RESUMO
Quinoa downy mildew, caused by Peronospora variabilis, is the most devastating disease of quinoa globally. Rapid, sensitive diagnostic methods are needed to detect and quantify this pathogen in seeds and plant tissue. A hydrolysis probe-based quantitative real-time PCR (qPCR) assay including a competitive internal control was developed for P. variabilis detection. This assay could detect as low as 20 ag of DNA or approximately 25 internal transcribed spacer (ITS) copies per reaction with efficiencies ranging from 93.9 to 98.2%. No non-target amplification was observed when tested against DNA from other downy mildew pathogens and related oomycetes. Peronospora variabilis strains from multiple countries were detected using this assay. The assay was successfully applied to quantify the pathogen in quinoa seeds from a field trial conducted in Washington State. Downy mildew disease was recorded on all 14 genotypes with the genotypes 104.88 and 106.49 recording the highest area under the disease progress values (3,236 ± 303 SE and 2,851 ± 198, respectively) while J6 and Dutchess recorded the lowest (441 ± 107 and 409 ± 129, respectively). Seed washes obtained from field samples were subjected to the qPCR assay, and the pathogen was detected in all samples. The highest pathogen ITS copy number recorded with 106.49 (194,934 ± 38,171 SE), while the lowest was observed in Pasto (5,971 ± 1,435) and Riobamba (9,954 ± 4,243). This qPCR assay could lead to improved detection and quantification of P. variabilis as well as increased understanding of quinoa-P. variabilis interactions and epidemiology.
RESUMO
Soil-biodegradable plastic mulch films are a promising alternative to polyethylene mulches, but adoption has been slow, in part because of uncertainties about in-field degradation. The international biodegradability standard EN-17033 requires 90% degradation within 2 years in an aerobic incubation at constant temperature (20-28 °C). However, in-laboratory biodegradability does not guarantee in-field degradation will follow the same timeframe. Field test protocols are needed to assess biodegradable mulches under a range of environmental conditions and collate site-specific information to predict degradation. Our objectives were to (1) monitor in-field degradation of soil-biodegradable plastic mulches following successive applications and incorporations, (2) quantify mulch recovery 2 years after the final incorporation, and (3) compare in-field degradation with the laboratory standard in terms of calendar and thermal times based on a zeroth-order kinetics model. A field experiment was established in spring 2015 in Mount Vernon, WA testing five biodegradable mulches laid each spring and incorporated each fall until 2018. Mulch recovery was quantified every 6 months until fall 2020, 2 years after the final incorporation. While mulches were incorporated annually, recovery of visible fragments (>2.36 mm) was constant or decreasing over time, indicating mulch deterioration kept pace with new additions. In fall 2020, mulch recovery was 4-16% of total mulch mass incorporated. A zeroth-order kinetics model was used to analyze mulch degradation after the final application. Model extrapolations indicate it would take 21 to 58 months to reach 10% recovery (90% degradation), exceeding the laboratory standard's 24-month benchmark by up to a factor of 2.4. However, when the analysis is done with thermal time, better agreement between in-field and laboratory degradation rates is observed. While other factors, including soil type, soil moisture, and mulch fragment size are also at play, thermal time, rather than calendar time, will be more applicable for assessing site-specific, in-field mulch degradation.
