Exploring the Mycovirus Sclerotinia sclerotiorum Hypovirulence-Associated DNA Virus 1 as a Biocontrol Agent of White Mold Caused by Sclerotinia sclerotiorum.

Sclerotinia sclerotiorum is a necrotrophic fungal pathogen causing white mold on many important economic crops. Recently, some mycoviruses such as S. sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV-1) converted S. sclerotiorum into a beneficial symbiont that helps plants manage pathogens and other stresses. To explore the potential use of SsHADV-1 as a biocontrol agent in the United States and to test the efficacy of SsHADV-1-infected United States isolates in managing white mold and other crop diseases, SsHADV-1 was transferred from the Chinese strain DT-8 to United States isolates of S. sclerotiorum. SsHADV-1 is readily transmitted horizontally among United States isolates of S. sclerotiorum and consistently conferred hypovirulence to its host strains. Biopriming of dry bean seeds with hypovirulent S. sclerotiorum strains enhanced resistance to white mold, gray mold, and Rhizoctonia root rot. To investigate the underlying mechanisms, endophytic growth of hypovirulent S. sclerotiorum in dry beans was confirmed using PCR, and the expression of 12 plant defense-related genes were monitored before and after infection. The results indicated that the endophytic growth of SsHADV-1-infected strains in plants stimulated the expression of plant immunity pathway genes that assisted a rapid response from the plant to fungal infection. Finally, application of the seed biopriming technology with SsHADV-1-infected hypervirulent strain has promise for the biological control of several diseases of wheat, pea, and sunflower.

The D-galacturonic acid catabolic pathway genes differentially regulate virulence and salinity response in Sclerotinia sclerotiorum.

Sclerotinia sclerotiorum causes white mold disease on a wide range of economically important crops such as soybean, canola, tomato, pea and sunflower. As one of the most successful plant pathogens, S. sclerotiorum has the unique ability of adapting to various environmental conditions and effectively suppressing or evading plant defense. Notably, S. sclerotiorum secretes an array of plant cell-wall degrading enzymes (CWDEs) to macerate host cell wall and utilizes the liberated monosaccharides and oligosaccharides as nutrients. One of the major plant cell wall constituents is polygalacturonic acid in pectin, with D-galacturonic acid being the most abundant component. In this research, we identified four S. sclerotiorum genes that encode the enzymes for the D-galacturonic acid catabolism, namely Ssgar1, Ssgar2, Sslgd1 and Sslga1. Gene-knockout mutants were created for all four catabolic genes. When cultured on pectin as the alternative carbon source, Sslgd1- and Sslga1-deletion mutants and Ssgar1/Ssgar2 double deletion mutants exhibited significantly reduced growth. The D-galacturonic acid catabolic genes are transcriptionally induced by either polygalacturonic acid in the culture media or during host infection. Virulence tests of the knockout mutants revealed that Ssgar2, Sslgd1 and Sslga1 all facilitated the effective colonization of S. sclerotiorum to the leaves of soybean and pea, but not of tomato which has the lowest D-galacturonic acid contents in its leaves. In addition to their positive roles in virulence, all four enzymes negatively affect S. sclerotiorum tolerance to salt stress. SsGAR2 has an additional function in tolerance to Congo Red, suggesting a potential role in cell wall stability of S. sclerotiorum. This study is the first report revealing the versatile functions of D-galacturonic acid catabolic genes in S. sclerotiorum virulence, salinity response and cell wall integrity.

Image-Based Phenotyping of Flowering Intensity in Cool-Season Crops.

The timing and duration of flowering are key agronomic traits that are often associated with the ability of a variety to escape abiotic stress such as heat and drought. Flowering information is valuable in both plant breeding and agricultural production management. Visual assessment, the standard protocol used for phenotyping flowering, is a low-throughput and subjective method. In this study, we evaluated multiple imaging sensors (RGB and multiple multispectral cameras), image resolution (proximal/remote sensing at 1.6 to 30 m above ground level/AGL), and image processing (standard and unsupervised learning) techniques in monitoring flowering intensity of four cool-season crops (canola, camelina, chickpea, and pea) to enhance the accuracy and efficiency in quantifying flowering traits. The features (flower area, percentage of flower area with respect to canopy area) extracted from proximal (1.6-2.2 m AGL) RGB and multispectral (with near infrared, green and blue band) image data were strongly correlated (r up to 0.89) with visual rating scores, especially in pea and canola. The features extracted from unmanned aerial vehicle integrated RGB image data (15-30 m AGL) could also accurately detect and quantify large flowers of winter canola (r up to 0.84), spring canola (r up to 0.72), and pea (r up to 0.72), but not camelina or chickpea flowers. When standard image processing using thresholds and unsupervised machine learning such as k-means clustering were utilized for flower detection and feature extraction, the results were comparable. In general, for applicability of imaging for flower detection, it is recommended that the image data resolution (i.e., ground sampling distance) is at least 2-3 times smaller than that of the flower size. Overall, this study demonstrates the feasibility of utilizing imaging for monitoring flowering intensity in multiple varieties of evaluated crops.

Functional Dissection of the Chickpea (Cicer arietinum L.) Stay-Green Phenotype Associated with Molecular Variation at an Ortholog of Mendel's I Gene for Cotyledon Color: Implications for Crop Production and Carotenoid Biofortification.

"Stay-green" crop phenotypes have been shown to impact drought tolerance and nutritional content of several crops. We aimed to genetically describe and functionally dissect the particular stay-green phenomenon found in chickpeas with a green cotyledon color of mature dry seed and investigate its potential use for improvement of chickpea environmental adaptations and nutritional value. We examined 40 stay-green accessions and a set of 29 BC2F4-5 stay-green introgression lines using a stay-green donor parent ICC 16340 and two Indian elite cultivars (KAK2, JGK1) as recurrent parents. Genetic studies of segregating populations indicated that the green cotyledon trait is controlled by a single recessive gene that is invariantly associated with the delayed degreening (extended chlorophyll retention). We found that the chickpea ortholog of Mendel's I locus of garden pea, encoding a SGR protein as very likely to underlie the persistently green cotyledon color phenotype of chickpea. Further sequence characterization of this chickpea ortholog CaStGR1 (CaStGR1, for carietinum stay-green gene 1) revealed the presence of five different molecular variants (alleles), each of which is likely a loss-of-function of the chickpea protein (CaStGR1) involved in chlorophyll catabolism. We tested the wild type and green cotyledon lines for components of adaptations to dry environments and traits linked to agronomic performance in different experimental systems and different levels of water availability. We found that the plant processes linked to disrupted CaStGR1 gene did not functionality affect transpiration efficiency or water usage. Photosynthetic pigments in grains, including provitaminogenic carotenoids important for human nutrition, were 2-3-fold higher in the stay-green type. Agronomic performance did not appear to be correlated with the presence/absence of the stay-green allele. We conclude that allelic variation in chickpea CaStGR1 does not compromise traits linked to environmental adaptation and agronomic performance, and is a promising genetic technology for biofortification of provitaminogenic carotenoids in chickpea.

A pulse crop dataset of agronomic traits and multispectral images from multiple environments.

Crop yield potential in breeding trials can be captured using unmanned aerial vehicle (UAV) based multispectral imagery. Several digital traits or phenotypes such as vegetation indices can represent canopy crop vigor and overall plant health, which can be used to evaluate differences in performance across varieties in crop breeding programs. This dataset contains agronomic data for named cultivars and breeding lines of spring-sown dry pea and chickpea, and over 275 multispectral images from advanced and preliminary breeding trials. The breeding trials were located at three locations in the "Palouse" region of Eastern Washington and Northern Idaho of the United States across 2017, 2018 and 2019 cropping seasons. The multispectral images were captured using a UAV integrated with a 5-band multispectral camera at multiple time points from early vegetative growth through pod development stages during each cropping season. This dataset details seed yield information from trials of dry peas and chickpea that were obtained from each location, as well as additional agronomic and phenological data recorded at one location (mostly Pullman, WA) for each cropping season. The dataset also includes 20-78 megabytes (MB) Tagged Image Format (TIF) uncalibrated stitched orthomosaic images generated from the photogrammetric software. The images can be processed using any convenient image processing algorithm to obtain vegetation indices and other useful information.

Genome Sequence of Sclerotinia sclerotiorum Hypovirulence-Associated DNA Virus 1 Found in the Fungus Penicillium olsonii Isolated from Washington State, USA.

We report the discovery of a Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV-1) isolate, named SsHADV1_PO, from the fungus Penicillium olsonii isolated from Washington state, USA. The genome of SsHADV1_PO is 2,166 bp and contains two open reading frames, with more than 98% nucleotide identity with respect to reported SsHADV-1 isolates.

Crop Performance Evaluation of Chickpea and Dry Pea Breeding Lines Across Seasons and Locations Using Phenomics Data.

The Pacific Northwest is an important pulse production region in the United States. Currently, pulse crop (chickpea, lentil, and dry pea) breeders rely on traditional phenotyping approaches to collect performance and agronomic data to support decision making. Traditional phenotyping poses constraints on data availability (e.g., number of locations and frequency of data acquisition) and throughput. In this study, phenomics technologies were applied to evaluate the performance and agronomic traits in two pulse (chickpea and dry pea) breeding programs using data acquired over multiple seasons and locations. An unmanned aerial vehicle-based multispectral imaging system was employed to acquire image data of chickpea and dry pea advanced yield trials from three locations during 2017-2019. The images were analyzed semi-automatically with custom image processing algorithm and features were extracted, such as canopy area and summary statistics associated with vegetation indices. The study demonstrated significant correlations (P < 0.05) between image-based features (e.g., canopy area and sum normalized difference vegetation index) with yield (r up to 0.93 and 0.85 for chickpea and dry pea, respectively), days to 50% flowering (r up to 0.76 and 0.85, respectively), and days to physiological maturity (r up to 0.58 and 0.84, respectively). Using image-based features as predictors, seed yield was estimated using least absolute shrinkage and selection operator regression models, during which, coefficients of determination as high as 0.91 and 0.80 during model testing for chickpea and dry pea, respectively, were achieved. The study demonstrated the feasibility to monitor agronomic traits and predict seed yield in chickpea and dry pea breeding trials across multiple locations and seasons using phenomics tools. Phenomics technologies can assist plant breeders to evaluate the performance of breeding materials more efficiently and accelerate breeding programs.

Detection of Erysiphe necator in Air Samples Using the Polymerase Chain Reaction and Species-Specific Primers.

ABSTRACT A polymerase chain reaction (PCR) assay employing species-specific primers was developed to differentiate Erysiphe necator from other powdery mildews common in the northwest United States. DNA was extracted from mycelia, conidia, and/or chasmothecia that were collected from grape leaves with a Burkard cyclonic surface sampler. To differentiate E. necator from other erysiphaeceous fungi, primer pairs Uncin144 and Uncin511 were developed to select unique sequences of the internal transcribed spacer regions of E. necator. Using these primers in PCR amplifications, a 367-bp amplicon specific to E. necator was generated, but no amplicons were generated from other erysiphaceous species collected from 48 disparate hosts representing 26 vascular plant families. The PCR limit of detection was one to five conidia of E. necator placed directly into reaction mixtures or 100 to 250 conidia placed on glass rods coated with silicon grease. During field studies, this PCR assay facilitated the detection of E. necator inoculum in air samples within hours of sample rod collection and prior to disease onset. Amplification of E. necator DNA did not occur when the PCR assay was conducted on vineyard air samples collected while grapes were dormant or during periods when vine growth occurred but E. necator remained dormant. The initial PCR detection of E. necator of the season occurred during seasonal ascospore releases caused by precipitation events between bud burst and the prebloom period during the 3 years of the study. Detection ceased for 7 to 11 days following ascospore release and then resumed several days prior to the observance of microscopic symptoms and signs of powdery mildew in the field. Results of this study represent the initial step toward the goal of incorporating an inoculum availability component into current and future grapevine powdery mildew risk assessment models.

Heritability of Resistance to Verticillium Wilt in Alfalfa.

Verticillium wilt of alfalfa, caused by Verticillium albo-atrum, may reduce forage yields by up to 50% in alfalfa-producing areas of the northern United States and Canada. It has been suggested that cultivars require at least 60% resistant plants to afford maximum protection against disease. Our objective was to calculate heritability estimates of resistance to Verticillium wilt in alfalfa. Estimates were generated for two alfalfa populations developed from the cvs. Affinity + Z and Depend + EV. Heritability on a half-sib progeny means basis was calculated based on data from greenhouse pathogenicity tests. Estimates based on repeated experiments conducted for single years (2004 and 2005) were high for both populations, ranging from 0.86 to 0.92. The heritability estimate based on data collected over 2 years was 0.26 for Affinity + Z and 0.66 for Depend + EV. Disease was more severe in 2005 than in 2004. However, the Spearman rank correlation between mean disease severity index values for half-sib families over 2 years was positive and significant for both populations. Results of pathogenicity tests suggested that neither cultivar had resistance levels approaching 60%. The heritability estimates suggest that resistance levels in both Affinity + Z and Depend + EV could be improved further through selection.

Genotyping Common Bean for the Potyvirus Resistance Alleles I and bc-1(2) with a Multiplex Real-Time Polymerase Chain Reaction Assay.

ABSTRACT A multiplex real-time polymerase chain reaction (PCR) assay was developed to simultaneously genotype plants for the I and bc-1(2) alleles, which condition resistance in beans to Bean common mosaic virus and Bean common mosaic necrosis virus. A segregating F(2) population was derived from the cross between pinto bean breeding line P94207-189A (bc-1 bc-1 I I) and Olathe (bc-12 bc-1(2) i i). Real-time PCR assays were developed that were specific for each allele, and a multiplex PCR reaction could unambiguously assign F(2) plants to one of nine genotypes. Remnant F(1) plants were used as a comparative reference sample. PCR results among this sample fit a normal distribution for both real-time PCR assays, and 99% probability distributions were determined for heterozygotes. F(2) plants were genotyped based on results relative to the probability distributions for heterozygotes. F(2) plants also were genotyped for the I and bc-1(2) alleles by performing F(3) family progeny tests for virus resistance. Agreement between the two methods was 100% (198/198) for the bc-1(2) allele, and 92.4% (183/198) for the I allele. Erroneous genotyping was due to recombination between the amplicon and the I allele. Realtime PCR assays provide a robust method for genotyping seedlings and, in some cases, may eliminate the need for progeny testing.

Genetic Relationship in Cicer Sp. Expose Evidence for Geneflow between the Cultigen and Its Wild Progenitor.

There is a debate concerning mono- or poly-phyletic origins of the Near Eastern crops. In parallel, some authors claim that domestication was not possible within the natural range of the wild progenitors due to wild alleles flow into the nascent crops. Here we address both, the mono- or poly-phyletic origins and the domestications within or without the natural range of the progenitor, debates in order to understand the relationship between domesticated chickpea (Cicer arietinum L.) and its wild progenitor (C. reticulatum Ladizinsky) with special emphasis on its domestication centre in southeastern Turkey. A set of 103 chickpea cultivars and landraces from the major growing regions alongside wild accessions (C. reticulatum, C. echinospermum P.H Davis and C. bijugum K.H. Rech) sampled across the natural distribution range in eastern Turkey were genotyped with 194 SNPs markers. The genetic affinities between and within the studied taxa were assessed. The analysis suggests a mono-phyletic origin of the cultigen, with several wild accession as likely members of the wild stock of the cultigen. Clear separation between the wild and domesticated germplasm was apparent, with negligible level of admixture. A single C. reticulatum accession shows morphological and allelic signatures of admixture, a likely result of introgression. No evidence of geneflow from the wild into domesticated germplasm was found. The traditional farming systems of southeaster Turkey are characterized by occurrence of sympatric wild progenitor-domesticated forms of chickpea (and likewise cereals and other grain legumes). Therefore, both the authentic crop landraces and the wild populations native to the area are a unique genetic resource. Our results grant support to the notion of domestication within the natural distribution range of the wild progenitor, suggesting that the Neolithic domesticators were fully capable of selecting the desired phenotypes even when facing rare wild-domesticated introgression events.