colony in which nearly 90% were positive for A+B (the rest B). Plant and psyllid samples from the cages were collected 3 times (about every 2 weeks) beginning 6 weeks after infestation and tested for Lso haplotypes using qPCR and SYBR analysis. There was a highly significant difference among the hosts in incidence of the haplotypes (Chisq=15.3, P<0.0001). In peppers, 70% of the haplotypes were A, and 30% A+B. Haplotype B by itself was not detected. In potatoes, 75, 18, and 7% of haplotypes were A+B, B, and A, respectively. However, nearly all samples from tomatoes (96.7%) had haplotype A+B (the rest B) but A as a single haplotype was not detected. Haplotype frequencies in psyllids followed a similar detection pattern.

## Early detection of *Leifsonia xyli* subsp. *xyli*, the causal agent of ratoon stunting disease, in sugarcane seedlings

D. OLIVERA (1), A. Urashima (1)

(1) Federal University of Sao Carlos, Brazil

The first major change of the Brazilian sugarcane industry over the last decades occurred when mechanical harvest replaced the manual cuttings of burned canes. The second transformation has started with mechanization of planting materials thanks to new methodologies such as pre-sprouted cane (PSC). To ensure the success of this innovation, however, is paramount the use of clean seed cane especially against ratoon stunting disease, caused by the fastidious bacterium *Leifsonia xyli* subsp. *xyli* (Lxx), which can only be spread over new areas by contaminated propagative materials. Therefore, the purpose of this work was to develop a routine diagnostic test for early detection of Lxx in PSC seedlings. In the first assay, four previously published pair of primers, Cxx ITSf#5/Cxx ITSr#5, Cxx1/Cxx2, C2F/C2R, LX23SF/LX23SR, was challenged under the original conditions, as well as, our own modifications against adult diseased sugarcane with various concentrations of Lxx. In the second assay, two of the best primers/conditions were tested against PSC of 45 and 60 days of age derived from propagative materials with different levels of Lxx (10⁶ to 10⁹ UFC/ml). Our result showed primers Cxx ITSf#5/Cxx ITSr#5 run under our modified conditions was the most efficient since Lxx was detected in all 52 PSC seedlings of 45 days, including all 18 resulted from propagative materials contaminated with 10⁶ UFC/ml. Part of MSc dissertation by the second author at PPGPVBA, CAPES Scholarship.

## Development of a loop-mediated isothermal amplification assay for the detection of *Harpophora maydis*

N. GONZALEZ (1), G. Munkvold (1)

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

*Harpophora maydis*, a seedborne fungal plant pathogen that causes late wilt of maize, is an important pathogen that occurs in Africa, Europe and Asia, but not in the Americas. It is a high-priority quarantine pathogen listed on the APHIS-PPQ OPIS pest list. Thus, there is a need for effective pathogen detection assays that can be used to identify contaminated seeds. We have developed a loop-mediated isothermal amplification (LAMP) assay to detect *H. maydis* based on a specific AFLP sequence from GenBank. *H. maydis* isolates were grown on PDA and DNA was extracted with the Extract-N-Amp plant kit. A primer set (AFLP 17) consisting of 5 primers recognizing different regions of the target sequence was designed using Primer Explorer V4 software. The LAMP reactions were performed in a 25 µl reaction mix (1X Isothermal master mix) and incubated at 67°C for 25 minutes. The amplified products were detected by fluorescence and results were confirmed by gel electrophoresis. The LAMP assay showed specificity to the pathogen as shown by negative amplification when tested with other fungal species and was developed without the need to standardize DNA concentrations. The assay was effective for detecting the pathogen in seed samples containing inoculated seeds. This LAMP assay should be suitable as a fast, easy to perform and reliable method to detect *H. maydis*, providing a tool to help prevent the introduction of this potentially devastating pathogen into the USA.

## Using Color Spectrophotometry to Evaluate Disease Severity of *Macrophomina phaseolina* in Soybean

J. JORDAN (1), A. Mengistu (2), H. Kelly (1)

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

Charcoal rot is a soilborne disease of soybean caused by the fungus *Macrophomina phaseolina* (Tassi) Goid. The fungus is associated with hot dry weather and can dramatically reduce yields. Two current methods are used to evaluate disease severity in root tissues: colony forming units (CFU) and visual severity ratings. These methods are time consuming and expensive. Therefore, a more efficient methodology is needed to improve the screening process. We evaluated the use of color spectrophotometry as an alternative method for screening disease severity in 2006-2010 using 340 conventional and 893 roundup ready cultivars. Field plants were sampled at the R7 growth stage and evaluated using CFU, visual ratings (1-5 scale), and a color spectrophotometer. As previously reported, CFU values and visual ratings were predictive of each other across all years (F=7.66, p<0.05). Color data was not predictive of severity ratings (F = 2.70, p = 0.10), however they were negatively correlated across all years (r = -0.62, p<0.001). This association suggests that this method may be used as alternative in disease severity quantification of charcoal rot.

## Detection of *Erysiphe necator* fungicide-resistant alleles in leaf and air samples using novel molecular diagnostic techniques

J. YAMAGATA (1), B. Warneke (2), T. Neill (3), W. Mahaffee (4), L. Miles (5), A. Schilder (6)

(1) CSU Monterey Bay, Seaside, CA, U.S.A.; (2) Oregon State University, Corvallis, OR, U.S.A.; (3) USDA ARS, Corvallis, RI, U.S.A.; (4) USDA ARS, Corvallis, CA, U.S.A.; (5) Hartnell College, Salinas, CA, U.S.A.; (6) Michigan State University, Department of Plant, Soil and Microbial Sciences, East Lansing, MI, U.S.A.

*Erysiphe necator* (*En*) the causal agent of grapevine powdery mildew is managed using fungicides such as quinone outside inhibitors (QoIs). Unfortunately, *En* is known to develop resistance to QoIs by a single nucleotide polymorphism, resulting in an amino acid substitution (G143A) in cytochrome b. To monitor this allele, a TaqMan assay was developed and validated against powdery mildew DNA from strawberry, blueberry, snap pea, squash, Gerbera daisy, rose, and oak plants. Single spored DNA isolates (n = 103) were used to verify the accuracy of the TaqMan assay by comparing it to previous trifloxystrobin germination tests. *In-vitro* tests of single spored isolates identified 20% as the necessary proportion of the mutant allele in a mixed DNA sample to test positive for resistance. Leaf and air samples from Oregon (n = 130) were utilized in field validations. Digital droplet PCR was also utilized to quantify the allele distribution in a mixed DNA sample and lowered the detection limit the mutant allele for positive resistance to <5% of the total *E. necator* DNA present. This study resulted in novel allele-specific TaqMan and Digital droplet PCR assays that detect G143A in *En*. This assay could be used to significantly reduce the labor required in dealing with leaf and air samples of *En* in the laboratory and could be used to more closely monitor resistance development for a standard management program of grapevine powdery mildew.

## Investigation of primary infection period of apple blotch and circular leaf spot based on loop-mediated isothermal amplification assay in Korea

H. JUNG (1), S. Lee (2), S. Lee (2), J. Keum (2), S. Park (2), H. Woo (3), Y. Lim (4), I. Kang (2)

(1) College of Agricultural and Life Sciences, Kyungpook National University, Daegu, South Korea; (2) College of Agricultural and Life Sciences, Kyungpook National University, South Korea; (3) Bayer Crop Science, South Korea; (4) Gyeongsangbuk-do Agricultural Research & Extension Services, South Korea

Loop-mediated isothermal amplification (LAMP) method is widely used to detect various plant pathogens. In this study, we surveyed primary infection period of apple blotch (AB) on apple and circular leaf spot (CLS) on persimmon using developed LAMP method. The causal agent of AB and CLS; *Marssonina coronaria* and *Mycosphaerella nawae* have long incubation period to appear the typical symptoms. Up to now, primary infection period was predicted through conidia scattering period or measurement of number of conidia. To control the AB and CLS properly, primary infection periods should to be clear. Following the *M. coronaria* and *M. nawae* have long incubation period, we developed two different LAMP assay which can apply to apple and persimmon, and surveyed primary infection periods of AB and CLS in Korea using developed LAMP assay. As the results, not only infection was confirmed in healthy apple and persimmon leaves, but also the infection periods were gradually earlier than before. Although the relationship between
primary infection period and fungicide program should be studied, it is assumed that, to control and prevent economical losses of AB and CLS, fungicide spray program should be changed.

## A new paradigm for early diagnosis tool for major fungal disease in apple and persimmon

S. LEE (1), M. Jeon (2), J. Kim (3), H. Jung (1)

(1) College of Agricultural and Life Sciences, Kyungpook National University, Daegu, South Korea; (2) School of Electrical Engineering and Computer Science, Kyungpook National University, Daegu, South Korea; (3) School of Electrical Engineering and Computer Science, Kyungpook National University, South Korea

To diagnose the fungal disease, it can be diagnose when the visible symptoms or sign are appeared. Or molecular biological method, such as polymerase chain reaction (PCR) is widely used, however, it is hard to apply in field condition and takes long time. To overcome these problem, we applied optical coherence tomography (OCT) tool for diagnosis the major fungal disease; apple blotch (AB) and circular leaf spot (CLS) in apple and persimmon. The casual agents of AB and CLS is *Marssonina coronaria* and *Mycosphaerella nawae*, which has long incubation period in their host. Using these characteristics, we tried to find the differences between healthy and infected leaves in the incubation period stage. OCT is non-invasive method which can examine the leaf samples without treatment. A-scan analysis which showing signal intensity showed that there was difference between healthy and infected leaves. Furthermore, infected leaves which had any visible symptoms were appeared similar pattern of signal intensity with diseased leaves. And infection was re-confirmed using species-specific PCR assay. Based on these results, it is assumed that OCT can be a useful diagnostic tool for early diagnose of two major fungal disease in apple and persimmon.

## Inmunochemical detection of the Silverleaf disease of fruit trees caused by *Chondrostereum pupureum*

D. GRINBERGS (1), J. Chilian (1), A. France (1)

(1) Instituto de Investigaciones Agropecuarias, Chile

The Silverleaf disease affects numerous species, including important fruit crops such as apple, blueberry, cherry, kiwi, peach, pear and plum. The leaves silvering is caused by the acumulation of an enzyme (endoPG) produced by *Chondrostereum purpureum*, however, foliar symptoms are not visible untill the third year after the orchard plantation. Curative control is not effective, thus, an early detection is essential to avoid the fungus dissemination. Therefore, the objective of this work was to develop an early and non destructive method to detect the disease. Using the endoPG's aminoacidic sequence, monoclonal antibodies were synthesized and DAS-ELISA protocol developed. Mature leaves showing different silvering intensity were assessed, using healthy plants as negative control and purified endoPG as positive control. The presence of *C. purpureum* in the plants was confirmed through its isolation on 25% potato dextrose agar (PDA) and by the amplification of the large intergenic spacer (IGS) región, located between 5S and 18S rDNA. Results indicated that there was a positive correlation between foliar symptoms intensity and endoPG concentration, and that the method was able to discriminate among leaves from healthy and diseased but asymptomatic plants. This detection method was validated in apple, blueberry, peach and plum, as far, becoming a useful tool to prevent the dissemination of the Silverleaf disease.

## Evaluation of molecular diagnostic methods for *Monilinia* species of regulatory concern

S. HAYMES (1), K. Zeller (1), Z. Abad (1), L. Knight (1)

(1) USDA APHIS PPQ S&T Center for Plant Science and Technology, U.S.A.

Brown rot caused by *Monilinia* species is an extremely destructive disease of pome and stone fruits in many producing areas of the world, resulting in significant pre- and post-harvest losses. Diagnostics of species is difficult due to overlapping morphology, host-ranges, and symptoms. There are currently no molecular diagnostics methods that clearly discriminate all closely related species. *Monilinia fructicola* and *M. laxa* are native to or established in the US. *Monilinia fructigena* and *M. polystroma* are widely distributed in Europe and Asia and are exotic for the US. Both are listed as level "A" pests of economic importance to US agriculture in the 2015 Prioritized Offshore Pest List and the 2016 Cooperative Agricultural Pest Survey (CAPS) List. *Monilinia mumecola* and *M. yunnanensis* have been described recently (in 2004 and 2011, respectively) but are of less known impact, and neither of these species is known from the United States. The objective of this study is to evaluate two existing conventional PCR assays for distinguishing between native and exotic *Monilinia* for regulatory purposes. We have verified analytical sensitivity and specificity of these assays and determined the functional sensitivity with field-derived or spiked samples. A two-stage molecular diagnostic protocol that incorporates both assays allows for discrimination among all six of these *Monilinia* species, and should facilitate the accurate diagnosis for CAPS surveys.

## Identification of genomics-based detection assay for *Rhizoctonia solani* anastomosis groups

T. MILES (1), J. Cerda (1)

(1) CSU Monterey Bay, U.S.A.

*Rhizoctonia* is an important plant pathogen for many crops throughout the world causing plant tissue rot. Typically, diagnoses is accomplished using pure culture "tester" isolates and determining an anastomosis group. In order to develop new diagnostic tools, three mitochondrial genomes of *Rhizoctonia solani* were aligned with *Mauve* using the software Geneious 7.1. AG1 and AG3 had dramatically different mitochondrial genome sizes (160 Kb versus 250 Kb, respectively) due to the presence of introns. A region of the rRNA large subunit from the mitochondria was identified and several primers were designed. DNA was extracted from 5 isolates of *R. solani* followed by the polymerase chain reaction and sequencing. Sequence length for the large subunit of rRNA for each tested isolate varied significantly in size to what was expected. Difference in size is due to the presence and length of introns in the mitochondrial genome. Sequences were aligned for homology and differences. We are in the process of extracting purified genomic mitochondrial DNA from type isolates of *R. solani* to get a more complete picture of the significant diversity between AG groups. Even further research will be done to convert this method into a rapid field detection tool via recombinase polymerase amplification.

## Development, testing and pending deployment of a LAMP diagnostic screening method for the Citrus Black Spot pathogen, *Guignardia citricarpa*

K. ZELLER (1), K. Levin (2), G. Wei (2), G. Abad (2), H. Gomez (3), T. Riley (3), Z. Liu (2)

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

*Guignardia citricarpa*, the causal agent of Citrus Black Spot (CBS) was discovered in Florida during 2010. Today, three FL counties remain under quarantine. *G. citricarpa* causes unsightly lesions that reduce fruit marketability, and CBS is subject to national and international quarantine restrictions. The affected packing houses must monitor all fruits coming into the facility before they ship to the EU. If CBS symptoms are found on just a single fruit, entire shipments are put on hold where this perishable commodity's time is extremely valuable and a decision must be made. Current protocol requires that suspect fruit be shipped to PPQ or State Labs for confirmatory diagnosis which include a conventional and qPCR assay. We have developed a diagnostic tool that will enable inspectors to turn a 2-3 day decision into less than 1 hour using a LiNK extraction method followed by Loop-Mediated Isothermal AMPLification (LAMP). The LiNK column allows for rapid DNA extraction for use on the Genie III, a durable, sensitive, and user-friendly isothermal platform. Our LAMP screening assay is able to detect down to 30 fg of DNA with amplification times between 5-20 minutes. The Genie has been able to reproduce qPCR results on all 55 citrus samples tested from Florida as well as 32/33 CBS DNA diagnostic samples submitted to CPHST. Further testing on environmental samples and non-target hosts are underway. We will discuss the advantages and limitations of the method and deployment.
## Rapid diagnostic tools for soilborne pathogens of strawberry

A. BURKHARDT (1), T. Miles (2), M. Ramon (1), S. Koike (3), F. Martin (1)

(1) USDA, U.S.A.; (2) CSUMB, U.S.A.; (3) Cooperative Extension Monterey County, U.S.A.

The ability to rapidly identify and quantify strawberry pathogens in the soil allows growers to make faster decisions about what to plant and how to manage diseases a field. Therefore, we are developing rapid assays using TaqMan real-time PCR and recombinase polymerase amplification (RPA) to diagnose a variety of pathogens commonly infecting strawberry fields, including *Verticillium dahliae*, *Fusarium oxysporum*, *Phytophthora* spp., and *Macrophomina phaseolina*. These assays can provide results in hours (TaqMan) or in as little as 20 minutes (RPA) using DNA extracted from either infected plant tissue or soil. Species-specific TaqMan and RPA assays have been developed for *Phytophthora* and are very sensitive (0.2 pg) and specific. For *V. dahliae*, a species-specific TaqMan assay with a sensitivity of 1-2 microsclerotia per gram of soil is being converted to an RPA assay. Host-specific markers for *F. oxysporum f. sp. fragariae* are currently being developed for both TaqMan and RPA using a locus previously published by Suga et al. (Plant Dis. 2013). Additional loci for *F. oxysporum f. sp. fragariae* are being identified through sequencing and comparative genomics of multiple isolates in order to expand the breadth of detection. For *M. phaseolina*, sequencing of multiple isolates has identified a genotype-specific locus, and primers are being developed for RPA and TaqMan assays to detect isolates which have been found to only infect strawberry.

## BioPCR: An improved method for detection of *Colletotrichum acutatum* in asymptomatic strawberry nursery plants

P. SUDARSHANA (1), K. Mani (1), P. Randhawa (1), T. Walters (2)

(1) California Seed and Plant Labs, U.S.A.; (2) Walters Ag Research, U.S.A.

*Colletotrichum acutatum* infections in strawberry nurseries often stay symptomless. Infected plants usually collapse upon transplanting in grower fields resulting in economic losses to the growers. A routine method to detect *C. acutatum* in asymptomatic plants is to culture root and crown tissue on semi-selective agar media such as TPDA and BR and confirm identification by microscopic observations of spore morphology and PCR. In our experience, the culture method often fails to detect *C. acutatum* when non-target fungi such as ?Trichoderma sp., ?Alternaria sp. and *Aspergillus* sp. are present in a sample. The rapid growth of non-target fungi mask the pathogen in culture. To overcome this problem, we investigated BioPCR as an alternative method to detect infections in asymptomatic tissue. In BioPCR, tissue was cultured for four days on agar media to enrich *C. acutatum* in the plant tissue. After 4 days, tissue was removed from the culture plates and tested by PCR using primers specific for *C. acutatum* (1). Culture plates were still available for reading. During 2015, we analyzed 106 commercial samples from strawberry nurseries in California by culture and BioPCR method. BioPCR detected *C. acutatum* in 83% plants compared to 42% by culture method. We conclude that BioPCR method is superior to culture method and can be used to assay strawberry nursery plants for detection of *C. acutatum* in asymptomatic plants.

## Development of a quantitative PCR assay to quantify resistance to *Diaporthe helianthi* and *Diaporthe gulyae* in sunflower

T. OLSON (1), F. Mathew (1), A. Adhikari (1), B. Kontz (1), L. Marek (2)

(1) South Dakota State University, Brookings, SD, U.S.A., (2) Iowa State University, Ames, IA, U.S.A. / USDA-ARS

In the U.S., Phomopsis stem canker was first detected in 1983, but it was not until the 2010 epidemic in Minnesota, North Dakota, and South Dakota that two pathogens, *Diaporthe helianthi* and *Diaporthe gulyae* were identified as the causal agents. At this time, no commercial hybrids are available that have complete resistance to the two pathogens. The objectives of this study were to (1) develop quantitative polymerase chain reaction (qPCR) assays for quantification of *D. helianthi* and *D. gulyae* from sunflower and (2) evaluate sunflower germplasm for resistance to the two pathogens using qPCR assays. The primers and probes for the two qPCR assays were designed from the translation elongation factor region for amplification of *D. helianthi* and *D. gulyae*. To evaluate sunflower germplasm for resistance to *Diaporthe* spp., 54 sunflower genotypes from 9 different countries were screened for resistance using the stem-wound method in the greenhouse. Disease severity was evaluated 14 days after inoculation using a 0-5 rating scale from Mathew et al. (2015). Preliminary results suggest significant differences among genotypes in their resistance to *D. helianthi* and *D. gulyae*; PI 561918 (HA 378) appeared to be less susceptible to the two pathogens than the susceptible check 'HA 288'. The qPCR assays developed in this study will be used to compare genotypes for resistance to *D. helianthi* and *D. gulyae* by quantifying the levels of pathogen DNA present in them after inoculation.

## Phytogenetic identification of pathogenic and endophytic fungal populations in west coast Douglas-fir foliage

D. DANIELS (1), J. Kiser (1)

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

Douglas-fir provides social, economic and ecological benefits in the Pacific Northwest (PNW). Minor defoliation has been linked to Swiss Needle Cast, associated with *Phaeocryptopus gaeumannii*. Unprecedented defoliation has increased since the 1990s, leading to decreased growth and yield. Affected areas exceed 500,000 acres in Oregon. Recent symptoms are inconsistent with predicted effects of *P. gaeumannii*. We hypothesize that the entire endophytic community is relevant to disease ecology, though this has not been investigated to date. Dynamics of needle cast are explored and baseline inventories of endophytes established by Sanger and Next-gen Sequencing (NGS). Endophytes were evaluated along environmental gradients at three sites in the PNW. Probability of occurrence (presence or absence of any endophyte in needle) was hypothesized to be higher in cooler, wetter climates. Preliminary results suggest that the probability of endophyte occurrence at the cool, wet site was 2.6 (p<0.0007) times higher than at the warm, dry site, and 3.7 (p<0.0001) times higher than at the warm, wet site. Despite advances in technology, few studies have identified endophytes in Douglas-fir by molecular means. Traditional culturing is hypothesized to be less effective than NGS. More inclusive identification could lead to greater efficiency in forest management. It is vital to understand the complete etiology of SNC and the ecological implications of disease in this important species.

## Rapid diagnostic assay development for detecting patch diseases on turf

B. KARAKKAT (1), M. Franchett (1), M. Olson (1), C. Mullenberg (1), P. Koch

(1) Department of Plant Pathology, University of Madison, WI, U.S.A., (2) Department of Plant Pathology and Molecular and Environmental Toxicology Center, University of Wisconsin, Madison, Wisconsin, U.S.A.

Necrotic Ring Spot, Summer Patch, and Take-all Patch are turf root diseases caused by soil-borne plant pathogenic fungi. The infected turf shows aboveground symptoms that are broadly categorized as 'patch' diseases that ruin the aesthetic value of cool-season turfgrass on lawns/fields in North America from late spring to early summer. These fungal pathogens are notoriously difficult to isolate from roots. Therefore, we decided to develop a rapid detection assay that can be performed on suspected root samples. We are testing two assays: Loop mediated isothermal amplification (LAMP) and Recombinase polymerase amplification (RPA). Both LAMP and RPA do not require pure genomic DNA but only a solution of root sample. LAMP and RPA rely on DNA strand displacement activity for its rapid amplification and can be completed at 650C in an hour and 390C in 20 minutes incubation, respectively. Initial LAMP screening gave false positives repeatedly in almost all assays indicating that very pure conditions are needed, which is not ideal in a diagnostic laboratory. Hence, we have started to work on RPA methodology available as Agdia's discovery kit to eliminate any false positives. Once we achieve a consistent and specific amplification with these assays, we will proceed to screen samples that will be arriving in late Spring 2016. Assay development, primer-probe design and samples surveyed will be reported in this study.

## Rapid differentiation of *Claviceps* species occurring in Oregon and Washington using high resolution melting curve analysis

N. KAUR (1), R. Cating (2), J. Dung (3), S. Alderman (4), D. Walenta (5), P. Hamm (1), K. Frost (1)

(1) Oregon State University, Hermiston, OR, U.S.A.; (2) Oregon State University, Corvallis, OR, U.S.A.; (3) Oregon State University, Madras, OR, U.S.A.; (4) USDA-ARS National Forage Seed production Research Center, Corvallis, OR, U.S.A.; (5) Oregon State University, La Grande, OR, U.S.A.
Ergot disease of grass seed crops can be caused by *Claviceps purpurea* and *C. humidiphila*, a species recently reported to occur in certain regions of OR and WA. Routine identification of *Claviceps* species involves conventional PCR amplification and sequencing of ITS, β-tubulin or EF-1α genes. In this study, a high resolution melting (HRM) assay was developed to rapidly identify and differentiate *C. purpurea* and *C. humidiphila* based on the melting temperatures (Tm) of partial β-tubulin amplicons. PCR primers were designed to target and amplify a 110 bp region of the β-tubulin gene containing multiple single nucleotide polymorphisms generating amplicons with distinct melting profiles for each species. Forty-six fungal cultures, previously classified based on RAPD profiles, and ITS, β-tubulin, and EF-1α gene sequences, were used to validate the HRM assay. The Tm for *C. purpurea* and *C. humidiphila* ranged between 79.8 to 80.3 and 78.9 to 79.3, respectively. Classification of *Claviceps* species based on Tm using linear contrasts (P < 0.05) correctly grouped 96% (N=46) of samples previously classified using sequence data. In two cases, *C. purpurea* was misclassified as a mixture of the two *Claviceps* species. Therefore HRM analysis allows rapid detection and differentiation of these two *Claviceps* species and will be useful to study their distributions within OR and WA cool-season grass seed growing regions.

## Specific detection of the root-lesion nematode *Pratylenchus scribneri* using conventional and real-time PCR

G. YAN (1), D. Huang (2), G. Yan (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.

*Pratylenchus scribneri* is a plant-parasitic root-lesion nematode causing economic damage to various crops worldwide. Identifying root-lesion nematodes to species using traditional morphological method is an arduous task requiring extensive training on nematode taxonomy. Consequently, molecular methods were developed to identify *P. scribneri* by conventional and real-time PCR using a species-specific primer set that was designed based on the alignment of 20 sequences of internal transcribed spacer of rDNA of seven *Pratylenchus* spp. The primer set specifically amplified DNA of *P. scribneri* but not DNA from five other *Pratylenchus* spp. or ten non-*Pratylenchus* spp. A single melt curve was observed indicating a high specificity according to real-time PCR analysis. The capability of specific PCR was tested using DNA extracts of root-lesion nematodes from 23 soil samples collected from various fields with five crops in North Dakota and Minnesota. The PCR primers and protocols were successfully used in *P. scribneri* identification. The conventional PCR could detect DNA of 1:8 dilution of two nematodes equivalent to 1/4 of a nematode. The real-time PCR was more sensitive and able to amplify DNA of serial dilutions (2 nematodes - 1/128 nematode) with Cq values ranging from 25.4±0.0 to 33.6±0.4. The developed PCR assays provide a rapid and reliable identification of *P. scribneri*, suitable for use in diagnostic laboratories for detection of field infestations with this nematode.

## Real-time and conventional PCR assays for identifying the stubby root nematode *Paratrichodorus allius*

G. YAN (1), D. Huang (2), G. Yan (2)

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

Stubby root nematode (SRN), *Paratrichodorus allius*, is an important pest on many crops, particularly, on potato due to its ability to transmit *Tabacco rattle virus* causing corky ringspot disease on tubers. Nematode surveys revealed that SRN is present in many fields on different crops in North Dakota and Minnesota. Detection and identification of SRN are important for effective disease management. Protocols of *P. allius* identification by real-time and conventional PCR with species-specific primers were developed. Primers were designed from internal transcribed spacer of rDNA based on sequences of *P. allius* and six closely related *Paratrichodorus* spp. A single amplicon (246 bp) was produced from DNA of *P. allius* only, but not from DNA of two other SRN species and 21 non-SRN species. Detection sensitivity analysis indicated that an equivalent of 1/20 of a nematode could be detected by conventional PCR and an equivalent of 1/1000 of a nematode by real-time PCR with Cq value at 32.6±0.5. The PCR assays were tested with SRN from 20 soil samples collected from various fields with five crops in ND and MN, and they successfully amplified DNA from all samples. These SRNs were confirmed to be *P. allius* by sequencing of three genomic regions. The PCR assays are rapid and reliable and can be used in *P. allius* identification using nematode individuals. This finding will help develop a real-time PCR assay for quantification of *P. allius* directly from DNA extracts of field soils.

## Development of species-specific qPCR tests for detection and quantification of *Meloidogyne hapla*

A. GORNY (1), X. Wang (2), S. Pethybridge (1)

(1) Cornell University, School of Integrative Plant Sciences, Plant Pathology & Plant-Microbe Biology Section, U.S.A.; (2) USDA-ARS, Plant Protection Research Unit, Cornell University, U.S.A.

The Northern root-knot nematode, *Meloidogyne hapla*, is an important soilborne pathogen of potatoes and other vegetables grown in the United States and is common within New York State. Effective management of *M. hapla* and other plant-parasitic nematodes relies on the accurate identification and timely quantification of pathogen populations prior to planting. Identification and quantification may be substantially improved by pre-plant soil tests which utilize quantitative PCR (qPCR) techniques to target DNA sequences specific to the pathogen of interest. Here, qPCR primers were designed to target unique regions of an effector gene *16D10* in *M. hapla* and assayed for specificity against plant-parasitic nematodes using qualitative PCR. The primers developed in this study were shown to be specific to *M. hapla*. A qPCR tests was developed and the sensitivity of the primers determined.

## DNA-nanosensor based diagnostics of plant pathogens

A. BAETSEN-YOUNG (1), M. Vasher (1), E. Alocilja (1), B. Day (1)

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

Crop loss from plant pathogens is a leading threat to food security in the United States. Current pathogen detection assays have limited field operability, leading to improper disease management decisions. Recently, rapid diagnostic DNA-nanosensors have increased perception and aided precision care of human pathogens in resource limited locations. Here we demonstrate the application of DNA-based nanosensor to detect an obligate biotroph, *Pseudoperonospora cubensis*. The detection of *P. cubensis* is based upon primers highly specific to a targeted gene, and the physical properties of DNA and nanoparticles with sodium chloride. Diluted DNA was denatured and cooled in a solution with the primers. Then nanoparticles were added to the solution followed by sodium chloride to reveal primer-target interaction. Target DNA presence was quantified colorimetrically (i.e. naked eye) and by absorbance on a spectrophotometer over a 400 to 700 nm range. Current PCR amplified target can be detected to 5 pg/mL by the naked eye. Optimization of this DNA-nanosensor to detect extracted genomic DNA, and within environmental matrices will be discussed. The deployment of this rapid bioassay has the potential to assist growers in developing sustainable disease management decisions in a field based setting.

## Optimization of a fluorescence *in situ* hybridization assay for detection and visualization of *Plasmopara obducens* from plants and soil

C. SALGADO-SALAZAR (1)

(1) USDA-ARS, Rutgers University, U.S.A.

*Plasmopara obducens* is the biotrophic oomycete responsible for downy mildew of ornamental impatiens, a destructive disease that significantly impacts the production of *Impatiens walleriana* in the United States. To date, there are no methods to detect *P. obducens* from asymptomatic plants, which may be contributing to the spread of the disease. Furthermore, the extent that *P. obducens* uses oospores to colonize and persist in the soil is undetermined. Fluorescence *in situ* hybridization (FISH) is a sensitive and robust method that uses sequence-specific, fluorescence-labeled oligonucleotide probes to detect target organisms from the environment. A FISH assay for the impatiens downy mildew pathogen was designed by preparing DNA sequence alignments of the rDNA-ITS region of *P. obducens* and other related taxa to identify suitable regions for species-specific probe development. Because *P. obducens* cannot be propagated *in vitro*, we developed a custom *E. coli* expression vector that transcribes the rDNA-ITS gene sequence of *P. obducens*
for use as a control and to allow for standardized fluorescence signal quantification (clone-FISH). The optimized probe hybridization stringency resulted in good fluorescence signal compared to those of the negative controls. The standardized protocol developed using the clone-FISH reagents will be used in subsequent experiments to directly visualize *P. obducens* in situ from plant and soil samples.

## Developing an innovative molecular toolbox for identification of *Phytophthora* species with emphasis on species of regulatory concern

Z. Abad (1), J. Bienapfl (1), L. Knight (1), K. Jennings (1), M. Galvez (1), L. Schena (2)

(1) USDA APHIS PPQ S&T, U.S.A.; (2) Mediterranean University of Reggio Calabria, Italy

The genus *Phytophthora* consists of 155 species, many of which cause significant economic impact to crops, ornamentals, and forest ecosystems worldwide. Many species are also exotic and of regulatory importance for the USA. Accurate species identification can be extremely challenging due to the need for extensive training and experience, as well as many misidentified DNA sequences deposited in public databases. In order to facilitate correct identification of *Phytophthora* species, we are pioneering work with international collaborators for the implementation of a “Molecular Toolbox” containing DNA sequences generated from the type specimens used for the original descriptions. Ten nuclear genes and five mitochondrial genes currently used for identification and phylogenetic studies have been evaluated. Six nuclear genes, including the YPT1 gene that is amplified with new primers developed by Schena, and one mitochondrial gene have been selected for use in our “Molecular Toolbox” based on their utility in the robust identification and phylogenetic studies of *Phytophthora* species. However, many of the types lack sequences for these different genes (over 50%) and predominantly only have ITS sequences available. We will provide all files generated from the DNA sequencing, which will also demonstrate the high quality of data generated, to be used for identification purposes. Emphasis of this project is for species of high economic impact and regulatory concern for the USA.

## Evaluation of Recombinase Polymerase Amplification (RPA) Isothermal Amplification Diagnostic Assay for *Phytophthora ramorum*

J. BIENAPFL (1), L. Knight (1), Z. Abad (1)

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

*Phytophthora ramorum* was found as the causal agent of 'Sudden Oak Death' oaks and tanoaks in the coastal counties of California in the 1990s. Since then, the pathogen has been confirmed on numerous native hosts and nursery plants in California, Oregon, and Washington, with sporadic occurrences in other regions of the USA. The current methods of testing for *P. ramorum* include conventional and real-time PCR which are expensive and timely. The primary purpose of this work is to enhance Plant Protection and Quarantine efforts in the detection of *P. ramorum* by evaluating a field-deployable, simple, and rapid detection method. A previously published Recombinase Polymerase Amplification (RPA) Isothermal Amplification Assay for detection of *P. ramorum* was evaluated for both sensitivity and specificity using the Twista™ apparatus. DNA extracted from pure culture, infected and healthy rhododendron tissue, as well as environmental samples were used to validate the *P. ramorum* protocol for regulatory use. Preliminary data comparing the isothermal amplification assay to current confirmatory *P. ramorum* diagnostic assays, evaluating ITS and Elicitin qPCRs shows that this protocol was not as sensitive as the ITS qPCR, but was comparable to the Elicitin qPCR. We will present the value of RPA isothermal amplification as a regulatory diagnostic tool for the rapid detection of *P. ramorum*.

## Extracting DNA for qPCR amplification from plant tissue in ELISA buffer

N. OSTERBAUER (1), S. Navarro (2)

(1) Oregon Dept. of Agriculture, U.S.A.; (2) Oregon Dept. of Forestry, U.S.A.

USDA APHIS mandates testing protocols for *Phytophthora ramorum*. Diagnostic labs use an ELISA kit to screen for *Phytophthora* infection in leaves. If a leaf tests ELISA-positive, a second sample is collected from the leaf for DNA extraction and molecular testing. This may delay detection by a day. DNA extraction directly from macerated leaf tissue in ELISA buffer would speed testing. To determine if DNA extracted from such tissue would be adequate for molecular testing, 198 leaf samples from a nursery were tested with ELISA, with 30 testing positive. For each ELISA-positive sample, leaf tissue was collected and DNA extracted following WI-B-T-2-3 Rev. 1; DNA was also extracted from the macerated leaf tissue in ELISA buffer. Tissue-DNA and ELISA-DNA was then tested for *P. ramorum* following N-R-WI-008-0 using the reporter dyes FAM (*P. ramorum*-specific) and JOE (internal control); all DNA was tested undiluted and diluted 1:10. For all samples, the tissue-DNA and ELISA-DNA tested negative for *P. ramorum* (FAM Cq = 0.00) at both dilutions. For the internal control, the tissue-DNA tested positive for amplifiable DNA with a mean Cq of 27.30, whereas the ELISA-DNA tested positive with a mean Cq of 26.41. For diluted DNA, the mean Cq for tissue-DNA was 20.45 and for ELISA-DNA was 20.79. The results suggest amplification of the internal control was comparable between DNA extracted from leaf tissue and DNA extracted from macerated leaf tissue in ELISA buffer.

## Utilizing mitochondrial loci to develop TaqMan and recombinase polymerase amplification assays for the genus *Pythium*

T. MILES (1), A. Burkhardt (2), F. Martin (2)

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

Mitochondrial marker systems have been utilized in other oomycete systems to develop novel molecular diagnostic tools in both laboratory and field settings. For *Pythium* a mitochondrial gene order difference was identified that allowed for genus specific amplification of more than 75 species across a wide range of clades within the genus. PCR results for this locus tested negative on *Phytophthora* and several *Phytophthora* spp. A TaqMan diagnostic assay was developed using a FAM labeled probe and validated on environmental samples from over 40 host plants (nursery and field samples). A sequencing protocol was also developed to confirm a positive, however, if multiple species are present amplicon cloning will be needed to get clean data. In addition to the TaqMan assay an assay using the isothermal technology known as recombinase polymerase amplification (RPA) was developed. Using the same purified and field samples the RPA technique was effective at detecting *Pythium* species in less than 20 minutes without a laborious DNA extraction. The use of this marker system will not only help confirm *Pythium* infection in plants, it may also be useful in environmental samples and it will complement the other marker system developed for *Phytophthora*. Sequencing of additional species is in progress to evaluate species specific detection/identification capabilities. Due to the amplicon size, this locus may also be useful in future metagenomic studies of *Pythium* diversity.

## Development of molecular diagnostic tools for the invasive oomycete pathogen *Phytophthora tentaculata*

N. Luecke (1), S. Koenig (1), T. Miles (2)

(1) CSU Monterey Bay, U.S.A.; (2) California State University Monterey Bay, U.S.A.

*Phytophthora* basal rot (PBR) of plants is a disease recently observed in Central California Native plant nurseries and restoration locations. The primary causal agent of this disease is the oomycete pathogen *Phytophthora tentaculata*. Prior to 2012, *P. tentaculata* was among the ~30 *Phytophthora* species not known to be present in the US. Previous research had developed an isothermal diagnostic tool known as recombinase polymerase amplification (RPA), which had *Phytophthora* genus specific detection capability with results obtainable within as little as 15 minutes directly in the field without conventional DNA extraction. A *P. tentaculata* species-specific RPA assay was developed and specificity validated against pure DNA from 135 *Phytophthora* taxa. To test this technique for detection of the pathogen in PBR, 113 symptomatic samples were collected and evaluated with the new RPA assay as well as the previously validated TaqMan assay, Immunostrip and conventional culturing and baiting techniques. Results were similar across the various amplification platforms, with qPCR being the most sensitive in general. Finally, spatial models were created to test if elevation correlated with the presence of the pathogen. These results indicate that *P. tentaculata* is often present in low-lying areas when infected plant material is used for restoration. This information will assist in more efficient, rapid, specific pathogen detection for management decisions in a field.
### A CANARY Assay for *Phytophthora* spp.

Z. LIU (1), D. Kalkofen (1), A. Flannery (1), G. Wei (2), F. Nargi (3), Z. Liu (2), G. Abad (2), J. Bienapfl (2), M. Nakhla (2)
(1) PathSensors, Inc., Baltimore, MD, U.S.A.; (2) USDA-APHIS-PPQ-S&T-Center for Plant Health Science and Technology (CPHST), U.S.A.; (3) Bioengineering Systems & Technologies, MIT Lincoln Laboratory (MIT-LL), U.S.A.

*Phytophthora* is a genus of Oomycetes (water molds) that can cause serious plant diseases and considerable economic loss. A notable example is *P. infestans*, which was responsible for the potato late blight that caused the Great Irish Famine in the 1840s, and is still the most destructive pathogen of solanaceous crops. Another example is *P. ramorum*, the cause of Sudden Oak Death which has had devastating effects on forests in California and Oregon. Efficient management of *Phytophthora* diseases requires early detection and accurate identification of the pathogen. CANARY® (Cellular Analysis and Notification of Antigen Risks and Yields) is a fast, sensitive, and easy to use technology that has been successfully utilized for the detection of bacteria, viruses, and toxins. We are developing a CANARY® assay for *Phytophthora* spp. Using pure culture preparations, the assay was able to detect all species of *Phytophthora* tested, including *P. cactorum*, *P. cambivora*, *P. capsici*, *P. cinnamomi*, *P. citrophthora*, *P. kernoviae*, *P. infestans*, *P. nicotianae*, *P. niederhauserii*, *P. ramorum*, and *P. sojae*. Importantly, exclusivity studies showed that the assay did not cross-react with *Pythium* spp. Preliminary testing with infected plant material indicates that the assay takes less than 20 minutes to complete. A user-friendly software interface guides the user through all steps of the assay, providing a simple and rapid screening method for *Phytophthora* spp.

### Early detection and quantification of *Pseudoperonospora cubensis* airborne sporangia using real-time PCR

A. RAHMAN (1), L. Quesada-Ocampo (1)

(1) NCSU, U.S.A.

*Pseudoperonospora cubensis*, the causal agent of cucurbit downy mildew, recently reemerged as a major destructive pathogen of a broad range of crops belonging to the Cucurbitaceae. Airborne sporangia of *P. cubensis* are the most important source of inoculum to initiate disease as well as cause secondary plant infections. Furthermore, airborne sporangia of this obligate oomycete are known to be able to travel long distances and survive for prolonged periods of time to solar radiation. Therefore, early and rapid detection of the airborne sporangia could serve as a warning for potential disease outbreaks. By designing genome specific primers and using SYBR green-based qPCR method, a preliminary threshold detection level of target pathogen DNA was determined in extracted DNA from *P. cubensis* sporangia and infected plant tissue. While the method successfully detected up to 5.3 pg/ml of sporangia DNA, the minimum DNA concentration required from infected plant material for successful detection was found to be almost 10-fold higher (69 pg/ml). Using Next Generation Sequencing (NGS) and bioinformatics tools, several unique and conserved candidate genomic regions were also generated to serve as diagnostic genomic markers for *P. cubensis*. These candidate genomic regions were tested for their suitability as diagnostic markers for detecting and monitoring airborne *P. cubensis* sporangia inoculum level in the field using real-time qPCR following entrapment with spore traps.

### On-site detection of *Pythium ultimum* in potatoes using loop-mediated isothermal amplification (LAMP)

J. WOODHALL (1), P. Wharton (2), S. Dangi (2), K. Perkins (3), J. Azcona (3)

(1) University of Idaho, U.S.A.; (2) University of Idaho, U.S.A.; (3) Fera Science Ltd, United Kingdom

*Pythium ultimum* is a soil-borne plant pathogen that causes damping off and root rot diseases in a wide range of crops. In potatoes, Pythium leak caused by *P. ultimum* has been a major storage disease problem in the Pacific Northwest potato production areas for many years, but in recent years reported losses have increased. Since similar symptoms can be caused by other pathogens, rapid and robust tests are required to detect the pathogen. Loop-mediated isothermal amplification (LAMP) has great potential for diagnostic use offering fast, specific and sensitive detection. In this study a new LAMP assay was designed for *P. ultimum* and evaluated in terms of specificity and sensitivity. The assay was highly specific when tested with DNA from 30 other isolates consisting of closely related Pythium species and other potato pathogens. The assay was also highly sensitive detecting as little as 17.5 fg of DNA in less than 30 minutes when used with a Genie® II for fluorescence measurement. A simple, rapid on site DNA extraction method for tubers was developed for use with the assay which resulted in a highly sensitive test capable of detecting the pathogen in range of both naturally infected asymptomatic and symptomatic tubers. In conclusion, LAMP assays can offer a rapid, sensitive, on-site test for the presence of *P. ultimum* in potato tubers.

### Sensitive detection and discrimination of HPWMoV, WSMV and TriMV using multiplex RT-PCR and High Resolution Melting: Part II

A. LARREA-SARMIENTO (1), M. Arif (2), F. Ochoa-Corona (3), A. Olmedo-Velarde (4), J. Olson (3)

(1) Universidad de las Américas, Quito, OK, Ecuador; (2) Kansas State University, Manhattan, KS, U.S.A.; (3) Oklahoma State University, Stillwater, OK, U.S.A.; (4) Universidad de las Fuerzas Armadas ESPE, Sangolqui, Ecuador

*High Plains wheat mosaic virus* (HPWMoV), *Wheat streak mosaic virus* (WSMV) and *Triticum mosaic virus* (TriMV) cause disease in wheat crops in Central and Western USA, including Oklahoma and Kansas. Although these viruses are from different families, they are transmitted by *Aceria toisichella*, the wheat curl mite (Prostigmata: Eriophyidae). These viruses may infect simultaneously single plants. Melting curve analysis of RT-PCR products obtained by High Resolution Melting (HRM) enables to detect and to discriminate different virus species. The method is rapid, sensitive and allows discrimination among multiple samples. Specific primers for original single endpoint RT-PCR and reference positive controls from Agdia® were used as previously reported (Part I this research, 2013) to generate cDNA. uMELT<sup>SM</sup> analysis of expected PCR products was used to predict HRM scenarios. Special attention was given to PCR product size, GC percentage and melting temperature (Tm). Tm derived from dF/dT plots *in vitro* were similar to predictions by uMELT<sup>SM</sup> *in silico*. All primers sets showed a sensitivity down to 1 fg/uL from cDNA in single-endpoint RT-PCR reactions. HRM assays are sensitive and allows detection and discrimination of viruses causing diseases and significant economic losses in wheat and corn.

### Detection of *Apple scar skin viroid* in apple fruits by reverse transcription loop-mediated isothermal amplification (RT-LAMP)

S. KWON (1), I. Cho (2), S. Choi (1), J. Yoon (1), G. Choi (1)

(1) National Institute of Horticultural and Herbal Science, South Korea; (2) National Institute of Horticultural and Herbal Science, South Korea

*Apple scar skin viroid* (ASSVd) is one of the most important pathogen causing scar skin, dappling or cracking on the surface of apple fruits. A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for detection of ASSVd. A set of two primers were designed inner primers (FIP and BIP) and outer primers (F3 and B3) from sequences of the Korean strain of ASSVd. The LAMP reaction was optimized using *Bst* DNA polymerase for 30 min at 65°C. The RT-LAMP products were evaluated as ladder-like bands by electrophoresis and visualized in the reaction tube by staining with SYBR Green I dye. RT-LAMP was more sensitive than that of conventional RT-PCR for the detection of ASSVd. The RT-LAMP assay developed in this study is a simple, rapid and sensitive and can be applied as a practical molecular diagnostic tool.

### Three genomic regions of *Cilevirus* allows detection and discrimination of two related species infecting citrus

W. TURIZO (1), O. Oliveros (1), F. Ochoa-Corona (2)

(1) Universidad Nacional de Colombia, Colombia; (2) Oklahoma State University, U.S.A.

Citrus leprosis disease (CLD) is caused by diverse etiological agents such as *Citrus leprosis virus C* (CiLV-C) and a newly proposed Citrus leprosis virus cytoplasmic type 2 (CiLV-C2), both in the genus *Cilevirus* and transmitted by Tenuipalpidae (false spider mites) *Brevipalpus* spp. CLD is also caused by Citrus leprosis virus nuclear type (CiLV-N) a possible *Dichorhavirus* (*Dichorhabdovirus*) and Hibiscus green spot virus 2 (HGSV 2) a *Higrevirus*
infecting citrus trees in Hawaii. The presence of CiLV-C in the western plains of Colombia was reported in 2005 using reverse transcription polymerase chain reaction (RT-PCR). A second report of CiLV-C2 sequenced from symptomatic citrus samples using next generation sequencing was made in 2013. These two reports had raised questions about distribution of CiLV-C and CiLV-C2 in Colombia and whether both viruses coinfect citrus groves. This study analyzed genomic sequences of CiLV-C and CiLV-C2 seeking target sequences for detection and discrimination using reverse transcription multiplex PCR. The developed assay is fast, sensitive and specific for detection and discrimination of these two species of *Cilevirus* infecting citrus. Only CiLV-C2 and no coinfections were found in samples collected in the citrus growing region of Meta and Casanare, Colombia.

## Development of conventional and Real-time RT-PCR assays for *Strawberry necrotic shock virus*

T. THEKKE VEETIL (1), T. Ho (2), V. Whitaker (3), I. Tzanetakis (2)

(1) University of Arkansas, U.S.A.; (2) University of Arkansas, U.S.A.; (3) University of Florida, U.S.A.

*Strawberry necrotic shock virus* (SNSV) is one of the several viruses reported in strawberry and *Rubus* as a component of viral complexes that cause plant decline. Highly sensitive conventional and real-time RT-PCR detection assays were developed; able to detect all sequenced isolates of the virus. Primers and probes were designed from conserved regions of the virus coat protein gene and reactions were optimized to ensure both specificity and sensitivity. Comparison between the two assays revealed an at-least 100-fold higher sensitivity for the latter. Both tests were successfully validated using samples collected from several areas in the United States as well as a collection of isolates from around the world. These assays could be integrated into virus screening programs in certification, nursery or commercial field settings in both strawberry and *Rubus*.

## Rose rosette virus detection using loop-mediated isothermal amplification (LAMP)

A. SALAZAR AGUIRRE (1), S. Molina Cárdenas (1), F. Ochoa-Corona (2), J. Olson (2)

(1) Universidad de las Fuerzas Armadas ESPE, Ecuador; (2) Oklahoma State University, U.S.A.

*Rose rosette virus* (RRV) is an *Emaravirus* transmitted by *Phyllocoptes frutiphilus* strongly associated with Rose rosette disease (RRD). Visual diagnosis maybe confusing and specific, sensitive, easy to use with visual detection methods are required. LAMP is an isothermal amplification that combines these criteria. Alignment of 20 RRV P4 (RNA 4) accessions allowed primer design seeking broad detection of reported isolates. Optimal amplification was obtained with Bst 2.0 WarmStart® DNA polymerase (0.32 u), outer primers F3 and B3 (0.2 µM), internal primers FIP and BIP (0.8 µM) and MgSO₄ (4 mM) at 66.5°C for 1 hour. Bovine serum albumin (BSA) (4 mg/mL) and polyvinylpyrrolidone (PVP) (1%) were added as isothermal amplification enhancers. The detection limit was 1 pg/µL with plasmid carrying the targeted sequence. Products were visualized by electrophoresis. The visual detection limit of plasmid in colorimetric reactions using Hydroxynaphthol blue (HNB) (120 µM) without BSA and PVP was 0.01 ng/µL. No cross-reactions with cDNA from ten frequently rose co-infecting or related viruses (HPV, MSV, INSV, TSWV, GRSV, ApMV, ArMV, PNRSV, TRSV and TMV) was detected. Healthy tissue and non-template controls were included in all reactions. RRV-LAMP tested successfully with 25 tissue samples of symptomatic RRD roses from Oklahoma. The method has potential applications in biosecurity, microbial forensics and nursery virus-free monitoring of germplasm.

## Development of a field-based detection technique for *Rose rosette virus* using isothermal reverse transcription-recombinase polymerase amplification

B. BABU (1), B. Washburn (2), S. Miller (2), F. Ochoa-Corona (3), G. Knox (1)

(1) North Florida Research and Education Center, University of Florida, Quincy, FL, U.S.A.; (2) Department of Biological Science, Florida State University, Tallahassee, FL, U.S.A.; (3) Department of Entomology and Plant Pathology, Oklahoma State University, Stillwater, OK, U.S.A.

Rose rosette disease, caused by *Rose rosette virus* (genus *Emaravirus*) is a major threat to the rose industry in the U.S. The only strategy currently available for disease management is early identification and eradication of the infected plants, thereby limiting its potential spread. Current RT-PCR based diagnostic methods for RRV are time consuming and is inconsistent in detecting the virus even from symptomatic plants. Hence a novel probe-based isothermal reverse transcription-recombinase polymerase amplification (RT-exoRPA) assay, based on the nucleocapsid gene of the RRV, has been developed. The assay is highly specific as tested against other common rose infecting viruses (both inclusive and exclusive). Dilution assays using the in vitro transcript showed that the assay is highly sensitive, with a detection limit of 1 fg. Moreover a rapid technique for the extraction of viral RNA (< 5 min) has been standardized from RRV infected tissue sources, using a single buffer which facilitates virus adsorption, followed by denaturation to release the RNA. RT-exoRPA analysis of the infected plants indicated that the virus could be detected from leaves, stems, petals, primary roots and secondary roots. The entire process including the extraction can be completed in less than 15 min, with less sophisticated equipments. The developed assay is currently evaluated for efficiency in large scale field testing for rapid detection of RRV in commercial nurseries and landscape.

## Development and characterization of polyclonal and monoclonal antibodies to Rose rosette virus

R. JORDAN (1), M. Guaragna (1), J. Hammond (1)

(1) US National Arboretum, USDA-ARS, U.S.A.

Garden roses, which form the cornerstone of the multi-billion dollar landscape industry, annually generate wholesale US domestic production valued at ~$400 million. Over the past few decades Rose rosette disease, caused by *Rose rosette virus* (RRV; genus *Emaravirus*), has become a major threat to the rose industry in the U.S. The only strategy currently available for disease management is early identification and eradication of the infected plants, thereby limiting its potential spread. Key to this effort is the development of efficient diagnostic tools to enable sensitive, rapid, user-friendly and accurate detection of the virus; currently limited to nucleic acid-based methods, such as PCR. With the aim of developing a serological diagnostic tool, rabbit polyclonal and several mouse monoclonal antibodies have been developed to the nucleocapsid protein (NP) of RRV. These antibodies were evaluated against NP peptides, cloned NP expressed in bacteria and plants, and RRV-infected plants. These results and those using the antibodies in various serological assay formats, including double- and triple-antibody sandwich ELISA and membrane-based assays, for lab and field detection of *Rose rosette virus* in infected sources, will be presented.

## Validation of RT-PCR High Resolution Melting as a detection method of *Tombusvirus*

F. OCHOA-CORONA (1), Y. Carrillo Tarazona (1)

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

Plant waterborne viruses, such as some in the genus *Tombusvirus*, spread as colloids in water and also by mechanical transmission. Broad detection and simultaneous species discrimination of viruses in this genus is needed for testing field samples. RT-PCR coupled to High Resolution Melting (HRM) has been used for different forensic applications and allows species differentiation as well as SNP detection. *Tombusvirus* complete genomic sequences retrieved from the NCBI-Genbank were used to design primers for broad detection using this method. Ten different virus species, *Carnation italian ringspot virus* (CIRV), *Cucumber necrosis virus* (CNV), *Cymbidium ringspot virus* (CymRSV), *Grapevine Algerian latent virus* (GALV), *Neckar river virus* (NRV), *Pelargonium leaf curl virus* (PLCV), *Pelargonium necrotic spot virus* (PNSV), *Petunia asteroid mosaic virus* (PetAMV), *Tomato bushy stunt virus* (TBSV) and *Pelargonium line pattern virus* (PLPV) were sourced by the DSMZ (Germany) and AGDIA (USA) and used for nucleic acid extraction. All viruses tested positive to broad detection endpoint RT-PCR with primers designed to react when coupled to HRM. Melting curve predictions calculated by *uMelt<sup>SM</sup>* algorithms were compared with data obtained using three DNA dyes (SYBR® Green, SYTO®9 and LCGreen®). Results with LCGreen® are closer to the predicted data calculated by *uMelt<sup>SM</sup>* when the amplicon size is between 50-200bp.
# Virus detection of *Tobamovirus* with wide spectrum degenerate oligonucleotides by TD RT-PCR High Resolution Melting

J. GARCIA SUAREZ (1), S. Dobhal (1), F. Ochoa-Corona (1)

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

Tobamoviruses cause notable plant diseases worldwide in *Solanaceae*, *Cucurbitaceae*, orchids and some are reported to be waterborne. Molecular tools exist for detection of this genus, however, a detection method combining genus detection and species discrimination is needed. Eleven reference positive controls from the DMSZ repository (Germany) and AGDIA (USA), were studied. Complete *Tobamovirus* genomes were aligned and a single antisense primer was designed to target the RNA dependent RNA polymerase (RdRp). This antisense primer co-reacted with five sense primers for selective subgroup detection by RT-PCR and HRM discrimination of eleven species within five subgroups. A protocol for detection and discrimination based on Touchdown Reverse transcription-Polymerase Chain Reaction (TD RT-PCR) coupled to High Resolution Melting (HRM) was developed. Species specific *dF/dT* Tm curves ranged 75-85°C and were generated for each primer groups as follow: I (BpEMV, PMMoV, TMV, ToMV), II (PaMMV, TMGMV), III (SFBV, RMV), IV (ORSV, CGMMV) and V (SHMV). The method allows the incorporation of new groups of tobamoviruses to the assay by designing additional group specific sense primers. The resulting 11 diagnostic sequences showed 98-100% identity regarding the targeted viruses. HRM facilitates a reliable discrimination of species specific profiles for each virus species. The method is amenable to microbial forensics diagnosis and biosecurity applications.

# Discriminating *Potexvirus* species by RT-PCR coupled to High Resolution Melting

A. OLMEDO-VELARDE (1), F. Ochoa-Corona (2)

(1) Universidad de las Fuerzas Armadas ESPE, Ecuador; (2) Oklahoma State University, U.S.A.

*Potexvirus* is a cosmopolitan plant virus genus and most of its thirty-seven confirmed species are mechanically transmitted, causing mosaic and ringspot symptoms on herbaceous hosts. High Resolution Melting analysis (HRM) allows the discrimination at species and strain levels and has application in diagnostics. Complete genome sequences for *Alternanthera mosaic virus* (AltMV), *Clover yellow mosaic virus* (CIYMV), *Lettuce virus X* (LeVX), *Opuntia virus X* (OpVX), *Papaya mosaic virus* (PapMV), *Pepino mosaic virus* (PepMV), *Potato aucuba mosaic virus* (PAMV), *Potato virus X* (PVX), *Schlumbergera virus X* (SchVX), *Strawberry mild yellow edge virus* (SMYEV) and *Hosta virus X* (HVX), were retrieved from NCBI Genebank and aligned using Mega 6 and screened for conserved domains. Primer set Potex F3/R3 was selected to discriminate among eleven potexviruses by a melting temperature prediction using uMelt<sup>SM</sup>, a melting curve prediction software program. Reference positive controls for these eleven viruses were obtained from DMSZ (Germany) and Agdia, Inc. (USA) and tested RT-PCR coupled to HRM with Potex F3/R3. All viruses tested positive and showed different melting temperatures ranging from 77.9°C to 82.4°C allowing for their discrimination through HRM analysis. The combination of RT-PCR-HRM can be used in diagnostic laboratories to discriminate eleven *Potexviruses with* high confidence.

# A preemptive detection system to screen water for the presence of plant viruses

J. Daniel (1), B. Gallucci Mazziero (1), B. Dunn (1), F. Ochoa-Corona (1)

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

Growers are expanding cultivation into non-favorable cropping areas pumping water from nearby lakes, rivers, wells, aquifers, and runoff, increasing the need for waterborne plant pathogen monitoring. Particulate matter dilution factors due to high water volumes, make detection and identification of potential pathogens a challenge because of the extremely low virion numbers. Current plant pathogen detection methods are passive, and sampling and testing occurs after disease symptoms appear. In the event of highly virulent pathogen incursions, detection may be too late to limit losses. This project seeks to develop a preemptive detection system that will easily sample large volumes of water to screen for the presence of plant pathogens. Three waterborne plant viruses: *Tomato bushy stunt virus*, *Pepino mosaic virus*, and *Pepper mild mottle virus* were used as models for this purpose. Previously developed multiplex detection protocols, were modified to detect these three waterborne viruses. Recovery of viruses using polyethylene glycol from 250 ml, 500 ml, and 1 L of water was demonstrated. Adaptation of an inexpensive, durable, and field-ready sampling device is presented. The testing of waterborne virus movement in two different microcosms is described. Monitoring of microbial loads in agricultural irrigation systems, and other water sources, is essential for effective surveillance and disease prevention for plant health.

# Multiplex RT-PCR detection of eight viruses infecting cucurbits and discrimination using High Resolution Melting analysis

L. PEÑA ZUNIGA (1), F. Ochoa-Corona (1), A. Ali (2)

(1) Oklahoma State University, U.S.A.; (2) University of Tulsa, U.S.A.

Cucurbits are one of the largest horticultural commodities in United States and Oklahoma largely contributes with watermelon production. However, the cucurbit cultivated acreage of Oklahoma had decreased, in part, due to viral infections. This research targets eight disease-causing viruses: *Cucumber mosaic virus* (CMVI and CMVII), *Cucurbit yellow stunting disorder virus* (CYSDV), *Melon necrotic spot virus* (MNSV), *Papaya ringspot virus* (PRSV), *Squash mosaic virus* (SqMV), *Watermelon mosaic virus* (WMV), and *Zucchini yellow mosaic virus* (ZYMV). A rapid detection and discrimination method is needed for biosecurity and breeding for resistance against these viruses. Sequences from the polymerase protein gene (RNA) from the six genera were retrieved from NCBI and aligned using MEGA 6. Primer sets were designed from conserved regions using the web application PrimerQuest® and the thermodynamic features of each primer were tested using Oligonalyzer 3.1. A Multiplex RT-PCR was developed to be coupled to High Resolution Melting (HRM) analysis for rapid detection of the targeted viruses. The obtained melting profiles allowed a clear discrimination among these viruses. This Multiplex RT-PCR High Resolution Melting method has potential to be applied in epidemiological studies of viral diseases in cucurbit crops, microbial forensics and biosecurity.

# Toward broad detection of emaraviruses: Endpoint RT-PCR

A. OLMEDO-VELARDE (1), F. Ochoa-Corona (2), T. Elbeaino (3)

(1) Universidad de las Fuerzas Armadas ESPE, Ecuador; (2) Oklahoma State University, U.S.A.; (3) Istituto Agronomico Mediterraneo di Bari, Italy

*Emaravirus* has six confirmed species and two unclassified species. Rapid detection and an effective screening of existing and new emaraviruses is needed for detection, identification, plant virus discovery and biosecurity applications. Available sequences of RNA dependent RNA polymerase (RNA 1) for *European mountain ash ringspot-associated virus* (EMARaV), *Fig mosaic virus* (FMV), *Pigeonpea sterility mosaic virus* (PPSMV), *Pigeonpea sterility mosaic virus 2* (PPSMV2), *Rose rosette virus* (RRV), *High Plains wheat mosaic virus* (HPWMoV) formerly High plains virus (HPV), *Raspberry leaf blotch virus* (RLBV) and Redbud yellow ringspot-associated virus (RYRSaV), were aligned using Mega 6. A primer set (EMARA F&R7/8) was designed from conserved domains after strand sense normalization. The sensitivity of EMARA F&R7/8 was found to be 100 fg/reaction assessed using a reference positive control of HPWMoV (Agdia). The multiple *Emaravirus* detection was confirmed with three rose samples showing symptoms caused by RRV from three locations in Stillwater, OK, a HPWMoV reference positive control, and cDNA of EMARaV and FMV. All samples were screened by endpoint RT-PCR and tested positive for HPWMoV, RRV, EMARaV and FMV. Other emaraviruses (RLBV, RYRSaV, PPSMV and PPSMV2) were detected *in-silico* using Primer-Blast. This method addresses the need for sensitive molecular detection methods and discovery of novel emaraviruses.

# Waterborne plant virus sampling and detection in aqueous environment

B. GALLUCCI MAZZIERO (1)

(1) Oklahoma State University, U.S.A.
The aim of this research is studying water sampling as a critical tool for detection of unwanted plant pathogenic virus that can seriously damage agriculture and environment. Water sources, such as reservoirs, lakes, farm ponds, tanks, and irrigation systems are vulnerable to inadvertent (most frequent) and intentional (rare) contamination by microbial pathogens. Pathogen detection in large bodies of water is complex because volume, dilution and water dynamics. This project assesses the development of a method for water sampling and detection of waterborne plant viruses of concern for water-security. Rapid detection and monitoring of high consequence plant pathogens in water systems, where may be present at extremely low titers present a significant challenge. The project seeks statistically validation of a water sampling model that uses quantitative-PCR as the method to detected *Potexvirus*, a genera of plant virus reported to have water borne members. In order to determine the minimum amount detectable in water, sensitivity tests will be conducted by ELISA and qPCR. Different pH will be tested during a process of virus filtration using a filtration system based on glass-wool. The capture is based on the isoelectric point affinity of the virus capsid protein with glass wool at an acid-neutral pH. This research is needed to support decision-making and will be useful for survey analysis and prevention, detection, and mitigation of unwanted waterborne plant viruses.

## Occurrence of endornaviruses in non-cultivated plant species

R. HERSCHLAG (1), E. Rodrigues de Souto (2), R. Valverde (1)

(1) Louisiana State University Agricultural Center, U.S.A.; (2) Universidade Estadual de Maringa, Brazil

Large dsRNAs have been extracted from plants infected with acute and persistent viruses and from fungi infected with mycoviruses. These extracted dsRNAs have been used for viral disease diagnosis and virus identification. We used a modified version of the non-phenol batch method, in combination with reverse-transcription PCR (RT-PCR), to test symptomless, non-cultivated, plant species for endornaviruses. Foliar tissues were collected from different plant species from various locations within the city of Baton Rouge, Louisiana. Tissues were desiccated with silica gel and used for dsRNA extractions. After testing 132 plant species, belonging to 60 families, six yielded dsRNAs similar in size (14-15 kb) to reported endornaviruses. Putative endornaviruses were detected in *Alternanthera philoxeroides* (alligator weed), *Geranium carolinianum* (wild geranium), *Sonchus asper* (spiny sow thistle), *Sorghum halepense* (Johnson grass), and *Hydrocotyle umbellata* (dollar weed). The same endornaviruses were also detected in at least 10 individual plants of the same species. RT-PCR tests using endornavirus-degenerate primers were conducted with the dsRNAs of the six putative endornaviruses. Results of this investigation suggest that endornaviruses occur at a rate of approximately 5% in non-cultivated plant species and that like endornaviruses of cultivated plants, their vertically transmission rate is high.

## Development and validation of a quadruplex real-time RT-PCR assay for simultaneous detection of three *Citrus leprosis* viruses in plants

G. WEI (1), A. Roy (2), W. Schneider (3), R. Brlansky (2), M. Nakhla (1)

(1) USDA-APHIS-PPQ-S&T-CPHST, U.S.A.; (2) University of Florida, IFAS, Plant Pathology Department, Citrus Research and Education Center, U.S.A.; (3) USDA-ARS-FDWSRU, U.S.A.

*Citrus leprosis virus* (CiLV) is the causal agent of Citrus leprosis - one of the most destructive diseases of citrus. CiLV is an RNA virus transmitted by *Brevipalpus* mite species. There are two major types of CiLV being described – one is the cytoplasmic type residing in the cytoplasm of infected cells and the other is the nuclear type primarily residing in the nucleus of infected cells. In the last decade CiLV spread swiftly from South America to Central America and recently has been detected in Mexico. The proximity and the potential threat to the multi-billion dollar US citrus industry have caused concerns in the United States where the disease has not been found since the 1960s. Molecular identification and early detection of each the cytoplasmic and nuclear types are essential to prevent CiLV from introduction and establishment in the US. In order to reduce testing time, cost and labor, we are developing and validating a quadruplex real-time RT-PCR assay for simultaneous detection of cytoplasmic CiLV-C, CiLV-C2, and nuclear CiLV-N with a Nad5 plant RNA internal control. From our repeated experiments, the results showed that sensitivity and specificity for the quadruplex RT-PCR assay were comparable to the level of monoplex RT-PCR detecting CiLV-C, CiLV-C2, and CiLV-N respectively. Further validation for the quadruplex RT-PCR assay including tests with additional field samples is in progress.

## A sociological assessment of *Potato virus Y* in western Washington: Barriers and bridges to adopting new management practices

A. BEISSINGER (1), C. Benedict (2), J. Goldberger (3), D. Inglis (1)

(1) Washington State University Northwestern Washington Research and Extension Center, Mount Vernon, WA, U.S.A.; (2) Washington State University Whatcom County Extension, Bellingham, WA, U.S.A.; (3) Washington State University, Pullman, WA, U.S.A.

Seed and commercial potatoes are grown in separate geographic areas of western Washington (WWA). In 2012, recombinant strains of *Potato virus Y* (PVY), PVYNTN and PVYNWi, were reported in WWA potato fields. The strains are largely asymptomatic on potato foliage and difficult to detect by visual field inspections and winter greenhouse grow-out tests. The problem led to tuber yield and quality losses of ~40% for commercial producers who purchased inaccurately certified seed. In 2015-16, most (N=6) of the seed potato growers in WA and approximately one-third (N=6) of the large-scale commercial potato growers in WWA were interviewed about the barriers and bridges to adopting updated PVY detection approaches such as ELISA and PCR-based laboratory tests, tropical winter field grow-outs, and PVY recognition trainings. The major barriers to adoption were logistical learning curves (67%), uncertainty that updated techniques would be advantageous (67%), and distrust of laboratory results (50%). The major bridges included premiums paid by customers for reliable PVY-free seed (75%), customer demand for more sensitive PVY testing methods (67%), and faster grow-out results (33%). The study results can guide researchers and extension educators who make PVY management recommendations that must be relevant to the needs and perceptions of growers. Further, they suggest that sociological studies of plant pathogens can provide a valuable tool for assisting in plant disease management.

## Rapid and sensitive detection of tomato chlorotic dwarf viroid in crude plant extracts using isothermal RT-recombinase polymerase amplification

R. HAMMOND (1), S. Zhang (2)

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

Tomato chlorotic dwarf viroid (TCDVd) is a serious threat to tomato production worldwide. Infection leads to reduced plant vigor, small and deformed fruit, and yield loss. TCDVd is easily transmitted mechanically from plant to plant once introduced and through seed; seed treatments do not control transmission of disease. The most effective control is the use of certified viroid-free planting materials. We developed a molecular diagnostic assay utilizing reverse transcription-recombinase polymerase amplification (RT-RPA) at an isothermal constant temperature of 39°C and target-specific primers and probe for the rapid, sensitive, and specific detection of TCDVd in crude extracts of infected leaf and seed tissues. The performance of the AmplifyRP® Acceler8™ RT-RPA diagnostic assay, utilizing a lateral flow strip contained within an amplicon detection chamber, was evaluated and the results were compared with a standard RT-PCR assay. The AmplifyRP® Acceler8™ assay was specific for TCDVd and its sensitivity was comparable to conventional RT-PCR. The assay does not require extensive sample purification, specialized equipment, or technical expertise. It is easy to perform and can be used under both laboratory and field conditions.

## Distribution and vertical transmission of Southern tomato virus in tomato

S. COSKAN (1), R. Alcalá-Briseño (1), J. Polston (1)

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

*Southern tomato virus* (STV) is a recently identified virus that infects tomato cultivars worldwide. STV has is a monopartite virus with a double-stranded RNA genome (3.5 kb) containing two overlapping open reading frames (ORFs) and is the type species of the genus *Amalgavirus* (family
*Amalgaviridae*). A reverse-transcription polymerase chain reaction (RT-PCR) using virus-specific primers was used to detect STV in 21 tomato cultivars. STV was detected in plants produced by a number of seed companies which indicates that the virus is widely distributed in the vegetable seed industry. STV was detected for the first time in four cultivars: 'Agriset 761', 'Mexico Midget', 'Roma' and 'Sweet Hearts'. 'Sweet Hearts' tomato plants were evaluated for the presence of STV using RT-PCR at different developmental stages of tomato plants. STV could be detected in the 1 to 2 week-old germinated seedlings at a frequency similar to that of older plants from the same seed lot. The presence or absence of STV was consistent across all four developmental stages. Seed transmission studies of progeny produced from crosses of different combinations of parents that were STV-positive or negative, revealed that STV could be transmitted to the next generation by either ovule or pollen.

## Emergence of a novel white stripe disease pathogen *Stenotrophomonas maltophilia* strain Sia5 in rice fields of Punjab under the climate change scenario

K. RIAZ (1), K. Riaz (2), A. Dilferoze (3), M. Alam (3), A. Riaz (4), S. Jameel (5), S. Khan (6), A. Hannan (7)

(1) Department of Plant Pathology, University of Agriculture Faisalabad, Pakistan; (2) University of Agriculture Faisalabad, Pakistan; (4) Plant Protection Division NIAB, Faisalabad, Pakistan; (5) Department of Plant Pathology, University of Agriculture, Faisalabad-38040, Pakistan; (6) Centre of Agricultural Biochemistry and Biotechnology, University of Agriculture, Faisalabad-38040, Pakistan; (7) Department of Botany, Ghazi University, Dera Ghazi Khan, Dera Ghazi Khan, Pakistan

Under severe fluctuations in temperature and rainfall patterns several previously unimportant pathogens are gaining importance especially in chief cash crops in many parts of the world. Recent surveys over the last three years in the rice Kallar belt districts of Punjab (Kala Shah Kaku, Hafizabad, Sheikhpura, Narowal, Shakargarh, and Sialkot) depicted the presence of a novel disease and its associated pathogen which is responsible for the appearance of white stripe symptoms on leaves that start from the leaf tip and extend along the midrib through the leaf lamina towards the base of the leaf sheath. Under severe infection leaf size got reduced, whole leaves got dried and panicles matured very late. Diseases incidence ranged from 25-30% inflicting 20-25% yield losses. Pathogen was isolated on nutrient agar that formed dark to pale yellow raised colonies with fluffy appearance and was reconfirmed through pathogenicity testing on susceptible rice variety. The most aggressive and most virulent isolate belonged to Sialkot region and was characterized as *Stenotrophomonas maltophilia* strain Sia5 through the BLAST searches and phylogenetic analysis of its PCR amplified 16srDNA sequence (GB: KT924441.1) and other biochemical characteristics. This is the first report on the occurrence of white stripe disease in rice from Pakistan.

## An invasive lethal phytoplasma disease threatening stone fruit production: Detection, epidemiology and management

Y. A. JAWDAH (1), M Jawhari (2), P. Tawidian (2), E. Choueiri (3), F. Quaglino (4), P. Bianco (4), A. Alma (4), M. Moilno-Lova (5)

(1) American University of Beirut, Beirut, Lebanon, (2) FAFS, AUB, (3) LARI, (4) UNIMI, Milan, Italy, (4) IDISAFA, Univ. Di Torino, Torino, Italy, (5) NNIMI, Milan, Italy

Introduced into North Lebanon in the early 1990s, almond witches' broom (AlmWB) disease spread rapidly in Lebanon killing about 200,000 almond, peach and nectarine trees. The disease is associated with 'Candidatus Phytoplasma phoenicium' that belongs to 16S rRNA subgroup IX-B. The major symptoms are proliferation, small chlorotic leaves, development of witches' broom followed by dieback and death of almond trees. So far, AlmWB has been reported in Lebanon and Iran. In Lebanon, AlmWB was considered of quarantine importance and official containment measures were initiated in major stone fruit production areas. Specific, sensitive and reliable PCR and real-time PCR detection methods were developed. Preliminary data revealed that a leafhopper and planthoppers are vectors of the disease and two weed species are suspected to act as alternative hosts. An integrated disease management approach is considered for highly infested regions. Grafting experiments are in progress and preliminary results look encouraging, a recovery phenomenon was observed. Finally, regional and international cooperation are highly recommended to continue studies on the epidemiology of this invasive disease and to adopt phytosanitary control measures and strategies in order to contain the further spread of the disease.

## Root and shoot susceptibility of peach/almond rootstock to pistachio bushy top syndrome isolates of *Rhodococcus* spp.

E. Fichner (1), S. Dhaouadi (2)

(1) University of California Division of Agriculture and Natural Resources, U.S.A.; (2) University of California Division of Agriculture and Natural Resources, U.S.A.

Approximately 30,000 acres of UCB-1 clonal pistachio rootstock planted in California from 2011-2014 were affected with pistachio bushy top syndrome (PBTS) caused by two phytopathogenic bacteria highly related to *Rhodococcus fascians* (*Rf*) and *Rhodococcus corynebacterioides* (*Rc*). Orchards with high disease incidence were removed and replanted to pistachio or almond. The susceptibility of almond rootstock to PBTS isolates is unknown. Objectives include determination of the susceptibility of peach/almond rootstock to PBTS isolates and the influence of root and shoot infections on symptom development and host colonization. Either roots or shoots of clonal 'Hansen 536' rootstock were dipped in a bacterial suspension containing both PBTS isolates. Uninoculated control plants were dipped in buffer solution. Plant growth metrics and shoot and root biomass were determined 8 weeks after inoculation, and leaves were sampled for endo- and epiphytic populations of *Rf* and *Rc*. Root-inoculated plants were stunted and exhibited increased node density in comparison to control plants. Foliar-inoculated plants remained asymptomatic, but foliar inoculation resulted in endophytic colonization of leaves. Root-inoculations resulted in aboveground plant colonization. The results demonstrate that 'Hansen 536' rootstock is susceptible to the PBTS isolates. The results indicate a need for field evaluation for disease in PBTS-affected orchards replanted to almond.

## Xylella fastidiosa isolates differ in the ability to undergo genetic recombination

P. KANDEL (1), R. Almeida (2), L. De La Fuente (1)

(1) Department of Entomology and Plant Pathology, Auburn University, U.S.A.; (2) Department of Environmental Science, Policy and Management, UC Berkeley, U.S.A.

*Xylella fastidiosa* (*Xf*) is a bacterium causing incurable diseases in economically important crops such as grapevines, citrus, and olives. Historically assumed to be restricted to the Americas, *Xf* diseases have been reported in both new locations and new crops in recent years. Inter-subspecific recombination (ISR) has been proposed to contribute to host shifts based on genetic diversity studies. Natural competence, as a mode of recombination, has been shown to occur in vitro in systems mimicking natural habitats. However, little is known about the recombination potential of *Xf* isolates, which are believed to be somewhat host specific. Therefore, in this study we compared the recombination frequencies of twelve *Xf* isolates belonging to two subspecies (*fastidiosa* and *multiplex*), using plasmids containing antibiotic markers flanked on either side by *Xf* homologous regions. The recombination frequency varied greatly (3.14 × 10⁻² – 2.3 × 10⁻⁸) among isolates. Generally, subsp. *fastidiosa* isolates had higher recombination frequencies than *multiplex* isolates, and within a subspecies, the most virulent isolates had higher frequencies. When combinations of marker-tagged, heat-killed donor and live recipient cells from two subspecies were mixed, ISR occurred with a genomic region of ~10kb transferred from the donor to the recipient cell. This study demonstrates different recombination abilities, and therefore suggests different evolutionary potentials among *Xf* isolates.

## Soil survival potential of pistachio bushy top syndrome isolates of *Rhodococcus* spp.

E. FICHTNER (1), S. Dhaouadi (1), E. Molina (2), R. Stamler (2), J. Randall (2)

(1) University of California Division of Agriculture and Natural Resources, U.S.A.; (2) New Mexico State University, U.S.A.

Over 35,000 acres of pistachio planted from 2011-2014 in California were affected by pistachio bushy top syndrome (PBTS), a disease caused by bacteria related to *Rhodococcus corynebacterioides* (*Rc*) and *Rhodococcus fascians* (*Rf*). Because the survival potential of PBTS isolates in soil is unknown, growers are reluctant to replant pistachios in affected orchards. The goal of this work was to assess the longevity of *Rc* and *Rf* in field soils
exposed to environmental conditions and in potting medium under greenhouse conditions. An in situ field study was established in a PBTS orchard with each isolate embedded on wooden sticks and buried at various depths. Another study utilized excavated field soils incubated outdoors to investigate the influence of soil type and moisture on survival. In a greenhouse study, potting medium was infested with *Rf* in the presence and absence of dried pistachio plant material. After three months, populations of *Rf* in potting medium remained similar to initial populations, regardless of incorporation of plant tissue. After one month, *Rf* populations increased in moist, non-saline field soil, but *Rc* was almost eliminated. Both isolates were inhibited by salinity and drying, but depth did not influence survival. The results suggest saprophytic competence of *Rf*, and a consequent concern for persistence of *Rf* in affected orchards and nurseries and illustrate the need for vigilance in sanitation from the nursery to the field.

## Bacterial black spot of mango, Mangifera indica, caused by Xanthomonas citri pv mangiferaeindicae, is confirmed in the Western Hemisphere

G. SANAHUJA (1), R. Ploetz (1), P. Lopez (1), J. Konkol (1), A. Palamateer (1)

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

During the summer of 2015, symptoms of what appeared to be bacterial black spot (BBS) were observed on mature fruits of mango, *Mangifera indica*, in the vicinity of Boynton Beach, FL. Star-shaped lesions, up to 2 cm in diameter, erupted from the surface of 'Keitt' and other cultivars, and oozed a sticky, clear exudate. From lesions, a cream-colored bacterium was recovered on YPGA and characterized with housekeeping genes used by Bui Thi Ngoc et al. (2010) (*efp* and *dnaK*). Amplicons from a representative isolate were sequenced (KU746336 and KU746337) and were 100% homologous with strains of *Xanthomonas citri* pv *mangiferaeindicae* (Xcm) in GenBank. ML analysis (Tamura-Nei model) with MEGA 6.06 placed the isolate among other strains of Xcm. In incubator studies (30°C day, 26°C night, 12h light), the mesophyll of leaves on potted 'Keitt' and 'Haden' plants were injected with a hypodermic needle and $1 \times 10^5$ CFU mL$^{-1}$ of the isolate. After 7 days, black lesions developed on inoculated leaves, but not on leaves injected with sterile deionized $H_2O$. Diagnostic amplicons for Xcm were generated for isolates recovered from the lesions, and the experiment was conducted twice. BBS is widespread in the Eastern Hemisphere, but this is the first report of the disease in the Western Hemisphere. Environmental conditions in southern Florida appear to be ideal for an increased importance of BBS in the region.

## Survey on pathogenic Xanthomonas recovered from different lettuce cultivars in Canada

V. TOUSSAINT (1), D. Xu (1), P. Hébert (1), M. Cadieux (1), M. Ciotola (1), H. Van der Heyden (2), D. Rekika (3), S. Jenni (1)

(1) Agriculture and Agri-Food Canada, St-Jean-sur-Richelieu, QC, Canada; (2) Compagnie de recherche Phytodata, Sherrington, QC, Canada; (3)

Fondation pour l'amélioration génétique de la litié et des légumes feuilles, Napierville, QC, Canada

Bacterial leaf spot, caused by *X. hortorum* pv. *vitians* (*Xhv*), is an important lettuce disease. In Canada, the main difficulty for disease control is the absence of registered chemicals. Thus, the most sustainable way to reduce yield losses is using resistant cultivars. Preliminary studies showed that different isolates did not share the same lettuce host range. Therefore, different *X. hortorum* pathotypes should be considered in a breeding program for resistance. The objective was to determine which pathotypes are present in Canada by sampling diseased lettuce plants in commercial production. In 2014 and 2015, 376 lettuce samples were collected in the main lettuce production area. *Xanthomonas* isolates were recovered from leaf lesions and characterized based on DNA sequences of four housekeeping genes (*dnaK*, *fyuA*, *gyrB* and *rpoD*). Concatenated DNA sequences of these genes for the isolates and reference strains were analyzed with Neighbor-Joining method using MEGA v6.04 to generate a phylogenetic tree. The 363 isolates formed two major clusters. Cluster I includes 17 strains representing 358 isolates and grouped with the *Xhv* reference strain BS3046. This Cluster can be separated into two sub-groups and preliminary evidence shows that they shared different lettuce host range. The Cluster II, a distinct group of *X. hortorum*, contains eight new strains, but the pathotype has to be determined. Further investigation is needed on the epidemiology of this unknown pathotype.

## Wheat blast is caused by multiple Pyricularia species, including Pyricularia graminis-tritici sp. nov.

V. CASTROAGUDIN (1), S. Moreira (2), D. Pereira (3), S. Moreira (4), P. Brunner (3), J. Maciel (5), P. Crous (6), B. McDonald (3), E. Alves (7), P.

Ceresini (4)

(1) University of Sao Paulo State, Ilha Solteira, Brazil; (2) Federal University of Lavras, Lavras, Brazil; (3) Institute of Integrative Biology, ETH Zurich, Zurich, Switzerland; (4) University of Sao Paulo State, Ilha Solteira, Brazil; (5) Brazilian Agriculture Research Corporation-Wheat (EMBRAPA-Trigo), Passo Fundo, Brazil; (6) CBS-KNAW Fungal Biodiversity Centre, Utrecht, Netherlands; (7) Federal University of Lavras, Lavras, Brazil

Presently, *Pyricularia oryzae* pathotype *Oryza* (Po-O) is considered the widely distributed rice blast pathogen, whereas *P. oryzae* pathotype *Triticum* (Po-T) is the South American wheat blast pathogen. In this study, we investigated whether Po-O and Po-T were distinct at species level. We also described a new *Pyricularia* species causing blast on several Poaceae, including wheat, from Brazil. Bayesian phylogenetic analyses were conducted on 10 genes from an extensive sampling (N = 128) of populations of *Pyricularia* adapted to rice, wheat and other Poaceae. Isolates were grouped into two major clusters (Clusters I and II, P=0.99). Cluster I contained isolates from wheat and another Poaceae (P=0.98). Cluster II was sub-divided in three clades (P>0.75). Clades 1 and 2 grouped wheat-derived isolates, and Clade 3 contained rice-derived isolates. The Clusters I and II corresponded to two distinct species: *Pyricularia graminis-tritici* sp. nov. (Pgt), newly described here, and *Pyricularia oryzae* (Po). The clades of Po concurred with Po-T and Po-O, and were not distinct at species level. Among species, morphological differences were not recorded; however, a distinctive pathogenicity spectrum was observed. Pgt and Po-T were highly virulent on wheat, barley, signal grass, and oats. Po-O was highly virulent on rice, wheat, barley, and oats, but not on signal grass. We demonstrated that blast disease on wheat and other Poaceae from Brazil is caused by multiple *Pyricularia* species.

## Unraveling Silverleaf disease: Reversion of foliar symptoms and plants recovery

D. GRINBERGS (1), A. France (1), J. Chilian (1)

(1) Instituto de Investigaciones Agropecuarias, Chile

Silverleaf, caused by the Basidiomycete *Chondrostereum purpureum*, is an important disease in fruit trees which produce wood necrosis and leaves silvering. In apple has been increasing due to changes in management, reducing orchards viability. Plants deteriorate their physiological condition, being water uptake and chlorophyll content the most affected parameters. The latter alters photosynthesis, reducing fruit yield and quality. Field studies have shown the spontaneous reversion of foliar symptoms, thus, the objective was to compare the physiological and sanitary condition of Brookfield plants before and after reversion. The sanitary condition was determined assessing symptoms severity, isolating the fungus on PDA, amplificating the large intergenic spacer (IGS) rDNA región and measuring foliar endopoligalacturonase concentration using DAS-ELISA. Physiological condition was measured through xylem water potential, chlorophyll fluorescence, a, b and a + b chlorophyll content and stomatal conductance. Besides, yield and fruit quality parameters such as color and starch were assessed. Isolation, PCR and ELISA indicated that the fungus was present in symptomatic plants and after reversion. Symptomatic plants presented alterations in all physiological parameters (P<0,05%), reduction of yield (38%), maduration disorders and lack of color. However, after reversion of silvering, physiological condition, yield and fruit quality were restored.

## Fungi in the wood: The fungi associated with the ambrosia beetle, *Xylosandrus germanus* and its galleries in *Malus domestica*

S. VILLANI (1), K. Ayer (2), D. Breth (3), K. Cox (2)

(1) North Carolina State University, U.S.A.; (2) Cornell University, U.S.A.; (3) Cornell Cooperative Extension, U.S.A.

The ambrosia beetle, *Xylosandrus germanus*, is considered an emerging threat to Western New York apple orchards. There are concerns that fungal farms established by *X. germanus* within vascular tissue play a role in the collapse of young apple trees. To gain further insight into the identity and relationship between fungi associated with *X. germanus* and decline of *Malus x domestica*, fungal isolations were performed from *X. germanus* wood
galleries and adjacent vascular tissue, and beetles recovered from galleries. Fungal identity was confirmed by sequencing the 2 internal transcribed spacer regions and the 5.8S gene of ribosomal DNA. Five distinct fungal species were recovered from the exterior of 36 beetles, while only 2 fungal species were recovered from the beetles' interior. *Nectria haematococca* (an. *Fusarium solani*) was most frequently isolated from the exterior and interior of *X. germanus*, while *Ambrosiella xylebori*, another common food source of ambrosia beetles, was only isolated from the interior and exterior of one beetle. Interestingly, however, *A. xylebori* was predominantly recovered from infested wood tissue. Our results suggest that *X. germanus* can establish fungal gardens of *N. haematococca* and *A. xylebori* within vascular tissue of apple trees, however it is unclear if tree decline is likely due to direct tunneling or colonization of *A. xylebori*. Additional investigations are ongoing to understand the implications of *A. xylebori* colonization of apple.

## High diversity in the population of an emerging blackberry pathogen, *Fusarium oxysporum*

A. PASTRANA (1), S. Kirkpatrick (1), T. Gordon (1)

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

A wilt disease of blackberry, caused by the soilborne fungus *Fusarium oxysporum*, has become a more widespread problem in California over the past two years. This emerging pathogen is severely reducing fruit production in affected fields. To improve our understanding of the structure of pathogen population, isolates collected from symptomatic blackberry plants in California and Mexico were evaluated for morphology, pathogenicity and vegetative compatibility. The translation elongation factor 1 alpha (TEF1-α), the intergenic spacer (IGS) and the β-tubulin regions were also sequenced. The combination of TEF1-α, β-tubulin and IGS sequences identified clusters of isolates that corresponded to vegetative compatibility groups (VCGs). A high number of distinct populations were founded both in California and Mexico. However, one VCG included isolates from both countries. No relation was found between VCG and cultivar or geographic origin. Diversity in the pathogen population suggests that the capacity to cause disease on blackberry is not a recent development.

## Mucor Rot – An Emerging Postharvest Disease of Mandarin Fruit Caused by *Mucor piriformis* and other Mucor spp. in California

S. SAITO (1), T. Michailides (2), C. Xiao (1)

(1) USDA-ARS, San Joaquin Valley Agricultural Sciences Center, U.S.A.; (2) Kearney Agricultural Research & Extension Center, UC-Davis, Division of Agriculture and Natural Resources, U.S.A.

In recent years, an emerging, undescribed postharvest disease was observed on mandarin fruit after extended storage in California. We collected decayed mandarin fruit from three citrus packinghouses in the Central Valley of California in 2015 and identified this disease as Mucor rot caused by *Mucor* spp. Mucor rot occurred in 11 of the 15 grower lots sampled, and the incidence of the disease in the total decayed fruit varied among affected grower lots, ranging from 3.3 to 93.1% with an average of 49.2%. In total, 197 isolates of *Mucor* spp. were obtained from decayed mandarin fruit and identified based on ITS sequence and morphological characteristics. Of the 197 isolates, 92.4%, 3.6%, 2.0%, 1.5%, and 0.5% were identified as *M. piriformis*, *M. circinelloides*, *M. racemosus*, *M. hiemalis*, and *M. mucedo*, respectively. Pathogenicity tests on mandarin fruit showed that *M. piriformis* was the most virulent among all tested species. Our results indicated that Mucor rot of mandarins in California is caused by *Mucor* species consisting of *M. piriformis*, *M. circinelloides*, *M. racemosus*, *M. hiemalis*, and *M. mucedo*, with *M. piriformis* being the most prevalent species. Previously, *M. racemosus* was reported on citrus. This is the first report of Mucor rot on citrus caused by *M. piriformis*, *M. circinelloides*, *M. hiemalis*, and *M. mucedo*.

## Botrytis californica, a new *Botrytis* species causing gray mold in blueberries and table grapes in California

S. SAITO (1), D. Margosan (2), T. Michailides (3), C. Xiao (1)

(1) USDA-ARS, San Joaquin Valley Agricultural Sciences Center, Parlier, CA, U.S.A.; (2) USDA-Agricultural Research Service, San Joaquin Valley Agricultural Sciences Center, Parlier, CA, U.S.A.; (3) Kearney Agricultural Research & Extension Center, Parlier, CA, U.S.A.

The *Botrytis cinerea* species complex comprises two cryptic species, originally referred as Group I and Group II based on *Bc-hch* gene RFLP haplotyping. Group I was described as a new cryptic species *B. pseudocinerea*. During a survey of *Botrytis* spp. causing gray mold in blueberries and table grapes in the Central Valley of California, six isolates, three from blueberries and three from table grapes, were placed in Group I but showed a distinct morphological character with conidiophores significantly longer than those of *B. cinerea* and *B. pseudocinerea*. We compared these isolates with *B. cinerea* and *B. pseudocinerea* by examining morphological and physiological characters, sensitivity to fenhexamid, and phylogenetic analysis inferred from sequences of three nuclear genes. Phylogenetic analysis using the three partial gene sequences encoding glyceraldehyde-3-phosate dehydrogenase (G3PDH), heat-shock protein 60 (HSP60), and DNA-dependent RNA polymerase subunit II (RPB2), supported the proposal of a new *Botrytis* species, *B. californica*, which is genetically closely related to *B. cinerea*, *B. pseudocinerea*, and *B. sinoviticola*, all known as causal agents of gray mold of grapes. *B. californica* caused decay on blueberry and table grape fruit inoculated with the fungus. This study suggests that *B. californica* is a cryptic species sympatric with *B. cinerea* on blueberries and table grapes in California.

## Multiple *Pestalotiopsis* and *Neopestalotiopsis* species cause flower blight of macadamia in Australia

O. AKINSANMI (1), S. Nisa (1), O. Jeff-Ego (1), A. Drenth (2)

(1) University of Queensland, Brisbane, Australia; (2) University of Queensland, Australia

Incidence of 'dry flower' the disease, has been increasing and now occurs in different production regions in Queensland and New South Wales, Australia. Although yield losses range between 10% and 30%, total crop failures have occurred in certain cultivars. Therefore, this study examined the etiology of the disease in Australia. Results from a survey of over 240 macadamia racemes in different orchards in Australia revealed that all the four developmental stages of macadamia racemes including the florets and the rachis were affected. Several fungi genera including *Alternaria*, *Epicoccum*, *Fusarium*, *Botrytis*, *Cladosporium*, *Penicillium*, *Aspergillus* and *Diaporthe* were obtained from tissue samples, however, their occurrence was irregular between samples and significantly (P <0.05) less than *Pestalotiopsis*-like isolates, which constituted 41% of the total isolates. Based on the concatenated partial sequence of three gene loci (Internal transcribed spacer, β-tubulin and translation elongation factor 1-alpha) the phylogenetic analysis of 32 *Pestalotiopsis*-like isolates showed they formed two novel taxa in the *Neopestalotiopsis* and *Pestalotiopsis* genera. The grouping of the isolates was regardless of location or plant part. Koch's postulates were confirmed with the different *Neopestalotiopsis* and *Pestalotiopsis* species and this is the first report of these two genera as causal agents of raceme blight of macadamia.

## Cytospora leucostoma is the most virulent and prevalent causal agent of Cytospora Canker on peaches on the Western Slope of Colorado

J. Stewart (1), K. Otto (1), I. Minas (2), S. Miller (1)

(1) Bioagricultural Sciences and Pest Management, Colorado State University, Fort Collins, CO, U.S.A.; (2) Department of Horticulture and Landscape Architecture; Western Colorado Research Center Colorado State University, Grand Junction, CO, U.S.A.

Cytospora canker on peaches (*Prunus persica* (L.) Batsch) is known to be caused by three species, *Cytospora cinctum*, *C. leucostoma*, and *C. paraleucostoma* within growing areas of the United States. In Colorado, recent surveys show that over 90% of the trees on the Western Slope are infected with Cytospora canker, causing significant economic impacts to peach growers. Understanding how these species differ phenotypically and in disease cycle and progression is important for disease management. The objectives of this study were to identify *Cytospora* isolates collected from high and low elevation sites, examine population structure and recombination, and assess each species/population for phenotypic characters such as virulence, temperature growth and culture morphology. The ITS region was sequenced to identify 106 isolates. Both *C. leucostoma* and *C. paraleucostoma* were recovered, although *Cytospora leucostoma* was the most prevalent. Additional sequencing of 4 genetic loci (ca. 1950 bps), suggested that *C. leucostoma* and *C. paraleucostoma* recombine and that well-supported clades do not exist for 3 of the 5 loci. Phenotypic characterization indicates overlapping
variation in temperature, growth, culture morphology, and virulence. These data suggest that isolates are genetically distinct populations of *C. leucostoma* rather than cryptic species.

## New Botrytis species affecting strawberry on the east coast

G. SCHNABEL (1), M. Dowling (1), M. Hu (1)

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

We detected a new species of *Botrytis* infecting strawberries in the United States. While monitoring fungicide resistance of 459 *Botrytis* isolates collected from South Carolina (211), Maryland (123), North Carolina (101), Virginia (17), and Ohio (7), we observed isolates with phenotypes different from typical *Botrytis cinerea*. These isolates produced fluffy, white mycelium and sporulated poorly on potato dextrose agar, minimal medium, strawberries, apples, and tomatoes. These isolates were found in 11 different locations across South Carolina (20), and North Carolina (4). The G3PDH gene was sequenced for these isolates and nucleotide BLAST search revealed that they formed a cluster separate from *Botrytis* species previously described. Further sequencing of RPB2, Hsp60, NEP1, and NEP2 genes for three representative isolates revealed that these isolates were identical to isolates of *Botrytis fragariae*, a species recently discovered infecting strawberry in Germany, but never before observed in the United States. In order to determine the distribution and prevalence of this species in the eastern United States, primers were developed that distinguish *Botrytis cinerea* and *Botrytis fragariae*. Preliminary data suggest that *Botrytis fragariae* isolates are more likely to have reduced sensitivity to the new fungicide polyoxin D. The discovery of a new species of *Botrytis* causing gray mold of strawberry may require adjustments in gray mold management strategies.

## Laurel wilt, caused by *Raffaelea lauricola*, is detected for the first time outside the southeastern USA

R. PLOETZ (1), Y. Thant (2), M. Hughes (3), T. Dreaden (4), J. Konkol (1), A. Kyaw (2), J. Smith (3), C. Harmon (3)

(1) University of Florida, Homestead, FL, U.S.A.; (2) Winrock International, Farmer-to-Farmer Program, Yangon, Myanmar, Yangon, Burma; (3)

University of Florida, Gainesville, FL, U.S.A.; (4) U.S. Forest Service, Lexington, KY, U.S.A.

Agriculture is an important industry in Myanmar (aka Burma), accounting for 60% of the GDP and employing 65% of the work force. In October 2014, a survey for diseases and pests of an emerging fruit crop in the country, avocado (*Persea americana*), was conducted in Southern Shan State. Around Tuanggyi, monocultures of up to 20 hectares have been established, whereas avocado is an over-story tree where coffee is grown in the Ywangan area. Symptoms of laurel wilt, including sapwood discoloration, leaf necrosis, defoliation, and tree mortality were observed during the survey in both areas. In Myanmar, isolates that resembled the laurel wilt causal agent, *Raffaelea lauricola*, were recovered from symptomatic sapwood on ½ strength potato dextrose agar + streptomycin sulfate. In the USA, *R. lauricola*-specific amplicons were generated for isolates from eight different trees with PCR primers for two taxon-specific microsatellite loci. In a quarantine greenhouse in Gainesville, FL, three of the isolates caused laurel wilt in each of two tests on avocado and swamp bay (*P. palustris*), with disease severities similar those caused by an isolate of *R. lauricola* from the USA. Although *R. lauricola* was probably introduced from Asia to the southeastern USA with its ambrosia beetle symbiont, *Xyleborus glabratus*, it had previously been reported only in the southeastern USA. This is the first report of laurel wilt in the Asian homeland of *R. lauricola* and *X. glabratus*.

## Pathogenicity tests confirm *Macrophomina phaseolina* as an aggressive fungal pathogen of flowering dogwood (Cornus florida L.)

L. MACKASMIEL (1)

(1) Tennessee State University, Nashville, TN, U.S.A.

Pathogenicity tests where seedlings of flowering dogwood (*C. florida* L.) were challenged with extracted spores of *Macrophomina phaseolina* from 10 day-old cultures, while control were mocked inoculated with plain malt extract agar in sterile deionized water, demonstrated the capability of the pathogen to cause severe root rot in *C. florida*. Roots of juvenile seedlings vernalized in sterilized vermiculite; then grown on large water agar petri plates overlaid with sterile, moisten-filter paper, after being inoculated with the pathogen, showed high percentage of necrotic tissue in the first 10 mm of root tips. Concurrently, juvenile seedlings grown in large containers after similar treatment exhibited presence of fungal structures in roots from challenged plants that were not observed in the control. On 35 day-young dogwood seedlings grown in sterile soil under greenhouse conditions and inoculated on wounded stems, fungus-treated plants produced decay and large canker-like lesions that were not on the control. The extent of fungal growth in millimeter (mm) as the index to evaluate the host's response(s) showed marked difference between treatments and control. The level of aggressiveness of the pathogen was determined by measuring the duration and number of dead tissues or plants per given time, and cell death confirmed the amount of infection inside the roots.

## Powdery mildews on native plants in Utah

E. LAURITZEN (1)

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

Many people want to grow more native plants in their yards. However, there is little information on plant diseases of native plants of the western United States. Powdery mildew is very common in Utah on many cultivated plants. The purpose of this project was to determine the presence of powdery mildew on native plants and identify the powdery mildew species using morphological and molecular techniques. Plants from the families Asteraceae, Geraniaceae and Polemoniaceae with powdery mildew were collected from a high elevation mountain valley (2,750 meters) in Northern Utah in fall 2015. Chasmothecia and conidia were collected from the plant tissue for morphological identification using a compound microscope. Identification to genuse and/or species was confirmed using polymerase chain reaction (PCR) amplifying the internal transcribed spacer (ITS) region and DNA sequencing. The resulting sequences were compared in a BLAST search to powdery mildew sequences in the NCBI GenBank database. Several species of powdery mildew including *Podosphaera* sp. on sticky purple geranium (*Geranium viscosissimum*) which to our knowledge is the first report of powdery mildew on this species in the United States and *Golovinomyces magnicellulatus* on *Polemonium* sp., the first report of this powdery mildew in Utah.

## Diaporthe and spruce decline: Incidence, pathogenicity, and population genetics

C. MCTAVISH (1), D. Fulbright (2), A. Jarosz (1)

(1) Michigan State University, U.S.A.; (2) Michigan State University, U.S.A.; (3) Michigan State University, U.S.A.

Spruce decline, characterized by needle loss and branch dieback, is found throughout Michigan. A survey of fungi from cankers in the Lower Peninsula of Michigan found *Diaporthe* (formerly *Phomopsis*) to be the most common, disease-causing fungus. Sequencing *Diaporthe* isolates using ITS1 and ITS4 established 5 different groups. Pathogenicity studies were performed on these groups by inserting mycelial agar plugs containing a representative isolate of each group into a small wound, sealing the wound with Parafilm, and measuring the canker area 9 weeks post-inoculation. Groups 4 and 5 were the most virulent, while group 3 was the least virulent group. Groups 1 and 2 were intermediate on the virulence spectrum. Colorado blue spruce (*Picea pungens*) was the most susceptible species, and 9 different seed sources of *P. pungens* did not differ in susceptibility. Other tree taxa tested, in order from most to least susceptible, were Norway (*P. abies*), white (*P. glauca*), Black Hills (*P. glauca* var. *densata*), Serbian (*P. omorika*), and Meyer (*P. meyeri*) spruce. Group 1 was more common in the eastern Michigan, and fell into a *Diaporthe eres* clade. Group 3 was more common in the central region, and also fell into a *D. eres* clade. Group 2 was most common in the central region, and was unresolved taxonomically. Groups 4 and 5 were common throughout the Lower Peninsula and were also unresolved. Future work includes sequencing more gene regions to resolve *Diaporthe* groups.
## New canker disease of Incense-cedar in Wisconsin caused by *Phaeobotryon cupressi*

J. WEILAND (1), R. Sniezko (2), M. Wiseman (3), M. Serdani (3), M. Putnam (3)

(1) USDA-ARS, Horticultural Crops Research Laboratory, Corvallis, OR, U.S.A.; (2) USDA Forest Service, Dorena Genetic Resource Center, Cottage Grove, OR, U.S.A.; (3) Oregon State University, Botany and Plant Pathology Department, Corvallis, OR, U.S.A.

Incense-cedar (*Calocedrus decurrens*) is a tree native to Oregon and California. Since the early 2000's, a canker disease has been observed with increasing frequency on ornamental and windbreak trees planted in the Willamette Valley of Oregon. Symptoms appear as dead, flagging, small-diameter (<1 cm) branches that are scattered throughout the crown. *Phaeobotryon cupressi* was consistently isolated from symptomatic trees located along the length of the Willamette Valley. Identification was based on morphology and on sequences from the internal transcribed spacer (ITS) and the translation elongation factor 1-α (EF1-α) region. Six isolates were tested for pathogenicity on potted 0.6-1 m tall incense-cedar saplings. Approximately 1 to 1.5 months after inoculation, inoculated branches began to turn brown and die, while control branches that received no inoculum remained healthy. *P. cupressi* was described as a new species in 2009, and was originally found causing cankers on Italian cypress (*Cupressus sempervirens*) in Iran. The pathogen was also detected once in the U.S. from *Juniperus squamosus* in Kansas. It is unknown whether *P. cupressi* affects native stands of incense-cedar, where similar symptoms have been observed, but studies are underway to evaluate the extent of this disease in Oregon.

## The impact of laurel wilt caused by *Raffaelea lauricola* on clonal populations of pondberry (*Lindera melissifolia*)

S. BEST (1), S. Fraedrich (1)

(1) USDA Forest Service, Athens, GA, U.S.A.

Pondberry is a rhizomatous, clonally reproducing, woody shrub species that is federally endangered. Like other members of the Lauraceae that occur in the United States (US), pondberry is highly susceptible to laurel wilt, a disease caused by *Raffaelea lauricola* and vectored by the redbay ambrosia beetle (*Xyleborus glabratus*). A study was initiated in 2012 to assess the potential impact of the disease on clonal populations of pondberry. Individual pondberry plants were planted in nursery beds near Athens, Georgia and grown for approximately 3 years, and during this time individual plants produced many ramets. The original plant in two populations were inoculated with *R. lauricola* in July, 2015 and plants were monitored regularly. After 12 wk, 55 ramets died in one population at distances up to 172 cm from the original inoculated plant, and in the other population 22 ramets died at distances up to 337 cm. *R. lauricola* was reisolated from 84% of the wilted ramets. The study confirms that pondberry is highly susceptible to laurel wilt and that the disease can spread rapidly through rhizomes of infected plants. Although pondberry is a small shrub that is not often attacked by X. glabratus, when infections occur they could be detrimental to populations. Additional studies are being conducted to examine the feasibility of using trenching around infected plants to prevent disease transmission, and the use of chemicals to protect plants from infection.

## The laurel wilt story: Introduction and impact of an exotic vector (*Xyleborus glabratus*) and pathogen (*Raffaelea lauricola*)

M. HUGHES (1), J. Riggins (2), A. cognato (3), F. Koch (4), C. Anderson (5), T. Dreaden (6), J. Formby (2), R. Ploetz (7), J. Smith (8)

(1) University of Florida, School of Forest Resources and Conservation, Gainesville, FL, U.S.A.; (2) Mississippi State University, Department of Biochemistry, Molecular Biology, Entomology, and Plant Pathology, U.S.A.; (3) Michigan State University, Department of Entomology, MI, U.S.A.; (4) U.S. Forest Service, Southern Research Station, Eastern Forest Environmental Threat Assessment Center, NC, U.S.A.; (5) Research School of Biology, Australian National University, Australia; (6) U.S. Forest Service, Southern Research Station, U.S.A.; (7) University of Florida, Department of Plant Pathology, FL, U.S.A.; (8) University of Florida, School of Forest Resources and Conservation, U.S.A.

Laurel wilt is a lethal vascular disease that affects plants in the Lauraceae family. Since the exotic vector (*Xyleborus glabratus*) and its pathogenic fungal symbiont (*Raffaelea lauricola*) were introduced to the USA in 2002, laurel wilt has killed redbay (*Persea borbonia*) and related trees in southern forests. Many questions remain about the disease in the USA, including the number of introduction events, the genetic diversity of the vector and pathogen, and how many host trees have died. To infer the number of introduction events and assess genetic variability, Amplified Fragment Length Polymorphism (AFLP) markers were used to screen a collection of *R. lauricola* isolates, and a portion of the cytochrome oxidase I (COI) gene was sequenced and compared for *X. glabratus* samples. Results suggest that *R. lauricola* and *X. glabratus* are both clonal organisms in the USA that were likely introduced in a single event. Forest Inventory and Analysis (FIA) data were used to obtain pre- and post-epidemic redbay population levels, which were then used to calculate redbay mortality due to laurel wilt. The FIA data suggest that laurel wilt has spread over 250,000 km² and killed nearly half a billion redbay trees. These data highlight the devastating impact that invasive organisms can have on native forests.

## Susceptibility of eight untested species of Cucurbitaceae to gummy stem blight under field conditions

G. RENNBERGER (1), A. Keinath (1)

(1) Coastal Research and Education Center, Clemson University, U.S.A.

To date, 23 species of 12 genera in the Cucurbitaceae are known to be affected by the cucurbit disease gummy stem blight (GSB), caused by the fungus *Stagonosporopsis* spp. (*Didymella bryoniae*). Eight different species representing seven genera of Cucurbitaceae were selected for taxonomic and geographic diversity. Muskmelon (*Cucumis melo* 'Athena'), which is highly susceptible, was included as a control. A field trial was set up as a randomized complete block design with four replications and seven inoculated plants per replication to assess the susceptibility of these species. The severity of the foliar blight phase and the incidence of the crown canker phase of GSB were rated. *Echallium elaterium* showed the highest severity to foliar blight, followed by *Apodanthera sagittifolia* and *Kedrostis leloja*. These species were more susceptible than the control. A group of five species, *Acanthosicyos horridus*, *Cucurbita digitata*, *Cucurbita ecuadorensis*, *Cucumis zambianus* and *Melothria scabra*, were as susceptible to foliar blight as the control. The highest incidence of cankers was observed on crowns of *K. leloja* and 'Athena' muskmelon, followed by *C. zambianus*, *A. sagittifolia* and *E. elaterium* in decreasing order. The pathogen was recovered from crowns of all species. This is the first report of susceptibility of these eight cucurbit species to GSB.

## *Raffaelea arxii* may be the primary symbiont of *Xyleborus affinis*

J. SAUCEDO (1), R. Ploetz (2), D. Carrillo (2), J. Konkol (2), J. Smith (1), J. Rollins (1), S. Ochoa (3)

(1) University of Florida, U.S.A.; (2) University of Florida, U.S.A.; (3) Universidad Michoacana de San Nicolás de Hidalgo, Mexico

Symbioses between fungi and ambrosia beetles are poorly understood. Most of the ambrosia beetles that have been studied have a primary fungal symbiont that predominates in a species' mycangium and natal gallery, where it is cultivated as a food source. However, there is increasing evidence that species of fungal symbionts can be laterally transferred to other beetle species (are not uniquely associated with a single species). For example, the primary symbiont of *Xyleborus glabratus*, *Raffaelea lauricola* (cause of laurel wilt), has been found in at least nine other species of ambrosia beetle. Symbiont composition was examined in several of the latter species. Mandibular mycangia of one of the species, *X. affinis* reared from laurel wilt-affected logs of avocado (*Persea americana*), were assayed for fungal communities. Partial sequences of LSU of rDNA regions identified high concentrations of phenotypically variable populations of *R. arxii* in 95%, whereas *Raffaelea* sp., *R. lauricola*, and *Ambrosiozyma platypodis* were recovered from, respectively, 45, 20 and 15% of 20 assayed individuals. Although *R. arxii* was first reported in South Africa as the primary symbiont of *X. torquatus* (syn. *X. volvulus*), the abundance and persistent association between *R. arxii* and *X. affinis* suggests that *R. arxii* may also be the primary symbiont of *X. affinis*. Primary and secondary symbionts of *X. affinis*, *X. volvulus* and other potential vectors of *R. lauricola* will be discussed.
## Web blight of spinach in the desert southwest caused by *Pythium aphanidermatum*

C. FENG (1), B. Liu (1), M. Matheron (2)

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

A unique foliar disease of spinach caused by *P. aphanidermatum* was observed in spinach in the Yuma, AZ/Imperial Valley, CA spinach production areas in October of 2015. The foliar symptoms of the disease included water soaked foliage, rapid collapse of young plants, with evidence of white cottony mycelium. The disease was associated with hot and wet conditions under high density (>3.0 M seed/A) plantings. Pure cultures were isolated from symptomatic spinach leaves and isolates were sequenced using ITS1/ITS4 primers. BLAST searches showed 100% homology with ITS sequences of *P. aphanidermatum* in GenBank. The objective of this research was to determine the effect of the *P. aphanidermatum* recovered on damping-off and foliar web blight on a broad range of hosts. Pathogenicity was tested in the greenhouse on spinach, cotton, soybean, pepper, tomato, cucumber, melon, squash, lettuce, corn, wheat, and rice, by either infesting the soil or doing foliar inoculations with colonized plugs of PDA. *P. aphanidermatum* caused severe damping off of all dicots tested, but only caused some seedling size reduction in corn and wheat. Web blight symptoms were severe with spinach, and all other dicot hosts. No web blight symptoms were observed on lettuce, corn, wheat, or rice. Metalaxyl provided complete protection for damping off of spinach and protected spinach plants for 10-20 days for U.S. treatment rates or 20-30 days after planting for E.U treatment rates, respectively.

## Stone fruit survey efforts in Texas monitoring for Plum Pox Virus, European Stone Fruit Yellows & Light Brown Apple Moth: 2015-2016

K. ONG (1), S. Rhodes (1)

(1) Texas A&M AgriLife Extension Service, U.S.A.

The Texas Plant Disease Diagnostic Lab continues participation in the National Stone Fruit Survey in FY15, monitoring for pathogens of concern for the stone fruit industry in Texas. The Texas A&M stone fruit breeding plots and four (4) commercial peach orchards in 3 counties were tested for the presence of Plum Pox Virus (PPV) and European Stone Fruit Yellows (ESFY) phytoplasma in 2015. A total of 667 foliar peach and plum samples were tested for PPV. For virus detection, each sample was tested by ELISA using Agdia ELISA kits and following USDA APHIS PPQ and National Plant Diagnostic Network protocols. All 667 samples tested negative for all five strains of PPV. ESFY was not observed or detected in any of the surveyed peach orchards. This survey effort will continue through 2016, focusing on PPV, ESFY and the Light Brown Apple Moth (LBAM). This survey is monitor for possible introduction of these pests and allows for early detection.

## Red rust of bromeliads caused by a parasitic alga in Florida

G. Sanahuja (1), P. Lopez (1), A. Palmateer (1)

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

*Cephalouros* is a genus of plant parasitic thalloid green algae containing at least two species *C. virescens* and *C. parasiticus* that are reported to cause disease on ornamental plants. Florida has 165 plant species belonging to 53 families that have been reported as hosts of *Cephalouros*. Recent outbreaks of a "red rust" disease affecting bromeliads in production nurseries and landscapes led to an investigation of the causal agent, which was determined to be a *Cephalouros* species. Morphological characteristics from *Cephalouros* isolates recovered from *Neoregelia* hybrids match with isolates of *C. parasiticus* collected from leaves of *Psidium guajava* (guava). Molecular analyses were performed from 18S ribosomal RNA and ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) genes. Pathogenicity tests were conducted using *Cephalouros* isolates collected from leaves of *Neoregelia* hybrids and isolates of *C. parasiticus* from guava. Both produced symptoms consistent with those originally described as "red rust" on *Neoregelia* hybrids and the causal agent of red rust on *Neoregelia* hybrids provisionally will be *Cephalouros parasiticus*.

## First detection of the stubby root nematode *Paratrichodorus allius* on potato in North Dakota and on sugarbeet in Minnesota

G. Yan (1), A. Plaisance (2), D. Huang (1), Z. Handoo (3)

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

(3) USDA-ARS, Nematology Laboratory, U.S.A.

Stubby root nematodes (SRN) are migratory ectoparasites that feed on roots, transmit tobraviruses and cause significant crop loss. In 2015, 49 soil samples were collected from a potato field in Sargent County, ND of which seven contained SRN with population densities ranging from 135 to 300/kg soil. Subsequently, 13 soil samples were collected from a sugarbeet field in Clay County, MN and six of them had SRN from 200 to 1,000/kg soil. Individual SRN were examined morphologically and molecularly for species identification. The specimens from both potato and sugarbeet fields were identified as *Paratrichodorus allius*. Observations of female morphological characteristics critical for identification included rod like/oval and well separated vaginal sclerotization, onchiostyle 42-45µm, absence of lateral body pores, and small ventral pharyngeal overlap. DNA was extracted from single nematodes. D2/D3 region of 28S rRNA, partial 18S rRNA and ITS1 rDNA were amplified and PCR products were cloned and sequenced. The consensus sequence from each region was deposited into GenBank and compared with known sequences. The sequence information supported the identity as *P. allius*. *P. allius* is known to be the prevalent vector of Tobacco rattle virus causing potato corky ringspot disease. Until now, problems with infestations of SRN on sugarbeet have been confined to parts of Europe, California and Idaho. This represents the first occurrence of *P. allius* in North Dakota and Minnesota.

## Phytophthora nicotianae causing foliar blight of *Codiaeum variegatum* and *Zamioculcas zamiifolia* in Florida

G. Sanahuja (1), P. Lopez (1), A. Palmateer (1)

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

*Phytophthora* is an oomycete plant pathogen, commonly known as a 'water mold' that attacks all parts of the host plant, causing root, crown and stem rot and foliar blight. *Phytophthora* diseases affect numerous ornamentals in nurseries, garden centers and landscapes generally during the hot and humid rainy season in south Florida. *Phytophthora nicotianae* was isolated for the first time from diseased *Codiaeum variegatum* ('variegated' croton) and *Zamioculcas zamiifolia* ('ZZ' plant) plants. Initially the leaves appeared water soaked and eventually turned dark brown to black in both plants. Morphological characteristics, molecular analysis of the ITS region, and pathogenicity test revealed that *P. nicotianae* was the causal agent. Tropical foliage plants typically account for the greatest percentage of sales within the ornamental industry and we anticipate these diseases will have a substantial impact on tropical foliage in south Florida. Local tropical foliage producers lost 10% and 35% of croton and ZZ plant production, respectively. Therefore, it is important to enlist the use of effective, registered pesticides to provide preventative control of *Phytophthora* blight during the periods where consistent outbreaks occur.

## Characterization of viral particles formed by *Grapevine vein clearing virus* (GVCV)

Y. ZHANG (1), M. Adhab (1), J. Schoelz (1)

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

*Grapevine vein clearing virus* (GVCV) is closely associated with a grapevine vein clearing syndrome observed in Missouri and surrounding states. The virus was initially characterized through deep sequencing of small RNAs isolated from symptomatic grape tissue. Three overlapping fragments of GVCV genomes were cloned and sequenced, from which the GVCV genome was assembled. The nucleotide sequence revealed that GVCV belonged to the *Badnavirus* genus. However, GVCV virions have never been observed under electron microscope. A GVCV infectious clone has been constructed in order to characterize GVCV virions and to complete Koch's postulates. The GVCV infectious clone was infiltrated into *Nicotiana benthamiana* young
leaves, and leaves were collected at 7 dpi, followed by a virion purification procedure involving ultracentrifugation through sucrose gradients. In contrast to the expected bacilliform virions, a flexuous rod structure was observed that had a 15nm diameter and varied in length from 83 to 930 nm. The same virion purification procedure was applied to GVCV-infected grape leaves showing typical vein clearing symptoms and two types of virion particles were observed from a single virion prep: a flexuous rod 15nm in width and up to 8,025 nm in length, as well as the typical bacilliform structure. Antibodies targeting the GVCV coat protein were designed from the GVCV coding sequence and will be applied to test the nature of these two potential GVCV particles.

## Survey for viruses of grapevine (Vitis vinifera L.) in coastal vineyards of Croatia

D. VONCINA (1), M. Al Rwahnih (2), A. Rowhani (2), R. Almeida (3)

(1) Department of Plant Pathology, University of Zagreb Faculty of Agriculture, Zagreb, Croatia; (2) Department of Plant Pathology, University of California, Davis, Davis, CA, U.S.A.; (3) Department of Environmental Science, Policy and Management, University of California, Berkeley, Berkeley, CA, U.S.A.

Viticulture in Croatia has a very long tradition representing an important branch of the national economy. Approximately 70 grapevine (Vitis vinifera L.) cultivars are considered to be autochthonous and are mostly grown in Croatian coastal region. Virus diseases of grapevines generally have a worldwide distribution and some have significant impact on vineyard longevity as well as quality and quantity of the fruit. This survey was initiated due to the limited data about the presence of economically most important viruses in Croatia. A survey was done on 10 autochthonous grapevine cultivars on a total of 48 vines from 22 vineyards. Extraction of total RNA was done from leaf petioles collected in August 2015 and vines were tested on the presence of 32 viruses by RT-qPCR. The most common virus found was GLRaV-3 (91,7%), followed by GVA and GRSPaV (81,3%), GFkV (68,8%), ArMV and GLRaV-1 (33,3%), GVB (10,4%), GPGV (8,3%) and GLRaV-2 (4,2%). Six viruses were detected for the first time in Croatia: GRVFV (14,6%), GRGV (12,5%), viruses from GLRaV-4 group, GVD and GVF (6,3%) and GSyV-1 (2,1%). Mixed infections were also common in individual grapevines. The survey indicates a poor sanitary status of autochthonous grapevine cultivars, demonstrating the necessity to extend work on sanitary selection programs.

## Blueberry green mosaic symptoms are associated with the presence of a new vitivirus

T. THEKKE-VEETIL (1), T. Ho (1), J. Polashock (2), I. Tzanetakis (1)

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

A virus was discovered in blueberry plants showing green mosaic symptoms, distinct symptomology from that associated with mosaic disease and was provisionally named as Blueberry green mosaic virus (BGMV). The genome was amplified using overlapping PCRs and has the typical organization of members of the genus *Vitivirus*, family *Betaflexiviridae* including five open reading frames (ORFs) and a 3' polyadenylated terminus. The ORFs code for the viral replicase, a 16 kDa protein of unknown function, the movement, coat and nucleic acid binding proteins, the latter presumably involved in RNA interference. Pairwise comparisons of the polymerase conserved domains of BGMV and established members of the genus showed amino acid identity ranging from 57-61% whereas phylogenetic analysis placed the virus in a distinct clade within the genus. The virus has been found in all plants showing green mosaic symptoms and its distribution and role in symptomology are currently being evaluated.

## Genome sequence analysis of a legume-infecting Tomato chlorotic spot virus isolate

R. ADEGBOLA (1), R. Kemerait (2), S. Adkins (3), R. Naidu (1)

(1) Washington State University, U.S.A.; (2) Department of Plant Pathology, The University of Georgia, U.S.A.; (3) USDA ARS USHRL, U.S.A.

*Tomato chlorotic spot virus* (TCSV, genus *Tospovirus*, family *Bunyaviridae*) has emerged as a significant constraint to economically important horticultural crops in Florida and the Caribbean region. In this study, we have determined the full genome sequence of a TCSV isolate from peanut (*Arachis hypogea*) collected in Haiti. Total RNA isolated from virus-infected leaves was used to generate sequences of the S-, M-, and L-RNA genomic segments by a combination of conventional Sanger sequencing and next-generation sequencing (NGS). The consensus sequences generated from Sanger sequencing were compared against those derived from *de novo* assembly of the filtered NGS reads to confirm the quality and accuracy of the complete genome sequences of the three RNA segments. The 8,873 nucleotide (nt) L-RNA shared 98% nt identity with the L-RNA sequence of a TCSV isolate from Brazil. The 4,847 nt M-RNA shared 99% nt identity with the M-RNA sequence of a *Groundnut ringspot virus* and TCSV reassortant from Florida. The 3,310 nt S-RNA shared 32.5 to 73.8% nt identity with S-RNA sequences of other tospoviruses. Phylogenetic analysis of proteins encoded by individual TCSV genomic RNA segments with corresponding proteins encoded by other tospoviruses is in progress. This study demonstrates advantages of NGS methods for high throughput, insightful analysis of genome segment sequences of a multi-partite negative-stranded RNA virus directly from field infected tissue with no viral enrichment.

## Viral metagenomics of Caladium x hortulanum (Araceae) in Florida

R. ALCALA BRISEÑO (1), M. Londoño (1), J. Polston (1)

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

Viral metagenomics and high-throughput sequencing were used to identify novel viruses present caladiums (*Caladium x hortulanum*, Araceae), collected from Lake Placid, Florida in 2013. Caladium cultivars showing viral-like symptoms were sequenced, reads were assembled and contigs were analyzed using BLASTX showing homology with viral species in the *Potyviridae*, *Rhabdoviridae* and *Caulimoviridae*. The bioinformatic results were validated by reverse transcription (RT)-PCR for RNA viruses and PCR for DNA viruses. Only two potyvirus species, *Dasheen mosaic virus* (DsMV), and *Konjac mosaic virus* (KoMV) were able to be confirmed by RT-PCR amplification and sequencing of the coat protein region (CP). DsMV was previously reported from caladium in Florida, however this is the first report of KoMV in Florida as well as North America. In order to explore the abundance and diversity of these viruses, 315 plants from 15 caladium cultivars were screened using RT-PCR with CP primers. DsMV was present in 69.5% of tested plants and in all cultivars. KoMV was present in 11% of tested plants, and in eight out of 15 cultivars. Sixty-one and thirteen CP sequences of DsMV and KoMV were sequenced, respectively. Pairwise comparison analysis indicated sequence similarity of 87.4% and 96.5% among isolates of DsMV and KoMV, respectively. The low diversity, abundance and phylogenetic analysis are consistent with a single introduction event for both DsMV and KoMV.

## Sequence variation between isolates of Plantago asiatica mosaic virus from lily and other hosts

J. HAMMOND (1), M. Reinsel (1)

(1) USDA-ARS, USNA, Floral and Nursery Plants Research Unit, U.S.A.

*Plantago asiatica mosaic virus* (PIAMV) was reported from *Plantago asiatica* in the Russian Far East, and Nandina mosaic virus was reported from heavenly bamboo (*Nandina domestica*) in California, both in the 1970's. Nandina mosaic virus has not been reported to naturally infect any other hosts and was recognized as a strain of PIAMV (PIAMV-NMV) only when the full sequence was determined. In contrast, PIAMV was reported to affect lilies (*Lilium* spp.) and primrose (*Primula seiboldii*) in Japan, and in lilies in the Netherlands, Italy, Hungary, Spain, the UK, Taiwan, Chile, and the USA. Cut-flower Oriental and Asiatic hybrid lilies suffer significant losses of foliar and flower quality, or are even rendered unmarketable by PIAMV infection, showing foliar mosaic and necrosis, and streaking or distortion of the flowers. We have sequenced the coat protein (CP) gene and 3' non-coding region of PIAMV isolates from multiple imported lily cultivars, and compared these sequences to isolates from other countries and hosts, including PIAMV-NMV. Sequences from all imported lilies fall within the same clade as PIAMV isolates from Europe; lily and primula isolates from Japan fall into another clade; whereas PIAMV-type (*P. asiatica*) and PIAMV-NMV form monophyletic clades. PIAMV-type and PIAMV-NMV share multiple CP
amino acids differentiating them from other isolates; other residues are uniquely conserved within the 'European' clade, or within the 'Japanese' (lily and primrose) clade.

## Detection and characterization of a novel potyvirus, Crinum mosaic virus, in ornamental Crinum

R. JORDAN (1), A. Khawaja (1), M. Guaragna (1)

(1) US National Arboretum, USDA-ARS, U.S.A.

Ornamental flower bulbs (including true bulbs, bulbils, corms, tubers and rhizomes) are increasingly important floriculture crops. *Crinum* is a large genus of herbaceous perennial flowering bulbs in the family Amaryllidaceae and most of the species are grown in the summer and have large, showy, fragrant flowers. Only two viruses have been reported to infect cultivated *Crinum*, both potyviruses - *Nerine yellow stripe virus* (NeYSV; in India) and *Crinum mosaic virus* (CriMV; in Australia, FL and HI). CriMV has not been characterized and infection has only been noted by the presence of potyvirus particles or characteristic potyviral inclusions. Leaf samples from several *Crinum* plants growing at the US National Arboretum exhibiting mosaic symptoms tested positive for the presence of Potyvirus in an RT-PCR screen using several universal potyvirus primers yielding the expected 1,600 bp product corresponding to the partial NIB gene, full-length coat protein (CP) gene and 3'UTR. The RT-PCR amplicon was cloned and sequenced and NCBI BLAST analysis of the consensus sequence revealed highest similarities, but not identity, with isolates of NeYSV. Pair-wise analyses of the 258 amino acid sequence of the predicted CP had 65-68% sequence identity with the NeYSV isolates from *Crinum*, *Hymenocallis*, *Nerine*, *Stenomesson*, and *Vallota*; whereas the seven NeYSV isolates share 85-98% CP identity. We propose to associate this unique potyvirus sequence with the aforementioned CriMV.

## Viruses of brugmansia in Mississippi

N. ABOUGHANEM-SABANADZOVIC (1), A. Lawrence (1), S. Sabanadzovic (1)

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

Brugmansia (*Brugmansia* spp.), or Angel's trumpet, is an ornamental woody plant/shrub originating from South America and belonging to the family Solanaceae. Because of production of attractive large and fragrant flowers, they are now commonly grown in the temperate climate worldwide, including USA. As any other plant, it is susceptible to different pathogens including viruses. During the late summer/fall of 2015, several symptomatic brugmansia samples of different cultivars were brought to our attention by a local plant enthusiast. Symptoms varied among samples and, generally speaking, consisted of leaf mosaics and distortion. Samples were examined separately by biological, serological and molecular methods, including deep sequencing, and resulted in identification of several potyviruses, such as Colombian datura virus (CDV) and Brugmansia mosaic virus (BruMV). CDV was previously reported from brugmansia collected in Florida and few other states in the USA; however, this study represents the first state record for this virus in Mississippi. BruMV was not previously known to occur in the United States and was probably recently introduced into the country by infected plant material.

## Molecular Detection of Ti ringspot associated virus, a novel emaravirus associated with ti ringspot disease of Cordyline fruticosa (L.) in Hawai'i

M. MELZER (1), A. Park (1)

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

An emerging virus-like disease of ti plant (*Cordyline fruticosa* L..) is spreading quickly throughout the Hawaiian Islands. Foliar symptoms include chlorotic lesions constricted by secondary veins or circular ring-spots that can coalesce into amorphous lesions. Tissue samples were collected from various locations across Hawaii and RNA was extracted. Degenerate RT-PCR primers targeting the polymerase gene of known emaraviruses amplified a ~400 bp product from the RNA of symptomatic plants. The RT-PCR products were sequenced and found to be >97.5% identical, indicating they represent a single virus species, designated Ti ringspot associated virus (TiRaV). When translated into amino acid sequences, this virus was found to be 70% identical to *Raspberry leaf blotch virus*, 55% to *European mountain ash ringspot-associated virus*, 53% to Redbud yellow ringspot virus, 50% to *Rose rosette virus*, 49% to *Fig mosaic virus* (FMV), and 46% to *Pigeon pea sterility mosaic virus*. These sequences and the *C. fruticosa* large rubisco subunit sequence were used to create specific primers and probes for a multiplex RT-qPCR detection assay. This assay was able to reliably detect TiRaV in symptomatic samples collected from various locations across Hawaii, and did not react to asymptomatic samples or cross react with an isolate of FMV.

## Is there resistance to Rose Rosette Disease among cultivated roses?

K. ONG (1), J. Olson (2), T. Evans (3), M. Windham (4), E. Roundey (5), J. Lau (5), D. Byrne (5)

(1) Texas A&M AgriLife Extension Service, College Station, TX, U.S.A.; (2) Oklahoma State University, Stillwater, OK, U.S.A.; (3) Department of Plant and Soil Sciences, University of Delaware, Newark, DE, U.S.A.; (4) Entomology and Plant Pathology Department, University of Tennessee, Knoxville, TN, U.S.A.; (5) Department of Horticultural Sciences, Texas A&M University, College Station, TX, U.S.A.

Rose Rosette Disease (RRD) is caused by the *Rose Rosette Virus* (RRV) and transmitted by a wind-blown, eriophyid mite (*Phyllocoptes fructiphilus*). Little is known about resistance of roses to the virus or the mite vector. A few rose species, such as *Rosa palustris*, *R. setigera*, *R. carolina*, *R. virginiana*, and *R. spinosissima* have been reported to be resistant to RRD, but resistance in cultivated roses is unknown. Although it is possible to genetically introgress disease resistance from a wild species into the cultivated rose, the development of commercial roses would be quicker if there was a good source of resistance within the cultivated germplasm. Preliminary observational data on over 400 rose accessions indicates that there is resistance to RRD among the cultivated rose germplasm. Large field trials to assess RRD resistance of about 300 rose accessions have been planted in Tennessee and Delaware. The disease pressure in these trials is being augmented by inter-planting RRD infected roses among and by placing mite infested shoot tips from infected plants onto the plants to be tested for their resistance. In addition, smaller trials are being planted in Oklahoma and Texas to assess the level of RRD resistance of selected roses. Since disease symptoms may not appear for many weeks to months after inoculation with the virus, we anticipate this will be a long term study and it may be 1 or more years before we know if resistance occurs in cultivated roses.

## Survey for Grapevine Pinot gris virus infecting Grapevine in the Foundation Plant Services Vineyards at the University of California, Davis

M. AL RWAHNIH (1), N. Westrick (2), D. Golino (2), A. Rowhani (2)

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

Grapevine Pinot gris virus (GPGV) is a new member of the genus *Trichovirus* in the family *Betaflexiviridae*. The virus was first discovered by high-throughput sequencing from an Italian Pinot gris grapevine (*Vitis vinifera*) showing chlorotic mottling, leaf deformation and stunting symptoms. Since then, GPGV has been reported in symptomatic and asymptomatic plants of other cultivars in many countries. Recent transmission studies indicated that the eriophyid mite *Colomerus vitus* (Pagenstecher) is the suspected vector. This vector infests grapevine wherever they are grown and it has been reported in California. To address the possible presence and prevalence of GPGV in the collections of the Foundation Plant Services (FPS, University of California, Davis), 2,014 vines from the collection were screened including 23 vines of Pinot gris. Among all the vines tested, only one asymptomatic vine cv. Touriga Nacional was positive for GPGV. This grapevine cultivar has been imported from Portugal in 1981and planted in the FPS collection in 2001. This is believed to be the first reported detection of GPGV in the U.S. Twenty samples in close proximity to the GPGV-positive vine were also collected and tested. These vines were all tested negative for GPGV. As a leading source of orchard and vineyard planting stock in the U.S., in addition to being a central component of the Grape Clean Plant Network, these negative results indicate that FPS stock is not a major source of GPGV.
## Identification and proposed species demarcation for *Cacao* swollen shoot virus and previously unidentified badnaviruses of *Theobroma cacao*

N. CHINGANDU (1), M. Zia-Ur-Rehman (2), T. Sreenivasan (3), S. Surujdeo-Maharaj (4), P. Umaharan (4), K. Kouakou (5), R. Aka (5), G. Ameyaw (6), O. Gutierrez (7), J. Brown (1)

(1) University of Arizona, Tucson, AZ, U.S.A.; (2) IAGS, University of Punjab, Lahore, Pakistan; (3) Cocoa Research Centre, The University of the West Indies, St. Augustine, Trinidad; (4) Cocoa Research Center, The University of the West Indies, St. Augustine, Trinidad; (5) Centre National de Recherché Agronomique (CNRA) Programme Cacao, Divo, Ivory Coast; (6) Cocoa Research Institute of Ghana, Tafo, Ghana; (7) USDA-ARS Subtropical Horticultural Research Station, Miami, FL, U.S.A.

Sustained production of cacao (*Theobroma cacao*) in West Africa, which supplies over 70% of the world's bulk cacao, is hindered by virus-like diseases. To date, *Cacao swollen shoot virus (CSSV) (Caulimoviridae, Badnavirus)*, endemic to West Africa, is the only known cacao-infecting badnavirus. In West Africa, CSSV-like symptoms became prevalent during 2000-2003, and continue to persist. Since the 1940s, virus-like symptoms in cacao of suspect badnaviral etiology have been recognized in Trinidad at the International Cocoa Genebank, one of the custodians of cacao germplasm resources. To identify the suspect viruses, total DNA isolated from symptomatic cacao leaves collected in Ghana, Cote d'Ivoire and Trinidad was subjected to rolling circle amplification and sequenced using Illumina HiSeq. DNA sequences were assembled using DNASTAR, and annotated using the NCBI ORF Finder, and the results were validated by sequencing PCR-amplified full-length genomes. Genome characterization revealed seven previously unidentified badnavirus isolates, each with four open reading frames, characteristic of other badnaviruses. Pairwise comparisons using Standard Demarcation Tool (SDT) of the RT-RNaseH sequence (80% nucleotide identity) with analogous badnavirus sequences indicated that the isolates represented three new badnavirus species. However, SDT analysis of the full-length genome sequences suggested at least five previously unidentified badnavirus species are associated with cacao.

## First report and characterization of *Taro bacilliform virus* in the USA

M. MELZER (1), K. Dey (1), A. Chan Borges (1), M. Long (1), W. Borth (1), N. Wichitrnithed (1), J. Hu (1), R. Li (2)

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

*Taro bacilliform virus (TaBV)* is a member of the genus *Badnavirus* (Family *Caulimoviridae*) and a pathogen of taro (*Colocasia esculenta*), an important starch staple in tropical regions. TaBV is transmitted by mealybugs and appears to be widely distributed throughout the tropical Pacific. Taro has cultural, economic and nutritional importance in Hawaii, but only *Dasheen mosaic virus* and *Taro vein chlorosis virus* have been reported in the state. We surveyed 328 taro plants from 35 sites on the islands of Kauai, Oahu, Molokai, Maui, and Hawaii for the presence of TaBV by PCR using degenerate badnavirus and TaBV-specific primers. Of these, 171 samples (52%) tested positive using the degenerate primers, but only 153 tested positive using TaBV-specific primers (47%), indicating the presence of novel badnaviruses and/or strains of TaBV in Hawaii's taro. The 7825 bp genome of one isolate of TaBV was sequenced and found to be 80% identical and 367 bp larger than the only complete TaBV genome in GenBank (accession AF357836), indicating that considerable genetic diversity exists for this virus. PCR, RT-PCR and Southern hybridization assays were conducted and provided evidence that TaBV is integrated into the genome of some Hawaiian taro varieties. TaBV was mechanically inoculated onto eight herbaceous plant species, and after 25 days, TaBV could be detected by PCR in *Cucurbita pepo*, *Pisum sativum*, *Cucumis sativus*, *Vigna unguiculata*, and *Phaseolus vulgaris*.

## Association of a mixed infection of *Lettuce chlorosis virus*, *Papaya ringspot virus*, and *Tomato yellow leaf curl virus-IL* in a Texas papaya orchard

O. ALABI (1), M. Al Rwahnih (2), J. Brown (3), J. Jifon (4), J. Park (5), L. Gregg (6), M. Sétamou (7), A. Idris (3)

(1) Dept. of Plant Pathology & Microbiology, Texas A&M University AgriLife Research & Extension Center, Weslaco, TX, U.S.A.; (2) Department of Plant Pathology, University of California, Davis, Davis, CA, U.S.A.; (3) School of Plant Sciences, University of Arizona, Tucson, AZ, U.S.A.; (4) Department of Horticultural Sciences, Texas A&M AgriLife Research and Extension Center, Weslaco, TX, U.S.A.; (5) Texas A&M University, Kingsville Citrus Center, Weslaco, TX, U.S.A.; (6) Texas A&M AgriLife Research and Extension Center, Weslaco, TX, U.S.A.; (7) Department of Agriculture, Agribusiness, and Environmental Sciences, Texas A&M University, Kingsville Citrus Center, Weslaco, TX, U.S.A.

Severe virus-like symptoms consisting of mosaic, distortion, yellowing, and brittleness were observed on papaya plants in a 50-ha orchard in South Texas during the 2014-2015 growing season. Disease incidence increased from ~40 to 100% within six-months of the outbreak and severely affected plants were stunted with reduced fruit loads and poor fruit quality. cDNA library constructed from DNAse-treated total nucleic acids from symptomatic plants was subjected to high-throughput sequencing (HTS). Based on BLASTn analysis of the assembled HTS contigs, the plant viruses detected were validated by Sanger sequencing of the complete genome sequences. The collective sequencing results indicated the presence of *Papaya ringspot virus (PRSV; Potyvirus)*, *Lettuce chlorosis virus (LCV; Crinivirus)*, and *Tomato yellow leaf curl virus (TYLCV-IL; Begomovirus)*. RT-PCR analyses of leaves from 51 randomly sampled papaya plants indicated the presence of PRSV, LCV, and TYLCV-IL in 100, 39.2 and 15.7% of the samples, respectively. Plants with mixed infections of PRSV, in combination with LCV and/or TYLCV-IL, exhibited more severe symptoms compared to plants with PRSV alone. The association of LCV and TYLCV-IL with symptomatic plants represents first report of these whitefly-transmitted viruses in papaya. Papaya, a perennial crop, could serve as an over-seasoning reservoir for TYLCV-IL and LCV presenting new challenges to disease management in papaya and other crop hosts of these viruses.

## Seed-borne virome in cucurbits

S. SABANADZOVIC (1), N. Aboughanem-Sabanadzovic (1)

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

In this work we investigated the 'community' of seed-borne viruses in more than 50 cultivars and PI lines/accessions belonging to different botanical species of cucurbits by the next-generation sequencing approach. To this aim, double stranded RNAs (dsRNAs) were extracted from plant tissue collected at the first true leaf stage from a pool of young seedlings belonging to the same genotype and grown under greenhouse conditions in order to prevent contamination by horizontally transmitted viruses. Polyacrylamide gel electrophoreses (PAGE) revealed the presence of single or multiple dsRNA bands in the majority of tested plants indicating ongoing infections by vertically transmitted viruses. The dsRNA extracts were reverse transcribed, tagged, and submitted to Illumina sequencing. Results of the sequence analyses demonstrated the widespread presence of persistent viruses in tested cucurbits, mainly those belonging to the families *Partitiviridae*, *Amalgaviridae*, *Endornaviridae* and *Totiviridae*. In this study, we identified and characterized numerous viruses, representatives of new species and confirmed the prevalence of Cucumis melo endornavirus and Lagenaria siceraria endornavirus in tested melons and bottle gourds, respectively. The importance of widespread presence of persistent viruses in some cucurbits and their possible effects to the host are yet to be understood.

## Reemergence of the torradovirus, *Tomato necrotic dwarf virus*, in processing tomatoes in the Central Valley of California

O. BATUMAN (1), M. Vasquez-Mayorga (1), J. Nunez (2), L. Hladky (3), W. Wintermantel (3), R. Gilbertson (1)

(1) University of California Davis, Davis, CA, U.S.A.; (2) University of California Cooperative Extension, Bakersfield, CA, U.S.A.; (3) United States Department of Agriculture, Salinas, CA, U.S.A.

In the fall of 2015, processing tomato plants (*Solanum lycopersicum*) in a late-planted field in Kern County, California had high populations of whiteflies and developed virus-like symptoms including stunting and distorted growth; leaf curling, crumpling and necrosis; and stem and shoot necrosis. The symptoms resembled those induced by *Tomato spotted wilt virus (TSWV)*, but immunostrip and RT-PCR tests were negative for TSWV and other tospoviruses. RT-PCR tests were then performed for *Tomato mosaic virus (ToMV)*, *Alfalfa mosaic virus (AMV)*, *Tomato necrotic spot virus (ToNSV)*,
and torradoviruses (family Secoviridae) were performed. The expected size DNA fragments were amplified for ToMV and torradovirus (degenerate primers for RNA-1 and RNA-2), whereas negative results were obtained for AMV and ToNSV. Analysis of the DNA sequences of the PCR-amplified torradovirus RNA-1 and RNA-2 fragments revealed that these plants were infected with a torradovirus that was most similar (~94%) to RNA-1 and RNA-2 of *Tomato necrotic dwarf virus* (ToNDV). This is the first report of ToNDV in the Central Valley of California, although it was previously reported from the Imperial Valley of California in the 1980s. Surveys to determine inoculum sources and further sequencing and biological characterization of ToNDV isolates in California are in progress.

## Discussion on calculation of disease severity index values from scales with unequal intervals

K. CHIANG (1), H. Liu (1), C. Bock (2)

(1) Division of Biometrics, Department of Agronomy, National Chung Hsing University, Taiwan; (2) USDA-ARS-SEFTNRL, U.S.A.

When estimating severity of disease, a disease interval (or category) scale comprises a number of categories of known numeric values – with plant disease this is generally the percent area with symptoms (e.g., the Horsfall-Barratt (H-B) scale). Studies in plant pathology and plant breeding often use quantitative interval scales. The severity is estimated as an interval scale value and is used to determine a disease severity index (DSI). However, very few studies have investigated the effects of different scales on accuracy of the DSI. Using an interval scale designed for pecan scab (*Fusicladium effusum*), we investigated performance of the DSI. We used interval scale values, the scale data converted to midpoint values of each category, and nearest percent estimates (NPEs). Results of the pecan scab data indicate that there is a consistent bias of the mean value when applying the interval scale values directly. The mean value of the DSI per cultivar is underestimated as compared with that of NPEs; results of the mid-point conversion are akin to those of the NPEs. Furthermore, based on the data used, the simulation studies were executed to explore the mean value of DSI for three different parameters. The results of these simulation studies are consistent with those of studies which used real data. Finally, a new disease index, which uses equal intervals with additional grades at low severities, was developed to minimize the effect of the scale on error.

## Predicting Robust Candidates for a Boxwood Blight Immunoassay: An Automated Computational Workflow with Broad Applications for Phytopathology

D. VELTRI (1), D. Luster (2), M. McMahon (2), J. Crouch (1)

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

The boxwood blight fungal pathogens Calonectria pseudonaviculata and C. henricotiae have emerged as a serious threat to natural and cultivated plant populations around the world. These pathogens can persist as latent infections in asymptomatic plant tissue even after fungicide treatment. Therefore, a fast and field-deployable diagnostic test based on immunological detection would provide a useful tool to help minimize spread of the pathogen. Although several nucleic acid-based approaches are available to detect boxwood blight pathogens, none are field-deployable solutions. We are developing an integrated computational pipeline using recent genomic and RNA-Seq data to identify quality candidate sequences for use in designing a diagnostic immunoassay. The pipeline first identifies proteins likely to be secreted using SECRETOL. To robustly test the putative secreted proteins for solubility, we developed a new classifier with improved recognition performance over other methods using a publicly-available dataset of secreted proteins. The candidate proteins are further narrowed down through a comparative genomics analysis that identifies proteins with predicted antigenicity that are specific to the target pathogens yet share low similarity to outgroup species. This pipeline stands to help speed up immunoassay development and lower costs by providing a starting set of candidate proteins with a higher likelihood of success in further wet laboratory experiments.

## Identification of conducive temperatures for decision support modeling of sugarcane rusts in Florida

B. CHAULAGAIN (1), M. Hincapie (1), S. Sanjel (1), C. Fraisse (2), R. Raid (1), P. Rott (1)

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

Most sugarcane varieties grown in Florida are currently susceptible to either brown (*Puccinia melanocephala*) or orange (*Puccinia kuehnii*) rust, two diseases that have been reported to cause economic losses in sugarcane industries worldwide. In Florida, sugarcane rusts are controlled using fungicides and application schedules are solely based on disease scouting. In order to develop a forecasting system to optimize fungicide applications, disease severity data were collected during the entire 2014 crop season. Percent rust affected area of the top visible dewlap leaf was recorded every two weeks from seven sugarcane varieties susceptible to brown or orange rust and planted in October, November and December 2013. Brown and orange rust progress curves were generated using maximum disease severity values at each scoring date, whatever variety and planting date. Because relative humidity and leaf wetness does not appear to be a limiting factor for development of sugarcane rusts in Florida, efforts were focused on temperature that is also known to affect rust progress. Curves of cumulative average daily temperatures between 17°C and 29°C were compared with disease progress curves. Temperature ranges of 17-25.5°C and 17-26.5°C appeared to be the best fit for progress of brown and orange rust, respectively. Weather and disease data from additional crop seasons need to be analyzed to confirm these first results and to develop a decision support model for rusts in Florida.

## Comparing epidemics of unique *Phytophthora infestans* clonal lineages with biometrically enhanced spatio-temporal modeling tools

K. MOREY GOLD (1), Z. Zhang (2), D. Rouse (1), A. Gevens (1)

(1) University of Wisconsin-Madison, U.S.A.; (2) University of Wisconsin-Madison, U.S.A.

The oomycete *Phytophthora infestans* causes late blight, a plant disease notorious for giving rise to the Irish Potato Famine of the 1840s. In 2009, a pandemic caused by a newly emerged clonal lineage of *P. infestans* (US-22) destroyed gardens and small farm tomatoes and potatoes across the US. In the following years, three clonal lineages (US-22, -23, -24) comprised national epidemics, with US-23 predominating from 2013 onward. US-23 is of noteworthy aggressiveness, has small sporangial size compared to previous lineages, and is highly pathogenic on both potato and tomato. Previously, two disease dispersal models were fit using Markov chain Monte Carlo (MCMC) for late blight epidemics in central Wisconsin caused by clonal lineage US-8. In our current work, we validated and enhanced these models using the late blight epidemic caused by US-23 in this region for 2009-2015 and we fit a spatial-temporal semivariogram for both epidemics. Disease data were collected from scouting records of a cooperating crop consultant and were manipulated using ArcGIS and RStudio. The power law model predicted disease dispersion accurately with R² values between 0.2 and 0.9. Sporangial size greatly influenced dispersion rate which played a role in disease progression. Modeling of specific clonal lineage epidemics in high-risk production regions can aid in developing prescriptive, risk-averse late blight management plans to reduce disease perpetuation and economic loss.

## Impacts of climate change on wheat powdery mildew epidemics in China

Y. Zhou (1), X. Tang (1)

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

Epidemics of wheat powdery mildew (*Blumeria graminis* f. sp. *tritici*) have been highly destructive to winter wheat in China. We analyzed trends in the regions of wheat powdery mildew epidemics in China from 1970 to 2012 and PA (percent area affected by wheat powdery mildew) in response to climate change. Using PA and temperature for pathogen oversummering and overwintering periods, a model was constructed and subsequently used to predict changes in PA for 15 Global Climate Models under low (B1), medium (A1B), and high (A2) climate emission scenarios for the 2020s, 2050s, and 2080s relative to the 1970–2009 period. Our results showed a significant increase in T (mean monthly air temperature) (P < 0.001), while SH (sunshine hours) and RH (relative humidity) decreased. Using the original value method, there was a significant positive relationship between T and PA (P < 0.05). Using the first-differences time-series value method, ΔT (first-differences in T) in oversummering and late spring periods were negative for ΔPA (first-differences in PA) while positive relationships were recorded in the rest of the periods. The results using the first-differences time-series value method
were more precise for disease epidemics. The results of model prediction indicate that the values for the percent area affected by powdery mildew will increase in future. These findings will aid in decision making for management strategies of wheat powdery mildew.

## Quantifying the potential effects of regional climate change on wheat leaf rust disease in the Grand-Duchy of Luxembourg

M. EL JARROUDI (1), L. Kouadio (2), J. Junk (3), B. Tychon (1), P. Delfosse (3)

(1) University of Liege, Belgium; (2) University of Southern Queensland, Australia; (3) Luxembourg Institute of Science & Technology, Luxembourg

Climate change will affect wheat crop production both in the main processes of plant growth and development and in the occurrences and severities of plant diseases. We assessed the potential infection periods of wheat leaf rust (WLR) in a changing climate at two climatologically different sites in Luxembourg using a threshold-based model for WLR. Field experiments were conducted during 2003–2013 at the selected sites to test the model. Projected climate data, from a multi model ensemble of regional climate models (spatial resolution 25 km) as well as an additional projection with a higher spatial resolution of 1.3 km, were used to investigate the potential WLR infection periods for two future time spans. The model simulated satisfactorily WLR infection periods at both sites during 2003–2013: probabilities of detection were close to 1 and the critical success index ranged from 0.80 to 0.94. Regional climate projections indicate an increase in temperatures over the 2014-2050 and 2091-2100 periods compared to the reference period 1991–2000. Annual precipitation is also expected to increase slightly in the future. Moreover, trends in increased favourable days of WLR infection would likely occur at both sites due to projected climatic conditions more conducive than in the reference period. Our methodology can be easily transferred to other regions and other fungal diseases by adjusting the meteorological threshold values essential for a specific disease development.

## Screening some rust universal-susceptible wheat varieties for ozone tolerance and some applications

A. MASHAHEET (1), D. Marshall (2), K. Burkey (2), A. Abdelrimi (1), F. Ullah (1)

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

Elevated ground-level ozone associated with climate change and the rapidly evolving rust races are major challenges to wheat production worldwide. Because of higher susceptibility, rust universal-susceptible varieties are used as susceptible hosts to maintain and increase inoculum for most of the rust races. These varieties could also be used to study the interactive effects of ozone on different rust races. A first step is to assess the O₃ responses of these wheat varieties. In this research, we screened the wheat lines Chinese Spring, CSA, Line E, Little Club, LMPG-6, McNair 701, Morocco, Rusty and Thatcher for O₃ tolerance at four O₃ concentrations. Results showed that Thatcher is the most sensitive and Chinese Spring is the most tolerant among the nine lines tested. For future studies of the effects of ozone on different stem rust races, LMPG-6, Rusty and McNair 701 could be used as O₃-sensitive if compared to the more ozone-tolerant CSA or Chinese Spring. Similarly, Thatcher is a suitable O₃-sensitive line that could be used with either Little Club or Morocco for studying O₃ effects on different races of leaf rust. Identification of lines having differential ozone response will enable further genetic studies on ozone tolerance.

## Distribution models of common bean dry root rot in Brazil: Current and predicted disease distribution in climate change scenarios

R. Macedo (1), L. Abud (1), F. Yoshida (1), L. Sales (1), E. Barbosa (2), M. Lobo (2), R. Macedo (1)

(1) Universidade Federal de Goiás, Brazil; (2) Brazilian Corporation for Agricultural Research (Embrapa), Brazil

Dry root rot is an ubiquitous problem on common beans in Brazil, with yearly yield losses attributed to the *Fusarium solani* species complex. Species distribution models (SDMs) based on 21 field disease records were developed to assess dry root rot current geographical distribution, and estimate disease range expected in IPCC scenarios A1B and A2 by the year 2030. Projections were adjusted by an ensemble of three modelling methods: Generalized Aditive Models, Surface Range Envolop and Artificial Neural Networks, built with “biomod” and “raster” packages from R statistical software. Also, we verified if three inoculum threshholds (1200, 3700 and 4500 propagules of *F. solani* per soil, in 2623 records) could work as disease spatial proxies, supported by Sperman’s correlations between inoculum thresholds and disease records. The best disease × soil inoculum density correlation was found with 3700 propagules per soil gram (r=0.85, P<0.001). The current projection shows high climatic suitability for common bean dry root rot in the Brazilian central region. According to IPCC scenarios, the geographical area affected by the disease will be reduced by almost 23%, but highly favorable areas will move towards Brazil South Region, which represents 40% of the country’s common bean yields. Implications and consequences of these results will be discussed to support future plans on root rot management.

## Discovery of biological drivers of pitch canker disease in a changing climate

T. QUESADA (1), T. Quesada (1), C. Staub (1), M. Marsik (1), K. Shin (1), K. Smith (2), J. Hughes (1)

(1) University of Florida, U.S.A.; (2) US Forest Service / University of Florida, U.S.A.

Natural and agricultural ecosystems are affected by climate change through increased temperature, humidity, and unusual weather patterns. They are increasingly threatened by invasions by non-native pests and pathogens. The fungus *Fusarium circinatum* causes pitch canker disease in several pine species, leading to high economic losses in the timber industry. Favored by high temperatures and humidity, future outbreaks may become more frequent under predicted changes in environmental conditions over the next 50-100 years. A better understanding of *F. circinatum* biology is needed to inform predictions of which pathogen variants would likely cause outbreaks in future climate settings. We evaluated growth, spore production and germination among *F. circinatum* isolates cultured at various temperatures (25, 28, and 31°C). We also implemented spore trapping experiments in three north-Florida sites to survey spore release throughout the spring and summer, and to test if weather events trigger increased spore discharge. Results show significant temperature effects (p<0.05) in mycelium growth and sporulation among isolates. Though spore trapping experiments need to be followed across several growth seasons, we present preliminary trends on spore release in the environment. These results will constitute the baseline for future research on fungal diversity, spatial distribution patterns and disease prediction, aimed at mitigating adverse effects of climate change on forest health.

## Potential species distributions of *Armillaria solidipes* and *Pseudotsuga menziesii* under contemporary and changing climates in the interior western USA

J. HANNA (1), M. Warwell (2), H. Maffei (3), M. Fairweather (4), J. Blodgett (5), P. Zambino (6), J. Worrall (7), K. Burns (8), J. Jacobs (9), S. Ashiglar (2), J. Lundquist (10), M. Kim (11), A. Ross-Davis (2), C. Hoffman (12), R. Mathiasen (12), R. Hofstetter (12), J. Shaw (13), E. Pitman (2), E. Nelson (12), G. McDonald (2), M. Cleary (14), S. Brar (15), B. Richardson (16), N. Klopfenstein (2)
(1) USDA Forest Service - RMRS, Moscow, ID, U.S.A.; (2) USDA Forest Service, RMRS, Moscow, ID, U.S.A.; (3) USDA Forest Service, FHP Region 6, Bend, OR, U.S.A.; (4) USDA Forest Service, Region 5 Regional Office, Vallejo, CA, U.S.A.; (5) USDA Forest Service, FHP Region 2, Rapid City, SD, U.S.A.; (6) USDA Forest Service, FHP Region 1, Coeur d'Alene, ID, U.S.A.; (7) USDA Forest Service, FHP Region 2, Gunnison, CO, U.S.A.; (8) USDA Forest Service, FHP Region 2, Lakewood, CO, U.S.A.; (9) USDA Forest Service, FHP Region 3, Albuquerque, NM, U.S.A.; (10) USDA Forest Service, FHP Region 10/PNWRS, Anchorage, AK, U.S.A.; (11) Department of Forestry, Environment and Systems, Kookmin University, Seoul, South Korea; (12) School of Forestry, Northern Arizona University, Flagstaff, AZ, U.S.A.; (13) USDA Forest Service, Interior West Forest Inventory and Analysis, Ogden, UT, U.S.A.; (14) Swedish University of Agricultural Sciences, Uppsala, Sweden; (15) SCION/Massey University, New Zealand; (16) USDA Forest Service, RMRS, Provo, UT, U.S.A.

*Pseudotsuga menziesii* and *Armillaria solidipes* are native forest host/pathogen species of the interior western USA that have co-evolved over millions of years. Armillaria root disease caused by *A. solidipes* is a major limitation for growth, survival, and economic/environmental benefits of *P. menziesii*. Historically, *A. solidipes* played a beneficial ecological role by preferentially attacking maladapted trees. However, under increasing climate change, more forest trees will likely become progressively maladapted, leading to increased damage from Armillaria root disease. We developed species-
distribution models using Maximum Entropy (MaxEnt) to predict area suitable for *A. solidipes* and *P. menziesii* under changing climatic conditions. We compared contemporary (1950-2000) predicted suitable climate for *A. solidipes* and *P. menziesii* versus year 2070 (2061-2080) predictions using the "business as usual" greenhouse gas-emissions pathway 8.5 (RCP8.5) paired with the HadGEM2-ES global circulation model. Our predictions of suitable climate for *A. solidipes* and *P. menziesii* are highly correlated, and the predicted suitable climate space moves dramatically northward for year 2070 under the RCP8.5 scenario. In many areas of the inland west, *A. solidipes* is predicted to persist under climate change, and it will likely cause major damage where maladapted forest trees remain.

## Effects of vernalization on the differential ozone response of two winter wheat genotypes

A. MASHAHEET (1), D. Marshall (2), K. Burkey (2)

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

Elevated ground-level ozone, associated with climate change is a limiting factor to wheat production. Evaluating winter wheat for ozone responses could be accomplished more rapidly at the seedling stage using unvernalized seedlings. However, there is no information available on the effects of vernalization on the expression of ozone injury responses, and the association of these responses to yield losses at adult plant stage. Twenty five winter wheat genotypes (including historical and recent varieties and breeding lines) were evaluated for ozone tolerance (at CF, 50, 70, 90 and 110 ppbv, 8hrs/day for 1 week), and two lines with differential ozone response were selected, Coker-9553 (O3-sensitive) and MD01W28-08-11 (O3-tolerant). These two genotypes were used to study the effects of vernalization on the differential ozone response, and the validity of results if unvernalized seedlings are used. Foliar Injury results at the seedling stage showed that vernalization has no significant effect on the differential ozone responses, and the two genotypes could be separated using vernalized or non-vernalized plant material. The differential response obtained at the seedling stage was validated at the adult-plant stage. Yield losses under elevated ozone treatments in open-top chambers were associated with increased foliar injury observed at the seedling stage.

## Screening the monosomic lines of the Chinese Spring wheat variety for ozone tolerance

A. MASHAHEET (1), D. Marshall (2), K. Burkey (2), R. Ullah (1), A. Abdelrimah (3)

(1) North Carolina State University, U.S.A.; (2) USDA-ARS, North Carolina State University, U.S.A.; (3) North Carolina State University, U.S.A.

Elevated tropospheric ozone is currently a major challenge to wheat production worldwide and is anticipated to be more challenging in the future. Breeding wheat for ozone tolerance is needed to maintain or increase wheat yield. However, there is limited information on the genetic control of ozone tolerance in wheat. The current understanding is that genome A and genome B are responsible for tolerance, whereas genome D is responsible for ozone sensitivity of modern wheat. In this study, we investigated the contribution of each of the 21 chromosomes of the Chinese Spring variety towards its ozone tolerance. We screened the 21 monosomic lines of Chinese Spring, at four ozone concentrations (CF, 50, 70 and 90 ppbv, 12 hr daily average, for 2 weeks). Ozone-induced foliar injury results showed that the absence of chromosome 7A reduces ozone tolerance. In contrast, the absence of chromosome 4A increases tolerance. None of the chromosomes of the genome D were found to individually reduce ozone tolerance. Future studies are needed to identify the genetic basis of ozone tolerance and the associated DNA markers to facilitate the incorporation of ozone tolerance into wheat breeding programs.

## Spatio-temporal brown eye spot progress of irrigated coffee by central pivot

M. SILVA (1), M. Silva (2), E. Pozza (2), E. Chaves (3), G. Vasco (2), P. Paula (3), G. Dornelas (3), M. Silva (3), M. Resende (3)

(1) UFLA, Brazil; (2) Lavras Federal University, UFLA, Brazil; (3) Lavras Federal University, UFLA, Brazil

The brown eye spot of coffee (*Cercospora coffeicola*), reduces both productivity and quality of the beverage. Thus, the objective was to evaluate the spatio-temporal disease progress in coffee farm irrigated by center pivot. The pivot area, located in the southern state of Minas Gerais in Brazil, was georeferenced with 50 points in 17 ha to proceed the geostatistical analysis. The disease incidence was evaluated in 60 leaves, in five plants/point at 60-days intervals, from August 2012 to December 2014, totaling 15 evaluations. Climatic variables were obtained by weather station installed in the area and the data were correlated with disease incidence. The production of the years 2013 and 2014 were correlated with the area under the incidence progress curve (AUIPC). The disease progress curves were plotted and the adjustment of semivariogram models was done. Then, the data were interpolated by ordinary kriging, and the maps of disease were constructed (ArcGIS 9.2®). The incidence reaching in August 2013 and 2014, 23 and 21%, respectively. The exponential model was adjusted to the semivariogram. In over 50% of the area, the intensity was high, exceeding 20%, with spatial gradient. There was negative correlation between incidence of the disease with minimum and medium temperatures and AUIPC and productivity in 2014.

## Effect of pre-anthesis rainfall patterns on fusarium head blight and deoxynivalenol in wheat: A multi-state study

W. BUCKER MORAES (1), K. F. Andersen (1), C. Cowger (2), R. Dill-Macky (3), L. V. Madden (1), P. Anderson Paul (1)

(1) Department of Plant Pathology, The Ohio State University, Wooster, OH, U.S.A.; (2) Department of Plant Pathology, North Carolina State University, Raleigh, NC, U.S.A.; (3) Department of Plant Pathology, University of Minnesota, Saint Paul, MN, U.S.A.

Although there is often a correlation between visual symptoms of Fusarium head blight (FHB) and deoxynivalenol (DON) in wheat, under certain conditions, DON levels may be higher than one would expect based on visual symptoms. Between 2012 and 2015, field experiments were conducted at three locations (Minnesota, North Carolina, and Ohio), representing different wheat growing regions and market classes, to investigate the effects of intermittent rainfall during the 7-8-day pre-anthesis window on FHB and DON in wheat. Plots were inoculated with corn kernels colonized by *Fusarium graminearum* at jointing (Feekes GS 6). The experimental design was a randomized complete block with five pre-anthesis simulated rainfall treatments: 1) rainfall every day (Rain_1); 2) rainfall only on the first and last two days of the window (Rain_2); 3) rainfall only on the middle 3-4 days of the window (Rain_3); 4) rainfall every other day (Rain_4); and 5) check. Plots that received simulated rain had numerically higher mean FHB incidence (INC), index (IND), Fusarium damaged kernels (FDK), and DON than the check at all locations. Rain_1 consistently resulted in higher mean INC and IND than Rain_2, Rain_3 and Rain_4. However, Rain_2 and Rain_4 had comparable mean FDK and DON to Rain_1. Thus, dry-wet moisture cycles may be associated with disproportionately high DON for a given level of IND.

## Changes in frequency of *Leptosphaeria maculans* avirulence genes in North Dakota

S. Mansouripour (1), K. Chittem (1), Z. Liu (1), L. del Rio Mendoza (1)

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

Blackleg, caused by *Leptosphaeria maculans* is resuming as a threat to North Dakota canola industry. A shift in the virulence profile of *L. maculans* populations is thought to be responsible for it. Estimating the prevalence of specific avirulence genes in *L. maculans* populations could be used to infer the relative value of its interacting resistance genes for a plant breeding program. The objective of this study was to determine the frequency of avirulence genes in *L. maculans* isolates from North Dakota. Single-spore cultures of *L. maculans* isolates collected from commercial grower's fields in 2004, 2007 and 2014 were used. Presence of avirulence genes *AvrLm1*, *AvrLm6*, *AvrLm4-7* and *AvrLm11* was determined using PCR assays. The results indicated that the frequency of *AvrLm1* increased from 3% among isolates collected in 2004 to 45% among isolates collected in 2014 while the frequencies of *AvrLm6* and *AvrLm11* remained unchanged (91 to 100% and 91 to 95%, respectively) between the same two periods. *AvrLm4* and *AvrLm7* alleles were present in 90 and 100% of the isolates, respectively in 2014. Determination of frequencies of *AvrLm4* and *AvrLm7* in 2004 and 2007 is underway. The higher frequency of isolates carrying *avrLm1* in 2014 suggests its corresponding resistance gene, *Rlm1*, can no longer provide effective
protection. In contrast, the high prevalence of avirulence genes Avrlm4, AvrLm6, AvrLm7, and AvrLm11 indicates their interacting resistance genes could be safely used.

### Transmission of *Rhodococcus* spp. causing pistachio bushy top syndrome in California

E. FICHTNER (1), S. Dhaouadi (1), T. Kapaun (2), J. Cardwell (3)

(1) University of California Division of Agriculture and Natural Resources, Tulare, CA, U.S.A.; (2) University of California Division of Agriculture and Natural Resources, Exeter, CA, U.S.A.; (3) Companion Animal Clinic, Visalia, CA, U.S.A.

Pistachio bushy top syndrome (PBTS) is a new pistachio disease caused by two *Rhodococcus* spp., highly related to *Rhodococcus fascians* (*Rf*) and *Rhodococcus corynebacteriodes* (*Rc*). Determination of potential modes of pathogen transmission is necessary for protection of replants and asymptomatic trees in orchards, as well as prevention of similar future epidemics. The objectives of this study were to assess the potential for PBTS isolates to be transmitted to healthy plants via naturally-infested soil at replant sites, using pruning tools, or by rodents. Clonal UCB-1 (*Pistacia atlantica* × *Pistacia integerrima*) plants were planted in naturally-infested soil; positive and negative control treatments were established by placing root-inoculated and uninoculated plants in steam-disinfested soil. Pruning transmission studies were conducted by cutting into symptomatic tissue in advance of cutting healthy plants; positive and negative control treatments were established by artificial infestation and flame sterilization of pruners, respectively. Mice trapped in the greenhouse were evaluated for internal and external populations of *Rf* and *Rc*. The results demonstrate that naturally infested soil and infested pruning tools may transmit the bacteria to healthy plants. Mice were also found to carry *Rhodococcus sp.* on fur and in their digestive tract. The results underscore the need for vigilant sanitation, both in the orchard and in nursery propagation systems.

### Quantification of airborne inoculums of *Magnaporthe oryzae* by using TaqMan Real-time PCR

X. CHEN (1), K. Yuan (1), S. Wang (2), F. Guo (2), B. Wu (2)

(1) China Agricultural University, China; (2) China Agricultural University, China

Rice blast caused by *Magnaporthe oryzae* is one of the most devastating diseases in China and worldwide. Accurate and timely quantification of airborne spores of the pathogen is important to disease forecast and management of the disease. In this study, airborne spores were trapped on the tape with a Burkard sporewatch spore samplers mounted in a rice field in Panjin, Liaoning province, China from July 1 to October 13, 2015. The spores were washed off the tape and followed by DNA extraction by using Mobio Power Soil DNA Isolation Kit, and quantified by TaqMan Real-time PCR. Specific primers and probe were designed to target the 28S rDNA of *Magnaporthe oryzae* and reaction system was optimized. qPCR results of field samples revealed two sporepeaks during the study period. They were 9.46 spores/m³ from July 18 to 20 and 2.95 spores/m³ from August 15 to 17, which were followed by a high incidence of leaf blast on August 4 and first observation of neck blast on August 22. This suggested that quantity of airborne spores of *Magnaporthe oryzae* could be useful in disease forecasting of rice blast. The qPCR system combined with spore samplers provided an efficient way to quantify airborne pathogen for disease forecasting and guiding disease management of rice blast.

### The seasonal dynamics of *Phyllosticta* spp. in Floridian citrus grove leaf litter

K. ZHANG (1), J. Rollins (2), M. Dewdney (3)

(1) Plant Pathology Department, Citrus Research and Education Center, University of Florida, Lake Alfred, FL, U.S.A.; (2) Plant Pathology Department, University of Florida, Gainesville, FL, U.S.A.; (3) Plant Pathology Department, Citrus Research and Education Center. University of Florida, Lake Alfred, FL, U.S.A.

The causal agent of Citrus Black Spot (CBS), *Phyllosticta citricarpa*, is a restricted pathogen in Europe and quarantined in the US after it was found in Florida in 2010. Additionally, an endophytic fungus *P. capitalensis*, associated with a wide range of hosts including citrus, is globally distributed and commonly found in Floridian citrus. These two species cannot be distinguished morphologically; therefore, previous CBS epidemiology studies included both endophyte and pathogen. Both fungi overwinter and produce ascospores and conidia in leaf litter. Understanding the seasonal dynamics of these two species in leaf litter can provide knowledge to control CBS inoculum in Floridian groves. 1000 leaves were collected biweekly from 40 arbitrarily selected trees in a grove in south Florida since May 2014. The number of leaves with fructifications, the number of *Phyllosticta* spores collected from the leaf litter, and the average leaf degradation level from each sample were recorded. The quantity of *P. citricarpa* and *P. capitalensis* DNA in leaf litter and spore suspensions was determined via qPCR. The results showed that the fructification abundance is correlated with leaf degradation level (pseudothecium: *P* = 0.0008, pycnidium: *P* = 0.0333), conidia were always present, and ascospores were occasionally absent. The quantity of *P. capitalensis* DNA was constant while the quantity of *P. citricarpa* DNA increased throughout the two-year sample collection period.

### Field observations of ascospore discharge of *Monilinia vaccinii-corymbosi* in northern highbush blueberries

G. Dabbah (1), A. Schilder (2)

(1) CSU Monterey Bay, U.S.A.; (2) Michigan State University, Department of Plant, Soil and Microbial Sciences, U.S.A.

*Monilinia vaccinii-corymbosi* (*Mvc*), is the causal agent of a disease known as mummy berry in highbush blueberries (*Vaccinium corymbosum* L.). The primary inoculum of *Mvc* is discharged from apothecia grown from mummified blueberry fruits that fall to the ground in previous seasons. In order to better predict the release of primary inoculum a study of ascospore discharge was conducted using aerial spore trapping and several weather variables. A Burkard spore trap was used to collect ascospores within infested blueberry fields in 2002, 2003, 2004, and 2009. A previously developed degree-day model was also assessed in order to compare the amount of chill-hours and degree-days required for southwest Michigan grown blueberries. Peak ascospore discharge was inversely related to daily relative humidity and the majority of ascospores were discharged between 67-107 growing degree-days and after 3000 chill-hours were attained. Additionally, peak ascospore discharge was reached when apothecial cup size was between 2-7mm in diameter which correlated with previous laboratory studies. The information in the study may be used to better understand the conditions needed to predict mummy berry apothecia emergence and ascospore discharge with the aim of improving future disease management practices.

### A leaf litter and fruit brown rot life style of *Phytophthora syringae* in California citrus

W. HAO (1), H. Förster (1), T. Miles (2), F. Martin (3), G. Browne (4), J. Adaskaveg (1)

(1) University of California, Riverside, Riverside, CA, U.S.A.; (2) California State University, Monterey Bay, Seaside, CA, U.S.A.; (3) USDA-ARS, Salinas, CA, U.S.A.; (4) University of California, Davis and USDA-ARS, Davis, CA, U.S.A.

In recent years, *Phytophthora syringae* has become a major pathogen causing brown rot of citrus fruit in California during the winter season. This is a serious concern for the citrus industry because *P. syringae* is a quarantine pathogen in some export countries. The source of *P. syringae* inoculum infecting citrus fruit has never been clearly established. Roots and rhizosphere soil from trees where brown rot was found to be caused by *P. syringae* were sampled in three orchards. Samples were plated onto a *Phytophthora*-selective medium and TaqMan qPCR and Droplet Digital PCR were performed. *P. citrophthora* and *P. parasitica* were consistently detected by isolation in roots and soil and by PCR in roots. *P. syringae* was not found causing root rot and was not detected in the soil using these methods. Leaf litter on the ground and soil were sampled from the same locations and immersed in water. Mature green pears were used as baits and incubated at 12°C for 1 to 2 wk. Brown lesions that developed on pears at the water line were plated onto selective medium. *P. syringae* was detected in most leaf litter samples but only in two soil samples. Thus, leaf litter is a major inoculum source for *P. syringae* and changes our understanding of the disease cycle of this important pathogen and separates it from other *Phytophthora* species that cause citrus root and brown rots. Recognition of the distinct epidemiology of the disease opens the opportunity for new cultural management practices.