four applications. While yield did not differ statistically between any of the treatments, an experimental formulation significantly reduced disease severity by 40.5% relative to the commercial product and 55.3% relative to the untreated control. From the positive field results achieved by enhancing leaf cuticle penetration, further field and lab testing to optimize performance will be completed.

## Season long management of potato silver scurf caused by Helminthosporium solani in Wisconsin

S. MACCHIAVELLI-GIRÓN (1), S. Jordan (1), J. Crane (2), A. Gevens (1)
(1) University of Wisconsin-Madison, U.S.A.; (2) University of Wisconsin-Madison, U.S.A.

Silver scurf, caused by the fungus *Helminthosporium solani*, is a tuber blemish disease of increasing concern in potato production. The disease affects periderm quality and appearance, impacting marketability as well as storability. Cultural management strategies are limited and often impractical for large-scale growers, making fungicide applications an important component of integrated silver scurf management. *Helminthosporium solani* initially infects tubers in the field, and has secondary infection cycles during storage. Because inoculum can be reduced at multiple times during potato production, we conducted a full life cycle study in 2015 to evaluate the aggregated impact of fungicide selection and timing on silver scurf severity in 'Dark Red Norland' potatoes. Fungicides were applied at three times: at planting as seed or in-furrow treatments; in-field as a series of 3 foliar sprays; and post-harvest immediately before storage. Non-treated comparisons were included. The at-planting applications had the greatest impact on symptom severity, although the post-harvest treatments also contributed to disease reductions. The lowest severity resulted from the Maxim MZ (fludioxonil + mancozeb) seed treatment with the Stadium (fludioxonil + azoxystrobin + difenoconazole) post-harvest treatment. Along with cultural methods, at-plant and post-harvest fungicide treatments together were necessary for silver scurf control.

## Greenhouse characterization of hydrazone-copper mixtures as low copper treatments to control early and late blight on tomato

C. AVILA-ADAME (1), D. Young (2), J. Webster (2), O. Brian (2), D. Ouimette (2)
(1) Dow AgroSciences LLC, U.S.A.; (2) Dow AgroSciences LLC, U.S.A.

Copper-based fungicides remain critical tools in crop disease management programs to control a broad spectrum of fungal and bacterial plant pathogens. However, because of the high rate required to control plant pathogens, there is concern regarding their potential negative environmental impact. Certain salicylaldehyde benzoylhydrazone (SBH) compounds bind copper and show strong synergism with copper when tested for inhibition of fungal growth under *in vitro* conditions. Experiments were conducted to characterize synergism for Cu-hydrazone mixtures in controlling *Phytophthora infestans* and *Alternaria solani* on tomato under greenhouse conditions. Treatments included a SBH alone, three copper materials, and Cu-SBH mixtures. The SBH alone was weakly active against both pathogens with 26 and 33% disease control at 400 micromolar for *P. infestans* and *A. solani*, respectively. The copper materials, which included Cu(OH)₂ in the form of Kocide® 2000, CuSO₄, and CuCl₂, provided a similar level of disease control (*p* > 0.3) when applied alone. The copper-SBH mixtures at various molar ratios produced synergistic interactions in nearly all combinations and were superior to the commercial fungicide Kocide® 2000 at equivalent rates of copper.

## Yield loss and management of orange cane blotch of blackberry, caused by the alga *Cephalouros virescens*

F. BROWNE (1), P. Brannen (1), L. Scherm (1), L. Fall (1), J. Taylor (2), J. Shealey (3), E. Beasley (1)
(1) University of Georgia, U.S.A.; (2) University of Georgia, U.S.A.; (3) University of Georgia, U.S.A.

The alga *Cephalouros virescens* causes orange cane blotch (OCB), a serious disease of blackberries in the southeastern United States. OCB impact on blackberry growth and yield was assessed. Neither cane diameter nor fruit size were significantly impacted by severity of OCB; however, maximum fruit number per cane decreased by 80% with increasing OCB severity, thereby resulting in reduced yields. Field efficacy trials with diverse algicides, disinfectants, and fungicides were conducted over a 3-year period from 2013 to 2015 at two locations on a thornless blackberry cultivar, 'Ouachita'. Treatments, applied during the summer and fall, included ametoctradin + dimethomorph, calcium polysulfide, calcium polysulfide + surfactants, captan, chlorothalonil, copper hydroxide, copper hydroxide + hydrogen dioxide, fluazinam, fluopicolide, hydrogen dioxide, mancozeb, mancozeb + copper hydroxide, mandipropamid, mefenoxam, mefenoxam + copper hydroxide, mefenoxam + mancozeb, potassium phosphite, potassium phosphite + captan, potassium phosphite + copper hydroxide, oxathiapiprolin and diluted sodium hypochlorite. Potassium phosphite, generally active on both oomycetes and fungi, was the only chemical that provided significant suppression of the disease, with 48-80% reduced lesion coverage of canes. Management of OCB will be difficult in light of the limited number of labeled applications of potassium phosphite allowed and the potentially long infection period of the pathogen.

## Evaluation of Nematicides for Control of Southern Root-Knot Nematodes in Lima Bean

J. JONES (1), G. Johnson (1), N. Kleczewski (1), J. Desaeger (2)
(1) University of Delaware, U.S.A.; (2) DuPont Crop Protection, U.S.A.

Southern root-knot nematodes (*Meloidogyne incognita* -RKN) significantly reduce lima bean (*Phaseolus lunatus*) yields. Chemical control options for RKN are limited. We evaluated the efficacy of new nematicides on RKN in lima bean experiments conducted in greenhouse (GH) and microplot (MP) settings. Treatments included Nimitz at low and high labeled rates, Luna, Movento, Luna+Movento, Vydate, Mocap and an untreated, RKN infested control. GH treatments were arranged in a factorial design, with application of nematode eggs (0, 6,000, or 30,000 eggs pot-1) crossed with nematicide treatment. MP treatments were arranged in a randomized complete block design and all plots were infested with 4.80 kg of RKN infected tomato root tissue and soil. Root galling, RKN eggs and juveniles, and aboveground plant dry masses were determined and statistically analyzed using Mixed Model ANOVA. In the GH study, Nimitz at both rates provided the greatest reduction in RKN galling compared to the controls, whereas Movento treatments were not significantly different than the controls. In the MP study, all treatments except Movento significantly reduced RKN populations and had significantly greater yields relative to controls. Vydate, Mocap, and Nimitz (high and low rate) treatments had the highest yields in the MP study. Based on these results, several new nematicides show potential for managing RKN in lima beans.

## Evaluation of soil conditioner OR079 for the optimization of metalaxyl in control of *Phytophthora nicotianae* in Texas citrus orchards

P. DUBERNEY (1), J. Hernandez (1), V. Ancona (1)
(1) Texas A&M Kingsville Citrus Center, U.S.A.

The Texas citrus industry faces a great problem with the control of foot and root rot disease caused by *Phytophthora nicotianae*. *P. nicotianae* infects feeder roots and the trunk of trees causing poor water and nutrient uptake leading to yield loss, fruit size reduction and tree decline. Treatments for *Phytophthora* diseases are limited, therefore optimization of available products is of great importance. We hypothesized that the effectiveness of metalaxyl in reducing *P. nicotianae* soil propagules can be increased when applied in combination with the soil conditioner OR079. To test our hypothesis we applied metalaxyl, OR079, and metalaxyl plus OR079 to mature grapefruit trees with *Phytophthora* infection and quantified *Phytophthora* propagules in the soil. *Phytophthora* propague counts were assessed at 4, 8, and 12 weeks post treatment and compared to an untreated control. Results showed that all treatments had significantly reduced *Phytophthora* counts in the soil at 4 and 8 weeks post application compared to that of the untreated control, but were not statistically different to the control by 12 weeks. Moreover, OR079 did not increase metalaxyl effectiveness in reducing *Phytophthora* propagules but reduced its activity when mixed. In addition, application of OR079 and metalaxyl significantly increased citrus fruit size. These results indicate that timely treatment for *Phytophthora* infection improves root health and water uptake leading to increase citrus fruit quality.
## Efficacy of Oxathiapiprolin toward *Phytophthora sojae* and *Phytophthora sansomeana*

A. VARGAS (1), M. Eyre (1), A. Dorrance (1)

(1) The Ohio State University, U.S.A.

Poor crop stand establishment caused by soil-borne pathogens is very common in soybean fields in Ohio. Metalaxyl has been the most commonly used fungicide to control *Phytophthora* and *Pythium* species in soybean. However, several studies have reported an increase of insensitivity to metalaxyl in *Phytophthora* and *Pythium* populations in Ohio. Therefore, alternative fungicides should be tested for efficacy towards *Phytophthora* and *Pythium* species. The objective of this experiment was to determine the efficacy of oxathiapiprolin in reducing the growth of *Phytophthora sojae* and *Phytophthora sansomeana* in soybean. A total of 28 isolates collected from infested fields were evaluated using amended agar Petri dishes with 5 different concentrations of oxathiapiprolin. Sensitivity of each isolate was evaluated by estimating the diameter of mycelial growth on each Petri dish and comparing it to the control. Results from preliminary data show that oxathiapiprolin treatments reduced *P. sojae* and *P. sansomeana* mycelial growth and mean values varied among isolates. Eight isolates did not grow at 0.01 µg active ingredient/ml. Based on these results could be used to manage seed rot caused by *P. sojae* and *P. sansomeana*.

## Efficacy of three soybean fungicide seed treatments against *Pythium* species in seed plate and growth chamber assays

K. SCOTT (1), A. Vargas (1), M. Eyre (1), A. Dorrance (1)

(1) The Ohio State University, U.S.A.

Recent studies have reported a wide range of sensitivity to metalaxyl in *Pythium* species in Ohio, thus new active ingredients are important for management of seed and seedling blight. The objective of this study is to determine the efficacy of three seed treatments with a combination of active ingredients for oomycetes against a diverse population of *Pythium* species. Commercial formulations of Intego™ Suite, Cruiser Maxx® Advanced, and Acceleron® were applied to seed at labeled rates and evaluated with *Pythium* isolated from fields across Ohio in both a plate assay and a cup assay. In the cup assay, the mean root score was significantly lower in all fungicide seed treatments compared to the control, but there were no significant differences between treatments. In the seed plate assay, there was a significant difference for mean seed rot score between all treatments. Acceleron® had the lowest seed rot score towards all *Pythium* species, followed by Intego™ Suite, Cruiser Maxx® Advanced, and the non-treated check, respectively. These seed treatments may provide adequate protection against *Pythium* infection in soybean seed and seedling stages. In addition, the seed plate assay may be used to efficiently test for isolate pathogenicity and the efficacy of seed treatments.

## Preventative fungicide applications in production and their impact on residual efficacy against impatiens downy mildew in the landscape

S. SUAREZ (1), P. Lopez (1), A. Chase (2), A. Palmateer (1)

(1) University of Florida, U.S.A.; (2) Chase Agricultural Consulting, LLC, U.S.A.

Since the emergence of impatiens downy mildew researchers have come to the conclusion that chemical control is the only effective short-term management option available. The greatest challenge is identifying cost effective management options for impatiens downy mildew in the landscape. Some of our first trials conducted early in the spring of 2012 revealed that impatiens transplanted into landscape beds incorporated with a granular form of mefenoxam remained healthy for 60 days. Subsequent experiments evaluating products for long residual (> 30 days) control have identified several additional chemistries including fluopicolide, various phosphonates and oxathiapiprolin as promising. Further studies investigated the potential for eradicative control and found phosphonate containing products effective even after infection. Most recent studies have focused on fungicide rotations targeting long term residual control of impatiens downy mildew. Weekly preventative fungicide spray rotations on potted impatiens in production have included cyazofamid, dimethomorph, fluopicolide, mefenoxam, potassium phosphite and oxathiapiprolin. To date, oxathiapiprolin has provided stellar control of impatiens downy mildew when used alone and in every rotation study. Results obtained from fungicide efficacy trials conducted over the past four years indicate that some chemistries provide long enough residual control for impatiens to remain untreated in the landscape.

## Combined Application of *Bacillus amyloliquefaciens* ZY-9-13 with Fungicides for Control of Tobacco Black Shank

W. MU (1), J. XI (1), C. XUE (1), L. HU (1), J. SONG (1)

(1) Zhengzhou Tobacco Research Institute of CNTC, China

Tobacco black shank is a devastating soil borne disease caused by *Phytophthora parasitic* var. nicotianae, which greatly reduces tobacco yield and quality. Fungicides, such as metalaxyl, dimethomorph and mancozeb, have been widely used to prevent and control this disease. Intergrated biological and chemical control can release pressures from environment and delay emergence of fungicide resistance. *Bacillus amyloliquefaciens* ZY-9-13 has good control effect to tobacco black shank in our previous studies. In this research, we 1) assessed sensitivities of *P. nicotianae* to metalaxyl and dimethomorph, 2) mixed *B. amyloliquefaciens* ZY-9-13 and fungicide metalaxyl, dimethomorph, mancozeb in order to provide an alternative disease control option. The baseline sensitivities of *P. nicotianae* to metalaxyl and dimethomorph were described by unimodal curves, with mean EC50 values of 0.0664(±0.0282) and 0.3502(±0.0332) µg/ml, respectively, which can be used for detection of resistance isolates. Laboratory tests showed that fungicide mixture of metalaxyl, dimethomorph and mancozeb had additive effect, and the inhibition ratio of ZY-9-13 to fungicide mixture was less than 10% even at high concentration. The intergrated biological and chemical control showed a synergistic action. In the field trial, disease control with the combination of *B. amyloliquefaciens* ZY-9-13 and fungicide mixture was 79.76%~86.90%, which was greater than with ZY-9-13 or fungicide alone.

## Laboratory and field evaluation of fungicides for management of *Phytophthora* blight of pumpkin, caused by *Phytophthora capsici*

M. BABADOOST (1), J. de Souza (2), T. Reboucas (2), Y. Xiang (1)

(1) Department of Crop Sciences, University of Illinois, U.S.A.; (2) Department of Plant Science, Southwest Bahia University State, Brazil

*Phytophthora* blight, caused by *Phytophthora capsici*, is a destructive disease of cucurbits and peppers in Illinois and worldwide. Fungicide use is an important component of management strategies of *Phytophthora* blight of cucurbits. Studies were conducted in laboratory and field to evaluate efficacy of selected fungicides for management of *Phytophthora* blight of pumpkin in Illinois. In the laboratory, sensitivity of five *P. capsici* isolates from Illinois was evaluated to three registered fungicides (cyazofamid, dimethomorph, mandipropamid) for use on cucurbits and two experimental fungicides (A20941OD, A20942SC) for control of *Phytophthora* spp. Development of colony and production of sporangia and zoospores of the isolates were assessed at different concentrations of 0 to 10 µg/ml for registered fungicides and from 0 to 200 µg/ml for experimental fungicides on V8 juice agar medium in Petri plates. The isolates were highly sensitive to mandipropamid, intermediate sensitive to cyazofamid and A20941OD, and less sensitive to dimethomorph and A20942SC. In the field experiment in 2014, incidence of pumpkin fruit rot caused by *P. capsici* was 31% in control plots and 5, 10, 2, and 11% in the plots treated with cyazofamid, dimethomorph, mandipropamid, and A20942SC, respectively. In 2015, incidence of pumpkin fruit rot caused by *P. capsici* was 69% in control plots and 42, 21, and 18% in the plots sprayed with cyazofamid, dimethomorph, and mandipropamid, respectively.

## Fungicide rotation schemes and Melcast for managing *Phytophthora* fruit rot of watermelon in Southeastern United States

C. KOUSIK (1), D. Egel (2), P. Ji (3), L. Quesada-Ocampo (4)

(1) U.S. Department of Agriculture, Agricultural Research Service, U.S. Vegetable Laboratory, Charleston, SC, U.S.A.; (2) Purdue University, Vincennes, IN, U.S.A.; (3) University of Georgia, Tifton, GA, U.S.A.; (4) North Carolina State University, Raleigh, NC, U.S.A.

*Phytophthora* fruit rot (*Phytophthora capsici*) limits watermelon production in most states in the Southeastern US. We identified several effective fungicides (Revus, Presidio, and Zampro) to manage *Phytophthora* fruit rot in our previous studies. In this study, the effectiveness of fungicide rotations
and spray applications based on Melcast for managing Phytophthora fruit rot were conducted in *P. capsici* infested fields at three locations in southeastern US (NC, SC, GA) in 2014 & 2015. The triploid seedless cv. Wonder was used at all locations with diploid cv. Mickylee as the pollenizer. Five weekly foliar application of fungicides were made at all locations. Significant fruit rot was observed in non-treated check plots in both years at all three locations (Mean=68%). All the fungicide rotation schemes significantly reduced Phytophthora fruit rot compared to non-treated check. Overall the rotation scheme of Zampro (dimethomorph+ametoctradin) alternated with Orondis (oxathiapiprolin) was very effective across three locations. Revus (mandipropamid) alternated with Presidio (fluopicolide) was similarly effective in SC. MELCAST reduced one spray application in SC in 2014 but not in NC or GA, and one spray in GA and SC in 2015 with reduction in fruit rot similar to other rotation schemes. Fungicides in different FRAC groups are currently available for use in rotations schemes to manage Phytophthora fruit rot and Melcast may also be useful for scheduling spray treatments.

## Characterization of Zinkicide™ in plant tissues

L. TETARD (1), M. Soliman (1), M. Young (1), A. Towers (1), T. Washington (1), P. Rajasekaran (1), S. Das (1), E. Johnson (2), A. Gesquiere (1), S. Santra (1)

(1) University of Central Florida, Orlando, FL, U.S.A.; (2) University of Florida, Lake Alfred, FL, U.S.A.

The unprecedented crop losses caused by the high incidence of huanglongbing (HLB) vectored by the Asian citrus psyllid (ACP) in Florida citrus groves calls for the rapid development of an effective product available to growers for HLB management. Zinkicide™ is a prototype plant nutrient-based nanoparticle developed to provide growers with a new and effective systemic bactericide that will translocate into plant tissues to target the phloem-limited bacterium, *Candidatus* Liberibacter asiaticus (Las), responsible for infection and damage of internal phloem tissues in the plant associated with HLB. Unlike traditional bactericidal management, we show that the new Zinkicide™ treatment allows systemic movement to reach phloem tissue and interact with Las with ongoing assessment of tree health responses in field trials. To complement green house and field trial data, we explore the fundamental properties of Zinkicide™ formulations and their interactions with bacteria and citrus plant tissues. Morphology, structure and composition of the formulations were determined using a combination of electron microscopy, UV-Vis, fluorescence and infrared spectroscopy measurements. In addition, correlations with antibacterial efficacy, phytotoxicity, particle uptake and evolution in plant were established by monitoring the fluorescence, lifetime and infrared signatures of the particles as they penetrate into the plant and interact with the phloem tissues.

## Monitoring Pennsylvania orchards for the presence of streptomycin resistant *Erwinia amylovora*

H. LAUKAITIS (1), B. Lehman (2), K. Peter (2), M. Baer-Lehman (1)

(1) Shippensburg University, U.S.A.; (2) Pennsylvania State University Fruit Research and Extension Center, U.S.A.

Within the last 40 years, streptomycin resistant strains of *Erwinia amylovora* have emerged in apple orchards throughout the United States. To date, resistant strains of *E. amylovora* have not been identified within Pennsylvania; however, they have been found within neighboring states such as New York, raising concerns of resistant strains soon surfacing in Pennsylvania orchards. The purpose of this study was to determine if streptomycin resistant strains are present in apple orchards located throughout Pennsylvania. In 2015, isolates were collected from grower submitted fire blight samples to the Pennsylvania State University Fruit Research and Extension Center in Biglerville, Pennsylvania. Initial identification was performed using Crosse Goodman specific media and apple inoculation. *Erwinia* identification was confirmed with direct colony PCR using primers specific to plasmid Ea29. Positive isolates were screened for resistance using a disk diffusion assay (100 µg/ml and 2500 µg ml of streptomycin sulfate respectively). Resistance was evaluated by observation of the presence or absence of a zone of inhibition. Of the samples processed to date, none have demonstrated streptomycin resistance. Screening is ongoing and any resistant isolates obtained will be further evaluated for the presence of plasmid mediated and chromosomal genes involved in resistance.

## Effect of kasugamycin application on bacterial resistance to kasugamycin and cross resistance with other antibiotics

S. GEBBEN (1), S. Gebben (2), G. McGhee (2), G. Sundin (2)

(1) Michigan State University, U.S.A.; (2) Michigan State University, U.S.A.

Kasugamycin (Ks) is an alternative option to the use of streptomycin for the control of bacterial plant diseases and it has been shown to be as effective as streptomycin in controlling fire blight, caused by *Erwinia amylovora*. However, there remains a concern that Ks application in orchards will select for Ks resistance that could be linked with other resistance genes that are active against antibiotics used in human medicine. To monitor for this possibility, we assessed the effect of the use of Ks (Kasumin 2L) in orchard systems on the level of resistance to Ks and five other antibiotics (streptomycin, ampicillin, gentamicin, cefotaxime, and tetracycline). Two sets of leaf and soil samples were collected (treated and non-treated with Ks) from a total of 23 orchards (5 different tree hosts from 7 states). Samples were processed in the laboratory and dilution plated onto King's B medium with or without Ks amendment. Bacterial population sizes were determined per sample and up to 15 gram-negative colonies growing on Ks-amended medium per sample (a total of 1,038 isolates) were used in antibiotic resistance screening. Although the bacterial population sizes were larger on Ks-amended medium from soil compared to leaf samples, there were no differences in populations from Ks treated vs. non-treated sites. Similarly, there was no difference in levels of resistance to the five antibiotics tested between Ks-treated and non-treated sites.

## Fungicide sensitivity in the wild rice pathogen *Bipolaris oryzae*

C. CASTELL-MILLER (1), D. Samac (2)

(1) PLPA/UMN, U.S.A.; (2) USDA ARS, U.S.A.

In recent years the occurrence of fungal brown spot, caused by Bipolaris oryzae has increased in cultivated wild rice (Zizania palustris) paddies in spite of the use of fungicides. To implement an efficient integrated disease management system, we are exploring whether field isolates have developed reduced sensitivity due to the recurrent use of strobilurin (i.e. inhibitors of the quinone outside site of the mitochondrial cytochrome *b*, QoI) and azole (i.e., C14 demethylation inhibitors, DMI) fungicides. We conducted in vitro spore germination assays to determine the EC₅₀ of two sets of isolates collected in 2008 and 2015 using 0, 0.10, 0.15, 0.25, 0.35, 0.5, 1.0, 5.0, 10.0, and 100 ug/ml of the active ingredient azoxystrobin (QoI), and 0, 0.1, 0.15, 0.25, 0.5, 1, 5, 10, 35 ug/ml propiconazole (DMI). All isolates tested were found to express the mitochondrial alternative oxidation pathway (AOX) allowing the fungus to germinate at all the doses of azoxystrobin tested. When the AOX was blocked, an average EC₅₀ value of 0.206 ug/ml was obtained for 2015 isolates and 0.158 ug/ml for isolates collected in 2008. Up to now, there is no evidence that isolates have become less sensitive to QoI fungicides. The EC₅₀ obtained for propiconazole for all strains tested until now was lower than the dose-equivalent used in the field. Molecular and in planta tests are underway to test the efficacy of the AOX pathway in spore germination.

## High-throughput strobilurin sensitivity testing of *R. solani* on Louisiana rice confirms reduced azoxystrobin but continued trifloxystatin sensitivity

A. LUNOS (1), C. Hollier (1), L. Brooks (1), S. Harding (1)

(1) Dept. of Plant Pathology & Crop Physiology, LSU AgCenter, U.S.A.

Azoxystrobin resistance in the rice sheath blight pathogen, *Rhizoctonia solani* AG 1-IA, has compromised a widely effective disease management tool for rice production in southwestern Louisiana. We previously characterized azoxystrobin, pyraclostrobin, and trifloxystrobin sensitivity of 40 *R. solani* isolates, one from each of 40 rice fields, to identify locations with azoxystrobin resistance and to assess cross-resistance. A high-throughput fungicide sensitivity assay was developed and used to characterize 122 additional isolates from these 40 fields. Serial dilutions of commercial grade fungicides were made in potato dextrose broth and added to 96-well microtiter plates. Fungal mycelial growth was measured based on optical density. Growth
curves were generated and used to determine EC₅₀ values, resulting in sensitivity distributions that agree with previous results. The distributions of azoxystrobin and pyraclostrobin EC₅₀ values were bimodal while trifloxystrobin was not. This and internal control isolates provide significant evidence of azoxystrobin resistance, reduced pyraclostrobin sensitivity, and continued sensitivity to trifloxystrobin in the sampled *R. solani* population. Growers need to prepare for the advent of azoxystrobin-resistant *R. solani* and could potentially substitute trifloxystrobin in fungicide rotations.

## Resistance to quinone outside inhibitor fungicides in *Cercospora sojina* in Ohio

L. WEBER (1), A. Dorrance (1)

(1) The Ohio State University, U.S.A.

Quinone outside inhibitor (QoI) fungicides are a common class of fungicides used to manage the fungus *Cercospora sojina*, the causal agent of frogeye leaf spot in soybean. Soybean leaf samples exhibiting symptoms of frogeye leaf spot were sent to the Soybean Pathology lab from across the state of Ohio during the summer of 2015. *Cercospora sojina* conidia were recovered from lesions to test their sensitivity to QoI fungicides. Potential *C. sojina* samples were confirmed by ITS sequencing then tested for the presence of the G143A mutation in the cytochrome b gene, the most commonly observed mutation in QoI-resistant fungi. QoI fungicide resistance was detected in two *C. sojina* samples in a mixed population of sensitive and resistant spores, while all other samples were sensitive to QoI fungicides. Fast and efficient identification of fungicide resistant *C. sojina* in Ohio will be useful in minimizing potential soybean yield loss as frogeye leaf spot incidence in Ohio increases in the warm, moist conditions and mild winters that favor the pathogen.

## Evaluating fungicide sensitivity of regional populations of *Blumeria graminis* f.sp. *tritici* in the United States

E. MEYERS (1), C. Cowger (2)

(1) North Carolina State University, U.S.A.; (2) USDA-ARS Plant Science Research Unit & North Carolina State University, U.S.A.

*Blumeria graminis* f.sp. *tritici* (Bgt), the causal agent of wheat powdery mildew, is primarily controlled by resistant wheat varieties and foliar fungicides. However, both control strategies risk being defeated by the large, rapidly adapting pathogen population. In 2013 and 2014, over 350 Bgt isolates were sampled from 27 fields in 15 states. The fungicide sensitivity of these single-spored isolates was evaluated using a detached leaf assay of fungicide-treated susceptible wheat leaf pieces. Five different fungicides, representing three major fungicide classes, were evaluated using 11 concentrations per fungicide. Detached leaves were inoculated with individual isolates and after 10 days leaf pieces were rated for sporulation. Bgt isolates from Mid-Atlantic states (NC, VA), Great Lakes states (MI, NY, OH, PA), and Deep South states (AL, FL, GA, MS) exhibited decreased sensitivity to the Demethylation Inhibitor (DMI) fungicides tebuconazole and prothioconazole compared to isolates from MO, AR, and the Plains states (KS, NE, OK). Plains isolates exhibited decreased sensitivity to the succinate dehydrogenase inhibitor (SDHI) fungicide fluxapyroxad compared to Deep South isolates. Sensitivity to the Quinone outside Inhibitor (QoI) fungicides pyraclostrobin and picoxystrobin did not differ by region. Genotypic evaluation of phenotypically insensitive isolates will further clarify the extent and nature of potentially fungicide-resistant isolates in this collection.

## Baseline sensitivities of *Rhizopus stolonifer* and first effective fungicides for management of almond hull rot in California

S. HAACK (1), H. Förster (1), J. Adaskaveg (1)

(1) University of California, Riverside, U.S.A.

Almond hull rot, caused primarily by *Rhizopus stolonifer* (*Rs*) and sometimes *Monilinia fructicola* (*Mf*), is an increasingly important disease in California almond production. These fungi infect the hull either during late fruit development (*Mf*) or at hull split (*Rs*). Fruit colonization leads to production of toxins that translocate into the fruiting wood, causing twig and branch dieback. Hull rot has been managed culturally by deficit irrigation and by limiting nitrogen application, but this opposes high-density, high-input farming that increases disease pressure. Fungicide treatments add to an integrated management approach. Baseline sensitivities to six fungicides representing DMI (difenoconazole, metconazole), SDHI (fluopyram, fluxapyroxad), and QoI (pyraclostrobin, trifloxystrobin) FRAC groups for inhibition of *Rs* mycelial growth or spore germination were determined using the spiral gradient dilution method. Mean EC₅₀ values for these fungicides were 0.102, 0.082, 0.292, 0.850, 0.002, and 0.002 mg/L, respectively. Sensitivities to each fungicide were within a ten-fold range, and thus, all isolates are considered sensitive. In field efficacy and timing studies, a single application of these fungicides and selected pre-mixtures at the beginning of hull split timed with a standard insecticide application was sufficient to reduce the disease by 60 to 80% when *Rs* was the primary pathogen. This represents the first fungicide-based field management strategy for hull rot.

## Sensitivity of *Neofabraea* spp strain to newly registered fungicides

P. SIKDAR (1), M. Mazzola (2)

(1) Washington State University, U.S.A.; (2) USDA-ARS, U.S.A.

Bull's eye rot, incited by a group of closely related fungi in the genus *Neofabraea*, causes significant economic loss to the Washington State apple industry. The pathogen complex is a significant limitation as it has been listed as a quarantine pathogen with a zero tolerance level for fruit shipped to China and Israel. Access to export markets for Washington State apples relies on the development of effective bull's eye rot control methods. Previous experiments indicate that fungicide treatments containing thiabendazole and thiophenate-methyl provide effective disease control, however use of this treatment is not recommended due to the potential for development of resistance in populations of *Penicillium* spp., which incites the common postharvest disease, blue mold. Fludioxonil (Scholar) did not demonstrate consistently effective control of bull's eye rot in fruit and did not limit *in vitro* growth of the pathogen. Additional analyses were conducted to screen *Neofabraea* spp. isolates for sensitivity to newer commercially available fungicides that include multiple active chemistries. Results from these trials suggest that fungicide containing fluopyram and trifloxystrobin inhibits *in vitro* growth of *Neofabraea* at a level equivalent to that of thiabendazole. In contrast, fungicide treatment containing cyprodinil with difenoconazole, or solatenol alone failed to inhibit mycelial growth or spore germination *in vitro*.

## Baseline sensitivities to SDHI fungicides and characterization of the *sdhB* gene in *Venturia inaequalis*

S. VILLANI (1), K. Ayer (2), K. Cox (2)

(1) North Carolina State University, U.S.A.; (2) Cornell University, U.S.A.

The succinate dehydrogenase inhibitor (SDHI) fungicides are a class of broad spectrum, single-site fungicides that interfere with fungal respiration in the mitochondrion. The recent registration of next-generation SDHIs for apple scab management prompted an investigation into the baseline sensitivity of *Venturia inaequalis* to penthiopyrad, fluopyram, and benzovindiflupyr. Thirty-five to 70 isolates with no prior exposure to single-site fungicides were used to determine the effective concentration at which conidial germination was inhibited by 50% (EC₅₀). Mean EC₅₀ values for penthiopyrad, fluopyram, and benzovindiflupyr were 0.086, 0.176 and 0.0016 µg ml⁻¹, respectively. Linear correlation analysis revealed a significant and positive correlation in sensitivity between fluopyram and penthiopyrad (P ≤ 0.0001, r = 0.66) and fluopyram and benzovindiflupyr (P = 0.0014, r = 0.52). Genomic sequencing was performed using the Illumina MiSeq platform to identify and characterize the *sdhB* gene for 3 isolates collected from a research, baseline, and commercial orchard population. No common mutation sites commonly associated with SDHI resistance were discovered in these isolates, or in other isolates that were recovered following field applications of SDHIs. Results suggest that SDHIs are highly effective against conidial germination and provide a basis for phenotypic and genotypic monitoring of SDHI resistance in populations of *V. inaequalis*.
## Assessment of QoI Resistance in *Colletotrichum* spp. Isolated from Boxthorn and Apple in Korea

S. KIM (1), J. Min (1), D. Back (1), H. Kim (1), S. Lee (1), K. Kim (1)

(1) Chungbuk National University, Korea

Strobirulin fungicides including azoxystrobin, krezoxim-methyl, and pyraclostrobin are inhibitors of mitochondrial respiration; they inhibit the cytochrome *bc*₁ enzyme complex at the Qo site (quinone outside inhibitors, QoIs). QoI resistance has been reported in many pathogens such as *Blumeria graminis* f. sp. *tritici*, *Plasmopara viticola* and so on. The primary molecular mechanism of QoI resistance is a point mutation at codon 143 based on the substitution of glycine by alanine(GGT to GCT, G143A) in the mitochondrial-encoded cytochrome *b* gene. We examined the sensitivity of *Colletotrichum* spp. isolated from boxthorn fields and apple orchards to pyraclostrobin through the mycelial growth inhibition test on PDA amended with the fungicide. The resistance of *C. acutatum* isolates from boxthorn was determined with values of EC₅₀ (effective concentration reducing the mycelial growth by 50%). The EC₅₀ of resistant isolates was more than 1.7µg/ml. On the other hand, The EC₅₀ of sensitive isolates of *C. gloeosporioides* from apples was determined as less than 0.24µg/ml, whereas resistant isolates were more than 66.9µg/ml. The codon 143 of cytochrome *b* gene was substituted from GGT to GCT in all of resistant isolates regardless of *Colletotrichum* species. For the successful control of anthracnose in boxthorn fields or apple orchards in Korea, it is required a monitoring of the QoI resistance with an allele-specific PCR with primers generated from the 143 codon substitution.

## Characterization of fungicide resistance phenotypes in *Botrytis cinerea* populations from blueberry in Florida

A. AMIRI (1), A. Zuniga (2), P. Harmon (2), N. Peres (2)

(1) Washington State University, U.S.A.; (2) University of Florida, U.S.A.

Botrytis blossom blight caused by *Botrytis cinerea* is an important blueberry disease and is mainly controlled by fungicide applications prior and during bloom. In this study, we evaluated the sensitivity of 624 *B. cinerea* isolates collected from 11 blueberry fields in central Florida between February and May of 2013, 2014, and 2015. Six fields were near strawberry and 5 fields were located 40 km or more from strawberry fields. Isolates were grown on malt yeast extract agar and tested for their sensitivity to pyraclostrobin (Pyra), boscald (Bosc), penthipyrad (Penth), fludioxonil (Flud), fenhexamid (Fenh), cyprodinil (Cyp), and iprodione (Ipr) using a spore germination inhibition assay and previously determined discriminatory doses. Overall, disease incidence was higher in samples collected from blueberry fields planted next to strawberry fields compared to blueberry fields alone. In 2013, resistance frequencies were 1.7, 14.9, 46.3, 49.5, 57.8, 57.9 and 92.6% for Flud, Penth, Ipr, Cyp, Bosc, Fenh, and Pyra, respectively. Similar resistance frequencies were observed in 2014 and 2015 with some significant differences across locations and fungicides. Resistance frequencies were higher in blueberry fields next to strawberry fields for all fungicides except Flud, Penth and Ipr. Our findings indicate that resistance to multiple fungicides in *B. cinerea* is widespread in blueberry fields and that resistant populations selected on strawberry may spread to adjacent crops.

## Baseline sensitivity of *Phyllosticta citricarpa* isolates from Florida to succinate dehydrogenase inhibitor fungicides in vitro study

K. NICOLETTA (1), M. Dewdney (1)

(1) University of Florida, Citrus Research and Education Center, U.S.A.

In 2010, Citrus Black Spot (CBS), caused by *Phyllosticta citricarpa*, was first found in Florida. CBS has a negative impact on fresh market fruit and yield production causing fruit lesions and premature fruit drop. This study assessed 116 monoconidial *P. citricarpa* isolates from 'Valencia' and 'Hamlin' with 39 isolates from Collier, 76 from Hendry and 1 from Polk Counties. Isolate sensitivity to the succinate dehydrogenase inhibitor (SDHI) fungicides: boscald (0, 0.01, 0.1, 1, 5, 10 and 25 µg/ml), fluxapyroxad (0, 0.001, 0.01, 0.05, 0.1, 1 and 5 µg/ml), and fluopyram (0, 0.001, 0.01, 0.1, 0.5, 1 and 5 µg/ml) was evaluated by mycelial growth inhibition assays. The mean EC₅₀ values for boscald and fluxapyroxad were 0.179 and 0.026 µg/ml, respectively. These EC₅₀ values were significantly different for each fungicide (*P* <0.001). It is expected the EC₅₀ value of fluopyram will fall between boscald and fluxapyroxad's values. In the baseline assay all the isolates tested are sensitive to SDHI fungicides, therefore we expect no mutations in molecular analyses. Developing the baseline sensitivity of *P. citricarpa* to SDHI fungicides allow for resistance monitoring. In previous studies, *P. citricarpa* isolates were found to be sensitive to both quinone outside inhibitor (QoI) fungicides and demethylation inhibitor (DMI) fungicides. Thus, SDHI fungicides could potentially be used as rotational sprays with QoI and DMI fungicides to reduce the risk of resistance developing.

## Screening for QoI resistance among *Colletotrichum* species associated with ripe rot of grape in Virginia vineyards

M. NITA (1), A. Bly (1)

(1) Virginia Tech, U.S.A.

Ripe rot of grape is a fungal disease caused by two *Colletotrichum* species complexes, *C. acutatum* and *C. gloeosporioides*. This disease can result in significant crop loss and off-flavors in wine. Among recommended fungicides against ripe rot are the QoI group, or strobilurins. We examined the prevalence of QoI insensitivity among Colletotrichum isolates collected throughout Virginia during 2013. For the fungicide-amended medium assay, petri dishes containing potato dextrose agar amended with 100 ppm azoxystrobin (Abound, 22.9% a.i. azoxystrobin, Syngenta Crop Protection) and 1,000 ppm SHAM (salicylhydroxamic acid) were inoculated with a mycelial plug of each of the isolates and then incubated at 25°C. The presence or absence of growth on azoxystrobin-amended medium was examined after 6 days. PCR-RFLP was utilized to detect a point mutation (G143A) of the cytochrome *b* gene. Primers GCCBF1 and RSCBR2 were used to amplify a 120-bp fragment, which was digested with restriction enzyme *Fnu4HI* to detect the G143A mutation. The results showed that among 283 isolates tested, 28% of isolates was insensitive to azoxystrobin *in vitro*, and 16% of the isolates had the G143A mutation. The higher percentage with the fungicide-amended medium assay was expected, since there are multiple mutations that can lead to insensitivity. High prevalence of QoI-insensitive isolates in Virginia vineyards warrants the modification of our management recommendations.

## Detection of quinone outside inhibitor resistant isolates of *Erysiphe necator* in Oregon vineyards

B. WARNEKE (1), J. Yamagata (2), T. Neill (3), T. Miles (2), W. Mahaffee (3)

(1) Oregon State University, U.S.A.; (2) California State University Monterey Bay, U.S.A.; (3) USDA-ARS, U.S.A.

Grape Powdery Mildew (GPM, caused by *Erysiphe necator* (*En*)) is the most persistent disease in the west coast grape industry due to a long and dry growing season which favors its fecundity and inoculum dispersal via airborne conidia. GPM control failures in 2015 led to the exploration of the possibility of quinone outside inhibitor (QoI) fungicide resistance present in Oregon *En* populations. A survey of spatially distinct vineyards in Oregon was performed in which leaf and fruit samples infected with *En* were collected. Isolation and DNA extraction of *En* samples and subsequent quantitative PCR analysis using competitive TaqMan probes detected the presence of the G143A mutation. Isolates with the G143A mutation were considered resistant to QoIs. Conidia germination bioassays using Trifloxystrobin and Kresoxim-Methyl in 1.5% water agar confirmed genetic results of 28 isolates examined. Of the 28 isolates examined, 22 were resistant to both QoIs (EC₅₀>100µg/ml), while 6 were sensitive (EC₅₀ from 0.004 to 0.06µg/ml). Using a discriminatory dose of 0.1µg/ml, 19 of 19 more isolates were resistant to QoIs and agreed with results from the qPCR assay.

## Characterization of resistance to DMI fungicides in *Colletotrichum* spp. isolates from peach

S. CHEN (1), C. Luo (1), M. Hu (2), G. Schnabel (2)

(1) Huazhong Agricultural University, China; (2) Clemson University, U.S.A.
Isolates of the *Colletotrichum acutatum* species complex were collected from South Carolina and Georgia peach orchards. Phylogenetic analysis of the combined ITS, glyceraldehyde-3-phosphate dehydrogenase, and beta-tubulin gene sequences separated the isolates into two species, *Colletotrichum nemphaeae* and *Colletotrichum fioriniae* subgroups 1 and 2. The sensitivity of these and three other *Colletotrichum* species from peach (*Colletotrichum fructicola*, *Colletotrichum siamense*, and *Colletotrichum truncatum*) to demethylation inhibitor (DMI) fungicides difenconazole, propiconazole, tebuconazole, metconazole, flutriafol, and fenbuconazole were determined. *C. truncatum* was resistant to tebuconazole, metconazole, flutriafol, and fenbuconazole. *C. nemphaeae* was resistant to flutriafol and fenbuconazole. *C. fructicola* and *C. siamense* were sensitive to all DMI fungicides, and *C. fioriniae* subgroup 2 was reduced-sensitive to DMI fungicides compared with *C. fioriniae* subgroup 1. Difenconazole and propiconazole provided the best control efficacy in vitro to all five species. Tebuconazole and metconazole were effective against all *Colletotrichum* species, except for *C. truncatum*. The 14-α sterol demethylase genes *CYP51A* and *CYP51B* from resistant isolates revealed point mutations associated with resistance to DMI fungicides. The strong in vitro activity of some DMI fungicides against *Colletotrichum* species may be exploited for improved anthracnose disease management of peach.

## In-vitro azoxystrobin sensitivity of *Colletotrichum gloeosporioides* isolates from blueberry in north and central Florida

M. VELEZ-CLIMENT (1), P. Harmon (1)

(1) University of Florida, U.S.A.

Anthracnose pathogens cause destructive diseases of blueberry fruits, leaves, and stems in Florida. Growers in central Florida have reported a lack of efficacy from QoI fungicides, specifically for anthracnose stem cankers on the southern highbush blueberry variety Flicker. Twenty-two single-spore isolates collected from symptomatic stems, leaves, and berries were identified and tested for sensitivity to QoI fungicides. Percent inhibition (PI) of conidial germination on PDA plates amended with 10µg mL⁻¹ of azoxystrobin demonstrated all isolates from central Florida farms were insensitive to azoxystrobin. Isolates from north Florida farms were sensitive. Internal transcribed spacer region (ITS) sequence comparisons grouped all isolates within the *C. gloeosporioides* species complex, and the diverse morphology of the colonies and conidia agreed with this placement. These data suggest the fungicide failures reported by growers in central Florida were due to selection of QoI fungicide insensitivity in stem-infecting populations of *C. gloeosporioides*. Furthermore, insensitive isolates from fruits and leaves indicate that the fungicide azoxystrobin may no longer be a viable management option for ripe rot and anthracnose leaf spot, either. Additional efforts are needed to determine the nature and distribution of the resistance, to develop alternative management options, and to reinforce good resistance management practices for Florida's blueberry industry.

## Fungicide sensitivity of *Monilinia vaccinii-corymbosi* in highbush blueberries in Michigan

K. FITZGERALD (1), J. Gillett (1), A. Schilder (1)

(1) Michigan State University, U.S.A.

Mummy berry in blueberries, caused by the fungus *Monilinia vaccinii-corymbosi* (*Mvc*), is usually controlled with fungicides, in particular sterol inhibitors which have been used for almost 20 years. Fungicide sensitivity of *Mvc* isolates was studied across Michigan. Isolates were collected from various field sites and grown in pure culture: 72 isolates were tested in poison agar assays using six concentrations of three commonly used fungicides in the sterol inhibitor class (fenbuconazole, metconazole, and prothioconazole). Commercial formulations of these products were used. Colony diameter was measured after an incubation of 14 days at 22 degrees C in the dark and compared to growth on non-amended PDA. EC₅₀ values were calculated for all isolates using a four-parameter logistical curve fit in the computer program R. EC₅₀ values ranged from 0.0016 to 0.0146 ppm for fenbuconazole, 0.0007 to 0.0104 ppm for metconazole, and 0.0021 to 0.0986 ppm for prothioconazole. A slight shift towards reduced fungicide sensitivity was detected in more intensively managed fields compared to baseline sites; however, highly resistant isolates were not detected. The ITS region of the ribosomal DNA of the isolates was sequenced to confirm their identity and assess species-level genetic diversity. All isolates were nearly identical. Although the risk of sterol inhibitor resistance in *Mvc* appears to be relatively low, fungicide resistance management is still imperative.

## Induced overexpression of the gene *MfCYP51* may reveal molecular mechanisms associated to tebuconazole resistance of *Monilinia fructicola* in Brazil

P. LICHTEMBERG (1), Y. Luo (1), T. Michailides (1), L. May De Mio (2)

(1) University of California - Davis, U.S.A.; (2) Universidade Federal do Paraná, Brazil

The aim of this study was to investigate the mechanism associated with *Monilinia fructicola* resistance to demethylation inhibitor fungicide (DMI) tebuconazole in Brazil. In this study 10 isolates (8 DMI-R and 2 DMI-S) with broad tebuconazole sensitivity (0,003 - >10 µg/ml) were used to quantify the expression levels of the genes *MfCYP51* and *MfABC1*, respectively encoding the target enzyme 14 α-demethylase and mediating the transport of substances across biological membrane. After the mycelial exposure to sub-lethal doses of tebuconazole, the 2⁻⁶⁻⁸CI method revealed increased *MfCYP51* mRNA levels for 6 out of 7 DMI-R isolates (ranging from 1.06 to 9.08), while DMI-S revealed small expressions levels of 0.14 and 0.46. The induced expression of the *MfABC1* gene with tebuconazole on isolate mycelium not always increased the levels of mRNA. The Mona element which likely trigger the elevated expression of *MfCYP51* was not present on 58 DMI-R isolates from Brazil including those with increased expression, suggesting that other genetic change yet to be discovered may be triggering this mechanism. Full *MfCYP51* sequencing is currently underway. This is the first report on molecular mechanisms for DMI resistance identification for *M. fructicola* isolates from Brazil; providing an important advancement for risk assessment of DMI fungicides used on brown rot management.

## Profiling of Resistance to SDHI Fungicides in *Botrytis cinerea* from Strawberry Fields

M. HU (1), D. Fernández-Ortuño (2), G. SCHNABEL (1)

(1) Clemson University, U.S.A.; (2) Universidad de Málaga, Spain

From 2012-13 to 2014-15, a total of 2,570 *B. cinerea* isolates were collected from strawberry fields in the eastern United States and their sensitivity to the SDHI fungicides including boscalid, fluopyram, fluxapyroxad, and penthiopyrad was assessed. Assays were based on visual assessment of presence or absence of mycelial growth on media amended with discriminatory doses of fungicides in microtiter plates. Overall frequencies of isolates resistant to boscalid, fluopyram, fluxapyroxad and penthiopyrad increased over the three years to 30.0, 1.0, 5.5, and 7.4%, respectively. Four resistance patterns designated A, B, C or D were found. Pattern A isolates were resistant to boscalid with the allele H272R at locus *sdhB*; Pattern B isolates were resistant to boscalid and penthiopyrad with the allele H272R or H272Y at locus *sdhB*; Pattern C isolates were resistant to boscalid, fluxapyroxad, and penthiopyrad with the allele H272Y at locus *sdhB*; and pattern D isolates were resistant to boscalid, fluopyram, fluxapyroxad, and penthiopyrad with alleles P225F or N230I at locus *sdhB*. Isolates with alleles H272R, H272Y, N230I or P225F were sensitive to a new SDHIIs, benzovindiflupyr, with mean EC₅₀ values less than 0.5 µg/ml for each genotype, suggesting that this fungicide may be useful for resistance management.

## Resistance of *Colletotrichum gloeosporioides* from strawberry to azoxystrobin and reduced-sensitivity to thiophanate-methyl

M. OLIVEIRA (1), M. Chamorro (1), N. Peres (1)

(1) University of Florida, U.S.A.

*Colletotrichum gloeosporioides* causes Colletotrichum crown rot (CCR) of strawberry in the southern United States. Symptoms start with wilting of the plants which consequently collapse. A discoloration can be observed when the crown is cut open. CCR is managed with the use of disease-free transplants and fungicide applications. Our objective was to evaluate the sensitivity of *C. gloeosporioides* to two common fungicides used to manage CCR in Florida strawberry fields. Sixty-three *C. gloeosporioides* isolated from strawberry crowns from 1995 to 2015 were evaluated in a conidial
germination assay for sensitivity to thiophanate-methyl and azoxystrobin at discriminatory concentrations of 3 and 100 µg/ml. An isolate collected in 1999 was found resistant to azoxystrobin. After that, resistance was observed sporadically only in isolates collected after 2007. Most of the isolates recently collected during the 2015-16 FL strawberry season were resistant to azoxystrobin. A reduced sensitivity to thiophanate-methyl was observed for all isolates in both fungicide concentrations tested, and none of the isolates was completely sensitive. We conclude that *C. gloeosporioides* isolates resistant to azoxystrobin have been selected after the introduction of this fungicide in the market in 2001. However, it appears that *C. gloeosporioides* isolates from strawberry have a natural reduced sensitivity to thiophanate-methyl.

## Increased frequency of multifungicide resistance in *Botrytis cinerea*

M. HU (1), G. Schnabel (1)

(1) Clemson University, U.S.A.

Over four consecutive strawberry production seasons, resistance to 7 chemical classes of fungicides was determined for 2130 isolates from commercial strawberry fields located in South Carolina and 10 other states of the eastern United States. The overall frequencies of isolates resistant to thiophanate-methyl, pyraclostrobin, fenhexamid, boscald, cyprodinil, iprodione, and fludioxonil increased over the four seasons to 84.7, 73.0, 32.2, 29.9, 29.7, 12.6, and 11.3%, respectively. Resistance to thiophanate-methyl was found virtually in every location. Resistance to fludioxonil was rarely found during the first three seasons, but increased to 11.3% in 2014-15. Our latest data from the 2015-16 season shows the frequency of resistance to fludioxonil has increased to 12.3%, indicating a strong selection for fludioxonil resistance in strawberry fields in the eastern United States. We hypothesize that the more FRAC codes are rotated into the spray program, the more we will select for isolates with increasing numbers of resistance determinants.

## Resistance of *Botrytis cinerea* to fungicides in California strawberries

S. COSSEBOOM (1), G. Schnabel (2), K. Ivors (1), G. Holmes (1)

(1) Cal Poly State University, U.S.A.; (2) Clemson University, U.S.A.

Botrytis gray mold, caused by *Botrytis cinerea*, is a very destructive pre- and post-harvest fruit rot of strawberry that affects production worldwide. Resistance to foliar applied fungicides has been reported in many regions, including California. The objective of this study is to characterize the fungicide resistance of *B. cinerea* isolates from California. Approximately 800 isolates of *B. cinerea* were collected from four strawberry fruit production regions of California (Oxnard, Santa Maria, Salinas, Watsonville) (10 isolates per ranch, 10 ranches per growing region). Isolates were collected early season (minimal selection pressure) and late season (maximum selection pressure) during the 2015-2016 growing season. Using a previously described technique, sensitivity of each isolate was determined with a discriminatory dosage of each active ingredient: boscalid, cyprodinil, fenhexamid, fludioxonil, fluopyram, iprodione, penthio pyrad, and thiophanate-methyl. Preliminary data show individual isolates with resistance to one or more active ingredients and modes of action. While previous work in California has documented resistance to a few active ingredients in a few fields, this is the first comprehensive study of *B. cinerea* resistance to commonly used fungicides and modes of action. Results of this study will help identify ineffective fungicides and improve gray mold management programs.

## Monitoring *Colletotrichum acutatum* Resistance to Quinone-outside Inhibitor Fungicides in Strawberry

B. FORCELINI (1), B. Forcelini (1), N. Peres (1)

(1) University of Florida, U.S.A.

*Colletotrichum acutatum*, the causal agent of anthracnose fruit rot (AFR) and anthracnose root necrosis (ARN), is a major fungal pathogen affecting strawberry. Control of AFR and ARN has been dependent on the use of Quinone-outside Inhibitor (QoI) fungicides. Resistance of *C. acutatum* isolates to QoI fungicides was reported for the first time in 2013 in Florida, and was linked to five specific strawberry nurseries from Canada and North Carolina. In 2016, *C. acutatum* isolates were collected after outbreaks of ARN in Florida and coastal California. Ninety isolates from four nurseries were tested for QoI sensitivity using a discriminatory dose mycelial growth assay. Forty-two isolates were not inhibited at 3 nor at 100 µg azoxystrobin per ml and were considered QoI-resistant. Resistant isolates originated from two of the four nurseries tested. *C. acutatum* resistance to QoI fungicides is becoming a widespread problem in strawberry nurseries located in different regions. This situation warrants immediate changes in anthracnose management strategies that integrate non-chemical strategies to limit selection and prevent spread of QoI-resistant populations.

## Sensitivity of *Sclerotinia sclerotiorum* to thiophanate-methyl, fluazinam, and procymidone

M. Lehner (1), R. Silva (1), T. Paula Jr. (2), E. Mizubuti (1)

(1) Universidade Federal de Vicos a, Brazil; (2) EPAMIG, Brazil

The sensitivity of 282 Brazilian isolates of *Sclerotinia sclerotiorum* to thiophanate-methyl (TM), fluazinam, and procymidone was assessed using discriminatory doses and concentration that results in 50% mycelial growth inhibition (EC50). No isolate was resistant to fluazinam or procymidone. The EC50 values varied from 0.003 to 0.007 and from 0.11 to 0.72 mg/ml for fluazinam and procymidone, respectively. One isolate collected in 2010 was resistant to TM (EC50 > 100 mg/mL). The 2010 TM-resistant isolate had a L240F mutation in the b-tubulin gene. This is the first report of mutation at codon 240 conferring resistance to benzimidazole in *S. sclerotiorum*. The high-resolution melting analysis allowed the distinction of TM-sensitive and TM-resistant isolates by specific melting peaks and curves. The 2010 TM-resistant isolate had mycelial growth, sclerotia production, and aggressiveness comparable with that of the sensitive isolates. In 2015, 8 isolates were collected from the same region where the 2010 TM-resistant isolate was obtained. All 2015 isolates were sensitive to fluazinam and procymidone, but resistant to TM. The 2015 TM-resistant isolates exhibited considerable instability in mycelial growth rates after consecutive transferring on PDA. Resistance to fluazinam and procymidone was nonexistent or rare. Resistance to TM may occur, but at this point no robust inference can be made about the persistence of TM-resistant isolates in the field.

## Sensitivity of *Sclerotinia sclerotiorum* to iprodione and propiconazole in Bangladesh

M. ISLAM (1), C. Vrisman (1), S. Miller (1)

(1) The Ohio State University, U.S.A.

*Sclerotinia sclerotiorum* is a destructive plant pathogen causing white mold of mustard. The pathogen was first reported in Bangladesh in 2007. Iprodione and propiconazole have been used to control the disease, but the sensitivity of *S. sclerotiorum* isolates to these fungicides in Bangladesh has not been determined. One hundred eighteen *S. sclerotiorum* isolates from Bangladesh and six from Ohio were tested for sensitivity to the two fungicides. Isolates were collected from infected mustard, soybean and cauliflower plants, plated on Potato Dextrose Agar medium (PDA) and purified by mycelial tip culture. Five-millimeter mycelial discs from 2-day-old cultures were plated on non-amended PDA or PDA amended with iprodione (0.0001, 0.001, 0.01, 0.1, 0.3, 1.0 or 10.0 ppm) or propiconazole (0.001, 0.01, 0.1, 0.3, 1.0, 10.0 or 100.0 ppm). Cultures were incubated at room temperature and radial mycelial growth was measured after 48 hours of incubation. EC₅₀ (50% mycelial growth inhibition) values were calculated by regression of mycelial growth relative to the control against log₁₀ fungicide concentrations. EC₅₀ values for iprodione and propiconazole ranged from 0.11 to 0.55 and 0.12 to 0.78 ppm respectively. Since *S. sclerotiorum* has appeared recently in Bangladesh and fungicide use has been relatively infrequent, these values will serve as baseline data for monitoring their efficacy going forward.

## Presence of fungicide resistant *Botrytis* strains in Norwegian forest nurseries

G. STRØMENG (1), V. Talgø (1), I. Fløistad (1)

(1) NIBIO, Norway
Approximately 30 million seedlings of Norway spruce (Picea abies) are planted in Norwegian forests each year, and the main part of these plants are produced by forest nurseries in Norway. Among the greatest challenges in the seedling production is the control of gray mold, caused by *Botrytis*. Fungicides used in Norwegian forest nurseries to control gray mold are thiophanate methyl, fenhexamid, iprodione, and a mixture of cyprodinil and fludioxonil. Increasing disease problems in newly planted seedlings over the last few years, made it necessary to investigate the possible presence of fungicide resistant *Botrytis* strains in forest nurseries. Isolates were collected from spruce seedlings and germ tube growth was measured on nutrient agar amended with different concentrations of the fungicides. We tested fludioxonil and cyprodinil separately, by using fungicides that contained fludioxonil or pyrimethanil (same chemical group as cyprodinil, cross resistance expected) as single active ingredients. Thus far, we have tested 35 isolates, and 54, 26 and 6% of these were resistant to thiophanate methyl, fenhexamid, and iprodione, respectively. Furthermore, 46, 26, 6, and 3% were moderately resistant to thiophanate methyl, pyrimethanil, iprodione, and fludioxonil, respectively. Nine isolates were resistant to two fungicides. We recommend that the nurseries avoid use of thiophanate methyl in the future, and that fenhexamid resistance is carefully monitored.

## Fungicide resistance in populations of *Fusarium proliferatum* causing bulb rot of onion in the Pacific Northwest

K. Fairchild (1), S. Windes (1), A. Malek (1), J. Woodhall (2), P. Wharton (1)

(1) University of Idaho, U.S.A.; (2) University of Idaho, U.S.A.

Fusarium bulb rot, caused by the fungus *Fusarium proliferatum*, is an emerging disease on onion in Idaho. This bulb rot occurs in storage and shows symptoms similar to neck rot. Symptoms usually start at the neck and progress down along the fleshy scales, but the basal plate is not affected. Infected scales become translucent yellow and water-soaked, later turning tan-brown and soft. Frequently, the rot appears to progress down from the neck along a single fleshy scale in the bulb. In most cases the infected bulbs do not show any pronounced external symptoms or signs of infection. Currently, there are no control measures known for this disease as the mode of infection and conditions required for disease development are poorly understood. Fungicide sensitivity testing was done on a range of fungicides which have shown potential for the control of *F. proliferatum* using spiral plate dilution gradients. Results showed that there was a range of sensitivities in the populations of *F. proliferatum* to the fungicides tested. A knowledge of fungicides showing effective control of this pathogen in vitro is important in the development of effective management strategies to control this disease.

## Sensitivity of *Podosphaera xanthii* isolates from Illinois to fungicides

Y. XIANG (1), M. Babadoost (2)

(1) University of Illinois, Urbana-Champaign, U.S.A.; (2) Department of Crop Sciences, University of Illinois, U.S.A.

Powdery mildew causes more than 50% yield losses in pumpkin and squash fields in Illinois, if it is not managed effectively. Since 2010, growers in Illinois have complained about failure of some fungicides to effectively control powdery mildew in cucurbit fields. This research was conducted to evaluate sensitivity of powdery mildew fungi from Illinois to major powdery mildew fungicide groups, including demethylation inhibitors (DMI), strobilurins or quinone outside inhibitor (QoI), succinate dehydrogenase inhibiting (SDHI), and cyflufenamid. In 2014 and 2015, 34 isolates of powdery mildew fungi were collected from cucurbits fields in 10 counties in Illinois. All of the collected isolates were identified as *Podosphaera xanthii* by using universal fungi primers ITS1F and ITS4 to amplify the internal transcribed spacer (ITS) region of nuclear ribosomal DNA (nrDNA). Twenty of the isolates from different locations and different hosts were tested for their sensitivity to the four fungicides, including triflumizole (DMI), azoxystrobin (QoI), penthio pyrad (SDHI), and cyflufenamid. Our preliminary studies in the laboratory showed that the isolates were highly sensitive to triflumizole, penthio pyrad, and cyflufenamid, but less sensitive to azoxystrobin. The sensitivity of the isolates to azoxystrobin varied. Similarly, severity of powdery mildew in the field plots treated with azoxystrobin was significantly higher than those in the plots received the other three fungicides.

## *Podosphaera xanthii* strains with resistance and reduced sensitivity to several fungicides may challenge control of cucurbit powdery mildew

M. McGrath (1)

(1) Cornell University, U.S.A.

Fungicides at risk for resistance development are important for effectively managing powdery mildew (PM) in cucurbit crops because they can move to lower leaf surfaces where the pathogen develops best. Isolates were collected from research and commercial plantings on Long Island, NY, on 16 Sep to 1 Oct 2015, which was after several fungicide applications for PM. A leaf disk bioassay was used to determine fungicide sensitivity. All but 1 of the 57 isolates tested were determined to be resistant to QoI fungicides (FRAC 11) and 79% of the isolates were resistant to boscalid, an SDHI fungicide (FRAC 7). Many grew on leaf disks treated with 500 ppm boscalid as well as on untreated disks. Isolates with very limited or no growth on 500 ppm boscalid disks tended to also be slightly tolerant or sensitive to 40 ppm myclobutanil (FRAC 3) and 40 ppm quinoxyfen (FRAC 13); most of these were collected from research plants at LIHREC not treated with any at-risk fungicides (organically-managed and non-treated plots in a fungicide evaluation). Many boscalid-resistant isolates also grew well on disks with 40 ppm myclobutanil and on disks with 40 ppm quinoxyfen. Some of these exhibit tolerance to higher doses. These results document strains of *Podosphaera xanthii* with resistance and reduced sensitivity to multiple fungicide chemistries can be selected during one growing season, including where not exposed to all 3 chemistries. Such strains could foil alternation fungicide programs.

## Fungicide resistance profiles and virulence of *Stagonosporopsis* species isolates from watermelon and melon in eastern China

M. NEWARK (1), N. Dufault (1), M. Paret (2), L. Pingfang (3), X. Yang (3)

(1) University of Florida, U.S.A.; (2) University of Florida, U.S.A.; (3) Jiangsu Academy of Agricultural Science, China

*Stagonosporopsis* spp. (syn: *Didymella bryoniae*) is a major pathogen on cucurbits. China is the largest producer of watermelon in the world with 22% of the seed imported to the US is coming from China. The purpose of this study was to examine fungicide resistance and virulence profiles of *Stagonosporopsis* spp. isolates in China as a first step in understanding potential global movement of novel strains. Sixty-nine mono-conidial isolates from 4 provinces in east China were collected between 2011 and 2015. Fungicides azoxystrobin, boscalid, difenoconazole, tebuconazole, and thiophanate M were tested using an amended agar assays at the rates of 0.096, 0.034, 0.032, 0.128, and 100mg/L, respectively. Virulence was examined using a detached leaf assay with the watermelon (*Citrullus lanatus*) var: 'Sugar Baby'. Leaves were inoculated with the pathogen and evaluated using a disease scale. Statistical analysis was done using R version 3.2.3 examining frequencies and distributions of fungicide resistance and virulence. All isolates found in East China were pathogenic on a US variety with many isolates having moderate (67%) or even high virulence (29%). In total, 92% of the isolates were resistant to two or more classes of fungicides. Based on these findings, it is apparent that fungicide resistance is present in eastern China and could pose a risk to the global movement of resistant strains.

## Investigating dynamics of QoI fungicide resistance in potato early blight complex pathogens in Wisconsin

S. DING (1), K. Meinholz (1), A. Gevens (1)

(1) University of Wisconsin-Madison, U.S.A.

The potato early blight complex (EBC), caused by *Alternaria solani* and *A. alternata*, is a perennial and destructive fungal disease that can lead to loss in yield and quality. Without cultivar resistance, management is with weekly-applied fungicides which can result in pathogen resistance in commercial fields treated with single site mode of action fungicides. One such group is the QoIs, which target the cytochrome *b* of pathogens. In this 2015 study, the F129L mutation which leads to QoI partial resistance was detected in all *A. solani* isolates collected from 4 farms representing unique potato production regions of Wisconsin. Three of the farms conducted conventional production practices; one farm was certified organic. QoI resistance in *A. alternata* was
due to the G143A mutation and was detected in 64% of the isolates in early July, and 91% in late August. At the organic farm, the percentage of *A. alternata* isolates with QoI resistance increased from 0 to 13% over the season. It is possible that *A. alternata*, with a broad host range, may have a wild type preserved in alternative hosts which can move into potato. *Alternaria solani*, which is more specifically a potato pathogen, may have been subjected to QoI selection more thoroughly. Our results suggested that there was rapid selection for QoI resistance within a single production season, indicating the need for modified and integrated management responses for long term management of the EBC.

## Sensitivity of *Alternaria* spp. causing diseases in tomato and potato to boscalid and azoxystrobin

C. VRISMAN (1), M. Islam (1), S. Miller (1)

(1) The Ohio State University, U.S.A.

Tomato and potato growers in Ohio have recently reported reduced efficacy of fungicides used to control diseases caused by *Alternaria*. We aimed to assess the sensitivity of *Alternaria* isolates to two commonly used fungicides. Thirty isolates were collected from symptomatic tomato and potato plants in 2015, 28 of which were identified as *A. alternata* and two as *A. solani*. Isolates were screened for sensitivity to commercial formulations of azoxystrobin and boscalid. Mycelial discs were transferred to potato dextrose agar plates containing 0.0, 0.01, 0.1, 1, and 10 µg boscalid/mL; or 0.0, 0.01, 0.1, 1, 10, and 100 µg azoxystrobin/mL amended with SHAM (100 µg/mL). The diameter of fungal colonies was measured after 8 days of incubation. The effective dose for 50% inhibition (EC₅₀) of mycelial growth was calculated for each isolate by regressing the relative growth against the log₁₀ of fungicide concentration. The *A. alternata* isolates were sensitive or slightly reduced in sensitivity to boscalid. One *A. solani* isolate was highly resistant to boscalid. More than 90% of the *A. alternata* isolates were resistant to some degree to azoxystrobin; both *A. solani* isolates were highly resistant. Overuse of azoxystrobin may have resulted in a shift in *Alternaria* populations to higher resistance to this fungicide. Resistance to boscalid was rare, but fungicide resistance management programs should be followed to reduce the risks of resistance development in *Alternaria* species.

## Fungicide sensitivity of *Pythium* species from the North Central Region of the United States

R. MATTHIESEN (1), A. Robertson (1), M. Chilvers (2)

(1) Iowa State University, U.S.A.; (2) Michigan State University, U.S.A.

Pre- and post-emergence damping-off of soybean in the North Central Region (NCR) can be caused by several *Pythium* spp. Various fungicide seed treatments are used to manage damping-off. Little is known about the effect of temperature on fungicide sensitivity of *Pythium* spp. in the NCR. The goal of this study was to assess fungicide sensitivity of *Pythium* spp. from the NCR to metalaxyl, ethaboxam, and azoxystrobin by assessing mycelial inhibition in vitro at two temperatures. Isolates of four *Pythium* spp. (*P. lutarium*, *P. oopapillum*, *P. sylvaticum*, and *P. torulosum*) from the Midwest, collected through the USDA funded Oomycete Extension Network, were assessed for fungicide sensitivity using five concentrations (0, 0.1, 1.0, 10, and 100 µg a.i./ml) at 13°C and 23°C in an agar medium plate assay. All species from all states, except AR, were more sensitive (>50% mycelia inhibition) to metalaxyl and ethaboxam than to azoxystrobin. At 13°C isolates of *P. oopapillum* from all states, except AR, were less sensitive to all fungicides compared to 23°C. Isolates of *P. sylvaticum* from IA, ND, NE, SD, and WI were less sensitive to all fungicides at 23°C compared to 13°C. At 13°C isolates of *P. torulosum* from IA, IN, ND, NE, and WI were less sensitive to all fungicides tested compared to 23°C. An improved understanding of fungicide sensitivity of *Pythium* spp. could improve our ability to develop and implement effective fungicide seed treatments in the NCR.

## *Pythium* species causing damping-off of alfalfa in Minnesota: Identification, pathogenicity and fungicide sensitivity

D. Samac (1), L. Berg (2), L. Radmer (2)

(1) USDA-ARS, U.S.A.; (2) University of Minnesota, U.S.A.

Damping-off and seed rot is an important disease of alfalfa, severely affecting stand establishment when conditions favor the disease. Globally, 15 *Pythium* species cause damping-off and seed rot of alfalfa, although surveys of species causing disease on alfalfa in Minnesota are lacking. Eight species were isolated by a seedling baiting technique from soil of five alfalfa fields in Minnesota with high levels of damping-off. Three species, *P. sylvaticum*, *P. irregulare*, and *P. ultimum* var. *ultimum*, were pathogenic on germinating alfalfa seedlings at 21 C. Strains of seven species isolated from infected soybean, *P. irregulare*, *P. intermedium*, *P. sylvaticum*, *P. recalcitrans*, *P. conidiophorum*, *P. ultimum* var. *sporangiiferum*, and *P. ultimum* var. *ultimum*, were pathogenic on alfalfa. Assays with Apron XL (mefenoxam) treated seed showed that fungicide sensitivity varied between and with species with approximately 56% of strains insensitive. In Apron XL amended medium, hyphal density was reduced at all concentrations but all strains had a similar growth rate as on non-amended medium. Insensitivity to Stamina seed treatments (pyraclostrobin) occurred in 94% of strains tested. The presence of broad host range species and fungicide resistance suggests that crop rotation and these widely used seed treatments are not effective tools for managing this disease. These results indicate that resistant cultivars are needed for managing damping-off in alfalfa production systems.

## G143A mutation in cytochrome *b* is found in azoxystrobin resistant *Phytophthora cactorum* but not in resistant *Phytophthora nicotianae* from strawberry

T. SEIJO (1), E. Zuchelli (2), N. Peres (1)

(1) University of Florida - GCREC, U.S.A.; (2) Universidade de Passo Fundo, Brazil

*Phytophthora cactorum* and *P. nicotianae* cause leather rot of fruit and Phytophthora crown rot (PhCR) in strawberry. Leather rot occurs sporadically, but can cause up to 50% fruit loss during conducive weather. In Florida's annual strawberry winter production system, PhCR can be severe resulting in stunting, mortality and severe yield loss. Currently, azoxystrobin is labelled for control of leather rot but not for PhCR. *P. cactorum* and *P. nicotianae* isolates from both leather rot and PhCR were collected from multiple strawberry fields in Florida between 1997 and 2016. Isolates were tested for sensitivity to azoxystrobin at 0, 0.01, 0.1, 1.0, 10, and 50 mg/L amended with SHAM (100 mg/L). The isolates were separated into two groups, sensitive isolates, with EC₅₀ values of less than 1.0 mg/L, and resistant isolates having EC₅₀ values greater than 50 mg/L. Resistant *P. cactorum* and *P. nicotianae* isolates were found among both leather rot and PhCR isolates collected after 2010. The first 450 bases of the mitochondrial cytochrome *b* gene were sequenced from a selection of resistant and sensitive isolates of both species. The G143A mutation reported to cause azoxystrobin resistance was found in all three resistant *P. cactorum* isolates but not in resistant *P. nicotianae* isolates or in any sensitive isolate. This is also the first report of resistance to azoxystrobin in *P. nicotianae* from strawberry.

## Prevalence of metalaxyl resistance in *Pythium* populations in dryland agriculture in the US Pacific Northwest

W. CHEN (1), S. Guy (2), R. McGee (3), T. Paulitz (1), L. Porter (4), K. Schroeder (5), G. Vandemark (1)

(1) USDA ARS, Washington State University, Pullman, WA, U.S.A.; (2) Washington State University, Pullman, WA, U.S.A.; (3) USDA ARS, Pullman, WA, U.S.A.; (4) USDA ARS, Washington State University, Prosser, WA, U.S.A.; (5) University of Idaho, Moscow, ID, U.S.A.

Metalaxyl (mefenoxam) has been the primary fungicide used in managing *Pythium* damping off of chickpea and other pulse crops. Metalaxyl resistance among *Pythium* spp. has been reported from the irrigated Columbia basin in the state of Washington where metalaxyl has been applied multiple times annually to manage potato diseases, but not from dryland agriculture where metalaxyl has been used only once per year in seed treatments. However, in the spring of 2014 and 2015, severe seed rot and damping-off of metalaxyl-treated chickpea seeds were found in the dryland agriculture region in southeastern Washington and northern Idaho, and were associated with *Pythium* isolates with high levels of resistance to metalaxyl. High densities of metalaxyl-resistant *Pythium* populations were found in areas of severe chickpea seed rot and damping-off, and were observed in seven commercial chickpea fields in southeastern Washington and northern Idaho. *P. ultimum* var. *ultimum* dominated the populations that were highly resistant to metalaxyl. EC₅₀ values for metalaxyl-resistant isolates were significantly higher than those of metalaxyl-sensitive isolates. Under controlled conditions,
metalaxyl treatments failed to protect chickpea seeds from seed rot and damping-off following inoculation with metalaxyl-resistant *Pythium* isolates. An effective alternative measure is urgently needed to successfully manage metalaxyl-resistant *Pythium* in chickpea production.

## Screening for phenylamide fungicide insensitivity in Wisconsin hop downy mildew (*Pseudoperonospora humuli*) populations

M. MARKS (1), A. Gevens (1)

(1) University of Wisconsin-Madison, U.S.A.

*Pseudoperonospora humuli* is the oomycete pathogen causing hop downy mildew, an important disease concern in WI. The disease is primarily managed by fungicides since varietal resistance is unavailable. Phenylamides have been shown to be systemic and highly effective in control, but due to resistance, particularly in the Pacific Northwestern (PNW) hop producing region, their use has dwindled. To date, the use of phenylamides, namely mefenoxam, has been extremely limited in WI due to the high cost and uncertainty of efficacy. In 2015, nineteen *P. humuli* isolates were collected from a commercial hop yard in Dane County, WI and screened for mefenoxam insensitivity using a leaf disk assay. Fourteen leaf disks were placed onto either 1% water agar (WA) or 1% WA amended with 25 µg/ml of mefenoxam (Ridomil Gold SL, Syngenta). The disks were inoculated at 3 sites with sporangial suspensions from each isolate and the incidence of sporulation at each site was tallied after 5-7 days. The percentage of total sporulating sites was calculated and isolates were considered insensitive if the incidence of sporulation on amended media was at least 50% of that on nonamended media. All isolates showed no sporulation on WA plates amended with mefenoxam, and were sensitive. Mefenoxam may be highly effective in managing hop downy mildew in this location. Additional locations will be tested to better assess the phenylamide sensitivity status and aid in grower fungicide decision-making.

## Production of *Phytophthora nicotianae* isolates resistant to oxathiapiprolin

R. BITTNER (1), A. Mila (1)

(1) North Carolina State University, U.S.A.

Black shank, caused by *Phytophthora nicotianae* (*Pn*), is a devastating disease of tobacco throughout the world. Oxathiapiprolin (OXA), a newly discovered fungicide, has shown efficacy against numerous oomycetes including *Pn*, but the potential for OXA resistance is unknown. The generation of resistance to OXA was attempted using UV light mutagenesis and mycelial adaptation with four *Pn* isolates. UV mutagenesis generated two isolates (EdgeB7-M1 and YadA28-M1) with mycelial resistance to OXA. Sporangia production of both UV mutants also showed increased insensitivity to OXA. Resistance produced by UV mutagenesis was stable after five consecutive transfers on non-amended medium. In greenhouse studies, EdgeB7-M1 remained pathogenic to tobacco, while YadA28-M1 was nonpathogenic. For mycelial adaptation, 48 colonies, twelve replicates from each of the four isolates, were transferred fifteen times on OXA medium. After the 15th transfer, twelve colonies from the initial 48 had a significant increase in mycelial insensitivity to OXA, compared to the wild-type isolates. Increased insensitivity from mycelial adaptation was not stable. Sixty-six percent of the isolates lost their insensitivity after fifteen successive transfers on a non-amended medium. Of the twelve adapted isolates, one remained pathogenic on tobacco. Our results show that the generation of OXA resistant *Pn* isolates is possible; however resistance may be associated with fitness penalties.

## Management of mefenoxam-resistant isolates of *Pythium ultimum* causing pythium leak on potato in the Pacific Northwest

I. CARRILLO (1), P. Wharton (1), K. Fairchild (1), L. Porter (2), K. Frost (3), P. Hamm (3)

(1) University of Idaho, U.S.A.; (2) USDA-ARS, U.S.A.; (3) Oregon State University, U.S.A.

Mefenoxam is a fungicide used to manage oomycete pathogens on potato. Resistance to mefenoxam has developed in *Phytophthora infestans* in all potato growing regions of the U.S. and is becoming more common in *P. erythroseptica*. In a 2006 survey, many fields in the Columbia Basin were found to have mefenoxam-resistant isolates of *P. ultimum*. The percentage of resistant isolates to sensitive isolates for these fields ranged from 0 to 92.5%. Despite the risk of resistance and the cost of mefenoxam, it is still widely used in potato production in the Pacific Northwest (PNW) due to its efficacy in managing pythium leak and the lack of effective alternative compounds. In the PNW, pythium leak is most often seen at harvest or during storage. However in recent years, particularly in fields with high levels of resistance, extensive damage has been found weeks ahead of harvest, suggesting a higher disease risk for resistant isolates than sensitive ones. In this study, a field survey was undertaken to evaluate the extent and persistence of mefenoxam resistance in populations of *Pythium* in the PNW and determine potential alternative fungicides that can be used for the control of mefenoxam resistant *P. ultimum* isolates. Results showed that the levels of mefenoxam resistance in PNW *Pythium* isolates had decreased since 2006. Several new fungicides were also identified which had good efficacy against *P. ultimum*.

## Oxathiapiprolin baseline sensitivity distribution of *Phytophthora capsici* isolates

G. OLAYA (1), R. Linley (2), K. Edlebeck (2), S. Kousik (3), P. Kuhn (4)

(1) Syngenta, U.S.A.; (2) Syngenta, U.S.A.; (3) USDA-ARS, U.S. Vegetable Laboratory, U.S.A.; (4) Syngenta, U.S.A.

Oxathiapiprolin is the only active ingredient in a new class of fungicides (piperidinyl-thiazole-isoxazoline) that is active against downy mildews and *Phytophthora* species, and generally weak against *Pythium* species. It is active against multiple developmental stages of Oomycete fungi including inhibition of mycelial growth, sporangial germination, zoospore release, and zoospore germination. The proposed site of action is the inhibition of an oxysterol binding protein (OSBP). FRAC has assigned oxyathiapiproline to group U15 and is considered to have a medium to high risk for resistance development. The oxathiapiprolin baseline sensitivity of *Phytophthora capsici* has been constructed using 40 isolates never exposed to this fungicide. *P. capsici* isolates were obtained from pepper, zucchini, squash, tomato, watermelon, pumpkin, smooth gourd, bitter gourd and an unknown host. Sensitivity tests were conducted *in vitro* using V8 agar media amended with oxathiapiprolin. Mycelial growth inhibition was quantified using the following oxathiapiprolin concentrations: 0, 0.000001, 0.00001, 0.001, 0.01 and 0.1 mg.ai/L. The baseline sensitivity distribution (ED50 values) of the *P. capsici* isolates ranged from 0.00006090 to 0.0005169 with a geometric mean of 0.00019716 mg oxathiapiprolin/L and a range of 8.5X. Oxathiapiprolin baseline information is critical for the early detection of resistant isolates and the establishment of effective resistance management recommendations.

## Fungicide sensitivity of *Phytophthora infestans* isolates collected in Colombia

M. PARRA-BASTIDAS (1), L. Cabrera-Villamizar (1), J. Chirivi-Salomon (1), C. Ñútez (2), S. Restrepo (1), G. Danies (1)

(1) Universidad de los Andes, Colombia; (2) Universidad Nacional de Colombia, Colombia

*Phytophthora infestans*, is a major constraint to potato production in Colombia. The disease is present in all regions where potatoes are grown and may cause losses of around 265,000 tons of potatoes per year. Control of late blight in Colombia is mainly achieved by the use of fungicides. Due to the specific mode of action of some of these fungicides and the high selection pressure exerted by the continued use of these products it is likely that some *P. infestans* strains in Colombia have already developed resistance against some of these systemic fungicides. Therefore, the aim of this study was to determine the sensitivity of isolates collected in potato-producing regions of Colombia to the systemic fungicides dimetomorph, fluopicolide, and propamocarb. Baseline sensitivity was tested *in vitro* on pea agar media amended with a range of concentrations of each fungicide tested. Dose-response curves and values for the median effective concentration, at which 50% of mycelial growth was inhibited, were determined for each isolate.

## Assessment of acquired resistance of originally sensitive isolates of *Phytophthora infestans* to the systemic fungicides fluopicolide and propamocarb

J. GONZÁLEZ-TOBÓN (1), J. Chirivi-Salomon (1), M. Parra-Bastidas (1), R. Childers (2), S. Restrepo (1), G. Danies (1)

(1) Universidad de los Andes, Colombia; (2) Harvard University, U.S.A.
*Phytophthora infestans*, a plant pathogenic oomycete, is the causal agent of late blight disease of potatoes and tomatoes. This disease causes devastating economical losses each year and control is mainly achieved by the use of fungicides. Previous studies have reported that sensitive isolates to the phenylamide fungicide, mefenoxam, become tolerant after a single passage on mefenoxam-containing medium. The acquired resistance of the sensitive isolates appeared to have a cost on the rate of mycelial growth and declined slightly with subsequent culturing on medium free of mefenoxam. The aim of this study was to investigate whether the same phenomenon is observed using two different systemic fungicides: fluopicolide and propamocarb. Susceptible isolates of *P. infestans* were exposed to three different concentrations of fungicide-amended medium: 0, 5, and 10 µg ml⁻¹ for fluopicolide and 0, 1 and 100 µg ml⁻¹ for propamocarb. To assess if the isolates had acquired resistance to each of the fungicides tested, mycelia from isolates growing at 5 µg ml⁻¹ for fluopicolide and 1 µg ml⁻¹ for propamocarb were transferred once again to media with the same concentrations mentioned above. This study provides further insights into the *in vitro* adaptability of *P. infestans* against antifungal stress.

## Fertigation and soil acidification sustain root density of huanglongbing-infected trees in Florida citrus groves

J. GRAHAM (1), E. Johnson (1), K. Gerberich (2), D. Bright (1)

(1) University of Florida, U.S.A.; (2) University of Florida, Canada

Huanglongbing (HLB) is a devastating citrus disease caused by the phloem-limited bacterium *Candidatus* Liberibacter asiaticus (Las). Early symptoms of HLB include fibrous root loss and leaf blotchy mottle, followed by yield decline, leaf drop, and canopy dieback. As a consequence of initial bacterial infection of fibrous roots, a 30-50% reduction in fibrous root density and elevated soil Phytophthora populations were detected in field surveys. Continued sampling of Hamlin and Valencia orange trees on Swingle citrumelo rootstock in different stages of HLB decline revealed that root loss occurs in two stages. The second phase of root loss (70-80%) begins at the early stage of tree canopy thinning resulting from leaf drop and branch dieback. Managements have been implemented to reduce soil, nutrient and water stress, and Phytophthora root rot. They include frequent irrigation cycles, fertigation and acidification of irrigation water and soil to reduce rhizosphere pH, and fungicides. Root density of trees under these practices fluctuates seasonally and annually but has not declined over the last 3 years. Trees managed with soil acidification and fertigation have steadily recovered in health and yield.

## Status of Huanglongbing (HLB)

### (citrus greening) Multi Agency Coordination (MAC) projects on thermal treatment of citrus trees with citrus greening

D. KOMM (1), D. Komm (2), R. Ehsani (3)

(1) USDA PPQ CPHST, U.S.A.; (2) USDA, U.S.A.; (3) University of Florida, U.S.A.

Citrus trees infected with ‘*Candidatus* Liberibacter asiaticus’, subjected to heat treatments, have shown an improvement in citrus yields and vigor. Thermal treatment equipment for all sizes of citrus trees is being developed and improved for citrus trees infected with ‘*Ca*. Liberibacter asiaticus’. Two MAC projects are contributing to the development and improvement of thermal treatment equipment for all sizes of citrus trees infected with this pathogen. Researchers involved in the first project have developed a mobile physical system using steam to prolong the productive life of citrus trees eight feet tall and smaller. This system uses a tent canopy that goes over the tree followed by steam treatment. Second project has developed a mobile thermotherapy system for citrus trees as tall as thirty feet. The entire system supports a tent canopy that continuously treats as it progresses in the row without stopping the steam treatment. Both projects have successfully heat-treated grove trees infected with HLB. There have been improvements in the thermal equipment to provide a fully functional mobile steaming system for immediate use on all sizes of trees for commercial treatments.

## Canopy and root response of HLB-affected citrus trees to steam-generated thermotherapy

N. Thapa (1), S. Commerford (1), R. Ehsani (1), E. Johnson (1), M. Dewdney (1)

(1) CREC, University of Florida, U.S.A.

Thermotherapy is an old technique used to control plant pathogens. Citrus huanglongbing (HLB) threatens Florida’s multibillion dollar citrus industry and has no effective control measures. The phloem-limited bacterium, *Candidatus* Liberibacter asiaticus (*Las*), is the causal agent of HLB and infects both canopy and root system. Thermotherapy can reduce *Las* titer in infected trees and this study explores steam as a heat source for rapid treatment to extend the productivity of HLB-affected citrus trees. Optimum temperature-time duration is important to reduce *Las* titer without affecting tree productivity. A randomized split-plot design field trial was laid out with the main plots at 3-month intervals to determine optimum time-of-year to reduce *Las* titer and the subplots at 6 temperature-time combinations (58°C/15s; 58°C/60s; 58°C/90s; 58°C/120s; 60°C/30s and untreated control, UTC) with 3 replications per treatment. Four leaves/tree from 10 trees/rep were collected quarterly and tested for *Las* titer via standard qPCR. Roots were assessed for *Las* titer as roots are an untreated reservoir. Preliminary data show an overall reduction in titer a month after the July treatment. *Las* titer increased in all leaves, but more in the UTC than the treatments, whereas root *Las* titer was reduced more in treatments than the UTC. This may be due to change of phloem flow from root to the treated canopy along with *Las*. Seasonality may be critical for successful suppression of *Las*.

## Inoculum sources of Xanthomonas fragariae in strawberry nursery packing houses: Presence, viability and transmission

H. WANG (1), C. Gigot (2), N. McRoberts (2), W. Turechek (3)

(1) EDISTO Research and Education Center, Clemson University, U.S.A.; (2) University of California, U.S.A.; (3) U.S. Horticultural Research Laboratory, USDA, U.S.A.

*Xanthomonas fragariae* causes strawberry angular leaf spot, an important disease in strawberry nursery production. When plants are dug and harvested in the field, they are transported to a "trim shed" where the leaves are removed and the roots are trimmed with table-mounted steel blades. Maintaining constant and stringent phytosanitary conditions is nearly impossible because of the sheer numbers of plants handled each day as part of this sorting and packaging process. To determine the likelihood of *X. fragariae* transmission during this process, PBS-soaked cotton balls were used to swab the blades and tables at nursery trim shed stations after processing strawberry plants harvested from infected fields. *X. fragariae* presence and percent viability were determined by squeezing PBS from the cotton balls and testing it by PMA-qPCR. The likelihood of disease transmission was estimated by rub-inoculating strawberry plants with the cotton balls. *X. fragariae* was detected in ~70% of the samples and ~30% of these contained living bacteria. Disease symptoms were observed in 9 of 62 strawberry plants inoculated. Results indicate that contamination of healthy plants is likely to occur during the post-harvest trimming process in nurseries and identified points where sanitation can be improved for better disease control.

## Assessment of in-vitro efficacy of different disinfectants against common bacterial diseases on woody ornamentals

P. LIYANAPATHIRANAGE (1), S. Dawadi (1), M. Kabir (2), T. Simmons (1), F. Baysal-Gurel (1)

(1) Tennessee State University, U.S.A.; (2) Tennessee State University, U.S.A.

Plants are continuously exposed to disease causing agents, which enter to the nurseries in many ways. Cutting tools and other equipment that are used to work with infected plants is considered as a major source for bacterial disease spread. Various products are available in the market to use as disinfectants to clean cutting tools. This study was conducted to evaluate the effect of commercial disinfectants on *Xanthomonas axonopodis*, *X. campestris* and *Pseudomonas syringae* pv. *syringae*. We determined the contact time required to kill those bacterial pathogens by disinfectants in different chemical classes. The relative shelf-life stability of disinfectants was also determined. Replicated *in-vitro* experiments were conducted in which pure cultures of bacterial pathogens were exposed to disinfectants for 1, 30 and 60 sec, then 100 µL aliquots were withdrawn, plated on NBYA medium, and incubated at 25°C for 3 days. Clorox regular bleach (10%), Virkon-S (0.5, 1 and 2%), Last rinse sanitizer-beer clean, Seventh generation multi surface, Seventh
generation daily shower, Seventh generation all purpose, alcohol (70%), vinegar (100%), Lysol, ZeroTol 2.0 and Green-Shield killed all three pathogens at all exposure times during 3 weeks shelf-life of products. Further studies will be done to determine efficacy of these disinfectants in preventing plant-to-plant spread of bacterial diseases through cutting tools.

## Effective treatments for eradication of *Xanthomonas cucurbitae* in pumpkin seed

X. ZHANG (1), M. Babadoost (2)

(1) Department of Crop Sciences, University of Illinois, U.S.A.; (2) Department of Crop Sciences, University of Illinois, U.S.A.

Bacterial spot, caused by *Xanthomonas cucurbitae*, has become a serious disease of pumpkin in Illinois and other states in the Midwest. *X. cucurbitae* infects leaves and fruit. Fruit infection often results in fruit rot. More than 90% of fruit infection with *X. cucurbitae* in pumpkin fields has been reported. *X. cucurbitae* is considered as a seed-borne pathogen, hence developing an effective seed treatment to eradicate the pathogen in seed is essential. This study was conducted to develop reliable treatments for eradication of *X. cucurbitae* in seed. Hot-water, HCl, and NaClO treatments for eradication of *X. cucurbitae* in 'Howden' and 'Dickinson' pumpkins were assessed. Seed were treated in water with 49, 50, 51, 52, 53, 54, 55, and 56°C, for 5, 10, 15, 20, 25, and 30 min at each of the temperatures. Treatment at 55°C for 10 min eradicated *X. cucurbitae* in both naturally-infected and artificially-inoculated seed without significant adverse effects on seed germination or seedling vigor. HCl treatments included 0.5, 1.0, and 2.0% HCl solutions for 10, 20, 30, 40, 50 and 60 min. The most effected HCl-treatment was 0.5% concentration for 40 min, which eradicated *X. cucurbitae* in the seed without any negative effect on seed germination or seedling vigor. For NaClO treatment, pumpkin seeds were soaked in NaClO solution with concentration of 0.5, 1, 1.5 and 2% for 1, 2, 3 and 4 min. None of the NaClO treatments completely eradicated *X. cucurbitae* in pumpkin seed.

## Silicon Augments *Xanthomonas gardneri* symptoms in Tomato cv. Early Girl

W. ZELLNER (1), C. Krause (2), J. Locke (1), J. Boldt (1), S. Leisner (3)

(1) USDA-ARS, U.S.A.; (2) USDA-ARS, U.S.A.; (3) The University of Toledo, U.S.A.

Silicon (Si) is a plant nutrient that enhances stress tolerance. Tomato accumulates a moderate amount of Si in foliar tissue, but its effect on *Xanthomonas gardneri* infection has not yet been determined. To test this, 'Early Girl' tomatoes were grown hydroponically in nutrient solution with (Si+) or without (Control) 2 mM potassium silicate. Plants were dip-inoculated with *X. gardneri*. Symptoms developed within 24 hrs post-inoculation. Total symptomatic leaf area was quantified 10 dpi, using the Assess 2.0 Program. Si-treated plants had significantly more lesions and higher symptomatic leaf area (11.7%) compared to controls (6.5%). Symptoms present on Si+ plants were spread throughout the leaf, while lesions on control plants were concentrated around the leaf margins. This suggested entry by *X. gardneri* in Si+ plants was through both stomates and hydathodes, while control plants were infiltrated mainly via hydathodes. To determine if differences in symptom formation between Si+ and control plants was simply due to changes in stomatal opening, stomatal conductance was directly measured between Si+ and control plants that were removed from hydroponics solution for 2 min. At 570s, stomatal conductance in Si+ plants compared to control was slightly, but significantly lower. This suggests that differences in *X. gardneri* symptoms in 'Early Girl' are not due to changes in the stomatal conductance influenced by the dip-inoculation method.

## Study of the influence of winter rye on soybean seedling and root rot diseases

G. ARALDI-DA-SILVA (1), T. Kaspar (2), M. Helmers (1), D. Mueller (1), L. Leandro (1)

(1) Iowa State University, U.S.A.; (2) USDA, U.S.A.

Cover crops can enhance or suppress plant diseases, but little is known about the effect of cover crops on soybean diseases. In 2015, the effect of winter rye (*Secale cereale* L.) cover crop on soybean seedling and root rot was studied at two experimental sites, Boyd and ISUAG-USB, in Ames, IA. Both sites were under a no-till corn-soybean rotation. Cover crop treatments were rye and no rye, at Boyd; and early termination rye, late termination rye, and no rye, at ISUAG-USB. Variables assessed were soybean stand, root rot severity, dry plant weight, healthy green leaf area (HGLA), and yield. Root rot severity was visually assessed on a percent scale, and HGLA was measured as canopy percent reflectance using a hand-held radiometer. At Boyd, soybean stand (*P*=0.68) and yield (*P*=0.61) were not affected by treatment, but healthy green leaf area (HGLA) was higher in rye plots (*P*<0.001) compared to plots with no rye. At ISUAG-USB, no rye plots had more soybean stand (*P*=0.08), greater yield (3105 ± 304 kg/ha) (*P*=0.07), and higher HGLA (*P*<0.001) compared to plots with late termination rye. Early termination rye did not differ from late termination and no rye treatments for any variables. No difference in dry plant weight and root rot severity were observed between treatments at both sites (*P*>0.2). Site variables, as well as cover crop treatments, can influence soybean crop response. This study is going to be repeated in 2016, and additional research is underway to quantify *Fusarium* and *Pythium* populations at both sites.

## Reduced *Macrophomina phaseolina* colonization of soybean by supplementing with the secondary nutrients calcium and magnesium

T. WILKERSON (1), M. Tomaso-Peterson (2), B. Golden (3), A. Brown (4), T. Allen (3)

(1) Mississippi State University, Stoneville, MS, U.S.A.; (2) Mississippi State University, Starkville, MS, U.S.A.; (3) Mississippi State University Delta Research and Extension Center, Stoneville, MS, U.S.A.; (4) Mississippi State Cemical Lab/ Mississippi State University, Starkville, MS, U.S.A.

*Macrophomina phaseolina* (Tassi) Goid (*Mp*) is a ubiquitous soilborne fungal pathogen that causes charcoal rot (CR) of soybean. Charcoal rot annually limits profitability, especially in years when hot and dry environmental conditions persist. Throughout the southern United States, CR consistently remains one of the top five soybean diseases and has resulted in an estimated 11 million metric ton reduction in yield over the past 20 years. The objective of this research was to reduce *Mp* colonization of field grown soybean by supplementing secondary nutrients, specifically calcium (Ca) and magnesium (Mg). In 2014 and 2015 non-irrigated, *Mp*-inoculated field trials were conducted with a CR-susceptible and a CR-moderately-resistant cultivar. Treatment applications consisted of 1,120 kg/ha rate of Ca and Mg alone and in combination at each of three timings: pre-plant, at plant, and pre-plant fb at plant. Control plots consisted of a completely non-treated non-inoculated in addition to an *Mp*-inoculated non-treated. Root samples were taken at the R3, R5, R7, and R8 growth stages. Colony forming units (cfu) were enumerated from ground, disinfested root tissue using a semi-selective medium. Reductions in cfus were observed to be approximately 35% in the moderately-resistant cultivar and a 45 to 50% reduction in the susceptible cultivar, compared to the non-treated. Reduction in colonization was observed in some cases although not across all treatments.

## Evaluating biodegradation treatments for the reduction of inoculum and incidence of citrus black spot *Phyllosticta citricarpa* in Florida citrus groves

K. RODRIGUES (1), T. Hobbs (1), M. Dewdney (1)

(1) CREC, University of Florida, U.S.A.

The fungal disease citrus black spot (CBS) has become an increasing problem in Florida citrus groves. We conducted a 3-year study to evaluate the effect of 5% urea (9.4 kg/190 l water/0.4 ha), Soil-Set (142 ml/190 l water/0.4 ha), and a water control on the level of the *P. citricarpa* or the morphologically similar citrus endophyte *P. capitalensis* in the leaf litter and subsequent CBS incidence on fruit in a Valencia grove in Immokalee, FL. The experiment was a randomized complete block design with three rows per treatment (trt) and four blocks. Leaf litter samples were collected from arbitrarily selected trees at time of treatment and then three times biweekly post-treatment application. For every collection, 100 leaves/trt/block were inspected for fungal sporocarp presence and each fungus was quantified via qPCR. The disease incidence on fruit was collected for 50 fruit/tree on 125 trees/trt/block. Fungal DNA levels were analyzed with a 3-way ANOVA and fruit disease incidence by contingency table analysis and chi-square tests. Leaf litter DNA had a significant interaction (*P* < 0.0001) between block x trt x collection date. For the incidence of disease on fruit, significant
treatment effects were seen in 2015 (P = 0.002) and 2016 (P < 0.0001) with Soil-Set reducing disease incidence by 29.6% and 26.5%, respectively but not with urea. Soil-Set may enhance the efficacy of fungicide applications for the management of CBS.

## Utility of compost tea for disease management in grapes

G. KOTAMRAJU (1), R. Sysak (1), J. Gillett (1), A. Schilder (1)

(1) Michigan State University, U.S.A.

Compost tea is a fermented watery extract of compost used for disease suppression and plant nutrition. A replicated trial was conducted in a 'Niagara' vineyard in Clarksville, Michigan to evaluate the efficacy of compost tea for disease control. Non-aerated manure-based compost tea was prepared over a 2-wk period, and foliar applications were made with a handheld sprayer every 7 or 14 days from 10 June through 27 Aug, 2016. Undiluted compost tea with NuFilm-P adjuvant applied every 7 days provided good control of black rot, powdery mildew, downy mildew, and Phomopsis cane and leaf spot. In general, less frequent and more dilute applications were less effective. 'Niagara' leaves were sampled daily for 7 days after application of aerated manure-based compost tea. Bacterial populations were higher on leaves sprayed with compost tea compared to non-treated leaves but declined to almost background levels after a week. Both yeast and fungal populations were reduced immediately after the application, but later increased over the control. Bacteria from various manure- and plant-based compost teas were isolated and evaluated for antagonism against *Botrytis cinerea* by dual plating. The majority of isolated bacteria belonged to the Bacillaceae family as determined by 16s rRNA sequencing. This study shows that compost tea may be used for disease suppression in grapes but further evaluation is warranted.

## Evaluation of disinfectant treatments to reduce transmission of *Didymella bryoniae* during watermelon grafting

A. KEINATH (1), V. DuBose (2), C. Conrad (2)

(1) Clemson University, U.S.A.; (2) Clemson University, U.S.A.

Gummy stem blight can develop in greenhouses on watermelon seedlings grown as transplants or as scions and on cucurbit rootstock seedlings used in grafting. When diseased seedlings are cut during grafting, the pathogen, *Didymella bryoniae* (*Stagonosporopsis* sp.), may be transferred to healthy seedlings. The objective of this study was to evaluate disinfectant treatments to prevent or reduce transmission. In a first experiment, Kleengro (didecyl dimethyl ammonium chloride), Physan 20 (benzyl ammonium chloride) and Virkon S (potassium peroxymonosulfate) were ineffective when sprayed onto entire watermelon seedlings before or after cutting hypocotyls with a blade contaminated with mycelium or conidia. In a second experiment, applying 0.6% sodium hypochlorite, 0.4% Physan 20 or 70% and 95% ethanol to a contaminated blade or heating it before cutting watermelon hypocotyls reduced incidence of gummy stem blight (P=0.01); water, 3% hydrogen peroxide, 1% Virkon S and 7.5% Kleengro were ineffective. In a third experiment, both the watermelon scion and the interspecific hybrid squash rootstock were cut with contaminated and treated blades before grafting. After 7 days in a humid healing chamber, heating the contaminated blade reduced incidence (11%) more than ethanol (45%), and both treatments reduced incidence compared to Physan 20 (100%) and water (100%) (P=0.01). Using heat or ethanol to disinfest cutting tools may reduce transmission of *D. bryoniae* during grafting.

## Scab severity in relation to hedge pruning pecan trees in the Southeastern USA

C. BOCK (1), M. Hotchkiss (1), T. Brenneman (2), K. Stevenson (2), B. Goff (3), M. Smith (4), L. Wells (5), B. Wood (1)

(1) USDA-ARS SE Fruit & Tree Nut Research Laboratory, Byron, GA, U.S.A.; (2) Department of Plant Pathology, University of Georgia, Tifton, GA, U.S.A.; (3) Department of Horticulture, Auburn University, Auburn, AL, U.S.A.; (4) Department of Horticulture, Oklahoma State University, Stillwater, OK, U.S.A.; (5) Department of Horticulture, University of Georgia, Tifton, GA, U.S.A.

Scab is the most damaging disease of pecan in the Southeastern USA. Pecan trees are tall (up to 30+ m), and managing disease in the upper canopy is problematic. Hedge pruning trees to ~12 m is being explored to facilitate efficacy of ground-based fungicide sprays, but resulting vigorous shoot growth could result in more severe scab. Nine experiment compared scab severity on trees receiving hedge pruning regimes vs no hedging. All trees received fungicide treatments (ground-based air-blast sprays and up to 3 aerial applications). Results showed that different hedging methods either had no effect, increased or decreased scab severity on immature fruit in one experiment (P<0.0001, assessed before mid-August [immature fruit were assesses in 8 of the 9 experiments]), and on mature fruit in 6 of 9 experiments (P<0.0001, assessed after mid-August), but in all cases differences were small. However, height in the canopy invariably had a significant effect on scab severity, with up to 18-fold more severe scab on fruit at ≥12 m compared to 5 m. Immature fruit high in the canopy had more severe scab in all experiments (P<0.0001-0.03), and mature fruit had more severe scab in 6 of 9 experiments (P<0.0001). In trees that received the same fungicide program the effect of hedging in increasing scab severity was negligible and not consistent, but scab was consistently more severe at greater heights in the canopy of both hedge pruned and non-hedged trees.

## Potential of heat treatment for management of *Botrytis cinerea* resistance on strawberry

A. ZUNIGA (1), N. Peres (1)

(1) University of Florida, U.S.A.

*Botrytis cinerea* is the causal agent of gray mold, an important disease of strawberry in Florida. The use of fungicides to control gray mold has been exploited over the past years and as a result, resistance to many fungicide groups has been reported. In this study, we are aiming to determine the effect of a heat treatment that has proven to reduce systemic infection with *Xanthomonas fragarie* in strawberry propagating material on the survival of fungicide-resistant *B. cinerea* isolates. For this, four different *B. cinerea* isolates were used to evaluate survival at four temperatures (44, 48, 52 and 56°C) and seven different duration times (1, 5, 10, 30, 60, 120 and 240 min). After treatment, the spores were transferred to a growth medium to determine the percentage of spore survival. Although, *B. cinerea* spores survived for 1 min for all temperatures, germination was reduced by 28 to 50% at the highest temperature. Spores of all isolates exposed to 44°C for 30 min did not survive and spore germination was reduced by 7 to 16% between 48 and 52°C for 1 min, with no spores survived for all other duration times. Previous experiments have determined that most strawberry cultivars can tolerate 44°C for up to 4 h. Thus, heat treatment of strawberry plants is a feasible strategy to produce disease-free strawberry plants and to reduce fungicide-resistant inoculum.

## Eradication of the boxwood blight pathogen, *Calonectria pseudonaviculata*, in compost

R. HARVEY (1), D. Davis (1), J. Pecchia (1)

(1) Penn State University, U.S.A.

*Calonectria pseudonaviculata*, the causal agent of boxwood blight, is a serious threat to boxwood growers across the country. Boxwood blight is currently present in at least 15 US states, however this number is likely to increase as more infections are found. Due to the production of microsclerotia by the pathogen, there is concern that if infected plants are composted, the pathogen could survive the composting process and possibly cause further infections if the compost is used around healthy boxwoods, or other susceptible plants. To determine survivability during the composting process, discreet microsclerotia were inoculated into compost within a bioreactor system and subsequently sampled at set time/temperature intervals. Survival was rated as a plus/minus system based on the presence of microsclerotia germination. The data presented here are a refinement of data previously presented previously, and suggest that *C. pseudonaviculata* microsclerotia will not survive composting at greater than 50°C for over 24 hours. Further work needs to be done to define the minimum time/temperature that is sufficient to ensure complete eradication. In addition, similar research is needed to assess pathogen survival within infected plant material and to confirm the time/temperature combinations *in vivo*.
**Thermal inactivation of *Calonectria pseudonaviculata*, the causal agent of boxwood blight**

M. MILLER (1), M. Cubeta (2)

(1) North Carolina State University, Department of Plant Pathology, U.S.A.; (2) North Carolina State University, U.S.A.

Thermotherapy is an effective method for eliminating viruses and microorganisms from seeds and bulbs, but little information is available on the use of this approach to inactivate pathogenic fungi in leaves of woody plants. In this study, we investigated the utility of hot water treatments for killing *Calonectria pseudonaviculata* (Cps), a fungal, foliar pathogen of boxwood (Buxus) during propagation. Experiments predicting the thermal death kinetics of Cps conidia, and experiments determining the ability of commonly grown, commercial boxwood cultivars to survive exposure to hot water were performed. Conidia of three field isolates of Cps were suspended in water and exposed to temperatures ranging from 45°C to 55°C for 0 to 20 min. Suspensions were then spread on potato dextrose agar, and percent spore germination was assessed after 24 h of incubation. Approximately 16 min were required to kill 90% of conidia exposed to 45°C, but only 4.5 min were required to kill 90% of spores when heated to 55°C. Cuttings from six partially resistant boxwood cultivars were submerged in water heated to 45°C, 50°C, and 55°C for 0 to 60 min. Treated cuttings were placed under mist and their rooting responses were rated 86 to 91 d later. The ability of boxwood cuttings to produce roots after hot water treatment at 45°C and 50°C varied by cultivar, whereas no cultivars survived treatment at 55°C. Further experiments will determine whether hot water can eliminate the pathogen *in planta*.

**Using soil amendments to reduce survival of *Botrytis cinerea* sclerotia in Mid-Atlantic diversified farming systems**

E. KOIVUNEN (1), E. Koivunen (1), C. Swett (1)

(1) University of Maryland, U.S.A.

*Botrytis cinerea* causes major pre-harvest losses in a wide range of crops in the Mid-Atlantic, including strawberries, blueberries, raspberries, and grapes. Management challenges have escalated with the loss of soil fumigants as well as increased land-use intensity associated with crop diversification. In a summer 2015 field survey, Maryland fields had up to 350 *B. cinerea* colony forming units per gram of soil and 20% yield losses from gray mold. The objective of this project was to evaluate the efficacy of soil amendments in degrading *B. cinerea* sclerotia – the most resilient source of inoculum. In potted studies, sclerotia viability decreased in crab meal and leaf compost treatments by 91.7% (± 5%) and 96.7% respectively, compared to 71.7% (± 5%) in untreated soil sixty days after burial. Percent of sclerotia colonized by hyphal fungi was highest in the crab meal (86.7% ± 3.3%) and leaf compost treatments (93.8% ± 6.3%). Similar trends between sclerotia viability and colonization were seen in Maryland field trials: sixty days after burial, the leaf compost + crab meal treatment had the lowest sclerotia viability and the highest colonization by bacteria or yeasts. These studies suggest that soil amendments may hold promise in reducing *B. cinerea* survival and dispersal in diversified farming systems in the Mid-Atlantic, in part by promoting microbial colonization of sclerotia.

**Nutrients and in vitro growth of *Phaeocryptopus gaeumannii*, a fungus causing the Swiss needle cast disease on *Pseudotsuga menziesii***

M. Yañez-Morales (1), O. Salgado-Feregrino (1), J. Velázquez-Mendoza (1), M. Jiménez-Casas (1)

(1) Colegio de Postgraduados, Mexico

The hyphae intercellular development of *Phaeocryptopus gaeumannii*, a foliage pathogen in Douglas-fir trees, is affected by the nutritional composition of the needles. The objective was to examine the effect of different nutrients and silicon, Si, on the *in vitro* growth of the pathogen. Two experiments were established in a completely random design with a fungus isolate (JN204508). The first experiment was by plating the isolate aliquot (1 mm, 0.002 g) in 2% MEA amended with different nutrients, 10 treatments, and incubated at 21°C in darkness for 42 days; diameter colony growth was measured and instantaneous relative rate growth was estimated. The second experiment was in liquid medium, potato extract-dextrose, and amended with nutrients, 21 treatments; incubation was under shaking conditions at 250 rpm at 23°C for 35 days. Mycelium growth was measured by their weight in wet and dry stage. The macronutrients (N P K Ca Mg S) favored the growth of *P. gaeumannii*; in contrast the micronutrients (Fe Cu Zn Mn Mo B) severely inhibited the development of the fungus. The complete nutrient solution plus Si at 1000 ppm reduced colony growth in the agar medium, and at 250 ppm in the liquid medium. Thus, the addition of Si inhibited growth of the fungus and this was attributed to a possible toxic effect of the element. The results provide a basis for designing a fertilization formula for Douglas-fir and allowing to partially inhibit intercellular fungal growth in needles.

**Adapting to water insecurity: Co-managing oomycete pathogens and water use using deficit irrigation sensor networks**

J. DEL CASTILLO MÚNERA (1), C. Swett (1), B. Belayneh (1), A. Ristvey (2), J. Lea-Cox (1)

(1) University of Maryland, U.S.A.; (2) University of Maryland, U.S.A.

Circumglobally, the agricultural sector consumes the highest percentage of available fresh water, and with increases in water insecurity, agricultural demands are becoming unsustainable. Sensor-driven deficit irrigation networks use less water by allowing a mild stress of the crop. The objectives of this study were to evaluate (i) deficit irrigation (DI) as means to reduce root disease in annual and perennial nursery/greenhouse crops; and (ii) effects of DI under various infection scenarios using tomato and *P. capsici* as pathosystem model, to address grower risks hindering technology adoption. In a two year study, Rhododendron were inoculated with *P. cinnamomi* and placed under four different irrigation treatments. Mortality incidence after 18 months of inoculation was significant (P = 0.0084), reflecting significantly lower mortality (21%) at 33.5% VWC compared to the 43, and 28% VWC treatments (29, 51% mortality). Preliminary studies on Poinsettia indicate a higher root rot incidence in the 48% VWC deficit irrigation treatment compared to saturated conditions (60% VWC), but differences were not significant. Studies underway in the model pathosystem are evaluating whether DI strategies can predispose plants to pathogen infection, and use of DI as a tool to prevent latent infection and disease. These studies will be used to develop recommendations for using deficient irrigation to co-manage water use, and disease losses in nursery and greenhouse production.

**Addition of supplemental spent lime to previously limed soils for control of Aphanomyces root rot on sugar beet**

J. BRANTNER (1) A. Chanda (1)

(1) University of Minnesota, U.S.A.

Application of sugar beet factory spent lime to soil in fields infested with the soilborne oomycete pathogen *Aphanomyces cochlioides* has been shown to decrease disease and increase sugar beet yield in Minnesota and North Dakota. Sugar beet growers are inquiring about the need to reapply lime in fields where lime has previously been applied. In a field trial where lime had originally been applied in April 2004 at 0, 6, 12, 18 and 24 Mg dry wt ha⁻¹, plots were split and an additional 7.8 Mg dry wt lime ha⁻¹ was added to half of each plot on October 31, 2014. The following spring, sugar beet was sown and data was collected on early season stand, Aphanomyces root rot and sugar beet yield. There was significant interaction between supplemental and original rates of lime for early season stand, root rot ratings, and yield (P ≤ 0.05). At 4 and 7 weeks after planting, sugar beet stands were higher in plots that received supplemental lime where original lime rates were 0 and 6 Mg ha⁻¹ (P ≤ 0.05), but not where original lime rates were 12 Mg ha⁻¹ or higher. Supplemental lime reduced Aphanomyces root rot and increased sugar beet yield only in plots where lime had not been previously applied (P ≤ 0.05). Results demonstrate a benefit from adding supplemental lime to fields previously limed at low rates, but do not support a need to add supplemental lime to fields previously limed with at least 12 Mg dry wt ha⁻¹ within the past ten years.

**Assessment of genetic diversity and virulence spectrum of *Magnaporte oryzae* population from sub-Saharan Africa**

DEVI GANESHAN (1), S. Mutiga (2), F. Rotich (3), D. Mwongera (4), J. Harvey (5), B. Zhou (6), L. Wasilwa (7), G. Wang (1), D. Silue (8), O. Ibrahima (9), J. Correll (3), N. Talbot (10), T. Mitchell (1)

(1) The Ohio State University, Department of Plant, (2) Biosciences eastern and central Africa (BecA) Hub, Nairobi, Kenya; (3) University of Arkansas, Department of Plant Pathology, AR, U.S.A.; (4) University of Exeter, United Kingdom; (5) Biosciences eastern and central Africa (BecA) Hub, Kenya;
(6) International Rice Research Institute, Philippines; (7) Kenya Agricultural and Livestock Research Organization, Kenya; (8) AfricaRice Center, Ivory Coast; (9) INERA, Burkina Faso; (10) University of Exeter, Department of Biosciences, United Kingdom

Rice Blast Disease (RBD), caused by the fungus *Magnaporthe oryzae* is a devastating disease found throughout the world and is particularly damaging to rice production in sub-Saharan Africa (SSA). Yield losses of 50-80% are common not only leading to net imports from abroad but also invasion of new pathotypes. An effective strategy to control RBD is to utilize resistant cultivars, but *M. oryzae* evolves rapidly due to genome instability, frequent loss/break of resistance genes occurs, overcoming resistance within few years of deployment. Breeding for durable resistance requires knowledge of pathogen's genetic diversity and virulence spectrum. In our study, genetic diversity of *M. oryzae* field isolates from 9 SSA countries (Benin, Burkina Faso, Ghana, Kenya, Nigeria, Tanzania, Mali, Togo, and Uganda) were assessed by SNPs using GBS technology. Phylogenetic analysis of SNPs from 78 isolates, revealed 7 distinct clades differing in virulence. PCA showed that isolates from East were genetically distinct from West Africa. Further, standard least square regression analysis of virulence spectrum in the 7 clades revealed 4 major groups which differed in disease scores highlighting the association between genetic diversity and virulence. GWAS will be performed to further strengthen the association between genetic diversity and virulence and map the putative virulence (AVR) genes for functional characterization facilitating an informed deployment of RBD resistance programs.

## Quantitative resistance loci identified for the bacterial blight causative agent, Xanthomonas oryzae pv. oryzae

A. HUERTA (1), E. Delorean (2), A. Bossa-Castro (2), C. Raghavan (3), R. Corral (2), V. Verdier3 (4), H. Leung (5), J. Leach (2)

(1) Colorado State University, Fort Collins, CO, U.S.A.; (2) Department of Bioagricultural Sciences and Pest Management, Colorado State University, Fort Collins, CO, U.S.A.; (3) International Rice Research Institute (IRRI), Los Baños, Philippines; (4) Institut de Recherche pour le Développement IRD-CIRAD-UM2, Montpellier, France; (5) 2 International Rice Research Institute (IRRI), Los Baños, Philippines

Any factor that limits or reduces attainable agricultural yield, including plant disease, will impact global food security. *Xanthomonas oryzae* pv. *oryzae* (*Xoo*) causes the most important bacterial disease of rice, bacterial blight (BB). Deployment of resistance varieties is the gold standard for managing plant diseases, having the strongest impact with minimal environmental effects and cost. However, resistance breeding depends on harnessing genetic diversity in available germplasm for identification of effective and durable disease resistance. To identify durable resistance, we screened 330 advanced inbred lines (AILs) from the *indica* Multi-Parent Advanced Generation Inter-Cross (MAGIC) rice population for resistance to Xoo strains, PXO99A and PXO99A harboring TAL-effector TAL7b (+TAL7b). TAL7b, a virulence factor for *Xoo*, allowed us to test the hypothesis that resistance targeted to important virulence factors will be more durable when deployed. Transgressive segregation for resistance was observed to both PXO99A and PXO99A +TAL7b, suggesting the presence of resistance QTL for BB. Genome-wide association (GWA) and interval mapping analysis, revealed 13 disease resistance QTL, six specific to PXO99 +TAL7B and seven to PXO99A. Future exploitation of the MAGIC AILs and screening a new subset of lines selected based on the knowledge acquired from this study will refine the resistance loci, and help elucidate the molecular basis for the resistance phenotypes identified.

## Accelerating the pace of developing FHB resistant two-row malting barley: Genomic selection approach

D. FERNANDO (1), J. Tucker (2), A. Badea (2), W. Legge (2)

(1) University of Manitoba, Canada; (2) Brandon Research and Development Centre, Agriculture and Agri-Food Canada, Canada

Fusarium head blight (FHB) incited by *Fusarium graminearum* is a major disease of barley (*Hordeum vulgare*) in Canada, which significantly impacts seed quality through production of mycotoxins in the grain. While breeding efforts have been ongoing at the Agriculture and Agri-Food, Brandon Research and Development Centre for a couple decades, progress has been limited by requirements for labour-intensive screening nurseries and quantitative based resistance. With advent of affordable genomic tools, breeding for crop characters that are difficult to measure and/or display low heritability are suitable for development through application of genomic selection techniques. A large population of 400 two-row barley lines has been assembled based on diverse reaction to FHB. This collection has been phenotyped in 2014 and 2015 at three locations in Manitoba (Brandon, Carberry, and Carman), and deoxynivalenol content in mature grains was measured by enzyme linked immunosorbent assay (ELISA). This data will be used in combination with genomic information to make predictions of genomic estimated breeding values. Genomic markers will be validated in breeding populations segregating for FHB resistance.

## Analysis of Resistance to Blast Isolates and Mapping of Rice Blast Resistance Gene in T2

Q. YANG (1), D. Sun (2), G. Chen (2), J. Zhang (2), D. Zhou (3), X. Zhu (2), H. Wang (3), Z. Chen (3)

(1) Institute of Plant Protection, Guangdong Academy of Agricultural Sciences, Guangzhou, China; (2) Institute of Plant Protection, Guangdong Academy of Agricultural Sciences, China; (3) South China Agricultural University, China

To understand the genetic mechanisms of space-induced rice mutants' resistance to blast, T2, an *indica* rice mutant from TaiHang68, was subjected to resistance spectrum using 39 blast isolates collected from Guangdong Province, China along with main cultivars from Guangdong province. The results showed that T2 conferred broader resistance spectrum than most of main cultivars from Guangdong province. Genetic analysis and gene mapping were also applied in this study. Isolate GD0193 was used for inoculating a population of $F_2$ derived from the cross between T2 and highly susceptible variety LTH. The results showed that the segregation of resistant (R) and susceptible (S) progenies matched a 3:1 ratio in the $F_2$ population (1585R:537S, $\chi^2$=0.77, P>0.75), indicating that this R gene was controlled by a single dominant gene, temporarily designed as *PiTH* gene. The R gene was mapped between T5 (1.4 cM) and K10 (0.29 cM) at chromosome 11 using SSR and InDel markers, and it was co-segregating with marker K7. *PiTH* may be a novel resistance gene in the *Pik* cluster, which contributes greatly to the blast resistance of T2. Thus, T2 could be used as a new germplasm to facilitate rice blast resistance breeding.

## Exploring gene loci controlling bakanae disease resistance by genome-wide association mapping

S. CHEN (1), K. Huang (2), Y. Kuo (2), M. Lai (3), D. Wu (3), C. Chung (1)

(1) Department of Plant Pathology and Microbiology, National Taiwan University, Taipei, Taiwan; (2) Department of Bio-Industrial Mechatronics Engineering, National Taiwan University, Taipei, Taiwan; (3) Crop Science Division, Taiwan Agricultural Research Institute, Taichung, Taiwan

Bakanae disease, caused by *Fusarium fujikuroi*, is a rice seed-borne disease and widely distributed in rice growing areas. Infected rice seedlings may show complex symptoms including abnormal elongation of stem or internodes, wider leaf angle, slenderness, and even die, causing considerable yield and quality loss. In recent years, studies have reported the rising incidence and severity of the disease in Asia. Even though the disease has been known over a century, little is known about the resistance genetics and resources in rice. In this study, we used genome-wide association mapping approach to identify gene loci controlling bakanae disease resistance. A set of 413 diverse rice varieties from 82 countries (rice diversity panel; containing 44,100 SNPs available from open-source) was artificially inoculated and disease levels were evaluated by visual observation using a 0-3 rating scale, detailed image analysis using a newly developed MATLAB-based software and quantification of *F. fujikuroi* colonization in rice seedlings with a selective medium. A total of 24 QTLs associated with disease severity and/or pathogen colonization were mapped by the mixed linear model in TASSEL software. Defense- and hormone-related candidate genes identified within the QTLs will be further validated.

## QTL Mapping of Resistance to Tan Spot in a Winter Recombinant Inbred Line Population Derived from Cross between Harry and Wesley

G. KARIYAWASAM (1), W. Hussain (2), A. Easterly (2), M. Guttieri (2), V. Belamkar (3), J. Venegas (2), S. Baenziger (2), J. Poland (4), J. Faris (5), S. Xu (5), J. Rasmussen (1), Z. Liu (1)
(1) North Dakota State University, Fargo, U.S.A.; (2) University of Nebraska, Lincoln, U.S.A.; (3) University of Nebraska, U.S.A.; (4) Kansas State University, Manhattan, KS, U.S.A.; (5) USDA-ARS, Northern Crop Science Laboratory, Fargo, ND, U.S.A.

Tan spot, caused by the *Pyrenophora tritici-repentis*, is an important disease on both common and durum wheat worldwide. The disease system is known to involve three necrotrophic effectors (NEs) that interact with corresponding host sensitivity genes, including Ptr ToxA-*Tsn1*, Ptr ToxB-*Tsc2* and Ptr ToxC-*Tsc1*. To investigate the role of NE-host sensitivity interactions in disease, we conducted the QTL mapping of tan spot resistance in a recombinant inbred line population derived from a cross between two US winter wheat cultivars 'Harry' and 'Wesley'. Harry was sensitive to Ptr ToxA and highly susceptible to tan spot while Wesley was insensitive and highly resistant. QTL mapping revealed two major loci: the one corresponding to the *Tsn1* locus, and one on 1AS, which likely corresponded to the *Tsc1* locus. Both the *tsn1* and *tsc1* loci were significantly associated with resistance to race 1, but only *tsn1* was significantly associated with race 2, and only *tsc1* was associated with race 3. Neither locus was significantly associated with disease caused by a race 2 *ToxA* knockout strain. Additional QTL were also identified in this population, but their effects on disease were relatively minor. This work indicates that the Ptr ToxA-*Tsn1* and Ptr ToxC-*Tsc1* interactions play important roles in conferring tan spot susceptibility in winter wheat, and breeding programs should remove these susceptibility loci in order to improve tan spot resistance.

## Molecular mapping of effective stripe rust resistance genes in wheat germplasm PI 182126

X. CHEN (1), J. Feng (2), M. Wang (3)

(1) USDA-ARS and Washington State University, U.S.A.; (2) Department of Plant Pathology, Washington State University, U.S.A.; (3) Department of Plant Pathologist, Washington State University, U.S.A.

Stripe rust, caused by *Puccinia striiformis* f. sp. *tritici*, is one of the most damaging diseases of wheat. Spring wheat landrace PI 182126, originally from Sind, Pakistan, has shown a high level of resistance to stripe rust in field experiments for more than 10 years and in greenhouse tests with several predominant races. The objective of this study was to map the resistance gene(s) in PI 182126. A population of 120 F<sub>5</sub> recombinant inbred lines was developed from the cross between PI 182126 and susceptible genotype Avocet S through single-seed decent. The population was tested with US predominant races PSTv-14 and PSTv-37 at seedling stage under greenhouse conditions and at adult-plant stage in fields under natural infection of the pathogen near Pullman and Mount Vernon, Washington in 2014. The population was genotyped using microsatellite markers. Two resistance genes were mapped to chromosomes 5B and 7B. Using Chinese Spring nulli-tetrasomic and 7B deletion lines, the gene on 7B was mapped to the 0.14 bin of the long arm. Based on the chromosomal location, tightly linked markers, and effective throughout growth stages against all races tested, the gene on 7B is different from *Yr39*, *Yr52*, *Yr59*, *YrZH84*, and *YrC591* previously mapped to the long arm of chromosome 7B, and is useful for diversifying stripe rust resistance genes used in breeding programs.

## SNP variation in switchgrass disease resistance genes

B. BAHRI (1), G. Daverdin (2), J. Cheng (3), K. Barry (3), S. Smith (4), K. Devos (5)

(1) Dept. of Plant Protection, National Agronomic Institute of Tunisia/ Dept. of Crop and Soil Sciences, University of Georgia, U.S.A.; (2) Marucci Center for Blueberry & Cranberry Research and Extension, Rutgers University, Chatsworth, NJ, U.S.A.; (3) DOE Joint Genome Institute, Walnut Creek, California, CA, U.S.A.; (4) Dept. of Plant Pathology, University of Georgia, Athens, GA, U.S.A.; (5) Institute of Plant Breeding, Genetics and Genomics, Dept of Crop and Soil Sciences, University of Georgia, Athens, GA, U.S.A.

Switchgrass (*Panicum virgatum* L.), an allotetraploid perennial C4 grass native to North America, is an important feedstock for cellulosic biofuel production. Its high genotypic and phenotypic variation yield substantial potential for genetic improvement of bioenergy-related traits, including disease resistance. We studied SNP variation in 276 switchgrass genotypes, belonging to 36 accessions from mainly the Southern USA, across ~1 kb NBS-LRR regions of 4 classes of putative resistance (R) genes. PCR amplicons for an R gene with homology to the barley powdery mildew resistance gene Mla from 150 switchgrass accessions mapped to two homeologous regions in the allotetraploid switchgrass genome. Reads from individual accessions aligned preferentially against only one locus. Fifty-eight SNP positions and 78 haplotypes were recorded. Twelve SNPs were present in more than 35% of the accessions. Although switchgrass is largely an obligate outcrossing species, 95% of the SNPs were homozygous. The results indicate that selection for some potentially beneficial SNPs occurred in natural switchgrass population which were subsequently fixed in homozygous condition at the locus level.

## Development of molecular makers for spinach resistance genes to downy mildew disease

C. FENG (1), J. Correll (1)

(1) University of Arkansas, U.S.A.

Spinach is an increasingly popular vegetable crop. Downy mildew, caused by *Peronospora farinosa* f. sp. *spinaciae* (Pfs), is the most economically important disease of spinach and the main focus of all spinach breeding programs. Resistant cultivars are the most economical way to manage downy mildew, particularly for organic spinach production, which is approaching 50% in the US. Resistance to downy mildew is typically controlled by single dominant genes, and 11 resistant loci have been hypothesized in spinach. Three resistant loci, RPF1, 2 and 3 have been genetically characterized; each provides resistance to more than 10 of the 16 described races of Pfs. A molecular SCAR marker, linked to RPF1, has been shown to be useful in selecting this locus for resistance. Bulked segregant analysis and genotyping by sequencing approaches were employed to develop markers for RPF2 and 3. One out of 900 10-mer random primers examined generated a fragment specific to resistant plants, and the derived SCAR marker co-segregated with the RPF2 locus. For RFP3, a SNP linkage map was constructed with two SNP markers closely linked (1.1 and 4 cM) to the RPF3 locus. The RPF2 and RPF3 markers have been shown to be effective in selecting the respective resistance loci outside of the original mapping populations demonstrating their utility. These markers could be used to pyramid resistant loci to improve the durability of downy mildew resistance in spinach.

## Genome-wide association study of transmission of Soybean mosaic virus through seed

Q. Liu (1), H. Hobbs (1), L. Domier (2)

(1) University of Illinois, U.S.A.; (2) USDA-ARS, University of Illinois, U.S.A.

Soybean mosaic virus (SMV), an aphid- and seed-transmitted member of the *Potyviridae*, causes significant reductions in soybean yield and seed quality worldwide. In North America, seedborne infections serve as the primary sources of inoculum for SMV infections. Therefore, host-plant resistance to seed transmission of SMV provides a means to limit the impact of SMV on soybean production. In this study, populations of 200 and 477 plant introductions from the United States Department of Agriculture Soybean Germplasm Collection were evaluated for their transmission of SMV through seed. To identify loci associated with resistance to SMV seed transmission, the phenotypic data and single nucleotide polymorphism (SNP) information for each line from the SoySNP50K resource were combined and analyzed using a compressed mixed linear model as implemented in the GAPIT package in R. The loci identified were compared to the results from previous biparental mapping studies of resistance to SMV seed transmission. This study provides a comprehensive analysis of loci associated with seed transmission of an economically important plant virus.

## Identification of resistance genes and resistance gene analogs by si-RNA sequencing of the sweet potato leaf phytobiome

A. ALLEYNE (1), A. Alleyne (2), M. James (3), D. Gutierrez (4), S. Fuentes (4)

(1) The University of the West Indies- Cave Hill Campus, Bridgetown, Barbados; (2) The University of the West Indies-Cave Hill Campus, Bridgetown, Barbados; (3) Ministry of Agriculture, Food and Water Resources, Plant Pathology Section, Graeme Hall, Barbados, Barbados; (4) International Potato Center (CIP), Virology Laboratory, Lima, Peru
Sweet potato (*Ipomoea batatas* L. (Lam) is affected by single and synergistic virus co-infections manifested as biotic stress and severe disease. Consequently, breeding for disease resistance is used in disease management. However, there are few reports of resistance genes (R-genes) against specific viruses in sweet potato virus disease complex (SPVD). This study sought to identify R- genes in the sweet potato leaf phytotbiome using Next Gen-sequencing. RNA-seq has been used to characterize viruses causing SPVD while discovery of R-genes traditionally involves cloning and characterization of resistance proteins and resistance gene analogs (RGA). In this study, RNA was extracted from three sweet potato cultivars (CBS 49, CBS 32 and C-104) symptomatic with *Sweet potato feathery mottle virus* (SPFMV), *Sweet potato leaf curl virus* (SPLCV) and *Sweet potato badnavirus* A and B. These were then subjected to RNA-seq analysis and bioinformatics analysis of the assembled contigs for resistance genes. We report the presence of transcripts of a total of thirty-one such genes, and putative proteins representing several classes of resistance genes including nucleotide-binding site leucine-rich repeat (NBS-LRR) and RGA from sweet potato such as AB125881, DQ903328, and DQ903597 in the sweet potato phytotbiome. These results provide a genome resource of R-genes for development of tagged molecular markers useful for mapping disease resistance in specific sweet potato cultivars.

## Differential effects of stalk rot diseases on photosystem II photochemical efficiency among resistant and susceptible sorghum lines

A. BANDARA (1), D. Weerasooriya (1), T. Tesso (1), C. Little (1)

(1) Kansas State University, U.S.A.

Stalk rots are among the most injurious sorghum diseases worldwide. Understanding the plant physiological changes that occur during these diseases will help to uncover potential resistance mechanisms. The objective of this study was to test the effect of stalk rots on sorghum (Sorghum bicolor (L.) Moench) leaf chlorophyll content and photosystem II photochemical efficiency (Fv/Fm). Two resistant (SC599, SC35) and susceptible (Tx7000, BTx3042) lines were field evaluated in 2014 and 2015. Plants were inoculated with *Fusarium thapsinum* (Fusarium stalk rot), *Macrophomina phaseolina* (charcoal rot), and phosphate-buffered saline (control) at 14 d after flowering. Flag leaf chlorophyll and Fv/Fm were measured at soft-dough, hard-dough, and physiological maturity using a Soil and Plant Analytical Development meter (SPAD) and a chlorophyll fluorometer, respectively. ANOVA was performed with appropriate modeling for repeated measures. Compared to control, both pathogens significantly reduced SPAD (P < 0.0001) across genotypes and growth stages and the reduction was greater in 2014. Across years and growth stages, both pathogens significantly reduced the Fv/Fm (P < 0.016) of susceptible lines while no such effect was evident for the resistant lines. Despite the apparent chlorophyll reduction during disease development, the ability to maintain higher photosynthetic activity at the tested growth stages appeared to contribute to physiological resistance against stalk rot diseases.

## Two SREBP Transcription Factors are Required for Ergosterol Biosynthesis and DMI Resistance in Penicillium digitatum

R. RUAN (1)

(1) Institute of Biotechnology, Zhejiang University, China

Application of imazalil is a common method for control of citrus green mold caused by Penicillium digitatum. As a consequence of continuous use, imazalil-resistant P. digitatum strains have emerged worldwide. Our previous study showed that overexpression of imazalil target genes CYP51A and CYP51B, induced by the insertion mutations in their promoter regions, is responsible for the resistance. However, the regulators inducing overexpression of CYP51s are unclear. In this study, we demonstrated two SREBP homologs, designated as PdSrbA and PdSrbB. Both ?PdsrbA and ?PdsrbB showed markedly reduced ergosterol contents and increased sensitivities to imazalil and other DMIs, while double deletion led to hypersensitive to DMIs. ?PdsrbA but not ?PdsrbB is hypersensitive to terbina?ne and tridemorph, two SBIs targeting to ERG1 and ERG24, respectively. In addition, PdsrbA also mediated responses to iron-depletion and hypoxia. However, no change was observed in growth rate or virulence for those mutants. Expression of CYP51s and other genes involved in ergosterol biosynthesis was significantly down-regulated in ?PdsrbA and ?PdsrbB. These results suggested that these two SREBP homologs co-regulate ergosterol biosynthesis as well as DMIs resistance in P. digitatum, and PdsrbA may be with wider functions.

## Chemical association of potato susceptibility to *Rhizoctonia solani*

X. Y. ZHANG (1, 2), H. H. Jiang (2), J. J. Hao (2)

(1) College of Agronomy, Inner Mongolia Agricultural University, Hohhot, China; (2) School of Food and Agriculture, University of Maine, Orono, ME, U.S.A.

*Rhizoctonia solani* is the causal agent of stem canker and black scurf of potato. To investigate chemical factors related to potato resistance to *R. solani*, 17 cultivars of potato were selected for a two-year field trial. Sixty-day-old plants were dug from field, and wax content of underground stems was measured. For other chemical measurement, potato seedlings grown in Murashige and Skoog medium to an approximate height of 12 cm, were transferred into greenhouse pots containing vermiculite and soil (v:v = 1:1). After 30 days, plants were dug and the underground stems were used to determine the silicon content. Leaves were used to determine the content of soluble sugar, soluble starch, free amino acid and soluble protein. Black scurf was measured based on 0 to 5 scale, which was used for disease index calculation. Wax content of underground stems of field-grown potato was negatively correlated with disease index ($r = -0.532 P < 0.05$), but silicon content did not have a significant correlation ($r = 0.042 P > 0.05$), indicating wax-enhanced potato resistance to *R. solani*. The correlation between soluble starch, soluble protein, soluble sugar and free amino acid and disease index were 0.476 (*P* < 0.05), 0.458 (*P* < 0.05), 0.300 (*P* > 0.05) and 0.219 (*P* > 0.05), respectively. This showed that lower levels of soluble starch and higher levels of soluble protein enhanced disease resistance, while soluble sugar and free amino acid had no significant effects on disease resistance.

## Genetic characterization of quantitative resistance to *Bremia lactucae*, the causal organism of lettuce downy mildew

L. Parra (1), I. Simko (2), R. Michelmore (3)

(1) UC Davis- Genome center, U.S.A.; (2) USDA-ARS, U.S.A.; (3) UC Davis-Genome Center, U.S.A.

Lettuce (*Lactuca sativa*) is one of the most valuable vegetable crops in the United States. Downy mildew (DM), caused by *Bremia lactucae*, is the most important foliar disease of lettuce worldwide, which decreases the quality of the marketable portion of the crop. The use of resistant varieties carrying dominant genes (*Dm* genes) is the most effective method for controlling this disease. However, the high pathogen variability leads to the defeat of cultivars containing resistance genes by new isolates of the pathogen. Some lettuce varieties such as Iceberg and Grand Rapids exhibit field resistance that cannot be attributed to *Dm* genes. This resistance trait, which is manifested in adult stages of development, has shown to be quantitatively inherited. To determine the genetic basis of field resistance, two populations of recombinant inbred lines originated from crosses between a field resistant cultivar and a susceptible cultivar (Grand Rapids x Salinas and Iceberg x PI491224) have been evaluated for DM severity and genotyped using Genotyping-By- Sequencing for quantitative trait loci (QTL) analysis. Marker-assisted gene pyramiding of multiple *Dm* genes in combination with QTLs for field resistance provides the opportunity for more durable resistance to *B. lactucae*.

## Exserohilum turcicum races causing northern leaf blight of corn in the North Central United States

J. Weems (1), C. Bradley (1)

(1) University of Kentucky, U.S.A.

Northern leaf blight of corn, caused by *Exserohilum turcicum*, is a yield- reducing foliar disease that commonly occurs across the North Central U.S. Previous race population distribution studies identified five physiological races in the U.S. prior to 1995. For this study, 156 *E. turcicum* isolates from Illinois, Indiana, Iowa, Minnesota, North Carolina, Ohio, and Wisconsin were screened against corn differential lines containing *Ht1*, *Ht2*, *Ht3*, *Htm1*,
and *Htn1* resistance genes. Isolates were collected between 1979 and 1985 (n=13) and between 2007 and 2014 (n=143). Twenty physiological races were observed based on the responses of the differential corn lines. Races 0, 1, and 1mm were the most prevalent races, comprising 21%, 27%, and 13% of the isolates, respectively. Races were diverse within all states and years. Virulence to multiple *Ht* resistance genes within individual isolates was observed in 47% of those tested, with 3% of the isolates conferring virulence to all *Ht* resistance genes tested. Virulence to the *Ht1*, *Ht2*, *Ht3*, *Htm1*, and *Htn1* resistance genes was present in 64%, 20%, 18%, 32%, and 27% of the *E. turcicum* isolates, respectively. Virulence to *Ht* resistance genes was fairly evenly distributed across states in isolates collected after 2008. Although *Ht* genes were still effective against several isolates, the use of quantitative resistance in conjunction with *Ht* genes would likely improve management of northern leaf blight in the North Central U.S.

## Wheat yellow rust dynamics in Tunisia since 2013 and resistance genes in durum wheat

B. BAHRI (1), M. Leconte (2), S. Hamza (1), C. de Vallavicaille Pope (2)

(1) National Agronomic Institute of Tunisia, Tunisia; (2) UMR BIOGER, INRA, AgroParisTech, Université Paris-Saclay, France

Wheat yellow rust epidemics, caused by *Puccinia striiformis* f. sp. *tritici* (PST) have increased worldwide with the emergence in Western Europe and North Africa of an aggressive strain and tolerant to high temperature (PstS2) since 2004 and exotic strains (Triticale 2006, 2015, Warrior and Kranich) recently. Even though resistant varieties bring to agriculture effective solutions to reduce the use of pesticides, races of the pathogen quickly overcome introduced resistance genes. In this study we determined the virulence combinations using the European and world differential sets and the simple sequence repeat diversity of 94 PST isolates collected in Tunisia since 2013 from the major wheat growing areas. In addition, 39 local Tunisian durum wheat accessions and 109 recombinant inbred lines RILs (F9) were evaluated at the seedling stage against old and new races of PST in order to identify resistant genotypes and markers associated with resistance genes. The results underlined the rapid change in wheat yellow rust population where 3 predominant races, Warrior1 (W1), Warrior- (W-) and Triticale 2006 were detected since 2013. In addition, 10% of the durum wheat accessions were resistant to W1, W- and PstS2. The evaluation of the RILs issued from a cross between Tunisian durum wheat Khiar and Agili39 revealed one major resistance gene against W1 and two minor genes against PstS2. The use of these genes in breeding can improve durability of the genetic control against PST.

## Effectiveness of adult plant resistance to wheat stem rust is specific to *Puccinia graminis* f. sp. *tritici* race

M. ROUSE (1), J. Briggs (2)

(1) USDA-ARS, U.S.A.; (2) University of Minnesota, U.S.A.

Adult plant resistance (APR) in wheat to stem rust caused by *Puccinia graminis* f. sp. *tritici* is considered to be conferred by multiple loci that are race non-specific. Our objective was to test the race-specificity of individual and combinations of wheat stem rust APR loci. We evaluated a biparental wheat population (Thatcher/McNeal) that is segregating for six APR loci with minor effects in response to four races of the stem rust pathogen (QTHJC, TPMKC, RCRSC, and QFCSC), in single-race field nurseries over three years. QTL mapping and ANOVA identified the significance and effectiveness of the six minor effect loci across the years and races. The QTL with the largest effect, coincident with the *Sr12* locus, was effective to virulent races QTHJC and TPMKC, but conferred a variable, sometimes nonsignificant, response to race RCRSC. The other five QTL varied in effectiveness between years and were generally less effective in response to race RCRSC in particular. The data from QTL mapping suggested that some of the QTL could be race specific, such as *QSr.cdl.1AL*, because they were detected in response to races such as TPMKC and QTHJC, but not to race RCRSC. This observation has implications specifically for wheat stem rust resistance breeding because it suggests that resistance observations to a single race may not be indicative of resistance to another race, even if the mechanism of resistance is multiple minor effect loci.

## Lack of interaction between *Fusarium oxysporum* f. sp. *niveum* and *Meloidogyne incognita* on cucurbit rootstocks resistant to *Fusarium wilt* of watermelon

A. KEINATH (1), P. Agudelo (1)

(1) Clemson University, U.S.A.

Interspecific hybrid squash (*Cucurbita maxima* × *C. moschata* 'Strong Tosa') and bottle gourd (*Lagenaria siceraria* 'Macis') rootstocks are resistant to *Fusarium oxysporum* f. sp. *niveum* (FON) but susceptible to *Meloidogyne incognita*, Southern root-knot nematode. Co-infection with root-knot nematode has rendered Fusarium resistance ineffective in other hosts. The objectives of this study were to determine if such an interaction occurred between *M. incognita* and FON races 1 and 2 on 'Strong Tosa,' 'Macis,' and watermelon 'Fascination' (resistant to race 1) and 'Tri-X 313' (susceptible to both races). The four hosts were inoculated in a greenhouse with one of four pathogen treatments: FON applied as 2 × 10⁷ microconidia per pot, *M. incognita* applied as 2000 J2 per pot, both pathogens, or neither pathogen. Each race was tested twice with 10 replicate plants per host and inoculation treatment. Plants not inoculated with FON did not wilt. After 4 weeks, severity of Fusarium wilt and recovery of *F. oxysporum* did not differ when hosts were inoculated with FON alone or with FON and *M. incognita* together (host-by-treatment interactions not significant, P≥0.08 and P≥0.17 for races 1 and 2, respectively). Galling was present on ≥10% of the root system of 90% of the plants inoculated with *M. incognita*, and no non-inoculated plants had galls. In conclusion, resistance to FON in cucurbit rootstocks is not compromised when *M. incognita* infects the roots.

## Adaptation to quantitative resistance in the hop cultivar Cascade by *Podosphaera macularis*

D. GENT (1), D. Gent (1), M. Twomey (2), S. Wolfenbarger (2)

(1) USDA Agricultural Research Service, U.S.A.; (2) Oregon State University Department of Botany and Plant Pathology, U.S.A.

In the hop powdery mildew pathosystem, the cultivar Cascade has not been substantially affected by powdery mildew despite being produced on a relatively broad scale for decades. Beginning in 2012, development of powdery mildew on this cultivar under field conditions has become increasingly common in Washington State. Surveys conducted during 2013 to 2015 indicated increasing occurrence and severity of powdery mildew on Cascade, concomitant with increases in the number of fungicide applications made growers. Nearly all isolates of *Podosphaera macularis* sporulated when inoculated to Cascade, although the latent period was shortest, infection frequency greatest, and the fungus most fecund on Cascade when inoculum was originally derived from this cultivar as compared to other cultivars. Only 4.9% of isolates obtained from Cascade were able to infect cv. Nugget, which possess the resistance gene termed R6, indicating that Cascade-adapted strains of the fungus are distinct from strains that attack cultivars possessing R6. In growth chamber experiments, powdery mildew levels on various cultivars were similar when inoculated with Cascade-adapted strains of *P. macularis* as compared to non-adapted strains, further supporting a specific adaptation by the fungus to the quantitative resistance in Cascade. Multiple R-genes are predicted to provide resistance to Cascade-adapted strains of the fungus, as is quantitative resistance found in other germplasm.

## Gene-For-Gene in Fusiform Rust Disease of Loblolly Pine

K. SMITH (1), D. Ence (2), D. Nelson (3), M. Yandell (2), J. Davis (4)

(1) USDA Forest Service, U.S.A.; (2) University of Utah, U.S.A.; (3) USDA Forest Service, U.S.A.; (4) University of Florida, U.S.A.

The rust fungus *Cronartium quercuum* f.sp. *fusiforme* (*Cqf*) incites fusiform rust disease on southern pines causing loss of stem quality or death. Given that fusiform rust is a coevolved, gene-for-gene pathosystem there is potential for resistance to be overcome in the field. Nine resistance genes (*Fr1-Fr9*) and one avirulence gene (*Avr1*) have been placed on the genetic maps of loblolly pine and *Cqf*. More recently genomic references have become available for both the host and the fungus. Allele-level sequence data for resistance genes and their corresponding avirulence genes would make it possible to monitor virulence in the field, guide deployment and understand local outbreaks. Toward that end work is underway in both the loblolly host and *Cqf* pathogen. A candidate for the *Fr1* resistance gene was identified in the 22 gigabase pair, loblolly pine reference genome. This gene remains a candidate
and fine mapping is currently being done to improve the loblolly genetic map in this region, as well as, identify additional resistance gene candidates from elite pine families. Research on the *Cqf* fungus has located a genomic interval that contains the *AvrI* gene using both traditional marker sequences and high-throughput re-sequencing of progeny from of an *AvrI* segregating cross. Using an improved *Cqf* assembly and re-sequencing of field isolates, we are attempting to define the *AvrI* gene and the causative mutation that led to virulence in the *AvrI* mapping population.

## Melampsora rust running amuck: Exploring willow rust population diversity in the Northeast United States

C. CROWELL (1), M. Bekauri (1), C. Carlson (2), F. Gouker (2), L. Smart (2), C. Smart (1)

(1) Cornell University Plant Pathology and Plant-Microbe Biology Section, U.S.A.; (2) Cornell University Horticulture Section, U.S.A.

Shrub Willow (*Salix* spp.) has proved to be a reliable short-rotation coppice (SRC) biofuel feedstock in the Northeast United States. However, the willow rust pathogen *Melampsora* spp. can cause up to a 50% reduction in yield, making some growing operations economically limited. This has spurred investigation into both the identification of resistance within *Salix* spp. and the diversity of *Melampsora* spp. in the Northeast. Approximately 200 single pustule isolates were collected from MI, NY, PA, VT, and WV, and are being characterized by rDNA sequencing. Preliminary data shows that both *M. americana* and *M. paradoxa* are present in our regions of interest. Further investigation of *Melampsora* spp. diversity throughout the Northeast will be conducted using genotyping-by-sequencing, a genome wide high-throughput SNP identification method, providing thousands of markers for population studies targeting un-methylated coding sequences. Additionally, we explored disease resistance by characterizing association and linkage mapping populations of *Salix purpurea* with the aim of identifying QTL and tightly-linked SNPs for marker-assisted selection. Preliminary data show a continuum of resistance, suggesting promising breeding opportunities. This information will lead to further understanding of the population biology of *Melampsora* willow rust, while providing crucial information necessary for the success of future willow breeders.

## Marker development and fine-mapping for the Phytophthora crown rot resistance locus, *Pc1*, in strawberry

Y. NOH (1), J. Mangandi (1), S. Verma (1), V. Whitaker (1), S. Isobe (2), J. Cha (3), S. Lee (1)

(1) University of Florida, U.S.A.; (2) Kazusa DNA Research Institute, Japan; (3) Chungbuk National University, South Korea

Phytophthora crown rot (PhCR) caused mainly by *Phytophthora cactorum* is a destructive disease and can cause economic losses to strawberry growers in US. In our previous study, a major QTL for PhCR resistance, *Pc1*, located on the linkage group 7D, was discovered in complex and multi-family populations using the whole-genome SNP genotyping (IStRAW90 Axiom® SNP Array) and pedigree-based marker-trait association analysis (FlexQTL™). For the fine-mapping of *Pc1*, a total of 25 SNP-based high resolution melting (HRM) markers and 63 simple sequence repeat (SSR) markers were selected in the *Pc1* region. All markers were tested with five strawberry varieties and 20 advanced breeding selections that inherited putative functional allele combinations of *Pc1pc1* (heterozygous resistant) or *pc1pc1* (susceptible). Total five markers were found to co-segregate with the resistance locus, *Pc1*. In addition, we developed three populations (n=339) by crossing the resistance parent (*Pc1pc1*), '12.55-220', with three susceptible accessions (*pc1pc1*), '12.82-44', '11.116-56', and '11.98-41'. All populations were planted to the field (Wimauma, FL) and evaluated for the PhCR disease resistance. The HRM and SSR markers linked to the *Pc1* resistance locus were tested for the PhCR fine-mapping population, and the linkage analysis was conducted. The precise location of *Pc1* and DNA markers developed in this study will be utilized for the PhCR resistance breeding program.

## Mitigating the effects of shoot blight caused by *Erwinia amylovora* by enhancing plant systemic resistance

B. LEHMAN (1), C. Bower (1), K. Peter (1)

(1) Penn State University, U.S.A.

Fire blight, caused by *Erwinia amylovora*, is one of the most devastating diseases of apple and pear and continues to cause large economic losses for tree fruit producers. Antibiotics are largely used to control blossom blight where bacterial resistance is absent, but the need exists for alternative control strategies where resistance occurs, and where shoot and canker blight are prevalent. One strategy is through controlled activation of the plant immune system by the application of materials containing plant defense activating compounds. Our goal was to test the effectiveness of an experimental resistance activating compound in reducing shoot blight severity. We applied three different concentrations of the chemical as foliar sprays on potted 'Gala' apple trees grafted on M.26 rootstocks. Three concentrations were applied at three, two, and one week before shoot tip inoculation and three, two and one day before shoot tip inoculation; Acibenzolar-S-methyl (Actigard) was also applied at three, two, and one week before shoot tip inoculation. Evaluations of shoot severity were made at one and two weeks after inoculation. Treatments started three days before inoculation reduced severity by 9, 26, and 35% for the three concentrations tested. Treatments started three weeks before inoculation reduced severity by 27, 21, and 44%, for the concentrations tested. Acibenzolar-S-methyl applications reduced severity by 75%. The testing of other alternatives is ongoing.

## Induction of resistance in cultivated peanut against peanut early leaf spot by salicylic acid

K. BOWEN (1), L. Gong (2)

(1) Auburn University, U.S.A.; (2) Auburn University, U.S.A.

Cultivated peanut (*Arachis hypogaea* L.) is an economically important crop for the United States and throughout the world. *Cercospora arachidicola* (Ca), the causal agent of early leaf spot (ELS) on cultivated peanut, can cause yield losses of up to 50%. Since earlier work reported inconsistent results with salicylic acid (SA) relative to peanut pathogens, we sought to examine the ability of SA to induce resistance against ELS on a high-oleic cultivar. We evaluated the effect of spraying three rates of SA (100, 200, and 300 µM) at 3, 5, and 7 days before inoculation (DBI) with ELS in whole peanut plants (cultivar GA-09B) in a growth chamber. Each treatment was replicated three times. At 15 days after inoculation (DAI), all SA treatments had significantly fewer lesions (3.1, 2.8, and 2.5 lesions with increasing SA rates; and 1.9, 3.0, and 3.6 lesions with increasing DBI), compared with the control (12.7). At 42 DAI, all sporulating lesions from SA treated plants tended to produce fewer spores compared with the control (170.0); for example, 27.2 with 300 µM SA, 54.5 with 3 DBI, and 31.0 with 7 DBI. Although there were fewer lesions on SA treated plants, these lesions were larger than those on the control. Results of this study suggest that SA plays at least a transient role in ELS resistance in peanut cultivar GA-09B.

## Development of a molecular marker platform for the breeding of blast resistance varieties in Taiwan

W. CHEN (1), W. Shen (1), F. Chang (2), W. Chang (3), Z. Yu (1), J. Liao (1), M. Lai (4), C. Wu (2), C. Chung (1)

(1) National Taiwan University, Taiwan; (2) Kaohsiung District Agricultural Research and Extension Station, Taiwan; (3) Taoyuan District Agricultural Research and Extension Station, Taiwan; (4) Taiwan Agricultural Research Institute, Taiwan

Rice blast is the most important disease commonly occurring in the first cropping season in Taiwan. To facilitate marker-assisted backcrossing, this study aims to identify effective blast resistance (*R*) genes in Taiwan and develop *R* gene and background selection markers for the use of IRRI-bred blast resistant lines (IRBLs) as donors. The resistance spectra of known *R* genes were evaluated by inoculating 31 IRBLs with 18 *M. oryzae* isolates collected from six rice-growing areas in Taiwan in 2009-2013. The *R* alleles at *Pi2/9*, *Pik*, and *Pita/Pita2* were resistant to more isolates. Genotyping-by-sequencing technology was used to analyze 13 IRBLs with broader-spectrum resistance and 20 Taiwan high-quality rice varieties. Based on the 131,942 SNPs between IRBLs and Taiwan varieties, a set of 11 polymorphic *R* gene markers and 96 Fluidigm markers evenly distributed on rice genome were developed. Two *M. oryzae* isolates 12YL-TT4-1 and 13TN-HB1-3 were used to determine which Taiwan varieties would be suitable recipients for the 13 *R* alleles. The result showed that Taikeng 4, Taikeng 9, Taikeng 10, Taikeng 14, Taikeng 15, Tainung 71, Taiyuan 3, Tainan 11, Kaohsiung 145, and Kaohsiung 147 do not carry the *R* alleles in IRBL9-W, IRBLz5-CA, IRBLz5-CA (R), IRBLz-Fu, IRBL1-CL, IRBL7-M, IRBLk-Ka, IRBLkp-K60, IRBLKm-Ts, IRBLkh-K3, IRBL20-IR24, IRBLta2-Pi, and IRBLta2-Re. The 13 IRBLs and newly developed markers can be used to improve blast resistance of the 10 high-quality rice varieties.
## Introgression of crown rot resistance into cultivated squash *Cucurbita moschata*, *C. maxima* and *C. agyrosperma*

S. ZHANG (1), Y. Fu (1), P. Moon (1), C. Waddill (1)

(1) Tropical Research and Education Center, University of Florida, IFAS, U.S.A.

*Phytophthora capsici* incites crown rot in squash and often causes significant losses to growers worldwide. No resistance is available in commercial squash varieties, but resistance has been found in the wild species *Cucurbita lundelliana* and introduced into *C. moschata*, resulting in several resistant lines. However, the yields of these resistant lines are too poor to warrant their commercial release. To overcome this, four of these resistant lines were crossed with commercial varieties of *C. moschata*, *C. maxima* and *C. agyrosperma* in single or three-way crosses. The F₁ progeny of *C. moschata* 'Butterbush' and *C. moschata* resistant lines were resistant to *P. capsici* and showed improved yield. Interspecific cross of 'Buttercup' (*C. maxima*) x 399 (432) (a resistant line) was made. The F₁ progeny were completely resistant to *P. capsici*, and showed strong heterosis in plant growth and fruit size, but were male sterile. Backcross progeny exhibited low seed settings. Three-way crosses of *C. agyrosperma* x F₁ (*C. moschata* x *C. moschata*) were obtained. The hybrids showed improved seed settings with higher percentage of normal seeds (41.2%) compared to the F₁ interspecific progeny of *C. maxima* x *C. moschata* (8.8%). Embryo rescue was employed to overcome interspecific barriers. Further backcrossing with recurrent parents and pedigree selection for *P. capsici* resistance, high fruit yield, and flesh quality will be performed.

## Evaluating soybean breeding lines developed from different sources of resistance to Phomopsis seed decay

S. LI (1), J. Smith (1)

(1) USDA-ARS, Crop Genetics Research Unit, U.S.A.

Phomopsis seed decay (PSD) causes poor soybean seed quality worldwide. The primary causal agent of PSD is *Phomopsis longicolla* (syn. *Diaporthe longicolla*). Breeding for PSD-resistance is the most effective long-term strategy to control this disease. To develop soybean lines with resistance to PSD, multiple breeding populations were developed from crosses between PSD-resistant and high-germination lines, as well as between PSD-resistant lines. Based on the seed plating assay, the range of percent seed infected by *P. longicolla* among all lines tested was from 0 to 52%, with an overall line mean of 7.5. There were significant differences in the reaction to PSD among lines derived from the same source of resistance, as well as among lines from different sources of resistance. More important for making future populations, the mean PSD score of the lines derived from the PI 424324B source was significantly lower than that of those derived from the PI 80837/SS93-6181 source. The PSD of lines from PI 417050 source were not significantly different from those from the other sources. These results indicate an apparent superiority for using the PI 424324B source of resistance for breeding improved lines. Seeds for many of the lines derived from both PI 424324B and PI 417050 had no incidence of PSD. Therefore, it is likely that some of them will be released in the future as resistant to PSD and thereafter utilized in breeding programs to develop PSD-resistant cultivars.

## Transgenic expression of pattern recognition receptor EFR in tomato leads to effective field resistance to bacterial wilt

S. KUNWAR (1), S. Kunwar (1), E. Evaristo da Silva (2), F. Iriarte (2), L. Ritchie (2), D. Clark (2), J. Freeman (2), R. Stall (3), J. Jones (3), J. Minsavage (3), C. Zipfel (4), D. Horvath (5), M. Paret (2)

(1) University of Florida, Gainesville, FL, U.S.A.; (2) North Florida Research and Education Center, University of Florida, U.S.A.; (3) University of Florida, U.S.A.; (4) The Sainsbury Laboratory, Nowich, UK, United Kingdom; (5) Two Blades Foundation, U.S.A.

Bacterial wilt of tomato caused by *Ralstonia solancearum* is a major problem in tomato production in the tropics and sub-tropics worldwide and can cause 50-80% yield losses. We investigated whether transgenic tomato (*Solanum lycopersicum*) expressing the resistance gene *EFR* (Elongation Factor Tu-Receptor) from *Arabidopsis thaliana* or *Bs2* gene from *Capsicum annuum* or both provides improved resistance to bacterial wilt disease in a field with *R. solanacearum*. The presence of *EFR* and *Bs2* or *EFR* alone on the highly susceptible FL 8000 background, significantly reduced the bacterial wilt incidence compared to *Bs2* line (carrying only *Bs2*) or non-transformed control. Although yields of marketable fruit from *EFR* or *EFR/Bs2* lines were statistically not different than those from *Bs2* lines or non-transformed control, the total yield was significantly improved in the *EFR* and *EFR/Bs2* lines. Following harvest, the basal and middle stem sections of the remaining living plants in the field were assessed by diluting plating on modified SMSA media. The results revealed no significant differences in the populations at the basal stem. Interestingly, in the middle stem, no bacteria could be recovered from *EFR* lines or *EFR/Bs2* lines but *R. solanacearum* populations were recovered from *Bs2* and non-transformed control lines at 10⁴ CFU/g and 10⁵ CFU/g tissue, respectively. This finding indicates the potential of *EFR* gene for field management of tomato bacterial wilt.

## Plant defensins inhibit growth of pathogens in the alfalfa crown rot disease complex

A. SATHOFF (1), D. Samac (2)

(1) University of Minnesota, U.S.A.; (2) USDA-ARS, U.S.A.

Alfalfa crown rot is a disease complex that severely limits alfalfa stand density and productivity in all alfalfa-producing areas. Currently, there are no viable methods of control. Plant defensins are small cationic antimicrobial peptides with a conserved signature of cysteines. Defensins have a gamma-core motif, a cluster of positively charged residues, which is essential for antimicrobial activity. The core motifs of five synthetic defensins were tested for antimicrobial activity against the pathogens in the alfalfa crown rot disease complex. In a 96-well microplate, each well contained half strength potato dextrose broth, approximately 2000 spores, and concentrations of defensin peptide up to 30 ug/mL in a total volume of 100 uL. After 48 h of incubation at 25°C in the dark, absorbance of the wells was measured at 595 nm on a microplate reader to quantify the inhibition of fungal growth. The amount of defensin needed to inhibit growth of pathogen strains by 50% (EC₅₀) was calculated. The core motif of MtDef4 was shown to be the most effective peptide with EC₅₀ values of 5.3 uM against *Phoma medicaginis* and 6.9 uM against *Fusarium oxysporum* f.sp. *medicaginis*. In addition, MtDef4 had activity against *Pseudomonas syringae* pv. *syringae* and *Xanthomonas alfalfa* but not the oomycete *Aphanomyces euteiches* in *in vitro* assays. These results indicate that transgenic expression of plant defensins in alfalfa has the potential to lead to improved crown rot resistance.

## A novel vector system to engineer begomovirus resistance in transgenic plants by transcriptional and post-transcriptional gene-silencing mechanisms

C. TAI (1), J. Chen (2), F. Jan (1)

(1) Department of Plant Pathology, National Chung Hsing University, Taiwan; (2) Department of Agronomy, National Chung Hsing University, Taiwan

To generate transgenic plants that are highly resistant to whitefly-transmitted *Tomato leaf curl Taiwan virus* (ToLCTWV) and *Tomato yellow leaf curl Thailand virus* (TYLCTHV), plasmid vectors were constructed to induce transcriptional gene silencing (TGS) and/or post-transcriptional gene silencing (PTGS) in a transgenic plant. The intergenic region (IR) of begomovirus containing inverted repeat sequences was inserted into the intron of a tomato gene encoding a RNA-directed RNA polymerase 1 (RDR1) or a proline dehydrogenase (PDH). Expression of a GFP-RDR in construct carrying a TYLCTHV IR (IRhp), tomato intron and GFP led to nuclear localization of IRhp RNAs in plant cells after intron was spliced out and induced TGS. A C1C2C3 construct carrying the 3' end of C1 ORF and the overlapping region of C2 and C3 ORFs of TYLCTHV triggered PTGS in the cytoplasm. Infection assays revealed that constructs inducing TGS or PTGS alone displayed moderate resistance to TYLCTHV. However, C-RDRin-IR constructs carrying an IRhp, intron and C1C2C3 ORFs induced both TGS and PTGS and displayed strong resistance to TYLCTHV. Constructs carrying RDR1 introns could be easily spliced out and triggered TGS after transient expression in tobacco plants. Constructs carrying PDH intron3 had lower efficiencies in splicing and in triggering TGS. Our results indicated that a combination of inducing both TGS and PTGS in a construct shows great promise in developing resistance against plant viruses.
Exploiting the approaches of biotechnology in sweetpotato for virus diseases resistance

Y. MENG (1), Y. Meng (2), C. Zhang (2), V. Njiti (2)

(1) Alcorn State University, U.S.A.; (2) Alcorn State University, U.S.A.

Sweetpotato (*Ipomoea batatas* (L.) Lam.; *Convolvulaceae*), a plant widely grown in all tropical and subtropical areas, is among the 10 most important food crops worldwide. As a crop produced by vegetative propagation, "cultivar decline" due to viral infections significantly reduces sweetpotato yield and storage root quality. In the United States, *Sweet potato feathery mottle virus* (SPFMV) and *Sweet potato leaf curl virus* (SPLCV) are two of the most prevalent sweetpotato viruses that cause devastating diseases and yield reductions. Even though resistance to virus infections remains the most cost effective way of disease management, to date, immune resistance to these viruses has not been identified in *I. batatas*. In this study, we explored a novel biotechnological method to develop transgenic sweetpotato plants with resistance to these two viruses. We engineered part of coat protein (CP) gene of SPFMV and the replication origin region of SPLCV into a binary vector for *Agrobacterium*-mediated genetic transformation for transgene expression. The SPFMV CP gene and SPLCV replication origin sequence are designed in antisense for the optimal induction of RNA silencing in transgenic sweetpotato. Research results showed that expression of foreign genes has been achieved by using *Agrobacterium tumefaciens* strain EHA105 harboring the expression cassette. Investigations on transgenic plant regeneration and resistance to virus infection under control conditions will be discussed.

Identifying onion cultivars with reduced susceptibility to center rot

J. MAZZONE (1), B. Gugino (2), M. Mansfield (2)

(1) The Pennsylvania State University, U.S.A.; (2) The Pennsylvania State University, U.S.A.

Onion growers in Pennsylvania (PA) are continually challenged by losses from center rot disease caused by the bacterial pathogens *Pantoea ananatis* and *P. agglomerans*. The objective of this research is to identify onion cultivars that are less susceptible to center rot than the commercial standard cv. Candy but still meet the criteria of the PA Simply Sweet Onion Marketing Program. Thirteen onion cultivars were evaluated between 2015 and 2016 for center rot susceptibility and select horticultural characteristics, including neck and bulb diameter. Cultivars were arranged in a randomized complete block design and plots were split by proximity to inoculated plants. Two onion leaves of select plants were inoculated with a mixture of *P. ananatis* and *P. agglomerans* isolates. At harvest, onions were graded by size, marketability and disease incidence. Sub-samples of asymptomatic onions were evaluated for soluble solids, pungency and postharvest disease incidence. In 2015, six cultivars had marketable yields comparable to cv. Candy. Center rot incidence significantly differed between cultivars at harvest (*P*=0.002) but not after 3-months in storage (*P*=0.241). A positive, linear relationship was observed between late season onion neck diameter and center rot incidence. All cultivars met the minimum soluble solids and pungency criteria for the marketing program. Evaluations in 2016 will further investigate cultivars that show promise in sweet onion production.

Early detection of adult plant resistance to oat crown rust using a qPCR assay

B. YIMER (1), B. Yimer (2), T. Gordon (2), J. Bonman (2)

(1) USDA-ARS, U.S.A.; (2) USDA-ARS, U.S.A.

Oat crown rust caused by the fungus *Puccinia coronata* f. sp. *avenae* is a major production constraint of oat in North America. There are over 100 major genes identified from various oat accessions, but their effectiveness is generally short lived. Adult plant resistance is the alternative and best option for durable crown rust resistance in oats. In field grown small grain cereals, adult plant rust resistance is usually measured at the flag leaf stage using a visual assessment of disease severity and infection type. Quantitative PCR-based assays have the potential to provide a less subjective measure of host resistance at an earlier stage in plant development. Such quantitative assay techniques provide a measure of the amount of fungus within infected plants which, for some rusts, can be directly extrapolated to host resistance. Here we report the use of a qPCR assay to detect adult plant resistance to crown rust in oats before the flag leaf stage in an experiment conducted in a growth chamber environment.

Response of sweet sorghum lines to stalk pathogens *Fusarium thapsinum* and *Macrophomina phaseolina*

D. FUNNELL-HARRIS (1), P. O'Neill (1), S. Sattler (1)

(1) USDA-ARS; Grain, Forage and Bioenergy Research Unit, U.S.A.

Sweet sorghum [*Sorghum bicolor* (L.) Moench] has potential for bioenergy. It is adapted to a variety of U.S. locations and the extracted juice can be directly fermented into ethanol. However, little research on fungal stalk rots has been reported, even though these diseases pose serious constraints for yield, juice quality and biomass usability. A greenhouse bioassay was designed to assess charcoal rot (*Macrophomina phaseolina*) and Fusarium stalk rot (*Fusarium thapsinum*) susceptibility at maturity, when these diseases are manifested in the field. Multiple plantings of a susceptible grain line, RTx430, were used as a control for variation in flowering times amongst sweet sorghum lines. Lesion length measurements in inoculated peduncles were used to quantify disease severity. Sweet sorghum lines 'Rio' and M81E exhibited resistance to *F. thapsinum* and *M. phaseolina*, respectively, and in contrast, line 'Colman' exhibited susceptibility to both pathogens. Lesion development over time was monitored in Colman. These results will enhance molecular and biochemical analyses of sweet sorghum responses to pathogens, and breeding stalk rot resistant sweet sorghum.

Development and evaluation of germplasm lines resistant to Sclerotinia stem rot

M. MCCAGHEY (1), M. McCaghey (1), J. Willbur (1), C. Grau (2), S. Chapman (2), B. Diers (3), C. Groves (2), A. Ranjan (2), M. Kabbage (2), D. Smith (1)

(1) University of Wisconsin- Madison, Madison, WI, U.S.A.; (2) University of Wisconsin- Madison, U.S.A.; (3) University of Illinois- Urbana-Champaign, U.S.A.

*Sclerotinia sclerotiorum*, the causal agent for Sclerotinia stem rot (SSR), is a devastating fungal pathogen in soybean and reduced yield by 59 million bushels in 2009. Incomplete resistance in commercial cultivars can be improved through additional breeding efforts and understanding the genetic basis of resistance. SSR-resistant soybean germplasm was developed by crossing two sources of resistance, W04-1002 and AxN-1-55, with lines exhibiting resistance to other pathogens such as *Heterodera glycines*. After multiple screenings, 31 lines were selected for advanced SSR in field screens in 2014 and were down-selected to 16 lines with a high-level of multi-disease resistance in 2015. Germplasm lines 91-38, 51-23, and SSR51-70 exhibited SSR resistance with minimal lodging. Line 91-38 yielded 2605.5 kg/ha, 51-23 yielded 3156.2 kg/ha, and SSR51-70 yielded 2801.8 kg/ha. The highest yield achieved was 3748.8 kg/ha for 52-82B. While many of these experimental lines have excellent physiological resistance, yield and agronomic performance of these lines are not at the level of an elite commercial cultivar. For example, 91-38 yielded over 1000 kg/ha less than the best line in the trial, however it exhibited one of the lowest disease severity rankings. Germplasm lines such as 91-38, 51-23, SSR51-70, or 52-82B could be improved through further breeding to generate high-yielding commercial soybean cultivars with a high level of physiological resistance to SSR.

Exploring the slow rusting potential of wheat genotypes against leaf and stripe rust of wheat

M. RAZA (1), M. Khan (2), M. Yaseen (3), F. Nutter (4)

(1) Department of Plant Pathology and Microbiology, Iowa State University of Science and Technology, Ames, IA, U.S.A.; (2) Department of Plant Pathology, University of Agriculture Faisalabad, Pakistan; (3) Department of Math & Stat, University of Agriculture Faisalabad, Faisalabad, Pakistan;

(4) Department of Plant Pathology and Microbiology, Iowa State University of Science and Technology, Ames, IA, U.S.A.

The focus of current wheat research is to achieve durable rust resistance in Pakistan. We explored slow leaf and stripe rusting potential in 37 wheat genotypes by studying different slow rusting parameters, e.g., average coefficient of infection (ACI), latent period, pustule size, pustule density, infection
rate (IR), area under disease progress curve (AUDPC) and yield assessment (1000 grain weight and yield/ha). Multivariate statistical analysis techniques (Biplots and Heatmaps) were used to analyze and visualize the slow rusting parameters for identification of slow rusting genotypes. Genotypes including Lasani-2008, Chris, Crow, AS-2002, Punjab-2011, Frontana and FSD-85 demonstrated lower ACI, pustule density, IR, AUDPC, small pustule size, longer latent period, and higher 1000 grain weight and yield/ha against leaf rust. Genotypes encompassing Chris, AS-2002, Punjab-85, Yecora and Lasani-2008 revealed lower ACI, pustule density, IR, AUDPC, small pustule size, longer latent period, and higher 1000 grain weight and yield/ha against stripe rust. Combined analysis recognized genotypes including Uqab-2000, Yecora, Lasani-2008, Chris, Crow, Frontana and AS-2002 as slow leaf and stripe rusters. Hence, these slow rusters can be cultivated in fields for sustainable wheat production, and can be included in further breeding programs to produce durable rust resistant varieties.

## Resistance to *Claviceps purpurea* (Fr.) Tul. in the winter wheat lines “Robigus” and “Solstice”

J. MENZIES (1), A. Gordon (2), D. O'Sullivan (3)

(1) Agriculture and Agri-Food Canada, Canada; (2) National Institute of Agricultural Botany, United Kingdom; (3) University of Reading, United Kingdom

Ergot, caused by *Claviceps purpurea*, is an important disease of cereals which can result in extensive financial losses to producers because of the presence of sclerotia containing toxic alkaloids in the grain. The severity of ergot in Canada and parts of Europe has increased over the past 20 years, and the development of wheat lines with resistance is being explored. The objective of this work was to determine if the winter wheat varieties "Robigus" and "Solstice" possess physiologic resistance to *C. purpurea*. Two winter wheat lines ("Robigus" and "Solstice"), two spring wheat lines ("Paragon" and "Cadillac") and one durum wheat line ("Avonlea") were inoculated with 5 Canadian and 4 UK isolates of *C. purpurea*. Nine spikes per wheat line were needle inoculated prior to anthesis for each pathogen isolate using a conidial suspension. Each spike was assessed for honeydew production 14 days after inoculation using a scale of 1 to 4; 1 = no honeydew to 4 = large drops running down the spike. The weight and number of sclerotia per spike were determined at plant maturity. "Robigus" had significantly lower honeydew production than "Solstice", which had lower production than the other three lines. "Robigus" and "Solstice" also had significantly lower sclerotial weight and number per spike than the other three lines. "Robigus" and "Solstice" showed resistance to infection by *C. purpurea* compared to the other three wheat lines.

## Monitoring of Leaf rust in seventeen Egyptian wheat cultivars during 2013/2014 and 2014/2015 seasons

A. ABDELRHIM (1), M. Abou-Zeid (2), H. Abd-Alla (3), C. Cowger (4), A. El-Bana (3), M. Ismail (3)

(1) North Carolina State University, Raliegh, NC, U.S.A.; (2) Agriculture research center, Giza, Egypt; (3) Minia University, El-Minya, Egypt; (4) North Carolina State University, Raleigh, NC, U.S.A.

Wheat cultivars' response to leaf rust caused by *Puccinia triticina* Eriks. vary between seasons; some cultivars lose resistance over time due to factors such as appearance of new pathogen races that have new virulence genes which overcome Lr-genes. This study monitored leaf rust development for adult plant stage of 17 Egyptian wheat cultivars, expressed in terms of infection type, final rust severity, area under disease progress curve and rate of disease increase (r- value) during 2013/2014 and 2014/2015 growing season in Sids research stations, Egypt. According to the results, only two varieties (Sakha94 and Misr1) exhibited adult plant resistance, three varieties (Gemmiza9, and 10, and Misr2) were moderately susceptible, and twelve varieties (Sakha93, Giza168, and 171, Gemmiza7, and 11, Sids1, 12, and 13, Shandwile1, and Bani-Swifl, 5, and 6) were susceptible during 2013-2014 and 2014-2015 growing seasons. However, nine varieties (Sakha94, Misr1, and 2, Giza 168, and 171, Gemmiza 9, and 10, Sids 12 and 13) recorded low AUDPC up to 310 and the rest varied between 320:1120. Only five varieties (Sakha94, Misr1, Giza 168, and 171, and Gemmiza 9) recorded r-value up to 0.092, while the rest ranged from 0.11 to 0.195 in both seasons. These data may help researchers and breeders by shedding light on the response of some Egyptian varieties to leaf rust in the last two growing seasons, and helping track the virulence genes in P. triticina during the same seasons.

## Scab susceptibility of a provenance collection of pecan

C. BOCK (1), L. Grauke (2), P. Conner (3), S. Burrell (1), M. Hotchkiss (1), D. Boykin (4), B. Wood (1)

(1) USDA/ARS SE Fruit & Tree Nut Research Laboratory, Byron, GA, U.S.A.; (2) USDA/ARS Pecan Breeding and Genetics, Somerville, TX, U.S.A.;

(3) University of Georgia, Tifton, GA, U.S.A.; (4) USDA-ARS, Stoneville, MS, U.S.A.

Scab (caused by *Fusarium effusum*) is the most economically destructive disease of pecan in the Southeast US. Epidemics are favored by rainfall and high humidity. A provenance collection of ~950 pecan trees from 19 locations representing the native range of the species is located in Byron, Georgia, and was assessed for pecan scab severity in 1998, 2013 and 2014. Foliar scab severity was estimated visually. There were consistent significant differences among the 19 provenances (F = 5.6 to 62.5, P<0.0001), with those from wetter locations (generally north of Texas) with the greatest proportion of scab resistant trees, while those provenances from the drier southern areas (Texas and Mexico) tended to be the most susceptible. However, within each provenance there was a range in susceptibility of trees. An association of provenance susceptibility with precipitation at the provenance source location bore out the relationship (r=-0.625 to -0.823 (P<0.0001 to 0.004)). Estimates of heritability were not entirely consistent among years, but different methods were used to assess scab severity in 1998 (a 1-5 category scale) compared to 2013 and 2014 (the percent ratio scale). Despite using different methods, there was generally good agreement among years in regard to severity of disease on individual trees. In conclusion, trees from more northern populations (in areas with greater annual rainfall) are most likely to provide valuable and diverse sources of resistance to scab.

## Effectiveness of a seed plate assay evaluating charcoal rot resistance in soybean

M. DA SILVA (1), P. Chen (1), J. Rupe (1)

(1) University of Arkansas, U.S.A.

Charcoal rot (*Macrophomina phaseolina*) of soybean (*Glycine max* (L.) is a disease of economic significance in the United States. While there is moderate host resistance in soybean cultivars, identifying and quantifying that resistance are difficult. Current assay methods, such as cut-stem inoculation and colony-forming unit index (CFUI), can be time consuming, and data may vary between tests. The objective of this research was to compare results of a seed plate assay to published results of the cut-stem and CFUI assays. Ten surface disinfested seeds of eight differential soybean genotypes, including LS94-3207, LS98-0358 and Pharaoh, susceptible and DT97-4290, DT98-7553, DT99-16864, DT99-17554 and DT99-17483 as resistant, were placed on water agar plates that had been inoculated with an isolate of *M. phaseolina* five days before. Plates were incubated at room temperature in the dark for 7 days and the number of seeds that germinated were determined. Noninoculated plates served as controls. In general, germination results from the plate assay were in agreement with the cut-stem and CFUI assays. Germination of resistance lines range from 58% to 84% and susceptible lines from 46% to 54%. Addition data will be presented later.

## Screening soybean germplasm for resistance to multiple *Fusarium* species

P. OKELLO (1), L. Hyronimus (1), A. Weber (1), A. Singh (2), F. Mathew (1)

(1) South Dakota State University, U.S.A.; (2) Iowa State University, U.S.A.

Fusarium root rot caused by multiple *Fusarium* spp. continue to cause increased soybean [Glycine max L.] yield losses in the United States. Commercial soybean varieties with complete resistance to *Fusarium* spp. are not available, thus necessitating the need for continued search for sources of resistance. The objective of this study was to identify screen soybean germplasm for resistance to four *Fusarium* species (*F. proliferatum*, *F. graminearum*, *F. sporotrichioides*, and *F. subglutinans*), which were identified as aggressive *Fusarium* spp. based on a preliminary study on soybean in South Dakota. To evaluate soybean germplasm for resistance to the four *Fusarium* spp., 44 genotypes belonging to maturity groups I from 10 different countries were