Laboratory-based assays for screening germplasm for resistance to Sclerotinia blight in peanuts can be conducted year-round, and thus may accelerate progress in breeding for resistant plants. Three previously proposed inoculation methods (using main stems of intact plants, detached main stems, or detached leaflets) were compared on six peanut genotypes known to represent range of resistance to Sclerotinia blight in the field or laboratory. The intact plant and detached main stem assays identified the most resistant and susceptible genotypes, but different results were obtained from either assay with Red River Runner, a cultivar with intermediate resistance to Sclerotinia blight. No differences among genotypes were observed with the detached leaflet assay. The sensitivity ratio was used to compare the three inoculation methods and identified the intact plant assay as the method with the smallest error variance. These results help identify the most efficient method for assaying physiological resistance to Sclerotinia blight in peanut.
Recent progress in sequencing
Field evaluations conducted over multiple years and in multiple locations are the ideal standard for evaluating resistance to diseases including Sclerotinia blight (
Several methods for evaluating peanut genotypes in the laboratory for resistance to Sclerotinia blight have been published. Some assays used main stems and lateral branches of whole, intact plants (
Several assays used excised main and lateral stems (
Detached leaf assays have been used to identify quantitative trait loci for resistance to
Determining the optimum resistance assay among methods which use different approaches can be problematic due to differences in scale and unknown relationships among these scales (
Six runner peanut genotypes were used to compare the three inoculation methods: the resistant cultivar Georgia-03L (
One virulent isolate of
The six runner genotypes were arranged in randomized complete blocks on 35.6 cm × 45.7 cm cafeteria trays. Petri dishes with colony diameters of 4 cm or greater were used to inoculate plants, and one Petri dish was used to inoculate all plants within each block (tray). Plugs were cut from the actively growing margin of the colony with a sterile cork borer (5 mm diam.); plugs cut from sterile PDA were used for the mock-inoculated control plants. A sterile metal spatula was used to transfer each plug from the plate to a capless 0.2 ml PCR tube so that the mycelia-covered side of the plug was on the top of the tube. Desiccation of plugs was prevented by covering each tube with Parafilm and cutting a small incision (ca. 6 mm) in the Parafilm. The first true leaf that did not subtend a vegetative branch from the bottom of the main stem was removed with a razor blade so that approximately 15 cm of the petiole remained attached to main stem. Plants were inoculated by placing the tube over the severed petiole, making sure that the plug was in contact with the freshly cut surface.
Each block/tray was placed in a growth chamber (PGR15; Controlled Environments Ltd., Winnipeg, MB) set at 22 C. High humidity (98%) was maintained by lining the growth chamber shelf with saturated bath towels which were rewetted every three days. Additional humidity was provided by a Cyclone fog machine placed inside the chamber (FutureGarden, North Lindenhurst, NY). Plants received fluorescent light set at 14-hr photophase. The length of the lesion on the main stem was measured daily from three to seven days after inoculation using a digital caliper (Mitutoyo America, Aurora, IL). PCR tubes were removed from the plants if petioles collapsed after infection.
Detached main stems were prepared for inoculation by using a razor to remove the main stem immediately above the first node, cutting perpendicular to the stem. Side branches arising from the second node were also removed, using cuts parallel to the main stem. A sterile foam plug (1.8 cm long × 1.6 cm diam.), which was previously cut lengthwise ca. 1 cm deep, was used to secure the main stem into a 16 × 150 mm test tube (VWR, Radnor, PA) filled with 20 ml of sterile, ½-strength Hoagland’s nutrient solution (Sigma-Aldrich, St. Louis, MO;
A leaf from the next node not subtending a vegetative branch (usually the third node), was inoculated as described for the intact plant assay. Six test tubes containing one main stem of the six cultivars were arranged in a randomized complete block design in a 25 × 10 × 8 cm test tube rack. The six racks (blocks) were placed directly on the saturated bath towels in the growth chamber. A seventh rack contained stems mock-inoculated with PDA. Environmental conditions (temperature, light, humidity) were maintained, and lesion data were collected, as for the intact plant assay. Additional Hoagland’s solution was added to the test tubes as needed, generally two to three days after inoculation.
A modification of the detached leaflet assay of (
Leaflet chamber boxes were placed in a growth chamber set at 22 C without light. Brown and necrotic lesions, rather than water-soaked areas, were measured daily on days 2 to 5 after inoculation. Lesions that were irregular in shape were measured by recording the longest length, following the protocol of
Experiments were conducted between 13 Dec. 2013 and 14 May 2014. One inoculation method was evaluated per week and the three methods were alternated sequentially. The intact plant assay and leaflet assays were conducted five times, and the detached stem assay was evaluated six times. Blocks were removed from the analyses if the plate used to inoculate each block contained a less aggressive culture, i.e. less than 50% of the leaflets or stems were infected.
For the intact plant and detached stem assays, the response variable was stem lesion length. For the leaflet assay, mean lesion lengths from the four leaflets were analyzed. Each inoculation method was analyzed separately. Hypotheses regarding cultivar differences in resistance to Sclerotinia blight were tested using repeated measures ANOVA with an autoregressive covariance structure (TYPE = AR(1)) in PROC MIXED of SAS (SAS, ver. 9.3, SAS Institute, Cary, NC). When a relevant interaction was significant, the SLICE option of the LSMEANS statement was used to examine simple effects. In each set of multiple comparisons, experiment-wise type I error was controlled at α = 0.05 using ADJUST = TUKEY option. Disease progression for each cultivar within each inoculation method was estimated by calculating the area under the disease progress curve (AUDPC;
No symptoms were observed on the non-inoculated control plants for any of the inoculation methods. A total of three blocks each from the detached stem and detached leaflet assays were not included in the analyses due to poor infection rates. Although
Lesion length was significantly affected by cultivar (
Bar graph of mean lesion length (±SE) over time on runner peanut genotypes Georgia‐03L, ARSOK‐R35, Red River Runner, Tamrun 96, Okrun, and Tamrun OL02 inoculated with
Differences in area under the disease progress curves were observed among cultivars (
Line graph of mean lesion length (±SE) over time on runner peanut genotypes Georgia‐03L, ARSOK‐R35, Red River Runner, Tamrun 96, Okrun, and Tamrun OL02 inoculated with
Disease progression in runner genotypes inoculated with
As observed in the intact plant assay, cultivar (
In the detached leaflet assay, lesion length was not affected by cultivar (
Using the sensitivity ratio, the intact plant assay was the most efficient assay of the three inoculation methods. Approximately three ( = [1/0.59]2;
Sensitivity ratio for methods evaluating peanut resistance to Sclerotinia blight.
In this study, the detached leaflet assay was unable to discriminate among the peanut entries even though lesions on all cultivars increased in size over time. Others have found the detached leaflet assay useful for identifying resistant genotypes (
Results from the intact plant and detached stem assays were relatively similar and both reflected resistance levels observed in the field better than the detached leaflet assay. Georgia-03L and ARSOK-R35, peanut entries known to be resistant in the field, had the smallest lesions and slowest disease progression. However, Red River Runner, a cultivar that is intermediate in resistance to Sclerotinia blight in the field, grouped among the resistant cultivars in the intact plant assay, but was more similar to susceptible entries in the detached stem assay. In another pathosystem, different responses were observed in assays using detached plant parts than in assays using intact plants. The hemibiotrophic pathogens
When pairwise comparisons were made among the three methods using the sensitivity ratio, the intact plant assay had the smallest error variance and required the fewest replications than the detached stem and leaflet assays. Although precise times for setup and maintenance were not recorded, the intact plant assay also requires less time than the detached stem assay because test tubes of Hoagland’s solution do not need to be prepared or refilled. Although the intact plant assay requires more space and is destructive, this method may be preferable if seed availability is not strictly limited.
The authors thank Angela Harting for technical assistance. This research was supported by USDA-ARS CRIS Project No. 3072-21220-007-00D. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. USDA is an equal opportunity provider and employer.