 1996 (Test 1), 1997 (Test 2), and 1998 (Test 3). Planting was delayed to the second week in June in all three field experiments to favor disease development. A 0.5-m row spacing and 6-m row length were used for the four-row plots. The outer two rows were planted with a white mold susceptible spreader (`Midland'), and the inner two rows were planted with the experimental genotypes. The soil type at the Montcalm Research Farm sites is a combination of Eutric Glossoboralfs (coarse-loamy, mixed) and Alfic Fragiorthods (coarse-loamy, mixed, frigid). Standard agronomic practices for tillage, fertilization, and herbicide were applied to ensure good crop growth and development. Plots were irrigated during initial flowering with 13 mm of water at 3-d intervals, depending on rainfall, in order to promote uniform disease pressure across the field. The field experiments were irrigated with an overhead sprinkler system five times in 1996, three times in 1997, and six times in 1998. Uniform infection of white mold in dry bean at the Montcalm Research Farm was identified in previous field studies. Plots were rated for disease severity and disease incidence (DI) (Steadman, 1997; Kolkman and Kelly, 1998; Steadman et al., 1998) using a "quarter scale" (Hall and Phillips, 1996), shortly before harvest, when the majority of plants had reached physiological maturity. Thirty plants per plot were each given a rating from 0 to 4, where 0 = no disease present, 1 = 1 to 25% of the plant with white mold symptoms, 2 = 26 to 50% of the plant with white mold symptoms, 3 = 51 to 75% of the plant with white mold symptoms, and 4 = 76 to 100% of the plant with white mold symptoms. A disease severity index (DSI) was calculated for each plot on a percentage basis, using the following formula:

Disease incidence was calculated as the number of plants with white mold infection out of the thirty individuals, based as a percentage. Plots were harvested after disease rating.

All greenhouse experiments were analyzed as RCBDs, using PROC GLM (SAS Institute, 1995). The three field experiments were analyzed separately using PROC LATTICE (SAS Institute, 1995). The 1996 field experiment was analyzed as a rectangular lattice, and the 1997 and 1998 field experiments were each analyzed as a partially balanced triple lattice. The 27 common genotypes were analyzed across all three tests (greenhouse) and years (field) as a RCBD, using PROC GLM (SAS Institute, 1995). Environments were considered as a random effect and genotypes as a fixed effect.

	Results and Discussion

TOP ABSTRACT INTRODUCTION Materials and Methods Results and Discussion REFERENCES

 
Significant genotypic differences were identified in the response to oxalate (Tables 1 and 2) . Wilting symptoms in the control container in each experiment were negligible and not significant, indicating the importance of oxalate in the appearance of wilting symptoms. The temperature in the three oxalate tests ranged from 24 to 40°C (Test 1), 21.5 to 26°C (Test 2), and 22.5 to 26.5°C (Test 3). Significant differences between tests (Tables 1 and 2) indicate the influence of the environment in affecting the estimate of resistance to oxalate, and the importance of including known resistant and susceptible control cultivars in each experiment. Significant correlations between the oxalate test ratings and field disease ratings were identified in each experiment (Table 3) . The highest correlation between the oxalate test and the DSI and DI ratings in the field was observed with the 27 genotypes across 3 yr.

Resistance to oxalate is a specific resistance mechanism that may work singly, or more likely in a combination with a number of plant avoidance mechanisms or alternative physiological mechanisms, to provide consistent levels of resistance to white mold in the field. Mechanisms can provide plant avoidance to white mold, in which the plant escapes the initial infection of the pathogen. Favorable conditions for the formation of apothecia, the corresponding onset of flowering for inoculation via ascospores, and appropriate temperatures following infection are critical components of the epidemiology of S. sclerotiorum (Boland and Hall, 1987). Plant avoidance mechanisms, such as early flowering or maturity, or an open porous canopy may limit the initial inoculation and subsequent infection of white mold. Physiological resistance mechanisms may not be restricted to resistance to oxalic acid. Alternative resistance mechanisms at the cellular level, such as phytoalexins (Sutton and Deverall, 1984), may be important to white mold resistance in the field.

Any genotype that escapes infection in the field can significantly skew the correlation between the greenhouse oxalate test ratings and field disease ratings. OAC Laser, an upright navy bean cultivar with a porous canopy, does not have high levels of resistance to oxalate in the greenhouse tests (Table 2), yet is very resistant to white mold infection in the field (Table 4) . Plant avoidance mechanisms and moderate to low levels of resistance to oxalate in OAC Laser most likely work in combination to provide excellent resistance in the field. Two early-flowering cultivars, Isles and Othello, can have low incidence of white mold in the field (Table 4) but exhibit high oxalate ratings (Table 2). The high oxalate test ratings indicate that both Isles and Othello have little physiological resistance to oxalate. Alternatively, less-adapted germplasm, such as I9365-19 and I9365-3 (Miklas et al., 1998), were identified to be resistant to oxalate, yet had high disease ratings in the field (Table 4). I9365-19 and I9365-3 represent useful sources of physiological resistance for introgression into adapted germplasm. Unadapted germplasm has been shown to carry putative physiological resistance by the straw test (Miklas et al., 1999). The success of the oxalate test confirms the segregation of responses of resistant and susceptible common bean cultivars to oxalate (Tu, 1985, 1989) and pathogen filtrate (Miklas et al., 1992b). The oxalate test indirectly identifies genotypes that have physiological resistance to white mold via oxalate resistance, bypassing the need for field testing where the detection of physiological resistance is confounded by plant avoidance mechanisms.

The oxalate test is useful for determining physiological resistance in the greenhouse. Photoperiod-sensitive unadapted germplasm can be tested for physiological resistance since plants are tested at the seedling stage (second trifoliate emerging) and are therefore not influenced by flowering (reproductive) traits. A large number of lines can be evaluated in a relatively short time period. Inoculation of the cut seedlings into a common solution of oxalate reduces variability that may be observed when using agar plugs of S. sclerotiorum. The inherent variability within a single isolate (Maxwell and Lumsden, 1970) or isolate variability from test to test is reduced (Miklas et al., 1992a). The time between inoculation of seedlings and rating of the response to oxalate is very short (12–15 h after inoculation), reducing the potential variability in environmental conditions that exist in a greenhouse during a longer period of time. The rating scale in the oxalate test was designed to effectively quantify the degree of damage to a genotype using a quick visual estimate. Extreme high temperatures can limit the ability to screen effectively using the fungus (Abawi and Grogan, 1975; Boland and Hall, 1987). In the oxalate test, temperatures up to 40°C were encountered that did not adversely affect the correlation between greenhouse and field results. The differential response of common bean genotypes exposed to an oxalate solution has a highly significant correlation with corresponding white mold field ratings for DSI and DI, and a highly significant negative correlation with yield (Table 3). Screening genotypes for resistance to oxalate, a primary pathogenicity factor for S. sclerotiorum, is an efficient indirect method to test for physiological resistance to white mold in common bean.

Received for publication March 29, 1999.

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