Peanut fields (107) were selected without bias as to diseases in Cochran, Crosby, Dawson, Gaines, Hockley, Lubbock, Terry, and Yoakum counties in the Texas Southern High Plains. Fields were identified by driving through multiple (paved and dirt) roads in a county and marking the presence of peanut fields, and by flying over counties and marking the presence of peanut fields. Each field in a county received an identification number; then a series of random numbers were drawn and the subset of fields to be surveyed was selected. Each field was sampled in an identical manner regardless of size. Fields ranged from 12 to 99 ha. Sampling began in the last week of August and continued throughout September of 2003. Fields sampled included Runner, Spanish, Virginia, and Valencia types. Varieties in some fields could be identified and included Flavorunner 458 (most frequent), Florunner, NC7, Spanco, Tamspan 90, Valencia 101, VC2, and Viraguard. Maturity at the time of sampling ranged from only small, immature pods all the way to mature, ready for harvest pods.

Four transects were made across each field, with 10 locations sampled per transect. The sample location was selected by pacing even distances along the transect. At each sample location, approximately 1m of plants in a row were pulled up and all the discolored pods were collected. The soil underneath the plants was searched for any pods that had been dislodged from the plants. Pods with any discoloration in the 40 sample locations of a field were placed in a paper bag and refrigerated until the sample was processed. Processing (conducted within 1 wk of sampling) consisted of examining each pod for necrosis, weighing all necrotic pods, and selecting up to 16 pods for assay. In some cases, there were less than 16 pods from a field with necrosis. Pods selected for assays represented the range of symptoms expressed for a field (black, brown, white, rotted, and sunken type lesions) were washed, dried, and two small pieces (approximately 0.5 to 1 cm) were plated out on water agar and a Pythium selective medium (Lewis and Filonow, 1990). Plates were incubated at room temperature (21 to 23C) and monitored daily for mycelial growth, for seven days. Mycelia growing from these pod pieces were transferred to potato dextrose agar and identified to the genus level. Twenty-six of 85 isolates identified as belonging to the genus Pythium (no more than 4 isolates from a given field) were sub-cultured on corn meal agar (CMA) + 10 µg rifampicin/ml, and identified to species based on the descriptions from Plaats-Niterink (1981). There were 50 unidentified Pythium isolates that did not produce any structures that were useful for taxonomic purposes and nine identified as the nonpathogenic P. oligandrum. Pythium isolates that were selected for species identification represented all the fields where isolates were found, that did not quickly become contaminated by bacteria. The pathogens present within each field were divided into four classes: 1. neither Rhizoctonia nor Pythium present; 2. Rhizoctonia present without Pythium; 3. Pythium present without Rhizoctonia; or 4. Rhizoctonia and Pythium both present.

Isolates of Pythium spp. were maintained on CMA + 10 µg rifampicin/ml. One cm² plugs of mycelium were placed on CMA plates containing the following concentrations of mefenoxam : 0, 0.0096, 0.096, 0.96, and 9.6 µl/ml of medium. The source of mefenoxam was Ridomil Gold EC (47.6% ai, Syngenta Crop Protection, Greensboro, NC). The size of the colony was measured approximately 40 hrs later. Percent inhibition was expressed as a percentage of the colony diameter in the absence of mefenoxam (Ridomil Gold EC). Plates were grown on a laboratory bench at room temperature which varied from 18–23 C. The plates were not kept in the dark, since mefenoxam is resistant to sunlight (Sukul and Spiteller, 2000). Each isolate was tested on a given date with five concentrations of fungicide (including the 0 concentration) and three replications per isolate/concentration, arranged in a randomized complete block design. Each isolate was tested at least two times. Isolates were run in subgroups from November 03 through February 04.

Azoxystrobin was tested in a fashion similar to that of mefenoxam, except that higher concentrations were used (10 µl, 100 µl, 1,000 µl, and 4,000 µl/ml). The source of azoxystrobin was Abound FL (22.9% ai, Syngenta Crop Protection, Greensboro, NC).

The concentration that effectively reduced colony diameter by 50% (EC₅₀) was estimated for all isolates. A number of different models were tested to estimate EC₅₀. However, the natural log transformation of both fungicide concentration and percentage inhibition of fungal growth provided the most consistent fit for data.

Both Pythium and Rhizoctonia spp. were isolated from rotted pods in similar percentages. The classes representing Rhizoctonia alone, Pythium alone, and Rhizoctonia and Pythium together were found in 19, 23, and 16% of the fields surveyed, respectively. In 42% of the fields, neither Rhizoctonia nor Pythium were found. In those cases, either no pod rot was identified, or only superficial necrosis was present which is typical of the black hull fungus Thielaviopsis basicola. Thielaviopsis basicola was not isolated from the rotted pods, but would have required different selective media. Sclerotium rolfsii was isolated from pods in 4% of the fields. Fusarium was isolated from pods in all fields. The overall condition of the pods was good, but when there was substantial rot, then typically either Rhizoctonia or Pythium were isolated. The 2003 summer was dry, particularly during the time of pod formation, so the environment was not conducive for pod rot. All of the surveyed peanut fields were irrigated to some degree. The frequency of Pythium and Rhizoctonia spp. isolated from rotted pods in a survey can change from year to year (Hollowell et al., 1998). In North Carolina, both Pythium spp. and Rhizoctonia spp. were the two pathogens most frequently isolated in a survey of peanut fields, but Rhizoctonia spp. predominated in one year, and Pythium spp. predominated in two years of a three year survey (Hollowell et al., 1998). It is possible that the pod rot percentage in west Texas will increase in years where the environment is more conducive for disease, and that the relative percentages of disease due to Pythium and Rhizoctonia may change. However, it is clear that this region has pod rot associated with both Rhizoctonia spp. and Pythium spp., although pod rot overall during a dry summer is relatively low.

Pythium oligandrum was frequently isolated from rotted peanut pods, but is not considered a plant pathogen (Benhamou, et al., 1999). Twenty-six Pythium isolates were identified to the species level, not including P. oligandrum. The species of Pythium most frequently isolated in west Texas were P. ultimum (9 isolates), P. myriotylum (10 isolates), and P. irregulare (6 isolates). One isolate was identified tentatively as P. heterothallicum. In a North Carolina survey, P. irregulare and P. spinosum were the species most frequently identified (Hollowell et al., 1998). Other species identified in North Carolina on peanuts were P. myriotylum, P. dissotocum, P. ultimum, P. vexans, P. dimorphum, and P. paroecandrum. The “classic” Pythium species on peanut is considered to be P. myriotylum (Porter et al., 1982).

The pathogenic Pythium isolates (those associated with pod rot, but not including P. oligandrum) had EC₅₀ which ranged from 0.001–0.270 µl ai mefenoxam/ml. Only one isolate was identified as P. heterothallicum, and it was quite sensitive to mefenoxam (EC₅₀  =  0.011 µl ai of mefenoxam/ml). Five isolates of P. irregulare that were obtained from three fields had EC₅₀ that ranged from 0.049–0.173 µl ai of mefenoxam/ml, with an average EC₅₀ of 0.119 µl/ml. Seven isolates of P. myriotylum that were obtained from five fields, had EC₅₀ that ranged from 0.012–0.163 µl ai of mefenoxam/ml and averaged 0.074 µl/ml. Three isolates of P. oligandrum from three fields had EC₅₀ that ranged from 0.008–0.099 µl ai of mefenoxam/ml, and averaged 0.042 µl/ml. Two isolates of P. ultimum from two fields had EC₅₀ of 0.005 and 0.006 µl ai of mefenoxam/ml. Within the same field, isolates of Pythium could have very similar EC₅₀ (G31A, B, and C ranged from 0.001 to 0.008 µl ai of mefenoxam/ml), or very different EC₅₀ (L3A and L3C were 0.033 and 0.270 µl ai of mefenoxam/ml, respectively, Table 1). None of the isolates would be considered resistant to mefenoxam. Mefenoxam resistance has been defined as EC₅₀>100 µl/ml (Moorman et al., 2002; Taylor et al., 2002) and the least sensitive isolate (L3C) had an EC₅₀ of 0.27 µl/ml. The EC₅₀ of P. ultimum var. sporangiiferum from sugar beet ranged from 0.050–1.3 µl/ml (Branter and Windels, 1998). The EC₅₀ of P. irregulare obtained from apple roots was much higher (0.200–0.790 µl/ml) than from P. ultimum (0.010–0.170 µl/ml) (Mazzola et al., 2002). Pythium irregulare that was obtained from plants taken from commercial greenhouses (a mixture of plant species) was frequently (21 of 57 isolates) resistant to mefenoxam, while no resistant P. myriotylum, and only 1 of 13 resistant P. ultimum isolates were found (Moorman et al., 2002). The sensitivity of mefenoxam in the Pythium isolates examined could be attributed to their lack of exposure to this fungicide. Previous exposure was probably limited to cotton seed treatments, which would have placed minimal selection pressure on Pythium populations.

Sensitivity of Pythium spp. to azoxystrobin was much lower than to mefenoxam. However, some fungi use an alternative respiratory pathway that can interfere with the activity of azoxystrobin in a petri dish assay (Stevenson et al., 2004). This means that these results cannot be compared directly to assays where salicylhydroxamic acid is used to inhibit Pythium from using an alternative respiratory pathway, though no published in vitro studies with azoxystrobin and Pythium were found. Sensitivity of different Pythium isolates to azoxystrobin in this study ranged from 1 to 103 µl ai of azoxystrobin/ml. Pythium heterothallicum had an EC₅₀ of 12 µl ai of azoxystrobin/ml. The EC₅₀ for P. irregulare ranged from 39–103 µl ai of azoxystrobin/ml and averaged 62. The EC₅₀ for P. myriotylum ranged from 5–25 µl ai of azoxystrobin/ml and averaged 14.7. The EC₅₀ for P. oligandrum ranged from 10–42 µl ai of azoxystrobin/ml and averaged 23. The EC₅₀ for P. ultimum was 14 for one isolate and no acceptable model could be fitted to the second isolate (Y6C). Isolate Y6C had a slight decrease in growth from 10 µl to 1,000 µl azoxystrobin/ml and no growth occurred at 4,000 µl azoxystrobin/ml. All the isolates of P. irregulare had less sensitivity to azoxystrobin than any of the other pathogenic Pythium isolates that were identified to species. Of the isolates that were not identified to species, the most sensitive P. irregulare isolate (G27A) was less sensitive than 93% of the remaining isolates.