Diagnosis of
Spotted wilt is a systemic disease in peanut (
Current standard determination of TSWV intensity in the field relies on a visual disease rating that represents both disease incidence and severity (
The efficacy of ELISA and RT-PCR for detection of TSWV was compared in two separate experiments. In both experiments, peanut root crown samples were used for analysis based on reports of higher incidence of TSWV in root crown compared to foliar tissue in different peanut cultivars (
Peanut root samples were collected at the Bolton Farm (Terrell County, GA). The crop was planted on 19 April 2006. In 2007, the crop was planted on two dates: 20 April and 22 May. Three peanut cultivars, Georgia Green (
For RT-PCR analysis, root samples (∼100 mg) were ground to a fine powder in liquid nitrogen using a pre-chilled mortar and pestle. Total RNA was extracted and DNased using RNeasy Plant Mini Kit (Qiagen, Valencia, CA) according to the manufacturer's instruction. RNA concentrations were measured using a spectrophotometer (Thermo Fisher, Waltham, MA) and the quality of RNA was determined by RNA gel electrophoresis. After RNA quality analysis was performed, first strand cDNA was synthesized using 0.75 µg total RNA. Total RNA was denatured at 95 C for 5 minutes in the presence of TSWV 722 and 723 primers (50 µM each) (
Peanut (cv. Georgia Green) root samples were collected in a grower field in Bulloch County, GA. The peanut crop was planted on the week of 22 May 2006. In this experiment, a long-term storage technique was utilized in which roots were cut horizontally into slices and allowed to dry in weigh boats at room temperature for two to three days. Dried root slices were placed into 15 ml storage tubes preloaded with Drierite Absorbent (Fisher, Pittsburg, PA) and stored at −20 C. DAS-ELISA was performed as described above. RT-PCR analysis was identical as that used in Experiment 1 except using the dried samples for analysis.
Comparison of TSWV infection rates as detected by RT-PCR and ELISA were analyzed using PROC FREQ procedure in SAS version 9.1 (SAS Institute, Cary, North Carolina) with the CHISQ TESTP option in the Model statement utilizing chi-square goodness of fit test. The ELISA observed results were used as the standard for expected values.
In 2006 at the Bolton Farm, 11 out of 24 total samples (45.8%) tested positive for TSWV by RT-PCR and 9 (37.5%) by ELISA (
Comparison of RT-PCR and ELISA assays for TSWV detection in peanut root tissues. Results are shown for total samples collected in Experiment 1, Bolton 2006 (E = early planting) and Bolton 2007 (E and N = normal planting); Experiment 2, Bulloch 2006 (N) and all samples combined. Experiment 1 used fresh root tissue and experiment 2 used dried, frozen root tissue.
From both planting dates at Bolton in 2007, a total of 48 samples were collected and tested. A total of 5 samples (10.4%) tested TSWV positive by RT-PCR and 4 (8.3%) tested positive by ELISA (
In 2006 at the Bulloch County, GA site, a total of 52 samples were tested. Forty-one samples (78.8%) tested TSWV positive by RT-PCR and 37 (71.2%) tested positive by ELISA (
ELISA has become the standard assay for TSWV diagnosis in peanut (
The results reported here are similar to previous reports comparing ELISA and RT-PCR for detection of
The quality of RNA was evaluated for the extraction from fresh root tissues (experiment 1) and dried, frozen root tissues (experiment 2). The result indicates that both types of tissue can be efficiently processed for both ELISA and RT-PCR assays without loss to the relative quality of viral proteins and viral RNA, respectively. The ability of dried, frozen roots to maintain testing consistency would eliminate the need to specially treat samples prior to processing and reduce the need for −80 C freezer space.
Serological kits are commercially available and fairly economical. Two formats are currently available, ImmunoStrips or DAS-ELISA (Agdia). The ImmunoStrips are easy to perform, can be completed in the field within minutes, do not require lab equipment, and the cost per sample is reasonable. DAS-ELISA requires minimal laboratory facilities and an operator with laboratory experience, but is more economical for large number of samples. On the other hand the RT-PCR assay is estimated to cost over 20 times more than ELISA and requires expensive equipment and an operator with a high level of laboratory experience. The advantage of this assay is that the purified RNA samples can be further processed using real-time RT-PCR to actually quantify viral RNA in specific tissues. However, because of the relative ease and inexpensive cost of ELISA versus RT-PCR, and the results of the current study showing no difference in detection of TSWV, ELISA remains the best choice for routine evaluation of TSWV infection in peanut.
We thank Larry Powell and Kathy Gray for technical assistance in the field and in the laboratory. We thank Ben Mullinix for statistical analysis and consultation.
1Biochemist, Plant Physiologist, and Research Agronomist, USDA-ARS, National Peanut Research Laboratory, PO Box 509, 1011 Forrester Dr. SE, Dawson, GA 39842. 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 USDA.