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Fungicide resistance profiles and virulence of Stagonosporopsis species isolates from watermelon and melon in eastern China
M. Newark1, N. Dufault1, P. Li3, X. Yang3, and M.Paret2; 1University of Florida, Gainesville, FL; 2 University of Florida, Quincy, FL; 3Jiangsu Academy of Agricultural Sciences Nanjing, China
Introduction:
Nearly one-quarter of all watermelon seeds planted in the U.S. are imported from China. Thus, there is a risk that novel isolates of watermelon pathogens, such as Stagonosporopsis spp., could be introduced into U.S. production systems. Assessing various disease factors (e.g. fungicide resistance and virulence) in Chinese production systems is critical to determining the likelihood that novel pathogen populations can be spread across international borders. It was hypothesized that fungicide resistant phenotypes in Chinese watermelon production areas as well as pathogen virulence would be similar to U.S. phenotypes. The objectives of this study were to phenotype isolates from major Chinese fresh-market growing regions for fungicide resistance and assess pathogenicity on a US variety.
Table 1: A list of the fungicide products examined in this study with their respective FRAC codes ( and amended media concentration.
Trade Name
Active Ingredient
FRAC
EC50 Value
Topsin® M
thiophanate-methyl 1
100mg/L1
Folicur®
tebuconazole 3
0.128mg/L2
Inspire®
difenoconazole
0.032mg/L2
Endura®
boscalid 7
0.034mg/L2
Quadris®
azoxystrobin 11
0.096mg/L3
1 EC50 (Effective Concentration that kills 50% of the pathogen) are based on values from Keinath and Zitter 1998
2 EC50 (Effective Concentration that kills 50% of the pathogen) are based on values from Thomas and Stevenson 2011
3 EC50 (Effective Concentration that kills 50% of the pathogen) are based on values from Keinath 2009 A B D C
Fig. 4: Breakdown of phenotypes based on mean percent inhibition. Sensitive phenotypes were characterized by a 40% or greater inhibition compared to the control. Reduced sensitivity was characterized by a 10-39% inhibition. Resistant phenotypes exhibited a 10% or less inhibition compared to the appropriate control. Numbers inside the bars represent the total number of isolates observed in each category.
*Azoxystrobin treatment was amended with 100mg/L SHAM and compared to SHAM amended control media.
Results:
A majority (> 85%) of the isolates were resistant to boscalid and thiophanate-M (Fig. 4)
A majority (> 85%) of the isolates were sensitive to difenoconazole and azoxystrobin (Fig. 4)
Tebuconazole had varying proportion in each sensitivity category (Fig. 4)
All isolates infected ‘sugar baby’ with a majority (67%) having a moderately aggressive phenotype (Fig 3)
Fig. 1: Regions marked in green are major fresh market production regions of China that were sampled for Stagonosporopsis spp. The region marked in blue is the major seed-production region.
Fig. 2: An example of the detached leaf virulence assay using the watermelon variety ‘Sugar Baby’. A.) Weakly pathogenic isolate causing 20% contiguous necrosis. B) A moderately aggressive isolates causing 60% contiguous necrosis. C) Highly aggressive isolate causing 90% contiguous necrosis. D) Actively growing mycelium and 6mm agar plugs of Stagonosporopsis citrulli.
Materials and Methods:
Isolate Collection
69 total isolates
56 from melon (Cucumis melo)
13 from watermelon (Citrullus lanatus)
Collections from in 4 Chinese provinces (Fig. 1)
Fungicide resistance
Pure cultures transferred to fungicide-amended media (Table 1)
Linear growth assessed using 2 radii measurements per plate
Measured at 48, 72, and 96 hours post-inoculation
Cultures stored at room temperature (25°C)
Percent inhibition was calculated with proper controls
Repeated in triplicate
Pathogenicity and aggressiveness
Modified from Gusmini et al. 2005
2-4 week old watermelon (Citrullus lanatus var: ‘sugar baby’) leaves
Actively growing mycelium plugs transferred to leaves (Fig. 2)
0-10 rating based on percent contiguous necrosis at 120 hours post-inoculation (Fig. 2)
Discussion:
In this study, we observed that a significant proportion of the Chinese isolates tested had a resistant phenotype to boscalid and thiophanate-methyl. Both of the aforementioned phenotypes have been observed in the U.S. (Keinath and Zitter 1998, Stevenson et al. 2012). However, the sensitivity of the isolates to azoxystrobin is different from what has been previously reported in the U.S. (Stevenson et al. 2004). While there are similar trends between the 2 countries, there also appears to be some differences based on phenotypes.
Future work:
We plan to examine phenotypic patterns further by comparing these results with those of an isolate collection from Florida. Then the genetic diversity of both isolate groups will be assessed using microsatellite markers developed at the University of Georgia.
Fig. 3: The number of the various isolates that were classified into the 3 aggressiveness phenotypes of weakly, moderately and highly aggressive. Weakly aggressive isolates had an average rating of 1-3.9, moderately aggressive isolates had an average rating of between 4 and 7 and highly aggressive isolates had average ratings of above 7.1 based on the scale by Gusmini et al All isolates examined were pathogenic on the leaves of the watermelon variety ‘sugar baby’.
Selected references
Gusmini, G., Wehner, T, and G. Holmes. Disease Assessment Scales for Seedling Screening and Detached Leaf Assay for Gummy Stem Blight in Watermelon. Cucurbit Genetics Cooperative Report 25: (2002).
Stevenson, K. L., Langston, D. B., and Seebold, K. W Resistance to Azoxystrobin in Gummy Stem Blight Pathogen Documented in Georgia. Plant Health Progress.
Thomas, A., Langston, D. B., and Stevenson, K. L Baseline Sensitivity and Cross-Resistance to Succinate-Dehydrogenase Inhibiting and Demethylation-Inhibiting Fungicides in Didymella bryoniae. Plant Disease 96(7): S1 S2
Supplemental figures S1 and S2
S1-An electronic copy of this poster
S2-My Linkedin profile.