Management of Soybean Cyst Nematode and Sudden Death Syndrome with Nematode-Protectant Seed Treatments Across Multiple Environments in Soybean.

As soybean (Glycine max) production continues to expand in the United States and Canada, so do pathogens and pests that directly threaten soybean yield potential and economic returns for farmers. One such pathogen is the soybean cyst nematode (SCN; Heterodera glycines). SCN has traditionally been managed using SCN-resistant cultivars and rotation with nonhost crops, but the interaction of SCN with sudden death syndrome (SDS; caused by Fusarium virguliforme) in the field makes management more difficult. Nematode-protectant seed treatments have become options for SCN and SDS management. The objectives of this study were to evaluate nematode-protectant seed treatments for their effects on (i) early and full season SCN reproduction, (ii) foliar symptoms and root-rot caused by SDS, and (iii) soybean yield across environments accounting for the above factors. Using a standard protocol, field trials were implemented in 13 states and one Canadian province from 2019 to 2021 constituting 51 site-years. Six nematode-protectant seed treatment products were compared with a fungicide + insecticide base treatment and a nontreated check. Initial (at soybean planting) and final (at soybean harvest) SCN egg populations were enumerated, and SCN females were extracted from roots and counted at 30 to 35 days postplanting. Foliar disease index (FDX) and root rot caused by the SDS pathogen were evaluated, and yield data were collected for each plot. No seed treatment offered significant nematode control versus the nontreated check for in-season and full-season nematode response, no matter the initial SCN population or FDX level. Of all treatments, ILEVO (fluopyram) and Saltro (pydiflumetofen) provided more consistent increases in yield over the nontreated check in a broader range of SCN environments, even when FDX level was high.

First report of Phyllachora maydis causing tar spot on corn in Delaware.

In October 2023, lesions consistent with descriptions of tar spot (Phyllachora maydis) were observed on corn (Zea mays) in Kent and Sussex County, Delaware (DE). Black, raised stromata were observed on leaves of commercially grown corn hybrids. Plants were at physiological maturity and disease severity was low with symptoms present on 1 to 10% of plants. In collected tissue, individual leaf severities ranged from 1 to 3% of leaf area with lesions. Hyaline conidia measuring approximately 15.5 µm in length and 0.5 µm in width were observed microscopically (n=5). Stromata were excised and sterilized in a 0.825% sodium hypochlorite solution for 30 s, rinsed in sterile deionized water for 30 s, and dried on a sterile paper towel for 30 s. Tissues were ground in a 1.5 mL microcentrifuge tube with a sterile plastic pestle. DNA was extracted using a DNeasy Plant Mini Kit (Qiagen). DNA was amplified at the internal transcribed spacer (ITS) region with ITS4 and ITS5 primers using polymerase chain reaction (PCR). NCBI BLAST search results yielded 100% sequence homology and 100% query cover (350/515 bp) to P. maydis accession MG881848.1 (Moura et al. 2023). Koch's postulates could not be completed due to the obligate nature of P. maydis. Tarspot was initially discovered in the United States in 2016 in Indiana and Illinois (Ruhl et al. 2016).This is the first report of tar spot on corn in DE. Yield losses from P. maydis can range depending on time of infection, environmental factors, and hybrid susceptibility and have been recorded up to 100% (Rocco da Silva et al. 2021). Because the disease did not enter the area until the end of the season, no yield impact was observed for 2023. Monitoring for the progression of disease will be crucial for future seasons (Telenko et al. 2020). High humidity and moisture levels favor disease development. Approximately half of DE corn acreage is irrigated due to sandy soils, current irrigation timing strategies may need to be reevaluated. Fungicide efficacy trials for management of tar spot have been conducted in other regions, but continued research will be needed to assess management options and optimize application timing for farmers in DE and the Mid-Atlantic region.

Seed Treatments for Management of Soybean Cyst Nematode, Heterodera glycines, in Mid-Atlantic Soybean Production.

Soybean Cyst Nematode (SCN), Heterodera glycines Ichinohe, is the most important pathogen of soybean in the Mid-Atlantic region. In recent decades, a decline in the effectiveness of genetic resistance has been observed and additional management approaches are needed. Seed treatments are of rising interest, but no local data on product response exists for the region. In 2020-2021, two experiments were conducted to observe the effects of chemical and biological seed treatment options. In one experiment, chemical seed treatments pydiflumetofen (Saltro®) and fluopyram (ILEVO®) were screened against nontreated plain seed for SCN suppression. In a second experiment, pydiflumetofen, fluopyram and four biological nematode-protectant seed treatments with a standard base insecticide and fungicide treatment were compared to nontreated plain seed and seed with only the standard base treatment to test product efficacy against SCN. Seed treatments increased the percent emergence over plain seed. Nematode reproductive factors and female counts from roots were collected, but did not statistically differ between seed treatments or plain seed. Yield differences were observed in one of the five trials, where pydiflumetofen + base seed treatment yielded the highest (p < 0.001) at 3813.1 kg/ha. Response from seed treatments varied, with no specific seed treatment consistently reducing SCN populations or increasing yield across trials. Seed treatments may have potential as an element of an integrated management approach for SCN.

First report of Rhizoctonia solani AG 4 causing brown bean of lima bean in Delaware.

Lima bean production has been an economically valuable staple in Delaware agriculture for almost a century, with annual revenue approaching 8 million dollars (USDA-NASS, 2019; Evans et al. 2007). From 2019-2021, lima beans displaying symptoms of brown discoloration, referred to as "brown bean" were observed in the green baby lima variety 'Cypress' across multiple commercial and research fields. Symptoms were present in approximately 1-5% of beans and not visible until pods were opened for harvest. Thirty-seven symptomatic beans were collected and surface disinfested in 0.85% sodium hypochlorite for 30 s, rinsed in sterile deionized water for 30 s, sectioned into four pieces and plated onto potato dextrose agar (PDA) amended with 50 µg/ml penicillin G and streptomycin sulfate. Petri dishes were incubated at 23ºC and observed for colony morphology. Pure cultures were obtained with tan colonies that had mycelia with right angle branching and septations near the branch, consistent with the description of Rhizoctonia solani Kuhn (Sneh et al. 1991). DNA extraction and pathogen identification was confirmed by sequencing of the internal transcribed spacer (ITS) region of nuclear ribosomal DNA using primers ITS4/ITS5 (White et al. 1990) for thirty-seven isolates collected in 2019 and 2020. Isolates were identified as Rhizoctonia solani AG 4 (99.9% sequence identity with GenBank Accession [MN106359.1].) A representative isolate was selected to complete Koch's postulates and the sequence was deposited in GenBank as accession number MW560551. To observe colonization ability, 10 detached pods were sterilized in 75% EtOH for 60 s, then rinsed in Milli-Q water. The detached pods were divided among two 150 mm Petri dishes containing a single 150 mm filter paper saturated with Milli-Q water. Five 1 mm2 agar plugs colonized with the representative R. solani isolate were placed 0.5 cm apart along the length of the pod. Plates were sealed with parafilm and left at room temperature. Control pods were kept in identical conditions but inoculated using clean agar plugs. The trial was repeated and a second trial was conducted on 12 attached asymptomatic pods from C-Elite Select lima bean plants at the succulent seed stage to complete Koch's Postulates. Pods were surface disinfested with 70% ethanol. Three attached pods were wounded with the tip of a sterile scalpel blade where a colonized agar plug was placed and loosely wrapped with a thin parafilm layer to maintain contact. Three attached pods not wounded were also inoculated with a colonized agar plug and wrapped by parafilm. Three wounded and non-wounded pods received clean agar plug controls. Both attached and detached pods were kept at room temperature for one week until symptoms began to manifest on the pod surfaces, at which point the beans from infected pods were removed and placed on PDA, three to a plate. In the attached assay, all beans of both wounded and non-wounded pods developed symptoms. The plates were stored in identical conditions and monitored for 5 days until tan colonies were observed. Culture morphology was consistent with the original isolate in all beans. Sequencing of the ITS region confirmed identity as R. solani AG-4. No symptoms were observed on control pods or seeds. Rhizoctonia solani is most frequently associated with symptoms of root rot (Sharma-Poudyal et al. 2015), but no stem symptoms are associated with the late season "brown bean" that has been observed throughout production in recent years. To our knowledge, this is the first report of Rhizoctonia solani AG 4 causing symptoms of brown bean of lima bean in Delaware. In preliminary observations, symptoms seem to be worse in pods that could have had contact with the soil directly or via rain splash. This disease cannot be detected until pods are split open, which has potential to reduce lima bean quality at harvest. Further monitoring should be conducted to quantify yield impacts and develop appropriate preventative and curative techniques.

First report of Curvularia lunata causing Curvularia leaf spot of corn in Delaware.

In mid-August 2020, small circular tan-colored lesions were detected in leaves of corn, Zea mays, in Sussex county Delaware. Symptoms were observed in many fields, affecting multiple hybrids that were at approximately the dough growth stage (R4). Lesions were present on most plants and individual ear leaf severity ratings ranged from 3 to 14 %. Symptomatic corn leaves were selected from the field and kept in a moist chamber for 24 hrs at 25 °C. In lesions, melanized spores were observed ranging from 21.94 µm to 30.51 µm in length and 7.83µm to 11.70µm in width (n=20). One cm2 leaf sections were extracted and sterilized in a 0.85% sodium hypochlorite solution for 30s followed by a sterile water rinse for 30s. Leaf pieces were then plated onto potato dextrose agar amended with 50µg/ml penicillin-G-sodium salt and streptomycin sulfate. Petri dishes were incubated at 25 °C with a 12-hr photoperiod for 14 days. Colonies were brown-black to lighter gray in color. Media was stained from orange, to dark brown, or not at all. Conidia were melanized and curved with three transverse septa matching the size above. Colony and spore morphology were consistent with the description of Curvularia lunata (Garcia-Aroca et al. 2018). Pure cultures were obtained, and a representative isolate was sequenced to confirm fungal identity by amplifying the internal transcribed spacer (ITS) region with primers ITS4 and ITS5. BLAST search results confirmed 100% similarity (483/531 bp) to the C. lunata reference sequence (MK623264). The sequence was deposited in GenBank as accession number MW794323. To complete Koch's postulates, corn (hybrid: Hubner H6187RCSS) was planted into 10 cm pots and maintained at 25°C and a 12 hr photoperiod. A conidial suspension was made by soaking a Petri dish of a 3-week-old fungal colony with 20 ml of sterile water and a drop of Tween-20 solution then, scraping mycelia to dislodge spores. The conidial concentration was calibrated to 5 x 105 spores/ml and 15 ml were applied to each whorl of four three-collar stage plants using a Preval sprayer (Nakoma Products, Bridgeview, IL). Four plants were inoculated with 15 ml sterile DI water as a control. This experiment was repeated twice. Each plant was moved to an incubator and covered with a plastic bag for 24 hr to maintain humidity. Conditions in the incubator were maintained at 25 °C with a 12-hr photoperiod. Inoculated plants displayed small, oval-shaped lesions within four days. No symptoms were observed on the control plants. Symptomatic leaves were harvested and placed into a moist chamber for 24 hrs to sporulate and lesions were plated onto PDA as described above. Culture morphology was consistent with the original isolate, with spores slightly larger ranging from 22.35 µm to 39.29 µm in length, and 8.36 µm to 11.69 µm in width (n=20). Isolates obtained from inoculated plants were sequenced and maintained 100% identity with the reference sequence described above. C. lunata was first reported in Louisiana in 2017 (Garcia-Aroca et al. 2018) and in Kentucky in 2018 (Anderson et al. 2019). This is the first report of Curvularia leaf spot on corn in Delaware. Symptoms developed late in the season, so it is unlikely that yield was affected in 2020. However, the economic impact of this disease in the United States is still unclear, it will be important to monitor potential impacts of this disease in Delaware corn production. References 1.Anderson NR, et al., 2019, Plant Disease, 103, 2692, doi: 0.1094/PDIS-03-19-0629-PDN. 2. Garcia-Aroca T, et al., 2018, Plant health progress, 19, 140, doi: 10.1094/PHP-02-18-0008-BR.

Diaporthe Seed Decay of Soybean [Glycine max (L.) Merr.] Is Endemic in the United States, But New Fungi Are Involved.

Diaporthe seed decay can compromise seed quality in soybean [Glycine max (L.) Merr.] in the warm and humid production areas of the United States during crop maturation. In the current study, 45 isolates of Diaporthe were recovered from seed sampled from soybean fields affected by Diaporthe-associated diseases in eight U.S. states in 2017. The isolates obtained belonged to 10 species of Diaporthe based on morphology and phylogenetic analyses of the internal transcribed spacer, partial translation elongation factor 1-α, and ß-tubulin gene sequences. The associated species included D. aspalathi, D. caulivora, D. kongii, D. longicolla, D. sojae, D. ueckerae, D. unshiuensis, and three novel fungi, D. bacilloides, D. flavescens, and D. insulistroma. One isolate each of the 10 species was examined for pathogenicity on seed of cultivar Sava under controlled conditions. Seven days postinoculation, significant differences in the percentages of decayed seeds and seedling necrosis were observed among the isolates and the noninoculated control (P < 0.0001). While the isolates of D. bacilloides, D. longicolla, and D. ueckerae caused a significantly greater percentage of decayed seeds (P < 0.0001), the isolate of D. aspalathi caused the greatest seedling necrosis (P < 0.0001). The observation of new fungi causing Diaporthe seed decay suggests the need for a more comprehensive survey in U.S. soybean producing areas since members of the genus Diaporthe appear to form a complex that causes seed decay.

Comparative genomic analysis reveals contraction of gene families with putative roles in pathogenesis in the fungal boxwood pathogens Calonectria henricotiae and C. pseudonaviculata.

BACKGROUND: Boxwood blight disease caused by Calonectria henricotiae and C. pseudonaviculata is of ecological and economic significance in cultivated and native ecosystems worldwide. Prior research has focused on understanding the population genetic and genomic diversity of C. henricotiae and C. pseudonaviculata, but gene family evolution in the context of host adaptation, plant pathogenesis, and trophic lifestyle is poorly understood. This study applied bioinformatic and phylogenetic methods to examine gene family evolution in C. henricotiae, C. pseudonaviculata and 22 related fungi in the Nectriaceae that vary in pathogenic and saprobic (apathogenic) lifestyles. RESULTS: A total of 19,750 gene families were identified in the 24 genomes, of which 422 were rapidly evolving. Among the six Calonectria species, C. henricotiae and C. pseudonaviculata were the only species to experience high levels of rapid contraction of pathogenesis-related gene families (89% and 78%, respectively). In contrast, saprobic species Calonectria multiphialidica and C. naviculata, two of the closest known relatives of C. henricotiae and C. pseudonaviculata, showed rapid expansion of pathogenesis-related gene families. CONCLUSIONS: Our results provide novel insight into gene family evolution within C. henricotiae and C. pseudonaviculata and suggest gene family contraction may have contributed to limited host-range expansion of these pathogens within the plant family Buxaceae.

Identification and characterization of Septoria steviae as the causal agent of Septoria leaf spot disease of stevia in North Carolina.

Stevia (Stevia rebaudiana) is an emerging perennial crop in the southeastern United States. A Septoria leaf spot disease of stevia was first identified on field plantings in Japan in 1978. The pathogen was named Septoria steviae based on a morphological characterization. In 2015, a species of Septoria with morphological characters of S. steviae was isolated from field and greenhouse-grown stevia plants with leaf spot symptoms in North Carolina. In this study, 12 isolates obtained from diseased stevia plants in 2015 and 2016 were characterized and compared with reference strains of S. steviae. Comparisons were based on conidial and pycnidial morphology and multilocus sequence analysis of actin (ACT), ß-tubulin (BT), calmodulin (CAL), nuc rDNA internal transcribed spacers (ITS1-5.8S-ITS2 = ITS), nuc rDNA 28S subunit (28S), RNA polymerase II second largest subunit (RPB2), and translation elongation factor-1α (TEF1). Measurements of conidia and pycnidia from symptomatic field leaves and 12 pure cultures grown on nutrient medium were consistent with those previously reported for ex-type strains of S. steviae. North Carolina strains formed a well-supported monophyletic group with ex-type strains of S. steviae. This study represents the first genetic characterization of S. steviae in the United States and provides an experimental framework to elucidate the genetic diversity and disease ecology of field populations of S. steviae.

Seasonal dynamics and fungicide sensitivity of organisms causing brown patch of tall fescue in North Carolina.

Brown patch, caused by multiple species of Rhizoctonia and Rhizoctonia-like fungi, is the most severe summer disease of tall fescue in home lawns across the southeastern United States. Home lawns were surveyed in central North Carolina from 2013 to 2015 to determine the organisms present during typical epidemics of brown patch in tall fescue. Isolates of Rhizoctonia and Rhizoctonia-like fungi were obtained by sampling 147 locations in July 2013 and May and July 2014. In addition, 11 sites were sampled once a week for 12 consecutive weeks from late May to the end of July 2015. All isolates were identified to species and anastomosis group with nuc rDNA internal transcribed spacer (ITS) sequence analysis. Isolations from brown patch lesions in May 2014 predominately yielded Ceratobasidium cereale (77% of the organisms recovered), whereas the organisms recovered in July 2013 and 2014 were R. solani AG 2-2-IIIB (44%), R. solani AG 1-IB (37%), and R. zeae (14%). In 2015, Ceratobasidium cereale was isolated from all 11 locations in May but was replaced by Rhizoctonia species in June and July. Sensitivity of the May 2014 isolates to multiple concentrations of the fungicides azoxystrobin, flutolanil, fluxapyroxad, and propiconazole was compared with sensitivity of isolates collected in 2003, to determine whether multiple years of exposure to fungicides applied for brown patch control had altered fungicide sensitivity. Historical isolates of R. solani, which had never been exposed to fungicide applications for brown patch control, were also included for comparison. Mean EC50 values (concentration of fungicide needed to inhibit mycelial growth by 50%) varied across fungicides and species, but no resistance was observed, and there was no apparent shift in sensitivity over the years. An additional 94 isolates from 2015 were screened against azoxystrobin, flutolanil, fluxapyroxad, and propiconazole, and fungicide insensitivity was not observed.