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United States’ sales of mungbean ( Vigna radiata L. Wilczek) products and other plant-based protein sources exceeded $1 billion in 2020. Most mungbean consumed in the United States is imported, but it has considerable potential as a domestic crop. Its heat and drought tolerance give U.S. farmers an alternative to soybean and corn crop rotation. Mungbean is a 60- to 90-day crop with similar equipment needs, chemical inputs, and storage requirements to those of soybean and has a developed market. Its vulnerability to diseases in the United States is unknown. Thus, we compared the susceptibility of 10 mungbean genotypes to 8 soybean diseases in the lab. We also monitored disease occurrence in sentinel field plots in Iowa. Mungbean accessions showed a wide range of susceptibility to Cercospora kikuchii, Colletotrichum gloeosporioides, Diaporthe caulivora, Fusarium oxysporum, F. virguliforme, Macrophomina phaseolina, Pythium sylvaticum, and Sclerotinia sclerotiorum. Symptoms of frogeye leaf spot, sudden death syndrome, and Rhizoctonia root and crown rot, common soybean diseases, were observed on mungbean in the field; isolates were identified as Cercospora sojina, F. virguliforme, and Rhizoctonia solani, and Koch's postulates were conducted. C. sojina and F. virguliforme are newly reported as causing disease in field-grown mungbean.

Field trials were conducted across Iowa from 2017 to 2020 to investigate the effect of fungicide application methods (traditional and undercover) on coverage, disease severity, and yield. Foliar fungicides were applied at the soybean (R3) growth stage by a traditional ground sprayer with an overhead spray boom or a ground sprayer with 360 undercover sprayers that hang down into the canopy. Fungicide coverage was measured as milliliters per liter of pyrenetetrasulfonic acid dye detected on soybean leaves and as a percent coverage of water-sensitive spray cards from three canopy zones (upper, middle, and lower). Both detection methods showed that the upper canopy received significantly more coverage through the traditional sprayer than the undercover sprayer. The percent coverage in the middle canopy was not different between the two application methods. The tracer dye detected greater fungicide distribution in the lower canopy by the undercover than the traditional method ( P = 0.034). Greater disease severity was observed in 2018 and 2019 than in 2017 and 2020. Regardless of the year or application method, no location in the canopy had a significant difference in foliar disease control, nor did fungicide application affect yield. Results from these trials show adequate coverage of fungicide, but disease severity was not reduced, and yield was not improved.

Brown spot, caused by Septoria glycines, is a common foliar disease of soybean (Glycine max). Applications of fungicide products that contain quinone outside inhibitor (QoI) active ingredients to soybean fields have contributed to the selection and development of QoI-resistant populations of S. glycines. We investigated the molecular mechanisms of QoI-resistance in these populations through targeted analysis of the cytochrome b gene. Isolates of S. glycines collected from several soybean fields over different seasons varied in sensitivity to QoI fungicides. Characterization of the cytochrome b gene revealed a mutation that changed an amino acid from glycine to alanine at codon 143 – one that is generally associated with QoI fungicide resistances. A PCR assay was developed that allowed successful discrimination of QoI-sensitive and -resistant isolates based on the G143A mutation. Results of this study demonstrated that 47.5% of S. glycines isolates tested were resistant to QoI fungicides. Accurate monitoring of this mutation will help slow the spread of QoI resistance and will be important for fungicide resistant management in this pathosystem.

Soybean (Glycine max) farmers in the Upper Midwest region of the United States frequently experience severe yield losses due to Sclerotinia stem rot (SSR). Previous studies have revealed benefits of individual management practices on SSR. This study examined the integration of multiple control practices on the development of SSR, yield, and the economic implications of these practices. Combinations of row spacings, seeding rates, and fungicide applications were examined in multi-site field trials across the Upper Midwest from 2017-2019. These trials revealed that wide row spacing and low seeding rates individually reduced SSR levels but also reduced yields. Yields were similar across the three higher seeding rates examined. However, site-years where SSR developed showed the highest partial profits in the intermediate seeding rates. This indicates that partial profits in diseased fields were negatively impacted by high seeding rates, but this trend was not observed when SSR did not develop. Fungicides strongly reduced the development of SSR, while also increasing yields. However, there was a reduction in partial profits due to their use at a low soybean sale price, but at higher sale prices fungicide use was similar to not treating. Additionally, the production of new inoculum was predicted from disease incidence, serving as an indicator of increased risk for SSR development in future years. Overall, this study suggests the use of wide rows and low seeding rates could be useful in fields with a history of SSR, while reserving narrow rows and higher seeding rates for fields without a history of SSR.

Frogeye leaf spot (FLS), caused by Cercospora sojina, is an economically important disease of soybean in the United States. Data from 66 uniform fungicide trials (UFTs) conducted from 2012 to 2021, across eight states (Alabama, Arkansas, Illinois, Iowa, Kentucky, Louisiana, Mississippi, and Tennessee), were gathered and analyzed to determine the efficacy and profitability of the following fungicides applied at the beginning pod developmental stage (R3): azoxystrobin + difenoconazole (AZOX + DIFE), difenoconazole + pydiflumetofen (DIFE + PYDI), pyraclostrobin (PYRA), pyraclostrobin + fluxapyroxad + propiconazole (PYRA + FLUX + PROP), tetraconazole (TTRA), thiophanate-methyl (TMET), thiophanate-methyl + tebuconazole (TMET + TEBU), and trifloxystrobin + prothioconazole (TFLX + PROT). A network meta-analytic model was fitted to the log of the means of FLS severity data and to the non-transformed mean yield for each treatment, including the nontreated. The percent reduction in disease severity (%) and the yield response (kg/ha) relative to the nontreated was the lowest for PYRA (11%; 136 kg/ha) and the greatest for DIFE+PYDI (57%; 441 kg/ha). A significant decline in efficacy over time was detected for PYRA (18 percentage points [p.p.]), TTRA (27 p.p.), AZOX + DIFE (18 p.p.) and TMET + TEBU (19 p.p.), by using year as a continuous covariate in the model. Finally, probabilities of breaking even were the greatest (>65%) for the most effective fungicide DIFE+PYDI, and the lowest (

Frogeye leaf spot (FLS), caused by Cercospora sojina, is an important foliar disease affecting soybean in the United States. A meta-analytic approach including 39 fungicide trials conducted from 2012 to 2021 across eight states (Alabama, Arkansas, Illinois, Iowa, Kentucky, Louisiana, Mississippi, Tennessee) was used to assess the relationship between FLS severity and soybean yield. Correlation and regression analyses were performed separately to determine Fisher's transformation of correlation coefficients (Zr), intercept (β0) and slope (β1). Disease pressure (low severity = ≤34.5 and high severity = >34.5%) and yield class (low = ≤3,352 and high = >3,352 kg/ha) were included as categorical moderators in the model. The Pearson’s r ̅, obtained from back-transforming the Z ̅r estimated by an overall random-effects model, showed a significant negative linear relationship between FLS severity and yield (r ̅ = -0.60). The Z ̅r was affected by disease pressure (P = 0.0003) but not by yield class (P = 0.8141). A random-coefficient model estimated a slope of -19 kg/ha for every % severity for a mean attainable yield of 3,719.9 kg/ha. Based on the overall mean (95% CI) of the intercept and slope estimated by the random-coefficients model, the overall relative damage coefficient was estimated to be 0.51% (0.36-0.69), indicating that a percent increase in FLS severity would result in a 0.51% yield reduction. The best model included yield class as a covariate and population-average intercepts differed significantly between low (3,455.1 kg/ha) and high (3,842.7 kg/ha) yield classes. We also estimated economic damage thresholds for a range of soybean prices, control costs and fungicide efficacies. As expected, the threshold values increased as the control efficacy also increased and were affected by different crop prices and fungicide simulated costs. The current results highlight the potential impact of FLS on soybean yield if not well managed and may be helpful in disease management decisions by applying fungicides before disease severity reaches economic damage thresholds.

Fungicide use is integral to reduce yield loss from Sclerotinia sclerotiorum on dry bean and soybean. Increasing fungicide use against this fungus may lead to resistance to the most common fungicides. Resistance has been reported in Brazil (Glycine max) and China (Brassica napus subsp. napus), however, few studies have investigated fungicide sensitivity of S. sclerotiorum in the U.S. The work presented here was conducted to determine if there was a difference in fungicide sensitivity of S. sclerotiorum isolates in the U.S. from: (i) dry bean versus soybean and (ii) fields with different frequencies of fungicide application. We further hypothesized that isolates with fungicide applications of a single active ingredient from tropical Brazil and sub-tropical Mexico were less sensitive compared to temperate U.S. isolates due to different management practices and climates. The EC50(D) fungicide sensitivity of 512 S. sclerotiorum isolates from the U.S. (443), Brazil (36), and Mexico (33) was determined using a discriminatory concentration (DC) previously identified for tetraconazole (2.0 ppm; EC50(D) ranged of 0.197 to 2.27 ppm), boscalid (0.2; 0.042 to 0.222), picoxystrobin (0.01; 0.006 to 0.027), and thiophanate-methyl, which had a qualitative DC of 10 ppm. Among the 10 least sensitive isolates to boscalid and picoxystrobin, two presented mutations known to confer resistance in the SdhB (qualitative) and SdhC (quantitative) genes; however no strong resistance was found. This study established novel DCs that can be used for further resistance monitoring and baseline sensitivity of S. sclerotiorum to tetraconazole throughout the world plus baseline sensitivity to boscalid in the U.S.

The asymptomatic host range of Fusarium virguliforme includes corn, a common crop rotated with soybean that we hypothesize may alter F. virguliforme population dynamics and disease management. A field-based approach explored the temporal dynamics of F. virguliforme colonization of corn and soybean roots under different tillage and residue managements. Experiments were conducted in Iowa, Indiana, Michigan, and Wisconsin, United States and Ontario, Canada from 2016 to 2018. Corn and soybean roots were sampled at consecutive timepoints between 1 and 16 weeks after planting. DNA was extracted from all roots and analyzed by real-time quantitative PCR for F. virguliforme quantification. Trials were rotated between corn and soybean, containing a two-by-two factorial of tillage (no-tilled or tilled) and corn residue (with or without) in several experimental designs. In 2016, low amounts (approximately 100 fg per 10 mg of root tissue) of F. virguliforme were detected in the inoculated Iowa, Indiana, and Michigan locations and noninoculated Wisconsin corn fields. However, in 2017, greater levels of F. virguliforme DNA were detected in Iowa, Indiana, and Michigan across sampling timepoints. Tillage practices showed inconsistent effects on F. virguliforme root colonization and sudden death syndrome (SDS) foliar symptoms among trials and locations. However, residue management did not alter root colonization of corn or soybean by F. virguliforme. Plots with corn residue had greater SDS foliar disease index in Iowa in 2016. However, this trend was not observed across the site-years, indicating that corn residue may occasionally increase SDS foliar symptoms depending on the disease level and soil and weather factors.

Soybean production in the upper midwestern United States is affected by Sclerotinia stem rot (SSR) caused by the fungal pathogen Sclerotinia sclerotiorum. Genetic resistance is an important management strategy for this disease; however, assessing genetic resistance to S. sclerotiorum is challenging because a standardized method of examining resistance across genotypes is lacking. Using a panel of nine diverse S. sclerotiorum isolates, four soybean lines were assessed for reproducible responses to S. sclerotiorum infection. Significant differences in SSR severity were found across isolates (P < 0.01) and soybean lines (P < 0.01), including one susceptible, two moderately resistant, and one highly resistant line. These four validated lines were used to screen 11 other soybean genotypes to evaluate their resistance levels, and significant differences were found across genotypes (P < 0.01). Among these 11 genotypes, five commercial and public cultivars displayed high resistance and were assessed during field studies across the upper midwestern United States growing region to determine their response to SSR and yield. These five cultivars resulted in low disease levels (P < 0.01) in the field that were consistent with greenhouse experiment results. The yields were significantly different in fields with disease present (P < 0.01) and disease absent (P < 0.01), and the order of cultivar performance was consistent between environments where disease was present or absent, suggesting that resistance prevented yield loss to disease. This study suggests that the use of a soybean check panel can accurately assess SSR resistance in soybean germplasm and aid in breeding and commercial soybean development.

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 β-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.

Plant stress phenotyping is essential to select stress-resistant varieties and develop better stress-management strategies. Standardization of visual assessments and deployment of imaging techniques have improved the accuracy and reliability of stress assessment in comparison with unaided visual measurement. The growing capabilities of machine learning (ML) methods in conjunction with image-based phenotyping can extract new insights from curated, annotated, and high-dimensional datasets across varied crops and stresses. We propose an overarching strategy for utilizing ML techniques that methodically enables the application of plant stress phenotyping at multiple scales across different types of stresses, program goals, and environments.

Tar spot is a foliar disease of corn threatening production across the Americas. The disease was first documented in Mexico in 1904 and is now present in 15 additional countries throughout Central America, South America, and the Caribbean. Researchers and growers in Central America, South America, and the Caribbean consider tar spot to be a disease complex caused by multiple fungal pathogens. When environmental conditions are conducive for infection, these regions have experienced yield losses that can reach up to 100%. In 2015, tar spot was detected in the United States for the first time in Illinois and Indiana. Since that time tar spot has spread across the U.S. corn-growing region, and the disease has been found in Florida, Illinois, Indiana, Iowa, Michigan, Minnesota, Missouri, Ohio, Pennsylvania, and Wisconsin. In 2020, tar spot was also found in southwest Ontario, Canada. Losses in the United States due to tar spot totaled an estimated 241 million bushels from 2018 to 2020. With the potential to continue to spread across the U.S. corn-growing states, much greater losses could result when environmental conditions are conducive.

Sclerotinia stem rot (SSR or white mold), caused by Sclerotinia sclerotiorum (Lib.) DeBary, is an economically important fungal disease of soybean. SSR routinely causes yield loss in the upper Corn Belt of the United States due to wet, humid conditions that coincide with moderate temperatures. This study investigated the novel cultural practice of mechanical cutting, or mowing, as an SSR management practice across multiple seeding rates. Mowing soybean during early vegetative growth alters plant architecture and growth habit. This results in a microclimate within the canopy less suitable for disease development. Field trials were conducted in Iowa, Illinois, Michigan, and Wisconsin in 2017 and 2018. Experimental design was a randomized complete block with four replications. Treatments included mowing (mowing and no mowing), seeding rate (197,684, 271,816, and 345,947 seeds/ha), and fungicide application (boscalid, Endura, and no fungicide). Soybean was mowed at approximately the V4 (four unfolded trifoliate leaves) growth stage. Mowing reduced disease in multiple locations; however, it also reduced yield in most of the locations. In general, there was less SSR in plots with lower seeding rates. Fungicide significantly reduced SSR in two of the five site-years for which disease was observed. Significant yield response to fungicide was also observed in two of the nine total field trials. Results indicate cultural practices such as mowing and reduced seeding rate can decrease SSR severity but also can impact potential yield. Additionally, yield response to SSR management practices may not be observed if disease is absent or at low levels.

Farmers may adjust seeding rate across or within fields for a variety of reasons. The objectives of this study were to determine if seeding rate affected two late-season stem diseases, anthracnose stem blight (ASB) and pod and stem blight (PSB), and yield of soybean. Disease incidence, severity, and yield were collected from eight field trials in Iowa and Wisconsin during 2016 to 2018. ASB incidence exceeded 80% in most of the trials and significantly differed across years with the highest incidence in 2016 and 2018 compared with 2017, the year with the least precipitation. A similar trend across years was observed for ASB severity. The incidence and severity of PSB also differed across years, although PSB incidence and severity were always less than ASB. PSB was greatest in 2017 compared with the other two years. Seeding rate did not affect either disease. A seeding rate of 198,000 to 395,000 seeds/ha provided statistically similar yield. Weather conditions each year affected late-season stem diseases.

Soybean (Glycine max [L.] Merrill) yield losses as a result of plant diseases were estimated by university and government plant pathologists in 29 soybean producing states in the United States and in Ontario, Canada, from 2015 through 2019. In general, the estimated losses that resulted from each of 28 plant diseases or pathogens varied by state or province as well as year. Soybean cyst nematode (SCN) (Heterodera glycines Ichinohe) caused more than twice as much loss as any other disease during the survey period. Seedling diseases (caused by various pathogens), Sclerotinia stem rot (white mold) (caused by Sclerotinia sclerotiorum [Lib.] de Bary), and sudden death syndrome (caused by Fusarium virguliforme O’Donnell & T. Aoki) caused the next greatest yield losses, in descending order. Following SCN, the most damaging diseases in the northern United States and Ontario differed from those in the southern United States. The estimated mean economic loss from all soybean diseases, averaged across the United States and Ontario, Canada was US$45 per acre (US$111 per hectare). The outcome from the current survey will provide pertinent information regarding the important soybean diseases and their overall severity in the soybean crop and help guide future research and Extension efforts on managing soybean diseases.

In total, 52 uniform field experiments were conducted in Illinois, Indiana, Iowa, Michigan, South Dakota, and Wisconsin in the United States and Ontario, Canada from 2013 to 2017 comparing crop protection products against sudden death syndrome (SDS) of soybean. Data were analyzed using meta-analytic models to summarize the relationship between foliar disease index (FDX) and yield. For each study, correlation and regression analyses were performed separately to determine three effect sizes: Fisher’s transformation of correlation coefficients (Z r ), intercept (β0), and slope (β1). Random- and mixed-effect meta-analyses were used to summarize the effect sizes. Study- and location-specific moderator variables FDX (low < 10% and high ≥ 10%), date of planting (early = prior to 7 May, conventional = 7 to 21 May, and late = after 21 May) cultivar (susceptible and partially resistant to SDS), study location, and growing season were used as fixed effects. The overall mean effect sizes of transformed correlation coefficient [Formula: see text] r was −0.41 and different from zero (P < 0.001), indicating that yield was negatively correlated with FDX. The [Formula: see text] r was affected by disease level (P < 0.01) and cultivar (P = 0.02), with a greater effect at higher disease levels and with susceptible cultivars. The mean [Formula: see text] 0 was 4,121 kg/ha and mean [Formula: see text] 1 was −21 kg/ha/% FDX and were different from zero (P < 0.01). Results from these data indicate that, for every unit of FDX increase, yield was decreased by 0.5%. Study locations and year affected the [Formula: see text] 0 , whereas none of the moderator variables significantly affected [Formula: see text] 1.

Brown spot, caused by

Soybean (

Annual reductions in corn (Zea mays L.) yield caused by diseases were estimated by university Extension-affiliated plant pathologists in 26 corn-producing states in the United States and in Ontario, Canada, from 2016 through 2019. Estimated loss from each disease varied greatly by state or province and year. Gray leaf spot (caused by Cercospora zeae-maydis Tehon & E.Y. Daniels) caused the greatest estimated yield loss in parts of the northern United States and Ontario in all years except 2019, and Fusarium stalk rot (caused by Fusarium spp.) also greatly reduced yield. Tar spot (caused by Phyllachora maydis Maubl.), a relatively new disease in the United States, was estimated to cause substantial yield loss in 2018 and 2019 in several northern states. Gray leaf spot and southern rust (caused by Puccinia polysora Underw.) caused the most estimated yield losses in the southern United States. Unfavorable wet and delayed harvest conditions in 2018 resulted in an estimated 2.5 billion bushels (63.5 million metric tons) of grain contaminated with mycotoxins. The estimated mean economic loss due to reduced yield caused by corn diseases in the United States and Ontario from 2016 to 2019 was US$55.90 per acre (US$138.13 per hectare). Results from this survey provide scientists, corn breeders, government agencies, and educators with data to help inform and prioritize research, policy, and educational efforts in corn pathology and disease management.

A study was conducted in five American states and Ontario, Canada, in 2015 and 2016 to determine the effects of fluopyram seed treatment and cultivar on the root rot and foliar phases of sudden dea...

Neonicotinoids are the most widely used insecticides worldwide and are typically deployed as seed treatments (hereafter NST) in many grain and oilseed crops, including soybeans. However, there is a surprising dearth of information regarding NST effectiveness in increasing soybean seed yield, and most published data suggest weak, or inconsistent yield benefit. The US is the key soybean-producing nation worldwide and this work includes soybean yield data from 194 randomized and replicated field studies conducted specifically to evaluate the effect of NSTs on soybean seed yield at sites within 14 states from 2006 through 2017. Here we show that across the principal soybean-growing region of the country, there are negligible and management-specific yield benefits attributed to NSTs. Across the entire region, the maximum observed yield benefits due to fungicide (FST = fungicide seed treatment) + neonicotinoid use (FST + NST) reached 0.13 Mg/ha. Across the entire region, combinations of management practices affected the effectiveness of FST + NST to increase yield but benefits were minimal ranging between 0.01 to 0.22 Mg/ha. Despite widespread use, this practice appears to have little benefit for most of soybean producers; across the entire region, a partial economic analysis further showed inconsistent evidence of a break-even cost of FST or FST + NST. These results demonstrate that the current widespread prophylactic use of NST in the key soybean-producing areas of the US should be re-evaluated by producers and regulators alike.

Sudden death syndrome (SDS), caused by Fusarium virguliforme, is an important soilborne disease of soybean. Risk of SDS increases when cool and wet conditions occur soon after planting. Recently, multiple seed treatment and foliar products have been registered and advertised for management of SDS but not all have been tested side by side in the same field experiment at multiple field locations. In 2015 and 2016, seed treatment fungicides fluopyram and thiabendazole; seed treatment biochemical pesticides citric acid and saponins extract of Chenopodium quinoa; foliar fungicides fluoxastrobin + flutriafol; and an herbicide, lactofen, were evaluated in Illinois, Indiana, Iowa, Michigan, South Dakota, Wisconsin, and Ontario for SDS management. Treatments were tested on SDS-resistant and -susceptible cultivars at each location. Overall, fluopyram provided the highest level of control of root rot and foliar symptoms of SDS among all the treatments. Foliar application of lactofen reduced foliar symptoms in some cases but produced the lowest yield. In 2015, fluopyram reduced the foliar disease index (FDX) by over 50% in both resistant and susceptible cultivars and provided 8.9% yield benefit in susceptible cultivars and 3.5% yield benefit in resistant cultivars compared with the base seed treatment (control). In 2016, fluopyram reduced FDX in both cultivars by over 40% compared with the base seed treatment. For yield in 2016, treatment effect was not significant in the susceptible cultivar while, in the resistant cultivar, fluopyram provided 3.5% greater yield than the base seed treatment. In this study, planting resistant cultivars and using fluopyram seed treatment were the most effective tools for SDS management. However, plant resistance provided an overall better yield-advantage than using fluopyram seed treatment alone. Effective seed treatments can be an economically viable consideration to complement resistant cultivars for managing SDS.

One hundred fifty-two Diaporthe isolates were recovered from symptomatic soybean (Glycine max) stems sampled from the U.S. states of Iowa, Indiana, Kentucky, Michigan, and South Dakota. Using morphology and DNA sequencing, isolates were identified as D. aspalathi (8.6%), D. caulivora (24.3%), and D. longicolla (67.1%). Aggressiveness of five isolates each of the three pathogens was studied on cultivars Hawkeye (D. caulivora and D. longicolla) and Bragg (D. aspalathi) using toothpick, stem-wound, mycelium contact, and spore injection inoculation methods in the greenhouse. For D. aspalathi, methods significantly affected disease severity (P < 0.001) and pathogen recovery (P < 0.001). The relative treatment effects (RTE) of stem-wound and toothpick methods were significantly greater than for the other methods. For D. caulivora and D. longicolla, a significant isolate × method interaction affected disease severity (P < 0.05) and pathogen recovery (P < 0.001). Significant differences in RTEs were observed among D. caulivora and D. longicolla isolates only when the stem-wound and toothpick methods were used. Our study has determined that the stem-wound and toothpick methods are reliable to evaluate the three pathogens; however, the significant isolate × method interactions for D. caulivora and D. longicolla indicate that multiple isolates should also be considered for future pathogenicity studies.