A Greenhouse Method to Evaluate Sunflower Quantitative Resistance to Basal Stalk Rot Caused by Sclerotinia sclerotiorum.

Resistance of sunflower to basal stalk rot (BSR) caused by the fungus Sclerotinia sclerotiorum is quantitative, controlled by multiple genes contributing small effects. Consequently, artificial inoculation procedures allowing sufficient throughput and resolution of resistance are needed to identify highly resistant sunflower germplasm resources and to map loci contributing to resistance. The objective of this study was to develop a greenhouse-based method for evaluating sunflower quantitative resistance to BSR that would be simple, space- and time-efficient, high throughput, high resolution, and correlated with field observations. Experiments were conducted with 5-week-old sunflower plants and Sclerotinia-infested millet seed as inoculum to assess the impact of pot size and temperature and to determine the most favorable inoculum rate and placement. Subsequently, an additional experiment was performed to assess the correlation of the greenhouse inoculation procedure with field results by using a panel of 32 sunflower genotypes with known field response to BSR previously determined in multiyear, multilocation artificially inoculated trials. Experimental observations indicated that the newly developed greenhouse inoculation procedure provided improved resolution to identify highly resistant genotypes and was strongly correlated with field observations. This method will be useful for screening of sunflower experimental and breeding materials, disease phenotyping of genetic mapping populations, and evaluation of resistance to different pathogen isolates.

Occurrence in Seeds and Potential Seed Transmission of Xanthomonas vasicola pv. vasculorum in Maize in the United States.

This paper reports original evidence regarding the potential role of seed transmission of Xanthomonas vasicola pv. vasculorum in the epidemiology of bacterial leaf streak (BLS) in maize. We evaluated the occurrence of the pathogen on seeds from diseased fields and its subsequent transmission to seedlings. In 2016 and 2017, X. vasicola pv. vasculorum was detected by TaqMan PCR from 22 of 41 maize seed lots harvested from naturally infected fields in Colorado, Nebraska, and Iowa. However, many of the PCR-positive samples did not yield culturable X. vasicola pv. vasculorum colonies. The highest levels of seed contamination were detected in dent maize and popcorn from NE and CO. Seed transmission was evaluated in greenhouse grow-outs from eight seed lots, totaling more than 14,000 plants. Putative seed transmission events from naturally contaminated seed lots, estimated from PCR results, occurred at a frequency between 0.1 and 0.5% in 10-seedling pooled samples and at a frequency of 2.7% from individual plant assays. However, no seedling symptoms were observed during these assays and live X. vasicola pv. vasculorum colonies were not recovered from PCR-positive seedlings. In contrast, seed transmission was readily demonstrated from artificially contaminated seed lots, including typical symptoms and recovery of live bacteria. Seed transmission consistently occurred from seeds soaked in bacterial suspensions with concentrations of ≥106 CFU/ml, suggesting that a threshold population of the bacterium is necessary for the development of BLS symptoms and recovery of live bacteria. The low bacterial populations on naturally contaminated seeds apparently were not sufficient to result in diseased seedlings.

Genomics-Informed Molecular Detection of Xanthomonas vasicola pv. vasculorum Strains Causing Severe Bacterial Leaf Streak of Corn.

Xanthomonas vasicola pv. vasculorum (syn. X. campestris pv. vasculorum) was initially identified as the causal agent of bacterial leaf streak of corn in South Africa. The pathovar vasculorum causes disease on sugarcane and corn, but a subset of these strains was noted for its increased disease severity in corn. This subset was reclassified as X. campestris pv. zeae in the early 1990s and was found to have slightly different biochemical and genetic properties than isolates from sugarcane. There has been an emergence of X. campestris pv. zeae-like strains of X. vasicola pv. vasculorum in both the United States and Argentina since 2010. We performed whole genome sequencing on U.S. isolates to confirm their identity. Informed by comparative genomics, we then developed specific TaqMan qPCR and loop-mediated isothermal amplification (LAMP) assays for the detection of this specific subset of X. vasicola pv. vasculorum strains. The qPCR 4909 assay was tested against 27 xanthomonads (diverse representation), 32 DNA extractions from corn leaves confirmed as positive or negative for the bacterium, 41 X. vasicola pv. vasculorum isolates from corn in the United States and Argentina, and 31 additional bacteria associated with corn, sugarcane, or sorghum. In all cases the assay was shown to be specific for the X. vasicola pv. vasculorum isolates that cause more severe disease on corn. We then tested the LAMP 166 assay against the 27 xanthomonads and 32 corn leaf DNA samples, and we found this assay was also specific for this subset of X. vasicola pv. vasculorum isolates. We also developed a live/dead cells distinction protocol using propidium monoazide prior to DNA extraction for analyzing seed washes using these assays. These two detection assays can be useful for both diagnosticians and researchers to specifically identify the X. vasicola pv. vasculorum isolates that cause more severe symptoms on corn.

A Real-Time PCR Differentiating Pantoea stewartii subsp. stewartii From P. stewartii subsp. indologenes in Corn Seed.

Stewart's wilt of corn caused by the bacterium Pantoea stewartii subsp. stewartii is a seed-borne disease of major phytosanitary importance. Many countries have imposed restrictions on corn seed imports from regions where the disease occurs to prevent the potential introduction of the pathogen. Current laboratory testing methods (enzyme-linked immunosorbent assay [ELISA] and polymerase chain reaction [PCR]) cannot readily distinguish P. stewartii subsp. stewartii from the closely related subspecies Pantoea stewartii subsp. indologenes. However, P. stewartii subsp. indologenes, a nonpathogen on corn, is occasionally found on corn seed as part of the resident bacterial population and can yield false positive test results. A real-time PCR targeting the cpsAB intergenic sequence was developed to specifically detect P. stewartii subsp. stewartii from corn seeds and distinguish it from P. stewartii subsp. indologenes. The assay successfully detected P. stewartii subsp. stewartii from corn seed, and P. stewartii subsp. indologenes-contaminated seed lots, which previously yielded false positives by ELISA and published PCR methods, were negative. The absence of P. stewartii subsp. stewartii and the presence of P. stewartii subsp. indologenes in this seed were confirmed by size differentiation of the cpsAB amplicons in a conventional PCR. By distinguishing the two subspecies, the assays described would avoid false positive results and help prevent unnecessary restrictions on international movement of corn seed.

Comprehensive Disease Survey of U.S. Sunflower: Disease Trends, Research Priorities and Unanticipated Impacts.

Between 2002 and 2015, a comprehensive survey of sunflower fields across seven Midwestern U.S. states was conducted 12 times and continues to be conducted every other year. The surveyors collected data on yield, agronomic management factors, disease, insect, weed, and bird damage. All surveyors were volunteers and came from universities (extension and research staff), USDA-ARS, and seed and chemical companies. In the 12 years the survey was conducted, data from 2,267 fields were collected. The results are presented annually at the National Sunflower Association Research Forum and are used to set sunflower research priorities. While 10 diseases are surveyed annually, we focus this article on the importance, findings, implications, and impacts of the five most important: downy mildew, Phomopsis stem canker, rust, Rhizopus head rot, and Sclerotinia head rot. This survey is unique among field crops in both scope and scale, and this manuscript discusses salient and clandestine benefits of intense and long-term disease surveys.

Re-evaluation of Seed Transmission of Clavibacter michiganensis subsp. nebraskensis in Zea mays.

The spread of Goss's bacterial wilt and leaf blight of corn (Zea mays), caused by Clavibacter michiganensis subsp. nebraskensis, to a wider geographic range in the early 2000s compared with the late 1960s has generated concern about the possible role of seed transmission in long-distance spread. The objectives of this research were: (1) to determine the percentage of seed infection found in seed harvested from inoculated and noninoculated plants of hybrids that varied in resistance to Goss's wilt; and (2) to estimate the seed transmission rate from these infected seed lots. The greatest percent seed infection was detected in seed from inoculated plants of the most susceptible hybrid and the least in seed from the most resistant hybrid. Seed lots with seed infection that ranged from 3.6 to 37.0% were planted in three field and three greenhouse trials. A total of 12 seed transmission events (Goss's wilt symptomatic seedlings) were identified among 241,850 plants examined, for a seed transmission rate of 0.005%. When the seed transmission rate was recalculated to consider only the infected seed portion of each seed lot, the rate increased to 0.040% (12 events from 30,088 potentially infected plants). Based on the low seed transmission rate observed and previous research on disease spread from a point source, it seems unlikely that seed transmission could introduce enough inoculum to create a serious disease outbreak in a single growing season. However, risk of seed transmission is relevant for phytosanitary restrictions and preventing the introduction of the pathogen to new areas. To date, Goss's wilt has not been detected outside North America, and while the risk of seed transmission is very low, the risk is not zero. Fortunately, the presence of C. michiganensis subsp. nebraskensis in corn seed is readily detectable by established seed health testing methods.

Genotyping-by-Sequencing Uncovers the Introgression Alien Segments Associated with Sclerotinia Basal Stalk Rot Resistance from Wild Species-I. Helianthus argophyllus and H. petiolaris.

Basal stalk rot (BSR), caused by Sclerotinia sclerotiorum, is a devastating disease in sunflower worldwide. The progress of breeding for Sclerotinia BSR resistance has been hampered due to the lack of effective sources of resistance for cultivated sunflower. Our objective was to transfer BSR resistance from wild annual Helianthus species into cultivated sunflower and identify the introgressed alien segments associated with BSR resistance using a genotyping-by-sequencing (GBS) approach. The initial crosses were made between the nuclear male sterile HA 89 with the BSR resistant plants selected from wild Helianthus argophyllus and H. petiolaris populations in 2009. The selected resistant F1 plants were backcrossed to HA 458 and HA 89, respectively. Early generation evaluations of BSR resistance were conducted in the greenhouse, while the BC2F3 and subsequent generations were evaluated in the inoculated field nurseries. Eight introgression lines; six from H. argophyllus (H.arg 1 to H.arg 6), and two from H. petiolaris (H.pet 1 and H.pet 2), were selected. These lines consistently showed high levels of BSR resistance across seven environments from 2012 to 2015 in North Dakota and Minnesota, USA. The mean BSR disease incidence (DI) for H.arg 1 to H.arg 6, H.pet 1, and H.pet 2 was 3.0, 3.2, 0.8, 7.2, 7.7, 1.9, 2.5, and 4.4%, compared to a mean DI of 36.1% for Cargill 270 (susceptible hybrid), 31.0% for HA 89 (recurrent parent), 19.5% for HA 441 (resistant inbred), and 11.6% for Croplan 305 (resistant hybrid). Genotyping of the highly BSR resistant introgression lines using GBS revealed the presence of the H. argophyllus segments in linkage groups (LGs) 3, 8, 9, 10, and 11 of the sunflower genome, and the H. petiolaris segments only in LG8. The shared polymorphic SNP loci in the introgression lines were detected in LGs 8, 9, 10, and 11, indicating the common introgression regions potentially associated with BSR resistance. Additionally, a downy mildew resistance gene, Pl17 , derived from one of the parents, HA 458, was integrated into five introgression lines. Germplasms combining resistance to Sclerotinia BSR and downy mildew represent a valuable genetic source for sunflower breeding to combat these two destructive diseases.