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Development of a Diagnostic Assay for Race Differentiation of Podosphaera macularis

    Authors and Affiliations
    • Mary Block1
    • Brian J. Knaus2
    • Michele S. Wiseman3
    • Niklaus J. Grünwald4
    • David H. Gent3 5
    1. 1Oregon State University, Department of Crop and Soil Science, Corvallis, OR 97331
    2. 2Oregon State University, Department of Horticulture, Corvallis, OR 97331
    3. 3Oregon State University, Department of Botany and Plant Pathology, Corvallis, OR 97331
    4. 4U.S. Department of Agriculture-Agricultural Research Service, Horticultural Crops Research Unit, Corvallis, OR 97330
    5. 5U.S. Department of Agriculture-Agricultural Research Service, Forage Seed and Cereal Research Unit, Corvallis, OR 97331

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    Hop powdery mildew (caused by Podosphaera macularis) was confirmed in the Pacific Northwest in 1996. Before 2012, the most common race of P. macularis was able to infect plants that possessed powdery mildew resistance based on the R-genes Rb, R3, and R5. After 2012, two additional races of P. macularis were discovered that can overcome the resistance gene R6 and the partial resistance found in the cultivar Cascade. These three races now occur throughout the region, which can complicate management and research efforts because of uncertainty on which race(s) may be present in the region and able to infect susceptible hop genotypes. Current methods for determining the races of P. macularis are labor intensive, costly, and typically require more than 14 days to obtain results. We sought to develop a molecular assay to differentiate races of the fungus possessing virulence on plants with R6, referred to as V6-virulent, from other races. The transcriptomes of 46 isolates of P. macularis were sequenced to identify loci and variants unique to V6 isolates. Fourteen primer pairs were designed for 10 candidate loci that contained single nucleotide polymorphisms (SNP) and short insertion-deletion polymorphisms. Two differentially labeled locked nucleic acid probes were designed for a contig that contained a conserved SNP associated with V6-virulence. The resulting duplexed real-time PCR assay was validated against 46 V6 and 54 non-V6 P. macularis isolates collected from the United States and Europe. The assay had perfect discrimination of V6-virulence among isolates of P. macularis originating from the western U.S. but failed to predict V6-virulence in three isolates collected from Europe. The specificity of the assay was tested with different species of powdery mildew fungi and other microorganisms associated with hop. Weak nonspecific amplification occurred with powdery mildew fungi collected from Vitis vinifera, Fragaria sp., and Zinnia sp.; however, nonspecification amplification is not a concern when differentiating pathogen race from colonies on hop. The assay has practical applications in hop breeding, epidemiological studies, and other settings where rapid confirmation of pathogen race is needed.

    The cultivated hop, Humulus lupulus L., is a dioecious, perennial climbing plant with annual shoots (Neve 1991). Hop has been grown for centuries for its female inflorescence known as a cone. The lupulin glands of hop cones contain aromatic compounds that impart bitterness along with unique aromas and flavors to beer. Hop plants are commercially grown in regions between 35° and 55° latitudes north and south due to the daylight requirements for flower development (Burgess 1964). The states of Idaho, Oregon, and Washington fall within the optimum growing range for hop and produce 98% of the hops harvested in the United States (USDA-NASS 2019). The demands of the brewing industry dictate which cultivars are planted (Haunold 1981), leading to an increased risk of disease susceptibility due to the preference for brewing quality over disease resistance in most instances (Neve 1991).

    Hop powdery mildew, caused by the fungus, Podosphaera macularis, is one of the most destructive diseases of hop. Decreases in photosynthetic activity from relatively small amounts of the disease are believed to have little impact on the hop plant due to the extensive size of the hop canopy. However, infection of the female inflorescence can have devastating effects on crop production by damaging the hop cones to the extent that they are no longer marketable (Gent et al. 2014, 2018). Hop powdery mildew can be managed through cultural methods or chemical interventions. However, the most cost-effective way for producers to prevent the proliferation of the pathogen is through the deployment of disease resistant cultivars. Hop plants are clonally propagated, so consequently, the cultivars used are genetically uniform within a field. Given the genetic uniformity and intense disease pressure, resistance has not proven to be durable in commercial production (Gent et al. 2017; Royle 1978; Wolfenbarger et al. 2016).

    In 1997, hop powdery mildew was first observed at damaging levels in commercial hop fields in the Pacific Northwest (Ocamb et al. 1999). In 1998, powdery mildew was documented in the hop growing regions of Oregon and Idaho. By 1999, the majority of hop acreage in Washington was affected by the disease (Ocamb et al. 1999). Growers were unprepared for the powdery mildew outbreak and experienced severe losses as a result. In the Pacific Northwest region, costs for disease management and yield losses due to hop powdery mildew in 1999 and 2000 were estimated to be 15% of the total crop revenue (Turechek et al. 2001).

    At the onset of the powdery mildew outbreak in the mid-1990s, it became imperative for hop breeders in the Pacific Northwest to select resistant cultivars to quell the disease, a breeding objective that had been pursued in international breeding programs since the early 1900s (Neve 1986; Salmon 1917). Known resistance genes to hop powdery mildew have been reported and designated as Rb, R1, R2, R3, R4, R5, and R6 (Darby 2013; Royle 1978). Prior to 2012, the most common race of P. macularis found in the Pacific Northwest was virulent on plants possessing the R-genes Rb, R3, and R5 (Gent et al. 2017). Widespread planting of cultivars with R6-based resistance led to the occurrence of new races of powdery mildew capable of overcoming resistance gene R6, termed V6 isolates (Gent et al. 2017; Wolfenbarger et al. 2016). Previous work characterizing V6 isolates in the Pacific Northwest found evidence of a fitness penalty of isolates that had overcome R6-based resistance, expressed as a decrease in the average number of colonies per leaf and an increase in the latent period (Wolfenbarger et al. 2016). The reduced fitness of V6 isolates suggests that there may still be utility in R6 resistance in hop as a quantitative trait locus (QTL) but this gene should not be relied on alone for suppression of the disease.

    V6 isolates of the hop powdery mildew fungus have become endemic in the Pacific Northwest region since 2012 (Wolfenbarger et al. 2016). These isolates can persist on hop cultivars that were susceptible prior to 2012, in addition to those cultivars that possess R6-based resistance. This means that powdery mildew found on previously susceptible hop cultivars must undergo screening on a differential set of cultivars in order to determine which race is present. Race determination is an important part of disease diagnosis as it guides the appropriate disease management response. Knowledge of what races of P. macularis are present in a region or a particular field could inform hop growers if their susceptible yards are at an increased risk for infection, enabling preemptive action. Additionally, a rapid detection method for race determination of the hop powdery mildew fungus could aid plant breeders with race-specific isolate selection for resistance screening of hop germplasm. Epidemiological studies would also benefit from hop powdery mildew race identification in that it could assist in determining the primary source of virulent races and tracking the spread of disease through a region.

    This research aimed to develop a rapid molecular assay to detect V6-virulent isolates of the hop powdery mildew fungus in the Pacific Northwest. A molecular assay would bypass the need for maintenance of a wide array of plant material and provide growers with results within hours rather than weeks.

    Materials and Methods

    Plant materials.

    Hop plants were propagated from softwood cuttings and maintained in a greenhouse free of powdery mildew by regular atomizing of sulfur. The powdery mildew-susceptible cultivar Symphony and the R6 cultivar Nugget were grown for isolate maintenance and V6-virulence identification, respectively. Plants were grown in Metro-Mix 840 (Sun Gro Horticulture, Hubbard, OR) for approximately 14 to 21 days and were watered daily, receiving Peter’s Professional 20-20-20 fertilizer (Sun Gro Horticulture) at each irrigation. The greenhouse was maintained at 20 to 25°C with a 14-h photoperiod. Isolates of P. macularis were maintained on detached hop leaves (Wolfenbarger et al. 2016). Briefly, young, unfurled leaves from the top two nodes were surface disinfested with 70% ethanol and rinsed for 30 s with water and dried. Disinfested leaves were detached and placed in a double Petri dish (Pearson and Gadoury 1987) with water in the lower Petri dish and inoculated with isolates. P. macularis isolates were transferred onto fresh leaves every 2 to 3 weeks.

    DNA sample preparation.

    Conidia and hyphae were collected from P. macularis colonies 2 to 3 weeks after inoculation by touching a 1-cm2 piece of office tape to the colony multiple times then placed in a 1.5-ml microcentrifuge tube. DNA was extracted from powdery mildew isolates using a Chelex extraction procedure (Brewer and Milgroom 2010). Extracted DNA was quantified using the Qubit dsDNA BR Assay Kit (Thermo Fisher Scientific, Waltham, MA) and a Qubit Fluorometer (Thermo Fisher Scientific) and diluted to 1 ng/µl. DNA was extracted using the Chelex method from powdery mildew fungi samples that were collected from different host species found in the Pacific Northwest. DNA was extracted using the FastDNA SPIN Kit (MP Biomedicals, Solon, OH) from other organisms associated with hop (Alternaria alternata, Botrytis cinerea, Diplodia seriata, Pseudoperonospora humuli, Fusarium sambucinum, Lecanicillium attenuatum, Lecanicillium lecanii, Phomopsis tuberivora, Phomopsis sp., and Verticillium nonalfalfae) that had previously been collected and preserved. DNA from hop plant tissue was extracted using DNeasy Plant Mini Kit (Qiagen) and was tested for cross reaction.

    Race characterization of P. macularis isolates.

    During 2019, 40 samples of leaves or cones with powdery mildew were collected from commercial hop yards in Oregon (21 isolates) and Washington (19 isolates) (Table 1). Isolates were obtained by bulk transfer of P. macularis (i.e., not reduced to a single conidial chain) onto detached leaves of cv. Symphony or Nugget as described by Wolfenbarger et al. (2016). Isolates were maintained through successive transfers onto Symphony, with routine transfers on Nugget to ensure selection for V6-virulence. Additionally, DNA was obtained from 60 isolates of P. macularis previously characterized for V6-virulence on cv. Nugget (Wolfenbarger et al. 2016). These were 25 isolates with confirmed V6-virulence and 35 lacking V6-virulence (Table 1). DNA was extracted as described previously and stored at –20°C until use in PCR assays as described below.

    Table 1. Podosphaera macularis isolates previously collected and their differentiation following a duplex real-time polymerase chain reaction run with two differentially labeled locked nucleic acid probes

    PCR primer design and synthesis.

    Gent et al. (2020) previously described identification of genetic variants in the transcriptome of isolates of P. macularis that are associated with V6-virulence in the fungal population in the Pacific Northwest. In the present study, we examined a subset of the 16 loci reported by Gent et al. (2020) for their utility as a target in an allele-specific PCR for differentiation of V6-virulence (Table 2). Primers were designed to flank single SNPs present in the highly differentiated loci and were sequence verified. Primers were designed using Geneious version R11.1 ( and the IDT OligoAnalyzer ( to be approximately 20 nucleotides in length with a Tm of 60°C. Isolates collected from Oregon and Washington with confirmed V6-virulence (HPM-609 and HPM-666) or lacking V6-virulence (HPM-663 and HPM-956) were used as positive and negative controls during the design process.

    Table 2. Primers used to amplify diagnostic variants on contigs for sequencing

    The primers were used in PCR assays carried out in 20-µl volumes containing 10 µl of 2× Prime Time Gene Expression Master Mix (1× final concentration; Integrated DNA Technologies [IDT], Coralville, IA), 500 nM of each forward and reverse primer, and 2 ng of template DNA. PCR was conducted using a Bio-Rad C1000 Touch thermal cycler (Bio-Rad, Hercules, CA) with the following reaction conditions: initial denaturation of DNA at 95°C for 2 min, followed by 40 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 60 s. Reactions were held at 72°C for 10 min for the final extension. The ITS region was also amplified using ITS primers ITS1 and ITS4 (White et al. 1990) as a further control to ensure amplification of the template DNA was possible. Primers were ordered through IDT. PCR products were verified by gel electrophoresis prior to bidirectional sequencing by Eurofins Genomics (Louisville, KY). Sequences of each region were aligned using Geneious to verify the presence of each diagnostic SNP. Primers that produced bright bands in conventional PCR reactions and were sequence-verified were selected to use in probe design, as detailed below.

    LNA probe and assay design, optimization, and testing.

    Locked nucleic acid (LNA) probes and qPCR assays were designed to differentiate isolates of P. macularis from the Pacific Northwest that possess SNPs associated with V6-virulence and the non-V6-virulent wild-type phenotype (Table 3). Assays were designed to meet the requirements of the minimum information for publication of quantitative real-time experiments (Bustin et al. 2009). PCR was conducted using a Bio-Rad CFX96 C1000 Touch thermal cycler (Bio-Rad). Samples were run in Hard-Shell 96-well plates and sealed with Microseal ‘B’ seals (Bio-Rad). Each reaction consisted of 10 µl of iTaq (1× final concentration; Bio-Rad), 500 nM forward and reverse primers (IDT), 250 nM of each LNA probe (IDT), and 2 ng of template DNA in a final volume of 20 µl. Reactions were held at 95°C for 3 min, followed by 40 cycles of 95°C for 30 s and 70°C for 30 s, with fluorescence measured after each 70°C step. A no-template control was included in each run. Quantification cycle (Cq) values were determined using Bio-Rad CFX Manager software version 3.0. Positive probe detection was defined as amplification occurring ≤38 Cq with greater than 2,000 relative fluorescence units (RFU).

    Table 3. Locked nucleic acid (LNA) probes and flanking primers for differentiating V6 from non-V6 isolates

    LNA reactions were run as previously described at varying annealing temperatures to optimize the reaction for race-specific differentiation. Six annealing temperatures were tested, ranging from 66 to 76°C. In each test of optimal annealing temperature, two isolates of each race were tested, along with a negative control that lacked a DNA template. Fungal isolates collected from Oregon and Washington with confirmed V6- virulence (HPM-609 and HPM-666) and lacking V6-virulence (HPM-663 and HPM-956) were used as positive and negative controls, respectively, during the design process. LNA reactions were performed using a Bio-Rad CFX96 C1000 Touch thermal cycler and the following reaction conditions: initial denaturation of 95.0°C for 3 min, followed by 39 cycles of 95.0°C for 30 s and 66°C (or varied temperatures during optimization tests) for 30 s, with fluorescence measured after each annealing and extension step. PCR reagents and conditions were as described previously. Each qPCR run included a negative control that lacked a DNA template. Cq values were measured and positive reactions were defined as described previously.

    Following reaction optimization, LNA probes and assays were tested for sensitivity and specificity to V6 isolates of P. macularis. Assay sensitivity was determined by running 10-fold dilutions of P. macularis DNA ranging from 2,000 to 2 pg/reaction. Two V6-virulent isolates were tested (isolate HPM-609 and isolate HPM-666) along with two non-V6 isolates (HPM-663 and HPM-956). The limit of detection was defined as the lowest concentration of template DNA that still resulted in detection by the target probe, as previously defined. Standard curves for each probe were generated using Bio-Rad CFX Manager software version 3.0. Mixed-isolate reactions were also conducted at varying DNA concentrations to determine the effect on each LNA probe to differentiate the races in a mixed sample. Two race combinations were tested (V6-virulent isolate HPM-1220 and non-V6 isolate HPM-1040), at each of eight DNA concentrations (Table 4). Reaction mixes were prepared as previously described, with the exception of 4 µl of template DNA (2 µl of HPM-1220 and 2 µl of non-V6 HPM-1040 DNA, each of variable concentration) being added to each reaction well.

    Table 4. Limit of detection of the duplex locked nucleic acid probe assay and threshold cycle (Cq) values for mixed-race samples run at varying DNA concentrations

    Assay specificity to V6 isolates was tested with 54 non-V6 isolates and 46 V6 isolates (Table 1). Each reaction consisted of 10 µl of iTaq (1× final concentration; Bio-Rad), 500 nM forward and reverse primers, 250 nM of each LNA probe, and 2 μl of template DNA brought to a final volume of 20 μl. Reactions were held at 95°C for 3 min, followed by 40 cycles of 95°C for 30 s and 70°C for 30 s, with fluorescence measured after each 70°C step. The potential for amplification of nontarget organisms also was tested with the above PCR conditions (Table 5). A negative control lacking template was included in each qPCR run. Cq values were measured and positive reactions were defined as described previously.

    Table 5. Duplex assay with powdery mildew fungi collected from other host species


    PCR primer design and synthesis.

    Fourteen primer pairs were designed from contigs that possess variants that differentiate V6 from non-V6 isolates based on the GʹST statistic (Gent et al. 2020; Hedrick 2005). No amplification occurred with primers designed from contigs 674, 1456, 3456, and 5050. Contig 2251 and 2407 yielded single amplicons in conventional PCR assays and contained SNPs at the predicted locations following sequence verification. BLASTX results for contig 2407 showed sequence similarity to a mitochondrial import inner membrane translocase protein while there were numerous (integrase, reverse transcription, retro virus-related Pol polyprotein) different BLASTX results for contig 2251. Therefore, we selected contig 2407 for probe and qPCR assay development due to the consistency of the BLASTX results.

    LNA probe and assay design, optimization, and testing.

    Two differentially labeled LNA probes, N2407.WT and N2407.P1, were designed to target a region of contig 2407 and differentiate V6 isolates from non-V6 isolates of P. macularis from the Pacific Northwest (Supplementary Fig. S1). At the optimized annealing temperature of 70°C, the LNA probes differentiated 43 V6 isolates from 54 non-V6 isolates derived from the Pacific Northwest (Table 1). Perfect discrimination was observed between Pacific Northwest-derived V6 isolates, detected by the FAM fluorophore in N2407.P1 probe, and non-V6 isolates, detected by the HEX fluorophore in the N2407.WT probe (Table 1). Cq values for the Pacific Northwest V6 isolates ranged from 28.10 to 37.43 (mean 33.25) with the FAM fluorophore. Pacific Northwest V6 isolates did not generate Cq values below the 38 Cq threshold with the HEX fluorophore. However, the V6 isolates from Germany were detected by the HEX fluorophore with Cq values ranging from 28.94 to 35.08 (mean 31.59). Non-V6 isolates generated Cq values of 27.66 to 36.83 (mean 32.11) with the HEX fluorophore but did not generate Cq values below the 38 Cq threshold with the FAM fluorophore. Negative controls lacking template failed to produce amplification in any LNA qPCR assays.

    In each of the three biological replicates, 20 pg was the minimum amount of DNA that could be detected by each LNA probe (Table 4; Supplementary Figure S2). When DNA concentrations of V6 and non-V6 isolates were equal, the LNA assay performed similarly whether running a two-isolate mixture of differing race or a reaction with only a single isolate (Table 4). During a single-race reaction, either the HEX or FAM fluorophore was detected per well, depending on the race of the template DNA added to that well. During the mixed-race reaction, both the FAM and HEX fluorophores were detected in each well. Cq values were comparable with those observed during the single-race limit of detection reactions (Table 4). The assay was able to detect DNA from both samples even when the DNA sample concentrations within the mixed-race sample differed by more than one order of magnitude.

    When the duplex assay was tested with DNA samples from other powdery mildew fungi, amplification occurred with all powdery mildews except those isolated from Eucalyptus. Samples taken from Plantago major had amplification with both probes with values below the 38 Cq threshold but with RFU values around 1,500. Powdery mildew collected from Vitis vinifera, Zinnia sp., and Fragaria sp. had nonspecific amplification with both probes with Cq values at or below the 38 Cq threshold (Table 5). The rest of the samples had weak amplification curves with less than 1,500 RFU and were regarded as potential background noise. DNA from other hop associated organisms and the hop plant tissue did not amplify (data not shown).


    The goal of this project was to develop a molecular diagnostic assay capable of distinguishing V6 isolates of P. macularis from other races of the hop powdery mildew fungus. At this time, V6 isolates of P. macularis have been predominately found in the Pacific Northwestern region of the United States where there has been broad deployment of hop cultivars containing R6-based resistance (Wolfenbarger et al. 2016). Quick and accurate race detection of plant pathogens, especially of powdery mildew fungi, can aid hop growers in disease management decisions as well as provide important information in epidemiological studies involving population structure dynamics and pathogen dispersal. This assay could offer a more rapid and accessible alternative to race detection than methods currently used for characterizing P. macularis that generally require multiple weeks for definitive results (Gent et al. 2017; Wolfenbarger et al. 2016).

    After analyzing the transcriptomes of 46 isolates of P. macularis collected in the Pacific Northwest, 16 candidate loci were identified that differentiate V6 isolates from the other two races widely prevalent in this region. Fourteen primer pairs were designed from these distinctive loci and tested with preliminary conventional PCR screening with four Pacific Northwestern P. macularis isolates with known virulence. We selected two contigs (contigs 2251 and 2407) that yielded single amplicons in conventional PCR assays and contained SNPs at the predicted locations following sequence verification.

    The assay was population-specific to P. macularis isolates collected from hop plants in the Pacific Northwestern region of the U.S. The assay did not predict V6-virulence in three isolates collected from Germany. Contig 2407 used for assay development contains an SNP that is associated with V6-virulence in the Pacific Northwest, but this SNP is not necessarily a causal mutation that confers virulence to the pathogen. As this assay was designed from isolates collected solely from the Pacific Northwest, it is not unexpected that V6 isolates originating from other countries were not detected. The mutation that confers V6-virulence to P. macularis isolates originating from the Pacific Northwestern U.S. likely originated from an extant isolate in this region (Gent et al. 2020) that subsequently became widespread following a widespread planting of R6-based resistant hop varieties in commercial fields. Multiple mutations or other mechanisms could lead to similar phenotypes in different populations.

    The assay we designed is constrained by the amount of fungal DNA needed to detect the presence of P. macularis, with the limit of detection approximately 20 pg. Therefore, this assay would not be appropriate to use in applications such as environmental sampling that requires sensitivity near the single conidium level (Gent et al. 2009; Mahaffee and Stoll 2016; Thiessen et al. 2016). Rather, this assay is appropriate for determining pathogen race from colonies growing on hop tissue. The amount of DNA extracted from one colony (approximately 0.5 cm in diameter) of P. macularis is approximately 730 pg, which would more than suffice for the race determination of samples collected from a hop yard.

    The differentially labeled probes can detect the presence of both V6 and non-V6 isolates simultaneously, which would be helpful in rapidly determining pathogen race when powdery mildew occurs on hop cultivars that do not possess R6-based resistance. This would not have been feasible without considerable effort using traditional inoculation-based race screening methods. Determining whether mixed infections are present requires inoculations onto multiple cultivars, a differential cultivar with R6, and also a susceptible cultivar that permits growth of non-V6-virulent isolates. Colonies that form on the latter would then need to be purified and reinoculated to isolate individuals that lack V6-virulence. This tedious process is not amenable to the scaling up required in a research context where hundreds of samples must be processed.

    Since 2012, the prevalence of V6 isolates of P. macularis has increased throughout the primary hop growing areas in the western U.S. (Wolfenbarger et al. 2016). Our assay has utility in epidemiological studies to quantify regional differences in prevalence of V6 isolates on certain cultivars. Wolfenbarger et al. (2016) previously observed regional differences in the prevalence of P. macularis isolates with V6-virulence on hop cultivars lacking R6 depending on overall disease severity in a region. Our assay could facilitate further investigation of factors influencing propagule density of V6-virulent isolates on certain cultivars.

    Aggressiveness of certain isolates may depend on the cultivar (Wolfenbarger et al. 2016). This has implications for hop breeding programs in that the assay could be used to identify pathogen race on germplasm and could lead to more directed selection with cultivars with R6-based resistance as parental stock. This assay can also be applied as a rapid diagnostic tool to detect and confirm the spread of V6 isolates from the Pacific Northwest to other regions on planting material, which appears to occur within and between production regions (Gent et al. 2020). Further, the primers developed for this assay have been adapted for an amplicon sequencing platform to massively scale genotyping (Weldon et al. 2021).

    While the assay can accurately detect and differentiate between V6 and non-V6 isolates of P. macularis derived from the Pacific Northwest, the assay does not differentiate between the pre-2012 and ‘Cascade’-adapted races of the fungus. The pre-2012 and ‘Cascade’-adapted races of the fungus may infect plants possessing Rb, R3, and R5, with the latter additionally overcoming the partial resistance in Cascade (Gent et al. 2017). There would be utility in further differentiation and identification of the three known races of P. macularis known to occur in the Pacific Northwest for the above-mentioned reasons. Future work will involve analyzing transcriptome variants for the development of an assay specific to the ‘Cascade’-adapted race of P. macularis.

    The author(s) declare no conflict of interest.

    Literature Cited

    Disclaimer: The use of trade, firm, or corporation names in this publication is for the information and convenience of the reader. Such use does not constitute an official endorsement or approval by the United States Department of Agriculture or the Agricultural Research Service of any product or service to the exclusion of others that may be suitable.

    The author(s) declare no conflict of interest.

    Funding: This research was conducted in support of USDA-ARS CRIS 5358-21000-040-00D and 2072-44822000-041-00-D.