screened in replicated tests using the inoculum-layer method. Disease severity was evaluated at 14 days after inoculation as a percentage of lesion produced on soybean roots by the causal pathogen. Preliminary results suggests one genotype (PI 603371) had significantly lower disease severity (P ≤ 0.05) than the susceptible check for all the four *Fusarium* species. This genotype will be useful as a parental material to develop commercial soybean cultivars with resistance to *Fusarium* species.

## Development of an *in vitro* bioassay to screen perennial Phlox germplasm for susceptibility to *Golovinomyces cichoracearum* (*Erysiphe cichoracearum*)

C. FARINAS (1), P. Jourdan (1), F. Peduto Hand (1)

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

Phlox is a genus of perennial and annual plants with colorful, long-lasting flowers, and a common inhabitant of gardens worldwide. In humid and warm areas, its cultivation is severely limited by powdery mildew. Published studies on resistance of Phlox to powdery mildew have mainly focused on the species *subulata*, *paniculata* and *drummondii*. However, the genus includes many more species, some of which could be valuable from both an ornamental and resistance standpoint. This project aims to develop a reliable laboratory bioassay to conduct *in vitro* screenings of Phlox germplasm for susceptibility to powdery mildew. The project is in collaboration with the OSU Ornamental Plant Germplasm Center, which allows access to more than 65 species of Phlox. For this purpose, detached leaves of healthy Phlox were disinfected and plated on water-agar medium. Variations of the host species, media type, disinfection process, and inoculation technique were evaluated. Disinfected leaf tissues remained in good status and contaminant-free for up to one month. Infection and growth of the pathogen on the host required specific conditions of light, temperature and relative humidity. To the best of our knowledge, this study represents the first attempt to develop a laboratory bioassay to screen Phlox resistance to powdery mildew. This assay has the potential to represent a useful tool for screening resistant lines and for phytopathological studies on the biology of the pathogen.

## Virulence of *Calonectria pseudonaviculata* and *C. henricotiae* isolates and screening for resistant boxwood cultivars in detached leaf assays

H. Guo (1), J. Crouch (2), M. Pooler (3)

(1) USDA-ARS and Rutgers University, U.S.A.; (2) USDA-ARS Systematic Mycology and Microbiology Laboratory, U.S.A.; (3) USDA-ARS US National Arboretum, U.S.A.

Boxwood blight caused by the two *Calonectria* species, *C. pseudonaviculata* and *C. henricotiae*, is a recently emerged threat to the annual $\$1.3 million$ boxwood industry. While fungicidal control can be effective, breeding resistant or tolerant cultivars is the preferred long-term strategy to control the disease in the landscape. Developing an effective assay to quickly screen potentially thousands of boxwood genotypes is an essential first step in such a breeding program. Therefore, this study was conducted to evaluate the virulence of different *Calonectria* isolates on a susceptible boxwood host. The virulence of 24 isolates was determined by inoculating detached leaves of *Buxus sempervirens* 'Suffruticosa' using two different inoculation techniques. All 24 isolates were virulent on 'Suffruticosa', but the virulence varied among isolates. The isolate NCBB-1 was the most virulent, and was used to compare relative resistance among eight diverse boxwood taxa. Preliminary results indicate that two boxwood taxa, *B. harlandii* and *B. sinica* var. *insularis* 'Nana', are the most tolerant to this isolate in detached leaf assays.

## Evaluating nine boxwood cultivars for susceptibility to *Calonectria pseudonaviculata* and *C. henricotiae*

N. SHISHKOFF (1)

(1) Foreign Disease Weed Science Research Unit/ARS/USDA, U.S.A.

Boxwood blight is caused by two species of *Calonectria*: *C. pseudonaviculata* (Cps) and *C. henricotiae* (Che). In North America, only Cps is known to occur, but the 2nd species is of concern since there is evidence that it differs in its temperature optimum, pesticide sensitivity and tolerance to sanitizers. US boxwood cultivars are currently being evaluated for tolerance to Cps and it would be useful to know if their reaction to Che is similar. Based on a previous assessment of susceptibility of 42 boxwood cultivars to Cps, nine were selected on a spectrum from highly susceptible to relatively tolerant. Cultivar sets were inoculated with spore suspensions (mixtures of 4 representative isolates) of each species and the experiment was repeated three times. While results showed some differences in cultivar behavior from trial to trial, there were cultivars that were consistently susceptible ('Dee Runk', 'Vardar Valley') or consistently tolerant ('SB17', 'Little Missy') and the interaction 'pathogen type'*'cultivar' was not significant. This suggests that breeding for tolerance for one species will confer tolerance for the other.

## Susceptibility of *Buxus* accessions to the boxwood blight pathogen *Calonectria pseudonaviculata*

J. LAMONDIA (1)

(1) The Connecticut Agricultural Experiment Station, U.S.A.

Forty *Buxus* accessions from the National Boxwood Collection of the U.S. National Arboretum provided as potted plants were evaluated for susceptibility to boxwood blight caused by *Calonectria pseudonaviculata* (Cps). Two experiments were conducted with 3 to 5 replicate plants each. Plants were inoculated with $1.5 \times 10^5$ Cps conidia in 2 ml coarse spray per plant and grown under high humidity with overhead irrigation. Leaf and stem lesions were recorded 3 or 4 wks after inoculation. *B. harlandii* (1 accession), *B. sinica* (3), *B. microphylla* (7) and *B. bodinieri* (1) had fewer lesions per plant than *B. microphylla* × *sempervirens* (6), which had fewer lesions than *B. sempervirens* (20 accessions). *B. wallichiana* (1 accession) was the most susceptible species (>160 lesions). Variation within species was also observed. *B. sinica* var. *aemulans* (accession 60705\*H), *B. sempervirens* (36365\*J) and *B. harlandii* (18834\*H) were the least susceptible with <10 lesions per plant. *B. sempervirens* 'Scupi' (9548\*H), *B. microphylla* 'Compacta' (4899\*CH), *B. sempervirens* 'Arborescens' (57953\*H), *B. sinica* var. *insularis* 'Pincushion' (51898\*H) and *B. microphylla* var. *japonica* 'Jim Stauffer' (72213\*H) each had < 20 lesions. These data differ from previous detached leaf and unrooted cutting assays. Plant size may affect inoculation and disease, normalizing did not result in significant differences in susceptibility ranking. These results may aid in the development of boxwood resistance to *Cps*.

## Identification of *Brassica napus* plant introductions with resistance to pathogenicity group 4 of *Leptosphaeria maculans*

S. MANSOURIPOUR (1), L. del Rio Mendoza (1)

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

Blackleg caused by *Leptosphaeria maculans* (Desmaz) Ces. & de Not is a serious disease of canola (*Brassica napus* L.) worldwide. In North Dakota, *L. maculans* isolates that overcome resistance genes *Rlm1*, *Rlm2*, and *Rlm3* are the most prevalent and have been classified in pathogenicity group (PG) 4 using a set of three differentials. The objective of this study was to identify sources of resistance against PG-4. Replicated greenhouse trials using mixtures of five PG-4 isolates were used to evaluate the reaction of *B. napus* plant introductions (PI) at the seedling stage. Twenty one PI materials identified as resistant were evaluated in field trials in 2014 and 2015 using a randomized complete block design with four replications. Cultivar "Westar" and two commercial canola hybrids were used as controls. Seedlings were inoculated repeatedly with a mixture of spores from the same five isolates used in the greenhouse and supplemented with naturally infested residues. Disease severity was estimated at harvest as percentage of internal stem crown tissue discoloration. Five PIs were considered highly resistant and behaved consistently better (P < 0.05) than the hybrids in both field trials with an average disease severity < 4% compared to 25% of the hybrids. Crosses between these PIs and canola breeding lines have been made to transfer the resistance into advanced germplasm. In addition, efforts to produce doubled haploid mapping populations from these sources are under way.
### Evaluating chemical fingerprinting as a tool to rapidly screen hybrid chestnut for resistance to pathogens

A. CONRAD (1), J. Westbrook (2), T. Zhebentyayeva (3), L. Rodriguez-Saona (4), P. Bonello (4), C. Nelson (5), A. Abbott (1)

(1) University of Kentucky, Lexington, KY, U.S.A.; (2) The American Chestnut Foundation, Asheville, NC, U.S.A.; (3) Clemson University, Clemson, SC, U.S.A.; (4) The Ohio State University, Columbus, OH, U.S.A.; (5) USDA Forest Service, Lexington, KY, U.S.A.

Chestnut blight, caused by the introduced pathogen *Cryphonectria parasitica*, continues to plague American chestnut (*Castanea dentata*) in the eastern US. Restoration is focused on breeding hybrids that morphologically resemble American chestnut but contain blight resistance from Chinese chestnut (*C. mollissima*). Compounding the problem for restoration efforts, *Phytophthora cinnamomi*, an agent of root rot, has emerged as a major pathogen in chestnut's southern range. Time and resource intensive inoculation-based screening methods are used currently to identify chestnuts resistant to these pathogens. Therefore, we evaluated if chemical fingerprinting (CF), a potentially less resource-intensive tool for phenotyping resistant/susceptible trees, can be used to screen chestnut for disease resistance. A Varian 3100 Fourier-transform infrared (FT-IR) spectrometer equipped with an attenuated total reflectance accessory was used to fingerprint methanol extracts from 0-1 year old chestnut stem tissue that differed in susceptibility to chestnut blight. Using Soft Independent Modeling of Class Analogy, chemical differences among disease rating classes were identified and used to develop class discriminating models. Early results suggest that CF may be a useful tool for screening seedlings for blight resistance; however, we are currently testing additional samples to refine, optimize, and validate models for both diseases.

### Screening soybean germplasm for resistance towards Pythium species

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

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

*Pythium* species are a major cause of soybean seedling root rot and seed rot in Ohio. One method of management is the use of resistant cultivars. Previous studies identified a "differential" resistance response to *Pythium ultimum* var. *ultimum* and *P. ultimum* var. *sporangiiferum* among soybean cultivars. The purpose of this experiment was to quantify the resistance level of several germplasm lines and to identify any potential "differential" effect. To do this, several soybean germplasm lines were evaluated for resistance to *Pythium* isolates collected from fields in Ohio using a seed plate assay. Seed plate assay results indicated that Conrad, Dennison, Lorain, Sloan, and Kottman all had varying levels of resistance towards 13 species of *Pythium*; more importantly, there was a highly significant (P<0.0001) cultivar x *Pythium* spp. interaction. All cultivars were very susceptible to *P. ultimum* var. *ultimum*, *P. ultimum* var. *sporangiiferum*, and *P. sylvaticum*. When inoculated with *P. conidiophorum*, *P. attrantheridium*, and *P. dissotocum*, Conrad, Dennison, and Lorain had a lower degree of seed rot than either Sloan or Kottman. These resistant cultivars may be used to provide a higher degree of protection against infection from *Pythium* species in soybean seeds and seedlings.

### Disease susceptibility screening for cold-climate wine grape cultivars

D. JONES (formerly Schreiner) (1), P. McManus (1)

(1) University of Wisconsin, Madison, Dept. of Plant Pathology, U.S.A.

The wine grape industry in the Upper Midwest has expanded rapidly in the past 15 years. Inconsistent reports on disease susceptibility in cold-climate wine grape cultivars may be leading to overspraying and underutilization of host resistance in the region. Inconsistencies in reports may stem from a lack of study on adult plants in a randomized, replicated, and fungicide free vineyard environment. Two vineyards containing eight cultivars were left unsprayed by fungicides in 2015. Severity of downy mildew, powdery mildew, and black rot was measured from bud break until 2 weeks post-harvest. There was a significant difference among cultivars for all three diseases at both sites. A second assay was conducted using potted grape vines as "trap plants" to capture inoculum present in the field and gain further information on disease susceptibility. Using trap plants and artificial humidification did not generate results consistent with the disease severity and progress observed in the field during the growing season. These preliminary data suggest that selection of certain cultivars has the potential to reduce sprays needed to manage diseases during the growing season in the Upper Midwest. Results further indicate differences exist between foliar and fruit susceptibility for all three diseases, a distinction that is not made in current rating systems. This work will continue in 2016 to gain additional susceptibility data and observe differences between growing seasons.

### Evaluation of stem inoculation technique for assessing resistance to *Phytophthora nicotianae* in hydrangea cultivars

M. KABIR (1), T. Simmons (1), P. Liyanapathiranage (1), C. Curry (1), L. Alexander (2), F. Baysal-Gurel (1)

(1) Tennessee State University, Nashville, TN, U.S.A.; (2) USDA-ARS, U.S. National Arboretum, McMinnville, TN, U.S.A.

Almost all hydrangeas, especially container-grown oakleaf hydrangea (*Hydrangea quercifolia*) are more susceptible to *Phytophthora* root rot disease. Most economic and trading hydrangea cultivars are propagated as rooted cuttings. Reliable and rapid stem cutting-based screening technique to evaluate hydrangea cultivars for resistance to *Phytophthora nicotianae* is needed toward disease management. Hydrangea cvs. Queen of Hearts, Ruby Slippers and Munchkin stem cuttings were screened in a laboratory procedure by point inoculation with 5-day-old culture, #1 size (outside diameter 5 mm) plugs of *P. nicotianae*. Each stem cutting was evaluated for lesion area and lesion severity after 3, 5 and 7 days of incubation at 25°C with 16-hr-light/8-hr- dark cycle. The experiment was repeated twice. After comparing the lesion development on three hydrangea cultivars, the results showed that Queen of Hearts is most susceptible to *P. nicotianae* than other cultivars and five days observation is the optimal time for stem cutting-screening technique. This study will facilitate the ongoing large-scale screening for *Phytophthora* root rot resistance among hydrangea cultivars very easily and it is clear that described stem cutting-inoculation technique has the potential to play a significant role for simple, efficient and reproducible screening.

### Cavity spot of carrots: Progress in screening for resistance

M. MCDONALD (1), M. McDonald (2)

(1) University of Guelph, Canada; (2) University of Guelph, Canada

Cavity spot of carrot is caused by several *Pythium* species, especially *Pythium violae* and *P. sulcatum*. Field trials were conducted from 2013 to 2015 to screen 60 carrot lines from the USDA breeding program at the University of Wisconsin lines for differences in susceptibility to cavity spot. Cultivars Atomic Red, Cellobunch and Envy were included as commercial checks. Carrots were seeded into 'muck' soil (pH 5.7-6.5, organic matter 65-78%) where the cavity spot pathogens occur naturally. Carrots were direct seeded (≈75 seeds/m²) in early June, onto raised beds, 66 cm apart and 6 m in length. Fifty carrots per replicate were harvested in late October, placed in cold storage for 6-8 weeks and washed and assessed for cavity spot incidence and disease severity (DSI). Cavity spot incidence and severity were very high in 2013 (97% and 69 DSI) and 2014 (96%, 54 DSI) and lower in 2015 (50%, 26 DSI). A wide range of susceptibility to cavity spot was found each year. Purple lines 6139B, 6244B, 7262B and 3497B were highly resistant (1-8%, 1-7 DSI). Two orange carrot lines, in the trials in 2014 and 2015, had consistently low disease. Line 1137B had 8% cavity spot in both years, with DSI of 7 in 2014 and 4 in 2015. Line 5367 B had 14 and 21% cavity spot in 2014 and 2015, respectively, with a DSI of 8 and 7. Field screening has identified breeding lines with promising levels of resistance to cavity spot.

### Screening of Wild Hop (*Humulus lupulus*) Germplasm for Identification of Resistance to *Pseudoperonospora humuli*

J. HAVILL (1), A. Orshinsky (1), D. Carter (1), S. Poulose (1)

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

*Pseudoperonospora humuli*, the causal organism of hop downy mildew, is considered the most economically-damaging pathogen to hop production. Host resistance, in addition to intensive cultural management practices and fungicide use, is the primary control measure. Wild hops (*Humulus* spp.) are
distributed across the North America and Eurasia. They may provide a resource for novel resistance genes and germplasm diversity for local hop breeding programs. Sixteen sites in Minnesota with wild populations of hops were identified. At 14 of these sites individual rhizomes were recovered from 1–27 unique individuals. One-hundred six wild and cultivated hop genotypes originating from the USA, England, Japan, and Kazakhstan were screened for foliar resistance to downy mildew (*Pseudoperonospora humuli*) using a detached-leaf assay. Each genotype was tested with three replicates. In total, 33 genotypes were as resistant as the current commercial standard (cv. 'Teamaker') which included five other cultivars, five Japanese hop (*H. japonicus*) genotypes, two experimental breeding lines, and 21 wild *H. lupulus* genotypes. We identified two geographically distinct locations in Minnesota containing previously unreported germplasm possessing high levels of foliar resistance that may be useful for breeding locally-adapted varieties. Work is currently underway to characterize the morphological characters and chemical attributes of the hop inflorescences of selected resistant wild plants.

## Barley yellow dwarf incidence and bird cherry-oat aphid preference in four wheat varieties in Idaho

M. Rashidi (1), J. Marshall (1), N. Bosque-Perez (2), A. Rashed (1)

(1) University of Idaho, Aberdeen Research and Extension Center, U.S.A.; (2) University of Idaho, Department of Plant, Soil and Entomological Sciences, U.S.A.

Barley/Cereal yellow dwarf viruses (B/CYDV) are aphid-borne pathogens that are transmitted by several species of cereal aphids, chief among them the bird cherry-oat aphid (BCOA), *Rhopalosiphum padi* (L.). This species has also been linked to the recent consecutive outbreaks of BYDV-PAV, between 2013 and 2015, in southern Idaho. The present study was conducted to evaluate the relative susceptibility of four commonly planted winter wheat varieties in Idaho, namely, SY Ovation, Brundage, Stephens, and WB-Junction, to BYDV and its BCOA vector. In choice experiments, viruliferous aphid preference for the four varieties was assessed by counting the number of aphids per plant 24 hours after infestation. BYDV titer within seedlings was quantified using qPCR, three weeks after inoculation. During a two-year field-cage study, BYDV symptom severity, aphid pressure, and root and shoot biomass were assessed for the four varieties. Root/shoot biomass data are currently being analyzed. BYDV in experimental cages was confirmed using ELISA. Although Stephens and SY Ovation were preferred relatively less frequently than WB-Junction and Brundage by BCOA, no significant differences were detected in aphid preference among the four varieties. Likewise, BYDV incidence in field cages was not statistically different among the four varieties, however SY Ovation had the lowest BYDV incidence, an observation consistent across both years of the study.

## Resistance in pea to the Bean leaf roll virus

L. PORTER (1)

(1) USDA ARS, U.S.A.

*Bean leaf roll virus (BLRV)* is a major luteovirus impacting dry pea production on an annual basis in Idaho and Washington. Symptomatic plants are often chlorotic, stunted and distorted. The virus is transmitted by aphids in a persistent manner and cannot be mechanically transmitted. Host resistance is an important economic and environmentally favorable means to manage this pathogen. Pea aphids were used to phenotype the genetic resistance of 36 pea genotypes, mostly consisting of dry pea cultivars, under controlled greenhouse conditions. Two pea genotypes, Journey and Stirling, were determined to have complete resistance to BLRV. Journey is a commercial forage green dry pea and Stirling is a formerly grown commercial green dry pea. Three commercial pea cultivars, Majoret, Granger and Spector were determined to be highly susceptible to BLRV with a 100% incidence of infection across three tests. Mapping populations will be established between the resistant and susceptible pea genotypes to identify molecular markers that can be used in marker-assisted selection of pea germplasm for genetic resistance to BLRV.

## The SimpleSynteny Server: Genome Comparison Simplified

D. VELTRI (1), M. Malapi-Wight (2), J. Crouch (1)

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

Visualizing syntenic relationships amongst gene clusters is a common task for studying organismal evolution. Here we present SimpleSynteny, a new comparative genomics web server which includes a number of helpful tools for the analysis of microsynteny across multiple genome assemblies. Requiring only FASTA files for genomes and gene targets of interest as input, SimpleSynteny's easy interface helps the user explore and customize individual genomes before combining them into a final high-resolution figure. The approach gives users the freedom to incorporate additional knowledge when editing contigs/scaffolds after computational predictions are made. A range of image formats and customization options are also provided to give additional control when saving results. In this presentation, we provide a quick overview of the tools provided and generate example figures from fungal genomes via a live demonstration of the site. SimpleSynteny stands to be a powerful new tool in a researcher's toolbox of comparative genomics software. The server is free to use and is currently available at: http://www.simplesynteny.com.

## Reducing Mycotoxin Levels in Corn via Lepidopteran Insect Control and Application of Atoxigenic Fungi

G. SCHUSTER (1), T. Mays (2), M. Sétamou (3), P. Porter (4), S. Nelson (5)

(1) Texas A&M University - Kingsville/Texas A&M AgriLife Extension, Kingsville, TX, U.S.A.; (2) Texas A&M AgriLife Extension Service, Brownfield, TX, U.S.A.; (3) Texas A&M University-Kingsville Citrus Center, Weslaco, TX, U.S.A.; (4) Texas A&M AgriLife Extension Service, Lubbock, TX, U.S.A.; (5) Texas A&M University-Kingsville, Kingsville, TX, U.S.A.

Mycotoxins are secondary fungal metabolites that have adverse health effects on animals and humans. Lepidopteran damage to corn is suspected to facilitate fungal colonization and infection to kernels and to increase the risk of mycotoxin production and accumulation in corn. This study was initiated to evaluate the application of AF-36 and Afla-Guard at two growth stages in reducing mycotoxin levels in corn. Treatments included an untreated control, insect control, AF-36 and Afla-Guard applied at the V8 or VT growth stage, and Afla-Guard applied at the V8 or VT growth stage with insect control. The application of AF-36 or Afla-Guard applied at the V8 or VT growth stage did not significantly reduce the concentration of either aflatoxin or fumonisin. In 2013, the insect control significantly reduced the fumonisin concentration, and in 2014 application of insecticide alone or with Afla-Guard applied at the V8 or VT growth stage reduced the concentration of fumonisin. Aflatoxin levels were not significantly reduced by the application of AF-36 or Afla-Guard at either application time or by the insecticide applications. Fumonisin concentrations in both years showed a positive relationship with the amount of ear damage caused by Helicoverpa zea and Spodoptera frugiperda. Aflatoxin concentrations in 2013 were positively correlated to the amount of ear damage, but no such correlation was observed between aflatoxin and insect damage in 2014.

## Deciphering soil and plant microbiomes associated with suppression of soybean diseases

H. PFAFFE (1), S. Marzano (1), L. Domier (1), A. Davis (1), D. Eastburn (1)

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

General disease suppression of soilborne pathogens can add to the toolbox of strategies for disease management. This study was undertaken to identify the microbiome indicators for soil suppressiveness to sudden death syndrome (SDS) of soybeans. Five cover crop treatments (rye, vetch, mustard, rye+vetch, fallow) and two tillage methods (chisel plow, ridge till) were compared. Each cover crop plot was divided to receive both tillage treatments, and there were four replications of this experiment. Soybean plants were evaluated for naturally occurring foliar and root diseases at the R6-R7 growth stage. Root, rhizospheric soil, and bulk soil samples were taken early in the season, and DNA was extracted for PCR amplification, targeting 16s bacteria and archaeal, and ITS genes, for subsequent high throughput sequencing. Our results have shown no significant differences in microbial populations of the cover crop treatments. However, there are several notable differences with the tillage treatments. Chisel plowed plots had increased levels of the
genera, *Verrucomicrobia*, *Glomeromycota*, *Proteobacteria*, and *Actinobacteria*. Ridge tilled plots showed increased levels of *Streptophyta*, *Firmicutes*, and *Crenarchaeota*. These results are being verified with a greenhouse assay to determine if it is possible to delineate microbiome profiles that correspond to general disease suppression.

## Effects of fungicide application timing and cultivar resistance on Fusarium head blight and deoxynivalenol in winter wheat

C. BOLANOS-CARRIEL1 (1), S. Wegulo (1), H. Hallen-Adams (1), D. Funnell-Harris (2), P. Baenziger (1)

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

*Fusarium graminearum* causes Fusarium head blight (FHB) in wheat. FHB reduces yield and quality and contaminates grain with the mycotoxin deoxynivalenol (DON). Effective management strategies are needed. The objectives of this research were to 1) Determine the effect of fungicide application timing at anthesis (the standard timing) and 6 and 12 days later on FHB and DON in the winter wheat cultivars Overley (susceptible) and Overland (moderately resistant) and 2) Compare the effects of a triazole and a strobilurin fungicide on FHB and DON in Overley and Overland. In 2015 two field trials (irrigated and rain-fed) were conducted in Nebraska, USA. The triazole Prosaro (prothioconazole + tebuconazole) and the strobilurin Headline (pyraclostrobin) were applied with a CO₂-powered backpack sprayer at anthesis and 6 and 12 days later. A split plot design in randomized complete blocks with 4 replications was used. Main plots were cultivars and subplots were fungicide treatments. FHB index and DON were significantly (P < 0.05) lower in Overland than in Overley. The window of fungicide application to control FHB and DON was widened from anthesis to 6 days later without loss of efficacy. Headline was less effective than Prosaro in controlling FHB and DON. Moderate resistance combined with a triazole fungicide most effectively reduced FHB and DON. The results indicate a wider fungicide application window and the effectiveness of combining resistance with a triazole fungicide.

## Impact of Agronomic Practices and Diseases on Wheat Profitability

J. SALGADO (1), P. Paul (1), L. Lindsey (1), R. Minyo (1)

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

Changes in nitrogen fertilizer rates, row spacing, and fungicide programs have been proposed as strategies for increasing wheat productivity and profitability in Ohio. The main goal of this study was to evaluate the economic benefit of these practices as influenced by diseases. Field experiments were conducted in 2013, 2014 and 2015 using a split-split-plot design with nitrogen rates (N-rate) as whole-plot, row spacing (19 and 38 cm) as sub-plot, and Prosaro fungicide treatments (TRT) as sub-sub-plot. Combinations of these factors represented separate management programs (PROG), and an economic analysis was conducted using yield (YLD), test weight (TW), grain prices and price discounts due to poor grain quality, as well as fertilizer, seed and fungicide costs to estimate net cash income for each PROG. Linear mixed model analyses showed that leaf rust and Fusarium head blight (FHB) were higher and YLD and TW lower in wide than in narrow rows. Leaf rust, YLD, and TW increased with increasing N-rates, but these effects varied with TRT. FHB severely affected the economic benefit of the different PROG. Compared to the standard PROG used in Ohio (100.9 kg N ha⁻¹, narrow rows and untreated), several of the modified PROGs only resulted in greater economic benefit ($3 to 215 ha⁻¹) when FHB levels were low. PROGs with narrow rows plus high N rates (> 134 kg N ha⁻¹) and a Prosaro treatment were among the most profitable, while wide-row PROGs were among the least profitable.

## Effect of cropping rotation and input level over 18 years on the northern Great Plains

B. GOSSEN (1), K. Bassendowski (1), E. Johnson (2), R. Lemke (1)

(1) Agriculture and Agri-Food Canada, Canada; (2) University of Saskatchewan, Canada

A cropping rotation study was conducted from 1994-2012 (three 6-yr cycles) at the Agriculture and Agri-Food Canada Research Farm at Scott, Saskatchewan, Canada in a split-split-plot design with four replicates. The main plots were three levels of inputs; high (HIGH), reduced (RED), and organic (ORG) The subplots were three levels of cropping diversity (rotations); fallow-annual grains (LOW), diversified annual grains (DAG), and diversified annuals and perennials (DAP). The sub-sub plots (each 13 × 40 m) were phases of the crop rotation. Foliar disease assessments were made shortly before crop maturity each year. Across treatments and years, there was no consistent pattern of response of final disease severity to treatment. For example, level of input and cropping diversity did not affect severity on the penultimate or flag leaf of wheat in 2007?2013. In those years where there were differences in severity, severity was often highest in the high-input treatments, but organic rotations were highest in some years. However, foliar disease severity differed substantially among years. This study demonstrated that weather conditions on the northern Great Plains had a much larger impact on foliar disease severity than input levels or cropping rotation in the moderate to highly diverse rotations assessed in this 18-year study.

## Effect of root pathogens *Bipolaris sorokiniana* and *Fusarium graminearum* on germination and seedling blight in wheat in South Dakota

S. ALI (1), N. Kaur (1), S. Sehgal (1), K. Glover (1), S. Kumar (1)

(1) South Dakota State University, Brookings, SD, U.S.A.

*Bipolaris sorokiniana* (Bs) and *Fusarium graminearum* (Fg) are important wheat root pathogens that effect seed germination and seedlings establishment thus ultimately impact the crop productivity. The objective of this study was to explore the effect of Bs and Fg infested seed on germination and seedlings establishment (blight) of 7 wheat cultivars, Advance, Briggs, Forefront, Oxen, Russ, Prevail, and SD4215 which were planted at Volga, SD in 2015. The treatments included, uninfested + untreated, uninfested + treated with fungicide, infested (Bs) + treated, infested (Bs) + untreated, infested (Fg) + treated, infested (Fg) + untreated. Seed germination and seedling blight data was recorded after the germination for 3 consecutive weeks. Wheat cultivars varied in seed germination and seedling blight to both pathogens; low seed germination (ranging from 35-71%) and high seedling blight (30-65%) was observed in Fg infested seed, whereas, in Bs infested seed the germination ranged 58-100% and seedling blight ranged 0-42%. The cultivars Oxen and Prevail had the lowest (33%) and the highest (71%) seed germination respectively. Also the treatments including the fungicide significantly increased the germination (ranging from 5-44%) and the seedling blight got reduced in almost all the cultivars.

## Evaluation of management programs of yellowing and wilting of blackberry (*Rubus* sp.) caused by *Fusarium oxysporum* in Michoacan Mexico

A. REBOLLAR-ALVITER (1), A. Hernandez-Cruz (2), A. Saldivia-Tejeda (3)

(1) Universidad Autonoma Chapingo, Mexico; (2) Instituto Tecnológico del Valle de Morelia, Mexico; (3) Universidad Autonoma Chapingo, Posgrado en Protección Vegetal, Mexico

Yellowing and wilting of blackberry (*Rubus* sp.), caused by *F. oxysporum* is one of the most devastating diseases in Michoacan, Mexico. The objective of this research was to evaluate different disease management programs using biological control agents (BCA), plant activators, and fungicides. The experiment was established in 2014 and 2015 in a plantation of blackberry cv. Tupy. A randomized complete block design with 4 blocks and 13 treatments and a control were used. Treatments consisted of sequential applications of fungicides (prochloraz, difenoconazole, thiabendazole, azoxystrobin and methyl thiophanate), plant activators (acibenzolar-S-methyl, potassium phosphite, oligosaccharin-glutathione), BCA (*Trichoderma harzianum*, *Streptomyces lydicus* strain WYEC 108) and microbial mixtures (Maya Magic® and Microsoil®). Area Under Disease Progress (AUDPC) of incidence, severity and plant mortality were assessed. There were significant differences between treatments. Treatments with the lowest AUDPC were the mixtures of microorganisms (Maya Magic®) without significant differences with treatments beginning with 2 to 3 applications of prochloraz or difenoconazole followed by Glutathione-oligosaccharins, and azoxystrobin, followed by acibenzolar S methyl applications. These results indicate that it is feasible to significantly reduce the severity and mortality in *F. oxysporum*-infected blackberry plantings, if disease management programs begin early in the growing season.
**Response of grafted seedless watermelon to root regeneration and inoculation with *Fusarium oxysporum* f. sp. *niveum***

Q. Liu (1), X. Zhao (1), T. Sanchez (1), Z. Black (1), N. Dufault (1), Q. Liu (1)

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

Grafting can be a successful tool in an integrated approach to manage soilborne diseases such as Fusarium wilt of watermelon caused by *Fusarium oxysporum* f. sp. *niveum* (FON). In cucurbit grafting, excision and regeneration of grafted seedling roots is often practiced by nurseries. The objectives of this greenhouse experiment were to assess the performance of grafted seedless watermelon plants under inoculation with FON, and compare grafted plants with original rootstock roots and with root regeneration. ‘Melody’ watermelon was grafted onto two squash rootstocks including ‘Marvel’ and ‘Super Shintosa’. Various plant growth parameters were measured. All the plants were plated on PDA to confirm the presence of FON colonies 32 days after transplanting. Non-grafted watermelon plants showed a significantly (P ≤ 0.05) higher percentage of recovered FON colonies (75%) compared to grafted treatments (0-8%). Plant growth measurements indicated less vigorous growth for non-grafted plants when compared to the grafted treatments. Root regeneration treatments had a numerically higher percentage of FON colonies (0-8%) compared with the regular grafting treatments (0-3%), while no differences were observed in plant growth measurements. These results showed that grafting can effectively prevent FON colonization in seedless watermelon, and supported the use of this practice as part of a watermelon production integrated management program.

**Novel approaches for the integrated control of the soilborne strawberry pathogens *Macrophomina phaseolina* and *Fusarium oxysporum* f. sp. *fragariae***

M. Carter (1), H. Smith (2), G. Holmes (3), K. Ivors (1)

(1) California Polytechnic State University, U.S.A.; (2) California Polytechnic State University, Statistics, U.S.A.; (3) California Polytechnic State University Strawberry Center, U.S.A.

*Macrophomina phaseolina* (Mp) and *Fusarium oxysporum* f. sp. *fragariae* (Fof) are emerging soilborne pathogens causing crown rot and Fusarium wilt in commercial strawberry fields in California. Fungicides representing eight active ingredients from four different FRAC groups were evaluated for their efficacy against each pathogen in greenhouse bioassays. Twenty-four fungicide treatments (eight fungicides at three rates) were drench applied to pathogen infested potting media two days prior to planting and 19 days after planting. Tea bags of buried inoculum were recovered two days after each fungicide application and plated on semi-selective medium to quantify each pathogen; disease severity was assessed weekly. Differences in the colony forming units (CFUs) and disease severity (AUDPC) for all treatments and the inoculated water-drench control for both Mp and Fof were not statistically significant, although there were differences among the CFUs for Mp. In addition, a strawberry plant extract was assessed for its ability to stimulate germination of Mp microsclerotia *in vitro*. Preliminary results showed that the number of germinating microsclerotia was significantly higher after the application of the strawberry extract. Germinated microsclerotia may be more sensitive to fungicides and offer a means of increasing fungicide efficacy against soilborne pathogens.

**Correlating the Effects of Glyphosate on Soybean Nutrition with Cercospora Leaf Blight and Septoria Brown Spot**

T. GARCIA AROCA (1), E. Silva (1), B. Ward (1), C. Robertson (1), P. Price (1), R. Schneider (1), R. Levy (2)

(1) Louisiana State University, Baton Rouge, LA, U.S.A.; (2) Louisiana State University, Alexandria, LA, U.S.A.

Glyphosate is linked to detrimental effects on soil microbiota and reduced levels of metal cations available for plant uptake. There is no agreement in literature regarding effects of glyphosate on plant diseases or micronutrient uptake. Some authors claim the use of glyphosate in Roundup Ready (glyphosate-resistant) soybeans leads to increased disease severity due to negative effects on uptake of manganese (Mn), iron (Fe) and other micronutrients, while others dispute these findings. This controversy is relevant to findings made by our laboratory regarding tissue concentrations of certain micronutrients, e.g. Mn and Fe, which substantially affect Cercospora leaf blight (CLB) in soybean. Therefore, we tested the hypothesis that glyphosate is affecting disease severity in soybean. Twelve Roundup Ready soybean varieties were planted at two locations in Louisiana in randomized complete block designs. Plots were either not sprayed or sprayed with Roundup. CLB and Septoria brown spot disease assessments were conducted in 2014 and 2015. Samples were taken for micronutrient analyses and real-time quantitative PCR (qPCR) for the CLB pathogen. Results showed significant increases of some elements, e. g. N and Mn, for Roundup-treated samples. CLB and Septoria brown spot severities were higher in non-treated plots. Results from qPCR analyses indicated more pathogen DNA (biomass) in Roundup-treated samples, however, these findings were not correlated with disease severity.

**Decision models for fungicide applications for frogeye leaf spot in soybean**

H. Kelly (1)

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

Frogeye leaf spot (FLS), caused by Cercospora sojina, is a common foliar pathogen of soybean in the United States and other soybean production areas in the world. While foliar fungicides can reduced disease severity and yield loss caused by FLS on susceptible cultivars, the appropriate application timing is essential for optimal fungicide efficacy and yield protection. To better manage FLS epidemics in Tennessee, fungicide application decision models were developed and evaluated across locations in 3 different counties in 2013 and 2014. Application models were developed based on growth stage of soybean and rain/irrigation events or temperature and relative humidity parameters. The number and timing of fungicide applications varied by location, year, and application model. All applications reduced disease severity, but only applications under moderate to severe disease pressure were necessary to protect yield. Results indicate models for fungicide application timing for FLS management need to incorporate additional disease parameters to more accurately trigger fungicide applications based on environmental factors and soybean growth stage.

**Controlling Asian Soybean Rust and Phytophthora Late Blight by Biologicals Alone and in Combination with Fungicides**

M. TWIZEYIMANA (1), E. Gachango (1), B. Espejo (1), K. Craig (1), P. Hammer (1)

(1) AgBiome, Inc., Research Triangle Park, NC, U.S.A.

Tremendous losses are associated with Asian soybean rust (*Phakopsora pachyrhizi*) and Phytophthora late blight (*Phytophthora infestans*) yearly. At AgBiome, isolates from our large collection of proprietary environmental microbes are screened in high-throughput assays against a broad range of fungal pathogens, insect pests, and plant-parasitic nematodes. Genome sequences from these isolates are analyzed for known families of proteins active against plant pests, and these proteins are assayed in bacterial expression systems and transgenic crops. Successful biological and trait candidates are tested, and confirmed active biologicals proceed to the fermentation and formulation. Nearly 1200 and 500 isolates have been screened against *P. pachyrhizi* and *P. infestans*, respectively. For both pathogens, the initial screening is done using leaf disks and hits are confirmed on whole plants in growth chambers. Selected leads undergo mouse toxicity testing and scale up by fermentation and formulation before they are evaluated in greenhouse and field. From all isolates screened for the control of Asian soybean rust, seven were selected and were evaluated in the greenhouse in Florida and Illinois and in the field in Florida and Argentina. Additional selected leads with activity against *P. infestans* will be sent to the field this year.

**Proactive Strategy for Management of Seedborne Pathogens of Pulse Crops in Montana**

B. AGINDOTAN (1), M. BURROWS (1)

(1) Montana State University, U.S.A.
Pulse crops (chickpea, lentil, and field pea) are grown for their health and nutritional benefits. In 2015, Montana led the U.S. in field pea and lentil production at 48% acreage. The pulse crop acreage projection by 2019 is 1.4 mil acres. Growers in Montana are aware of the fungal disease Ascochyta/Mycospharella blight and have been testing their seeds exclusively for this disease complex. Due to limited seed availability, seed is coming into the state from other long-term pulse producing areas with pathogens. To determine the prevalence of seedborne fungi in pulse crops from the 2015 growing season, seed samples of field pea (256), lentil (170), and chickpea (35) were tested by plating 400-600 seeds per sample on potato dextrose agar and incubating at 20°C (12h light: dark) for 10-14 d. Fungi were identified to genus level using microscopy and DNA sequencing. The most prevalent fungi isolated were *Alternaria* (71 - 83%), *Cladosporium* (53 - 67%), *Ascochyta* (18-56%), *Penicillium* (46 - 57%), *Rhizopus* (20-40%), and *Nigospora* (6-20%; 1st report). Others were *Fusarium* (14- 17%), *Aureobasidium* (7.4-11%; 1st report), *Botrytis* (8-14%), *Stemphylium* (9 - 16%), *Collectotrichum* (1-9%), and *Diaporthe* spp. (4-14%; 1st report). Growers can manage these fungal diseases if they know the health status of their seedlots, use seed treatment fungicides, and prevent planting seed lots infested with pathogens with long-lived survival structures on organic and conventionally managed acres.

## Production practices impact yield parameters and disease severity on sweet and forage sorghum grown as biomass feedstock

A. HAGAN (1), S. Scott (1), K. Bowen (1)

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

Sweet and forage sorghum are potential feedstocks for energy and chemical industries but the impact of production practices on diseases is largely unknown. Planting date (PD), nitrogen (N) rate, and variety impact were assessed in 2011 and 2012 on plant populations, above ground dry matter (ADM) yield, total fermentable sugar concentration (°Bx), and anthracnose severity. A factorial with year as the main plot, PD as the split-plot, N rate as the split split-plot, and sweet and forage sorghum variety as the split split split-plot was used. Disease assessment and harvest was at soft dough. Plant population was not impacted by any variable. ADM yield segregated by year, PD, and variety. Generally, similar ADM yield was noted at all N rates. M81-E sweet sorghum ADM yield trended higher than forage sorghum. °Bx varied by year, PD, and variety with values declining at the later PD. M81-E had a higher °Bx than either forage sorghum with 1990 having a higher °Bx in 2011 than SS405 in 2012. Anthracnose differed by year, PD, N rate and variety where disease severity was highest in 2012, when sorghum followed sorghum, and summer weather was wetter than 2011. In both years, M81-E had less anthracnose damage than either forage sorghum with SS405 having a higher disease rating in 2012 than 1990 in 2011 due to rotation and drier summer in latter year. Higher °Bx and anthracnose ratings were seen at 88 than 22 kg N ha⁻¹. Anthracnose ratings were lower at the April than June PD.

## Anthracnose impacts biomass and sugar yield of forage but not sweet sorghum

A. HAGAN (1), K. Bowen (1), H. Miller (1)

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

Impact of anthracnose on aboveground dry matter (ADM), estimated sugar yield (SY), and total fermentable sugar concentration (°Bx) of energy feedstock forage and sweet sorghum is undefined. A factorial study with irrigated SS405 forage sorghum and Topper 76-6 (2014) or M81-E (2015) sweet sorghums as whole plots, and 1, 2, 3, 5, 6, or 7 (2014 only) broadcast applications of Headline (12 fl oz A⁻¹) at 28, 35, 42, 49, 56, 64, and 72 DAP [days after planting] as the split-plot treatment was used. A non-fungicide treated control was included. Anthracnose intensity was recorded at 7 to 14 d intervals from 30 DAP until harvest at soft dough; areas under the disease progress curves (AUDPC) for anthracnose were calculated from these ratings. On SS405, anthracnose intensified from onset at 30 to 45 DAP through harvest at 111 DAP when > 75% leaf area loss was noted for all but the 6- or 7- application programs. While Topper 76-6 was anthracnose-free, this disease intensified on M81-E from onset at 90 DAP until harvest at 133 DAP when > 75% leaf area loss was observed for all Headline programs and the non-fungicide treated control. In 2015, anthracnose AUDPC increased as Headline application number declined on SS405 but not M81-E. AUDPC was regressed against ADM, SY, and °Bx. In both years, ADM, SY, and °Bx declined by up to 30% as anthracnose intensified on SS405. Headline application number did not impact ADM, SY, and °Bx of the disease-free Topper 76-6.

## Managing Rhizoctonia crown and root rot of sugar beets utilizing azoxystrobin-alternative fungicides in Nebraska

R. HARVESON (1), R. Harveson (2)

(1) University of Nebraska Panhandle Research & Extension Center, U.S.A.; (2) University of Nebraska Panhandle REC, U.S.A.

In Nebraska, Rhizoctonia root and crown rot, caused by *Rhizoctonia solani*, is generally considered to be the most commonly occurring and damaging sugar beet disease. Previous studies have demonstrated that applications of azoxystrobin will effectively reduce disease and improve yield parameters. However, the almost exclusive use of Quadris (azoxystrobin) for more than a decade raises concern for resistance development by the pathogen to this fungicide. Studies were conducted from 2013-2015 to evaluate the performance of alternative fungicides currently registered for this disease in sugar beets (Priaxor, Proline, Headline, and Vertisan) and compare their performance to Quadris. The study consisted of 16 treatments: 1) untreated control, 2-6) applications applied in-furrow at planting, 6-10) foliar applications after 4 inch soil temperatures averaged 65° F for three consecutive days, and 11-15) both in-furrow and foliar applications. Data collected included multiple disease counts during the season, and sucrose and root yield determinations at harvest. Our results indicated that Quadris, Priaxor, and Proline all performed similarly, significantly reducing disease incidence and increasing sugar yields compared with untreated controls. We further determined that better results were obtained with the combination of in-furrow and foliar applications later in the season than either treatment alone.

## Monitoring pomegranate pathogens towards developing effective disease management program

A. KC (1), G. Vallad (1)

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

Pomegranate (*Punica granatum* L.) is becoming a popular alternative crop in the Southeastern United States due to its nutritional benefit and emerging production potential in the area. Studies were conducted in 2015 to monitor pathogens prevalence from bud break to fruit maturity. Buds were collected from late January through March from three locations in Florida, two in Georgia, and one in South Carolina and were divided into six different stages from an unopened flower bud to fruit initials. The first, second and third stage buds were cut into halves whereas fourth, fifth, and sixth stages were cut into six longitudinal sections and were individually plated in V8 agar media and incubated at 21°C. Plates were monitored daily and the pathogen was identified based on morphology. Three orchards in Central Florida were also monitored every other week from late February to August and samples were collected to monitor pathogen prevalence. Several pathogens were recovered in the absence of any symptoms during the bud stages. The first symptoms observed on fruits were anthracnose caused by *Colletotrichum* sp. With increasing precipitation, the disease spread leading to spotting and blighting of leaves, stems and fruits. Further disease development, including the blighting of shoots and fruit mummification became more prevalent towards the mid to late season and was caused by combination of several pathogens.

## A meta-analysis of efficacy of anaerobic soil disinfestation on soil borne pathogen suppression

U. SHRESTHA (1), R. Auge (1), A. Saxton (2), D. Butler (1)

(1) Department of Plant Sciences, (2) Department of animal sciences, University of Tennessee, Knoxville, U.S.A.

Anaerobic soil disinfestation (ASD) is a widely applied pre-plant soil treatment practice to control soil borne pathogens under variable environmental conditions. A meta-analysis on multiple ASD studies (533 studies from 46 published/unpublished articles) on soil borne pathogens including bacterial, oomycete and fungal pathogens was conducted to determine the effect sizes and efficacy of ASD on pathogen control. We also examined various environmental conditions as moderator variables to explore the effectiveness of ASD against pathogens. Bacterial (*Ralstonia*) and oomycete (*Pythium*
and *Phytophthora*) pathogens were highly suppressed by ASD treatment (80%-88%), whereas across fungal pathogens (*Cylindrocarpon*, *Fusarium*, *Macrophomina*, *Rhizoctonia*, *Sclerotium*, *Verticillium*) suppression ranged from 59%-86%, with the exception of *Sclerotinia* (15%). Among different host specific *F. oxysporum* (*Fo*) pathogens (237 studies), spinach and tomato wilt caused by *Fo* f. sp. *spinaciae* (87%) and *Fo* f. sp. *lycopersici* (76%) were significantly suppressed by ASD. Under most environmental conditions, suppression of pathogen inoculum due to ASD treatment ranged from 62%-94%, that included study types, soil temperature, soil types and incubation period. Our meta-analysis indicates that ASD is effective against a broad range of soil borne pathogens and optimization to environmental conditions and cropping systems is likely to improve treatment consistency.

## Evaluation of the weather-based spray advisory program MELCAST, fungicide alternations and mulch to manage watermelon diseases in Missouri

Z. MERSHA (1), M. O'Connor (1)

(1) Lincoln University - MO, U.S.A.

Melons are economically important crops in Missouri. Each year, Alternaria leaf blight (ALB), anthracnose (ANT) and gummy stem blight (GSB) cause yield losses in melons. Most commercial growers use calendar-based fungicide spray schedule. MELCAST (MELon disease foreCASTer) is a weather-based disease-forecasting and spray advisory program that allows cantaloupe and watermelon growers to schedule sprays based on weather. From 2013-2015, there were 3-6 MELCAST sites in central, east and south east Missouri. Research plots were established at one of the sites in central Missouri at G. W. Carver Farm to examine MELCAST, mulches and fungicides in reducing diseases of watermelon using variety 'Crimson Sweet'. Fungicide sprays significantly (P<0.05) reduced diseases compared to non-sprayed plots. Alternating Bravo WeatherStik and Inspire Super or Quadris Opti and Luna Experience significantly reduced ALB, ANT and GSB. No significant difference was detected between these two plans. In 2014, total foliar biomass was significantly (P<0.05) higher with fungicide sprays (34.7 lb/plot) than without (9.1 lb/plot) for the plastic mulch plots. Disease suppression was not significantly different either between MELCAST and weekly sprays or between rye-vetch and plastic mulch. Use of MELCAST saved 1-2 fungicide sprays per season. Growers are encouraged to use mulch and to alternate fungicides in order to overcome risk of fungicide resistance and to improve productivity of watermelons.

## Temporal variation of soil biota in fumigated and non-fumigated potato fields

T. SMART (1), B. Geary (1)

(1) Brigham Young University, U.S.A.

Soil biota plays a prominent role in the regulation of soilborne pathogen incidence and severity. Thus, microbial community structure is of particular interest in commercial potato production as fumigation practices have been shown to alter soil biota. Despite long-term use of fumigation in potato production, there is still a limited understanding of soil biota and pathogen recovery and regeneration. In this study, soil cores were taken from neighboring fumigated and non-fumigated potato fields located in five different states: Washington, Oregon, Idaho, North Dakota, and Minnesota. Each core was assessed for pathogen incidence and severity and changes in soil biota over a temporal scale. Fungal and bacterial soil microbial diversity was assessed using meta-barcoded primers on an Illumina MiSeq platform. Our results demonstrate that fumigation creates disparity between pathogen populations and other soil biota which causes disequilibrium when compared to non-fumigation. Future research may establish predictive models in evaluating pathogen incidence and severity as part of an integrated disease management system that seeks to improve sustainable soil health for high-quality potato production.

## Effect of temperature and sugar beet growth stage on yellowing decline caused by *Fusarium secorum*

M. KHAN (1)

(1) North Dakota State University and University of Minnesota, U.S.A.

Fusarium yellowing decline caused by *Fusarium secorum* was recently reported in Minnesota. Conditions favorable for disease development were determined using three isolates (12-4P, 12-2P and 1189), three temperatures (55°C, 65°C, and 75°C), and three growth stages (3, 5 and 7 weeks old plants [WOP]). One *F. oxysporum* f. sp. *betae* isolate was used for comparison. Plants were inoculated using root dip (10⁵ microconidia or macroconidia ml⁻¹) for two minutes. Evaluation for foliar symptoms was done every two days until harvest 35 days after inoculation, and area under disease progress curve (AUDPC) was calculated. Temperatures of 75 and 65°C were more favorable for disease development than 55°C. The mean AUDPC were 38, 29, and 4 for 12-4P, 12-2P and 1189 isolates, respectively. 12-4P isolate had significantly higher AUDPCs at 75 and 65°C for 3 WOP compared to both 5 and 7 WOP. 12-2P isolate resulted in no significant differences between AUDPCs at 3, 5, and 7 WOP within each temperature. However, 12-2P isolate caused significantly more disease at 75°C and 65°C compared to 55°C for 3 and 5 WOP. Isolate 1189 was least aggressive with no significant differences in AUDPC among all combinations of temperature and growth stage. Isolates 12-4P and 12-2P resulted in higher AUDPC than *F. oxysporum* f. sp. *betae* at the same temperature and sugar beet growth stage. Results suggest early planting in cool soils may help to manage the disease.

## Management of wilt diseases on tomato by organically acceptable methods

M. RAHMAN (1), M. Rahman (1), L. Jett (1)

(1) West Virginia University, U.S.A.

Soilborne wilt disease caused by fungal pathogen *Verticillium* spp., *Fusarium* spp and bacterial pathogen *Ralstonia solanacearum* often cause serious losses to tomato growers either by stunting or totally killing the plants prematurely. Rotation and chemical fumigation of soil is not feasible to many growers either due to limited farm size or organic growing conditions. Biofumigation and/or biologically based options for managing these pathogens are critical needs to these growers. A study was conducted with heirloom tomato 'Mortgage lifter' at the WVU Organic Farm to investigate the efficacy of bio-fumigants, biological antagonists, and resistant rootstock in managing wilt disease. Thirty five days after planting in contaminated soil, grafted (on resistant rootstock "Maxifort") tomato plants showed significantly higher plant vigor compared with non-treated check. All treatments had significantly lower symptomatic leaves than that of non-treated check at 35 days after planting. Cumulative harvests for six weeks showed that yield from all treatments except mustard cover crop were significantly (P<0.001) higher compared with non-treated check. Grafted plants produced 20 lb tomatoes/plant compared with only 11 lbs by non-treated check. Results suggest that organically acceptable methods can provide significant yield advantage to heirloom tomatoes in soilborne pathogen infested soil.

## Cryotherapy and Mini-plant Biological Indexing: New Tools for use in Citrus Clean Stock/Certification Programs

C. RAMADUGU (1), M. Keremane (2), G. Volk (3), J. Hartung (4), G. McCollum (5), R. Lee (2)

(1) University of California Riverside, Riverside, CA, U.S.A.; (2) USDA ARS National Clonal Germplasm Repository for Citrus and Dates, Riverside, CA, U.S.A.; (3) USDA ARS National Center for Genetic Resources Preservation, Fort Collins, CO, U.S.A.; (4) USDA ARS Molecular Plant Pathology, Beltsville, MD, U.S.A.; (5) USDA ARS US Horticultural Research Laboratory, Fort Pierce, FL, U.S.A.

Shoot tip grafting (STG) followed by biological indexing using 10-14 month old indicator plants has been the mainstay for eliminating graft transmissible pathogens (GTP) from citrus accessions. We report the application of cryotherapy (freezing vegetative buds in liquid nitrogen followed by grafting onto seedling rootstocks) as an alternate, effective method for the elimination of certain GTPs. Because larger vegetative buds are used for cryotherapy, up to 1.5 mm, as compared to 0.1 mm of apical meristem buds used for STG, personnel become proficient after 1-2 weeks of training as compared to 6-12 months training needed for STG. We also have developed a biological indexing method using very young (mini) indicator plants, 60-90 days old. Side by side comparisons of 30 accessions using traditional and mini-plant biological indexing indicate the same level of sensitivity between
the two methods. The use of mini-plants for biological indexing requires much less time and greenhouse space, also GTP requiring cooler temperatures to produce symptoms may be indexed year-round by placing the mini-plants near the cooling pads in summer. The use of cryotherapy and mini-plant biological indexing will enable citrus clean stock and/or certification programs to be more productive while using fewer resources.

## Bacillus mycoides isolate J (BmJ): Plant protection through Systemic Acquired Resistance. A new option for plant disease management

S. OCKEY (1), B. Highland (2), B. Jacobsen (3), M. Dimock (2)

(1) Certis USA, U.S.A.; (2) Certis USA, U.S.A.; (3) Montana State University, U.S.A.

*Bacillus mycoides* isolate J (BmJ) induced resistance provides control of bacterial, fungal, oomycete and viral pathogens on a wide range of crop plants. BmJ induced resistance is signaled through the NPR-1 gene and involves several PR proteins and ethylene. Optimal systemic resistance induction occurs 3-5 days after application of live cells and provides disease control for 14-21 days depending on the plant induced. BmJ is compatible with pesticides including; triazole, EDBC, and QoI class fungicides and a wide range of insecticides. Disease control equal to commercial standards has been achieved with BmJ alone or in combination with fungicides where BmJ replaces half the fungicide used in fungicide alone programs. BmJ has also been demonstrated to be of benefit to triazole and QoI fungicide resistance management programs in Cercospora leaf spot of sugarbeet control programs. BmJ used in combination with insecticides and rouging has produced >50% control of potato PVY in multiple years in the field. Research results also support disease control in cucurbit crops, tomato, pepper, potato, pecans, spinach, lettuce and sugarbeet with comparisons to commercial pesticide standards. BmJ is licensed to CERTIS USA by Montana State University and will be sold as a WDG formulation. Application rates are based on $1 \times 10^7$cfu/ml (60-240 gm/A depending on spray volume). Registration is expected with Canada's Pest Management Registration Authority (PMRA), the USEPA.

## Comparison of carbon quantity in anaerobic soil disinfestation

J. HONG (1), F. Di Gioia (2), M. Ozores-Hampton (2), E. Rosskopf (1)

(1) USDA ARS, U.S.A.; (2) University of Florida, Institute of Food and Agricultural Sciences, South West Florida Research and Education Center, U.S.A.

Anaerobic soil disinfestation (ASD) is a pre-plant alternative to chemical fumigation, used to manage soil plant pathogens, nematodes, and weeds. Compared to chemical fumigation, ASD was as effective for controlling these pests, and fruit yield often exceeded. Application of ASD in Florida includes incorporating composted broiler litter, 22 Mg ha⁻¹, and molasses, 13.9 m³ ha⁻¹, tarping the soil, and saturating to capacity. The environment under the tarp becomes anaerobic during the treatment, allowing anaerobic microbes, which contribute to pathogen management, to thrive. The objective of this study was to compare various rates of molasses, 0-4 times the standard rate (SR), in greenhouse conditions. Treatments were compared using the following variables: soil conditions, plant vigor, soil microbiome, and weed count. Soil microbiome was investigated by extracting microbial DNA from the soil, taken periodically during the treatment, and using length-heterogeneity PCR. Shifts in the microbiome correlated to changes in soil pH and reduction potential. The microbiome fluctuated slightly in the no molasses treatment, while the microbiome of the SR changed throughout the treatment, and the pH temporarily decreased. Bacterial diversity decreased 7 day post treatment (dpt) for the treatments with molasses rates greater than the SR. The soil pH for these treatments dropped from pretreatment levels and remained acidic; pH for 4 times the SR was 1 dpt = 6.9 and 21 dpt = 5.3.

## Practical resistance to mefenoxam in *Pythium aphanidermatum* and its impact on managing *Pythium* root rot in poinsettia cultivars

E. Lookabaugh (1), B. Shew (1)

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

Mefenoxam (mef) resistance is widespread among populations of *Pythium* in NC greenhouses, but resistant (MR) and sensitive (MS) isolates often are found concurrently. The combined utility of host resistance and mef to manage root rot in plants exposed to mixed populations of *P. aphanidermatum* was tested on seven poinsettia cultivars (cvs). One MR and one MS isolate were used, giving four inoculum/fungicide combinations: MR+mef, MR-mef, MS+mef, MS-mef. Plants were treated twice with the labeled rate of Subdue Maxx. Plants were scored for root rot (RR, 1 = 0% rot; 5 = 100%) and aboveground disease severity (DS, 1 = healthy; 5 = dead plant) 10 wk after inoculation. MS-mef treated plants had the highest RR (mean = 4.0) and DS (mean = 3.9) in all cvs. Mef controlled RR in all cvs inoculated with the MS isolate (mean = 1.6) but not in 5 of 7 cvs inoculated with the MR isolate (mean = 3.4), demonstrating practical resistance to mef in *P. aphanidermatum*. MR-mef treated plants had lower DS severity than MS-mef treated plants (3.3 vs. 3.9), suggesting that the MS isolate was more aggressive than the MR isolate. Treatment with mef lowered DS in MR inoculated plants only slightly (mean = 2.6). If confirmed with other MS and MR isolates, this may explain why MS isolates persist in facilities that regularly use mef. Resistant poinsettia cvs could be used to offset loss of fungicide efficacy and reduce losses to Pythium root rot in facilities with mixed *Pythium* populations.

## Evaluation of Commercial Hot and Bell Pepper Cultivars for Resistance to *Phytophthora capsici*

C. PARADA ROJAS (1), C. Parada Rojas (2), L. Quesada Ocampo (2)

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

*Phytophthora blight* (caused by *Phytophthora capsici*) is an important disease with a broad host range including Solanaceous, Cucurbitaceous, and Fabaceous crops. In pepper, *P. capsici* causes crown, root, and fruit rot as well as foliar lesions. Disease management relies primarily on the application of fungicides, planting of tolerant cultivars, and cultural practices. Field trials were conducted over the summers of 2015 and 2016 to evaluate 32 commercial hot and bell pepper cultivars in North Carolina (NC). Cultivars Martha-R and Meeting were found to be the most resistant to a group of isolates from NC. Ebano-R, Revolution, Paladin, and Vanguard showed intermediate levels of resistance. Bastille, Red Knight, and Plato were highly susceptible to *P. capsici*. Greenhouse experiments were conducted to determine the virulence of three individual *P. capsici* isolates across 48 commercial hot and bell pepper cultivars. The isolates exhibited different levels of virulence to the pepper cultivars screened for crown and root rot resistance. Cultivars CM334, Martha-R, Meeting, and Intruder were resistant to the isolates tested. In addition, we phenotypically characterized a *P. capsici* isolate collection from NC for fungicide sensitivity and mating type. This information will be useful for NC growers in selecting resistant cultivars and for future population genetics analyses.

## Managing Potato Pink Rot Using Chemical and Non-Chemical Strategies

X. ZHANG (1), J. Hao (1), H. Jiang (1), N. Marangoni (1), X. Zhang (1)

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

Pink rot (*Phytophthora erythroseptica*) is a widespread disease on potatoes (*Solanum tuberosum*). Controlling pink rot becomes a challenge owing to the fast development of a fungicide-resistant *P. erythroseptica* population. To determine effective strategies for pink rot control, field trials were conducted in 2014 and 2015 using fungicides or crop rotation. Pink-rot-susceptible seed tubers (cv. 'Russet Norkotah') were used. *Phytophthora erythroseptica* inoculum was distributed in-furrow at planting prior to soil treatments. In fungicide trials, chemical and biological products were applied in-furrow at planting or through soil drenching after plant emergence. The crop rotation trial was conducted with "rotation crop-potato" sequences. Tuber yield was measured and disease severity was rated based on a "0 (healthy tuber) to 5 (>50% rot area)" scale at harvest. Fluopicolide, mefenoxam-oxathiapiprolin combinations, and fluopicolide/oxathiapiprolin plus *Bacillus* combinations significantly reduced pink rot. None of the tested biocontrol agents significantly reduced pink rot alone. Mefenoxam was effective when there was no mefenoxam-resistant population of *P. erythroseptica*. Oats, clover, and barley underseeded with ryegrass lowered pink rot severity levels, although it was not statistically significant. Soil microbial communities in rotation plots are being investigated using next generation sequencing technology.
## The Nærstad model for potato late blight is available on the open source license technology platform VIPS

B. NORDSKOG (1), A. Hermansen (1), V. Le (1), H. Eikemo (1), A. Hjelkrem (1), T. Skog (1), R. Nærstad (1)

(1) Norwegian Institute of Bioeconomy Research, Norway

The Nærstad model for potato late blight (*Phytophthora infestans*) is a weather driven forecast model based on validation trials with spore traps and trap plants under Norwegian field conditions. The model combines sub models for spore production, release, survival, and infection, with calculations derived from hourly values of temperature, relative humidity, global radiation, leaf wetness (LW), and precipitation. LW may also be calculated with additional wind and net radiation parameters. The model output is presented as hourly risk values for infection of *P. infestans*. The Nærstad model is currently provided to users in Norway, Sweden and Latvia through the open source licensed technology platform VIPS (www.vips-landbruk.no). Data from most online weather stations or public weather data networks can be implemented into the VIPS system, including virtual weather stations based on weather forecasts. In VIPS, models such as the Nærstad model can easily be tested and validated under local conditions. Model outputs from multiple weather stations can be displayed in a map to visualize regional differences, in addition to more detailed outputs for each location. The open source license allows users of VIPS to make adaptations and changes to accomodate local needs, test and validate models locally or as part of an international network, and eventually provide the models directly to end users through the same system.

## Control of tulip mild mottle mosaic disease and tulip streak disease based on soil diagnostic

M. KAZUMI (1)

(1) Toyama Prefectural Agricultural, Forestry and Fisheries Research Center, Japan

Tulip mild mottle mosaic disease, cause of *Tulip mild mottle mosaic virus*, TMMMV and Tulip streak disease, cause of Tulip streak virus, TuSV transmitted by *Olpidium virulentus* cause chronic damage and become one of the factors of avolition on produce of tulip bulb in Toyama, Japan. The management of both viral diseases depends on use of fungicide, a fertilizing efficiency-control type fertilizer and cultivation of late time. However, it is necessary to choose the appropriate control measures according to the level of both viral disease occurrence risks on farmer's field. In this study, we developed management of both viral disease consisting of risk evaluation of both viral diseases on tulip field by the quantification of TMMMV and TuSV from soil using real-time PCR, evaluation of individual control measures for both viral diseases by meta-analysis and selection of appropriate control measures according to the level of both viral diseases occurrence risks on tulip field.

## Management of *Tomato chlorotic spot virus*, an emerging tospovirus of tomato causing severe losses in south Florida

S. ZHANG (1), Y. Fu (2), X. Fan (2), D. Seal (2), Q. Wang (3), E. McAvoy (4)

(1) Tropical Research and Education Center, University of Florida, IFAS, Homestead, FL, U.S.A.; (2) Tropical Research and Education Center, University of Florida, IFAS, U.S.A.; (3) UF/IFAS Miami-Dade County Extension, U.S.A.; (4) UF/IFAS Hendry County Extension, U.S.A.

*Tomato chlorotic spot virus* (TCSV) is a devastating tospovirus transmitted by thrips. TCSV was first reported in the USA from tomato plants in Homestead, FL in 2012. Outbreaks of TCSV in Homestead area since 2014 have caused significant losses to growers. TCSV has become established in south Florida and it occurs more widely with greater frequency across this region, bringing a serious concern that TCSV may spread to other tomato production regions. Field trials have conducted in Homestead since 2015 to develop management strategies for TCSV. Fourteen tomato varieties resistant to *Tomato spotted wilt virus* (TSWV) were evaluated. In the spring trial in 2015, all tested varieties exhibited varying levels of resistance to TCSV (10-40% vs 100% infection in the commercial standard cv. 'FL 47') two months after transplanting. In the two repeated trials carried out in the fall of 2015, all these varieties had lower than 8% disease incidence 16 weeks after transplanting, whereas 50% plants of cv. 'FL 47' showed TCSV symptoms. In an insecticide trial conducted in the spring of 2015, only spinetoram (Radiant®) and cyantraniliprole (Exirel®) significantly (P<0.05) reduced TCSV incidence compared to the untreated control. In the fall trials of 2015, plant activator acibenzolar-S-methyl (Actigard®) numerically reduced TCSV incidence. The use of metalized UV-reflective plastic mulch failed to suppress the disease. Results from this study will be reported and discussed in detail.

## Variation within and among laboratories in detection of *Candidatus* Liberibacter asiaticus using qPCR

G. MCCOLLUM (1), C. Levesque (2), M. Keremane (3), M. Kunta (4)

(1) USDA-Agricultural Research Service, U.S.A.; (2) Citrus Research Board, U.S.A.; (3) USDA, ARS, U.S.A.; (4) Texas A&M University, U.S.A.

Huanglongbing (HLB) disease has ravaged the Florida citrus industry during the last decade and poses a serious threat to citrus production worldwide. *Candidatus* Liberibacter asiaticus (CLas) is the presumed causal agent of HLB in the United States. Diagnosis of CLas infection is based on PCR; qPCR for wide-scale surveys and conventional PCR for confirmation. The objective of our research was to compare results among multiple laboratories using slightly different qPCR protocols to confirm the sensitivity and diagnostic reliability of qPCR. An end point dilution series of CLas-positive nucleic acid was prepared and distributed to 4 laboratories in which CLas diagnostics are routinely performed. In each laboratory qPCR diagnostics were performed using the dilution series as template, however, specific protocols typical for each laboratory varied slightly. Results were compared within and between laboratories to determine linearity, amplification efficiency, end point and consistency. Excellent agreement in results was seen among protocols within individual laboratories as well as between individual laboratories. Regression analysis indicated that regardless of protocol, PCR efficiencies were between 95 and 105%. In addition, results confirmed a Ct value of 38 for the endpoint (i.e. a single copy of target). Our results support the robustness and diagnostic reliability of the currently approved qPCR protocol.

## Regulatory implications of a newly discovered transmission mechanism for the pathogens that cause huanglongbing (citrus greening disease)

S. HALBERT (1), M. Keremane (2), C. Ramadugu (3), W. Dawson (4), J. Lee (5), J. Keesling (5), B. Singer (6), R. Lee (7)

(1) Florida Department of Agriculture and Consumer Services, Division of Plant Industry, Gainesville, FL, U.S.A.; (2) USDA/ARS, National Clonal Germplasm Repository for Citrus and Dates, Riverside, CA, U.S.A.; (3) University of California, Riverside, Riverside, CA, U.S.A.; (4) Citrus Research and Education Center, University of Florida, Lake Alfred, FL, U.S.A.; (5) University of Florida Mathematics Department, Gainesville, FL, U.S.A.; (6) Emerging Pathogens Institute, University of Florida, Gainesville, FL, U.S.A.; (7) Retired, USDA/ARS, Peyton, CO, U.S.A.

Huanglongbing (HLB, citrus greening disease) is one of the most devastating citrus diseases in the world. In Florida, it is associated with a bacterium *Candidatus* Liberibacter asiaticus (Las) and transmitted by a psyllid, *Diaphorina citri* Kuwayama. Lee et al. (2015; PNAS 112: 7605-7610) documented a new transmission mechanism for Las. An adult female psyllid, or her mate, infects the new growth where she lays eggs. The localized infection becomes a source for developing nymphs. Progeny can repeat the process as soon as they become adults. Since each female can lay 750 or more eggs, and psyllids can complete development in 15 days, the potential for increase of infected insects is rapid. In the laboratory, plants colonized by infected psyllids usually develop disease, but symptoms may be delayed for months or years. Catastrophic spread of Las can occur long before symptoms manifest. This delayed expression of disease symptoms could allow for the disease to spread undetected.

## Known unknowns and unknown unknowns: Fungi intercepted at the U.S. borders

M. ROMBERG (1), J. McKemy (1)

(1) USDA APHIS PPQ, U.S.A.

Global trade in plants and plant products continues to increase yearly and has the potential to move fungi associated with poorly studied hosts around the world. USDA Animal and Plant Health Inspection Service (APHIS) plant pathology identifiers at ports across the U.S. are responsible for identification
of fungi on products arriving by sea, land and air. In 2015, over 18,000 interceptions were examined by USDA APHIS plant pathology identifiers. These interceptions covered about 600 genera of fungi identified on these hosts. The inspected hosts comprise ornamental, forestry, and horticultural plants and plant products. Nearly one quarter of the interceptions were from cargo awaiting entry decisions and required identifications in less than 48 hours. The rest were from plants and plant products seized from passenger baggage, mail, ship stores and other sources and represent some of the as yet unknown fungal/host combinations that could be arriving in the U.S. via these pathways. If a fungus not known to be present in the U.S. is intercepted on a shipment, that shipment is either destroyed or re-exported, which underscores the need to know which fungi have been reported in the US and also the need for systematic research in widespread fungal groups. An overview of intercepted fungi will be presented including fungi intercepted for the first time from various ports and not known to occur in the U.S.

## Functional genomic analysis of *Botrytis cinerea* isolates from Ohio

V. VIJAYAKUMAR (1), B. Cottrell (1), H. Reynolds (1), J. Slot (1), F. Hand (1), G. Valero (1), C. Tomashuk (1)

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

*Botrytis cinerea* is one of the most widely spread phytopathogenic fungi displaying a broad host-range. While *Botrytis* is generally considered a model necrotroph, systemic symptomless infections have also been reported. Previous studies have shown significant genomic variation between gray and rare bikaverin producing pink isolates, but their contribution to diversity and host/pathogen relationships is understudied. We aim to elucidate genomic features of symptomless and host specific infections, and biochemically characterize virulence determinants. For this, we sequenced the genomes of 4 isolates from Ohio: 1 gray, isolated as asymptomatic endophyte of apple blossoms; 1 pink and 1 gray, isolated from infected flowers of *Matthiola incana*; and 1 purple, obtained from distinct sectors of pink isolate. We compared all 4 genomes to B05.10 and T4 reference genomes. Results showed large polymorphisms within coding regions of predicted genes (52,710, missense; 418 nonsense and 80,430 silent) and deletion of gene clusters (GC) associated with virulence. In both pink and purple isolates the botcinic acid GC was deleted, while in the gray asymptomatic isolate the abscisic acid GC was deleted. Investigation of virulence determinants among isolates showed a fully functional sclerotia formation, while the pink and purple strains alone were impaired in oxalic acid formation. Our results suggest that genetic diversity plays a significant role in species complexity and lifestyle.

## Virus testing in certified strawberry nursery plants in Oregon

D. SHARMA-POUDYAL (1), S. Lane (1), J. Grant (1), N. Osterbauer (1)

(1) Oregon Department of Agriculture, U.S.A.

*Strawberry crinkle virus* (SCV), *Strawberry mottle virus* (SMoV), *Strawberry mild yellow edge virus* (SMYEV), and *Strawberry vein banding virus* (SVBV) are aphid-borne strawberry viruses frequently found in the Pacific Northwest. As such, they are excellent indicators for the effectiveness of nursery certification programs in maintaining virus free plants. Oregon nurseries import certified strawberry plants primarily from California and multiply these plants prior to sale to the final consumer. Testing for viruses in certified plants reveals the virus status of the plants and measures the effectiveness of the originating state's certification program. A survey for these four viruses was performed in a Klamath County nursery multiplying certified strawberry plants. A total of 59 plant samples were collected and tested for SCV and SMYEV while 118 samples were tested for SMoV and SVBV. SVBV was detected using PCR with SVBV-specific primers. SCV, SMoV, and SMYEV were not detected using RT-PCR with virus-specific primers. SVBV was detected in one out of 118 samples tested. These results demonstrate the continued need for virus testing of strawberries throughout the plants' entire life cycle in an official certification program.

## National Seed Health Accreditation Pilot Program: Monitoring seed health of seeds imported into the United States

T. BRUNS (1), G. Munkvold (2), A. McMellen-Brannigan (3), R. Dunkle (4)

(1) Iowa State University Seed Science Center, U.S.A.; (2) Iowa State University, U.S.A.; (3) USDA-APHIS, U.S.A.; (4) American Seed Trade Association, U.S.A.

The risk of introducing plant pathogens into new areas by seed movement is a critical issue in agriculture. Part of the mission of USDA-APHIS is to protect U.S. agriculture from foreign pathogens. In order to help fulfill this mission, a pilot program, the U.S. National Seed Health Accreditation Pilot Program (NSHAPP), has been authorized by USDA-APHIS. NSHAPP is a voluntary program for seed importers that is administered by the National Seed Health System at Iowa State University Seed Science Center in Ames, IA. NSHAPP aims to prevent the introduction of plant pathogens into the United States by organizing the testing of imported seed. The scope of program focuses on cucumber green mottle mosaic virus (CGMMV) in cucumber, melon and watermelon seed. CGMMV causes severe disease of cucurbit crops, and was first detected in the U.S. in 2013 in California field production. Testing for CGMMV under NSHAPP is done by an ELISA method validated by the Int. Seed Testing Assoc. Participants report data on tested seedlots monthly, but positive seed tests must be reported immediately. Positive seedlots must be destroyed or re-exported. This program represents a proactive approach to reducing the risk of pathogen introduction in imported seed and demonstrates effective collaboration between seed importers and USDA-APHIS. Successful implementation of the pilot program may lead to expansion of voluntary testing and reporting to include other pathogens in imported seed.

## Assessing Goss's bacterial wilt and leaf blight of corn in the High Plains of Texas with infrared aerial imagery

J. WOODWARD (1), R. French-Monar (2), J. Todd (3), O. Moore (4)

(1) Texas A&M AgriLife Extension, U.S.A.; (2) Texas A&M AgriLife Extension, U.S.A.; (3) Todd Ag Consulting, U.S.A.; (4) High Plains Consulting, U.S.A.

Outbreaks of Goss's bacterial wilt and leaf blight of corn, caused by *Clavibacter michiganensis* subsp. *nebraskensis*, were reported in the Texas High Plains from 2013 to 2015. The disease was identified based on symptomology, bacterial streaming, colony characteristics and Immunostrip test results. Impact of the disease on corn is poorly understood in this region, thus the objective of this study was to evaluate disease development using aerial imagery. Fields were photographed from a fixed-wing aircraft from mid-July to late-September of each year. Images were effective at differentiating resistant and susceptible hybrids and improved scouting efficiency when assessing incidence and severity. Disease onset differed by location but initial infections were easily identifiable. In general, infection foci were non-random or aggregated; however, edge effects were observed in two areas adjacent to fields where the disease was observed the previous year and reduced tillage was implemented. Yield reductions ranging from 12 to 53% were observed in areas of some fields; whereas, other fields exhibiting >85% incidence were abandoned for grain production and cut for silage. These results indicate that Goss's wilt has the potential to negatively affect corn production under favorable conditions. Increased awareness through educational materials, including information on hybrid selection, crop rotation and residue management has allowed for better management of the disease.

## Understanding fungal reservoirs: Tracking *Aspergillus* populations in air, soil, crop debris and the reproductive organs of Bt and non-Bt corn

H. ABBAS (1), M. Weaver (1), C. Accinelli (2), W. Shier (3), R. Zablotowicz (1)

(1) USDA-ARS, U.S.A.; (2) University of Bologna, Italy; (3) University of Minnesota, U.S.A.

*Aspergillus flavus* can colonize a wide range of environments. The advent of effective biocontrol agents makes it important to know where fungi overwinter, because the amount and toxigenicity in each reservoir may determine the most effective site(s) to apply biocontrol. *Aspergillus* propagules on leaf, husk, silk and tassel of Bt and non-Bt corn, and from soil and air/dust were quantified using dilution plating on dichloronitroaniline rose Bengal agar. There was no effect of Bt corn on the amount of *Aspergillus* propagules. *Aspergillus* propagules in air/dust varied dramatically from year to year (0-278 cfu/1000L). We studied corn hulls, cob and kernel fragments that accumulated near on-farm grain storage bins in normal farm operations and were
periodically moved to the edge of an adjacent field and disked into soil in fall tillage. The following March, *A. flavus* propagules in soil samples ~20, ~30 and ~40 meters from the bins were 1,500-28,000 CFU/g, with ~3 times as many at 20m as at 40m, but 24% aflatoxigenic at all distances. In January 2016 similar debris and dust accumulating near on-farm grain storage bins contained 14,500±5,400 CFU/g *A. flavus* propagules. Additional studies on *A. flavus* propagules from rice, sesame and Illinois corn field soil and plant debris will be discussed. The findings are consistent with a substantial plant debris-associated soil populations. These findings are very useful in selecting optimal biological control strategies for aflatoxin.

## Impact of stalk rot diseases on sweet sorghum sugar concentration and content

A. BANDARA (1), T. Tesso (1), D. Wang (1), K. Zhang (1), C. Little (1)

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

The sugar-rich nature of sweet sorghum [Sorghum bicolor (L.) Moench] makes it an attractive bioethanol feedstock. Fusarium stalk rot (Fusarium thapsinum, FT) and charcoal rot (Macrophomina phaseolina, MP) are high priority fungal diseases of sorghum, which can potentially affect sweet sorghum biofuel traits. The objective of this study was to test the effects of FT and MP on sweet sorghum sugar concentration and content. Seven parental lines and 12 hybrids were field evaluated in 2014 and 2015. At 14 d after flowering, plants were inoculated with FT, MP, and phosphate-buffered saline (control). Plants were harvested at 35 d after inoculation and measured for disease severity using stem lesion length. Juice volume (per plant) extracted from stalks was measured and subjected to HPLC to determine sugar concentration (mg/mL). ANOVA revealed non-significant pathogen main or simple effects on glucose and fructose concentration and significant genotype × pathogen effect on sucrose concentration. Pathogens did not significantly affect the total sugar concentration (Σ sucrose, glucose, fructose). However, when analyzed for content (g/plant), genotype × pathogen and environment × pathogen effects were evident for total sugars and sucrose. Pathogens affected glucose and fructose content in an environment-specific manner. Therefore, in general, stalk rot pathogens do not affect the concentration of major sweet sorghum sugars, but do affect sugar content.

## Effect of planting date and peanut cultivar on leaf spot epidemics and pod yield

B. JORDAN (1), A. Culbreath (2), W. Branch (3)

(1) University of Georgia, Dept. of Plant Pathology, U.S.A.; (2) University of Georgia, Dept of Plant Pathology, U.S.A.; (3) University of Georgia, Dept. of Crop and Soil Science, U.S.A.

Planting date can affect the risk of losses to early and late leaf spot caused by, *Cercospora arachidicola* and *Cercosporidium personatum*, respectively, of peanut, *Arachis hypogaea*, in both conventional and organic systems. The objective of this study was to characterize the effect of planting date on leaf spot epidemics and yield in new cultivars with moderate tolerance to these diseases. A field trial was conducted in 2015 in Tifton, GA. Treatments were six planting dates (24 and 27 April, 4, 11, 19, and 26 May) arranged factorially with two cultivars, Georgia-06G and Georgia-12Y. Experimental design was a randomized complete block design with 4 replications. No foliar fungicides were applied. Late leaf spot was the predominant disease. Epidemics were severe in plots planted at the later dates. Final leaf spot ratings (Florida 1-10 scale) and AUDPC increased linearly with later planting date (Julian day) for both cultivars. Yield of Georgia-06G decreased linearly, and yield of Georgia-12Y decreased according to a quadratic function with later planting. For most planting dates, final leaf spot severity and AUDPC were lower, and yield was higher for Georgia-12Y than for Georgia-06G. The combination of early planting with Georgia-12Y shows potential for reducing risks of losses, leaf spot, and maximizing yield in situations such as organic production where fungicide use would be minimal.

## Fungal profiles in ginger root and cayenne pepper powders from U.S. retail

V. TOURNAS (1), J. Kohn (2), E. Katsoudas (2)

(1) Center for Food Safety and Applied Nutrition/FDA, U.S.A.; (2) Northeast Regional Lab/Office of Regulatory Affairs/FDA, U.S.A.

A limited survey was conducted to determine the mycological quality (mould and yeast levels and profiles) of ginger and cayenne pepper powders sold in local stores in the Washington, D.C. area. Ginger powder samples were purchased from four companies (A, B, C, and D), while cayenne pepper was obtained from two companies (A and B). Powders packed in capsules as well as in bulk bottles and jars were examined. All ginger samples tested during this study contained live fungi. Mould levels ranged between 2.4 × 10³ and 6.2 × 10⁴ colony forming units/g (cfu/g). The most common species were *Aspergillus flavus* and *Aspergillus niger* followed by eurotia and *Fusarium* spp. No yeasts were found in any of the samples tested. Some variation in fungal species and contamination levels were apparent among different companies. Higher levels of fungal infestation were observed in cayenne pepper samples. Seventy one percent of the analyzed samples were carrying live moulds while only one sample tested positive for yeasts. Mould counts ranged between <100 and 6.9 × 10⁵ cfu/g. The most frequent fungal contaminants were A. niger, A. flavus, and eurotia (E. chevalieri and E. rubrum) followed by Aspergillus spp. and members of the Mucorales family. Variation in mould levels existed between the two companies and among lots of the same company.

## Wheat streak mosaic virus differentially affects soft red winter wheat cultivars

B. HODGE (1), L. Stewart (2), P. Paul (3)

(1) The Ohio State University, U.S.A.; (2) United States Department of Agriculture-Agricultural Research Service, U.S.A.; (3) The Ohio State University, U.S.A.

Wheat viruses are known to impact production throughout the world. A statewide survey conducted in 2012 identified several viruses in wheat samples taken from fourteen Ohio counties. Wheat streak mosaic virus (WSMV) was identified in samples from two counties, Auglaize and Champaign. WSMV is a positive sense, single-stranded RNA virus transmitted by wheat curl mites (Aceria tosichella), and is known to cause severe yield loss and stunting in hard red winter wheat. However, there is little information available about WSMV specific to soft red winter wheat cultivars grown in Ohio. To assess its potential impact, nineteen Ohio cultivars were mechanically inoculated with WSMV at Feekes stage 1. All cultivars supported systemic infection. Over 21 days post-inoculation, individual plants were rated for symptom severity, final height, and final wet weight. Across cultivars, inoculated plants exhibited an average 13.2% decrease in plant height, and a 32.1% decrease in wet weight. The cultivars differed in virus responses, as determined by generalized linear mixed model analysis. This experiment demonstrates that WSMV infection affects soft red winter wheat cultivars differentially, which may lead to the identification of cultivars that minimize yield loss.

## Occurrence of a Citrus canker strain of limited host specificity in Texas

M. KUNTA (1), J. da Graça (1), B. Salas (2), D. Bartels (2), J. Park (1), G. Santillana (3), V. Mavrodieva (3)

(1) Texas A&M University-Kingsville Citrus Center, Weslaco, TX, U.S.A.; (2) USDA APHIS PPQ CPHST Mission Laboratory, Edinburg, TX, U.S.A.;

(3) CPHST Beltsville Laboratory USDA APHIS PPQ, Beltsville, MD, U.S.A.

In October 2015, a Mexican lime exhibiting citrus canker-like symptoms was found by the USDA-APHIS-PPQ citrus disease survey in a residential property in Cameron County, Texas. The Texas A&M University-Kingsville Citrus Center detected citrus canker using a real-time PCR analysis. The USDA-APHIS-PPQ Lab in Beltsville, MD confirmed that it was a *Xanthomonas citri* subsp. *citri* isolate. A delimiting survey was initiated by USDA and by February 2016, 169 suspect leaf samples were sent to the PPQ Beltsville lab for analysis. Of 118 samples positive for citrus canker, all within a 5-mile radius, 116 were from Mexican lime, 1 was from Kaffir lime and 1 from Ponderosa lemon. Survey data collected on stem lesions suggested the oldest lesions to be between 4 – 6 years old. To examine the host range of the TX citrus canker strain, four pots each of 11 different citrus varieties, including the major commercial varieties grown in TX, were inoculated by syringe infiltration of symptomatic crude leaf extracts. The only citrus plants
that developed clear canker lesions were Mexican limes. A combination of serological and molecular tests also suggested that the Texas citrus canker isolate was different from the typical Asiatic strain (Xac A) and more similar to other known *X. citri* subsp. *citri* strains with limited host range such as Xac A<sup>W</sup> or A*. Xac A<sup>W</sup> strain was previously reported from Florida on Mexican lime and Alemow, a close relative of Kaffir lime.

### A new 'Candidatus Phytoplasma pini'-related strain associated with witches'-broom of Pinus spp. is detected in Maryland

S. COSTANZO (1), J. Rascoe (1), Y. Zhao (2), R. Davis (3), M. Nakhla (1)

(1) USDA-APHIS-PPQ-CPHST, U.S.A.; (2) USDA-ARS, U.S.A.; (3) USDA-ARS

Phytoplasmas are insect transmitted specialized bacteria that cause devastating diseases in crops and natural ecosystems. Pine trees (*Pinus* spp.) showing abnormal shoot branching and witches' broom symptoms in a wooded area of Laurel, Maryland, were examined for the presence of phytoplasmas. Nested PCR assays for amplification of the 16S ribosomal RNA gene (rDNA) sequences were used on pine tree DNA samples to confirm possible association of a phytoplasma with the disease. Comparison of restriction fragment length polymorphism (RFLP) patterns and nucleotide sequence analysis of 16S rRNA gene fragments revealed that several trees were infected by a 'Candidatus Phytoplasma pini' -related strain (MDPP). The 16S rDNA sequence of MDPP shares 98.4% similarity with that of the 'Candidatus Phytoplasma pini' reference strain reported from Spain (isolate P127). The collective 16Sr DNA F2nR2 RFLP pattern is distinct from the reference patterns of all previously delineated subgroups, and is most similar to that of subgroup 16SrXXI-A with a pattern similarity coefficient of 0.79. Since the similarity coefficient value is lower than the threshold (0.97) for new subgroup recognition, MDPP may represent a new subgroup within the group 16SrXXI-A. The new subgroup is hereby designated as 16SrXXI-B. To our knowledge, this is the first report of a 'Ca. Phytoplasma pini'-related strain associated with pine trees in North America and possibly the entire western hemisphere.

### The phytoplasma associated with purple woodnettle witches'-broom disease in Taiwan represents a new subgroup of the aster yellows phytoplasma group

F. JAN (1), Y. Tseng (1), C. Chang (2)

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

In October 2013, a new disease affecting purple woodnettle (*Oreocnide pedunculata*) plants was found in Miaoli County, Taiwan. Diseased plants exhibited leaf yellowing and witches'-broom symptoms. Molecular diagnostic tools and electron microscopic cell observation were used to investigate the possible cause of the disease with a specific focus on phytoplasmas. The result of PCR with universal primer pairs indicated that phytoplasmas were strongly associated with the symptomatic purple woodnettle. The virtual restriction fragment length polymorphism (RFLP) patterns and phylogenetic analysis based on 16S rDNA and ribosomal protein, *rplV-rpsC* region revealed that purple woodnettle witches'-broom phytoplasma (PWWB) belongs to a new subgroup of 16SrI and rplI group and was designated as 16SrI-AH and rplI-Q, respectively, herein. RFLP analysis based on *tuf* gene region revealed that the PWWB belongs to *tufI*-B, but phylogenetic analysis suggested that PWWB should be delineated to a new subgroup under the *tufI* group. Taken together, our analyses based on *16S rRNA* and *rplV-rpsC* region gave a finer differentiation while classifying the subgroup of aster yellows group phytoplasmas. To our knowledge, this is the first report of a *Candidatus Phytoplasma asteris*-related strain in 16SrI-AH, rplI-Q and *tufI*-B subgroup affecting purple woodnettle, and of an official documentation of purple woodnettle as being a new host of phytoplasmas.

### Three races of Xanthomonas campestris pv. vitians causing bacterial leaf spot on lettuce identified

C. BULL (1), M. Trent (2), R. Hayes (3)

(1) Department of Plant Pathology and Environmental Microbiology, Penn State University, U.S.A.; (2) USDA/ARS, U.S.A.; (3) USDA/ARS, U.S.A.

*Xanthomonas campestris* pv. *vitians* (Xcv), the causal agent of bacterial leaf spot on lettuce, reduces lettuce quality and yield worldwide. Although strain specific, host resistance is the most feasible method to reduce losses. Therefore, we screened lettuce germplasm for resistance using the broadest diversity within the pathogen population. A collection of 120 strains were assigned to one of six sequence types (A, B1, B2, C, D and E) by multilocus sequence typing (MLST) using concatenated sequences of *rpoD*, *dnaK*, *fyuA*, *gyrB*, and *gap1*. The abaxial sides of lettuce leaves were infiltrated with single bacterial isolates (1 × 10<sup>8</sup> CFU/ml) representing each sequence type. Tissue collapse beginning at 30 hr and resulting in light-brown papery lesions at 48 hr was considered an incompatible reaction. Replicated assays with strains were conducted with germplasm for which resistance was identified. This method was used to determine that the single dominant gene, named *Xanthomonas resistance 1* (*Xar1*), confers complete resistance to MLSTs B, D, and E. Subsequently, germplasm was identified with complete resistance to strains from sequence type A and a separate source of resistance was identified to strains from sequence type C. The genetics of resistance to sequence types A and C is being elucidated and we are completing analysis of all strains on resistant germplasm. However, the data gathered to-date indicate that there are at least three races of Xcv.

### Candidatus Liberibacter solanacearum associated to *Physalis philadelphica*, a new solanaceous host

H. SILVA-ROJAS (1), A. Contreras-Rendon (2), J. Sanchez-Pale (2)

(1) Colegio de Postgraduados, Mexico; (2) Universidad Autonoma del Estado de Mexico, Mexico

*Candidatus* Liberibacter solanacearum (CaLs) bacterium is an economically important plant pathogen of solanaceous is vectored by the psyllid *Bactericera cockerelli*. Recently, were observed in husk tomato (*Physalis philadelphica*) fields in Toluca Valley in the state of Mexico, Mexico. Plants exhibiting stunting, severe leaf chlorosis, shortened internodes, curling of leaves, aborted flowers, deformation and reduced fruit size were observed, affecting the productive period. Large numbers of the psyllid *B. cockerelli* were observed on the crop. The objective of this research was to detect CaLs in husk tomato plants with similar symptoms as described above and whether this bacterium was present in *B. cockerelli* populations in the field. In 2015, we collected 250 samples from symptomatic plants and five psyllid *B. cockerelli* per plant in commercial plots in Toluca Valley. The molecular detection of CaLs was performed using OA2/OI2c, CLipoF/OI2c and Lp Frag 1-25F/427R primers. The amplified fragments were sequenced using Sanger methodology. Sequence alignment was carried out with the sequences belonging to the haplotypes currently recognized in GenBank database. The SNPs were identified visually. Haplotype B of CaLs was found in 100% of the plants tested, whereas in the insect found A and B haplotypes. This information will enable the approach of management strategies to husk tomato production.

### Use of an unmanned aerial vehicle to assess tomato spotted wilt severity in large peanut mapping populations

S. PELHAM (1), C. Holbrook (2), B. Guo (2), Y. Chu (3), P. Ozias-Akins (3), C. Li (4), R. Xu (4), A. Cubreath (1)

(1) University of Georgia - Department of Plant Pathology, Tifton, GA, U.S.A.; (2) United States Department of Agriculture - Agricultural Research Service, Tifton, GA, U.S.A.; (3) University of Georgia - Department of Horticulture, Tifton, GA, U.S.A.; (4) University of Georgia - College of Engineering, Athens, GA, U.S.A.

Recombinant inbred line (RIL) populations of peanut (*Arachis hypogaea* L.) are being used to develop markers for resistance to several diseases, including tomato spotted wilt, caused by Tomato spotted wilt virus (TSWV). In efforts to develop molecular markers to assist in selection for resistance to TSWV, several populations have been developed from parents with varying levels of TSWV resistance. Mapping programs are faced with the challenge of phenotyping large numbers of genotypes in a timely manner with a limited number of people. Through the use of an unmanned aerial vehicle, imaging techniques, and ground truthing, we developed an analysis method to quickly assess spotted wilt severity in the field using selected RILs from four mapping populations of peanut. With adequate ground truthing, spotted wilt is easily differentiated from other diseases in the field by the color and the visible stunting of the plant. By using a greenness equation in Matlab to identify yellow pixels we can rate the severity of the disease in
many small plots in a short amount of time. Both Matlab greenness values and severity values from APS Assess analysis of aerial images correlated well with field disease ratings. This technique allows for quick acquisition of field response data from large mapping populations.

## Reporting the occurrence of *Ralstonia* based wilts of chillies in major vegetable growing areas of Punjab

A. HAMEED (1), S. Jameel (1), N. Liaqat (1), M. Ashfaq (2), R. Riaz (3), K. Riaz (1)

(1) Department of Plant Pathology, University of Agriculture Faisalabad, Pakistan; (2) Department of Plant Pathology, PMAS-Arid Agriculture University Rawalpindi, Pakistan; (3) Centre of Agricultural Biochemistry and Biotechnology, University of Agriculture, Faisalabad-38040, Pakistan, Faisalabad, Pakistan

*Ralstonia solanacearum* (Rs), is one of the most severe pathogens of solanaceous crops with a very wide host range. Recently, a survey was conducted in districts of Punjab Province including Okara, Chiniot and Faisalabad to determine the incidence of bacterial wilt of chillies. The maximum disease incidence (58%) was recorded in district Okara followed by Chiniot (43%). The least disease incidence was found in district Faisalabad with 20% occurrence. The samples were collected and after surface sterilization, placed on casamino acid-peptone-glucose-agar (CPG) medium. The colonies obtained on CPG medium were transferred on 2,3,5-triphenyltetrazolium chloride (TZC) medium. It was observed that the colonies on TZC were irregular, mucoid and white with pink centers. Out of all the isolates tested, 72% showed positive hypersensitive reaction (HR) with variable response. Only 40% of these HR positive isolates were confirmed as *R. solanacearum* through simple staining, streaming, KOH solubility, biochemical, pathogenicity and Plate Trapped Antigen-Enzyme Linked Immunosorbent Assay (PTA-ELISA) tests. Molecular confirmation of *R. solanacearum* was also done through genomic DNA using Rs-specific primers.

## Identification of the causal agents of soft rot of potato in Bangladesh and activity of biocontrol agents against the pathogens

F. ELAHI (1)

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

Potato is a staple food crop in Bangladesh. Bacterial soft rot is a major postharvest threat to potato, reducing yield approximately 37% in storage systems. Limited research has been conducted to identify and manage the causal agents of soft rot in Bangladesh. Eighteen bacterial isolates were collected from twelve potato-growing regions of Bangladesh in 2015. Sixteen of the isolates produced deep pits on Crystal-Violet Pectate (CVP) medium, were gram negative, oxidase negative, and hypersensitive response (HR) positive on tobacco, characteristic of *Pectobacterium*. Two isolates produced shallow pits on CVP medium, were gram negative, oxidase positive, fluorescent, and weakly HR positive, characteristic of *Pseudomonas*. All isolates caused soft rot on potato tubers. Partial 16s ribosomal DNA sequences from 16 presumptive *Pectobacterium* isolates exhibited >85% identity with *Pectobacterium* ssp. sequences and two presumptive *Pseudomonas* isolates exhibited 90% identity with *P. fluorescens* and *P. putida* sequences previously deposited in GenBank. Fifteen *Pseudomonas* sp. strains with known biocontrol activity were tested for *in vitro* antibiosis against the soft rotting isolates. The biocontrol agents inhibited the growth of all *Pectobacterium* isolates but neither of the *Pseudomonas* isolates. Biological control of potato soft rot by *Pseudomonas* spp. is promising but additional research is needed to identify potential biocontrol agents for soft rotting *Pseudomonas* spp.

## Race characterization of *Pyrenophora tritici-repentis* population from Lithuania and Romania

A. SIDRAT (1), S. Ali (2), Z. Liatukas (3), M. Ittu (4), S. Sehgal (2), N. Kaur (2)

(1) South Dakota State University, Brookings, SD, U.S.A.; (2) South Dakota State University, Brookings, SD, U.S.A.; (3) Institute of Agriculture, Lithuania Research Center for Agriculture and Forestry, Instituto al., Akademija 58344, Lithuania; (4) National Agricultural Research and Development Institute Fundulea, Calarasi, Romania

Tan spot, caused by *Pyrenophora tritici-repentis* (*Ptr*) is an economically important disease of wheat. Eight races exist of this fungal population. In this study, 223 single spore isolates from Lithuania (n=114), and Romania (n=109) were evaluated for their race structure through genotypic and phenotypic characterization. Molecular variation was identified through PCR with *Ptr ToxA* and *Ptr ToxB* genes specific primer. Of these 223 isolates genotyped, 110 isolates were further phenotyped by inoculating them individually on two-week old seedlings of tan spot wheat differentials Glenlea, 6B365, 6B365, and Salamouni. Nearly 86% (191/223) isolates genotyped contained *ToxA* genes. Phenotypic characterizations on 110 isolates identified 69, 6, 13, and 4 isolates *Ptr* race 1, 2, 3, and 4, respectively. Eighteen of 223 isolates lacking in *Ptr ToxA* gene could not fit into currently identified 8 races as they produced necrosis on Glenlea and chlorosis on 6B365. These results indicate that a diverse *Ptr* population exists in Lithuania and Romania. This information would help the regional wheat breeding programs in the development of durable tan spot resistance cultivars.

## Morphological and molecular identification of *Phoma medicaginis* var. *medicaginis* causing spring black stem and leaf spot of alfalfa in Nevada

R. Bomberger (1), S. Wang (1)

(1) Nevada Department of Agriculture, U.S.A.

In June 2015, an outbreak of foliar blight disease was observed in an alfalfa (*Medicago sativa* L.) field with more than 50% of plants affected. Individual plants exhibited rapid death of foliage with sporadic or coalesced lesions on both lower leaves and stems. To isolate the causative agent, necrotic stem and leaf tissue, as well as pycnidia, were surface sterilized and then placed onto potato dextrose agar (PDA) amended with streptomycin sulfate and incubated at 22°C in the dark. Colonies of *Phoma* species were obtained from all samples collected from 5 separate field locations. Ten single spore cultures (SSCs) were obtained from 10 isolates and compared morphologically. All SSCs and isolates produced almost black colonies with abundant hyaline, oval to cylindrical, single-celled conidia with no significant morphological variation observed. A 540 bp DNA fragment was amplified from the ITS regions of rRNA genes and it was sequenced directly from both strands. Sequence analysis revealed that all 10 SSCs had the same amplicon sequence (Accession No. KU695570), with 100% homology to the corresponding regions of *P. medicaginis* var. *medicaginis* (Accession No. AY504634). Thus, the pathogen isolated from diseased alfalfa plants in Nevada was determined to be *P. medicaginis* var. *medicaginis*, the only pathogen associated with the outbreak. This is the first confirmed outbreak of spring black stem disease in Nevada since 1977 when it was initially reported in the state.

## Identification of a new *Mastrevirus* of *Saccharum barberi*, *Saccharum officinarum* and *Saccharum spontaneum* in Florida

W. BOUKARI (1), R. Alcala-Briseño (2), S. Kraberger (3), E. Fernandez (4), D. Filloux (4), J. Comstock (5), A. Varsani (3), P. Roumagnac (4), J. Polston (1), P. Rott (6)

(1) University of Florida, Gainesville, FL, U.S.A.; (2) University of Florida, Gainesville, U.S.A.; (3) University of Canterbury, Christchurch, New Zealand; (4) CIRAD, Montpellier, France; (5) USDA-ARS, Canal Point, FL, U.S.A.; (6) University of Florida, Belle Glade, FL, U.S.A.

A metagenomic study was conducted to identify viruses present in 180 leaf samples of *Saccharum* species collected in 2013/2014 from plants in commercial fields in the Everglades Agricultural Area and the germplasm collection of the USDA-ARS Sugarcane Field station at Canal Point, FL. The objective of this study was to identify known and potentially new sugarcane viruses. In addition to previously reported sugarcane viruses in Florida, we identified the presence of a potential new *Mastrevirus* species. Since there are no *Mastrevirus* species reported from Florida, and species in this genus such as *Sugarcane streak virus* and *Maize streak virus* are known to cause diseases in sugarcane, efforts were made to obtain full length viral genome sequences from individual plants of *Saccharum* spp. Rolling circle amplification followed by enzymatic restriction and cloning was used to obtain nine full genome length sequences (each approximately 2.8 kb): three from *S. barberi*, two from *S. officinarum* and four from *S. spontaneum*, all from the germplasm collection. All nine genome sequences appeared to represent a new species of *Mastrevirus* after their pairwise comparison using the species
demarcation tool (SDTversion1.2). Sequence similarity among sequences also suggested the presence of two strains of this new virus. This new species was found in plants that were introduced to Florida more than six decades ago.

## Mycosphaerellaceae associated with greasy spot of citrus in different climatic regions

A. VICENT (1), V. Aguilera-Cogley (2), J. Armengol (3), M. Berbegal (3)

(1) Instituto Valenciano de Investigaciones Agrarias (IVIA). Moncada 46113, Valencia, Spain; (2) Laboratorio de Protección Vegetal, Instituto de Investigaciones Agropecuarias de Panamá (IDIAP), Los Canelos - Santa María, Panamá., Panama; (3) Instituto Agroforestal Mediterráneo, Universitat Politècnica de València. 46022, Valencia, Spain., Valencia, Spain

Citrus greasy spot is distributed in humid areas in the Caribbean and Central America. The disease is characterized by yellow-brown spots on leaves and premature defoliation. Symptoms similar to greasy spot were observed in semi-arid areas in the Mediterranean Basin, although with low severity. The fungus *Zasmidium citri-griseum* (= *Mycosphaerella citri*) has been identified as the causal agent of greasy spot in the Caribbean and Central America, but disease etiology in the Mediterranean is unknown. A selection of 42 isolates of *Mycosphaerellaceae* obtained from greasy spot symptoms in humid areas in Ghana and Panama and arid regions in Spain and Morocco was studied. Isolates were characterized based on morphology, growth rate at different temperatures, multi-locus analysis (ITS, EF-1α and RPB2), and pathogenicity tests. Isolates from Panama and Ghana (n=24) were identified as *Z. citri-griseum* whereas isolates from Spain and Morocco (n=20) were identified as *Amycosphaerella africana*. The optimum growth temperature of *Z. citri-griseum* was 27.9°C while *A. africana* grew better at 22.8°C. Isolates of *Z. citri-griseum* from Panamá were pathogenic to 'Valencia' sweet orange plants. None of the *A. africana* isolates from Spain induced symptoms in 'Ortanique' plants. These results confirmed *Z. citri-griseum* as the causal agent of greasy spot in Panama, being associated also with the disease in Ghana. Greasy spot symptoms in Spain and Morocco were associated with *A. africana*.

## First report of *Botrytis pseudocinerea* in blueberry fields in southern Chile

E. BRICEÑO (1), E. Briceño (2), S. Aguirre (3), A. Behn (2)

(1) Universidad Austral de Chile, Chile; (2) Universidad Austral Chile, Chile; (3) Univ austral Chile, Chile

*Botrytis cinerea* is the most important pathogen in blueberry crops (*Vaccinium* spp.) in southern Chile, where the rainy and windy weather are highly favorable to its reproduction. Due we had observed a high morphological variability on *Botrytis* isolates from different commercial fields, the aim of this work was to perform a molecular study of isolates of *Botrytis* associated with blueberry gray mold. To estimate the diversity of this pathogen, 39 monosporic cultures of *Botrytis* obtained from mature blueberry fruits from eight locations (Lat. 39°06'S to 41°28'S) and eight cultivars were genetically characterized. Their genotype was determinate by PCR detection of transposons Boty and Flipper. In addition, coding fragment for the MS457 gene was sequenced and a phylogenetic tree was built using the Neighbor-joining and UPGMA clustering method. The genetic studies showed the presence of four genotypes, predominating Vacuma (57%) in the geographic region, then Boty (18%), Flipper (15%) and finally Transposa (10%). MS457 gene only was present in 10/39 isolates, its homology and phylogenetic analysis showed the presence of two sympatric populations of *Botrytis* on blueberry, allowed us to identify eight isolates as *B. cinerea* and two isolates as *Botrytis pseudocinerea*. In our knowledge this is the first report de *B. pseudocinerea* in blueberries in Chile.

## Identifying and characterizing fungal pathogens causing seedling diseases of soybean through a multi-state survey

A. WARNER (1), J. Bond (2), A. Fakhoury (2)

(1) Southern Illinois University Carbondale, U.S.A.; (2) Southern Illinois University Carbondale, U.S.A.

Seedling diseases are common and very detrimental to soybean, *Glycine max*. They are caused by a variety of plant pathogens including fungi, bacteria, and oomycetes. The central goal of this project was to identify fungal pathogens causing seedling diseases of soybean in various soybean producing states. Two approaches were used for genus and species determination, including morphological features using microscopy and the sequencing of specific genetic loci. For sequencing, up to three different barcodes were targeted, the internal transcribed spacer (ITS), the elongation factor EF-1α, and the intergenic spacer (IGS). The data produced from this project included the identification of approximately 150 fungal isolates per state from eight different states: Arkansas, Indiana, Illinois, Iowa, Nebraska, Michigan, Minnesota, and Kansas. Isolates were collected in 2012 and 2013 to help determine the predominant causal agents of seedling diseases of soybean in those surveyed locations. A variety of genera were isolated from the fields surveyed, with *Fusarium* species constituting approximately 74% of the isolates. Data analyses also involved determining associations and correlations between different factors, such as cultural practices, soil physical and chemical properties, irrigation practices, cropping history, and the frequency of the incidence of the species in the surveyed locations.

## Fusarium canker on holly in Norway

G. STRØMENG (1), V. Talgø (1), A. Stensvand (1), J. Razzaghian (1)

(1) NIBIO, Norway

Holly (*Ilex aquifolium*) thrives well in the mild and wet costal climate of southwestern Norway, where it is commonly harvested for Christmas decorations. During the last decade, a *Fusarium* sp. has frequently been found to damage both natural and cultivated stands, causing leaf drop, dead twigs, canker wounds and berry rot. The most severe outbreaks of the disease ever occurring were observed in late autumn 2015. Isolates of the fungus are most similar to *F. acuminatum* (97% identical TEF sequences). On one occasion, the fungus was detected on dead shoots of Nordmann fir (*Abies nordmanniana*). Inoculation tests proved it pathogenic to both host plants. Many holly stands are no longer intensively cultivated, and they have become very dense and overgrown by other vegetation, thus creating an ideal microclimate for fungal pathogens. In an investigation in a natural stand in 2006-2009, the quality of the holly improved by letting more air and light into the dense plantation by different combinations of pruning, thinning, clearance of unwanted trees and bushes, and grazing with sheep. Thus, it is possible to gain control with the disease by sanitation and proper management, and thereby increase both the quality and quantity for marketing. There is a potential market in other Nordic countries where the climate is too harsh for holly, but earlier attempts of export fell through due to quality issues.

## Fungal Diseases of *Stevia rebaudiana* Grown in Eastern Tennessee

T. COLLINS (1), M. Dee (1), H. Korotkin (1), D. Hensley (1), B. Ownley (1)

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

Extract of *Stevia rebaudiana*, a plant native to Brazil and Paraguay, is a substitute for sugar to sweeten food and drink products. In the last five years, acreage of stevia for commercial production has increased significantly in the southeastern USA. Crops produced in large acreage monoculture are more susceptible to pathogens; potentially destroying whole crops or limiting crop yields. The aim of this study was to identify fungi recovered from diseased stevia field plants grown in Eastern Tennessee. Fungi were screened for pathogenicity in detached stevia leaf assays. Isolates that caused significant necrosis on detached leaves were identified and evaluated in replicated, greenhouse plant disease assays. Pathogens with significant potential to cause disease were identified as *Fusarium armeniacum*, *Botrytis cinerea*, and *Alternaria alternata*. Fungal identification was based on morphological characteristics and sequence identity of the internal transcribed spacer region of 18S ribosomal DNA, and translation elongation factor 1-alpha. Nucleotide amplicons obtained with PCR, were compared with published sequences in Fusarium-ID and the National Center for Biotechnology Information. In greenhouse assays, inoculated plants exhibited necrotic leaf lesions, similar to those observed in field plants. The three fungi were re-isolated from inoculated plants and identity was re-confirmed. Early identification and management of known stevia pathogens will help reduce crop loss.
**Interaction between a rust fungus and Colletotrichum truncatum**

J. DIANESE (1), E. Souza (1), H. Vale (1), R. Pereira-Carvalho (1)

(1) Departamento de Fitopatologia, Universidade de Brasília, Brazil

Mixed natural infection by *Uromyces* and *Colletotrichum* species in *Euphorbia hirta* lead to characteristic symptoms with rust pustules surrounded by black setose acervuli, formed exclusively in the presence of the rust fungus. Besides morphologically identified with precision, *U. euphorbiae*, was now characterized comparing sequences of the 28S rDNA with GenBank accesses of *Uromyces* species. Bayesian Inference (BI) and Maximum Likelihood (ML) analyses showed that *U. euphorbiae* is phylogenetically close to the type species, *U. appendiculatus*, and well distinguished from the remaining species considered, including those pathogenic to Euphorbiaceae. The *U. euphorbiae* type species reported on *E. hypericifolia* was collected in Albany, New York, USA in 1873. As the sequences from our specimen from Brazil, far from the type location, are not suited for its epitypification, it is here designated as a reference specimen, obtained from genomic DNA extracted from teliospores, and deposited in GenBank under number: KU133293, corresponding to our herbarium voucher: UB Mycol. Col. 22311. Two *Colletotrichum* isolates, one endophytic from symptomless areas of the leaves, and another from an acervulus close to rust pustules were isolated. For both isolates, sequences of the ITS, GAPDH, BTUB, and HIS were obtained and subjected to BI and ML analyses. The result clearly indicated, confirming the morphological analyses, that both isolates belong to the same species, *C. truncatum*.

**Thielaviopsis punctulata Causes Black Scorch Disease on Date Palm in the United Arab Emirates**

K. EL-TARABILY (1), S. AbuQamar (1), E. Saeed (1), A. Sham (1)

(1) United Arab Emirates University, UAE

Date palm (*Phoenix dactylifera* L.) is one of the most important plants grown in the arid and semi-arid land that tolerates extreme environmental desert conditions. The main associated pathogens causing palm diseases are attributed to fungi and phytoplasma. In the United Arab Emirates (UAE), the causal agent of black scorch on date palm was found to be the fungal pathogen, *Thielaviopsis punctulata*. The pathogen was isolated from all tissues of diseased trees with virulent *Thielaviopsis punctulata*. Depending on the severity of the infection, symptoms included tissue necrosis, wilting, neck bending, death of terminal buds, and eventually plant mortality. This fungus, which was consistently isolated on potato dextrose agar from infected tissues, produced two types of conidia: the thick-walled aleuroconidia (chlamydospores) and phialoconidia (endoconidia). In addition, all target regions of 5.8S rRNA (ITS), 28S rDNA, *β-tubulin* and transcription elongation factor 1-*α* (*TEF1-α*) genes were amplified using polymerase chain reaction. We also found that the fungicide Score® inhibited the mycelial growth of *Thielaviopsis punctulata* both *in vitro* and *in vivo*. Altogether, the morphology of the fruiting structures, pathogenicity tests and molecular identification confirmed that the causal agent of symptomatic tissues is *Thielaviopsis punctulata*. This is the first report of the black scorch disease and the fungus *Thielaviopsis punctulata* on date palm in the UAE.

**Fungal Species Associated with Olive Dieback of Branches and Evaluation of Cultivar Resistance to Botryosphaeriaceae species**

J. MORAL (1), C. Agustí-Brisach (2), M. Pérez-Rodríguez (2), C. Xavier (2), A. Rhouma (3), A. Trapero (2)

(1) University of California, Davis, Kearney Agricultural Research and Extension Center, Parlier, CA, U.S.A.; (2) Departamento de Agronomía, ETSIAM, Universidad de Córdoba, Campus de Rabanales, Edif. C4, 14071, Cordoba, Spain; (3) Lab. Improvement and protection of olive genetic resources, Olive Tree Institute, BP 208 Mahrajene 9 City– 1082, Tunis, Tunisia

Over two consecutive seasons, 16 olive orchards with trees exhibiting dieback symptoms of branches were surveyed in Southern Spain. The six most frequent fungal species recovered were morphologically, molecularly (by sequencing of the ITS, TUB, and LSU regions), and pathogenically characterized. The resistance of the 11 most important table cultivars to *Neofusicoccum mediterraneum* and *Botryosphaeria dothidea*, the causal agent of “escudete” (small shield) of fruit, was also studied by inoculation of branches and immature fruits, respectively. The species *Cytospora pruinosa*, *N. mediterraneum*, *Nothophoma quercina*, *Phoma incompta*, and *Phomopsis* sp. were identified. Only *N. mediterraneum* and *P. incompta* were able to induce the typical dieback symptoms and cankers that affected plants development. *N. mediterraneum* was the most virulent of the evaluated species although difference of virulence among their isolates were observed. The remaining fungal species were weakly pathogenic on potted plants. According to resistance tests, cv. Gordal Sevillana followed by the cvs. Manzanilla Cacereña and Manzanilla de Sevilla were the most susceptible to branch dieback caused by *N. mediterraneum*. Furthermore, the fruits of cv. Aloreña de Atarfe followed by those of cvs. Ocal and Manzanilla de Sevilla were the most susceptible to *B. dothidea*. Knowledge of the etiology and cultivar resistance of these diseases will help to establish better control measures.

**Identification of Alternaria species associated with tomato and potato early blight disease in Oman**

H. MAYTON (1), A. Al-Rubaii (2), S. Al-Kaabi (2), M. Al-Jabri (2), R. Al-Maqbali (2), A. Al-Adawi (2)

(1) Ministry of Agriculture and Fisheries Sultanate of Oman, Cornell University, Oman; (2) Ministry of Agriculture and Fisheries Sultanate of Oman, Oman

In Oman, approximately 1400 hectares of tomatoes are planted each year with an annual production that exceeds 74000 tons. Tomatoes are ranked as the most important vegetable crop grown by local farmers. Early blight is a devastating disease in Oman and affects both tomato and potato production. In order to manage the disease more effectively, the Ministry of Agriculture and Fisheries in Oman initiated a project to characterize the pathogen population. Samples were collected from infected tomato and potato plants in commercial production fields during the 2014 and 2015 growing seasons from states in the north and south Al-Batinah governates in Oman. Alternaria spp. were isolated from symptomatic tissues. Microscopic examination of cultures from single spore isolations revealed three distinct species: Alternaria tenuissima, A, alternata and A. solani. Molecular identification was achieved through amplification and sequencing of internal transcribed spacer (ITS) and histone 3 gene regions. Phylogenetic analysis of the ITS gene clearly separated Alternaria solani from the other two species. Partial coding of histone 3 gene revealed three clades representing A. alternata (440bp), A. solani (489bp) and A. tennusima (546bp). Pathogenicity tests of the sample isolates on detached tomato leaves of cultivar ‘Ginan’ showed brown lesions similar to symptoms observed in field. No symptoms were observed in water-inoculated control tomato leaves.

**Morphological and Molecular Characterization of Plant-Parasitic Nematodes Populations from Corn Fields in Ohio**

A. SIMON (1), T. Niblack (1), P. Paul (2), H. Lopez-Nicora (1)

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

Results from an Ohio survey in 2013 and 2014 showed that root-lesion nematode, among the most economically important plant-parasitic nematodes, was detected in more than 80% of the 425 fields sampled. Other plant-parasitic nematodes extracted were the lance, stunt, dagger, pin and spiral nematodes, found in 48, 37, 57, and 94% of fields, respectively; however, the impact of these nematodes on corn in Ohio remains unclear. Because damage caused by nematodes varies among genera and species within genera, it is necessary to characterize these nematodes to species. Nematodes were killed, fixed, and mounted before examination with a compound microscope, and identified to species based on quantitative and qualitative morphological characteristics with the aid of diagnostic keys. Three species with distinct morphological features of the genus *Pratylenchus* were identified as *P. penetrans*, *P. neglectus*, and *P. thornei*. Two species of *Tylenchorhynchus* were identified as *T. silvaticus* and *T. nudus*. Species of the lance, pin, spiral and dagger nematodes, namely *Hoplodaimus galeatus*, *Paratylenchus neoamblycephalus* and *Helicotylenchus pseudorobustus* were also identified based on morphology. Species identity will be further confirmed based on analyses of the D2-D3 regions of 28S rRNA gene and ITS rRNA gene. Findings from this study will be useful for establishing experiments to develop damage relationships and management programs for corn nematodes in Ohio.
## Vermiform plant-parasitic nematodes on soybean in North Dakota and their relationship with soybean cyst nematode

G. YAN (1), G. Yan (2), A. Plaisance (3)

(1) North Dakota State University, U.S.A.; (2) North Dakota State University, Department of Plant Pathology, U.S.A.; (3) North Dakota State University, Department of Plant Pathology, U.S.A.

Plant-parasitic nematodes (PPN) are an important group of pests on soybean. The occurrence and population densities of PPN other than soybean cyst nematode (SCN; *Heterodera glycines*) are unknown in soybean fields of North Dakota. A survey was conducted in 2015 in soybean fields or fields with history of SCN to ascertain the incidence and abundance of vermiform PPN and their possible association with SCN. Vermiform and cyst nematodes were extracted separately from each sample (100 cm³ soil) and then identified and quantified. Out of the 155 soil samples collected from 14 counties in North Dakota, 144 were found to be infested with PPN. Eight genera of vermiform PPN were detected including *Helicotylenchus* (incidence: 49%; highest density: 1,800; average density: 174), *Tylenchorhynchus* (41%; 340; 30), *Paratylenchus* (37%; 2,480; 151), *Pratylenchus* (19%; 245; 9), *Xiphinema* (7%; 180; 4), *Paratrichodorous* (4%; 60; 1), *Hoplolaimus* (3%; 140; 2), and *Mesocriconema* (1%; 300; 2). Both SCN juveniles (24%; 1,200; 46) from soil and SCN eggs (56%; 21,540; 501) from cysts were counted, but weak correlation was found between them for all samples ($r = 0.489$; $p < 0.0001$). Poor or no correlations between population densities of each genus of vermiform nematodes and SCN were observed with $r$ ranging from -0.307 to 0.248. More soil samples will be collected in 2016 to confirm the occurrence and abundance of nematodes in soybean fields and the relationship of vermiform nematodes with SCN.

## Occurrence of *Phytophthora syringae* causing Phytophthora fruit rot in apple in Chile

G. DÍAZ (1), M. Lolas (1), R. Méndez (1), J. Contreras (2), M. Caceres (1)

(1) Universidad de Talca, Chile; (2) Universidad de Talca, Chile

CCorean apple industry is one of the major exporter of fresh apples worldwide with an export volume of 628.247 ton. Maule Region (35°25′) is the most important apple production region in Chile with a 60% of production of fresh apple. The fungal diseases in postharvest, is one of the major problems for Chilean industry of fresh apple. Recently was described speck rot caused by *Phacidiopycnis washingtonensis* in apple in Chile. During survey for postharvest diseases of apple in 2014-2015, a postharvest rot was observed on stored apple cv. Jonagold and Pink Lady collected in packinghouses. Symptomatic apples (n=40) were characterized by an initial light brown skin discoloration with spongy to firm flesh texture. With the objective to isolate the causal agent, small fragments were collected from diseased tissues and aseptically placed on Corn Meal Agar and PDA. A *Phytophthora* sp. was consistently isolated. Eight representative *Phytophthora* isolates were characterized for morphological and molecular identification, pathogenicity tests and sensitivity to fungicide. Based on morphology and phylogenetic analyses, this fungus was identified as *Phytophthora syringae*. All isolates grew at 0°C. Inoculated apple fruits developed necrotic lesions. The isolates were sensitive to dimethomorph. To our knowledge, this study is the first description of *P. syringae* causing Phytophthora fruit rot on apple during postharvest in Chile.

## Phytophthora root and crown rot on field-grown lavender plants in 2015

S. JEFFERS (1), S. Sharpe (1), M. Williamson (2)

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

*Phytophthora nicotianae* has a very wide host range, including numerous ornamental plants. In Jun 2015, *P. nicotianae* was isolated from lavender plants and rooted cuttings (*Lavandula angustifolia* and *L. ×intermedia*) with symptoms of Phytophthora root and crown rot (PRCR). The plants recently had been planted in a field in northwestern SC. When this was brought to the attention of the US Lavender Growers Association (USLGA), it was discovered that other growers suspected problems with PPRC—particularly on recently-planted, nursery-grown plants. USLGA members were encouraged to send samples to Clemson University to test for *Phytophthora* spp. Between Jul and Nov 2015, plant and soil samples were received from 12 states; all plants were cultivars of *L. angustifolia* or *L. ×intermedia*. Samples from 11 states were diagnosed with PPRC; *Phytophthora* spp. were recovered from roots and crowns by isolation on selective media and from soil with a baiting bioassay. Isolates were tentatively identified by morphology, but species identification will be confirmed by DNA sequences. Three species of *Phytophthora* were isolated: *P. nicotianae* was isolated most frequently—from samples from 11 states; *P. palmivora* was found in samples from 3 states; and *Phytophthora* sp. was found in one sample. Previously, PPRC has been reported on English lavender, *L. angustifolia*, but not on hybrid lavender, *L. ×intermedia*. Lavender plants infected with *Phytophthora* spp. may have come from the nursery. Authors acknowledge CONICYT PhD fellowship (MV), CONICYT-GO (MV), USM 131562, FONDECYT 1151174& CBDAL grants (MS).

## Four species of *Phytophthora* recovered from roots of American and hybrid chestnut seedlings and associated soils in the southeastern US

S. HARPE (1), S. Jeffers (1), S. Clark (2)

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

Since Phytophthora root rot (PRR) was first described on American chestnut in 1932, the only species of *Phytophthora* reported as a causal agent has been *P. cinnamomi*. In Europe, however, multiple species of *Phytophthora* are involved in PRR of native chestnut trees. In 2009, the USDA Forest Service began evaluating American, Chinese, and hybrid chestnut seedlings in forest test plots in NC, TN, and VA, but PRR caused some seedlings to die prematurely. The objective of this research was to determine if multiple species of *Phytophthora* may be causing PRR of American and hybrid chestnut seedlings. A total of 260 root and 454 soil samples were assayed from 2010 to 2014. *Phytophthora* spp. were recovered by isolation from roots on PARPH-V8 selective medium and baiting soil with rhododendron and camellia leaf pieces. All isolates were tentatively identified based on morphology; species identifications will be confirmed by DNA sequences. *P. cinnamomi* was recovered most frequently—from 41 (16%) root and 109 (24%) soil samples. However, *P. cambivora* and *P. heveae* were also recovered—from 18 (7%) and 2 (<1%) root samples and 18 (4%) and 17 (4%) soil samples, respectively. At a separate test site in SC, *P. drechsleri* was isolated from roots of several hybrid chestnut seedlings. This is the first report of *Phytophthora* species other than *P. cinnamomi* associated with PRR of pure and hybrid American chestnut. These species are being tested for pathogenicity to American chestnut.

## Two new races and several novel strains of the spinach downy mildew pathogen *Peronospora farinosa* f. sp. *spinaciae*

C. FENG (1), J. Correll (1), K. Saito (1), K. Kammeijer (2), S. Koike (3)

(1) University of Arkansas, U.S.A.; (2) University of California, U.S.A.; (3) University of California Cooperative Extension—Monterey County, U.S.A.

Downy mildew disease, caused by *Peronospora farinosa* f. sp. *spinaciae* (Pfs), is the most economically important disease of spinach. Current high-density spinach production provides conducive conditions for disease development, particularly for the increasing acreages of organic spinach. The use of resistant cultivars to manage downy mildew exerts strong selection pressure on the pathogen and many new races of Pfs have emerged in recent years threatening spinach production worldwide. To monitor race diversity of Pfs, downy mildew samples were collected from major spinach production areas and tested for race identification with a standard set of differentials. Two new races (races 15 and 16, sanctioned by the IWGP) and several novel strains have been identified in the past 3 years. The disease reactions of Pfs15 (isolate UA4712) was identical to that of race 4 except the race 4 could not overcome the RPF9 locus resistance and race 15 could. The RPF1, 2, 4, and 6 loci provided resistance to Pfs15. Pfs16 (isolate UA201519B) could overcome the resistance of RPF2, 4, 5, 9, and 10 but not RPF1, 3, and 6. One novel strain (UA1014APLP) could overcome all known spinach resistant loci, but it only infects cotyledons and not true leaves of some cultivars. None of the 360 USDA spinach germplasm accessions tested has resistance to Pfs16 or UA1014APLP. Improved resistance is needed for protection of spinach to the highly variable spinach downy mildew pathogen.
**First report of tomato late blight disease in Oman**

H. MAYTON (3), S. Al-Kaabi (1), M. Al-Jabri (1), W. Al-Shibili (1), W. Fry (2), A. Al-Adawi (1)

(1) Ministry of Agriculture and Fisheries Sultanate of Oman, Oman; (2) Cornell University, Ithaca, NY, U.S.A.; (3) Ministry of Agriculture and Fisheries Sultanate of Oman, Cornell University, Oman

Prior to 2013, tomato late blight had not been reported from the Sultanate of Oman. Tomato is an important vegetable crop, with approximately 1400 ha of tomatoes in Oman. However, in 2013 a disease resembling late blight was observed in several states, with some farms reporting destruction of 100% of the area planted with tomatoes and decrease in production of 30-90% for other farms. The causal agent of late blight, Phytophthora infestans, was successfully isolated from infected plant tissues in February and March 2013. Identification of P. infestans was achieved through microscopic observation and molecular analyses. Sporangia were lemon shaped, semipapillate and caduceus ranging from 36 to 41 µm in length and 13 to 30 µm in width. Internal transcribed spacer (ITS) and cytochrome c oxidase 1 (Cox1) gene regions were amplified and sequenced. BLAST search for ITS and Cox1 sequences of isolates collected from Oman showed 100% similarity with sequences of P. infestans isolates available in genebank (NCBI). To confirm pathogenicity of P. infestans in tomato, three isolates were selected to fulfill Koch's postulates. A spore suspension with concentration of 2.5 × 10⁴ sporangia/ml of each isolate was inoculated on tomato leaves of the cultivar "Ginan" and incubated at 18 Co for 12 days. Symptoms of water soaked and greyish black lesions were observed on inoculated leaves while tomato leaves inoculated with sterile water remained healthy.

**Red clover vein mosaic virus (RCVMV), a recently reported disease agent from alfalfa, cultivated crops and weeds in Saudi Arabia**

I. ALSHAHWAN (1), I. AlShahwan (2)

(1) King Saud University, Saudi Arabia; (2) College of Food and Agric. Sciences, King Saud University, Saudi Arabia

During a two years survey (2012-2013), 1166 samples were collected from symptomatic alfalfa plants in the major alfalfa-producing regions in Saudi Arabia to detect RCVMV and other viruses. Fourteen samples were positive by DAS-ELISA to RCVMV. In 2014, a detailed study was performed to determine the status and to characterize RCVMV where 315 samples were collected from the three utmost producing regions. Fifty six samples were positive, which were confirmed by RT-PCR. Re-PCR products of 932 bp were obtained from the cDNA of RCVMV-infected plants. In the host range study five (alfalfa, black eye pea, cowpea, Gomphrena and glutinosa tobacco) out of 17 plant species resulted in variable symptoms which were confirmed by ELISA, PCR and nucleic acid hybridization assay. This study reports for the first time that cowpea, black eye pea and *Nicotiana glutinosa* are hosts for RCVMV. This is the first detailed report regarding RCVMV infecting alfalfa in Saudi Arabia and its characterization. Data regarding infection of field crops and weeds with this virus will be included. Sequence Homology tree revealed high CP sequence similarity between the Saudi isolates of RCVMV (99-100%), but low CP sequence similarity between the Saudi isolates and other foreign isolates of RCVMV (35%). Phylogenetic analysis of sequences of the Saudi isolates of RCVMV and 40 other CP sequences of Carlavirus species from GenBank grouped in the same clade though they had low homology percentages.

**Rubus yellow net virus: An integrated element in the raspberry genome**

A. Diaz-Lara (1), N. Mosier (2), K. Keller (2), R. Martin (2)

(1) Oregon State University, U.S.A.; (2) USDA-ARS Horticultural Crops Research Unit, U.S.A.

*Rubus yellow net virus* (RYNV) infects *Rubus* spp. causing a serious decline when present in mixed infections and a vein chlorosis in indicator plants. RYNV belongs to the family *Caulimoviridae*, which can exist as infectious particles (exogenous) or integrated elements (endogenous). It was observed that RYNV PCR-positive plants often did not induce symptoms in bioassays, suggesting an integrated element. Molecular techniques were used to discriminate between exogenous and endogenous RYNV sequences. Reverse transcription-PCR using RYNV specific primers after DNase treatment produced positive results for the virus in graft transmissible isolates only. To confirm these results, rolling circle amplification on DNA preparations from these plants resulted in amplicons whose sequence belonged to RYNV. This suggested that graft-transmissible isolates contained an exogenous version of the virus. In Southern blot analysis using *Rubus* genomic DNA digested with *Bam*HI (one recognition site in the RYNV genome), samples that were positive in bioassays for RYNV gave a single band the size of the RYNV genome, while plants with non-transmissible isolates produced three extra bands. Next generation sequencing and appropriate bioinformatics programs are being used to identify the specific integration sites. These results show that RYNV can be integrated into the raspberry genome and highlight the need to consider this phenomenon in future *Rubus* quarantine and certification programs.

**Raspberry leaf curl disease**

P. DI BELLO (1), A. Diaz-Lara (2), R. Martin (3)

(1) Oregon State University, U.S.A.; (2) Oregon State University, U.S.A.; (3) USDA-ARS Horticultural Crops Research Unit, U.S.A.

Raspberry leaf curl disease (RLCD) was first reported in the late 19th century in eastern Canada and northeastern U.S. and is a sporadic destructive disease of red raspberry. Symptoms include leaf curling, leaf distortion, leaf chlorosis, shoot dwarfing, and shoot proliferation. Infected plants produce crumbly, unmarketable fruit, show severe decline within a few years and are much more susceptible to winter injury, which can kill infected plants. RLCD is vectored by the aphid *Aphis rubicola* in a persistent manner. Based on transmission properties it is suspected RLCD is caused by a virus or virus complex. To determine the viral etiology of RLCD, total nucleic acids were extracted from two red raspberry plants showing typical symptoms of RLCD, digested with DNase, rRNA removed and finally subjected to next generation sequencing on the Illumina Miseq platform using the paired end 150 bp kit. Sequences were analyzed with the VirFind pipeline and specific primers were designed to validate potential virus-like sequences. RLCD infected plants were grafted to blackberries and raspberries, and vector studies with aphids were undertaken. The virome of the plants exhibiting RLCD-like symptoms will be presented.

**Current Status of Grapevine Virus Diseases in British Columbia**

S. POOJARI (1), J. Boulé (1), N. DeLury (1), T. Lowery (1), M. Rott (2), A. Schmidt (2), J. Úrbez Torres (1)

(1) Summerland Research and Development Centre, Canada; (2) Canadian Food Inspection Agency, Canada

Grapevine leafroll disease (GLRD), caused by Grapevine leafroll associated viruses (GLRaVs), is considered the most wide spread virus disease of grapevine (*Vitis vinifera* L.) worldwide. British Columbia (BC) is the second largest grape-growing region in Canada with most of the acreage planted in the Okanagan Valley. To understand the epidemiology of GLRaVs in BC vineyards, a comprehensive survey was conducted during the 2014 and 2015 growing seasons throughout all wine growing regions of BC. The status of GLRaVs in BC was determined by analyzing a total of 2740 random composite samples representing 12 white and 9 red cultivars. Both ELISA and PCR based diagnostic test results showed GLRaV-3 to be the most prevalent (23.9%) followed by GLRaV-2 (7.1%), GLRaV-1 (2.7%) and GLRaV-4 (2.7%). GLRaVs mixed infections were detected in different combinations from composite samples with GLRaV-1 + GLRaV-3 (2.5%) to be the most common, followed by GLRaV-2 + GLRaV-3 (2.1%) and GLRaV-3 + GLRaV-4 (0.9%). Our results also revealed the presence of *Grapevine fan leaf virus*, *Grapevine fleck virus*, *Grapevine Pinot gris virus*, Grapevine red blotch associated virus and *Rupestris stem pitting associated virus* (RSPaV). These results along with information generated on the disease progress and insect vectors in BC will emphasize the importance of a clean plan network program for sustainability of Canadian wine grapes.

**The prevalence of grapevine leafroll and red blotch diseases in Washington vineyards**

J. ADIPUTRA (1), P. Swamy (1), B. Donda (1), B. Bagewadi (1), N. Natra (1), R. Naidu (1)

(1) Washington State University, U.S.A.
Grapevine leafroll disease (GLD) is a significant constraint to the production of wine grapes (Vitis vinifera) in Washington State. Grapevine leafroll associated virus (GLRaV) 1, 2, 3, and 4 have so far been documented, with GLRaV-3 being the most widely distributed in Washington vineyards. However, recent reports of the occurrence of grapevine red blotch disease (GRBD) have raised concerns about the extent of its distribution in Washington vineyards. Since the symptomatology of GLD and GRBD is highly similar in wine grape cultivars, reliable information on the prevalence of these two diseases is necessary for deploying appropriate disease mitigation strategies. Towards this objective, surveys of vineyards were conducted during 2014 and 2015 seasons in seven American Viticultural Areas of Washington. A total of 1,469 samples collected from fourteen red-berried and five white-berried wine grape cultivars were tested for the presence of GLRaV-3 and Grapevine red blotch-associated virus (GRBaV) by polymerase chain reaction (PCR)-based diagnostic assays. The PCR results were further confirmed by cloning and sequencing of virus-specific amplicons. The combined data showed that 83.7% and 13.7% of samples were positive for GLRaV-3 and GRBaV, respectively, indicating that GLD is predominant relative to GRBD in Washington vineyards. A multiplex PCR assay has been optimized for high throughput testing of grapevine samples for the presence of GLRaV-3 and GRBaV.

## Association of *Grapevine rupestris* stem pitting-associated virus and *Grapevine Pinot gris* virus in declining grapevines on Schwarzman rootstock

H. MCCOWN (1), S. Bag (1), M. Eng (1), M. Sudarshana (2)

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

In the fall of 2014, in Napa County, California, four-year-old grapevines of Cabernet Sauvignon (Vitis vinifera) on Schwarzmann (V. riparia X V. rupestris) rootstock were found to exhibit red canopy and vine decline. Upon examination of the woody cylinder, the rootstocks, but not the scions, had extensive stem pitting. To investigate the causal agent(s), nucleic acid extracts from affected grapevines were analyzed for viruses by RT-PCR assays. These vines tested positive for the Syrah strain of Grapevine rupestris stem pitting-associated virus (GRSPaV-Syrah) and negative for five leafroll-associated viruses, three vitiviruses, and Grapevine red blotch-associated virus. Further examination by deep sequencing of a cDNA library, prepared from the dsRNA fraction of cambial scrapings from the affected trunks, and subsequent bioinformatics analysis, revealed the presence of Grapevine Pinot gris virus (GPGV) in addition to GRSPaV-Syrah. Canopy surveys conducted in the fall of 2015 indicated that >40% (n=342) of the vines had declined. It is likely that the association of GPGV and GRSPaV may have a synergistic effect resulting in the stem pitting symptom.

## Survey of *Arabis Mosaic and Grape Leaf Roll Associated virus* in Maryland vineyards

R. POKHAREL (1)

(1) Maryland Dept of Agriculture, U.S.A.

*Arabis mosaic virus* (ArMV) and *grape leaf roll associated virus* (GRLaV) cause significant crop loss in grapes. However, their natural incidence in Maryland vineyards is not known, and several growers have reported symptoms similar to those caused by these viruses. To find their distribution, 15 vineyards were surveyed during August to September 2015 in Maryland. Up to six varieties per vineyard were examined for virus symptoms. Up to five samples (symptomatic, if present) were collected from each variety. Two hundred seventy samples were tested for each virus group, including the three most symptomatic samples per variety. These samples were tested using a double-antibody sandwich (DAS)-enzyme-linked immunosorbent assay (ELISA) kit developed by Bioreba for GRLaV, and by Agdia for ArMV. Three groups of GRLaV (GRLaV-1+3 and GRLaV-2) were tested in two groups. Forty percent of vine samples collected were symptomatic for GRLaV and 29% were symptomatic for ArMV. Only ten percent of samples were found positive for ArMV. Similarly, only 1.1% of samples tested were positive for (GRLaV 1 + GRLaV 3) and 1.5% of samples were positive for GRLaV-2. This result indicated that many plants exhibiting symptoms similar to GRLaV and ArMV were negative for these viruses. This may be because symptoms observed in the field were produced by causes other than the virus tested. Further testing of such vines for other virus and virus-like pathogens may help growers to manage this problem.

## First report of *Watermelon chlorotic stunt virus* in rain-fed farms in Usfan, Saudi Arabia

N. CHINGANDU (1), M. Al-Saleh (2), A Idris (3), J. Brown (1)

(1) University of Arizona, Tucson, AZ, U.S.A.; (2) Department of Plant Protection, King Saud University, Saudi Arabia; (3) University of Arizona, School of Plant Sciences, Tucson, AZ, U.S.A.

Watermelon (*Citrullus vulgaris* L.), an important crop in Saudi Arabia, is cultivated in the lowlands and wadis following seasonal winter rainfall, along the western coast. Production of watermelon faces constraints imposed by abiotic stresses and biotic diseases, and insects, including whiteflies. During spring 2015, >95% of watermelon plants growing on a farm near Usfan exhibited foliar chlorosis and mottling, and overall stunting symptoms, reminiscent of those caused by *Watermelon chlorotic stunt virus* (WmCSV) (*Begomovirus, Gemniviridae*). Total nucleic acids were isolated from leaves of three symptomatic watermelon plants, and subjected to rolling circle amplification (RCA). Libraries prepared for the RCA amplicons were subjected to Illumina MiSeq, and reads were assembled using SeqMan NGen software. BLASTn analysis of assembled contigs revealed the presence of the DNA-A and DNA-B genomic components most closely related to the Old World bipartite WmCSV. The three DNA-A components, at 2752 bp, shared 99-100% nucleotide (nt) identity with each other, and 99% nt with a WmCSV isolate from Leith, Saudi Arabia [KJ939448]. The three DNA-B component sequences, at 2757 bp, shared 96-97% nt identity with each other, and 96% nt identity with the DNA-B component of an isolate, also from Leith [KJ939447]. This is the first report of WmCSV in the Leith, a farming community separated from Usfan by a vast, inhospitable desert.

## Necrotic Streaking of Asiatic Lilies caused by *Plantago asiatica mosaic virus* in Chile

X. BESOAIN (1), A. Vidal Takasaki (2), R. Camps (2)

(1) Pontificia Universidad Católica de Valparaíso, Chile; (2) Pontificia Universidad Católica de Valparaíso, Chile

During the spring of 2015 a survey was done in Quillota, in greenhouses with Asiatic hybrid lilies for cut flower. Symptomatic plants were found in four different cultivars. Symptoms were brown necrotic streaking associated with irregular leaf chlorosis, and severely infected plants also showed brown streaks in the stem and irregular necrosis in tepal tips. Only in cv. Litouwen it was observed that microbulbs replaced flower buds. Tissue extracts from symptomatic plants were obtained and RT-PCR of gene RdRp was performed employing specific primers for Potexvirus. The obtained sequences revealed 99% identity with the consensus sequences of PIAMV. After positive results for PIAMV, a pathogenicity test was performed on plants of two cultivars of Asiatic lilies. Two isolates of PIAMV were inoculated in three plants for each isolate, and other three plants were left as controls. Forty-five days after inoculation, plants from Asiatic cv. Indian Summerset showed a shorter stem, necrotic streaks in leaves and in the stems and chlorosis in irregular pattern. Litouwen plants showed symptoms of decreased growth, brown necrotic streaks in the leaves associated with chlorosis and microbulbs instead of flower buds. All symptomatic plants tested positive with specific primer for PIAMV and control plants tested negative. Koch's postulates were fulfilled for PIAMV on *Lilium* plants and the disease was called Necrotic Streaking of Lily caused by PIAMV affecting Asiatic cultivars.

## Trichosanthes latent virus (TrLV), a novel potyvirus from *Trichosanthes cucumeroides* in Japan

O. KIM (1), K. Kobayashi (2), K. Natsuaki (2), H. Shinohara (2), H. Negishi (2)

(1) Tokyo University of Agriculture (TUA), Japan; (2) Tokyo University of Agriculture (TUA), Japan

Only a few studies have been conducted on incidence of plant viruses on wild cucurbit weeds in Japan. As wild and horticultural cucurbits often share same viruses, epidemiological survey as well as precise identification of causal virus(es) is required for the efficient control strategy. We once reported un-common potyvirus tentatively named as JK virus isolated from *Trichosanthes cucumeroides* (Kim et al., 2011). By additional surveys, most of
collected *T. cucumeroides* in Kanto region were found to be infected with JK virus showing high co-incidence with *Papaya ringspot virus* (PRSV). On the contrary, as far as examined, JK virus was not detected from any wild cucurbit samples including *Trichosanthes* spp. in Okinawa Prefecture where *Papaya leaf distortion mosaic virus* (PLDMV) infection in cucurbit weeds have been reported (Maoka and Hataya, 2005). Mechanical inoculation of JK virus indicated a narrow host range limited to family *Cucurbitaceae* and showed symptomless or mild symptom when back-inoculated to *T. cucumeroides*. Compared with sequences of CI or CP regions from other potyviruses, JK virus shared less than 80% identity with PLDMV which was the most closely related potyvirus. Therefore, JK virus can be considered as a novel potyvirus species, for which the name as Trichosanthes latent virus (TrLV) is proposed.

## Identification and characterization of Zucchini tigre mosaic virus on creeping cucumber in Japan

K. KOBAYASHI (1), H. Shinohara (2), H. Negishi (2), O. Kim (2)

(1) Tokyo University of Agriculture (TUA), Japan; (2) Tokyo University of Agriculture (TUA), Japan

Creeping cucumber (*Melothria pendula*), a wild cucurbit weed, showing mottle and distortion symptoms was observed in Okinawa main island, Japan in September 2015. Total RNA using TRIzol reagent extracted from collected samples was used for reverse transcription-polymerase chain reaction (RT-PCR) with degenerate primers (CIFor and CIRev) for potyviruses (Ha et al., 2008) followed by cloning and sequencing. Obtained 637 nucleotides (nts) sequence of partial CI region from isolate Oki-47 showed 86% with corresponding sequence from Zucchini tigre mosaic virus (ZTMV; accession no. KC345554) as a highest identity. When the whole coat protein (CP) coding region was considered, the nt and amino acid (aa) sequence identities shared 94% and 97% with *Papaya ringspot virus* (PRSV; accession no. AB583208) respectively. The compared PRSV isolate was re-classified as one of ZTMV isolates by Romay *et al.* (2014). Serological test by western blotting assay revealed that isolate Oki-47 was closely related to PRSV, but not to *Papaya leaf distortion mosaic virus* (PLDMV). Absence of PRSV, PLDMV, and *Zucchini yellow mosaic virus* (ZYMV) were also confirmed which are known as major causal potyviruses infecting to cucurbits in Japan. This is the first report of ZTMV incidence in Japan as well as creeping cucumber for its natural host.

## Complete nucleotide sequences of a new bipartite begomovirus from *Malvastrum* sp. plants with bright yellow mosaic symptoms in South Texas

O. ALABI (1), C. Villegas (2), L. Gregg (3)

(1) Dept. of Plant Pathology & Microbiology, Texas A&M University AgriLife Research & Extension Center, U.S.A.; (2) South Texas College, U.S.A.;

(3) Texas A&M AgriLife Research and Extension Center, U.S.A.

Two isolates of a new bipartite *Begomovirus*, tentatively named as Malvastrum bright yellow mosaic virus (MaBYMV), were molecularly characterized from naturally-infected *Malvastrum* sp. plants showing bright yellow mosaic disease symptoms in South Texas. The host plant genus identity was determined by DNA barcoding of the non-coding *trnH-psbA* intergenic spacer region and the *matK* plastid gene. Restriction enzyme-digested rolling cycle amplification products from symptomatic plants were used as template in PCR to amplify complete virus genome segments with pairs of newly-designed abutting primers. The obtained ~2.7 Kb fragments were cloned and sequenced. Six complete DNA-A and five DNA-B genome sequences of MaBYMV obtained from the two isolates ranged in length from 2,608-2,609 nucleotides (nt) and 2,578-2,605 nt, respectively. Both genome segments share a 178-180 nt common region. In pairwise comparisons, complete DNA-A and DNA-B of MaBYMV were most identical (87-88% and 79-81%, respectively) and phylogenetically related to corresponding sequences of *Sida mosaic Sinaloa virus*-[MX-Gua-06]. Further analysis showed that MaBYMV is a putative recombinant virus, thus supporting the notion that malvaceous hosts may be influencing the evolution of several begomoviruses. The design of new diagnostic primers enabled the detection of MaBYMV in cohorts of *Bemisia tabaci* collected from symptomatic *Malvastrum* sp. plants, thus implicating whiteflies as potential vector of the virus.

## Infectivity analysis of strain L of chickpea chlorotic dwarf virus, a cotton mastrevirus, in *Nicotiana benthamiana* plants

M. MANZOOR (1), A. Bibi (2)

(1) Institute of agricultural sciences, University of the punjab, Pakistan; (2) institute of agricultural sciences, university of the punjab, Pakistan

Cotton is damaged by a group of viruses, responsible for cotton leaf curl disease complex, belongs to the genus *Begomovirus*; family *Geminiviridae*. The present study unveils another virus which belongs to genus of the same family causing leaf curl disease in cotton. Agrobacterium-mediated inoculation, of strain L of the chickpea chlorotic dwarf virus (CpCDV) (mastrevirus) isolated from cotton plants, to *Nicotiana benthamiana*, alone or in combination with other begomovirus showed a remarkable difference in symptoms from very mild symptoms to severe cup shaped with stunted growth. The variability in symptoms and their development in other parts of tested plants and subject viruses, belonging to different genera, perhaps owing to difference in ORFs of subject viruses.

## Nucleotide sequence and genome organization of a novel *Panicovirus* from Bermuda grass

M. TAHIR (1), B. Lockhart (2), S. Grinstead (1), D. Mollov (1)

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

Bermuda grass (*Cynodon dactylon*) is widely used in tropical and subtropical areas for golf greens, athletic fields, and landscapes. Samples of Bermuda grass displaying symptoms of decline were tested at the University of Minnesota plant virology lab. Spherical virus particles (28-30 nm) were observed by transmission electron microscopy. Total RNA was extracted and sent for Next Generation Sequencing (NGS) on an Illumina platform. Contigs were assembled using CLC Workbench, and analyzed using BLAST searches against reference genomes. A contig of about 4 kb contained four predicted ORFs in an arrangement typical of viruses in the family *Tombusviridae*. Overlapping primer pairs were designed from the consensus sequence for RT-PCR verification and sequence confirmation. All amplicons were cloned and the resulting sequences matched the original NGS contig. Amino acid sequence comparisons of the putative replicase (RdRp) and movement and coat proteins (CP) showed sequence similarities in the range of 37-66% compared to *Thin paspalum asymptomatic virus*, *Cocksfoot mild mosaic virus*, and *Panicum mosaic virus*. Phylogenetic analyses using the CP and RdRP of this newly identified virus place it in the genus *Panicovirus*. The sequence of this Bermuda grass virus is sufficiently different from other viruses to conclude it is a new member of the genus *Panicovirus*, family *Tombusviridae*.

## Identification of virus species infecting pepper in Guangdong province of China

Y. Tang (1), Z. He (1)

(1) Plant Protection Research Institute, Guangdong Academy of Agricultural Sciences, China

Viral disease is one of main diseases of pepper production in Guangdong province of China. During 2013-2015, we surveyed the occurrence of the viral disease and identified the viruses infecting pepper. The diseased samples of pepper were collected randomly from the main pepper-growing areas of Guangdong. The diseased samples of the same survey location were mixed and extracted total RNA for deep sequencing of small RNA. The viruses were verified by RT-PCR, respectively using the specific primers designed according to the assembling sequences. The diseased plants in fields exhibited diversiform symptoms, including stunting, mosaic, chlorosis, leaf narrowing and distortion, and mottled. The sequence analysis results showed that there were 16 species viruses infecting pepper, including *Chilli ringspot virus*, *Broad bean wilt virus*, *Cucumber mosaic virus*, *Pepper vein yellows virus*, *Chilli veinal mottle virus*, *Pepper veinal mottle virus*, *Pepper yellow leaf curl virus*, *Capsicum chlorosis virus*, *Tobacco mild green mosaic virus*, *Pepper cryptic virus*, *Potato virus Y*, *Pepper mild mottle virus*, *Watermelon silver mottle virus*, *Tobacco mosaic virus*, *Bell pepper endornavirus*, *Nicotiana tabacum pararetrovirus-like*. The co-infection with two, three and more than three viruses accounted for 6.02%, 21.29% and 69.88% in one
diseased sample, respectively. It is concluded that the number of virus species infecting pepper crop was abundant and co-infection was common in Guangdong of China.

## Beet curly top virus strains associated with sugar beet in Idaho, Oregon, and a survey collection

C. STRAUSBAUGH (1), I. Eujayl (1), B. Wintermantel (2)

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

Curly top of sugar beet is a serious yield limiting disease in semi-arid production areas caused by *Beet curly top virus* (BCTV) and vectored by the beet leafhopper. The primary means of control for BCTV is host resistance, but effectiveness of resistance can vary with BCTV strain. BCTV variation was last investigated in Idaho and Oregon during a 2006-2007 survey, but changes in disease severity suggested a need for reevaluation. Therefore, 406 leaf samples symptomatic for curly top were collected from sugar beet plants in commercial sugar beet fields in Idaho and Oregon from 2012 to 2015. DNA was isolated and the BCTV strain composition was investigated based on polymerase chain reaction (PCR) assays with strain specific primers for Severe (Svr) and California/Logan (CA/Logan) strains and nonspecific primers that amplified Worland (Wor)-like strains. The 2006-2007 ID/OR BCTV positive samples from sugar beet included the following strains: 87% Svr, 7% CA/Logan, and 60% Wor-like. BCTV strain distribution in the new survey averaged 2% Svr, 30% CA/Logan, and 87% Wor-like. Whole genome sequencing (GenBank accessions KT276895 to KT276920) with overlapping primers, suggests that the Wor-like strains included Wor, Colorado (CO), and a previously undescribed strain designated Kimberlyl (Kim1). These data indicate there was a shift from Svr being one of the dominant BCTV strains in commercial sugar beet fields in 2006-2007 to being undetected at times in recent years.

## Virus diseases: A major threat to high value vegetable crops in Bangladesh and Nepal

A. FAYAD (1), A. Fayad (1), R. Naidu (2)

(1) Virginia Polytechnic Institute and State University, U.S.A.; (2) Washington State University, U.S.A.

The Feed the Future Innovation Lab for Integrated Pest Management (IPM IL), a USAID-funded program managed by Virginia Tech, has been working on identifying major threats to vegetable production in Bangladesh and Nepal. Surveys have been conducted in major production areas including those of smallholder farmers. Results show a trend of emerging and re-emerging virus disease in cucurbits including cucumber, squash, pumpkin and other gourds, tomato, pepper, potato, and okra. Research is focused on identifying sources of virus, with a special emphasis on insect vector and seed transmission pathways. The IPM IL is working with farmers, researchers, and local and international NGOs in Bangladesh and Nepal to address management of these virus diseases, and developing ecologically-based and environmentally friendly IPM strategies that reduce the reliance on synthetic pesticides and improve yield while at the same time reducing damage to the environment and improving nutrition and health. IPM packages have been developed and are being disseminated through farmer participatory approaches including IPM demonstration plots and farmer groups.

## Tomato yellow mottle virus (ToYMoV): Characterization of a newly emergent begomovirus associated with tomato disease in Costa Rica

M. MALIANO (1), M. Rojas (1), T. Melgarejo (2), M. Macedo (3), N. Barboza (4), A. Inoue Nagata (3), R. Gilbertson (5)

(1) UC Davis, Davis, CA, U.S.A.; (2) Universidad Nacional Agraria La Molina, Peru; (3) University of Brasilia, Brazil; (4) Universidad de Costa Rica, Costa Rica; (5) UC Davis, CA, U.S.A.

Tomato production in Costa Rica is affected by diseases caused by whitefly-transmitted begomoviruses. Since the early 1990s, the main virus involved was the New World (NW) bipartite *Tomato yellow mottle virus* (ToYMoV), which was associated with stunting and yellow mottling. To fulfill Koch's postulates, putative full-length were obtained. *Nicotiana benthamiana* and tomato seedlings inoculated with the ToYMoV DNA-A and DNA-B clones developed stunting and yellow mottling of leaves, confirming the infectivity of the clones. Sequence analysis revealed that ToYMoV is closely related to bipartite tomato-infecting begomoviruses from Mexico and Guatemala. Multimeric clones were generated and used to assess the host range of ToYMoV. More recently, the NW bipartite *Tomato leaf curl Sinaloa virus* (ToLCSiV) and the invasive Old World (OW) monopartite *Tomato yellow leaf curl virus* (TYLCV) were introduced to Costa Rica where they have caused yield losses. Pseudorecombinants made between infectious DNA-A and DNA-B clones of ToYMoV and ToLCSiV did not induce symptoms. Tomato plants inoculated with cloned DNAs of TYLCV, ToLCSiV and ToYMoV alone or in mixtures revealed that symptoms of ToLCSiV and ToYMoV appeared earlier than those of TYLCV. However, within 14 days after inoculation, TYLCV symptoms became dominant, although ToLCSiV and ToYMoV were still detected at high levels. These results will be discussed in terms of synergy among these viruses.

## Validation of a novel gene target for detection and quantification of *Clavibacter michiganensis* subsp. *nebraskensis*, the causal agent of Goss's wilt

R. MCNALLY (1), C. Ishimaru (2), D. Malvick (2)

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

Goss's wilt of maize is a reemerging and significant disease caused by the bacterium *Clavibacter michiganensis* subsp. *nebraskensis* (*Cmn*). Despite its expansion in North America, molecular tools for diagnosing and studying the epidemiology of this disease remain limited. Here we report the identification of *CMN_01184* as a novel gene target and demonstrate its use for development of conventional PCR (cPCR) and quantitative PCR (qPCR) assays for detection and quantification of *Cmn*. Primers designed for cPCR and qPCR were evaluated for specific amplification of *CMN_01184* using a diverse collection of 129 bacterial and fungal isolates, including multiple corn pathogens, environmental organisms from agricultural fields, and all known subspecies of *C. michiganensis*. Of these, only isolates of *Cmn* tested positive for *CMN_01184*. To evaluate the utility of *CMN_01184* for *in situ* detection of *Cmn*, cPCR and qPCR based on this target gene was performed on infested field soil and naturally infected corn leaves containing *Cmn*. *CMN_01184* was amplified only in soil samples and leaves containing *Cmn*. Finally, in greenhouse studies, systemic infection of corn leaves by *Cmn* was quantified over a two-week infection period using the qPCR assay based on *CMN_00184*. The results demonstrate collectively that specific and sensitive PCR assays based on *CMN_01184* can aid in diagnosis as well as studies of the epidemiology and ecology of Goss's wilt.

## In-field detection of the select agent *Rathayibacter toxicus* using loop-mediated isothermal amplification

M. ARIF (1), G. Busot (2), R. Mann (3), B. Rodoni (4), J. Stack (5)

(1) Kansas State University, Manhattan, KS, U.S.A.; (2) Department of Plant Pathology, Kansas State University, Manhattan, KS, U.S.A.; (3) Department of Economic Development, Jobs, Transport and Resources, Latrobe University, Bundoora, Australia; (4) Department of Economic Development, Jobs, Transport and Resources, Latrobe University, Australia; (5) Department of Plant Pathology, Kansas State University, Australia

Early, accurate and sensitive detection of *Rathayibacter toxicus* by plant health officials would improve inspections of imported annual ryegrass hay and seed at ports of entry and enhance in-field detection by biosecurity surveillance personnel. The gram positive bacterium and select agent *R. toxicus* produces a toxin in annual ryegrass (*Lolium rigidum*). Livestock deaths in Australia due to toxin contaminated ryegrass have raised concerns about the global spread of *R. toxicus* in ryegrass hay and seed. Genomes from all available *R. toxicus* populations were explored to design target-specific loop-mediated isothermal amplification (LAMP) primers. Gene *rpoD* was selected for primers design. Internal LAMP primers targeting the ITS gene of host plant *L. rigidum* were also designed to enhance reliability and accuracy. Genie II, Sybr Green I and lateral flow device were used for LAMP assays. The developed assays were validated *in silico* against the NCBI GenBank nucleotide database and *in vitro* against the inclusivity and exclusivity panels for specificity. In-field assays were performed in South Australia; accurately detected *R. toxicus* from infected annual ryegrass samples. The developed
LAMP assays are sensitive, accurate and easy to implement. The proposed LAMP method will be helpful in diagnostics, surveys, disease management, and biosecurity decisions to support export/import of annual ryegrass.

# Development of recombinase-polymerase amplification assay for the detection of Western X phytoplasma ('Candidatus Phytoplasma pruni') in sweet cherry

D. VILLAMOR (1), H. Ferguson (1), K. Eastwell (1)

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

Resurgence of Western X disease (WX) of sweet cherry (*P. avium*), caused by WX phytoplasma ('Candidatus Phytoplasma pruni'), has been observed in Washington State (WA) in recent years. The disease presents a major economic impact in WA sweet cherry production as affected trees not only decline but produce unmarketable small, insidious fruits. In this study, an isothermal recombinase-polymerase amplification assay (RPA) for the detection of WX phytoplasma was developed. Using sequence data generated by high-throughput sequencing of three isolates of WX phytoplasma from WA along with sequence available in the GenBank database, primers and probe were selected within the immunodominant protein (idpA) coding region. The RPA assay system has three attributes that make it a very useful tool for detection of WX phytoplasma by cherry growers: (1) it is fast and simple – results are obtained in 20 minutes and because it uses only crude sap preparation, no sophisticated equipment for nucleic acid extraction is necessary; (2) it is sensitive – the RPA assay showed similar sensitivity with PCR both in temporal detection throughout the growing season and the amount of bacterial DNA detected; and (3) it is specific only to WX phytoplasma. Additionally, RPA detected WX phytoplasma from total DNA extracts of three known leafhopper vectors of the pathogen; this makes it a valuable tool in pathogen vector research.

# Viability of *Candidatus* Liberibacter asiaticus in grapefruit leaves at different stages of maturity and Huanglongbing disease symptom development

M. KUNTA (1), E. Louzada (1), P. Vedasharan (1)

(1) Texas A&M University-Kingsville Citrus Center, U.S.A.

Citrus Huanglongbing, associated with *Candidatus* Liberibacter asiaticus (CLas), is the most serious disease in the USA causing substantial economic losses. The real-time PCR (qPCR) assays used to detect CLas have a limitation that they cannot differentiate live and dead bacterial cells. Accurate quantification of live CLas cells in the plant tissue is essential to devise effective disease management strategies and to test the efficacy of new disease control methods. Several optimized quantitative real-time PCR methods using propidium monoazide (VqPCR), a DNA-binding dye, to quantify live CLas populations were described. However, it is unclear if live CLas populations vary with age of the leaf and varying degrees of HLB disease symptom development. To address this question, we have quantified the levels of live CLas populations in grapefruit leaves at different maturity stages and leaves with varying levels of symptoms collected from naturally infected field trees. VqPCR assay results obtained using two different chemistries including SYBR® and TaqMan® will be presented.

# Development of a multiplex real-time PCR for the improved detection of citrus canker-causing *Xanthomonas*

V. MAVRODIEVA (1), G. Santillana (1), G. Dennis (2)

(1) USDA APHIS PPQ S&T Beltsville Laboratory, U.S.A.; (2) USDA APHIS PPQ S&T, U.S.A.

To improve on the current assay for the detection of all citrus canker causing *Xanthomonas* which targets the *pthA* gene, a multiplex assay was developed to incorporate the 16S rRNA gene as a second target. Two new *pthA* amplisets designed to have better sensitivity than the current one, and two new 16S amplisets were evaluated individually, and in multiplex format. The PCR product of each amplisel was sequenced and verified to be the correct targets. Primers and probes interaction was determined and optimal concentrations selected followed by dNTP and MgCl₂ concentrations optimization for the different amplisel combinations. *In silico* and experimental specificity analyses were performed. All amplisets were found to have a 100% match with the target organism. The *pth*-targeted amplisets showed cross-reactivity to some non-citrus *Xanthomonas* but not to other citrus-infecting genera. The method being developed targets symptomatic citrus therefore cross-reactions of the primers with *Xanthomonas* spp. of other crops should not interfere because most xanthomonads exhibit highly restricted host range (Delcourt et al., 2013). One of the 16S amplisets showed insufficient specificity thus was excluded from further optimization studies. Initial comparability studies suggest that the new *pthA* amplisets (used in multiplex with 16S rRNA gene) may be more sensitive than the currently used uniplex (*pthA*), having lower Ct values for the sets of archived diagnostic samples tested.

# Development of a rapid and reliable isothermal AmplifyRP diagnostic assay for specific detection of *Xylella fastidiosa*

R. LI (1), P. Russell (2), N. Mcowen (2), B. Davenport (2), S. Zhang (2)

(1) Agdia Inc., U.S.A.; (2) Agdia Inc, U.S.A.

*Xylella fastidiosa*, infecting 309 plant species belonging to 63 families and 193 genera, is a regulated bacterial pathogen in many countries. Recently *X. fastidiosa* was associated with the devastating olive quick decline syndrome (OQDS) in southeastern Italy, resulting in an unprecedented concern to its spread in Mediterranean countries. In order to meet the needs for quarantine detection and field diagnostics, Agdia developed an advanced AmplifyRP assay for the detection of *X. fastidiosa*. The assay combined both endpoint (Acceler8) and real-time (XRT) detection in a single reaction PCR tube. This characteristic allows users to either collect the quantitative fluorescence data using a portable fluorometer or observe the qualitative test result in a disposable detection chamber. The assay is as sensitive as that of the published quantitative real-time PCR and has no cross-reaction to the closely related *Xanthomonas* species and numerous tested host plants including grape, citrus, and olive. The assay uses crude plant extracts and is applicable for use in both laboratories and fields. This assay provides users a fast and reliable tool to detect *X. fastidiosa* carried by plant materials and insect vectors from the contaminated regions.

# Recombinase polymerase-based diagnostics for in-field detection of *Pseudomonas syringae* pv. *actinidiae*

J. STACK (1), M. Arif (2), J. Rascoe (3), M. Nakhla (3), G. Busot (1)

(1) Kansas State University, U.S.A.; (2) Kansas State University, U.S.A.; (3) USDA APHIS PPQ CPHST, U.S.A.

In-field detection of the kiwifruit pathogen, *P. syringae* pv. *actinidiae* (Psa), will support plant biosecurity decisions regarding trade and production. To detect and discriminate among Psa strains (including the 2008 global outbreak strain, Psa-V), a Recombinase Polymerase Amplification (RPA) assay was developed for laboratory and field use. Primer sets targeting two effector genes (*hopZ3* and *hopZ5*) were designed and screened against isolates of highly virulent Psa and closely related *P. syringae* pathovars. All Psa strains can be detected by the presence of *hopZ3* amplicons but only the virulent type, Psa-V or biovar 3, is detectable by *hopZ5* amplicons. Because RPA assays were coupled to lateral flow device (LFD), reverse primer were labeled with either biotin or digoxigenin and the reaction was developed in the presence of a carboxyfluorescein (FAM) labeled probe. RPA assays had an amplification time of 10 minutes. Sensitivity determinations indicated a detection limit of 1 pg or 100 fg depending of the targeted gene. Isothermal assays are a rapid diagnostic assay that enables the early detection of pathogens and a valuable tool for in-field pathogen detection to support plant biosecurity decisions.

# Evidence for seed transmission of *Xylella fastisiosa* in pecan (*Carya illinoinensis*)

K. CERVANTES (1), D. Ray (1), R. Stamler (2), J. French (1), J. Soneji (3), R. Heerema (4), L. Grauke (5), J. Randall (4)

(1) New Mexico State University, Las Cruces, NM, U.S.A.; (2) New Mexico State University, U.S.A.; (3) NMSU, Las Cruces, NM, U.S.A.; (4) New Mexico State University, Las Cruces, NM, U.S.A.; (5) USDA-ARS Pecan Breeding, Somerville, TX, U.S.A.
*Xylella fastidiosa* is a gram-negative bacterial phytopathogen found exclusively in the xylem tissue of host plants. In pecan (*Carya illinoinensis*), *X. fastidiosa* was previously shown to be the causal agent of pecan bacterial leaf scorch (PBLS) in the United States and was shown to result in yield losses and a reduction in overall plant health. We observed that *X. fastidiosa* in pecan is difficult to detect through PCR due to non-specific amplification and inhibition from phenolic compounds, additional efforts are being made to improve PCR diagnosis of this pathogen in pecan tissue and to determine the location of bacterial populations in infected trees. In previous studies on pecan, *X. fastidiosa* was presumed to be solely transmitted via xylem-feeding insect vectors such as sharpshooters and spittlebugs. However, in this study, PCR and sequence analysis identified the presence of *X. fastidiosa* in pecan seed from multiple cultivars. Currently, greenhouse studies are in progress to determine if *X. fastidiosa* in pecan seeds is viable and able to colonize seedling tissues. In addition, a microbiome enrichment kit will be used to identify microbial populations present in the samples using next generation sequencing (NGS).

## A computer program for fast and easy typing of *Ralstonia solanacearum* species complex strains into genospecies and sequevars 1&2

M. STULBERG (1), Q. Huang (2)

(1) FNPRU, USDA/ARS & ORISE, U.S.A.; (2) FNPRU, USDA/ARS, U.S.A.

The phytopathogen *Ralstonia solanacearum* is a species complex that contains race 3 biovar 2 strains belonging to phylotype IIB sequevars 1 and 2 that are quarantined or select agent pathogens. Recently, the *R. solanacearum* species complex strains have been reclassified into three genospecies: *R. solanacearum*, *R. pseudosolanacearum* and *R. sygyzii*. An unidentified *R. solanacearum* strain is considered a select agent in the US until proven to be a non-race 3 biovar 2 (non-phylotype IIB sequevars 1&2). We wrote a computer program that analyzes a minimum 400-bp user-input *egl* sequence from a *R. solanacearum* species complex strain for *egl* homology and SNP content to determine 1) whether it belongs to the *R. solanacearum* species complex, 2) if so, to which genospecies, and 3) whether it is of the sequevar type (sequevars 1 and 2) associated with the select agent/quarantined *R. solanacearum* strain. The program correctly typed all 371 tested *egl* sequences with known sequevars. It also successfully typed 25 *R. solanacearum* strains with no prior sequevar information, as well as 4 strains from infected plant samples, using their partial *egl* sequences amplified and sequenced with primers designed in this study. The *Ralstonia solanacearum* typing program does not require expertise or specific knowledge to use, gives results in seconds, and provides data interpretation for the user regarding the genospecies and sequevar 1 or 2 status of the strain in question.

## Real-time PCR detection and discrimination of the parsley pathogens *Pseudomonas syringae* pv. *apii* and *Pseudomonas syringae* pv. *coriandricola*

J. REINTKE (1), G. Hiddink (2), D. Sanchez Mendez (2)

(1) Enza Zaden BV, Netherlands; (2) Enza Zaden BV., Netherlands

Bacterial leaf spot in parsley is caused by two different *Pseudomonas* syringae pathovars, *Pseudomonas* syringae pv. *apii* and *Pseudomonas* syringae pv. *coriandricola* (Koike, Bull, 2011 Bull et al, 2011). Both were identified as causal agents in outbreaks of bacterial leaf spot in parsley. Seed was claimed to be the initial source of infection. Current seed test methods include a pathogenicity tests but lack confirmation methods. Therefor a realtime PCR assay was developed to detect and discriminate the two pathovars causing leaf spot disease in parsley. The primers and probes were developed on sequence variation in the gap1-gene of 35 closely related *Pseudomonas* syringae pathovars. Both P.s. *apii* and P.s. *coriandricola* were amplified with the forward primer 5'-ACCCAGTCGATGATCCCGA-3', reverse primer 5'-AGCGAAACGTTGATCACCGG-3', while probes differentiated P.s. *apii* (5'-6FAM-CAGGCAAACTGACCGGCATGGCCGTT-BHQ1-3') from P.s. *coriandricola* (5'- HEX-CGGGCAAGCTCACGGGGATGGCTGTT-BHQ1-3'). Primers and probe showed sufficient discriminative power at the genospecies level when tested on 52 closely or non-related strains. Primers were used in an improved seed health tests for the detection of P.s. *apii* and P.s. *coriandricola* on parsley seeds. The seed health test was validated and test results were confirmed for biological relevance using grow-outs of all used seed lots.

## PCR Assays for the Detection of *Xanthomonas euvesicatoria*, Causal Agent of Bacterial Spot on Pepper and Tomato Plants

R. JEONG (1), S. Son (2), M. Kyeon (2), H. Lee (3), J. Cha (2)

(1) Chungbuk National University, Korea; (2) Department of Plant Medicine, Chungbuk National University, Korea; (3) Animal and Plant Quarantine Agency, Korea

*Xanthomonas euvesicatoria* (Xev) causes bacterial spot disease on pepper and tomato plants. A specific detection technique for this pathogen is important for the management of the disease and an efficient plant quarantine services. To obtain species-specific primers for PCR assays, low homologous ORFs of *Xanthomonas euvesicatoria* 85-10 to genes in NCBI GenBank were selected by BLASTn. Xev60 F/R primers that were designed from one of the select ORFs amplified specific DNA from all of tested strains of Xev including the 9 internationally collected strains and 67 Korean strains while the primers did not amplified any DNA from the 20 internationally collected strains of 3 other bacterial spot-causing Xanthomonads, *X. vesicatoria*, *X. perforans*, and *X. gadneri* and the other plant pathogenic bacteria. Taqman probe, Xev60-Tq was designed from the inside the PCR amplicon. Real-time PCR with Xev60 F/R and the Xev60-Tq probe generated Ct values with dose-dependent of target DNA () and Ct values of Xev strains were above of threshold Ct value for positive detection. PCR assays developed in this study can reliably detect and identify Xev.

## Loop-mediated isothermal amplification for the detection of soft rot causing *Dickeya* spp.

G. MARRERO (1), J. Yasuhara-Bell (2), M. Melzer (2), A. Alvarez (2)

(1) University of Hawaii at Manoa, U.S.A.; (2) University of Hawaii at Manoa, U.S.A.

*Dickeya* spp. cause soft rot, wilt and blackleg diseases in a wide range of plants and crops of agricultural importance. The emerging pathogen *D. solani* may be a contributing factor to the increased incidence of blackleg and soft rot diseases on potato in Europe, whereas *D. dianthicola*, a close relative, is currently listed on the European and Mediterranean Plant Protection Organization's list of pests recommended for regulation as quarantine pests. Presently, the North American Plant Protection Organization has placed *D. solani* on the U.S. list for emerging pest alerts. Symptoms caused by *Dickeya* spp. can be confused with those produced by *Pectobacterium* spp. in the field; differentiation of these two soft rot causing groups is time-consuming and requires multiple assays for confirmation. Therefore a robust rapid diagnostic test for the differentiation and detection of *Dickeya* spp. was developed using loop-mediated isothermal amplification (LAMP). In this study, a genus-specific LAMP assay was developed using whole genome data. The specificity of the assay was evaluated on six of the seven recognized *Dickeya* species and *Pectobacterium* spp. associated with potato; the LAMP assay only showed positive reactions with *Dickeya* spp. Direct detection of *Dickeya* from infected plant tissue was assessed and provided a result in less than 30 minutes. This LAMP assay offers a rapid and sensitive way for testing of *Dickeya* spp. from plant samples.

## Relative post-inoculation detection frequencies of 'Candidatus Liberibacter solanacearum' haplotypes in three solanaceous hosts

C. RUSH (1), L. Paetzold (1), F. Workneh (1)

(1) Texas A&M AgriLife Research, Bushland, TX, U.S.A.

'Candidatus' *Libreibacter solanacearum' (Lso), vectored by the potato psyllid (*Bactericera cockerelli*), is a putative bacterial pathogen, which causes potato zebra chip disease. The pathogen also infects all known solanaceous plants including peppers and tomatoes. Currently there are two haplotypes (A and B) of the pathogen identified from potatoes, and individual psyllids can carry either A or B or both. Greenhouse experiments were conducted to investigate post-inoculation relative incidences of the haplotypes in host plants (pepper, potato, and tomato) and in successive psyllid generations reared on the hosts. Plants were infested with psyllids in 5 cages each (12 psyllids/cage) with each cage containing 2 plants. Psyllids were obtained from a