Athelia rolfsii (Curzi) C.C. Tu & Kimbr. (= Sclerotium rolfsii Sacc.; Xu et al., 2010) is a cosmopolitan soilborne pathogen distributed primarily in warmer areas such as the southern U.S. (Aycock, 1966; Punja and Rahe, 2001). In addition to infecting more than 500 plant species including tomato, carrot, beet, sweet potatoes, and melon (Aycock, 1966; Jenkins and Averre, 1986), A. rolfsii is considered to be one of the most economically damaging pathogens of peanut in the U.S. (Backman and Brenneman, 1997). In Georgia's 2015 season alone, the value of control costs and yield losses was estimated to be $59.7 million (Kemerait, 2015). Substantial yield losses to A. rolfsii are also experienced in peanut-producing regions of Africa (Subrahmanyam et al., 1997; Cilliers et al., 2000), Asia (Mayee and Datar, 1988), the Middle East (Grinstein et al., 1979), and South America (Marinelli et al., 1998). Cultivars with high resistance to A. rolfsii such as Bailey and Georgia-12Y are available, but most commercial cultivars obtainable today are less resistant (Kemerait et al., 2018).

Despite the need for more resistant cultivars, few laboratory assays have been developed for evaluating resistance to A. rolfsii in peanut and other crops. Akem and Dashiell (1991) found differences in resistance among soybean genotypes by inoculating detached shoots from 6-week-old plants with mycelial plugs. In addition to measuring lesion length over time, they also counted the number of sclerotia and percentage of germinable sclerotia produced by each soybean genotype. Pratt and Rowe (2002) were able to discriminate between resistant and susceptible genotypes of alfalfa and obtained similar results from both excised leaflets and whole plants inoculated with mycelial plugs of A. rolfsii. A greenhouse assay for evaluating resistance in Jerusalem artichoke was developed in Thailand using inoculum produced in sorghum broth and seeds (Sennoi et al., 2010). More recently, Xie et al. (2014) used sclerotia to inoculate tomato, pepper, and peanut (cv. Georgia Green) with 19 isolates of A. rolfsii. However, tomato plants were more susceptible than pepper and peanut plants at the same age (8 weeks after germination), and the assay was adjusted so that tomatoes were inoculated using four sclerotia instead of the ten used for pepper and peanut (Xie et al., 2014). Bera et al. (2016) used sorghum seed colonized by A. rolfsii to screen 25 accessions of wild species in pots for resistance. Using this approach, two resistant accessions with less than 13 and 14% mortality (Arachis pusilla DGR 12047 and Ar. appresipila ICG 8945, respectively), and two moderately resistant accessions with 25 and 26% mortality (Ar. monticola ICG 8135 and Ar. duranensis ICG 8204, respectively), were identified (Bera et al., 2016). Finally, the most extensive work on developing a laboratory assay to evaluate A. rolfsii resistance in peanut was conducted by Shokes and colleagues (1996). Five inoculation methods, varying from mycelium slurries to colonized oat grains, were used to inoculate 7-week-old Florunner plants in the greenhouse and field. The most effective inocula were germinating sclerotia on agar disks and clothespins impregnated with mycelium and potato dextrose broth (PDB)(Shokes et al., 1996). The agar disk technique was later used to screen 11 genotypes in two locations for three years to successfully identify resistant germplasm (Shokes et al., 1998).

Multi-year, replicated field trials are generally the best way to evaluate disease resistance (Brenneman et al., 1990, 2014), but field trials are labor-, time-, and space-intensive. Unfavorable environmental conditions and uneven distribution of inoculum (Shew et al., 1984) may also negatively affect field results. In addition, it may be difficult to procure enough seed of non-standard peanut genotypes such as wild species for replicated field plots (Bera et al., 2016). In contrast, laboratory-based resistance assays cannot accurately reflect field conditions but may be useful for pre-screening material or for screening entries for which seed is limited. Because one of our long-term goals is to evaluate PI accessions of wild Arachis species for disease resistance, our objective was to develop an assay, first using cultivated genotypes, for screening intact peanut plants for resistance to Athelia rolfsii.

A total of thirteen peanut genotypes were used in the assays (Table 1). Cultivars included the resistant Georgia-03L (Branch, 2004; Woodward et al., 2008; Chapin et al., 2010; Culbreath et al., 2010) and Georgia-12Y (Branch, 2013; Branch and Brenneman, 2015); moderately resistant Florida-07 (Gorbet and Tillman, 2009), and Georgia-07W (Branch and Brenneman, 2008), and Tamrun OL02 (Simpson et al., 2006); and moderately susceptible FloRun '107' (Tillman and Gorbet, 2015) and Georgia-06G (Branch, 2007). Cultivars Georgia-03L, Georgia-12Y, Florida-07, Georgia-07W, FloRun '107', and Georgia-06G are rated 10, 10, 15, 15, 20, and 20, respectively, by Peanut Rx (Kemerait et al., 2018). Susceptible controls also included U.S. mini-core accessions CC038 (CC, core collection number; PI 493581), CC041 (PI 493631), and CC787 (PI 429420); these accessions appeared particularly susceptible to A. rolfsii over multiple years of field experiments (Bennett, unpublished data). Lastly, the following three mini-core accessions with unknown susceptibilities to A. rolfsii were also included: CC068 (PI 493880), CC384 (PI 155107), and CC650 (PI 478819). CC068 and CC650 are resistant to Sclerotinia minor in the field (Bennett et al., 2018).

Plants were grown in a greenhouse maintained at 22 to 32°C. Three seeds of each entry were planted in 11-cm-diam. pots filled with Metro-Mix 350 (Sun Gro Horticulture). Two weeks after planting, all but one seedling was culled. Plants were fertilized at 5 and 7 weeks after planting with 30 mL of 0.2% ammonium nitrate. At 8 weeks after planting, plants were inoculated with a virulent isolate of A. rolfsii (Ar-15-1A), which was originally collected in 2015 from a diseased plant in Fort Cobb, Oklahoma. The fungus was grown on 90-cm Petri plates filled with 15 mL of full-strength potato dextrose agar dispensed with a peristaltic pump (UniSpense, Wheaton). Cultures were incubated in the dark at 28.5°C (near optimum temperatures for A. rolfsii; Aycock, 1966) and 2-d-old cultures were used for inoculations.

New or old flowers present on the plants were removed prior to inoculation to reduce sporulation of other fungi. Plants were inoculated at the base of a side stem arising from the second node. A sterile, half-circle of a cotton cosmetic pad was soaked with sterile water. A 9-mm-diam. agar plug was taken from the margin of a colony and placed mycelium-side up on center of the cotton pad. The pad was placed below the side branch and wrapped loosely but securely, so that the mycelium and agar plug were in direct contact with the stem. Inoculated plants were misted with reverse osmosis water, were placed on bath towels saturated with water, and covered with large clear plastic storage bins to maintain high humidity. Pots were arranged in a randomized complete block design with three replications in a growth chamber set at 28.5°C and 14-h photophase with fluorescent lights. Towels were wetted daily. To monitor temperature and relative humidity, two HOBO sensors were placed inside the growth chamber. Data were collected at 4, 7, 10, and 13 days after inoculation. Lesion length was measured on the main stem, as well as on the side branches arising from the first node, using digital calipers (Mitutoyo America). In preliminary experiments, mycelial growth appeared to be greater in susceptible genotypes, so length of mycelium on the main stem and first node side stems was also measured. If the plant or branch died before the end of the experiment, the measurements were recorded as missing data. The experiment (trial) was conducted four times between January and March 2016.

All analyses were conducted in SAS Version 9.4 (SAS Institute, Cary, NC). For analyses of the side branch data, the mean of the two branches were used. Differences among entries in disease progression (lesion length) and mycelial growth were analyzed using repeated measures ANOVA in PROC MIXED with TOEP covariance structure. Trial and block(trial) were used as random variables in the model. The SLICE option was used to examine differences among and within entries at 4, 7, 10, and 13 days after inoculation. In addition, the area under the disease progress curve (AUDPC) for lesion length and mycelial growth was estimated using the formula of Shaner and Finney (1977). To examine consistency among trials in addition to differences among entries, AUDPC means were compared with PROC GLIMMIX using a split-plot design with trial as the whole plot and entry as the subplot. The SLICE option was used to check for differences among trials and entries. All pairwise comparisons were adjusted for Type I error with the ADJUST = SIMULATE option at 𝜶 = 0.05. Correlation analysis between lesion and mycelium lengths were conducted using PROC CORR.

A significant interaction was present between entry and time (P < 0.01) in all repeated measures analyses of main and side stems, indicating that differences among peanut entries in lesion length and mycelial growth were dependent on the d the measurements were taken. In addition, there were no significant differences among entries at days 4 or 7 after inoculation, regardless of stem type or response variable (P = 0.11 to 1) when interaction was examined by d (data not shown).