Alison E. Robertson

Corn yield loss estimates due to diseases in the United States and Ontario, Canada from 2012 to 2015.

Annual decreases in corn yield caused by diseases were estimated by surveying members of the Corn Disease Working Group in 22 corn-producing states in the United States and in Ontario, Canada, from 2012 through 2015. Estimated loss from each disease varied greatly by state and year. In general, foliar diseases such as northern corn leaf blight, gray leaf spot, and Goss’s wilt commonly caused the largest estimated yield loss in the northern United States and Ontario during nondrought years. Fusarium stalk rot and plant-parasitic nematodes caused the most estimated loss in the southernmost United States. The estimated mean economic loss due to yield loss by corn diseases in the United States and Ontario from 2012 to 2015 was

Oomycete Species Associated with Soybean Seedlings in North America—Part I: Identification and Pathogenicity Characterization

76.51 USD per acre. The cost of disease-mitigating strategies is another potential source of profit loss. Results from this survey will provide scientists, breeders, government, and educators with data to help inform and prioritize research, policy, and educational efforts in corn pathology and disease management. M U E L L E R E T A L . , P L A N T H E A L T H P R O G R E S S 1 7 (2 0 1 6 )

Temperature Affects Aggressiveness and Fungicide Sensitivity of Four Pythium spp. that Cause Soybean and Corn Damping Off in Iowa

Oomycete pathogens are commonly associated with soybean root rot and have been estimated to reduce soybean yields in the United States by 1.5 million tons on an annual basis. Limited information exists regarding the frequency and diversity of oomycete species across the major soybean-producing regions in North America. A survey was conducted across 11 major soybean-producing states in the United States and the province of Ontario, Canada. In 2011, 2,378 oomycete cultures were isolated from soybean seedling roots on a semiselective medium (CMA-PARPB) and were identified by sequencing of the internal transcribed spacer region of rDNA. Sequence results distinguished a total of 51 Pythium spp., three Phytophthora spp., three Phytopythium spp., and one Aphanomyces sp. in 2011, with Pythium sylvaticum (16%) and P. oopapillum (13%) being the most prevalent. In 2012, the survey was repeated, but, due to drought conditions across the sampling area, fewer total isolates (n = 1,038) were collected. Additionally, in 2012, a second semiselective medium (V8-RPBH) was included, which increased the Phytophthora spp. isolated from 0.7 to 7% of the total isolates. In 2012, 54 Pythium spp., seven Phytophthora spp., six Phytopythium spp., and one Pythiogeton sp. were recovered, with P. sylvaticum (14%) and P. heterothallicum (12%) being recovered most frequently. Pathogenicity and virulence were evaluated with representative isolates of each of the 84 species on soybean cv. Sloan. A seed-rot assay identified 13 and 11 pathogenic species, respectively, at 13 and 20°C. A seedling-root assay conducted at 20°C identified 43 species as pathogenic, having a significantly detrimental effect on the seedling roots as compared with the noninoculated control. A total of 15 species were pathogenic in both the seed and seedling assays. This study provides a comprehensive characterization of oomycete species present in soybean seedling roots in the major production areas in the United States and Ontario, Canada and provides a basis for disease management and breeding programs.

Oomycete Species Associated with Soybean Seedlings in North America—Part II: Diversity and Ecology in Relation to Environmental and Edaphic Factors

Damping off of soybean and corn, caused by Pythium spp., is favored by cool temperatures and wet soil conditions and is primarily managed using fungicide seed treatments. The goal of this research was to determine the effect of temperature on aggressiveness and fungicide sensitivity of Pythium spp. recovered from soybean and corn in Iowa. A total of 21 isolates of four of the most prevalent Pythium spp. in Iowa were screened. Seed and seedling assays were used to quantify the aggressiveness of P. lutarium, P. oopapillum, P. sylvaticum, and P. torulosum on soybean and corn at 13, 18, and 23°C. Isolates recovered from soybean or corn were equally pathogenic on both hosts. P. torulosum was more aggressive at 13°C compared with 18 and 23°C. Conversely, P. sylvaticum was more aggressive at 18 and 23°C than at 13°C. A plate assay was used to assess fungicide sensitivity to seven fungicides that are commonly used as seed treatments, and EC50 values at each of the three temperatures were determined and compared. EC50 values for P. torulosum were higher for all fungicides tested at 13°C, compared with 18 or 23°C, whereas EC50 values for P. sylvaticum were higher for all fungicides at 18 and 23°C compared with 13°C. These data contribute to our understanding of the effect of soil temperature on the risk of soybean and corn damping off, which may aid in the development of more effective management practices.

A Novel Phytophthora sojae Resistance Rps12 Gene Mapped to a Genomic Region That Contains Several Rps Genes

Soybean (Glycine max (L.) Merr.) is produced across a vast swath of North America, with the greatest concentration in the Midwest. Root rot diseases and damping-off are a major concern for production, and the primary causal agents include oomycetes and fungi. In this study, we focused on examination of oomycete species distribution in this soybean production system and how environmental and soil (edaphic) factors correlate with oomycete community composition at early plant growth stages. Using a culture-based approach, 3,418 oomycete isolates were collected from 11 major soybean-producing states and most were identified to genus and species using the internal transcribed spacer region of the ribosomal DNA. Pythium was the predominant genus isolated and investigated in this study. An ecology approach was taken to understand the diversity and distribution of oomycete species across geographical locations of soybean production. Metadata associated with field sample locations were collected using geographical information systems. Operational taxonomic units (OTU) were used in this study to investigate diversity by location, with OTU being defined as isolate sequences with 97% identity to one another. The mean number of OTU ranged from 2.5 to 14 per field at the state level. Most OTU in this study, classified as Pythium clades, were present in each field in every state; however, major differences were observed in the relative abundance of each clade, which resulted in clustering of states in close proximity. Because there was similar community composition (presence or absence) but differences in OTU abundance by state, the ordination analysis did not show strong patterns of aggregation. Incorporation of 37 environmental and edaphic factors using vector-fitting and Mantel tests identified 15 factors that correlate with the community composition in this survey. Further investigation using redundancy analysis identified latitude, longitude, precipitation, and temperature as factors that contribute to the variability observed in community composition. Soil parameters such as clay content and electrical conductivity also affected distribution of oomycete species. The present study suggests that oomycete species composition across geographical locations of soybean production is affected by a combination of environmental and edaphic conditions. This knowledge provides the basis to understand the ecology and distribution of oomycete species, especially those able to cause diseases in soybean, providing cues to develop management strategies.

Infection of Maize by Clavibacter michiganensis subsp. nebraskensis Does Not Require Severe Wounding

Phytophthora sojae Kaufmann and Gerdemann, which causes Phytophthora root rot, is a widespread pathogen that limits soybean production worldwide. Development of Phytophthora resistant cultivars carrying Phytophthora resistance Rps genes is a cost-effective approach in controlling this disease. For this mapping study of a novel Rps gene, 290 recombinant inbred lines (RILs) (F7 families) were developed by crossing the P. sojae resistant cultivar PI399036 with the P. sojae susceptible AR2 line, and were phenotyped for responses to a mixture of three P. sojae isolates that overcome most of the known Rps genes. Of these 290 RILs, 130 were homozygous resistant, 12 heterzygous and segregating for Phytophthora resistance, and 148 were recessive homozygous and susceptible. From this population, 59 RILs homozygous for Phytophthora sojae resistance and 61 susceptible to a mixture of P. sojae isolates R17 and Val12-11 or P7074 that overcome resistance encoded by known Rps genes mapped to Chromosome 18 were selected for mapping novel Rps gene. A single gene accounted for the 1:1 segregation of resistance and susceptibility among the RILs. The gene encoding the Phytophthora resistance mapped to a 5.8 cM interval between the SSR markers BARCSOYSSR_18_1840 and Sat_064 located in the lower arm of Chromosome 18. The gene is mapped 2.2 cM proximal to the NBSRps4/6-like sequence that was reported to co-segregate with the Phytophthora resistance genes Rps4 and Rps6. The gene is mapped to a highly recombinogenic, gene-rich genomic region carrying several nucleotide binding site-leucine rich repeat (NBS-LRR)-like genes. We named this novel gene as Rps12, which is expected to be an invaluable resource in breeding soybeans for Phytophthora resistance.

Dissemination of Goss’s Wilt of Corn and Epiphytic Clavibacter michiganensis subsp. nebraskensis from Inoculum Point Sources

Gosss bacterial wilt and leaf blight of maize is caused by Clavibacter michiganensis subsp. nebraskensis. Infested residue is the primary source of inoculum and infection occurs via wounds caused by sand blasting, hail, or wind damage. The pathogen survives as an epiphyte on maize leaves and, because the disease has been observed on plants in the field with no obvious wounding, we wondered whether infection by epiphytic C. michiganensis subsp. nebraskensis and disease development could occur in the absence of severe wounding. Consequently, greenhouse experiments were done to evaluate disease development in the absence of wounding in ambient and increased humidity conditions. Maize plants at the V4 to V5 crop development stage were spray inoculated with a suspension of C. michiganensis subsp. nebraskensis (108 cells ml-1). Leaf blight incidence was assessed on whole plants and individual leaves; epiphytic populations of C. michiganensis subsp. nebraskensis were monitored by dilution plating of leaf washes; and epiphytic C. michiganensis subsp. nebraskensis colonization was visualized using scanning electron microscopy (SEM). Gosss leaf blight symptoms were observed on nonwounded plants in ambient (37.0% plant incidence) and increased humidity conditions (60.0% plant incidence). Populations of epiphytic C. michiganensis subsp. nebraskensis survived and increased on maize leaves, particularly at increased humidity. We observed C. michiganensis subsp. nebraskensis colonizing maize leaves in localized sites that included epidermal junctions, cuticle depressions, in and around stomata, and at the base of trichomes. Single cells and aggregates of C. michiganensis subsp. nebraskensis were observed within substomatal chambers using SEM. These data indicate that severe wounding is not necessary for C. michiganensis subsp. nebraskensis infection of maize, and stomata or trichomes may serve as entry points for the bacterium.

Analysis of Extreme Phenotype Bulk Copy Number Variation (XP-CNV) Identified the Association of rp1 with Resistance to Goss's Wilt of Maize

Gosss wilt of corn, caused by Clavibacter michiganensis subsp. nebraskensis, has reemerged since 2006 as an economically important disease of corn in in the Midwestern United States. In 2012 and 2013, field plot studies were conducted with a pathogenic, rifampicin-resistant C. michiganensis subsp. nebraskensis isolate and a Gosss wilt-susceptible corn hybrid to monitor epiphytic C. michiganensis subsp. nebraskensis population densities and the temporal and spatial spread of Gosss wilt incidence originating from inoculum point sources. The randomized complete block trial included three treatments: noninoculated control, inoculum point sources established by wound inoculation, and inoculum point sources consisting of C. michiganensis subsp. nebraskensis-infested corn residue. Epiphytic C. michiganensis subsp. nebraskensis was detected on asymptomatic corn leaves collected up to 2.5 m away from inoculum sources at 15 days after inoculation in both years. The percentage of asymptomatic leaf samples on which epiphytic C. michiganensis subsp. nebraskensis was detected increased until mid-August in both years, and reached 90, 55, and 35% in wound-, residue-, and noninoculated plots, respectively, in 2012; and 50, 11, and 2%, respectively, in 2013. Although both growing seasons were drier than normal, Gosss wilt incidence increased over time and space from all C. michiganensis subsp. nebraskensis point sources. Plots infested with C. michiganensis subsp. nebraskensis residue had final Gosss wilt incidence of 7.5 and 1.8% in 2012 and 2013, respectively; plots with a wound-inoculated source had final Gosss wilt incidence of 16.6 and 14.0% in 2012 and 2013, respectively. Our findings suggest that relatively recent outbreaks of Gosss wilt in new regions of the United States may be the result of a gradual, nondetected buildup of C. michiganensis subsp. nebraskensis inoculum in fields.

Comparison of Susceptible and Resistant Maize Hybrids to Colonization by Clavibacter michiganensis subsp. nebraskensis

Gosss wilt (GW) of maize is caused by the Gram-positive bacterium Clavibacter michiganensis subsp. nebraskensis (Cmn) and has spread in recent years throughout the Great Plains, posing a threat to production. The genetic basis of plant resistance is unknown. Here, a simple method for quantifying disease symptoms was developed and used to select cohorts of highly resistant and highly susceptible lines known as extreme phenotypes (XP). Copy number variation (CNV) analyses using whole genome sequences of bulked XP revealed 141 genes containing CNV between the two XP groups. The CNV genes include the previously identified common rust resistant locus rp1. Multiple Rp1 accessions with distinct rp1 haplotypes in an otherwise susceptible accession exhibited hypersensitive responses upon inoculation. GW provides an excellent system for the genetic dissection of diseases caused by closely related subspecies of C. michiganesis. Further work will facilitate breeding strategies to control GW and provide needed insight into the resistance mechanism of important related diseases such as bacterial canker of tomato and bacterial ring rot of potato.

Perceptions of Midwestern Crop Advisors and Growers on Foliar Fungicide Adoption and Use in Maize

Clavibacter michiganensis subsp. nebraskensis causes Gosss bacterial wilt and blight on maize and is managed primarily with C. michiganensis subsp. nebraskensis-resistant hybrids. To understand the mechanisms of resistance to infection by C. michiganensis subsp. nebraskensis, leaves of a susceptible and a resistant maize hybrid at the V4 to V5 developmental stage were wound inoculated with the pathogen. Blight lesion length was monitored, C. michiganensis subsp. nebraskensis colonizing ability was determined, and structural changes were observed using microscopy. Bacterial colonization preceded lesion development that occurred 4 to 5 days postinoculation in both hybrids. Lesion expansion in the susceptible hybrid was associated with a faster rate of C. michiganensis subsp. nebraskensis spread and multiplication in the tissues. In the resistant hybrid, spread and multiplication was reduced (P < 0.0001) and, at 16 days postinoculation, became imperceptible. Initially, C. michiganensis subsp. nebraskensis showed a preference for colonization of the metaxylem vessels in both hybrids. Spread from cell to cell was accomplished through disruption of cell walls, presumably from abundance of bacterial cells or enzymatic activity. Morphological responses of the resistant maize hybrid to infection by C. michiganensis subsp. nebraskensis were similar to those reported in maize inbred lines that were resistant to Stewarts wilt caused by Pantoea stewartii. Resistance to C. michiganensis subsp. nebraskensis was associated with production of a dense matrix in the xylem that deformed and restricted movement of the bacterial cells.