Fantastic Downy Mildew Pathogens and How to Find Them: Advances in Detection and Diagnostics.

Downy mildews affect important crops and cause severe losses in production worldwide. Accurate identification and monitoring of these plant pathogens, especially at early stages of the disease, is fundamental in achieving effective disease control. The rapid development of molecular methods for diagnosis has provided more specific, fast, reliable, sensitive, and portable alternatives for plant pathogen detection and quantification than traditional approaches. In this review, we provide information on the use of molecular markers, serological techniques, and nucleic acid amplification technologies for downy mildew diagnosis, highlighting the benefits and disadvantages of the technologies and target selection. We emphasize the importance of incorporating information on pathogen variability in virulence and fungicide resistance for disease management and how the development and application of diagnostic assays based on standard and promising technologies, including high-throughput sequencing and genomics, are revolutionizing the development of species-specific assays suitable for in-field diagnosis. Our review provides an overview of molecular detection technologies and a practical guide for selecting the best approaches for diagnosis.

Assessing Fitness Costs and Phenotypic Instability of Fentin Hydroxide and Tebuconazole Resistance in Venturia effusa.

Sensitivity monitoring of Venturia effusa, cause of pecan scab, has revealed insensitivity to fentin hydroxide and tebuconazole, but recent research indicates that the insensitivity to fentin hydroxide is not stable. A study was undertaken to determine if a fitness cost may be responsible for this instability. In this study, experiments were conducted to evaluate fitness components and phenotypic stability of insensitivity of V. effusa to fentin hydroxide and tebuconazole. Conidial production, conidial germination, microcolony growth, sensitivity to osmotic stress, and sensitivity to oxidative stress in the absence of fungicide were compared for isolates with differing sensitivities to both fungicides. Percent conidial germination decreased linearly with increasing fentin hydroxide insensitivity, and microcolony growth on 1.0 mM H2O2 decreased linearly with increasing tebuconazole insensitivity. Stability of resistance was assessed on concentrations of 1.0, 3.0, and 10 µg/ml of both fungicides prior to and after five transfers on non-fungicide-amended medium. Tebuconazole insensitivity was stable after transfers, but fentin hydroxide insensitivity on 1.0 and 3.0 µg/ml decreased significantly after transfers, indicating instability. Here we provide evidence that in V. effusa tebuconazole insensitivity is stable and fentin hydroxide insensitivity is not. These results suggest that fentin-hydroxide-resistant V. effusa isolates have reduced conidial viability compared with sensitive isolates, which may allow the population to regain sensitivity in the absence of this frequently used fungicide.

Quantifying the Effects of a G137S Substitution in the Cytochrome bc1 of Venturia effusa on Azoxystrobin Sensitivity Using a Detached Leaf Assay.

The quinone outside inhibitor (QoI) fungicides are known for their inherently high resistance risk owing to substitutions in amino acid residues 129, 137, or 143 of the cytochrome b gene of phytopathogens. In Venturia effusa, cause of pecan scab, an intron adjacent to position 143 likely reduces this risk; however, the effects of a recently discovered substitution at position 137 (G137S) are unknown. Traditional in vitro assays are not useful for determining sensitivity of isolates of V. effusa to the QoI fungicides, owing to the fungitoxic effects of required alternative oxidase inhibitors. A detached leaf assay was developed to quantify the sensitivity of 59 isolates to azoxystrobin: 45 wild-type isolates and 14 carrying G137S. Isolate EC50 values ranged from <0.0001 to 0.3047 µg/ml; EC50 values for wild-type isolates ranged from <0.0001 to 0.2007 µg/ml (median 0.0023 µg/ml), whereas EC50 values for G137S isolates ranged from 0.0033 to 0.3047 µg/ml (median 0.0178 µg/ml). The median EC50 value for G137S isolates was significantly greater than that of the wild-type isolates; however, there was overlap between the two groups. This is the first report of sensitivity of V. effusa isolates to a QoI fungicide and evidence of G137S as a potential mechanism of partial resistance. However, although a complete control failure is unlikely, the impact of this substitution on QoI efficacy in Georgia pecan orchards remains to be determined.