One of the most important diseases in peanut is stem rot also known as southern blight, caused by
Knowledge about pathogen diversity is an important component for developing useful integrated management strategies, but so is monitoring the pathogen phenotype fungicide sensitivity (
Based on the
During the 2013 growing season, 15
Morphological characteristics of each isolate were assessed using a media growth assay. Actively growing mycelial plugs of 6 mm diameter were harvested from the edge of the growing colony, transferred to the center of a PDA petri plate and incubated for 2 days at 26 ± 2°C in complete darkness. Digital images of the plates with a scale were captured for intrinsic growth analysis. The total area of the colony growth was measured using the digital image measurement software KLONK (KLONK Image Measurement, Denmark, Copenhagen). The area of the inoculum plug was subtracted from the total colony area to find colony growth after inoculation. Petri dishes were placed back in the growth chamber (26 ± 2°C; complete darkness) for an additional 14 days to assess sclerotial characteristics. Again, digital images of the plates with a scale were captured for analysis (
Isolates were tested for mycelial compatibility by placing 2 agar plugs of the same isolate and 1 agar plug of a different isolate onto PDA (20 g PDA, Difco™ in 1 L of distilled water) plates equidistant (5 - 6 cm) from each other (
One representative isolate from each MCG identified within the peanut isolates was also compared to a selection of the established MCGs reported by Xie et al. (2014) which included
A mycelial growth assay was used to test for isolates sensitivities to the commercial fungicides Abound® (azoxystrobin 22.9%, Syngenta® U.S., Wilmington, DE), Proline® (prothioconazole 41%, Bayer® U.S., Research Triangle Park, NC), TebuStar® 3.6 L (tebuconazole 38.7%, Agristar® US, Ankeny, IA), Convoy® (flutolanil 40%, Nichino America, Inc. U.S., Wilmington, DE ), FontelisTM (penthiopyrad 20.4%, DuPont® U.S., Wilmington, DE), and Solatenol® (benzovindiflupyr, Syngenta® U.S., Wilmington, DE). The commercial fungicide products were diluted in distilled water to amend QPDA with the concentrations of 5, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, and 0.0001 μg of the fungicide's active ingredient per ml of medium. Fungicide solutions were added to sterile QPDA that had been cooled to 50°C using a water bath. Solutions were regularly (∼ every 1 to 2 min) swirled for homogenous mixing. For the azoxystrobin sensitivity test, a stock solution of salicylhydroxamic acid (SHAM) (Sigma Chemical Co., St. Louis, MO) with methanol was prepared by adding 100 mg of SHAM to 1 ml of methanol in a microcentrifuge tube and warming in a water bath at 50°C for 1-2 minutes to completely dissolve SHAM in methanol. 70 μl of this SHAM stock solution (7 mg) was added to every 100 ml of autoclaved liquid QPDA media that was cooled in a water bath to 60°C (
Mycelial inhibition was calculated by transferring an actively growing mycelial plug (6 mm diameter) of
Isolates were exposed to these 10 concentrations for two experimental repeats and analyzed using nonlinear regression as described below. The resulting regression model for each isolate was used to estimate the concentrations causing percentage inhibition of 5%, 20%, 35%, 50%, 65%, 80%, and 95% which were used in the final three repeats to estimate EC50 values. The concentrations of fungicide used for the final three repeats are listed in
Concentration of the commercial fungicide's active ingredient (a.i.) used for the final three runs of fungicide sensitivity assay. These concentrations were the estimated values that caused 5, 20, 35, 50, 65, 80, 95 percent growth inhibition of the isolates based on 2 previous runs using standard concentrations.
The growth inhibition at each fungicide concentration was calculated using the following equation:
All phenotypic data analyses were completed using SAS (version 9.4; SAS Institute Inc, Cary, NC, USA). Phenotypic data were analyzed as a completely randomized design using the PROC GLM command to conduct an analysis of variance (ANOVA) with
Isolates differed in their average colony growth on PDA two days after inoculation and sclerotial traits, including color, size and total number produced 14 days after inoculation (
Morphological characteristics of
A total of seven MCGs were identified among the 15 isolates of
Simpson's diversity index indicated the probability of randomly selecting two isolates of the same MCG in this study is approximately 0.28 and 0.09 when studies were combined with published data by Xie et al. 2014 (
Quantitative diversity indices for phenotypic results based on mycelial compatibility groupings (MCGs) observed in the current and previous studies.
Ranges of EC50, mean EC50 and overall inhibition response of the
The effective concentrations that limit the growth of
The average response of the
The distribution of EC50 values for the 15 isolates was not significantly different (p > 0.05) from a normal distribution for all the isolates tested except tebuconazole (
A significant correlation was observed between the EC50 values of flutolanil and penthiopyrad (r = 0.69, p-value = 0.004). No significant correlations (p-value > 0.100) were observed for the rest of the fungicide products tested; however, a positive correlation coefficient was noted between flutolanil and solatenol (r = 0.41, p-value = 0.134).
Phenotypic traits, such as morphological characteristics and MCGs, have been used to assess the diversity of
Despite the high level of diversity indicated by MCGs, other morphological characteristics, such as number, size and color of sclerotia, remained more consistent with each phenotypic grouping. Sclerotia that were dark brown tended to have larger and less numerous sclerotia produced on media when compared to light brown sclerotia. Also, all the sclerotia in the largest MCG, group 1, were dark brown compared with light brown sclerotia for the second largest MCG, group 2. These results are different from Xie et al. (2014) and
Fungicide sensitivity is another important phenotypic trait that can be used to better understand fungal populations exposed to selection pressures (
The efficacy of various fungicidal compounds on the inhibition of
Similar conclusions can be made about tebuconazole, in that the mean EC50 values reported in this monitoring study were generally lower than those observed by
Correlation analysis is commonly used to identify the possibility of cross-resistance between fungicide compounds. Positive correlation values were among between the SDHI compounds of flutolanil, penthiopyrad and solatenol, in which the greatest correlations were seen between flutolanil and the other two compounds. This is concerning since many new products being released for peanut disease control often contain a SDHI product. This means that there could be an increased selection pressure put on
A unique outcome observed while conducting the fungicide sensitivity assay was that sub-lethal concentrations of flutolanil, as well as four other fungicide products, produce apparent increases in mycelial growth compared to the non-amended checks. This process, called hormesis, has been documented with other fungicidal compounds, primarily pertaining to oomycete control (
Despite the high levels of phenotypic variation noticed in MCGs of
This research was supported by grants from the Florida Peanut Producers Association and the National Peanut Board as well as general support from the University of Florida Department of Plant Pathology. We want to acknowledge Kris Beckham for her assistance with instruction of basic laboratory techniques and maintenance of the
First, second, third and fourth author: Graduate student, Graduate student, Post-doctoral Scholar and Assistant Professor. Department of Plant Pathology, University of Florida, Gainesville, Florida, 32611-0680.