A Quantitative Real-Time PCR Method to Detect the Quinoa Downy Mildew Pathogen, Peronospora variabilis.

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.

Physical characteristics of soil-biodegradable and nonbiodegradable plastic mulches impact conidial splash dispersal of Botrytis cinerea.

Botrytis cinerea causes gray mold disease of strawberry (Fragaria ×ananassa) and is a globally important pathogen that causes fruit rot both in the field and after harvest. Commercial strawberry production involves the use of plastic mulches made from non-degradable polyethylene (PE), with weedmat made from woven PE and soil-biodegradable plastic mulch (BDM) as emerging mulch technologies that may enhance sustainable production. Little is known regarding how these plastic mulches impact splash dispersal of B. cinerea conidia. The objective of this study was to investigate splash dispersal dynamics of B. cinerea when exposed to various plastic mulch surfaces. Mulch surface physical characteristics and conidial splash dispersal patterns were evaluated for the three mulches. Micrographs revealed different surface characteristics that have the potential to influence splash dispersal: PE had a flat, smooth surface, whereas weedmat had large ridges and BDM had an embossed surface. Both PE mulch and BDM were impermeable to water whereas weedmat was semi-permeable. Results generated using an enclosed rain simulator system showed that as the horizontal distance from the inoculum source increased, the number of splash dispersed B. cinerea conidia captured per plate decreased for all mulch treatments. More than 50% and approximately 80% of the total number of dispersed conidia were found on plates 10 and 16 cm away from the inoculum source across all treatments, respectively. A significant correlation between the total and germinated conidia on plates across all mulch treatments was detected (P<0.01). Irrespective of distance from the inoculum source, embossed BDM facilitated higher total and germinated splashed conidia (P<0.001) compared to PE mulch and weedmat (P = 0.43 and P = 0.23, respectively), indicating BDM's or embossed film's potential for enhancing B. cinerea inoculum availability in strawberry production under plasticulture. However, differences in conidial concentrations observed among treatments were low and may not be pathologically relevant.

Development of a Decision Support System for the Management of Mummy Berry Disease in Northwestern Washington.

Mummy berry, caused by Monilinia vaccinii-corymbosi, is the most important disease of the northern highbush blueberry (Vaccinium corymbosum L.) in North America and can cause up to 70% yield losses in affected fields. A key event in the mummy berry disease cycle is the primary infection phase where ascospores are released by apothecia that infect emerging floral and vegetative tissues. Current management of mummy berry disease in northwestern Washington is predominantly reliant on the prevention of primary infections through prophylactic, calendar-based fungicide spray applications early in the growing season. To improve the understanding of risk during these periods and to help tailor management strategies, we developed a decision support system (DSS) based on field records spanning over five seasons and four locations in northwestern Washington. Environmental conditions across the region were highly uniform but different dynamics of apothecial development were observed under high- and low-management regimes. Based on our analysis, we suggest basing the initial iteration of the DSS on two sub-models. The first sub-model predicts the onset of apothecia based on chill-unit accumulation under high- and low-management regimes, and the second predicts primary infection risk, which provides opportunities to improve the timing of fungicide applications. The synoptic DSS proposed here is based on the current biological knowledge of the pathosystem and available data for the northwestern Washington region. We provide the analysis and the DSS implementation and evaluation as an open-source repository, providing opportunities for further improvements. Finally, we provide suggestions for future research and the operational efforts needed for improving the utility and accuracy of the mummy berry DSS.

Genetic Diversity of Phymatotrichopsis omnivora Based on Mating Type and Microsatellite Markers Reveals Heterothallic Mating System.

Phymatotrichopsis omnivora is a member of Pezizomycetes and causes root rot disease on a broad range of dicotyledonous plants. Using recently generated draft genome sequence data from four P. omnivora isolates, we developed simple sequence repeat (SSR) markers and identified both mating type genes (MAT1-1-1 and MAT1-2-1) in this fungus. To understand the genetic diversity of P. omnivora isolates (n = 43) and spore mats (n = 29) collected from four locations (Oklahoma, Texas, Arizona, and Mexico) and four host crops (cotton, alfalfa, peach, and soybean), we applied 24 SSR markers and showed that of the 72 P. omnivora isolates and spore mats tested, 41 were distinct genotypes. Furthermore, the developed SSR markers did not show cross-transferability to other close relatives of P. omnivora in the class Pezizomycetes. A multiplex PCR detecting both mating type idiomorphs and a reference gene (TUB2) was developed to screen P. omnivora isolates. Based on the dataset we tested, P. omnivora is a heterothallic fungus with both mating types present in the United States in a ratio close to 1:1. We tested P. omnivora spore mats obtained from spatially distinct disease rings that developed in a center-pivot alfalfa field and showed that both mating types can be present not only in the same field but also within a single spore mat. This study shows that P. omnivora has the genetic toolkit for generating sexually diverse progeny, providing impetus for future studies that focus on identifying sexual morphs in nature.

Enabling sustainable agriculture through understanding and enhancement of microbiomes.

Harnessing plant-associated microbiomes offers an invaluable strategy to help agricultural production become more sustainable while also meeting growing demands for food, feed and fiber. A plethora of interconnected interactions among the host, environment and microbes, occurring both above and below ground, drive recognition, recruitment and colonization of plant-associated microbes, resulting in activation of downstream host responses and functionality. Dissecting these complex interactions by integrating multiomic approaches, high-throughput culturing, and computational and synthetic biology advances is providing deeper understanding of the structure and function of native microbial communities. Such insights are paving the way towards development of microbial products as well as microbiomes engineered with synthetic microbial communities capable of delivering agronomic solutions. While there is a growing market for microbial-based solutions to improve crop productivity, challenges with commercialization of these products remain. The continued translation of plant-associated microbiome knowledge into real-world scenarios will require concerted transdisciplinary research, cross-training of a next generation of scientists, and targeted educational efforts to prime growers and the general public for successful adoption of these innovative technologies.

The First Genomic Resources for Phymatotrichopsis omnivora, a Soilborne Pezizomycete Pathogen with a Broad Host Range.

Phymatotrichopsis omnivora is a destructive plant pathogen causing root rot disease of alfalfa, cotton, pecan, grape, and many other important dicotyledonous species. A member of the family Rhizinaceae, in the class Pezizomycetes, P. omnivora is a soilborne ascomycete fungus that is difficult to maintain in culture, currently genetically intractable, and for which there are no publicly available genomic resources. We have generated draft genome sequences of four P. omnivora isolates obtained from cotton and alfalfa, growing in Texas and Oklahoma. These genome sequences will provide new insights into the biology of the fungus, including the factors responsible for its broad host range and pathogenicity.

Evidence for Sexual Reproduction: Identification, Frequency, and Spatial Distribution of Venturia effusa (Pecan Scab) Mating Type Idiomorphs.

Venturia effusa (syn. Fusicladium effusum), causal agent of pecan scab, is the most prevalent pathogen of pecan (Carya illinoinensis), causing severe yield losses in the southeastern United States. V. effusa is currently known only by its asexual (conidial) stage. However, the degree and distribution of genetic diversity observed within and among populations of V. effusa are typical of a sexually reproducing fungal pathogen, and comparable with other dothideomycetes with a known sexual stage, including the closely related apple scab pathogen, V. inaequalis. Using the mating type (MAT) idiomorphs from V. inaequalis, we identified a single MAT gene, MAT1-1-1, in a draft genome of V. effusa. The MAT1-1-1 locus is flanked by two conserved genes encoding a DNA lyase (APN2) and a hypothetical protein. The MAT locus spanning the flanking genes was amplified and sequenced from a subset of 14 isolates, of which 7 contained MAT1-1-1 and the remaining samples contained MAT1-2-1. A multiplex polymerase chain reaction screen was developed to amplify MAT1-1-1, MAT1-2-1, and a conserved reference gene encoding ß-tubulin, and used to screen 784 monoconidial isolates of V. effusa collected from 11 populations of pecan across the southeastern United States. A hierarchical sampling protocol representing region, orchard, and tree allowed for analysis of MAT structure at different spatial scales. Analysis of this collection revealed the frequency of the MAT idiomorphs is in a 1:1 equilibrium of MAT1-1:MAT1-2. The apparent equilibrium of the MAT idiomorphs provides impetus for a renewed effort to search for the sexual stage of V. effusa. [Formula: see text] Copyright © 2018 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .