Development, Validation, and Utility of Species-Specific Diagnostic Markers for Detection of Peronospora belbahrii.

Peronospora belbahrii is an oomycete and the cause of basil downy mildew, one of the most destructive diseases affecting basil production worldwide. Disease management is challenging due to wind-dispersed sporangia and contaminated seed; therefore, identifying P. belbahrii in seed lots before sale or planting or in the field before symptoms develop could allow for timely deployment of disease management strategies. In this study, a draft genome assembly and next-generation sequencing reads for P. belbahrii, as well as publicly available DNA-seq and RNA-seq reads of several other downy mildew pathogens, were incorporated into a bioinformatics pipeline to predict P. belbahrii-specific diagnostic markers. The specificity of each candidate marker was validated against a diverse DNA collection of P. belbahrii, host tissue, and related oomycetes using PCR. Two species-specific markers were identified and used as templates to develop a highly sensitive probe-based real-time quantitative PCR (qPCR) assay that could detect P. belbahrii in leaf tissue and seed samples. Both markers were capable of reliably detecting as low as 500 fg/µl of P. belbahrii genomic DNA and as few as 10 sporangia. The qPCR assay was then validated with seed samples collected from a basil cultivar experiment. In total, 48 seed samples were collected and tested; P. belbahrii was detected in samples of all cultivars at estimated concentrations of 600 fg/µl up to 250 pg/µl and at as few as 10 sporangia up to >1,000 sporangia. The markers and assays are valuable for diagnostics and identifying P. belbahrii-contaminated seed lots to mitigate the effects of future basil downy mildew epidemics.

Fungicide Resistance in Venturia effusa, Cause of Pecan Scab: Current Status and Practical Implications.

Pecan scab, caused by Venturia effusa, is the most economically damaging disease of pecan in the southeastern United States, and annual epidemics are most effectively managed through multiple fungicide applications. The fungicide applications can be the single greatest operating cost for commercial growers and the return on that investment is impacted by fungicide resistance. V. effusa produces multiple generations of conidia per season, exhibits substantial genetic diversity, overwinters as stromata in the tree, and is under immense selection from the applied fungicides, all of which lead to a high risk for developing fungicide resistance. Since the mid-1970s, resistance or reduced sensitivity has been observed in isolates of V. effusa to the methyl benzimidazole carbamates, demethylation inhibitors, quinone outside inhibitors, organotin compounds, and the guanidines. Over the last 10 years, several studies have been conducted that have improved both scab management and fungicide resistance management in V. effusa. The aim of this review is to summarize recent developments in our understanding of fungicide resistance in V. effusa in the context of scab management in southeastern pecan orchards. The history, modes of action, general use of the labeled fungicides, and mechanisms and stability of fungicide resistance in V. effusa are discussed; conclusions and future research priorities are also presented.

Clade-Specific Biosurveillance of Pseudoperonospora cubensis Using Spore Traps for Precision Disease Management of Cucurbit Downy Mildew.

Pseudoperonospora cubensis is an obligate oomycete and cause of cucurbit downy mildew (CDM), the most destructive foliar disease affecting cucurbit hosts. Annual epidemics develop throughout the United States as windborne sporangia travel great distances and survive prolonged exposure to solar radiation. Recent genomic evidence suggests that P. cubensis isolates display host adaptation based on their respective clade. Early detection is key for fungicide application timing, and identification of the host-adapted clade provides information on the risk of infection for specific cucurbit crops. In this study, a multiplex quantitative PCR assay was developed based on species- and clade-specific nuclear genomic markers. The assay detected as few as 10 sporangia or DNA at 100 fg/ml for both clades and was validated in the field by deploying rotorod spore samplers in cucurbit sentinel plots located at two research stations in North Carolina. Using this assay, sporangia DNA was detected in spore trap sampling rods before signs of P. cubensis or CDM symptoms were observed in the sentinel plots. Both clade 1 and clade 2 DNA were detected in late-season cucumber and watermelon plots but only clade 2 DNA was detected in the early-season cucumber plots. These results will significantly improve disease management of CDM by monitoring inoculum levels to determine the cucurbit crops at risk of infection throughout each growing season.

Spatial Variation and Temporal Dynamics of Fungicide Sensitivity in Venturia effusa Within a Pecan Orchard.

An 18-ha commercial pecan orchard was sampled over 3 years to study the spatial and temporal variation in fungicide sensitivity of Venturia effusa, cause of pecan scab. The orchard was divided into a two-dimensional, 8 × 8 grid of 64 quadrats, each containing nine trees (unless there were missing trees), and samples were collected once per year from each quadrat to be tested for sensitivity to fentin hydroxide, propiconazole, and thiophanate-methyl. Averaged across the orchard, insensitivity to all three fungicides was significantly lower in 2016 compared with 2015, but significantly greater for fentin hydroxide and thiophanate-methyl in 2017. Although significant spatial autocorrelation was observed for sensitivity to propiconazole in 2017 and for thiophanate-methyl in 2015 and 2017, indicating clustering, all other fungicide-by-year combinations were not significant. Omnidirectional spatial dependence was observed for sensitivity to propiconazole and thiophanate-methyl in 2017. In both instances, the semivariance increased linearly with lag distance; however, the range of spatial dependence was >276.5 m and could not be estimated accurately. Additionally, a separate sampling was conducted in all 3 years to identify an appropriate sampling size and pattern for fungicide sensitivity screening. A leaflet sample size of 165 in 11 groups of 15 allowed for accurate sensitivity testing for the three fungicides in all 3 years; however, a sample size of 45 leaflets in three groups of 15 was sufficient for quantifying sensitivity for propiconazole and thiophanate-methyl, in most cases. These results indicate that considerable biological variation in fungicide sensitivity exists in orchard-scale populations of V. effusa and that the spatial characteristics of those populations may differ in two-dimensional space depending on the growing season.

Disease and Yield Response of a Stem-rot-resistant and -Susceptible Peanut Cultivar under Varying Fungicide Inputs.

Peanut (Arachis hypogaea L.) producers rely on costly fungicide programs to manage stem rot, caused by Sclerotium rolfsii. Planting disease-resistant cultivars could increase profits by allowing for the deployment of less-expensive, lower-input fungicide programs. Field experiments were conducted to characterize stem rot and early and late leaf spot (caused by Passalora arachidicola and Nothopassalora personata, respectively), yield, and overall profitability of cultivars Georgia-06G (stem-rot-susceptible) and Georgia-12Y (stem-rot-resistant) as influenced by seven commercial fungicide programs. Stem rot incidence was consistently lower on Georgia-12Y for all fungicides when compared with Georgia-06G and was lowest for both cultivars in plots treated with prothioconazole plus a tank mixture of penthiopyrad and tebuconazole. Leaf spot severity was similar for both the resistant and susceptible cultivars, and the greatest reduction occurred in plots treated with prothioconazole plus a tank mixture of penthiopyrad and tebuconazole. Fungicide programs gave similar yield and net return on Georgia-12Y; however, plots of Georgia-06G treated with prothioconazole plus a tank mixture of penthiopyrad and tebuconazole had the greatest yield and net return. Yields and economic return from the highest level of fungicide inputs on Georgia-06G were numerically less than those of Georgia-12Y treated with only chlorothalonil. These results show the value of fungicides in peanut disease management with susceptible cultivars, as well as the benefits of planting stem-rot-resistant cultivars in high-risk situations.

Dynamics of Fungicide Sensitivity in Venturia effusa and Fungicide Efficacy under Field Conditions.

Venturia effusa, which causes pecan scab, has developed resistance to fungicides that were once effective. Over 2 years, laboratory-based sensitivity of fentin hydroxide (TPTH) and tebuconazole in V. effusa and their efficacy under field conditions were compared. Leaf and nut scab were assessed on pecan trees receiving 10 applications of TPTH, tebuconazole, azoxystrobin, azoxystrobin plus tebuconazole, TPTH plus tebuconazole, or no fungicide (NTC) per year. Sensitivity of V. effusa on leaflets collected from treated and nontreated trees was assessed in June and September, respectively. The mean relative germination (RGe) on TPTH at 30 µg/ml was 10.9 and 40.9% in 2016 and 4.2 and 0.6% in 2017. Mean relative growth (RGr) on tebuconazole at 1 µg/ml was 45.5 and 34.6% in 2016 and 69.3 and 56.3% in 2017. In both years, leaf and nut scab were significantly lower on trees treated with azoxystrobin, azoxystrobin + tebuconazole, or TPTH + tebuconazole when compared with NTC and tebuconazole-treated trees. Compared with the NTC, tebuconazole did not significantly reduce leaf scab in 2017 or nut scab in either year, indicating that an RGr value between 34.6 and 69.3% is likely to result in a control failure on tebuconazole-treated trees. Although better activity was expected, TPTH reduced scab with RGe values between 0.6 and 40.9%. These results are valuable for developing fungicide sensitivity thresholds to better predict fungicide performance.

Location of an Intron in the Cytochrome b Gene Indicates Reduced Risk of QoI Fungicide Resistance in Fusicladium effusum.

Pecan scab, caused by Fusicladium effusum, is most effectively managed using multiple fungicide applications, including quinone outside inhibitors (QoIs). However, QoIs have a high risk for resistance developing in phytopathogenic fungi. QoI resistance is generally associated with amino-acid substitutions at positions 129, 137, and 143 of the cytochrome b (cytb) gene. A substitution at position 143 confers complete resistance, while an intron immediately downstream of this position prevents the substitution. The objective of this study was to assess the risk of QoI resistance by characterizing a partial fragment of the F. effusum cytb gene. Sequence analysis of the 1,919-bp fragment revealed the presence of a 1,407-bp intron immediately downstream of position 143. This intron was identified in 125 isolates collected from 16 counties across the state of Georgia. No substitutions were identified at positions 129 or 143 but, in seven of the isolates, glycine was replaced with serine at position 137. The ubiquitous nature of the detected intron provided strong evidence that the G143A substitution may not occur in F. effusum isolates, although resistance could still develop through intron loss events or the selection of intron-lacking genotypes, or as the result of other mutations in the cytb gene.

Occurrence of QoI Fungicide Resistance in Cercospora sojina from Mississippi Soybean.

Frogeye leaf spot, caused by Cercospora sojina Hara, is a foliar disease affecting soybean (Glycine max (L.) Merr.), often managed by applications of quinone outside inhibitor (QoI) fungicides. In 2013 and 2014, 634 C. sojina monoconidial isolates were collected from soybean fields throughout Mississippi. Initially, in vitro bioassays were performed to evaluate the sensitivity of 14 of 634 isolates plus a baseline. Resistant and sensitive isolates were characterized by determining the effective fungicide concentrations at which 50% of conidial germination was inhibited (EC50). The molecular mechanism of resistance was determined for all 634 isolates, using a PCR-RFLP method and comparing nucleotide sequences of the cytochrome b gene. The state of Mississippi was divided into five distinct geographical regions (the Hills, Delta, Pines, Capital, and Coast) based on estimates of total soybean hectares. The greatest proportion (16.7%) of QoI-sensitive isolates was collected in the Hills while the Coast had no QoI-sensitive isolates. QoI-sensitive isolates from the Pines, Capital, and Delta ranged from 1.6 to 7.0%. Results of this study determined that more than 93% of C. sojina isolates collected in Mississippi carried the G143A amino acid substitution, indicating a shift to a QoI-resistant population throughout Mississippi soybean fields.