Sclerotinia blight, caused by Sclerotinia minor Jagger, is one of the most widespread and damaging peanut diseases worldwide. In the United States, Sclerotinia blight is a problem primarily in Virginia, North Carolina, Oklahoma, and Texas, capable of causing 50% yield loss in heavily infested fields (Porter and Melouk, 1997). For peanut growers, planting resistant cultivars is the most economical approach for managing this disease. Fungicides are frequently used to manage Sclerotinia blight in fields with high levels of disease, but treatments are costly. Sclerotia of S. minor are also viable in the soil for four to five years without a host (Melouk and Backman, 1995); therefore, cultural practices such as multi-year crop rotations may be difficult economically for growers to implement (Damicone and Jackson, 1994). Peanut cultivars with significant resistance to Sclerotinia are available (Chappell et al., 1995; Goldman et al., 1995; Hollowell et al., 2008), although none developed using traditional breeding methods are immune to the disease.

The time required to develop a new peanut cultivar may take more than ten years (Baring et al., 2006; Smith et al., 1991). Multiple years of field testing are needed to identify and verify disease resistance, but it may be possible to accelerate progress by augmenting field trials with greenhouse-based resistance assays. Greenhouse assays may be less limited by seasonality and physical space than field trials, and are thus suitable for screening a larger number of plants. Environmental conditions and pathogen inoculum levels, which frequently vary in the field (Hollowell et al., 2008), may also be controlled more easily in the lab. Lab-based resistance assays for Sclerotinia blight using detached stems (Brenneman et al., 1988; Melouk et al., 1992), whole plants (Cruickshank et al., 2002; Goldman et al., 1995; Hollowell et al., 2008), and leaflets (Hollowell et al., 2003) have been developed. Unfortunately, many of these assays are better at identifying highly resistant and highly susceptible cultivars, and less capable of discriminating among moderately resistant cultivars (Faske et al., 2006; Goldman et al., 1995). In addition, results from greenhouse-based assays may not be completely congruent with results from field trials (Hollowell et al., 2008, 2003). These assays cannot account for plant architecture characteristics that create favorable or unfavorable conditions for disease (Brenneman et al., 1988; Hollowell et al., 2008, 2003).

An alternative, potentially useful method for screening peanuts for resistance to Sclerotinia blight uses oxalic acid. Some fungal plant pathogens, including Sclerotinia spp. (Godoy et al., 1990; Livingstone et al., 2005) and Sclerotium rolfsii (Bateman and Beer, 1965; Kritzman et al., 1977), produce oxalic acid to infect plants. Oxalic acid was used to screen several crops, including common bean (Kolkman and Kelly, 2000), scarlet runner bean (Chipps et al., 2005), sunflower (Noyes and Hancock, 1981), and soybean (Cunha et al., 2010; Wegulo et al., 1995), for resistance to S. sclerotiorum. In these crops, response to oxalic acid exposure, as measured by wilt severity or discoloration in detached stems, discoloration in leaflets, or proportion of lysed cells, correlated with field resistance. However, the oxalic acid assay still shares the same disadvantage as other greenhouse-based assays in that it measures physiological resistance and cannot predict plant avoidance through canopy architecture (Brenneman et al., 1988; Hollowell et al., 2008, 2003). Response to oxalic acid was used to evaluate resistance in peanuts transformed with barley oxalate oxidase (Livingstone et al., 2005); however, this method has not yet been evaluated as a resistance assay for wider selection of conventionally-bred peanuts. The objective of this project was to determine if detached stems of runner and spanish peanut cultivars known to be resistant or susceptible to Sclerotinia blight in the field differed in response to oxalic acid.

Six runner and seven spanish peanut genotypes varying in susceptibility to Sclerotinia blight were used to evaluate responses to oxalic acid (Table 1). The following runner genotypes were included: susceptible cultivars Florunner (Norden et al., 1969) and Okrun (Banks et al., 1989); moderately resistant breeding line ARSOK-R143-3 and cultivars Red River Runner (Melouk et al., 2012) and Tamrun 96 (Smith et al., 1998); and the highly resistant breeding line ARSOK-R35. The following spanish peanut genotypes were used: susceptible cultivars Pronto (Banks and Kirby, 1983) and Spanco (Kirby et al., 1989); moderately resistant breeding lines ARSOK-S140-1OL and ARSOK-S1 (PI 670132; Chamberlin et al., 2014), and cultivars Tamnut OL06 (Baring et al., 2006) and Tamspan 90 (Smith et al., 1991); and the highly resistant breeding line ARSOK-S133-3. The relative resistance levels of the breeding lines were assessed on the basis of a minimum of 3 years of field evaluations.

Plants were grown in the greenhouse at 22 to 32 C in 15-cm pots filled with Metro-Mix 350 (Sun Gro Horticulture, Bellevue, WA). Supplemental lighting was supplied by 300-watt LED lights (Sunshine Systems, Wheeling, IL) set at a 14 hr photophase. After six weeks, the two lowermost lateral stems of each plant were cut near the main stem, and main stems were cut immediately below the third or fourth node. Cuts were made perpendicular to each stem using a razor blade, and detached stems were immediately placed in approximately 6 cm of reverse osmosis water. Stems were held in water 60 to 75 min until stems from all genotypes were prepared.

Detached lateral and main stems were rinsed with sterile reverse osmosis water, blotted dry, and placed individually into 5-ml tubes containing 3.5 ml of 0, 20, or 50 mM oxalate solution. Foam plugs were used to secure each stem so that at least 1.5 cm of the stem base was submersed in the solution. Tubes with detached stems were placed in the dark at room temperature (21 to 24 C). Plant response to oxalate exposure was rated after 12, 18, and 24 hr using a modification of the rating scale of Kolkman and Kelly (2000), where 1 = no wilt symptoms, 2 = one leaf wilted, 3 = two leaves wilted, 4 = one petiole collapsed, 5 = two petioles collapsed, and 6  = branch stem collapsed. The length of the bleached, lesion-like stem discoloration in response to oxalic acid exposure was also measured. Tubes were refilled with additional solution at 12 and 18 hr after immersion as needed.

Experiments evaluating the response of runner genotypes to oxalate solutions were conducted separately from the spanish genotypes. A randomized complete block design was used with three replications (three lateral stems and three main stems) per cultivar x oxalate concentration. Blocks were arranged so that the same replication for each cultivar was placed into the oxalic acid solution at approximately the same time. Test tube racks holding individual blocks were placed on separate trays. The experiment was conducted three times between December 2012 and February 2013. Separate analyses were conducted for each market type and stem type. Hypotheses regarding differences in lateral and main stem response (mean wilt scores and length of discoloration) of the differentially resistant genotypes to oxalate were tested using repeated measures ANOVA with an autoregressive covariance structure (TYPE = AR(1)) in SAS (v. 9.2, SAS Institute, Inc., Cary, NC). When a relevant interaction was significant, the SLICE option of the LSMEANS statement was used to examine simple effects.

No wilting or stem discoloration was observed at any time period for stems of either peanut market type immersed in water (0 mM oxalic acid). Consequently, these data were removed from the analyses.