Spatial Dependency in Stubble-Borne Pyrenophora teres f. teres and Influence of Sample Support Size on DNA Concentration and Fungicide Resistance Frequency.

Fungicide resistance in foliar fungal pathogens is an increasing challenge to crop production. Yield impacts due to loss of fungicide efficacy may be reduced through effective surveillance and appropriate management intervention. For stubble-borne pathogens, off-season crop residues may be used to monitor fungicide resistance to inform pre-planting decisions; however, appropriate sampling strategies and support sizes for crop residues have not previously been considered. Here, we used Pyrenophora teres f. teres (Ptt) with resistance to demethylase inhibitor fungicides as a model system to assess spatial dependency and to compare the effects of different sampling strategies and support sizes on pathogen density (Ptt DNA concentration) and the frequency of fungicide resistance mutation. The results showed that sampling strategies (hand-picked versus raked) did not affect estimates of pathogen density or fungicide resistance frequency; however, sample variances were lower from raked samples. The effects of differing sample support size, as the size of the collection area (1.2, 8.6, or 60 m2), on fungicide resistance frequency were not evident (P > 0.05). However, measures of pathogen density increased with area size (P < 0.05); the 60 m2 area yielded the highest Ptt DNA concentration and produced the lowest number of pathogen-absent samples. Sample variances for pathogen density and fungicide resistance frequency were generally homogeneous between area sizes. The pattern of pathogen density was spatially independent; however, spatial dependency was identified for fungicide resistance frequency, with a range of 110 m, in one of the two fields surveyed. Collectively, the results inform designs for monitoring of fungicide resistance in stubble-borne pathogens.

Field-scale gene flow of fungicide resistance in Pyrenophora teres f. teres and the effect of selection pressure on the population structure.

The effectiveness of fungicides to control foliar fungal crop diseases is being diminished by the increasing spread of resistances to fungicides. One approach that may help to maintain efficacy is remediation of resistant populations by sensitive ones. However, the success of such approaches can be compromised by re-incursion of resistance through aerial spore dispersal; although, knowledge of localized gene flow is lacking. Here, we report on a replicated mark-release-recapture field experiment with several treatments set up to study spore-dispersal-mediated gene flow of a mutated allele that confers demethylase inhibitor resistance in Pyrenophora teres f. teres (Ptt). Artificial inoculation of the host, barley (Hordeum vulgare), was successful across the 12-ha trial, where the introduced sensitive- and resistant-populations were, respectively, 6- and 13-fold the DNA concentration of the native Ptt population. Subsequent disease pressure remained low which hampered spread of the epidemic to such extent that gene flow was not detected at, or beyond 2.5 m from source points. In the absence of gene flow, plots were assessed for treatment effects; fungicide applied to populations that contained 14.3% of allele mutation increased in frequency to 24.5%, whereas sensitive populations had no change in structure. Untreated controls of native Ptt population remained genetically stable, yet untreated controls that were inoculated with sensitive Ptt had half the resistance frequency of the native population structure. The trial demonstrates the potential for management to remediate fungicide resistant pathogen populations, where localized gene flow is minimal; to safeguard chemical crop protection into the future.

Optimized Sample Processing Pipeline for PCR-Based Fungicide Resistance Quantification of Stubble-Borne Fungal Pathogens.

Globally, yield losses associated with failed crop protection due to fungicide-resistant pathogens present an increasing problem. For stubble-borne pathogens, assessment of crop residues during the off-season could provide early fungicide resistance quantification for informed management decisions to mitigate yield losses. However, stubble assessment is hampered by assay inhibitors that are derived from decaying organic matter. To overcome assay inhibition from weathered stubble samples, we used a systems approach to quantify the frequency of resistance to demethylase inhibitor fungicides of the barley pathogen Pyrenophora teres f. teres. The system canvassed (i) 10 ball-milling conditions; (ii) four DNA extraction methodologies; and (iii) three column purification techniques for the provision of sufficient yield, quality, and purity of fungal DNA for a PCR-based fungicide resistance assay. Results show that DNA quantity and purity differed within each of the above three categories, with the optimized pipeline being (i) ball-milling samples in a 50-ml stainless steel canister for 5 min using a 20-mm ball at 30 revolutions s-1; (ii) a modified Brandfass method (extracted 64% more DNA than other methods assessed); and (iii) use of silica resin columns for the highest DNA concentration with optimal DNA purity. The chip-digital PCR assay, which quantified fungicide resistance from field samples, was unaffected by the DNA extraction method or purification technique, provided that thresholds of template quantity and purity were satisfied. In summary, this study has developed molecular pipeline options for pathogen fungicide resistance quantification from cereal stubbles, which can guide management for improved crop protection outcomes.

Narrow windrow burning canola (Brassica napus L.) residue for Sclerotinia sclerotiorum (Lib.) de Bary sclerotia destruction.

BACKGROUND: Since the introduction of herbicide-tolerant varieties of canola (Brassica napus L.) in 1993, global plantings have increased resulting in an increased incidence of Sclerotinia sclerotiorum (Lib.) de Bary infections. Developments in narrow windrow burning techniques to destroy the seed of multiple herbicide-resistant weeds provide an opportunity to also intercept and heat-treat the S. sclerotiorum inoculum source, termed sclerotia, before it re-enters the soil to infect susceptible crop species in successive years. RESULTS: Preliminary kiln studies determined that a temperature of 264 °C for 10 s was needed to destroy S. sclerotiorum sclerotia viability (LT99 ) of sclerotia < 3 mm in diameter, whereas temperatures of 353 and 362 °C for the same duration were required to kill sclerotia (LT99 ) of 3-4 and > 4 mm in diameter respectively. In the field, temperatures > 500 °C were maintained in the centre of burning narrow windrows of canola residue for > 450 s and 300 °C was maintained consistently at either edge of the windrows for the same duration. The temperatures achieved when burning canola narrow windrows were sufficient to kill all sclerotia concentrated into the narrow windrow. CONCLUSION: As a technique, narrow windrow burning of canola residue provides the temperature and temperature durations required to kill S. sclerotiorum sclerotia, thus providing a non-fungicidal control option as part of a wider integrated disease management approach. © 2018 Society of Chemical Industry.