# Functional analysis of root-knot nematode (Meloidogyne javanica) virulence genes in rice

D. Fernandez, M. Grossi de Sa, I. Mezzalira, M. Beneventi, D. Amora, H.

Baimey, A. Petitot, Janice de Almeida, E. Saliba Albuquerque

► To cite this version:

D. Fernandez, M. Grossi de Sa, I. Mezzalira, M. Beneventi, D. Amora, et al. Functional analysis of root-knot nematode (Meloidogyne javanica) virulence genes in rice. Annual Meeting of the American-Phytopathological-Society (APS), Jul 2016, Tampa (Floride), United States. 163 p., 10.1094/PHYTO-106-12-S4.1. hal-01606195

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# 2016 APS Annual Meeting
## Abstracts of Presentations

Abstracts submitted for presentation at the APS Annual Meeting in Tampa, Florida, U.S.A., July 30–August 3, 2016. Recommended format for citing annual meeting abstracts, using the first abstract below as an example, is as follows: Harper, S., Cowell, S., and Dawson, W. 2016. Factors that influence the structure of *Citrus tristeza virus* populations. (Abstr.) Phytopathology 106:S4.1. http://dx.doi.org/10.1094/PHYTO-106-12-S4.1

The abstracts are published for citation purposes. They were not reviewed by the *Phytopathology* Editorial Board and were not edited by the APS editorial staff. Please send questions or comments to aps@scisoc.org.
http://dx.doi.org/10.1094/PHYTO-106-12-S4.1

© 2016 The American Phytopathological Society

### Factors that influence the structure of *Citrus tristeza virus* populations

S. HARPER (1), S. Cowell (2), W. Dawson (2)

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

A virus infection of a host is not a discrete, static entity, but a dynamic population whose members interact with one another and with their host to persist and spread. We have been using populations of *Citrus tristeza virus* (CTV) strains to examine this phenomenon, and have found that under constant conditions, the population structure will stabilize to an equilibrium defined by the host. This led us to ask what determinants, other than the host, can define a population, and what factors can cause a shift in population structure. Here we examined the effect of (a) inducing stress on the host through drought, high temperature, and by inducing systemic acquired resistance through the application of salicylic acid (SA), and (b) the addition of *Citrus tatter leaf virus* (CTLV) and *Citrus leaf blotch virus* (CLBV). We found that the structure of a CTV population comprised of three strains showed no significant change between treated and non-treated plants when exposed to drought or treated with SA, nor did the addition of CTLV or CLBV. Only exposure to approximate Florida summer temperatures (36°C day/25°C night) caused a change in structure, decreasing virus titer in a strain-specific and host-specific manner. Knowledge of the factors that determine population structure will aid in developing methods to manipulate populations and prevent disease.

### Virus populations associated with Maize lethal necrosis (MLN) in East Africa

L. STEWART (1), M. Redinbaugh (1), S. Wijeratne (2), G. Mahuku (3), C. Niblett (4), A. Kiggundu (5), G. Asea (5), A. Wangai (6), K. Willie (1), D. Massawe (7)

(1) USDA-ARS Corn, Soybean and Wheat Quality Research Unit, Wooster, OH, U.S.A.; (2) Molecular and Cellular Imaging Center, The Ohio State University, Wooster, OH, U.S.A.; (3) International Institute of Tropical Agriculture (IITA), Dar es Salaam, Tanzania; (4) Venganza, Inc, Augustine, FL, U.S.A.; (5) National Agricultural Research Organization, Entebbe, Uganda; (6) Kenya Agricultural and Livestock Research Organization (KALRO), Nairobi, Kenya; (7) Plant Pathology Department, The Ohio State University, Wooster, OH, U.S.A.

Maize lethal necrosis (MLN) has emerged as a devastating disease in East Africa in recent years. MLN is caused by co-infection of a maize-infecting potyvirus and maize chlorotic mottle virus (MCMV). Deep sequencing of MLN-associated samples collected in Kenya and Uganda indicate high conservation of MCMV sequences, whereas the associated potyvirus population is diverse and complex. Sugarcane mosaic virus (SCMV)-like sequences are abundant and variable across samples, and clustered into four groups with over 10% nucleotide sequence divergence. Sequence analyses indicate presence of other putative viruses in MLN-diseased tissue that require further characterization. These data are valuable to inform diagnostics, containment, and management of destructive maize viruses in East Africa.

### Investigating the spread of Grapevine red blotch-associated virus

E. Cieniewicz (1), M. Fuchs (1), K. Perry (2)

(1) Cornell University New York State Agriculture Experiment Station, U.S.A.; (2) Cornell University, U.S.A.

Red blotch is a recently recognized viral disease of *Vitis* spp. The causal agent, Grapevine red blotch-associated virus (GRBaV) has a single-stranded, circular DNA genome (3,206 nt), and is a proposed member of the family *Geminiviridae*. GRBaV has been detected in all major grape-growing regions of the United States and Canada, likely as a result of the dissemination of infected propagation and planting material. Of major concern to the grape industry is whether GRBaV is transmitted in vineyards. While there is no indication of spread in most areas where GRBaV has been detected, within-vineyard transmission is documented in California, likely due to insect vectors. To identify insect vectors of GRBaV, a detailed census of hemipteran insects in a selected California vineyard was performed. Of more than 50 species screened in 2015, only four species of the families Cicadellidae, Membracidae, and Cixiidae consistently carried genetic elements of GRBaV, as shown by multiplex PCR. The ability of vector candidate species to
transmit GRBaV to healthy grapevines is being assessed in greenhouse transmission assays. Understanding the mechanism of transmission will inform proper management strategies to control the insect vector and minimize spread of GRBaV.

### Epidemiological insights into an emerging virus disease: *Blueberry shock virus* on cranberry

S. THOMAS-SHARMA (1), L. Wells-Hansen (1), R. Page (1), V. Kartanos (2), E. Saalau-Rojas (3), P. McManus (1)

(1) University of Wisconsin-Madison, U.S.A.; (2) Bayer Crop Science, U.S.A.; (3) UMass Cranberry Station, U.S.A.

Berry scarring, where cranberry uprights (shoots) produce scarred, disfigured berries that turn prematurely red, is an emerging problem for growers in WI and MA. We detected *Blueberry shock virus* (BIShV) on symptomatic uprights using DAS-ELISA and PCR, and initiated epidemiological studies. Similar to BIShV in blueberry, symptomatic cranberry uprights 'recover' from symptoms in the following season. These recovered uprights continue to test positive for BIShV and had greater incidence of BIShV in seeds (71%), compared to symptomatic uprights (12%). Studies on yield in three WI marshes affected by BIShV revealed that flowering is comparable in healthy, recovered, and symptomatic uprights. By the time of harvest, symptomatic uprights produced fewer marketable fruit and had lower fruit weight when compared to healthy uprights. Yield components on recovered and healthy uprights were comparable. Phylogenetic analysis of the coat protein of cranberry isolates from WI (n=12) and MA (n=5) revealed 96-100% nucleotide similarity among isolates, and isolates did not group by geography. Analysis of the protein sequence revealed that BIShV on cranberry shares only 90% coat protein identity with BIShV on blueberry. Small RNA sequencing and assembly (sRSA) indicates genome-level single nucleotide polymorphisms in BIShV from cranberry, suggesting different strains on blueberry and cranberry. In-depth analysis of sRSA data to identify other known and unknown viruses is underway.

### Threat to control Canna Yellow Streak Virus; altered infectivity by associating with a Sub-viral element of novel Canna Yellow Mottle Virus

D. WIJAYASEKARA (1), P. Hoyt (1), B. Dunn (1), A. Gimondo (1), J. Verchot (1)

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

Canna Yellow Mottle Virus (CaYMV) poses a significant threat to the canna industry by causing unwanted virus-like symptoms to the foliage. CaYMV was first reported in 1979 and the full genome has not been reported. In order to determine the full CaYMV genome and characterize the disease, next generation sequencing was carried out using Virion DNA extracted from infected canna plants. The full CaYMV genome consisting of 6976 bp with two associated sub-viral elements (SVE), was recovered. The CaYMV genome and two sub-viral elements were cloned and sequenced using PCR primers. With initial diagnostic experiments using multiplex PCR reported in 2015, CaYMV was found to be associated with Canna Yellow Streak Virus (CaYSV). To determine the nature of this association, analysis of naturally infected canna varieties including greenhouse-grown and field-grown canna for the presence of CaYMV, CaYSV and SVEs was conducted using specific diagnostic primers for CaYMV and SVE 0036. Among six canna varieties, one variety "Australia" tested positive for only the SVE-0036 while another variety "Striped Beauty" tested positive for all three. Sap containing SVE0036, CaYSV, or CaYMV were mixed and used to inoculate bean or lupine plants. Infectivity of CaYSV with the presence of SVE0036 altered in bean and lupine plants. These data suggest that it is important to consider the presence of SVE0036 when testing for CaYSV infectivity.

### Trivapro™ fungicide, a new fungicide for broad spectrum control of diseases of corn, soybeans, and wheat

E. TEDFORD (1), E. Tedford (1), A. Tally (1)

(1) Syngenta, U.S.A.

Trivapro™ fungicide was registered late in 2015, and is now commercially available for use on corn, soybeans, and wheat. This fungicide contains three broad spectrum ingredients (a strobilurin, triazole, and an SDHI) that all have different modes of action. The SDHI component, Solatenol™ is an extremely potent active ingredient that provides long residual control of many important diseases. This combination of active ingredients provides both preventive and curative disease control. Trivapro is particularly effective against many of the rust pathogens. Results from corn and wheat trials in the US in 2015 demonstrated the grower benefits of longer lasting control. Research has also shown some crop enhancement benefits.

### Evaluation of fungicides for management of Fusarium wilt of watermelon

N. MILLER (1), M. Adams (1), L. Quesada-Ocampo (1)

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

Fusarium wilt of watermelon, caused by *Fusarium oxysporum* f. sp. *niveum*, is an economically important disease in the United States and worldwide. It has traditionally been managed with long crop rotations and resistant varieties. However, the development and increased prevalence of new pathogen races has reduced the number of available resistant cultivars. In light of this, new chemical control options would greatly assist growers in controlling Fusarium wilt of watermelon. This study evaluates new and existing chemical control options for their potential in limiting *F. oxysporum* growth in the lab and managing disease in the field. Fungicide efficacy *in vitro* using a fungicide-amended media assay with 10 single formulation products at four concentrations revealed that prothioconazole and ADEPIDYN™ fungicide were among the best performing fungicides. Sensitivity to these fungicides was determined for 100 isolates of *Fusarium oxysporum*. Field-testing of these two products at two concentrations and application methods indicated that both products significantly reduced disease incidence, but a drench treatment followed by a foliar spray treatment 14 days post transplant was superior in reducing Fusarium wilt incidence in comparison to either product used in only a drench treatment. These two products could effectively complement existing strategies in managing Fusarium wilt of watermelon.

### Pimaricin: A new post-harvest fungicide for selected citrus, stone, and pome fruits

D. Chen (1), H. Förster (1), J. Adaskaveg (1)

(1) Department of Plant Pathology and Microbiology, University of California, Riverside, U.S.A.

Pimaricin (natamycin) is a polyene macrolide antimycotic produced by *Streptomyces natalensis*. It has been demonstrated to block fungal growth by binding to ergosterol within cell membranes. In use as a food preservative for many years, no resistance to pimaricin has ever been observed in *Penicillium* spp. populations. Pimaricin has potential to be an effective naturally derived fungicide for postharvest management of fruit decays when used by itself (e.g., stone fruits) or in mixtures with conventional fungicides (e.g., citrus and pome fruits) for improved efficacy and as an anti-resistance strategy. We tested the in vitro toxicity of pimaricin against selected postharvest pathogens of fruit crops. Mean EC₅₀ values for inhibition of mycelial growth of isolates of *Alternaria alternata*, *Botrytis cinerea*, *Geotrichum citri-aurantii*, *P. digitatum*, and *P. expansum* were 0.92 mg/L, 0.79 mg/L, 3.20 mg/L, 1.47 mg/L, and 1.14 mg/L, respectively. Treatments of inoculated citrus (*G. citri-aurantii*, *P. digitatum*), stone fruits (*B. cinerea*, *Monilinia fructicola*, *Rhizopus stolonifer*), and pome fruits (*A. alternata*, *B. cinerea*, *P. expansum*, *Mucor piriformis*) with 1000 mg/L pimaricin were mostly effective with a reduction in decay incidence by 65 to 95% as compared to the control. However, reduced efficacy or treatment failures were observed in some pathosystems (e.g., *P. expansum* on pears). The mechanism for this unique selective performance is currently being investigated.

### Evaluation of new fungicides for management of Phytophthora root rot of citrus

W. Hao (1), M. Gray (1), H. Förster (1), J. Adaskaveg (1)

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

Phytophthora root rot (PRR), caused by *Phytophthora* spp., is an important disease of citrus in California. For its chemical control, only mefenoxam and potassium phosphite are currently registered. Mefenoxam resistance is widespread, and overuse of phosphonates may lead to resistance in *Phytophthora*
spp. Thus, we evaluated the efficacy of new chemicals belonging to different modes of action (i.e., ethaboxam, fluopicolide, mandipropamid, and oxathiapiprolin) against PRR. Navel orange trees on Carrizo rootstock were inoculated with *P. parasitica* at planting. Applications with 12 fungicide treatments were made five weeks after planting, in spring and fall 2014, and in spring 2015. Feeder roots and adjacent soil were collected before or after application. PRR incidence and pathogen populations were determined by plating root segments and soil suspensions, respectively, on a selective medium. All of the new fungicides reduced PRR incidence and soil populations significantly to very low levels as compared to the control starting with the first application. Mefenoxam was only effective when a high label rate was used in the fourth application. Most of the new fungicides also increased tree canopy size and trunk diameter compared to the control. Greenhouse studies confirmed the efficacy of these new fungicides. Once registered, these new compounds will provide highly effective treatment options and resistance management strategies in the control of PRR of citrus.

## Improving genomic resources for the detection of *Macrophomina phaseolina* infection of strawberry

A. BURKHARDT (1), K. Childs (2), M. Ramon (1), F. Martin (1)

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

*Macrophomina phaseolina* is the causal agent of charcoal rot, which is characterized by wilting, stunting, and crown discoloration. It has had an increasingly devastating impact on strawberry production in California as treatment methods have shifted from broadcast pre-plant fumigation with methyl bromide+chloropicrin to individual bed treatment with alternative fumigants. Hundreds of *M. phaseolina* isolates were collected from infected hosts in multiple countries and states, including many from California. An SSR analysis revealed that, with few exceptions, the strawberry isolates were a single genotype (isolates from other hosts were not pathogenic on strawberry). To quickly identify the strawberry genotype, genotype-specific primers were developed with the end goal of implementing a diagnostic assay from soil prior to planting. These primers were designed by sequencing multiple isolates and using comparative genomics to identify genotype-specific loci. However, the complexity of the differences in the genomes of strawberry and non-strawberry isolates cannot be fully resolved with this approach. Therefore, PacBio sequencing was used to assemble a strawberry isolate reference genome. The draft assembly has an N50 of 3.3 Mb with 143 contigs and ~150x genome coverage. Ongoing work to improve and annotate this assembly is being done so that it can be used to identify additional strawberry-specific loci and to understand host specificity through comparative genomics.

## Identifying sources of inoculum and timing of tissue infection by fungal pathogens associated with winterberry fruit rot

F. PEDUTO HAND (1), S. Lin (1), N. Taylor (1)

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

Winterberry is a valuable deciduous ornamental plant carrying brightly colored berries during the winter that is popular as a cut green for use in holiday decorations. In the past 3-4 years, a fruit rot has been challenging winterberry growers in the midwestern and eastern U.S., in some cases leading to complete crop loss. In this study, we monitored winterberry 'Bonfire' and 'Sparkleberry' throughout the 2015 growing cycle in two Ohio nurseries to identify sources of inoculum and assess periods of tissue infection. Twigs and mummified fruit were collected during plant dormancy to determine potential sources of primary inoculum. Flowers, leaves, and berries were collected weekly from June to December to record leaf spot and fruit rot incidence and severity, as well as pathogen recovery. Spore traps were used during the growing season to monitor spore abundance in the orchards. Based on the isolation results as well as counts from the spore traps, it appears that the disease may be due to a fungal complex, including species of *Alternaria*, *Cladosporium*, *Colletotrichum*, *Epicoccum*, *Fusarium*, *Phoma*, and *Phomopsis*. The major source of primary inoculum appears to be the mummified fruit. Leaf spots may serve as a source of secondary inoculum. A significant increase in the number of fungi recovered at bloom indicates that flowers could serve as a point of entry for fruit infections. The ultimate goal of this study is to provide growers with appropriate management strategies.

## Occurrence of foliar diseases of watermelon on commercial farms in South Carolina in 2015

G. RENNBERGER (1), A. Keinath (1), P. Gerard (2)

(1) Coastal Research and Education Center, Clemson University, U.S.A.; (2) Department of Mathematical Sciences, Clemson University, U.S.A.

The main foliar diseases of watermelon in the southeastern U.S. are anthracnose, downy mildew (DM), gummy stem blight (GSB) and powdery mildew (PM). A survey was conducted in South Carolina in 2015 to obtain current data on the occurrence and distribution of these diseases. A stratified two-stage cluster sampling design was used, with 6 pre-selected counties as strata, 17 randomly selected growers as the first cluster and 21 randomly selected fields as the second cluster. Diseased leaves (100 per field) were collected along 4 transects encompassing 2500 m² with 5 equidistant sampling points along each transect. All leaves were examined microscopically, and all pathogens present were recorded. Data were analyzed with SAS survey procedures for means and proportions, logistic regression and frequency analysis. The estimated statewide probability of occurrence (and range across counties) was 0.48 (0.32-0.78) for GSB, 0.21 (0.00-1.00) for PM, 0.15 (0.00-0.49) for DM and 0.06 (0.00-0.36) for Cercospora leaf spot (CLS). No anthracnose was found. Field size significantly influenced the probability of pathogen occurrence; with increasing field size, the probabilities decreased. More than 30% of leaves had two or more pathogens. Associations between pathogen pairs were analyzed. The only significant interaction was that CLS was more likely to occur when GSB was present on the leaf. This study will help to match fungicide recommendations to the most frequent diseases.

## Worldwide distribution of the sweet potato strains of *Ceratocystis fimbriata*

Q. LI (1), T. Harrington (2), J. Li (3)

(1) China Agricultural University/Postdoctoral Fellows Workstation of Haidian Science Park, China; (2) Iowa State University, U.S.A.; (3) China Agricultural University, China

*Ceratocystis fimbriata* is a complex of species with a wide host range and worldwide geographical distribution. However, a sweet potato strain appears to be host specialized to cause black rot of storage roots. Isolates from black rotted roots of sweet potato (*Ipomoea batatas*) in China, Japan, Indonesia, Papua New Guinea, New Zealand and the USA had identical ITS rDNA sequences and only minor variation in 14 microsatellite loci (only two alleles each for two loci, and the other loci monomorphic). Three *Ipomoea* microsatellite genotypes (C1, C2 and C3) were found among the 12 isolates from sweet potato in China, two genotypes were found in the three isolates from Japan, and the other 17 isolates had identical microsatellite markers. The DNA sequences of the mating type genes of sweet potato isolates were most similar to those of isolates of *C. fimbriata* from various host plants in Ecuador, a center of diversity for sweet potato. *Ceratocystis fimbriata* on sweet potato was first described in 1890 in eastern USA, where black rot has been known for 150 years. It is hypothesized that the fungus was introduced to Japan on sweet potato storage roots and introduced from there to China in the 1930s.

## Fungi associated with cankers and wounds of cultivated and wild olives in the Western Cape, South Africa

C. SPIES (1), W. van Jaarsveld (2), P. Moyo (3), L. Mostert (2), F. Halleen (4)

(1) ARC Infruitec-Nietvoorbij, South Africa; (2) Stellenbosch University, South Africa; (3) Stellenbosch University, South Africa; (4) ARC Infruitec-Nietvoorbij, South Africa

Olive (*Olea europaea* subsp. *europaea*) production in South Africa is centred in the Western Cape Province, where viticulture is the main agricultural enterprise. Wild olive trees (*Olea europaea* subsp. *cuspidata*) also occur naturally in this region. Trunk pathogens of grapevines such as *Eutypa lata*, *Neofusicoccum australe*, *Pleurostoma richardiae*, and species of *Phaeoacremonium* have been associated with olive trunk diseases in California, Italy and Spain, but the incidence of these on South African olive trees are unknown. The aim of this study was to identify fungi associated with cankers and wounds of cultivated and wild olives in the Western Cape Province of South Africa. Isolations from 125 olive and 27 wild olive trees sampled in nine
districts yielded a fungal collection dominated by an undescribed *Pseudophaeomoniella* species. This species has been identified from 54 olive (43%) and 15 wild olive trees (47%) in eight districts. Other fungi, including known olive trunk pathogens, occurred at incidences of 13% or less. This stands in contrast to high incidences of the Botryosphaeriaceae and *Phaeoacremonium* reported on olive trees in California and Italy. The high incidence and distribution of *Pseudophaeomoniella* sp. suggests this fungus plays an important role in olive trunk diseases in South Africa.

## Multiple mechanisms of nematode control conferred by seed and root applications of microbial biocontrol agents, *Trichoderma* and *Bacillus*

M. CADLE-DAVIDSON (1), G. Harman (1), R. Hendriks (2), W. Nosir (1)

(1) Advanced Biological Marketing, Inc, U.S.A.; (2) Advanced Biological Marketing, Inc South Africa Division, South Africa

Nematode diseases of plants are estimated to contribute $150B USD in annual crop losses around the world due to Cyst (Heterodera and Globodera spp) and Root knot nematode (Meloidogyne spp) infection in species ranging from Legumes to Solanaceae to Brassicaceae among others. Chemical and host resistance resources are available for control however a common recommended practice is rotation to nonhost crops. Biological control of nematode diseases has been shown in the literature with the main mechanism being direct antagonism. 2015 field observations of soybeans in Minnesota USA (strip plots) and Gauteng South Africa (replicated small plots) indicated significant control of Soybean cyst and Root knot nematodes using *Trichoderma* and *Bacillus* seed treatments. Subsequently, a split root greenhouse experiment was conducted to identify strain specific activities and gain insight into control mechanisms. Tomato plant root systems were split and transplanted into two pots, both infested with *M. javanica*, such that individual plants would be influenced by two pots each. One of the two pots was treated with an individual biocontrol strain or strain combination as a soil drench. Twelve treatments were tested each with six replicates. The results showed excellent control of nematodes by some strains and strain combinations, synergistic effects between strains, and multiple mechanisms of control. Mechanisms and implications for population control will be discussed.

## Enhanced nematicidal activity of abamectin via nanoparticle formulation

R. Guenther (1), C. Opperman (1), J. Zhu (1), D. Lindbo (2), J. Kerns (1), S. Lommel (1), T. Sit (1)

(1) North Carolina State University, U.S.A.; (2) USDA-Natural Resources Conservation Service, U.S.A.

Abamectin (Abm) has broad nematicidal activity but its usage is limited to seed coatings due to its high affinity for soils. To overcome this limitation, Abm was infused into plant viral nanoparticles ($PVN^{Abm}$) based on Red clover necrotic mosaic virus, a virus that's normally transmitted through the soil sans vector. An added benefit of $PVN^{Abm}$ formulation is the increased diffusion rate of Abm through the soil. $PVN^{Abm}$ was found to be stable and have low affinities for soils collected from commercial fields (planted with corn, soybeans and sweet potatoes) in North Carolina even in samples with greater than 3% organic content. $PVN^{Abm}$ was found to increase the duration that Abm is nematicidal by 2-4 fold in comparison to commercial formulations. In sterile and live soil greenhouse testing, $PVN^{Abm}$ provided a 50% reduction in galling and egg quantities per root for tomatoes challenged by root knot nematode (RKN). When applied to turf, $PVN^{Abm}$ is capable of moving Abm below the thatch layer and a single application was found to retain appreciable turf greening when tested on turf infested with sting nematode. Microplot field trials are under way to determine the performance of $PVN^{Abm}$ under different application protocols/conditions and to quantify its effectiveness in reducing RKN/soybean cyst nematode damage in row crops and sting nematode in turf.

## Interaction between *Fusarium* and soybean cyst nematode on soybean

P. OKELLO (1), A. Adhikari (1), A. Varenhorst (1), S. Osborne (2), E. Byamukama (1), F. Mathew (1)

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

The soybean cyst nematode (SCN), *Heterodera glycines*, and *Fusarium* spp. are soilborne organisms that constrain soybean [Glycine max L.] production in the United States. While *Fusarium* and SCN are known to coexist in soybean fields, our knowledge of how these organisms interact and the implications for management is limited. The objective of this study was to determine if any interaction occurs between *Fusarium* spp. and SCN on soybean under greenhouse conditions. Pre-germinated soybean seedlings of SCN-susceptible (Williams 82) and SCN-resistant (Jack) cultivars were grown in cone-tainers filled with a sand-clay soil mixture (2:1) infested with inoculum of either of the four *Fusarium* spp. (*F. proliferatum*, *F. virguliforme*, *F. sporotrichioides*, and *F. subglutinans*). A suspension of 2,000 egg/ml suspension of SCN HG type 0 were used for inoculations at planting. The experiment was set up as factorial design with five replications for each treatment (*Fusarium* alone, SCN alone, and a combination of SCN and *Fusarium*) and repeated twice. After 39 days, disease severity as percentage of lesion length on soybean roots and SCN egg counts from soybean roots were evaluated. For disease severity, significant effects of the treatment involving *Fusarium* spp. was observed on the two soybean cultivars (P = 0.01). Our results from the interaction study between *Fusarium* spp. and SCN illustrates the need for developing targeted approaches to manage the *Fusarium*-SCN complex on soybean.

## Effect of Seed Treatments on Root-Knot Management for Soybean and Cotton

W. ALJAAFRI (1), W. Aljaafri (1), G. Lawrence (1), V. Klink (1)

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

Biological control is being accepted as an alternative to chemicals methods. Experiments were conducted at Mississippi State University to determine the efficacy of potential biological control products for management of the root-knot nematode. Seed applied biological products were received from Albaugh, LLC. Cotton and soybean seeds were treated with nine and seventeen biological compounds, respectively. Seeds were planted in 500 cm³ of a steam sterilized sand: soil mix 10 cm Dia. clay pots and remained for 50 days. Each test included Abamectin and ILeVo as industry standards. Seed applied biological compounds significantly reduced root-knot nematode development on cotton and soybean. Seeds treated with Abamectin, and ALB-EXP5-1+ALB-SAR1 significantly reduced juvenile development and number of eggs produced on cotton. On soybean seeds treated with Abamectin and Bionematicide+thiabendazol+ALB-GG reduced root-knot juvenile development. Abamectin and SAR+Harpin protein+Thiabendazol reduced the number of eggs that developed on soybean roots. Seed applied biological products may have potential in nematode management.

## Adaptation of soybean cyst nematode populations to the PI 88788 source of resistance from 2000 through 2015 in Iowa and the effects on soybean yields

M. MCCARVILLE (1), C. Marett (2), M. Mullaney (2), G. Gebhart (2), G. Tylka (2)

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

Most soybean cultivars grown in Iowa with resistance to the soybean cyst nematode (SCN), *Heterodera glycines*, have SCN resistance genes from a breeding line named PI 88788. Each year, field experiments are conducted throughout Iowa to evaluate 60 or more SCN-resistant and three or more SCN-susceptible cultivars at up to nine locations. Data on SCN population density, SCN virulence (HG type), soybean yield, precipitation, and growing degree-days from the experiments conducted from 2000 through 2015 were compiled and analyzed to determine how these factors affected SCN reproduction and soybean yield. Reproduction of SCN was positively correlated with temperatures and negatively associated with precipitation. Reproduction of SCN on resistant soybean cultivars with resistance from either PI 88788 or another source, Peking, was positively correlated with increasing virulence on these resistance sources. Both virulence on PI 88788 and reproduction on PI 88788-derived cultivars increased from 2000 to 2015, but not so on Peking. Yields of susceptible, PI 88788-derived, and Peking-derived SCN-resistant soybean cultivars were negatively correlated with SCN reproduction. Results indicate that virulence to PI 88788 is increasing in SCN populations in Iowa, resulting in greater SCN reproduction and yield loss. This trend is problematic because almost all SCN-resistant soybean cultivars available for Iowa farmers have resistance genes from the PI 88788 breeding line.
## Spatio-temporal analysis of rice blast incidence in China

F. Guo (1), M. Lu (2), B. Wu (1), F. Guo (1)

(1) China Agricultural University, China; (2) National Agro-tech Extension and Service Center, China

Rice blast caused by *Magnaporthe oryzae* is a devastating disease worldwide. Knowledge on the spatial and temporal patterns of disease risk at regional scale is important to understand its epidemic. In this study, rice blast incidence was surveyed in the main rice production areas in China between June 10 and September 10 from 2009 to 2014. Rice blast often had two peaks each year, and was highest in 2012 among these years. For the major production areas other than north eastern China, the disease incidence was highest in the Yunnan-Guizhou plateau, followed by Sichuan Basin and southern China, and lowest in central and eastern China. Spatial autocorrelation analysis revealed a significant correlation at distances ranging from 110KM to 340KM. Results of iso cluster unsupervised classification conducted in GIS on interpolated incidence on different dates could classify rice production areas into low and high disease risk regions. Although the regions with high disease risk varied among years, some regions frequently had high disease risk, such as areas along the border of Yunnan and Guangxi, the border of Sichuan and Chongqing, the border of Fujian and Guangdong, and the junction of Jiangxi, Anhui, and Zhejiang. The results of hot spot analysis were consistent with those of cluster analysis. The spatial and temporal patterns of rice blast obtained from this study provided a reference to efficiently organize resources for better management of the disease in China.

## Association between temperature sensitivity and virulence of *Blumeria graminis* f. sp. *tritici*

Y. ZHOU (1), L. Han (1), Y. Zou (1), X. Duan (1), J. Fan (1)

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

Wheat powdery mildew caused by *Blumeria graminis* f. sp. *tritici* (*Bgt*) is a highly destructive disease in China, which is highly sensitive to meteorological factors, especially temperature. In order to clarify the co-evolution between temperature sensitivity and virulence of *Bgt*, we collected a total of 90 isolates from 5 provinces (cities), including Yunnan, Sichuan, Beijing, Henan and Shaanxi, and tested the virulence of isolates according to 32 wheat varieties or lines with known powdery mildew resistant genes and sensitivities to temperature by detached leaf segment method. The results showed that the mean ET50 of tested isolates was 23.22°C, with the highest 25.15°C and lowest 19.78°C, respectively. The ET50 value of 73.34% of isolates ranged from 22.0°C to 24.0°C, and 10% of isolates below 22°C, 16.67% of isolates above 24°C. The virulence gene diversity index of the 90 *Bgt* isolates was 0.2370. The highest index *H* of isolates which from Sichuan Province is 0.2212 while the lowest index *H* of isolates from Henan Province is 0.1975. The results showed that there was a significant negative correlation between temperature-sensitivity and virulence diversity of *B. graminis* f. sp. *tritici* populations.

## Spatio-temporal aspects of rust and nutrition of coffee plants in different irrigation systems

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

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

The rust (*Hemileia vastatrix*) is the most important disease of coffee and the intensity depends on productivity, cropping system, plant nutrition and water availability. Thus, the objectives were to evaluate the spatio-temporal disease progress and the relation with plant nutrition and soil fertility in coffee farms irrigated by drip and center pivot, using geostatistics. The drip and pivot areas were georeferenced with 52 points in 11ha and 50 in 17ha, respectively. The disease incidence was evaluated in 60 leaves, in five plants/point at 60-days intervals, from August 2012 to May 2015, totaling 17 evaluations. It was also evaluated at each point, productivity, mineral nutrition of the plants and soil fertility annually. These data were correlated with the rust incidence. 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 and nutrients were constructed (ArcGIS 9.2®). The disease incidence in dripping was less than 3% over the years. In the pivot, the incidence reaching in August 2013 and from June to August 2014, between 21 and 27%. There was correlation between soil fertility and plant nutrition with rust for Ca, P and K. The spatial distribution of the disease and nutrients changed over the years. The exponential model was adjusted to coffee rust over the space. The disease gradient was observed from the foci in the kriging maps.

## Validation and refinement of a predictive model for *Sclerotinia sclerotiorum* apothecial development in soybean fields

J. WILLBUR (1), H. Lucas (2), B. Mueller (2), S. Chapman (2), M. Kabbage (2), D. Smith (2)

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

Sclerotinia stem rot (SSR), caused by *Sclerotinia sclerotiorum*, is one of the most important yield-limiting diseases of soybean worldwide and is a significant concern in the Great Lakes region of the USA. Phenologically-based fungicide applications are often inefficient, and fungicide application is sometimes unnecessary when weather conditions are not conducive for infection. Weather-based risk assessment tools are sometimes used to more accurately predict the timing of fungicide applications, however, such tools are not currently available for soybeans. In 2014, a logistic regression model using virtual weather data as inputs was developed to predict apothecial germination. The model uses 30-day averages of mean air temperature (r = -0.59, *P* < 0.01) and maximum leaf wetness (r = 0.35, *P* < 0.01) to predict the probability of apothecial presence prior to and during the soybean flowering period. In 2015, model-based fungicide applications successfully decreased disease severity and prevented yield loss in field trials. An on-site weather station was also used to validate virtual weather data. Due to inaccuracies in virtual moisture variables, higher resolution virtual weather data was combined with data collected in Wisconsin and Michigan for model refinement. The model will be incorporated into a preliminary web-based user interface to assist growers in effectively timing fungicide applications for integrated SSR management.

## Weather-based risk assessment models for common leaf spot and black seed disease of strawberry caused by *Mycosphaerella fragariae*

O. CARISSE (1)

(1) Agriculture and AgriFood Canada, Canada

Common leaf spot of strawberry (*Mycosphaerella fragariae*) is a sporadic disease. On susceptible cultivars and under favorable weather conditions, *M. fragariae* causes black seed disease, reduced plant vigor, and reduced yield the following year. Logistic regression analysis was used to examine the relationship between weather and outbreaks of common leaf spot and black seed. Information collected at 100 location-years was used to develop the models. The response variables were fields with an average of more than 10 lesions per leaf at the beginning of flowering (10% flowering) or with black seed symptoms on more than 5% of the fruits at harvest. Nonparametric correlation and logistic regression analysis was used to identify combinations of temperature, relative humidity, and rainfall during the spring period (bud break to 10% flowering) as predictor variables. Based on model prediction accuracy, sensitivity, and specificity, only few models correctly classified fields (> 80% correct classifications). The best predictors were temperature and rain (frequency and duration). The best models were validated with an independent data set (n = 20) and prediction accuracy was similar than the accuracy for the original data sets. Considering that management actions such as fungicide applications are not needed every year and in every field, these models could be used to identify field at risk for common leaf spot and black seed diseases.

## Genetics behind avirulence: Identification of novel avr-genes in *Magnaporthe oryzae*

D. TATE (1), J. Hu (1), V. Ganeshan (1), T. Mitchell (1)

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

Rice blast disease, caused by *Magnaporthe oryzae*, accounts for 10-30% of yearly rice loss which is estimated to represent enough food to feed 60 million people. The study of interactions between *M. oryzae* and rice can be used to help mitigate one of the most devastating diseases plaguing a
primary food source and knowledge of this pathosystem can be used to manage related fungal diseases. The objective of this study is to find the corresponding avirulence (Avr)-gene to the Pi-2 resistance (R)-gene in hopes of better understanding the strong relationship between *M. oryzae* and rice. In previous studies we have identified five candidate AvrPi2 genes by *Agrobacterium tumefaciens*-mediated transformation; infection assays; and TAIL-PCR with the incompatible strain KJ201. These genes have been cloned into the compatible strain Chinos. By infecting Pi-2 R rice plants with the Chinos transformants, and assessing disease, we were able to identify and characterize the most probable cognate Avr-gene. Pathogenicity assays demonstrated that two transformed lines Chinos4842 and Chinos8832 produced a similar phenotype to KJ201, with Chinos4842 showing a nearly identical phenotype match. Further, the mutants' ability to induce a hypersensitive response (HR) was assessed using 3,3'-diaminobenzidine (DAB) staining in leaf sheath assays. Of the transformed lines, Chinos4842 was the only line able to produce a HR. These results strongly suggest that MGG_04842 is AvrPi2.

## Magnaporthe oryzae zinc finger effector1 exploits rice HIRA/AS1/AS2 complex to alter plant development and increase disease susceptibility

Y. Chen (1), W. Liu (2), Q. Wang (3), Y. Yang (2)

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

Rice blast, caused by *Magnaporthe oryzae*, is one of the most important rice diseases in the world. During the fungal infection, *M. oryzae* produces many effector proteins to manipulate rice cellular process and facilitate disease development. In this study, a C~2~H~2~ zinc finger effector1 (MoZFE1) protein was identified from *M. oryzae* and shown to enter rice cells. Overexpression of MoZFE1 in transgenic rice (MoZFE1-OX) was found to alter rice growth and development and increase disease susceptibility. RNA-seq analysis reveals suppressions of some development- and defense-related gene expressions in the MoZFE1-OX lines. Both *in vitro* and *in vivo* biochemical analyses demonstrate that MoZFE1 directly interacts with a histone chaperon HIRA and suppresses the AS2 gene expression. HIRA/AS1/AS2 is a conserved gene-silencing complex and plays a role during plant organogenesis. Loss of function mutants (*rice hira*, *as1*, and *as2* lines created with CRISPR/Cas9) was shown to cause developmental and differentiation defects with some phenotypes similar to MoZFE1-OX lines. Our study suggests that MoZFE1 exploits rice HIRA/AS1/AS2 complex to alter host gene expression, thereby manipulating plant growth and development as well as defense response. Our findings will help elucidate the molecular mechanism of the rice-*M. oryzae* interaction and facilitate the development of new strategies to improve rice disease resistance and food security.

## Comparative Genomics of Two Magnaporthe oryzae Field Isolates Uncovers Novel Candidate Effector Genes

J. HUANG (1), J. Huang (1), Y. Hong (1), L. Xu (1), M. Chen (1), Z. Zhong (1), X. Chen (1), H. Zheng (1), J. Bao (1), Z. Wang (1)

(1) Fujian-Taiwan Joint Center for Ecological Control of Crop Pests, Fujian Agriculture and Forestry University, Fuzhou, China

During the colonization process, rice blast fungus Magnaporthe oryzae secrets a repertoire of effectors into host tissue to overcome immunity and facilitate invasive growth. To better understand the biological functions of these complicated and diverse effectors, two field isolates Guy11 and FJ81278 were re-sequenced, and isolate-unique secreted proteins were identified through comparative genomics. When comparing with each other, 37 and 18 unique genes encoding secreted proteins were obtained in the genome of Guy11 and FJ81278, respectively. Consistent with the known effector features, the majority of these proteins are small, cysteine-rich, specifically expressed at the early stage of infectious process, and possessed presence or absence polymorphisms among different field isolates. Meanwhile, due to the different origin of Guy11 and FJ81278, the two sets of unique genes owned the diverse distribution patterns in the strains isolated from different rice varieties (Oryza sativa ssp. indica and japonica). Furthermore, we have identified two unique proteins could suppress BAX-triggered programmed cell death in Nicotiana benthamiana so far, which indicated they are novel effectors. In conclusion, our comparative genomics approach is effective to generate a list of unique genes which could be candidate effectors and identify the novel effector genes. *To whom correspondence may be addressed. E-mail: wangzh@fafu.edu.cn

## Molecular dissection of a complex gene locus conferring high virulence on barley cv. Bowman in the fungal cereal pathogen Cochliobolus sativus

S. ZHONG (1), Y. Leng (1)

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

*Cochliobolus sativus* (Anamorph: *Bipolaris sorokiniana*) is a fungal pathogen causing spot blotch, common root rot, and black point in cereal crops. For spot blotch, four pathotypes (0, 1, 2 and 7) have been identified based on their differential virulence patterns on three barley genotypes (Bowman, ND5883 and NDB112), but the molecular mechanism underlying host specificity of these pathotypes is not well understood. Our previous studies indicated that isolate ND90Pr (pathotype 2) has a complex gene locus (*VHv1*) conferring high virulence on barley cv. Bowman. *VHv1* is located in an ND90Pr-specific genomic region, which contains 43 predicted genes including two for nonribosomal peptide synthetases (NRPSs). Knocking out either of the two NRPS genes led to loss of high virulence on cv. Bowman, suggesting that both NRPSs are involved in the biosynthesis of a presumably secondary metabolite virulence factor in the pathogen. We further characterized other genes adjacent and between the two NRPS genes at the *VHv1* locus and found that the two genes encoding 3-hydroxyacyl-CoA dehydrogenase and short-chain dehydrogenase/reductase (SDR), respectively, were also required for the high virulence of isolate ND90Pr on barley cv. Bowman.

## A forward genetic screen in *Phomopsis longicola* provides unique insights into pathogenesis

M. ZACCARON (1), S. Sharma (2), W. Fagundes (2), W. Hawkins (2), N. Lawson (2), J. Ridenour (2), J. Smith (2), B. Dhillon (2), J. Rupe (2), B. Bluhm (2)

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

*Phomopsis longicola* (Hobbs) causes Phomopsis seed decay of soybean (*Glycine max*), and also causes lesions on soybean stems, pods, and petioles. *P. longicola* can also exist endophytically within soybean stems, which has given rise to the hypothesis that pathogenesis occurs when the pathogen transitions from endophytic or hemibiotrophic growth to necrotrophy. In this study, a forward genetic screen was performed to elucidate genetic mechanisms underlying necrotrophy, and a RAD-seq approach was adapted to characterize genomic lesions in selected mutants. A collection of 1114 tagged, insertional mutants was created via Agrobacterium-mediated transformation, and mutants were evaluated individually in soybean stems for their ability to induce necrosis and form pycnidia. Six mutants induced significantly less necrosis in soybean stems than the wild-type strain, and four formed significantly fewer pycnidia. No gain-of-function mutations (increased necrosis) were observed. A modified RAD-seq protocol developed to identify the site of insertion was applied to a mutant significantly reduced in its ability to cause stem necrosis; a single copy of the disruption cassette integrated into the presumed promoter region of a cellobiohydrolase. This study describes new tools to dissect the interaction between *P. longicola* and soybean, and provides new insight into conserved mechanisms underlying stem, pod, and seed necrosis caused by *P. longicola*.

## Oomycete species richness in cacao soil assessed by massive amplicon sequencing of the cytochrome oxidase II gene

M. RATTI (1), E. Goss (1), J. Cevallos-Cevallos (2), C. Arias (2)

(1) Plant Pathology Department, University of Florida, U.S.A.; (2) Centro de Investigaciones Biotecnologicas del Ecuador CIBE-ESPOL, Ecuador

Oomycetes are ubiquitous in both natural ecosystems and crops, however diversity studies have focused in temperate regions. Emerging diseases together with climate change highlight the need to study oomycetes in other potential disease hotspots. Our aim was to assess Oomycete species richness and composition in soil samples from cacao farms of Ecuador by using high-throughput multiplexed Illumina sequencing of the cytochrome oxidase II gene (cox2). We used these nucleotide data to test the hypothesis that different oomycete species richness and composition are found between soil from cacao farms and neighboring soils, which included weeds and other cultivated crops. Eight farms were sampled covering three zones of the Coastal Region of Ecuador. Total DNA was extracted directly from collected soil samples and used for library preparation. A positive control, consisting of
gDNA from three known Phytophthora species, was included in the analysis. Amplicons of ~580bp were sequenced by Illumina MiSeq 2x300. Resulting reads were filtered before conducting megaBLAST analysis against a custom Oomycete sequence database. Most of the identified species were Phytophthora and Pythium. The majority of known pathogens, such as P. palmivora, were not restricted to cacao farms, rather they were also found in the plant communities adjacent to farms. These results suggest that wild plants and neighboring crops contribute to the ecology of Oomycete pathogens affecting cacao.

## Genome sequence and effector repertoire of the hop downy mildew pathogen Pseudoperonospora humuli

L. CANO (1), M. Bowman (2), S. Withers (3), D. Gent (4), K. Childs (5), L. Quesada-Ocampo (3)

(1) University of Florida, Fort Pierce, FL, U.S.A.; (2) Van Andel Research Institute, Grand Rapids, MI, U.S.A.; (3) North Carolina State University, Raleigh, NC, U.S.A.; (4) Oregon State University, Corvallis, OR, U.S.A.; (5) Michigan State University, East Lansing, MI, U.S.A.

Oomycete plant pathogens secrete effector molecules that manipulate host physiology to achieve colonization. Effector proteins have been identified in various oomycete species but little is known about the sequence, function, and evolution of effectors of downy mildew plant pathogens.

*Pseudoperonospora humuli* is the causal agent of downy mildew of hop, an economically important disease that occurs worldwide and has gained importance in recent years in the United States due to an increase of hop production. The main goal of our study was to identify and make available to the oomycete community a set of candidate pathogen effectors from *P. humuli* using transcriptomics. Identified effector candidates can be employed in high throughput functional genomics screens, commonly referred to as effectoromics, to test hop germplasm for specific pathogen recognition by resistance *R* proteins. Here we report the identification of 296 sequences annotated as RXLR host-translocated effectors and the expression during *P. humuli-hop* interaction. Our findings highlight a set of *in planta* expressed RXLR candidate effectors for pathogen-assisted breeding in hop for resistance to downy mildew.

## What is a clone? Rethinking the ancestry of US clonal lineages of *Phytophthora infestans* using whole genome sequences

B. KNAUS (1), J. Tabima (2), Z. Kamvar (2), C. Davis (3), H. Judelson (3), N. Grünwald (1)

(1) USDA ARS, Horticultural Crops Research Laboratory, Corvallis, OR, U.S.A.; (2) Oregon State University, Botany and Plant Pathology, Corvallis, OR, U.S.A.; (3) Univ. California, Plant Pathology, Riverside, CA, U.S.A.

Identifying genetic boundaries of a clone or clonal lineage is not straightforward. Identical multilocus genotypes can arise independently from unrelated clones. Similarly, lineages derived from the same ancestral clone can accumulate variants that distinguish them. Populations of the potato and tomato late blight pathogen, *Phytophthora infestans*, are well known for emerging as novel clonal lineages. These successions of clones have historically been named within the United States as lineages US1-US24, in order of appearance. Hypothetically, these lineages can emerge through divergence from other US lineages, recombination among US lineages or as novel, independent lineages. We used 32 whole genome sequences including dominant US clonal lineages and available samples from global populations. Based on the core orthologous genes located on supercontigs 1.1 and 1.5 we observed substantial amounts of variation shared among strains within clonal lineages. We found no support for emergence of any of the US lineages from a common ancestor shared with any other US lineages. Each of the US clonal lineages fit a model where populations of new clonal lineages emerge via migration from a source population that is sexual in nature and potentially located in central Mexico or elsewhere. This work provides novel insights into patterns of emergence of clonal lineages in plant pathogen genomes and provides an evolutionary framework for definition of clones in *P. infestans*.

## Temperature and light effects on germination of *Peronospora effusa*

R. CHOUDHURY (1), S. Koike (2), N. McRoberts (1)

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

Spinach downy mildew, caused by the biotrophic oomycete *Peronospora effusa*, is an economically important disease that is found in all spinach growing regions of the US. To effectively predict disease risk we need to understand the response of *P. effusa* to different environmental conditions. However, compared with several other downy mildew pathogens, relatively little research has been done on the optimal environmental conditions for growth and sporulation of *P. effusa*. We conducted several germination assays, exposing *P. effusa* sporangia to different temperature and lighting conditions. Between 5 and 25°C under constant darkness, germination of *P. effusa* sporangia on water agar declined following an approximately logistic decay. These results were qualitatively different from a previous study of *P. effusa* germination that found a bimodal response curve, with increased germination at lower and higher temperatures. Time course studies revealed that most sporangia germinated within the first eight hours of plating, regardless of incubation temperature. Sporangia exposed to green or blue light had significantly reduced germination when compared with those exposed to red, yellow, or no light. Light intensity and color significantly impacted germination, although the effect of color varied by light intensity. The use of these results in the development of a disease risk model will be discussed.

## Effect of red light on morphology of *Peronospora belbahrii* sporangia

J. PATEL (1), S. Zhang (1)

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

Downy mildew of basil, caused by *Peronospora belbahrii*, has spread to more than 42 US states since it was first detected in Homestead, FL in 2007. Recently, our experiments showed that red light can suppress downy mildew in basil. The aim of this study was to investigate the effect of red light on biology of *P. belbahrii* sporangia. The red LED lights were hung 4 feet above the greenhouse bench and 5 feet between two adjacent LED lights. The LED in the spectrum of 625 nm wavelength illuminated at an average light intensity of 12 µmol.m⁻².s⁻¹. The LEDs were automatically turned on from 8 pm to 8 am each day. One set of inoculated basil plants was maintained under dark conditions and another set of inoculated basil plants were placed under the exposure of red light. Fresh sporangia were harvested from the basil plants under dark conditions and under red light. A total of fifty sporangia were examined under a microscope with 40x magnification lens. The sporangia produced under dark conditions were brown in color and had oval shaped sporangia, whereas sporangia produced under red illumination were white in color and had round shaped sporangia. The change in morphology of the sporangia is an indication of a possible direct effect of red lights on *P. belbahrii* sporangia production. These results warrant further investigations to determine the mechanisms underlying changes in biology of *P. belbahrii* sporangia affected by red light.

## Bean common mosaic virus isolate overcomes the *bc-3* resistance allele in common bean: Virus genetic determinants defining a novel pathogenicity group

X. FENG (1), J. Myers (2), A. Karasev (1)

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

*Bean common mosaic virus* (BCMV) exists as a complex of strains that can be distinguished biologically, on a set of bean differentials, into pathogenicity groups (PG) numbered I to VII. Resistance against BCMV in *Phaseolus vulgaris* is governed by six recessive resistance alleles at four loci. One of these alleles, *bc-3*, is able to protect *P. vulgaris* against all BCMV strains and against other potyviruses; *bc-3* was identified as the *eIF4E* allele carrying mutated eukaryotic translation initiation factor gene. We characterized a novel BCMV isolate, 1755a, that was able to overcome *bc-2* and *bc-3* alleles in common bean. Hence, it displayed a novel pattern of interactions with resistance genes in *P. vulgaris*, and was assigned to a new pathogroup, PG-VIII. The IVT7214 cultivar supporting the replication of BCMV-1755a was found to have the intact homozygous *bc-3* CAPS marker. The complete genome of the 1755a isolate was sequenced and found to be a recombinant between the BCMV strain NL1 (PG-I) and an unknown BCMV strain. Analysis of the recombination patterns in the genomes of NL1 and US1 (PG-I), NY15P (PG-V), US10 and RU1-OR (PG-VII), and 1755a (PG-VIII),
indicated that P1/HC-Pro cistrons of BCMV strains may interact with most resistance genes. This is the first report of a BCMV isolate able to overcome the *bc-3* resistance in common bean.

### Determination and characterization of a symptom determinant of *Grapevine fanleaf virus*

L. OSTERBAAN (1), L. Osterbaan (1), M. Fuchs (1)

(1) Section of Plant Pathology and Plant-Microbe Biology, Cornell University, New York State Agricultural Experiment Station, U.S.A.

*Grapevine fanleaf virus* (GFLV) -the causative agent of fanleaf degeneration- is one of the most devastating viral diseases of grapevines. GFLV is a bipartite (+) sense RNA nepovirus and is transmitted by the ectoparasitic nematode *Xiphinema index*. The five proteins encoded by RNA1 are associated with replication and protein maturation, while the three proteins encoded by RNA2 are involved in replication, cell-to-cell movement, and genome encapsidation. Infected vines exhibit a variety of symptoms, including shortened internodes, fan-like (deeply lobed) leaves, mosaic, and vein yellowing. Infected vines have decreased fruit yield and quality, and also a shortened productive lifespan. Little is known about the host-pathogen interactions leading to symptom development. Recent work with chimeras of symptomatic and asymptomatic strains of GFLV (strains GHu and F13, respectively) in the plant model species *Nicotiana benthamiana* has shown that symptom development in this host species is dependent on a 408-nucleotide sequence at the 3' end of RNA1 of GFLV-GHu. A reverse genetics approach was used to characterize the symptom determinant by using agroinoculation to establish infection with a series of recombinant GFLV genomes in planta and thus determine the minimal GFLV-GHu sequence necessary for symptom development in *N. benthamiana*. A better understanding of GFLV's biology will inform the mechanism by which the virus causes symptoms in its hosts.

### Understanding superinfection exclusion by complex populations of *Citrus tristeza virus*

S. KANG (1), M. Bergua (1), S. Folimonova (1)

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

Superinfection exclusion (SIE) is a phenomenon in which a primary viral infection restricts a secondary infection with the same or closely related virus. For plant viruses, SIE, referred to as cross-protection, was implemented as an agricultural practice to protect crops against aggressive viruses by pre-immunizing plants with mild virus isolates. Recently we showed that SIE by *Citrus tristeza virus* (CTV), an economically important virus of citrus, occurs only between isolates of the same strain, but not between isolates of different strains. This work, however, was done using "pure" isolates of CTV, each containing a single virus strain, while most citrus trees in the field harbor complex populations made up of different CTV strains. The objective of this study was to assess how SIE works in plants simultaneously pre-infected with several CTV strains. Co-inoculation of citrus trees with variants of T36, T68 and/or T30 strains demonstrated that multi-strain populations equilibrate soon after establishment of infection, show uniform intra-host distribution and maintain the established equilibrium over time. SIE experiments showed that exclusion of a secondary infection by a CTV variant was triggered by the presence of another variant of the same strain in the primary population, which was in the agreement with our previous observations. The same rule appeared to be in effect when SIE by mixed virus populations was tested in a series of different citrus varieties.

### Potato virus Y strains co-localize and compete in single epidermal leaf cells

S. MONDAL (1), S. Mondal (1), S. Gray (1), M. Ghanim (2)

(1) Cornell University, U.S.A.; (2) Volcani Center, Israel

In recent years, recombinant strains of *Potato virus Y* (PVY) have become predominant in U.S. potato crops; whereas the parental PVY strains have been disappearing. Often multiple PVY strains are found in the same field and occasionally within the same plant, but little is known about how they interact or compete within the plant. Here, we examine the spatial and temporal dynamics of PVY strain infection at the cellular and tissue levels in different hosts. Immunofluorescence and confocal microscopy was used to localize mixtures of PVY strains (PVY<sup>O</sup> and PVY<sup>NTN</sup>) in epidermal leaf cells of 'Samsun NN' tobacco or 'Red Maria' potato. We observed that both strains systemically infect tobacco and are co-localized in cells of all leaves; however their relative prevalence changes over time. Early in infection when mosaic symptoms are observed, PVY<sup>O</sup> predominates the infection although PVY<sup>NTN</sup> is present in some cells. As infection progresses and veinal necrosis is observed, PVY<sup>NTN</sup> becomes the prevalent virus in those tissues outcompeting PVY<sup>O</sup>. Co-localization of PVY<sup>O</sup> and PVY<sup>NTN</sup> was also found in epidermal cells of potato leaves with almost all cells being infected with both viruses. Co-localization of virus strains in single cells is not a widely used approach for understanding interactions among them. The apparent non-antagonistic interaction among PVY strains could explain the multitude of emerging recombinant PVY strains discovered in potato in recent years.

### Ethylene inhibition can prevent aphid attraction and Potato Virus Y spread

A. BAK (1), S. Chen (2), T. Antichera (2), L. Perilla (2), C. Casteel (2)

(1) UCDavis, U.S.A.; (2) UCDavis, U.S.A.

Vector-borne pathogens influence host characteristics relevant to host-vector contact, increasing pathogen transmission and survival. Potato Virus Y (PVY) is vector-borne and considered one of the most damaging viruses for potato worldwide. Previous studies have demonstrated aphid vectors have higher fecundity and preferentially settle on PVY-infected potatoes compared to controls. Here we demonstrate PVY infection increases ethylene production and we show that ethylene production is required for increased vector attraction to infected plants. Further, we demonstrate pathogen spread can be prevented by temporarily inhibiting ethylene signaling. Virus-induced changes in ethylene responses may mediate vector-plant interactions more broadly and thus represent a conserved mechanism for increasing transmission by insect vectors.

### Precision diagnostics and next-generation decision support systems

N. BOONHAM (1), J. Turner (2), I. Brittain (2), D. McCluskey (3), R. Kaye (3), D. Clarke (4), N. Morant (4), D. Langton (5), F. Salinari (5), W. Charlton (6), M. Andreou (7)

(1) Fera, York, United Kingdom; (2) Fera, United Kingdom; (3) University of Hertfordshire, United Kingdom; (4) Gensys Biotech Ltd, United Kingdom; (5) Agrii, United Kingdom; (6) Bayer Crop Sciences, United Kingdom; (7) Optisense Ltd, United Kingdom

The UK wheat crop is valued at £2 billion p.a., approximately 12% of which is lost to disease. Septoria leaf blotch (*Zymoseptoria tritici*) is the most prevalent and fungicide sprays estimated at £82million p.a. (GFK-Kynetec 2013) are used for control. Resistance is a significant problem, caused by continued overuse of chemicals with similar modes of action. Better control requires more precision in terms of spray timing as well as more careful selection of fungicides. Linking this information with risk prediction by use of disease forecasting systems is also critical. We have been developing an in-field, self-reporting, automated spore detection system, to monitor ingress of inoculum into fields. Based on cyclone air sampling technology linked to isothermal DNA amplification (LAMP) the system will sample and test air within crops, transferring data on inoculum movement wirelessly to the laboratory. In addition to inoculum, identifying resistant genotypes of a pathogen is critical to enable effective spray decisions to be made. Work has started on the development of LAMP assays to discriminate the SNPs (single nucleotide polymorphisms) responsible for azole resistance in the CYP51 gene of Septoria. The assays will be deployed on the handheld Genie III instrument, enabling decisions to be made rapidly in the field. Ultimately the two developments will converge, telling us not just when we should spray crops but also what active chemistry will be most effective.

### Next generation sequencing evaluation for detection of potential threats or emerging forest pest, entry point from spore and insect traps

G. BILODEAU (1), E. Tremblay (2), J. Béribé (3)

(1) Canadian Food Inspection Agency, Canada; (2) Canadian Food Inspection Agency, Canada; (3) Natural Resources Canada, Canada
International trade promotes the spread of exotic plant pathogens (fungi, oomycetes) that can act as entry points. Their spores that spread by wind, rain and through vectors (insects) can cause the introduction of exotic diseases responsible for the devastation of various plant species. Since it is difficult to trap and conduct investigations to these organisms, new technologies such as qPCR and metagenomics therefore can now be an option to detect without having to grow them. The DNA is extracted for metagenomics of spore trapping filters and insect traps conservation liquid collected in 2013-2014 and then sequenced with the Ion Torrent platform using fusion primers which we designed in order to three multiplex genic regions (350-400bp amplicons from ribosomal DNA region ITS1 for fungi and oomycetes and the mitochondrial DNA region ATP9-NAD9 for *Phytophthora* sp) and then deciphered sequences. Bioinformatic analyzes pipelines for analysis of millions of sequences are also made and compared with qPCR testing for multiple targets. Comparing the Next Generation Sequencing consistent qPCR for the detection of forest pathogens species and allowed to find several species related targets of *Phytophthora* and related Fungi. Based on the results obtained, it was possible to identify potential sources of entry, get access to contents abundance, biodiversity and phylogeny in order and so accelerate identification process of current risk to these species for biosurveillance.

## An inexpensive visual PCR method for field detection of *Candidatus* Liberibacter asiaticus associated with citrus huanglongbing

M. KEREMANE (1), C. Ramadugu (2), D. Hall (3)

(1) USDA ARS, U.S.A.; (2) University of California Riverside, U.S.A.; (3) US Horticultural Research Laboratory, U.S.A.

Huanglongbing (HLB) is threatening citrus cultivation in California and Texas. Testing for Liberibacter (Las, the HLB pathogen) by qPCR is labor-intensive and requires expensive instrumentation. The key to HLB management is large scale testing by many interested personnel. Development of on-site testing methods will increase the chance of pathogen detection by growers, homeowners and extension agents and enables timely implementation of disease management strategies. We describe an easy method to test psyllids (HLB vectors) for the presence of the Las pathogen. The initial equipment needed includes a heating block, table top centrifuge, tubes and reaction reagents. The cost for all of the equipment needed for testing by this method will be under $300. Psyllids are captured, stored in ethanol, air-dried, and DNA extracted by heating at 80°C in extraction buffer. Pathogen DNA is amplified using a loop-mediated amplification method at ambient temperatures (65°C) for 30 minutes. Samples positive for Las will appear yellow after the reaction and negative samples will remain pink. The whole reaction can be performed within an hour in a farmhouse by non-scientific personnel with very little training. Availability of methods for large scale testing will facilitate better disease management.

## Comparison of two deep sequencing methods for the detection of viruses in sweet potato

R. LI (1), M. Cao (2), P. Lan (3), J. Abad (4), C. Zhou (5)

(1) USDA-ARS, U.S.A.; (2) Southwest University of China, China; (3) Yunnan Agricultural University, China; (4) USDA-APHIS, U.S.A.; (5) Southwest University of China, China

Current methods used to detect viruses from imported accessions in plant germplasm quarantine programs face multiple challenges due to many pathogen targets, mixed infections and high genetic diversity of some pathogens. In this study, deep sequencing of small interfering RNAs (siRNAs) and RNAs (HiSeq) were compared using a sweet potato plant infected with five RNA viruses [*Sweet potato chlorotic stunt virus*, *Sweet potato feathery mottle virus*, *Sweet potato virus C*, *Sweet potato virus G*, *Sweet potato virus 2*] and two DNA viruses [*Sweet potato leaf curl virus* and *Sweet potato symptomless 1*]. Several bioinformatic approaches were used to resolve the viral genes and detect viruses and their quasispecies. Direct mapping of both read types against a crop-specific virus database resulted in rapid detection of the seven known viruses, whereas the blast search of assembled contigs from the HiSeq reads had low call on the DNA viruses. However, the genomic sequences of the viruses, especially the RNA viruses, were characterized to a high level of detail when analyzing the contigs from the HiSeq reads, enabling the detection of quasispecies. Deep sequencing is a powerful technique that can facilitate the detection and characterization of numerous pathogens in a single test, but both the sequencing strategy and data analysis aspects require careful consideration.

## Fluorogenic Detection of Plant Viruses by Helicase Dependent Amplification with Self-Quenched Primers

S. MOLINA CÁRDENAS (1), A. Salazar Aguirre (1), F. Ochoa-Corona (1), J. Olson (1)

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

Thermophilic helicase dependent amplification (tHDA) is an isothermal DNA amplification that does not require temperature cycling. A study of primers sharing similar thermodynamics, but different GC content in their targeted amplification products, was made to assess fluorescence using self-quenched primers (SqP) reacting in tHDA standard temperature and chemistry. Model viruses used were *Rose rosette virus* (RRV), *High plains wheat mosaic virus* (HPWMV) formerly High plains virus (HPV) and *Hosta virus X* (HVX). RT-PCR was used as the reference method. Temperature gradients (60 to 70°C) were performed to each set of primers. tHDA reactions with SqP were optimized assessing combinations of two primer concentrations (0.15 and 0.2 µM) with two MgSO₄ concentrations (4 and 4.5 mM). The sensitivity of each primer set was determined by quantitative tHDA (qtHDA) in real time. Subsequently the products amplified by qtHDA with SqP were visualized in 2% agarose gel electrophoresis. Detection limits using a plasmid DNA carrying the target sequences for RRV and HPWMV are 0.0001 ng. There was no amplification of the HVX targeted region due to its putative high GC content. This study explored primer design criteria for tHDA with SqP for sensitive detection of plant viruses. The use of SqP reduces the cost of qPCR and qtHDA and has acceptable levels of sensitivity. tHDA with SqP bring new possibilities for field deployment primer design in biosecurity and microbial forensics.

## Long-term rotation history and previous crop effects on corn seedling health

M. BENITEZ (1), S. Osborne (2), M. Lehman (2)

(1) USDA, North Central Agricultural Research Laboratory, U.S.A.; (2) USDA, North Central Agricultural Research Laboratory, U.S.A.

Diverse rotations provide benefits to agroecosystems through changes in the soil environment. A long term experiment was established to study four different four-year rotation sequences in which the crop phase prior to corn was sampled. Soils from rotations ending with soybean, sunflower, corn and pea were tested for effects on subsequent corn in the greenhouse. Corn was grown in a soil mix containing 12% of rotation soil either infested with corn root worms (CRW) or inoculated with *Fusarium graminearum*. Seedlings were scored for root damage and shoot vigor, rhizosphere samples collected for microbiome analyses and remaining roots scanned for root measurements. Seedlings grown in soils from rotations ending with pea and sunflower had greater vigor regardless of CRW infestation. Seedlings grown in non-inoculated soils from pea rotation had shorter roots but greater root volume and diameter. Differences in germination time and vigor were stronger between *Fusarium*-inoculated and non-inoculated soils originating from pea rotations, compared to other rotations. The response to the rotation ending in pea is consistent with field observations where corn grown after pea requires less fertilizer. Analyses of the rhizosphere microbiome and seedling nutrient content, together with soil physico-chemical characteristics will provide insight on the mechanisms through which corn respond positively to rotation sequences incorporating pea.

## Evidence for sexual reproduction in *Fusarium effusum*

N. CHARLTON (1), C. Mattupalli (2), B. Wood (3), C. Bock (3), C. Young (2)

(1) The Samuel Roberts Noble Foundation, U.S.A.; (2) The Samuel Roberts Noble Foundation, U.S.A.; (3) USDA-ARS-SEFTNRL, U.S.A.

*Fusarium effusum* is the causal agent of pecan scab, the most prevalent and often catastrophic disease of pecan in the southeastern USA. Despite earlier efforts to determine a sexual stage, reproduction in *F. effusum* has been observed only by asexually produced conidia. However, the degree and distribution of genetic diversity observed within and among populations of *F. effusum* are typical of a sexually reproducing fungus. The mating type locus was identified in a draft genome of *F. effusum* and was amplified and sequenced in 14 isolates collected from different geographic locations and
cultivars, revealing that 50% of the samples contained the *mtAA* idiomorph and are considered mating type A (*MTA*). The remaining samples contained *mtBA* and are mating type B (*MTB*). A multiplex PCR screen was developed to amplify *mtAA*, *mtBA* and *tubB*, and used to screen 1203 isolates of *F. effusum* from 13 pecan populations across the southeastern USA. Analysis of this collection revealed the frequency of the mating type idiomorphs is in a 1:1 equilibrium. Select isolates were crossed in pairwise combinations on oatmeal agar and after six months, organs typical of immature sexual fruiting bodies were observed, indicating existence of a putative sexual stage. Identification of a sexual stage will provide improved understanding of the disease epidemiology and pathogen population genetics, and potentially provide insight into novel approaches to manage this damaging disease.

## Ecological Impact of Signaling Chemicals on *Phytophthora erythroseptica*

H. JIANG (1), J. Hao (1)

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

*Phytophthora erythroseptica* causes pink rot of potato, mostly with zoospores as major primary inocula. Zoosporic germination and infection of host (potato) are regulated by signaling molecules with quorum sensing mechanism. This study was to determine if the signals were species specific and if they were impacted by the environment. Exudates were obtained from high concentrations of fungi, bacteria, and oomycetes, as well as soil and roots of potato varieties. The exudates were used to treat zoospores of *P. erythroseptica*. Results showed that several soil microorganisms, including *P. capsici*, *P. cactorum*, *Pythium irregulare*, *Serratia* sp., *Delftia* sp., and *Microbacterium* sp. induced zoosporic germination of *P. erythroseptica*; *Pseudomonas* spp. and *Bacillus amyloliquefaciens* inhibited zoosporic germination; exudates of *Dickeya dadantii* and *Pectobacterium carotovorum* had no effect on zoosporic germination. Root exudate from the pink-rot-sensitive variety, 'Dark Red Norland,' induced zoosporic germination, while tolerant variety 'Snowden' did not. These results indicate that the zoosporic behavior is not only self-regulated, but also affected by other environmental organisms, such as host plants and soil microorganisms through signaling molecules. Whether same signals are shared among organisms is being investigated. This result may improve our understanding of the initiation and development of potato pink rot and contribute to breeding resistant potato cultivars.

## Model for the geographic origin and spread of the globally distributed pathogen *Phytophthora palmivora*

J. Wang (1), E. Goss (1), M. Coffey (1)

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

The genetic structure and diversity of plant pathogens are shaped by their evolutionary history, including coevolutionary interactions with hosts, geographic spread, and long-distance migration events, often mediated by humans. In turn, diversity and dispersal of present populations impact the risk of disease epidemics and strategies for controlling disease. *Phytophthora palmivora* is a globally distributed oomycete that infects a broad range of cash crops and fruit trees in the tropics and subtropics, including cocoa and palms. The center of diversity of *P. palmivora* is in Southeast Asia, but it is an important and widespread pathogen in South America. Our multilocus sequence analysis showed that the centers of origin of the pathogen are located in the Philippines and Indonesia. We then tested alternative models for the regional and global movement of *P. palmivora*. We found genetic variation consistent with historical movement among Pacific Islands, likely associated with coconut. We also found evidence of a bridgehead effect in South America, in which the colonization of South America and host shift to cocoa led to further global dispersal, including gene flow back to Southeast Asia. We propose that the extensive genetic diversity in *P. palmivora* in Southeast Asia is the result of a complex history, including long-term co-evolution with native hosts, geographic isolation with migration, and re-introduction of genotypes from South America.

## The in planta regulon of the major *Ralstonia solanacearum* virulence regulator PhcA

D. KHOKHANI (1), T. Tran Minh (1), C. Allen (1)

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

PhcA, an important quorum sensing responsive virulence regulator of the bacterial wilt pathogen *Ralstonia solanacearum*. PhcA affects expression of virulence factors like extracellular polysaccharide, endoglucanase, and motility. However, the full extent of the regulon is unknown. We used RNAseq to test the hypothesis that the PhcA system enables Rs to transition from saprophytic to parasitic mode. An impressive 634 genes were differentially regulated at early stage of tomato wilt disease development in ΔphcA compared to the wild-type strain GMI1000, with false discovery rate of less than 0.005. Both, RNA-seq data and validation experiments indicated that multiple anion and sugar transporters are expressed at higher levels in the ΔphcA, suggesting that the pathogen uses this regulator to improve its survival at low cell densities. Increased expression of hemagglutinins and other adhesins in ΔphcA suggest that at low population densities *R. solanacearum* cells may adhere more to plant surfaces and to each other. Scanning electron microscopy revealed that ΔphcA cells form thick mats in tomato xylem vessels. These were strikingly different from the smaller, looser in planta aggregates of wild-type cells. Further, ΔphcA was largely restricted near the point of infection whereas wild-type colonized tissues beyond the infection site. Taken together, the study identified new targets of PhcA regulation and shed more light on their biological roles in the disease cycle.

## Determination of CsrA/RsmA regulon in *Xanthomonas citri* subsp. *citri*

M. ANDRADE (1)

(1) Dept. Microbiology and Cell Science, CREC- University of Florida, U.S.A.

In bacteria, it is poorly understood how post-transcriptional regulation mediated by RNA-binding proteins contributes to adaptation to environment changes during host infection. RsmA (Regulator of secondary metabolism) is a small RNA-binding protein that plays an important role in regulation of pathogenicity, motility, biofilm formation, endoglucanases and exopolysaccharide production in *Xanthomonas citri* subsp. *citri* (Xcc). In previous work, we showed that RsmA contributes to the pathogenicity by activating expression of genes encoding the type 3 secretion system (T3SS) and effectors in Xcc. However, the mechanistic understanding of Xcc RsmA in regulating motility, biofilm formation and production of exopolysaccharide and endoglucanases remains unclear. We have performed crosslinking and affinity purification followed by RNAseq analysis in order to determine the RsmA targets in Xcc cells in both rich medium and a T3SS-inducing medium. Our data revealed hundreds of putative binding mRNA targets in Xcc. Functional classification (Gene Ontology) of RsmA mRNA targets suggests Xcc RsmA is a global RNA-binding regulator that can positively and negatively affect the expression of genes in *Xanthomonas*.

## Construction of phosphodiesterase mutants in *Erwinia amylovora* to evaluate the role of cyclic di-GMP in virulence regulation

R. KHARADI (1), R. Kharadi (1), L. Castiblanco (1), G. Sundin (1)

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

The second messenger molecule cyclic-di-GMP (c-di-GMP) is a key regulator of biofilm formation, motility and virulence in bacteria. Diguanylate cyclase (DGC) enzymes function in the synthesis of c-di-GMP and phosphodiesterase (PDE) enzymes are involved in the degradation of c-di-GMP. We have previously studied the role of DGCs in regulating virulence traits in the fire blight pathogen *Erwinia amylovora*. Here we demonstrate that all three predicted PDE genes (*edp* genes, for *Erwinia* diguanylate phosphodiesterase), *edpA*, *edpB* and *edpC* negatively regulate amylovoran production and biofilm formation. This was correlated with a significant increase in the volume of ooze produced and the amylovoran content of ooze in an immature-pear infection model using the single-gene deletion mutants *DedpA*, *DedpB*, and *DedpC*. We also observed a significant decrease in motility, virulence in immature pears, and systemic infection levels in apple shoots in the triple-gene deletion mutant *DedpA/edpB/edpC*. Gene expression levels of *dspE* and *hrpL*, both indicators of type III secretion in *E. amylovora*, were significantly reduced in *DedpA/edpB/edpC* compared to the wild type. Thus, c-di-GMP seems to be primarily involved in the positive regulation of amylovoran production and biofilm formation; in addition, increased levels of c-di-GMP also negatively regulate motility and type III secretion during the pathogenesis of *E. amylovora*.
# Characterization of RpfB-dependent DSF-Family Quorum Sensing Signal Turnover System in the Phytopathogen Xanthomonas

L. ZHOU (1), X. Wang (1), Y. He (1)

(1) Shanghai Jiao Tong University, China

Molecules of the diffusible signal factor (DSF)-family are a class of quorum sensing (QS) signals used by the phytopathogens Xanthomonas. RpfB has recently been reported as a fatty acyl-CoA ligase (FCL) that activates a wide range of fatty acids to their CoA esters in vitro. Through genetic and biochemistry analysis, we found that RpfB represents a naturally occurring DSF-family signal turnover system in X. campestris pv. campestris (Xcc) and X. oryzae pv. oryzae (Xoo). RpfB effectively turns over DSF-family signals DSF and BDSF in vivo. RpfB FCL enzymatic activity is required for DSF and BDSF turnover in Xcc and Xoo. The expression of rpfB is growth phase-dependent and its expression is significantly enhanced when Xanthomonas cells enter the stationary phase. DSF regulates rpfB expression in a concentration-dependent manner. rpfB expression is also negatively regulated by the DSF signaling components RpfC, RpfG, and Clp. Although most of our findings have suggested that the DSF-family signal turnover systems are generally consistent in Xcc and Xoo, we also identified some discrepancies in bacterial pathogenesis and virulence factor production between the rpfB mutants of Xcc and Xoo. Therefore, our findings strongly indicated the complexity of the RpfB-dependent DSF-family signal turnover system, which is likely fine-tuned to facilitate infection process in different hosts (rice versus crucifers).

# Integration of plant host-derived signals to coordinate developmental processes involved in Pantoea stewartii subsp. stewartii biofilm maturation

M. ROPER (1), M. Roper (1)

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

Participation in complex, multicellular cooperative communities, known as biofilms, is a universal attribute of bacteria. Biofilm formation is now a major tenet of microbiology, yet we know little information about what environmental and physical cues drive biofilm formation in a natural context, such as during bacterial infection processes. Biofilms are encased in an extracellular matrix consisting primarily of exopolysaccharide (EPS), which holds these biofilms together, providing robustness to the architecture and making them difficult to clear from the host. The focus of this project is on the xylem-dwelling bacterium, Pantoea stewartii subsp. stewartii (Pss)/sweet corn pathosystem. We have placed our attention on the multi-component Rcs signal transduction system, an environmental sensing system found in Pss and other enteric bacteria. This system controls genes related to biofilm formation, namely EPS matrix production. We demonstrate that host plant-derived reactive oxygen species (ROS) are one environmental signal that modulates the Rcs system and this stimulation is dependent on the redox sensing transcription factor, OxyR. In addition, we demonstrate a link between a novel RTX-like, cytolytic protein in lysing host plant cells, releasing ROS and stimulating the Rcs system and subsequent EPS production, thus aiding in transition between the apoplastic and xylem phases of Stewart's wilt disease.

# Epidemiology and Population Biology of Grape Downy Mildew (Plasmopara viticola) in Georgia

C. HONG (1), P. Brannen (1), H. Scherm (1)

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

The epidemiology of *Plasmopara viticola* has been studied extensively in temperate and Mediterranean climates, but limited information is available for the southeastern U.S. where hot and humid conditions may affect survival, disease progression, and population structure of the pathogen. Downy mildew epidemic progress was monitored in 2015 in a North Georgia *Vitis vinifera* vineyard on cultivars Merlot and Chardonnay. Disease onset was 2 months later than predicted by a mechanistic model for oospore germination and primary infection, highlighting knowledge gaps relative to epidemiological processes in Georgia conditions. Since timing of disease onset may be related to the mode of pathogen overwintering, pairings among single-sporangium isolates were conducted to determine the potential for sexual reproduction. Oospores were observed in about half of the pairings, indicating that both mating types are present. To better understand the relative contribution of sexual and asexual reproduction and to determine the population structure of the pathogen, isolates of *P. viticola* were collected from 19 grape varieties in 10 vineyards throughout Georgia. Initial genotyping of 96 isolates identified only one of the previously described five cryptic species of *P. viticola*, *clade aestivalis*. This study confirmed sexual reproduction, revealed preliminary pathogen population structure, and identified knowledge gaps regarding primary infection by *P. viticola* in Georgia vineyards.

# Changes in Plasmopara obducens population structure corresponded with the emergence and rapid spread of the impatiens downy mildew epidemics

C. SALGADO-SALAZAR (1), Y. Rivera (1)

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

*Plasmopara obducens* is the newly emergent oomycete responsible for impatiens downy mildew disease (IDM) on cultivated *Impatiens walleriana*. Since IDM outbreaks began in 2004, the disease has reduced the $187 million annual production of impatiens in the U.S. by 40%. In this study, we evaluated if there was a change in *P. obducens* population structure associated with the U.S. epidemic. Over 900 pre- and post-epidemic *P. obducens* samples were genotyped using 20 polymorphic microsatellite markers developed from a *de novo* genome assembly. Population structure analysis identified five populations (P1-P5). Prior to IDM epidemics, P1 dominated, existing as pure P1 or admixed with the other four genotypes (~50%) on North American native *Impatiens* species. The first epidemic IDM specimen collected on *I. walleriana* in 2004 from Tennessee showed an almost pure P1 genotype. Approximately 50% of the post-epidemic samples showed admixed genotypes, but admixture between P1 and the other four genotypes was uncommon. Genetic diversity was high for the four most abundant populations P2-P5 responsible for the IDM epidemics (0.56-0.78), but lower for P1 (0.37). These data showed that the recent emergence of IDM in the U.S. corresponded with several major shifts in *P. obducens* populations, with endemic P1 genotypes supplanted by novel genotypes. The high level of diversity and genetic structure in *P. obducens* populations may present difficulties for durable disease control.

# Emergent rudbeckia downy mildew epidemics caused by Plasmopara halstedii are genetically distinct from the pathogen populations infecting sunflower

Y. RIVERA (1), C. Salgado-Salazar (1), T. Gulya (2), J. Crouch (3)

(1) USDA-ARS Systematic Mycology and Microbiology Laboratory and Rutgers University, Beltsville, MD, U.S.A.; (2) USDA ARS Sunflower and Plant Biology Research Unit, Fargo, ND, U.S.A.; (3) USDA-ARS Systematic Mycology and Microbiology Laboratory, Beltsville, MD, U.S.A.

The oomycete *Plasmopara halstedii* is regarded as the causal agent of downy mildew disease on 35 Asteraceae genera. The pathogen is also a significant problem of sunflower (*Helianthus annuus*) worldwide and more recently, a destructive pathogen of ornamental *Rudbeckia fulgida* (rudbeckia) in the U.S.A. As with other recent downy mildew epidemics on specialty crops, little is known about the pathogen causing modern outbreaks on rudbeckia. To determine if modern epidemics on rudbeckia are caused by the same *P. halstedii* genotypes as those historically found on native rudbeckia or to those causing disease on sunflower, we performed population genetic and phylogenetic analyses. Fifteen microsatellite markers were designed from a draft genome assembly generated from a *P. halstedii* isolate on sunflower. Phylogenetic and microsatellite analyses of 232 samples collected between 1883-2014 revealed two main groups corresponding to host origin. Isolates infecting sunflower hosts were further separated into eight subgroups with no obvious pattern by year, physiological race or geographical origin. Isolates infecting rudbeckia hosts were further separated into three subgroups, one corresponding to historical pre-epidemic specimens and two modern subgroups. Our results indicate that the emergent populations of *P. halstedii* infecting rudbeckia are a different species from those infecting sunflower and genetically distinct from pre-epidemic populations infecting native rudbeckia.
Pseudoperonospora cubensis on commercial and non-commercial cucurbits in North Carolina: Population structure determined by simple sequence repeats

E. WALLACE (1), L. Quesada-Ocampo (2)

(1) NCSU, U.S.A.; (2) NCSU, U.S.A.

The cucurbit downy mildew pathogen, *Pseudoperonospora cubensis*, is a major limiting factor in cucumber production in the United States (US). Each year, *P. cubensis* causes foliar destruction on cucumber, but it also infects approximately 60 host species in the Cucurbitaceae family. All major commercial cucurbits are hosts in the US. Many non-commercial hosts are grown on a small scale or occur as weeds and their contributions to the yearly *P. cubensis* epidemic are understudied. North Carolina (NC) has diverse cucurbit production and previous studies have found that NC has relatively high genetic diversity of *P. cubensis*. In this study, Simple Sequence Repeats (SSRs) were identified in silico from the *P. cubensis* predicted transcriptome. A subset of the identified markers were screened and evaluated for reliability and polymorphism. Eleven markers were selected and applied to *P. cubensis* isolates collected from six commercial and three non-commercial cucurbits across four time points and three regions in NC. Population analyses revealed that the greatest genetic differentiation occurs when isolates are grouped by host species more so than when grouped by location or time. These findings show host species are playing the most significant role in *P. cubensis* diversification in NC. The results suggest non-commercial cucurbits could be contributing to *P. cubensis* inoculum for certain hosts more so than others, thus influencing the population dynamics of the pathogen.

Species diversity and population structure of *Pythium* and *Globisporangium* in Long Island, NY ornamental greenhouses in 2014

M. PROANO (1), C. Ayala (2), A. Chiriboga (2), M. Daughtrey (3), C. Garzon (4), S. Marek (1), H. Melouk (1), S. Marek (4)

(1) Oklahoma State University, Stillwater, OK, U.S.A.; (2) Universidad de las Fuerzas Armadas ESPE, Sangolquí, Ecuador, Ecuador; (3) Cornell University, Long Island Horticultural Research & Extension Center, Riverhead, NY, U.S.A.; (4) Oklahoma State University, U.S.A.

*Globisporangium* and *Pythium* are plant pathogens that cause root rots, blackleg of cuttings, and damping off of various crops. Growth of the nursery and floriculture production industries, one of the fastest growing agricultural sectors in the United States, has contributed to the increase of the economic impact of ornamental plant diseases. Seasonal assessment of the diversity of species causing Pythium diseases on three ornamental greenhouses from Long Island, NY is providing information about the temporal dynamics of *Pythium* and *Globisporangium* species in these agricultural systems. DNA sequences of the ITS region were obtained to confirm the identity of 207 isolates collected seasonally from three ornamental operations in 2014. As in previous years, *G. irregulare* s.l. population. PCoA of ten SSR loci defined four groups based on genetic distances. AMOVA revealed significant genetic differentiation between greenhouse populations and between genetic lineages. The main lineage included most of the isolates present in two greenhouses, which suggest a potential common source of inoculum for those facilities.

ADEPIDYN™ fungicide: Cross resistance patterns in *Alternaria solani*

G. OLAYA (1), R. Linley (2), K. Edlebeck (2), T. Harp (3)

(1) Syngenta, U.S.A.; (2) Syngenta, U.S.A.; (3) Syngenta, U.S.A.

ADEPIDYN™ is the first member of a new chemical group among the succinate dehydrogenase inhibitor fungicides (SDHI, FRAC Group 7), the N-methoxy-(phenyl-ethyl)-pyrazole-carboxamides. The common name for ADEPIDYN™ is pydiflumetofen. ADEPIDYN™ has high binding properties to the complex II enzymes that provide a strong fungicidal potency combined with the control of a wide range of plant pathogen species. A variety of mutations are associated with resistance to SDHI fungicides. Cross resistance patterns across SDHI fungicides are complex due to the fact that some mutations confer high levels or resistance and others do not and mutations could lead to different sensitivity shifts in different SDHIs. In *Alternaria solani* isolates, the *in vitro* sensitivity revealed incomplete cross resistance patterns between ADEPIDYN™ and boscalid, pentiopyrad, SOLATENOL™ and fluopyram. ADEPIDYN™ exhibited the highest intrinsic activity among these fungicides and across isolates with or without mutations in the sdh subunits (SdhB-H277Y, SdhB-H277R, SdhC-H134R, SdhD-D123E abd SdhD-H133R). ADEPIDYN™ was able to control early blight disease development on inoculation studies done with *A. solani* isolates sensitive and with different levels of resistance to boscalid and Sdh mutations. Fluopyram was also able to control the disease using higher fungicide rates and disease control with SOLATENOL™, pentiopyrad and boscalid varied according to the presence or absence of Sdh mutations.

Fungicide resistance profiles of unique and clonal genotypes of gummy stem blight fungi from the southeastern United States

H. LI (1), K. Stevenson (2), M. Brewer (1)

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

Gummy stem blight (GSB) is a devastating disease of cucurbits that is usually managed with fungicide applications. However, GSB fungi have rapidly evolved resistance to multiple classes of fungicides. To better understand the evolution and persistence of fungicide resistance in field populations, we studied resistance profiles associated with unique and clonal genotypes of GSB fungi. Nineteen *Stagonosporopsis caricae* isolates and 120 *S. citrulli* isolates collected from watermelon fields in the southeastern U.S. were genotyped with 16 SSR markers and a *cytb* marker for azoxystrobin (QoI) resistance, and phenotyped for sensitivity to tebuconazole (DMI), boscalid (SDHI), and fluopyram (SDHI). *Cyp51* and *sdhB* genes of select isolates with varying sensitivity to DMI and SDHI fungicides, respectively, were sequenced. All *S. caricae* isolates were resistant to tebuconazole and azoxystrobin, and sensitive to boscalid and fluopyram. All *S. citrulli* isolates were sensitive to tebuconazole, sensitive to fluopyram with one exception, and resistant to azoxystrobin with two exceptions. Phenotypic differences in response to boscalid were detected among *S. citrulli* isolates, but the phenotype was not associated with clonal genotype, suggesting multiple evolution events. This study provides critical information for effectively managing both species of GSB fungi present in the southeastern U.S., while providing novel insights on the evolution of resistance in plant-pathogenic fungi.

Survey for Quinone Outside Inhibitor Resistant *Cercospora sojina* in Virginia Soybeans

T. ZHOU (1) H. Mehl (1)

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

Quinone outside inhibitor (QoI) fungicides control a wide spectrum of fungal diseases, but this class of fungicides has a high risk of developing resistance. QoI fungicides are used in the management of frogeye leaf spot (FLS) caused by *Cercospora sojina*. In 2014, FLS isolates from four fields in VA were tested, and QoI resistance was confirmed from two fields. In 2015, a statewide survey was conducted to estimate the prevalence of QoI resistant FLS in VA. Leaflet samples with FLS symptoms were collected from 55 soybean fields throughout the VA soybean growing region. Ten leaflets were randomly selected per sample and conidia were isolated from one lesion per leaflet. A total of 116 isolates were successfully cultured from 25 locations, and DNA was extracted from monoconidial cultures. A PCR-based assay was used to screen isolates for the G143A mutation in the cytochrome b gene that confers QoI resistance. Of the 116 isolates, 24% were positive for the G143A mutation. At the 10 locations with QoI resistant isolates, frequencies of the mutation ranged from 50 to 100%. DNA was extracted directly from leaves collected at all 55 locations, and a pyrosequencing assay was designed to quantify the frequencies of the G143A mutation within the *C. sojina* populations in each field. The survey confirms the presence of QoI resistant FLS in Virginia, so growers should use a different or mixed mode of action fungicides, plant resistant varieties, and rotate to non-host crops.
## Fitness and competition studies of QoI resistant and sensitive *Cercospora sojina* isolates, the causal agent of frogeye leaf spot

B. LIN (1), H. Kelly (1), H. Yu (2), A. Mengistu (3)

(1) University of Tennessee-Entomology & Plant Pathology, Jackson, TN, U.S.A.; (2) University of Tennessee-Entomology & Plant Pathology, U.S.A.;

(3) United States Department of Agriculture, Agricultural Research Service (USDA-ARS), U.S.A.

Frogeye leaf spot (FLS), caused by *Cercospora sojina*, is a foliar disease affecting soybean, often managed by applications of Quinone outside inhibitor (QoI) fungicides. Because *C. sojina* isolates have developed resistance to QoIs, a better understanding of the biology and aggressiveness of isolates is important. Spore suspension inoculum prepared from QoI-resistant and -sensitive isolates were mixed in six different ratios. Greenhouse plants (cultivar Blackhawk) were treated: water (control), Quadris, or Quadris Top. Results were:1) Disease severity increased as the proportion of QoI resistance increased, indicating that QoI-resistant isolates are more aggressive; 2) Quadris inhibited FLS caused by QoI-sensitive isolates only, and when the proportion of QoI-resistance was ≤ 10%; 3) Quadris Top provided more effective control of FLS by reducing symptoms and delaying disease development. Fitness parameters measured were: (i) mycelial growth and sporulation capacity, (ii) conidial germination and growth inhibition on fungicide amended media. No significant differences of the fitness parameters were observed between resistant and sensitive isolates, except for sporulation capacity and growth inhibition. Moreover, qPCR results showed resistant isolates dominated the population in all mixed inoculation tests. These results indicate there are differences in QoI resistant and sensitive *C. sojina* isolates in-planta that will influence effective disease management of FLS.

## Assessing potential virulence differences between QoI-sensitive and -resistant *Cercospora sojina* isolates from Mississippi soybean

N. BROCHARD (1), M. Tomaso-Peterson (2), T. Allen (3), R. Melanson (3)

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

Frogeye leaf spot (FLS) of soybean is caused by the fungus *Cercospora sojina* Hara. Quinone outside inhibitor (QoI) fungicides have historically been the standard for FLS management in MS. However, widespread QoI resistance has been reported in the MS soybean production system. QoI resistance may be associated with a fitness cost that includes a loss of virulence. A comparison of virulence was assessed between two *C. sojina* sensitivity groups based on the presence or absence of the G143A substitution and termed QoI-sensitive or -resistant. Twenty four *C. sojina* isolates, 11 QoI-sensitive and 13 QoI-resistant, were assayed in an *in planta* virulence assessment. Soybean seedlings were inoculated with conidial suspensions of each *C. sojina* isolate. Percent FLS severity assessments were made 21 days post inoculation. QoI-sensitive and -resistant *C. sojina* isolates were pathogenic to soybean seedlings based on the production of typical FLS lesions. Disease severity significantly differed among isolates within sensitivity groups. No differences in disease severity were noted between sensitivity groups; however, a 4.5% increase in severity was noted for the QoI-resistant group. This suggests QoI-resistant isolates are equally virulent as QoI-sensitive isolates, indicating no fitness cost associated with QoI-resistance. Failure to control *C. sojina* with QoIs dictates the need for alternative management practices when high-yielding FLS-susceptible cultivars are planted in MS.

## Characterization of Arabidopsis Defense-related Gene Homologues in Tomato and Strawberry

J. PEREIRA (1), K. Silva (2), M. Garald (3), J. Jeffrey (3), Z. Mou (3)

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

The United States is the largest strawberry producer, and second in tomato. Yield of these crops is threatened by a number of diseases. Durable measures for disease management have been difficult to develop. One strategy that is being pursued is utilizing defense-related genes from *Arabidopsis thaliana* and their homologues in other plants. The Arabidopsis Elongator complex plays an important role in plant immunity. Elongator is not specifically involved in pathogen recognition, subsequently, it may provide durable resistance. Our results show that overexpressing Arabidopsis Elongator genes in tomato and strawberry improves resistance to several bacterial and fungal pathogens. The predicted tomato and strawberry Elongator genes are highly homologous to those of Arabidopsis. Importantly, the tomato and strawberry Elongator gene homologues are able to complement the morphological and defense phenotypes in corresponding Arabidopsis Elongator mutants, indicating that they are functional. Generating transgenic plants with broad-spectrum disease resistance would be a promising alternative to conventional methods. However, recently there has been a strong anti-GMO propaganda aiming to reach the public. Since the tomato and strawberry Elongator gene homologues are functional, they could be utilized to generate tomato and strawberry plants with increased disease resistance, using alternative technologies that are not considered transgenic, such as cisgenesis and intragenesis.

## Speeding up the discovery of novel sources of leaf rust resistance in wheat

A. RIAZ (1), N. Athiyannan (2), S. Periyannan (3), O. Afanasenko (4), O. Mitrofanova (5), E. Aitken (6), E. Lagudah (3), L. Hickey (2)

(1) The University of Queensland, Queensland Alliance for Agriculture and Food Innovation, St Lucia, QLD 4072, Australia, Brisbane, QC, Australia;

(2) The University of Queensland, Queensland Alliance for Agriculture and Food Innovation, St Lucia, QLD 4072, Australia, Australia; (3)

Commonwealth Scientific and Industrial Research Organization (CSIRO), Australia; (4) Department of Plant Resistance to Diseases of All Russian Research Institute for Plant Protection, Pushkin, 196608, Russia, Russia; (5) N. I. Vavilov Institute of Plant Genetic Resources, Bolshaya Morskaya 42-44, Saint Petersburg, 190000, Russia, Russia; (6) The University of Queensland, School of Agriculture and Food Science, St Lucia, QLD 4072, Australia

Leaf rust (LR) caused by *Puccinia triticina*, is among the most important diseases of wheat (*Triticum aestivum* L.) crops globally. Deployment of cultivars incorporating genetic resistance, particularly the combination of adult plant resistance (APR) and all stage resistance (ASR), is considered to be the most effective and sustainable method to control rust pathogen. While scoring of ASR in the glasshouse is straight forward, identification of APR in the field is often challenging due to varying micro environments. To overcome this, here we report a novel screening procedure that involves application of a rapid phenotyping method performed under accelerated growth conditions (AGC; i.e. constant light and controlled temperature) and effective elimination of known APR genes using linked DNA markers. Using this approach, screening of 300 diverse wheat accessions from the N. I. Vavilov Institute of Plant Genetic Resources (VIR) identified a set of 50 wheat accessions as potential sources for new APR to LR. In the subsequent screening of the subset under field conditions, 13 accessions consistently displayed good levels of APR. Further, in the DNA marker screening of the 300 accessions, 88 were identified to carry known APR genes (*Lr34*, *Lr46* and *Lr67*). Interestingly, accessions carrying *Lr67* were mostly landraces from India and Pakistan, consistent with previous reports about the likely origin of this APR gene. This can accelerate discovery of new sources of resistance.

## Mapping of two QTL conferring adult plant resistance to *Puccinia graminis* f. sp. *tritici* race TTKSK (Ug99) and evidence for race specificity

J. BRIGGS (1), M. Rouse (2), S. Bhavani (3), C. Hiebert (4)

(1) University of Minnesota, U.S.A.; (2) USDA-ARS, U.S.A.; (3) CIMMYT, Kenya; (4) Agriculture and Agri-Food Canada, Canada

The durability of wheat stem rust (caused by *Puccinia graminis* f. sp. *tritici*) resistance is strengthened by the use of multiple broad-spectrum sources of resistance. Adult plant resistance (APR) is thought to provide partial effect, race non-specific resistance. APR was observed in the Ecuadorian bread wheat cv. Morocho Blanco (MB) in field tests at Njoro, Kenya. MB is susceptible to TTKSK (Ug99) at the seedling stage. A doubled haploid (DH) population was created from a cross between MB and the susceptible line LMPG-6 to identify loci associated with APR phenotypes. Eighty-eight DH lines were genotyped with approximately 90,000 SNPs using a custom Infinium assay from Illumina. Sixty-seven additional DH lines (MB x LMPG-6) and seventy-three recombinant inbred lines (MB x Faller) were used to verify SNPs associated with reduced levels of stem rust infection. Severity and infection type were assessed on adult plants in Njoro, Kenya from 2013-2015, Debre Zeit, Ethiopia in 2015, and in single race nurseries inoculated with races RCRSC, TPMKC, QTHJC, and QFCSC in Rosemount, USA from 2014-2015. Two QTL that reduce stem rust severity were identified on chromosome arms 2BS and 6AS in the three screening seasons in Kenya. However, in Ethiopia only 6AS was effective, and in the USA only 2BS was effective to race QFCSC. The current data indicates that a singular race and location should not be used to develop lines using APR as possible genotype x race interactions exist.
# Utilizing mapping-by-sequencing to identify the Dominant Net Form Net Blotch Resistance Gene from Barley Line CI 5791

P. TAMANG (1), R. Brueggeman (1), J. Richards (1), T. Friesen (2)

(1) North Dakota State University, U.S.A.; (2) Cereal Crops Research, USDA, U.S.A.

Net form net blotch (NFNB) caused by *Pyrenophora teres* f. *teres* is a destructive foliar disease in barley growing regions worldwide. The diverse pathogen populations and complex host-parasite interactions occurring in this pathosystem has hindered breeding efforts. A dominant NFNB resistance gene identified on chromosome 6H in the line CI5791, designated *RptCI5791*, provides resistance to diverse isolates and is tightly linked to the dominant susceptibility locus *Spt1*. CI5791 lacks *Spt1* as we were unable to amplify any allelic variants of *Spt1*. Thus, we hypothesize the high level of resistance in CI5791 is due to the dominant resistance gene *RptCI5791* coupled with the absence of the dominant susceptibility locus *Spt1*. To further identify and characterize this broad-spectrum resistance, we used a forward genetics approach by g-irradiation and identified five putative mutants CI5791-γ3, -γ6, -γ7, -γ8, and -γ9 that are susceptible. We are utilizing the barley SEQCAP exome capture array (Nimblegen) and Illumina sequencing to identify the gene using a mapping-by-sequencing method. Mutated gene/s that map to the centromeric region of barley chromosome 6H will represent a strong candidate *RptCI5791* gene. However, the possibility still exists that alleles of the CI5791 gene may not be present in the lines used in the exome capture, which would result in a failure to identify the gene. In this case we will continue pursuing the tried and true positional cloning approach.

# Development of molecular markers tightly linked to Potato virus Y resistance gene Rychc in a diploid potato population

A. FULLADOLSA (1), S. Jansky (2), D. Halterman (3), A. Charkowski (1)

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

In the last 15 years, *Potato virus Y* (PVY) has been the main plant pathogen limiting seed potato certification in North America. The emergence of necrotic and recombinant PVY strains that cause mild foliar symptoms has resulted in increased PVY incidence. The use of cultivars that do not express foliar symptoms has also contributed to the problem. Several PVY resistance genes have been identified in wild relatives of potato, but few have been introduced into North American cultivars. We identified a diploid *Solanum chacoense* clone (CHC 39-7) in accession 275138, carrying PVY resistance gene *Ryche*, and used it in a cross with the susceptible, diploid *Solanum tuberosum* US-W4. A heterozygous resistant F1 progeny, XD3, was self-pollinated and the resulting population was phenotyped and used to develop molecular markers for genotyping. The phenotypic and genotypic results showed skewed segregation ratios, suggesting that a lethal factor is associated with susceptibility in the population. In addition, the development of markers that are more tightly linked to resistance in CHC 39-7 has aided in fine mapping *Ryche* and elucidating gene organization on chromosome IX. Furthermore, XD3 was crossed with a high yielding, adapted diploid *S. tuberosum-S. chacoense* hybrid and several progeny were selected for their high yields and good tuber type. These breeding clones and molecular markers provide new tools for breeders to introduce PVY resistance into North American cultivars.

# Metabarcoding the soybean seed core mycobiome

R. PEDROZO (1), A. Jumpponen (1), C. Little (1)

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

The goal of this study was to explore the soybean seed core mycobiome using next-generation sequencing. Eight seed samples, representing four different locations in the state of Kansas and two soybean genotypes, were used. Seeds were surface disinfested with a 5% bleach solution (0.525% NaOCl v/v) for 1 min and dried overnight at room temperature. DNA was extracted from each ground soybean sample. After amplification of the ITS region, using ITS1F and tagged ITS4 primers, amplicons were sequenced using the Illumina MiSeq platform. Approximately 170,000 high-quality reads were produced from all samples analyzed in this study. Sequences from each sample were then taxonomically assigned using the Fungal ITS classifier from the Ribosomal Database Project (RDP). Initial analysis showed that both non-pathogenic fungi, such as *Alternaria* sp., *Epicoccum* sp. and *Cladosporium* sp., as well as pathogens such as *Cercospora* sp., *Phomopsis* sp. and *Fusarium* sp. were observed in all locations and genotypes. To better understand the soybean seed core mycobiome and its implication for global food security, additional experiments are underway to explore the presence of seedborne fungi, especially regarding the pathogenic groups, among individual soybean seeds as well as among three major soybean seed tissues including the seed coat, cotyledons, and embryo axis.

# Soil microbiomes associated with alternative strategies to manage Verticillium dahliae

P. INDERBITZIN (1), J. Ward (2), A. Barbella (2), N. Solares (2), B. Calderon (2), D. Chellemi (2), K. Subbarao (1)

(1) UC Davis, U.S.A.; (2) Driscoll's Strawberry Associates, U.S.A.

*Verticillium dahliae* is a fungal plant pathogen that causes Verticillium wilt, which results in significant losses in many crops. Control of *V. dahliae* is difficult as the pathogen can survive in soil without a host for over 10 years. Fumigation is an effective control, but the most viable option, methyl bromide, is no longer used and alternatives are expensive and subject to strict local regulations. Our goal is to develop environmentally-friendly and economically-viable alternatives to manage Verticillium wilt. These include organic soil amendments such as broccoli residue and a mixture of crab and feather meal, which have demonstrated disease suppressive effects in prior research, likely mediated by the soil microbiome. We performed greenhouse studies on a Verticillium sensitive host, eggplant, on which we assessed Verticillium wilt severity, and characterized soil microbial communities using 16S Illumina sequencing. The experimental setup involved a randomized complete block design with three soil amendments and 4-5 replicates for each amendment, and was performed separately for two soil types with different inoculum levels. The experiment was repeated once. We found that soil type, time of sampling, and amendment significantly impacted microbial communities; and that a sub-set of taxa was more abundant in disease suppressive treatments than in controls. Future studies will focus on the mechanistic bases of disease suppression by soilborne microbes.

# Root and rhizosphere microbiome responses to new tomato rootstock systems

R. POUDEL (1), L. Meyer (2), A. Jumpponen (3), M. Kennelly (4), C. Rivard (2), B. Cordova (5), J. Brisbane (5), K. Garrett (5)

(1) Plant Pathology Department, Institute for Sustainable Food Systems, and Emerging Pathogens Institute, Gainesville, FL, U.S.A.; (2) Horticulture, Forestry, and Recreational Resources, Kansas State University, Olathe, KS, U.S.A.; (3) Division of Biology and Ecological Genomics Institute, Kansas State University, Manhattan, KS, U.S.A.; (4) Department of Plant Pathology, Kansas State University, Manhattan, KS, U.S.A.; (5) Plant Pathology Department, Institute for Sustainable Food Systems, and Emerging Pathogens Institute, University of Florida, Gainesville, FL, U.S.A.

Grafting tomato scions onto rootstocks can increase yields by providing disease resistance, stress tolerance, and increased vigor. We hypothesized that grafting affects the rhizosphere microbiome, or rhizobiome. We analyzed impacts of grafting scion BHN589 with two rootstocks BHN1028, RST-04-106 on the rhizobiome of tomato plants using rRNA gene sequences, including non-grafted and self-grafted controls. We also compared the microbial communities in roots vs. rhizosphere. In the first year, there were no observed effects of grafting or rootstock on microbial diversity in the three farm sites. Many OTUs were shared (72% in bacteria, 65% in fungi) among treatments, while some taxa were treatment-specific. Across all grafting treatments, higher diversity was found in the rhizosphere compared to the roots (p<0.001). Composition of the rhizobiome varied with root-rhizosphere compartment and farm site. *Proteobacteria*, *Actinobacteria*, *Firmicutes*, *Bacteroidetes*, and *Planctomycetes* were most abundant in all treatments, with the latter three being less abundant in roots. *Pezizales*, *Cantharellales*, and *Xylariales* were enriched in roots, whereas *Pleosporales*, *Mortierellales*, and *Hypocreales* were enriched in the rhizosphere. Interestingly, community modularity was higher in the roots compared to those from the rhizosphere. Ultimately, understanding rhizobiome responses to rootstocks will support vegetable production, rhizobiome-based crop breeding, and disease management.
## Genetic determinants of bacterial adaptation to plants

A. LEVY (1), A. Levy (1)

(1) DOE Joint Genome Institute, U.S.A.

Plants tightly associate with an array of diverse bacteria, with which they share complex and fascinating interactions, including parasitism, mutualism, and commensalism. These plant-associated (PA) bacteria have evolved a large set of genes that enable them to adapt to the plant environment. The functions of these genes are in many cases poorly studied and lack sufficient mechanistic understanding to facilitate microbiome engineering. Here, we sequenced 485 genomes of bacterial isolates and single cells isolated from the root environments of Arabidopsis, poplar, and maize. We then employed a large-scale comparative genomics study to characterize PA genes, and in particular root-associated (RA) genes. This was done by comparing 18 million genes from 3902 high quality and phylogenetically diverse bacterial genomes that were classified based on their isolation site. Several genomic features are common to most PA bacteria, such as the expansion of carbohydrate metabolism regulons. Many of the PA genes are encoded within putative gene operons, some of which encode for novel complex functions. Novel PA genes include a rapidly evolving virulence factor in the *Acidovorax* genus, a phage-like secretion system, and bacterial proteins with plant-like domains. The large PA and RA gene repository and the novel sequenced RA bacterial genomes will enable engineering of beneficial interactions for improved crop productivity and agricultural sustainability.

## Unravelling the sugarcane microbiome to resolve the Yellow Canopy Syndrome in Australia

K. HAMONTS (1), P. Trivedi (2), A. Garg (2), P. Holford (3), J. Grinyer (2), I. Anderson (2), B. Singh (2)

(1) Hawkesbury Institute for the Environment, Penrith, Australia; (2) Hawkesbury Institute for the Environment, Western Sydney University, Australia;

(3) School of Science and Health, Western Sydney University, Australia

Yellow Canopy Syndrome (YCS) is a largely undiagnosed condition impacting sugarcane crops in Queensland, Australia, causing yield losses up to 30%. Since the first observation of YCS near Cairns in 2012, the condition has spread to all major cane growing areas and is a critical issue for the industry. Key YCS symptoms differ from leaf yellowing due to drought stress, phytotoxicity, insect attack, known diseases, nutrient deficiency or natural maturing. Despite elimination of some potential causes, the causal agent of YCS remains unknown. We aim to determine the involvement of biotic interactions and soil nutritional health in YCS development. A microbiome-based approach using Illumina MiSeq and HiSeq was applied on 598 samples to reveal the complexity of organisms (bacteria, fungi, other eukaryotes, viruses) present in YCS-affected sugarcane and rhizosphere soil. Soil nutrient status and microbial enzyme activity were also determined and did not significantly differ between affected and healthy fields. Bacterial (>25000 OTUs) and fungal (>11000 OTUs) assemblages of leaves, stalks, roots and rhizosphere soils differed between asymptomatic vs. symptomatic plants, as well as some rhizosphere microbial functions. Community shifts with YCS appeared variety-specific, and to date few consistent bacterial and fungal and no viral signals associated with YCS emerged. We envision ongoing data mining may reveal potential causal agent(s) of YCS or management practices for YCS.

## Illuminating the diversity of rust fungi infecting millets

J. DEMERS (1), L. Castlebury (1)

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

Millet are a diverse assemblage of grains frequently grown in subsistence agriculture or as forage crops, and their popularity as nutritional alternative grains has recently increased. Millet pathogens are not well known, and modern collections of fungi associated with millets are lacking. Rust fungi are a significant group of pathogens that cause economically important diseases of three commonly grown millets: pearl millet (*Pennisetum glaucum*), foxtail millet (*Setaria italica*), and teff (*Eragrostis tef*), a millet becoming popular as a health food. 28S rDNA sequences were analyzed from over 40 herbarium specimens of rust fungi on millets, and related grasses, housed at the U.S. National Fungus Collections. Three taxa of rust fungi were observed on pearl millet, *Puccinia substriata* var. *indica*, *P. substriata* var. *penicillariae*, and *P. cenchri*, previously unreported on this host. The foxtail millet rust pathogen, *Uromyces setariae-italicae*, showed a high degree of intraspecific genetic diversity and was present on grasses in the Paniceae subtribes Cenchrinae and Melinidinae. The teff rust pathogen, previously known as *U. eragrostidis*, is actually distinct from *U. eragrostidis* and is identified as *U. pedicellata*. Knowledge of the diseases of millets is increasingly important as they become more widely grown as human food sources and pressure to increase yields in the presence of the pathogens intensifies.

## Genetic diversity of *Colletotrichum* isolates from *Sorghum bicolor* and *S. halepense* in the Southeastern United States

K. XAVIER (1), C. Schardl (1), E. Buiate (1), M. Torres (2), M. Queiroz (3), S. Chopra (4), L. Vaillancourt (5)

(1) Department of Plant Pathology, University of Kentucky, Lexington, KY, U.S.A.; (2) Functional Genomics Laboratory, Cornell University/Qatar Foundation, Education City, Qatar; (3) Laboratório de Genética Molecular e de Micro-organismo, Viçosa, KY, U.S.A.; (4) Department of Plant Science, Pennsylvania State University, University Park, PA, U.S.A.; (5) Department of Plant Pathology, University of Kentucky, Lexington, KY, U.S.A.

Anthracnose caused by *Colletotrichum sublineola* is the most important disease of grain sorghum worldwide, and it is emerging as a significant production constraint for sweet sorghum grown in the Southeastern U.S. Anthracnose is also common on the wild sorghum relative johnsongrass (S. halapense). Marker analysis using repetitive fingerprinting probes revealed that isolates from cultivated sorghum were generally distinct from isolates from *S. halpense*. RFLP analysis based on probes against individual sequences presumed to encode effectors and secondary metabolism enzymes showed that most isolates from johnsongrass grouped separately from most isolates from cultivated sorghum. Isolates from cultivated sorghum were generally aggressive to sweet sorghum variety Sugar Drip, while isolates from *S. halapense* generally caused little or no disease. Phylogenetic trees were inferred based on isolates from both host species using portions of the DNA lyase gene, the manganese superoxide dismutase gene, and a region between the APN1 and the MAT2 genes (MATAPN) genes. These trees were found to be congruent, and to identify two distinct species, the first one associated with the cultivated *S. bicolor*, and a second, new species associated with *S. halepense*. Strong evidence was found that cross-infection of these two species does occur, and this could be expected to complicate efforts to develop and deploy resistant sweet sorghum varieties in areas where johnsongrass is common.

## Phylogenetic and population analyses of the brown root-rot pathogen (*Phellinus noxius*) highlight the existence of two distinct populations

J. STEWART (1), N. Sahashi (2), T. Hattori (2), Y. Ota (2), L. Shuey (3), R. Schlub (4), N. Atibalentia (5), F. Brooks (6), A. Tang (7), R. Lam (7), M. Leung (8), L. Chu (9), H. Kwan (9), A. Mohd Farid (10), S. Lee (10), C. Chung (11), H. Lee (11), Y. Huang (11), R. Liou (11), J. Tsai (12), P. Cannon (13), J. Hanna (14), N. Klopfenstein (14), M. Kim (15)

(1) Colorado State University, Fort Collins, CO, U.S.A.; (2) Forestry and Forest Products Research Institute, Tsukuba, Ibaraki, Japan; (3) Forestry & Agricultural Research Institute, University of Pretoria, South Africa; (4) University of Guam, ANR/CES/CNAS, Mangilao, Guam, U.S.A.; (5) American Samoa Community College-CNR, Division of Community & Natural Resources, U.S.A.; (6) Department of Plant and Environmental Protection Sciences, University of Hawaii-Manoa, Honolulu, HI, U.S.A.; (7) Muni Arborist Limited, Hong Kong, China; (8) Muni Arborist Limited, China; (9) School of Life Sciences, The Chinese University of Hong Kong, Hong Kong, China; (10) Forest Research Institute, Kepong, Selango, Malaysia; (11) Department of Plant Pathology and Microbiology, National Taiwan University, Taiwan; (12) Taiwan Agricultural Research Institute, Taiwan; (13) USDA Forest Service, Forest Health Protection, Vallejo, CA, U.S.A.; (14) RMRS, USDA Forest Service, Moscow, ID, U.S.A.; (15) Department of Forestry, Environment and Systems, Kookmin University, Korea

*Phellinus noxius* is a vastly destructive, fast-growing fungal pathogen that affects a wide range of woody hosts in pan-tropical areas, including Asia, Oceania, and Africa. This pathogen causes brown root-rot on diverse hosts, with little indication of host specificity. Pathogenic symptoms of *P. noxius*
infection can include reduced tree growth, defoliation, and branch dieback; however, *P. noixus* can survive as a saprophyte. Understanding the genetic diversity and evolutionary history of *P. noixus* populations worldwide well help assess the evolutionary origins, worldwide movement, and potential ecological differences within *P. noixus*. The objective of this study was to estimate a wide-scale genetic diversity and evolutionary history of *P. noixus* movement. A total of 158 isolates were included from Japan, Taiwan, Australia, China (Hong Kong), Malaysia, and the Pacific islands of American Samoa, Saipan, Guam, Palau, Pohnpei, and Kosrae were sequenced at four loci including ITS, LSU-rDNA, elongation factor-1 alpha, and RNA polymerase subunit II. The results show two distinct clades of *P. noixus*. Currently, sequencing data are generating by Illumina sequencing of double-digest reduced representation libraries (ddRAD). Continued analyses will assess regional population structure, gene flow among populations, diversity in recently observed populations, and potential suitable climate space for specific genotypes.

## Microsatellite genotyping reveals high genetic diversity in *Phytophthora infestans* from Mexico

S. SHAKYA (1), M. Larsen (2), M. Condoy (3), H. Lozoya-Saldana (3), N. Grunwald (2)
(1) Oregon State University, U.S.A.; (2) USDA-ARS, U.S.A.; (3) Universidad Autónoma Chapingo, Mexico

Toluca valley of Mexico is considered to be the center of origin of late blight pathogen, *Phytophthora infestans*. This hypothesis is supported by populations being sexual, mating type ratios 1:1 and a high standing genetic diversity in HWE. However, the population structure from other areas of Mexico is understudied. We hypothesize that migration of *P. infestans* outside of Toluca might have resulted in admixed population structure with decreasing diversity away from Toluca. To test this hypothesis we sampled and microsatellite genotyped more than 85 isolates from Mexico in the regions of Chapingo, Juchitepec, San Geromino, Tlaxcala and Toluca. Our analysis revealed high number of multi-locus genotypes and high allelic diversity. Juchitepec samples formed two distinct clusters suggesting two differentiated populations. Tlaxcala isolates seem to be uniformly distributed possibly indicating multiple migration events and admixture. Chapingo and San Geromino samples were more similar between them. In general, all regions showed no linkage among loci suggesting presence of sexual reproduction, except for Chapingo. Contrary to our hypothesis, diversity in other populations was even higher or comparable to Toluca. These results indicate that the center of origin is larger than previously thought. Our work provides a more refined understanding of the population structure of *P. infestans* in areas near Toluca where the pathogen has been well studied.

## Characterization of isolates of *Phytophthora capsici* from the mid-Atlantic region, U.S.

N. ABEYSEKARA (1), H. Hickman (2), S. Westhafer (2), N. Gregory (2), G. Johnson (2), T. Evans (2), N. Donofrio (2)
(1) University of Delaware, U.S.A.; (2) University of Delaware, U.S.A.

The heterothallic oomycete *Phytophthora capsici* causes disease under warm (25-28°C), wet conditions. This pathogen causes pod rot in lima bean (*Phaseolus lunatus* L.), an emerging disease in the mid-Atlantic region which reduces yields and limits production. In order to better understand sources of inoculum, isolates were collected from infected commercial plant samples, 13 irrigation water sources, potential weed hosts, and soil sediments in Delaware. Water filtration, baiting with fruits, or baiting with *Rhododendron* leaves, were used in an attempt to extract *P. capsici* from irrigation water. Species other than *P. capsici* were recovered in abundance from irrigation water, weed hosts, and soil sediments. A total of 38 isolates were recovered from symptomatic lima bean, pepper, pumpkin, and watermelon samples and positively identified as *P. capsici* both morphologically and molecularly. Isolates derived from single zoospores where characterized for mating type, mefenoxam sensitivity and virulence on eight commercial lima bean cultivars. The A2 mating type was prevalent among the collected isolates (78.9%) and seven isolates with resistance to mefenoxam were identified. All isolates were virulent on the eight lima bean cultivars tested. Two SSR markers were identified that co-segregate with mefenoxam sensitivity, with over 80% accuracy when used together, that could have potential use in disease management efforts.

## Epidemiology and management of bacterial leaf spot caused by novel *Pseudomonas syringae* strains on watermelon in Florida

E. NEWBERRY (1), L. Ritchie (1), B. Babu (1), T. Sanchez (2), K. Beckham (2), J. Jones (2), J. Freeman (1), N. Dufault (2), M. Paret (1)
(1) NFREC, University of Florida, U.S.A.; (2) Department of Plant Pathology, University of Florida, U.S.A.

Major disease outbreaks occurring on watermelon and squash caused by *Pseudomonas syringae* were widespread in Florida and Georgia during recent years. This was recognized as a new disease in the region, and affected approximately 6,500 acres of watermelon from Florida in 2013. Field trials were conducted during the 2014 and 2015 spring seasons in Quincy and Citra, FL to examine environmental conditions which may be conducive for disease progression, and to assess the efficacy of foliar application of copper with or without Ethylene bis-dithiocarbamate (ManKocide/Kocide 3000) and foliar/drip application of plant activator (Acibenzolar S-Methyl; Actigard) in managing both transplant and field level introductions of the pathogen. Pearson's correlation coefficient indicated that disease severity from the four trials was significantly correlated with average weekly temperature and total weekly rainfall in greenhouse inoculated (-0.67, p < 0.0006; 0.51, p < 0.01) and field inoculated plots (-0.72, p < 0.001; 0.58, p < 0.01) respectively. ManKocide and Actigard foliar applications were found to significantly reduce disease development over time, as indicated by the area under the disease progress curve (p < 0.05). The results of this study indicate that temperature and rainfall are key factors in determining disease development, and ManKocide and Actigard foliar applications are highly effective in controlling the disease with 3 to 4 applications early in the spring season.

## Whole genome sequencing of *Xanthomonas perforans* identifies effectors that influence breeding strategies

S. TIMILSINA (1), P. Abrahamian (2), N. Potnis (3), G. Minsavage (4), F. White (4), B. Staskawicz (5), J. Jones (4), G. Vallad (4), E. Goss (4)
(1) University of Florida, Gainesville, FL, U.S.A.; (2) Gulf Coast Research and Education Center, University of Florida, FL, U.S.A.; (3) United States Department of Agriculture, Charleston, SC, U.S.A.; (4) University of Florida, U.S.A.; (5) University of California-Berkeley, U.S.A.

The management of bacterial spot caused by *X. perforans* on tomato is a challenge due to historical changes in the pathogen populations. The availability of genomic sequence for hosts and pathogens provides new data for effector-based breeding strategies. We examined the stability and evolution of several effectors in *X. perforans*, based on genome sequence from representative strains collected in Florida since 1990. Effectors AvrXv3 and XopJ4 trigger resistance in tomato cultivars carrying the corresponding R genes. However, the Florida *X. perforans* population has shifted from tomato race 3 to tomato race 4, such that avrXv3 is non-functional in all tomato race 4 strains with mutations specific to different phylogenetic groups. The *X. perforans* effector genes *xopJ4* and *avrBs2* remain intact in all strains. The *avrBsT* gene is absent in race 3 strains collected in the 1990s, but present in race 3 strains collected in 2006 and the majority of race 4 strains. The conserved presence of XopJ4 and increased presence of AvrBsT among race 4 strains of *X. perforans* make them ideal targets for resistance breeding. Specific R-genes against these two effectors could be pyramided with Bs2 resistance to develop tomato cultivars with enhanced resistance against *X. perforans* for commercial production in Florida.

## Cybridization: A promising plant resistance for citrus canker control

M. MURATA (1), A. Omar (1), C. Chase (2), J. Grosser (1), J. Graham (1)
(1) University of Florida, U.S.A.; (2) University of Florida, U.S.A.

Cybridization is a somatic hybridization system that enables the construction of novel nuclear and cytoplasmic genome combinations. In this work, cybridization was used to study the role of mitochondrial (mt) and chloroplast (cp) genes in resistance to *Xanthomonas citri* subsp. *citri* (Xcc), the cause of citrus canker. Putative cybrids were created through the fusion between nucellar callus cells of Meiwa kumquat (*Fortunella crassifolia*), a citrus canker resistant, and mesophyll cells of citrus canker susceptible grapefruit (*Citrus paradisi*). Six EST-SSR markers demonstrated that all clones have a grapefruit nuclear genome; however, four CAPS markers showed variation in the cytoplasmic genomes. In 123 cybrids, kumquat mt and cp were identified, and in 19 cybrids, kumquat mt and grapefruit cp were present. Eighty-two cybrid clones were evaluated by detached leaf assay (DLA), and clones that performed better in the DLA were evaluated by syringe inoculation of attached leaves. Q-PCR was carried out to quantify the Xcc population.
Cybrid clones showed a range of citrus canker resistance, but all clones produced a significantly lower number of lesions and a one log unit reduction of the Xcc population compared to grapefruit. Cybrid clones with grapefruit cp were more susceptible to Xcc compared to clones with kumquat cp. In addition to citrus canker resistance, these cybrids provide a valuable model to study the role of cytoplasmic genomes in plant disease resistance.

## Biological control of plant pathogenic bacteria

G. KRITZMAN (1), G. Kritzman (2)

(1) Nobactra, Israel; (2) Nobactra, Israel

Bacterial diseases take a high economical toll in all areas of agriculture. Currently growers attempt to treat these outbreaks with copper compounds that have low efficacy and are detrimental to the environment. Nobactra brings a new concept to control a wide range of bacterial diseases, comprised of two key elements that when combined have a strong synergistic relationship. The first component is a cocktail of 10 antagonistic bacteria and the second component is a powder formulation of essential oils. The advantages of using a cocktail of bacteria rather than a single microbe include: a) consistency in a range of soil and climatic conditions; b) significantly lower chances of resistance development due to the multiple mechanisms of activity; c) wide spectrum of activity. The innovative oil formulation overcomes the high phytotoxicity of the essential oil and allows for effective use against bacterial diseases without harming the plant. Nobactra's solutions have shown high efficacy rates in the control of several diseases. The following are key examples: Potato scab (Streptomyces spp.) - seed treatment and soil treatment. Tomato canker (Clavibacter michiganensis subsp. Michiganensis - Cmm) Nobactra controls the distribution of Cmm in the greenhouse. Seed borne bacterial diseases (Pseudomonas, Xanthomonas, Acidovorax, Erwinia, Cmm.). Nobacta's products are officially registered in Israel.

## Investigation of the anti-bacterial activity of SERENADE® ASO against bacterial diseases of crops

T. SMITH (1), T. Knobloch (2)

(1) Bayer, Crop Science Division - Biologics Research, U.S.A.; (2) Bayer, Crop Science Division - Small Molecule Research, France

Bacterial diseases of agricultural crops are costly with losses estimated at $10 billion per year. Current management strategies often use a multifaceted approach, including biological control. The biological-based product, SERENADE® ASO, is a known tool in integrated foliar spray programs to combat fungal diseases in fruits and vegetables, based on fungal cell membrane disrupting properties of lipopeptide chemistry produced by *Bacillus subtilis* QST-713. In addition, SERENADE® ASO has proved helpful to growers in managing bacterial diseases, such as *Pseudomonas syringae* pv. *tomato* and *Xanthomonas campestris* pv. *vesicatoria*, causal agent of bacterial speck and spot, on crops such as tomato and pepper. Recent research using both *in vitro* and *in vivo* tools point to two main actions contributing to the antibacterial activity of SERENADE® ASO: a direct antibacterial effect from biological chemistry produced by QST-713 during fermentation and an indirect effect on the plant. Different approaches were applied to eliminate potential activity from living cells in SERENADE® ASO to examine the role of biological chemistry. Research with *Arabidopsis* treated with SERENADE® ASO showed up-regulation of several pathways associated with host defense, and microscopy studies using tomato seedlings revealed transient changes in the condition of stomata openings. Additional research is ongoing to better characterize the mechanism of SERENADE® ASO against plant pathogenic bacteria.

## Grass residues as a sustainable carbon source in application of anaerobic soil disinfestation for control of apple nursery replant disease

S. Hewavitharana (1), M. Mazzola (2)

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

Studies were conducted to assess the efficacy of anaerobic soil disinfestation (ASD) for control of replant disease in an apple nursery setting. Treatments applied in a field trial conducted at an experimental orchard in Washington State included ASD using orchard grass residues (GR; 20 t ha⁻¹) as the carbon source, Telone C35 (27 gal acre⁻¹) and no treatment control (NTC). The apple rootstocks G.935, G.41, and M.9, with respectively decreasing level of tolerance to replant disease, were planted into each of six blocks per treatment with five replicates per rootstock. Post-treatment weed biomass analysis, rootstock growth analysis and rhizosphere fungal community analysis were conducted. Prior to tillage that preceded rootstock planting, ASD yielded significant weed suppression but weed biomass in fumigated plots was comparable to NTC. During the first growing season trunk diameter of G.41 and G.935 rootstocks were significantly increased by ASD compared to the NTC and it was comparable to fumigation treatment. ASD enhanced growth of M.9 relative to NTC but was lower than that for the fumigation. According to TRFLP analysis, different soil treatments possessed unique rhizosphere fungal communities. Based on this study, an increase of 5-25% gross revenue was projected using ASD based on market value of different rootstocks and sizes. ASD with grass is a potential viable soil-borne disease management practice for the apple nursery industry.

## Management of Fusarium wilt Tropical race 4 in bananas

A. DRENTH (1), J. Henderson (2), A. Drenth (2)

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

*Fusarium oxysporum* f.sp. *cubense* Tropical race 4 (TR4) is one of the most important pathogens of banana which severely affects many banana varieties, including Cavendish. First identified in Taiwan, TR4 has spread rapidly over major banana production areas and has had a major impact on banana production. More recently it has been identified outside Southeast Asia in Mozambique, and in Jordan. The most recent incursion was detected in Australia's main banana growing region around Tully in North Queensland in March 2015. The continuous long distance dissemination of this fungus, as well as local spread to new plantations and fields, raises many questions concerning our ability for effective containment of this pathogen. The recent outbreak in Australia also highlighted a lack of understanding of many basic pathological and epidemiological facts of this important pathogen. Therefore, our aim was to improve detection and identification of the pathogen, destruction of infected plants, containment strategies and overall management of plantations. In the absence of high levels of resistance in economically acceptable banana varieties, containment may be a temporary but effective strategy to win valuable time to identify or develop banana varieties with high levels of resistance to Panama wilt TR4. A global program for evaluation of germplasm in multiple field sites linked with international collaborators is urgently needed.

## Pseudomonas sp associated with Smilax bona-nox display strong activity against Phytophthora spp

G. ALI (1), A. El-Sayed (2), D. Norman (2), M. Brennan (3)

(1) University of Florida, Apopka, FL, U.S.A.; (2) University of Florida/ Institute of Food and Agricultural Sciences, Apopka, FL, U.S.A.; (3) MREC/University of Florida/ Institute of Food and Agricultural Sciences, Apopka, FL, U.S.A.

*Smilax bona-nox* is a perennial weed, which is found in natural landscapes and forests throughout the southwestern U.S. *S. bona-nox* displays disease tolerance and vigorous growth, which could be partially attributed to beneficial plant-associated microorganisms. In this report, we recovered 44 bacterial isolates from roots and shoots of *S. bona-nox*. Based on 16S rRNA gene sequencing, these isolates belonged to 14 different genera. Antimicrobial screens showed that two of these isolates, EA6 and EA14, displayed strong inhibition of *Phytophthora parasitica*. Phylogenetic analyses based on the *rpoD* gene revealed that EA6 and EA14 belonged to the genus Pseudomonas. EA6 and EA14 did not produce biofilm, but produced bio-surfactants, volatile hydrogen cyanide and sulfur compounds. Biochemical fractionation of the volatile and secreted products of these two isolates for identifying the bioactive compound(s) revealed that volatile and organic extracts did not display any anti-Phytophthora activity. In contrast, salt precipitation of the aqueous phase revealed strong anti-Phytophthora activity, which was abolished by autoclaving or proteinase K treatments. Protein fractionation analyses suggest that the antimicrobial activity of EA6 is likely due to synergistic interaction of a mixture of glucanolytic enzymes. In conclusion, these two Pseudomonas isolates can be used for organically controlling Phytophthora diseases in crops.
## Efficacy of biopesticides on root diseases and pests in hydroponic production of vegetables

A. POLEATEWICH (1), R. Buitenhuis (1), M. Brownbridge (1), T. Cranmer (1), A. Summerfield (1)

(1) Vineland Research and Innovation Centre, Canada

Soil-borne pathogens cause significant losses in hydroponic greenhouse vegetable production, even when fungicides and water treatments are employed. Several microbes and microbe-derived products protect plants from pathogens. Many new products are available but comparative efficacy data is lacking. This research evaluated biopesticide efficacy under commercial conditions to identify implications for IPM. A small scale hydroponic system was constructed to evaluate efficacy of 10 bio-pesticides on commercial greenhouse tomato and cucumber cultivars. The effect of cultivar, grafting, and substrate type on product efficacy was also determined. Products were applied as a drench at transplant to rockwool blocks and at transplant to coco or rockwool slabs. Products were compared to fungicide and water controls. Plants were challenged with *Pythium* spp. 3-5 days after product application and root rot, plant height and weight was evaluated after 3 weeks. Several products reduced root rot equal to or greater than the fungicide Previcur. Disease reduction varied depending on the substrate, time of year and rootstock genetics. The best performing products in the disease trials were tested for potential positive or negative effects on performance of greenhouse pests *Trialeurodes vaporariorum* and *Tetranychus urticae*. Results from this research will allow growers to make informed management decisions and derive maximum benefit from their investment in disease control products.

## Organic rice disease management research in the United States

X. ZHOU (1)

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

Organic rice production has steadily increased in the U. S. over the last 20 years, with a majority of acreage being grown in the southern region. Due to warm and long growing seasons in the region, diseases are among the most important factors threatening the sustainability and productivity of organic rice. Field studies were conducted for the first time in the U. S. to evaluate the efficacy of cultivar resistance, cover crop, fertility, tillage and biocontrol agent for management of rice diseases on organically managed land in Texas in 2009 to 2014. Among 20 cultivars evaluated, Tesanai 2 had the highest level of resistance to both narrow brown leaf spot (NBLS) and brown spot followed by XL723 and XL753. Production following the incorporation of clover winter cover crop resulted in a consistent reduction in NBLS and brown spot than following winter fallow or annul ryegrass cover crop. An application of the soil amendment Nature Safe or Rhizogen at 90, 150 or 210 kg N/ha significantly reduced NBLS and brown spot. Use of conventional tillage practice prevented the occurrence of straighthead on all 20 cultivars evaluated in the clover winter cover crop plots. A foliar application of Serenade Max significantly reduced NBLS and sheath blight, resulting in an increase in yield. Cultivar resistance, cover crop, fertility, tillage and biocontrol can be effective tools for management of diseases in organic rice production.

## Elucidating the genetic basis of race differentiation in *Cercospora sojina* through genotype-by-sequencing approaches

S. SHARMA (1), B. Dhillon (1), A. Fakhoury (2), B. Bluhm (1)

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

*Cercospora sojina*, the causal agent of frogeye leaf spot (FLS), is a widespread pathogen of soybean in the United States. Recently, a race structure was proposed for *C. sojina* based on susceptibility vs resistance responses of 12 soybean differential cultivars. We hypothesize that the delineation of races in *C. sojina* is attributable to variation in effector gene complements among individual isolates. To test this hypothesis, we devised a genotype-by-sequencing strategy for *C. sojina* to facilitate association mapping of loci associated with race structure. We developed a restriction-site associated DNA sequencing (RAD-seq) approach to cost-effectively genotype large numbers of *C. sojina* isolates, with the ultimate goal of identifying single nucleotide polymorphisms (SNPs) for association mapping. Bulk segregant analysis was performed with two pools of *C. sojina* isolates (48 isolates per pool, selected based on resistant or susceptible reaction on soybean cultivar UA5612). Ion Torrent PGM sequencing identified 15,158 SNPs, and candidate pathogenicity genes were identified based on differences in SNP density and non-synonymous SNPs between the resistant and susceptible pools. Elucidating the genetic basis of race structure in *C. sojina* will accelerate the development of DNA-based assays to differentiate races of the pathogen, and contribute to the fundamental understanding of genetic mechanisms underlying FLS.

## RNA-Seq analysis of *stuA* mutants in *Fusarium verticillioides* indicates dramatic genomic wide transcriptional reprogramming

M. Rath (1), N. Crenshaw (2), S. Gold (2)

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

StuA, first discovered in *Aspergillus nidulans* and a member of the APSES class of transcription factors, regulates several essential developmental stages in fungi such as virulence, sporulation and toxin production in phytopathogenic fungi. *Fusarium verticillioides* (*Fv*), a maize phytopathogen, produces fumonisin mycotoxins in maize. Fumonisin-contaminated corn causes fatal toxicity in livestock, is associated with neural tube birth defects and growth stunting in children and is also a potential carcinogen to humans. Our objective is to explore the impact of the *stuA* homolog on morphogenesis and toxin production in *Fv*. *?stuA* mutants were generated via *Agrobacterium*-mediated transformation of *Fv* with an OSCAR deletion construct. Characterization of the mutants indicate slower vegetative growth on solid media, reduced sporulation and macroconidiation. A cracked corn assay on maize kernels and virulence assay on maize seedlings revealed reduced fumonisin accumulation and reduced virulence respectively in *stuA* mutant infected maize as compared to the wild-type pathogen infection. To characterize the mutants transcriptomally, RNA-sequencing of three of the mutants and the wild-type *Fv* strain M3125 was done via Illumina NextSeq and the Tuxedo pipeline is being utilized for differential expression analysis. Preliminary RNA-seq analysis indicates major alteration of numerous transcription factors, transporters and signaling in the *stuA* mutants.

## Enhancing our understanding of Fusarium maize stalk rot pathogenesis through gene association subnetwork module analyses

H. ZHANG (1), H. Zhang (2), M. Kim (2), H. Yan (2), B. Yoon (2), W. Shim (2)

(1) Texas A&M university, U.S.A.; (2) Texas A&M university, U.S.A.

*Fusarium verticillioides* is an important stalk rot pathogen of maize. While a select number of genes associated with virulence have been characterized, our knowledge of the genetic network underpinning this mechanism is very limited. Previously, our lab identified a striatin-like protein, Fsr1, that plays a key role in stalk rot pathogenesis. To further characterize genetic networks downstream of Fsr1, we performed RNA-Seq with maize B73 stalks inoculated with *F. verticillioides* wild type and *fsr1* mutant. Datasets were first used to infer *F. verticillioides* co-expression networks. Subsequently, we used a computationally efficient branch-out technique, along with an adopted probabilistic pathway activity inference method, to identify functional subnetwork modules likely involved in *F. verticillioides* pathogenicity. Through our analyses, potential pathogenicity-associated subnetwork modules were identified, each consisting multiple interacting genes with coordinated expression patterns, but whose collective activation level is significantly different in the wild type and the mutant. In this study we selected three putative pathogenicity genes, *FvSYN1*, *FvEBP1* and *FvCYP1*, from three predicted subnetworks. We generated gene-deletion mutants and performed pathogenicity assays. Furthermore, qPCR assays were performed to determine whether these genes serve as a hub gene and impose critical regulatory control on associated genes in each subnetwork.

## Genetic basis for pathogenicity in *Fusarium oxysporum f.sp. cepae* causing basal rot in onion

J. CLARKSON (1), A. Taylor (1), A. Jackson (1), A. Armitage (3), R. Harrison (3)

(1) Warwick Crop Centre, University of Warwick, United Kingdom; (2) Warwick Crop Centre, University of Warwick, United Kingdom; (3) East Malling Research, United Kingdom
Basal rot of onion, caused by *Fusarium oxysporum* f.sp. cepae (FOC), is a soilborne fungus and part of the *F. oxysporum* species complex, which includes other important pathogenic *formae speciales* (f.spp.) adapted to particular crop hosts, as well as non-pathogenic isolates. The genetic basis for pathogenicity in *F. oxysporum* is poorly understood, but ‘secreted in xylem’ (SIX) genes have recently been associated with virulence in several f.spp. A set of 32 *F. oxysporum* isolates were tested for pathogenicity on onion and whole genome sequencing and PCR approaches used to detect putative effector genes. Seven SIX genes and two other putative effectors were identified in highly pathogenic isolates which were all absent in non-pathogenic isolates. Expression of SIX genes and other putative effectors increased *in planta* over 96 hours following inoculation of onion seedlings. Different SIX gene complements were also identified in other f. spp. but none were found in *F. avenaceum*, *F. proliferatum* or *F. redolens*, which have also been associated with basal rot. Preliminary results from a comparative analysis of genomes from pathogenic and non-pathogenic *F. oxysporum* isolates will also be described as well as the potential use of SIX genes as a diagnostic to enable quantification of FOC and assessment of disease risk in field or store.

## Quantitative pyrosequencing for rapid detection and quantification of *Aspergillus flavus* atoxigenic active ingredients in complex fungal communities

K. SHENGE (1), B. Adhikari (1), K. Callicott (1), R. Bandyopadhyay (2), P. Cotty (3)

(1) USDA-ARS, U.S.A.; (2) International Institute of Tropical Agriculture (IITA), Nigeria; (3) USDA-ARS, U.S.A.

Use of atoxigenic *Aspergillus flavus* genotypes to displace aflatoxin producers is a highly effective method for reducing aflatoxin contamination in crops. However, there is a need for improved methods for rapid assessment of treatment efficacy and residual effects. To address this need, the current study designed twenty-four quantitative pyrosequencing assays for rapid post-harvest detection and quantification of *Aspergillus flavus* genotypes which serve as active ingredients in the biocontrol product Aflasafe™ that is registered for use in Nigeria. The fungi must be quantified in mixed fungal populations associated with crop surfaces. Sixteen of the assays are for individual genotypes and eight are for simultaneous detection and quantification of multiple active ingredients. Assay refinement with mixtures of DNA from target and non-target genotypes indicated both accuracy and sensitivity of the assays in detecting the active ingredients in fungal populations. Quantification of target alleles by single and multiple genotype assays was highly correlated with proportions of target DNA in the mixtures. Results from tests to validate the assays with DNA extracted from treated corn samples were not consistent among assays. The current results demonstrate that quantitative pyrosequencing is a robust and reliable tool for rapid detection, quantification, and monitoring of multiple genotypes within complex fungal communities associated with crops.

## Engineering mobile RNA in Carrizo to enhance plant defense responses to control citrus greening

S. Zhang (1), G. Perazzo (1), D. Gabriel (1)

(1) Integrated Plant Genetics, U.S.A.

Plants modulate plant defense responses against pathogens with both pro- and anti-programmed cell death (PCD) proteins. By suppressing expression of anti-PCD proteins, PCD can occur more rapidly and with stronger defense responses. There are many PCD triggers, including Reactive Oxygen Species (ROS) which activate plant defense signaling pathways over long distances. Citrus greening is caused by *Ca. Liberibacter asiaticus* (Las). Las produces a peroxidase that has been suggested to dampen the ROS signaling pathway. A gene silencing strategy was used to suppress an anti-PCD gene in Carrizo rootstocks. The expression levels of three citrus defense response genes (*NDR1*, *PRI* and *EDS1*) were significantly higher in silenced lines than in nontransgenic (NT) controls after inoculation with Las flagellin (flg22), used as a proxy for Las. Silenced Carrizo lines were also more tolerant to heat and cold stresses than NT controls. Challenge inoculations are currently underway using approach-grafted, Las infected citrus; all sampling was taken from leaves above the approach graft union. To date, three transgenic Carrizo lines failed to become infected over a 9 month challenge period as compared with two transgenic lines that became infected within two months. Following removal of the infected source tree, two NT scions that were consistently positive have now tested negative, possibly indicating movement of the silencing signal from transgenic rootstock to NT scion.

## Developing resistance to citrus huanglongbing

C. RAMADUGU (1), M. Keremane (2), T. McCollum (3), D. Hall (3), M. Roosevelt (1)

(1) University of California Riverside, U.S.A.; (2) USDA ARS National Clonal Germplasm Repository for Citrus and Dates, U.S.A.; (3) US Horticultural Research Laboratory, U.S.A.

Citrus huanglongbing (HLB) has caused significant damage to citrus industries in Florida and Brazil. The disease is associated with a fastidious bacterium, *Candidatus* Liberibacter asiaticus (Las) and transmitted by psyllids. Development of HLB resistant rootstock and scion cultivars will be beneficial to the industry. Sources of HLB resistance are not known in the genus *Citrus*. We have demonstrated HLB resistance in *Eremocitrus* and *Microcitrus* which are closely related genera sexually compatible with *Citrus*. Naturally occurring hybrids of *Citrus* with *Eremocitrus* and *Microcitrus* parentage were exposed to the HLB pathogen under greenhouse conditions and determined to be HLB resistant in controlled experiments. Since the resistance from these non-*Citrus* genera appears to be heritable, we have generated about 800 mandarin and trifoliate hybrids using *Eremocitrus* and *Microcitrus* as pollen parents. The hybrid seeds were raised in greenhouses in Florida and challenged by feeding with LAS-positive psyllids for four weeks. Preliminary results indicate the absence of Las in many hybrids. We are monitoring the putative resistant hybrid plants continuously by assaying for the presence of Las by qPCR and digital PCR, and also by observing the hybrids for expression of disease symptoms. We will confirm resistance after conducting no-choice psyllid feeding experiments.

## Proposed model for *Candidatus* Liberibacter asiaticus and solanacearum systemic invasion and multiplication in the psyllid host and vector

J. BROWN (1), T. Rast (1), J. Cicero (2), T. Fisher (1)

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

*Ca.* Liberibacter asiaticus and solanacterum are the causal agents of Huanglongbing and Zebra chip disease of citrus and solanaceous crops, respectively. Bacterial effectors that interact with psyllid vector proteins during invasion of and exit from the gut, and that facilitate entry into the salivary glands were identified using functional genomics, proteomics, yeast-2 hybrid (Y2H), co-immunoprecipitation (Co-IP), and electron microscopic analyses. *In silico* annotation and differential expression studies of psyllid nymphs and adults, and midgut and salivary glands identified transcripts and proteins with altered expression in response to *Ca.* Liberibacter infection. Differentially expressed transcripts were used as bait in Y2H studies to corroborate protein-protein interactions. Those positive by Y2H were subjected to verification by bait to prey co-transformation and Co-IP. Electron micrographs of the PoP midgut revealed the presence of CLso in the ventricular lumen, apical and basal epithelial cytosol, and in the filter chamber periventricular space. CLso were also prevalent in salivary gland pericellular spaces and head epidermal cell cytosol. Collectively, the results suggest a model for invasion in which *Ca.* Liberibacter- and prophage-encoded effectors manipulate the host endocytic pathway into a 'pathogen-mediated phagocytosis scenario' leading to bacterial exit from the gut into the hemocoel, and systemic invasion of other psyllid tissues and organs.

## Systemic infection of split root trees by *Candidatus* Liberibacter asiaticus suggests rapid movement between phloem sieve tubes

J. ORROCK (1), J. Orrock (1), E. Johnson (1)

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

Huanglongbing (HLB; syn. citrus greening), caused by the phloem-limited bacterium *Candidatus* Liberibacter asiaticus (Las), poses one of the largest threats to citrus production. In ten years it has spread to most citrus trees in Florida. Citrus production in Florida is down >70% from the pre-HLB era. Las infects a tree when an infected *Diaphorina citri* (Asian Citrus Psyllid) feeds on phloem sap in a leaf. After infection of the leaf, Las rapidly colonizes the root system, but remains sectored in the canopy. Canopy sectoring is thought to occur because of limited lateral connections of phloem sieve tubes.
To investigate if lateral movement of Las between sieve tubes is distance or tissue specific, root systems of grafted and seedling trees were split to three different heights: below crown, above crown, and above graft union (or equivalent on seedlings). The trees were then graft inoculated above one side of the split root system. Infection to both halves of the root system was monitored weekly. Contrary to our hypothesis; the graft union and crown do not play a unique role in lateral movement. Similar patterns of infection occurred in all tissue types of the grafted trees and seedling trees. Greater variability in the time between Las detection in the root systems on the inoculated side and the opposite side in all trees with the highest split suggests that vertical distance is important in lateral movement of the bacterium.

## Cultivar-specific wheat miRNAs during stripe rust infection in *Triticum aestivum*

S. RAMACHANDRAN (1), N. Mueth (1), P. Zheng (1), S. Hulbert (1)

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

Wheat represents approximately 30% of the world's production of grain crops with more than 220 million cultivated hectares. Productivity at harvest is mainly governed by genetic composition and environmental factors influencing growth and development. Understanding the genetic factors that regulate these processes will help in developing varieties with better yield potential and disease resistance. MiRNAs are regulators of gene expression in eukaryotes that control myriad processes from development to abiotic and biotic stress responses. Using 12 small RNA libraries prepared from two wheat cultivars infected with stripe rust fungus (*Puccinia striiformis*), we identified 43 previously-known miRNAs, 50 novel variants of known miRNAs and 93 candidate novel miRNAs. Digital gene expression revealed 20 cultivar-specific miRNAs, 6 miRNAs differentially expressed between the two cultivars and 4 miRNAs responsive to stripe rust infection. These results were validated using RT-PCR and qRT-PCR. Using different target prediction algorithms, 69 wheat miRNAs were found to target fungal genes- a majority of which coded for small, secreted, cellular proteins. These results suggest a trans-kingdom regulation of gene expression potentially involved in the cultivar-specific resistance to stripe rust. Overall, this study contributes to the current repository of wheat miRNAs and provides novel information on the as yet uncharacterized roles for miRNAs in wheat host-pathogen interactions.

## Antifungal mechanisms of a plant defensin MtDef4 are not conserved between ascomycete fungi, *Neurospora crassa* and *Fusarium graminearum*

K. ISLAM (1), K. El-Mounadi (2), D. Shah (3)

(1) Donald Danforth Center, U.S.A.; (2) Kutztown University of Pennsylvania, U.S.A.; (3) Donald Danforth Center, Bangladesh

Small cysteine-rich defensins are ubiquitous antifungal plant peptides which contribute to the innate immunity of plants. The plant defensin, MtDef4, inhibits the growth of the ascomycete fungi, *Neurospora crassa* and *Fusarium graminearum*, at micromolar concentrations. Previous studies have suggested that MtDef4 is transported into the cytoplasm of these fungi and exerts its antifungal activity on various intracellular targets. Here, we have investigated whether the antifungal mechanisms of MtDef4 are conserved in these fungi. We show that *N. crassa* and *F. graminearum* respond very differently to MtDef4 challenge. Membrane permeabilization is required for the antifungal activity of MtDef4 against *F. graminearum* but not against *N. crassa*. We find that MtDef4 is targeted to different subcellular compartments in each fungus. Internalization of MtDef4 in *N. crassa* is energy-dependent and involves endocytosis. By contrast, MtDef4 appears to translocate into *F. graminearum* autonomously using partially energy-dependent pathway. MtDef4 has been previously shown to bind to the phospholipid phosphatidic acid (PA). We provide evidence that the plasma membrane localized phospholipase D (PLD1), involved in the biosynthesis of PA, is needed for entry of this defensin in *N. crassa*, but not in *F. graminearum*. To our knowledge, this is the first example of a plant defensin which inhibits the growth of two ascomycete fungi via different mechanisms.

## Identifying genetic variation in the *Sorghum* PAMP response

D. CHEN (1), P. Balint-Kurti (2), G. Stacey (1)

(1) Divisions of Plant Science and Biochemistry, University of Missouri-Columbia, MO, 65201, U.S.A.; (2) USDA-ARS, Department of Plant Pathology, North Carolina State University, Raleigh, NC, U.S.A.

*Sorghum* is an important grain, forage and biofuel crop with a fully sequenced genome and a variety of available genetic resources. In comparison to studies of Arabidopsis, very little is known about the innate immunity response of crop species, including sorghum. Such studies could contribute to efforts to create crops with enhanced disease resistance. The plant innate immunity system is triggered by plant recognition of conserved pathogen molecules, so called pathogen-associated molecular patterns (PAMPs). We measured PAMP induced production of reactive oxygen species (ROS) as a quantitative measure of the immunity response in more than 50 different *Sorghum* genotypes. In addition, we also analyzed the production of nitric oxide (NO) as another measure of the plant response to PAMP addition. The results showed a significant variation among the phenotypes with a few responding very strongly with others showing essentially no response. This variation provides the basis for additional experiments focused on defining specific genetic loci that control the *Sorghum* PAMP response. We are also conducted a transcriptomic analysis of the *Sorghum* PAMP response with the goal to define expression QTL (eQTL) related to pathogen resistance. The goal is to use the information obtained to aid breeding efforts to increase biotic stress resistance in *Sorghum*.

## Understanding quantitative disease resistance in rice: Defense response gene regulation and promoter patterns

B. TONNESSEN (1), R. Mauleon (2), N. Alexandrov (2), J. Leach (3)

(1) Colorado State University, U.S.A.; (2) International Rice Research Institute, Philippines; (3) Colorado State University, U.S.A.

The potential yield of rice worldwide is reduced by one third due to disease. The extensive work in breeding rice varieties that harbor resistance genes has helped to mitigate this damage. The method is effective, but the selective pressure of a single host resistance gene can be overcome after only a few generations. Enhancing the expression of basal defense response (DR) genes that are activated against a broad spectrum of pathogens could be a more sustainable approach to breeding for resilience. DR gene promoters can affect timing and intensity of expression, and rice varieties with good basal resistance have been shown to contain polymorphisms that are beneficial in the activation of the defense response. Recently, the genomes of a diverse panel of 3000 rice varieties were sequenced. This information, along with transcriptomic analyses of resistant interactions, were utilized in this study to uncover the genome-wide patterns in DR gene promoters. These patterns are potential cis-elements, and DR gene-specific combinations of these elements in multiple loci infers their functionality in fine-tuning regulation during a resistant host-pathogen interaction. These results could refine marker assisted breeding for a stable, broad spectrum resistance.

## COS-OGA stimulates plant innate immunity in a cumulative process that involves salicylic acid

G. VAN AUBEL (1), P. Van Cutsem (2)

(1) University of Namur, Belgium; (2) University of Namur, Research Unit in Plant Cellular and Molecular Biology, Belgium

COS-OGA is a new patented active substance for plant protection registered in Europe against powdery mildew. COS-OGA contains chitosan oligomers (COS), plant non self-molecules combined with pectin-derived oligogalacturonides (OGA), plant self-molecules. COS-OGA mimics a plant-fungus interaction: during plant infection, fungal pathogens deacetylate their cell wall chitin into chitosan to escape plant chitin receptors. Chitosan fragmentation by plant enzymes then yields polycationic COS molecules while plant cell wall degradation by fungal polygalacturonases releases polyanionic OGA molecules. The plant defense stimulation was investigated on tomato plants after several foliar sprayings of COS-OGA. A proteomic study on leaves revealed that the elicitor treatment led mainly to an overexpression of Pathogenesis-Related proteins, among which P69 subtilisin-like proteases. Gene expression study by qRT-PCR revealed that COS-OGA significantly upregulated transcripts of salicylic acid (SA)-responsive genes while transcripts linked to ethylene and jasmonic acid pathways were not affected. The activation of SA-dependent plant defense reactions was
confirmed by SA quantification in the leaves. The results showed an increase of free SA levels in leaves, proportional to the number of COS-OGA applications. Finally, repetitive COS-OGA sprayings efficiently protected greenhouse-grown tomato against *Leveilla taurica*, with 90% leaf protection in terms of severity.

## Degradation of the master regulator of plant defense NPR1 by the type III effector HopAB2

Z. FU (1), H. Chen (1), J. Chen (2), Z. Shang (1), M. Li (1), F. Liu (3)

(1) University of South Carolina, Columbia, SC, U.S.A.; (2) Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, Nanjing, China; (3) Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, Nanjing, China

The plant hormone salicylic acid (SA) is required for systemic acquired resistance (SAR). NPR1, as an SA receptor, is required for SA-mediated plant defense. The expression of over 97% of SA-responsive genes is dependent on NPR1, therefore, NPR1 functions as a master regulator of plant defense. Many studies have shown that pathogen effectors suppress or disrupt MAMP-triggered immunity (MTI) and cell death. There are also isolated studies, which suggest that effectors suppress or disrupt SA-mediated plant defense. In order to provide strong and direct evidence on the targeting of SA-mediated plant defense by pathogen effectors, we tested the interactions between the SA receptor NPR1 and all type III effectors from the bacterial pathogen *Pseudomonas syringae* in yeast two-hybrid assays. Interestingly, we found that the type III effector HopAB2 interacts with NPR1 only in the presence of SA or its active analogs. Using Agrobacterium co-infiltration assays, we found that HopAB2 degrades GFP- tagged NPR1 protein, dependent on its E3 ligase activity and through the 26S proteasome. Using Dexamethasone (DEX)-inducible *Arabidopsis* transgenic plants expressing the hopAB2 gene, we found that HopAB2 degrades NPR1 native protein. Similar to *npr1-2* mutant plants, these transgenic *Arabidopsis* plants show sensitivity to SA-induced toxicity and are comprised in SAR. Our studies provide solid evidence that HopAB2 disrupts SA-mediated plant defense by degrading NPR1.

## Resistance of Carolina Gold Select rice to African strains of *Xanthomonas oryzae* pv. *oryzicola* is triggered by inactivated TAL effectors

L. TRIPLETT (1), S. Cohen (2), C. Heffinger (3), A. Bogdanove (4), J. Leach (2)

(1) The Connecticut Agricultural Experiment Station, New Haven, CT, U.S.A.; (2) Colorado State University, U.S.A.; (3) Yale University, U.S.A.; (4) Cornell University, Ithaca, NY, U.S.A.; (5) Colorado State University, Fort Collins, CO, U.S.A.

The rice pathogen *Xanthomonas oryzae* pv. *oryzicola* causes bacterial leaf streak of rice, a disease for which no single-gene resistance is available. The bacterium produces numerous transcription activator-like (TAL) effectors that activate host susceptibility genes. There are two common rice resistance mechanisms against TAL effectors: DNA polymorphisms that allow escape of TAL effector binding, and placement of TAL effector binding sites to cause specific TAL effectors to activate resistance genes. We found that the heirloom rice variety Carolina Gold Select has a third form of resistance, triggered by diverse TAL effectors. The resistance was triggered in a manner independent of the transcriptional activation domain and of the composition of repeat variable residues that specify host DNA target sequences. While the TAL effector central repeat region was critical to resistance, an effector with only 3.5 central repeats was sufficient for resistance activation. The resistance was mapped to a single dominant locus, *Xo1*, on chromosome 4. The *Xo1* interval confers complete resistance to strains in the African clade of *X. oryzae* pv. *oryzicola*, representing the first dominant resistance locus against bacterial leaf streak in rice. The TAL effector triggered resistance phenotype has strong similarity to that conferred by the tomato resistance gene *Bs4*, suggesting that monocots and dicots have converged on the structure of TAL effectors as a pattern triggering resistance.

## Xanthomonas effector kinase XopAU promotes disease symptoms by activation of host MAPKK

D. TEPER (1), E. Bosis (1), G. Popov (1), A. Girija (1), G. Sessa (2)

(1) Department of Molecular Biology and Ecology of Plants, Tel Aviv University, Israel; (2) Department of Molecular Biology and Ecology of Plants, Tel Aviv University, Israel

*Xanthomonas euvesicatoria* (*Xcv*) is the causal agent of bacterial spot disease of pepper. *Xcv* pathogenicity depends on a type III secretion system that delivers effector proteins into host cells to manipulate host signaling and promote disease. Transient expression of the *Xcv* effector XopAU in host and non-host plants promoted typical defense responses including phosphorylation of MAPKs, accumulation of pathogenesis-related (PR) proteins and elicitation of cell death. Genetic analysis of XopAU by insertional mutagenesis and overexpression revealed a role for this effector in the development of chlorotic disease symptoms. XopAU encode a catalytically active protein kinase and this activity is required for its biological function. Protein-protein interaction studies demonstrated that XopAU physically interacts with the plant immunity-associated MAPKK MKK2. In addition, MKK2 was shown to be directly phosphorylated by XopAU *in vitro* while expression of XopAU *in planta* promotes an increase in the phosphorylation of MKK2. Silencing of *MKK2* or overexpression of catalytically inactive MKK2<sub>2k99r</sub> in *Nicotiana benthamiana* reduced XopAU-mediated cell death and MAPK phosphorylation while silencing of MKK2 in pepper attenuated the *Xcv*-mediated chlorotic disease symptoms. Together, this study indicates that XopAU contributes to Xcv disease symptoms by manipulating host MAPK signaling through activation of MKK2.

## Dissecting the function and downstream targets of *CsLOB1* in citrus

S. DUAN (1), H. Jia (1), S. Gowda (1), N. Wang (1)

(1) Citrus research and education center, U.S.A.

Citrus canker caused by *Xanthomonas citri* pv. citri (*Xcc*) is a devastating disease affecting most citrus species. PthA4, one of Xcc Type III secretion system effectors, is responsible for citrus canker pustule formation on citrus leave. PthA4 binds to the effector binding element in the promoter of *CsLOB1* to activate its gene expression, which subsequently activates genes responsible for pustule formation. *CsLOB1* has not been functionally characterized and it remains unknown how CsLOB1 controls its downstream genes. *CsLOB1* was highly expressed in the citrus stem phloem and cambial zone in wild-type plants. We aim to study the function of *CsLOB1* and identify the putative downstream target genes of *CsLOB1*. To observe the physiological effect by suppressing *CsLOB1* expression, a Citrus Tristeza Virus-based RNAi vector, engineered with truncated anti-sense CsLOB1, was constructed for Virus-induced Gene Silencing (VIGS). Suppression of *CsLOB1* via VIGS caused decreased stem diameter and retarded plant growth. To further confirm this result, Agrobacterium-mediated transient gene expression method is being used to reveal the *CsLOB1* function during pustule formation post Xcc infection on citrus leave using small hairpin (shRNA) and artificial microRNA methods. Meanwhile, Dexamethasone-inducible *CsLOB1* over-expression transgenic plants were constructed to identify the putative downstream genes of *CsLOB1*.

## Trapping conidia spores of *Podosphaera aphaniis* in high tunnels and field strawberry plots

R. ONOFRE (1), D. Gadoury (2), N. Dufault (1), N. Peres (1)

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

Strawberry Powdery Mildew (SPM) caused by *Podosphaera aphaniis* can be severe on susceptible cultivars, especially in tunnel and glasshouse production. Epidemics of SPM are largely driven by intense sporulation and conidia dispersal, which are two highly relevant factors for management strategies. The objective of this study was to evaluate SPM aerial conidia spore concentrations in plastic tunnel and field environments of Florida. A 7-day recording volumetric spore trap was installed in both the field and high tunnel plots during the 2015-16 growing season. Airborne conidial dose at each site was recorded hourly for 5 weeks, as was temperature, relative humidity, leaf wetness and rainfall. In both plots, spore counts followed a diurnal pattern, with a peak in aerial concentrations between 10 am and 2 pm. Cumulative airborne dose in the tunnel (1488 spores/m³) was notably higher than in the field (82 spores/m³). Several coincident factors likely contribute to the airborne dose in tunnel growing systems, including an inherently more favorable environment for sporulation and infection, and subsequent increased disease severity. However, factors such as residence time of conidia
within the system after becoming airborne, and effects of visible light and UV radiation on inoculum production and dispersal may also contribute to higher inoculum dose. Further work is being conducted to explore the effects of various environmental factors on conidia production and dispersal.

## Detection and quantification of *Bremia lactucae* by spore trapping and quantitative PCR

S. KUNJETI (1), A. Anchieta (2), F. Martin (3), Y. Choi (4), M. Thines (5), R. Mitchelmore (1), S. Koike (6), C. Tsuchida (1), W. Mahaffee (7), K. Subbarao (1), S. Klosterman (3)

(1) University of California Davis, U.S.A.; (2) USDA Salinas, Salinas, CA, U.S.A.; (3) USDA-ARS, Salinas, CA, U.S.A.; (4) Korea University, Division of Environmental Sciences and Ecological Engineering, Seoul 136-701, Korea; (5) Biodiversity and Climate Research Center (BiK-F), Germany; (6) University of California Cooperative Extension, Monterey County, Salinas, U.S.A.; (7) USDA-ASR, U.S.A.; (8) University of California Davis, Salinas, CA, U.S.A.

*Bremia lactucae* adversely affects marketability of lettuce. The disease has been managed with a combination of host resistance and fungicide applications with mixed success over the years. Fungicide applications are routinely applied under the assumption that inoculum is always present during favorable environmental condition. This approach could lead to fungicide resistance in *B. lactucae* populations. Detection and quantification of airborne *B. lactucae* near lettuce crops may lead to a more judicious timing of fungicide applications. For specific detection of *B. lactucae*, we designed a qPCR assay based on a target sequence in *B. lactucae* mitochondrial DNA. PCR tests using DNA from 83 geographically diverse isolates, representing 14 *Bremia* spp., and amplicon sequencing confirmed that the primers developed for the TaqMan assays are species-specific and only amplify template from *B. lactucae*. Cq values >35 were considered as nonspecific but a single sporangium could be detected at a Cq of 34. The correlation (R²) for regression between sporangial density derived from flow cytometry and Cq values derived from the qPCR was 0.86. The assay was deployed using spore traps in the Salinas Valley, where nearly half the US lettuce is produced. The sensitivity of the *B. lactucae*-specific assay enabled detection of the pathogen within the two-week lettuce free period, and indicated that the assay can be useful for predicting inoculum load for timing of fungicide applications.

## Timing of primary inoculum release by *Monilinia vaccinii-corymbosi* infecting blueberries in the US Pacific Northwest

D. HARTEVELD (1), T. Peever (2), J. Pscheidt (3)

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

Mummy berry, caused by *Monilinia vaccinii-corymbosi*, infects highbush blueberry (*Vaccinium corymbosum*) and causes severe economic losses in the Pacific Northwest (PNW). Control is mainly based on calendar-based fungicide sprays. The primary inoculum source is ascospores produced from apothecia that develop on mummified berries, or pseudosclerotia, that overwinter on the soil surface. Timing of apothecial development and ascospore release varies among locations and seasons, and differences in environmental factors may be why control programs are not always effective. This study aimed to identify environmental factors involved in the timing of primary inoculum release in the PNW, and develop a predictive model for ascospore release. Apothecial development was monitored in a severely affected field from 2008-2013 in Corvallis, Oregon and in two fields in northwestern Washington during the 2015 season. Data for eleven environmental factors was obtained from in-field weather stations and Washington State University's AgWeatherNet. Soil temperature, solar radiation and precipitation were the most significant factors influencing apothecial development demonstrated by a generalized linear model regression analysis. A forecasting model developed to predict primary inoculum release is being validated for PNW locations during the 2016 season. Outcomes of this research will improve the timing of disease management practices and overall control of mummy berry in the PNW.

## Plant pathogens were collected over Florida up to 3500 m by a wing-mounted dust collection system called DART (Dust Atmospheric Recovery Technology)

A. SCHUERGER (1), B. Tench (2), T. Emmons (3), J. Palaia (4)

(1) University of Florida, U.S.A.; (2) Dept. of Plant Pathology, University of Florida, U.S.A.; (3) Dept. of Engineering, Univ. of Central Florida, U.S.A.; (4) 4Frontiers Corporation, U.S.A.

Asian dust plumes deliver up to 64 million tonnes of dust over the NW of the USA, and African dust storms deliver over 50 million tonnes of dust over Florida each year. Recent studies have demonstrated that plant pathogens associated with Asian and African aerosols can be transported to the USA in naturally occurring dust storms. In order to initiate a long-term monitoring program of plant pathogens in Asian and African dust plumes, we have developed a dust collection system called DART (Dust Atmospheric Recovery Technology). The DART dust sampler was mounted on a F104 Starfighter jet and separately on a T6 Texan propeller driven airplane and test flown over FL in 2013-2014. The DART system utilizes a high-volume pump to pass air through 4 separate filtration units where both aerosols and microbial cells are captured. Flow rates through filters are directly correlated to increased air speed, and are inversely correlated to increased altitude. The DART dust sampler has performed nominally up to 7600 m, 0.92 Mach, and 3.5 +G's. During initial test flights in 2013 and 2014, fungi recovered from the lower troposphere over FL (up to 3500 m) contained plant pathogens including species in the genera: *Acremonium*, *Aspergillus*, *Cladosporium*, *Curvularia*, and *Fusarium*. Numbers of recovered fungi, but not bacteria, increased significantly when 5 or 10 µm filters were used in the DART system compared to filter pore sizes ≤ 2 µm.

## Validation of the CDM ipmPIPE Forecasting System: Relating Aerial Transport of Pathogen Spores to Outbreaks of Cucurbit Downy Mildew

K. NEUFELD (1), A. Keinath (2), B. Dutta (3), B. Gugino (4), D. Langston (5), M. Lewis Ivey (6), M. McGrath (7), S. Miller (8), E. Sikora (9), P. Ojiambo (1)

(1) Department of Plant Pathology, North Carolina State University, Raleigh, NC, U.S.A.; (2) Clemson University, Charleston, SC, U.S.A.; (3) Department of Plant Pathology, University of Georgia, Tifton, GA, U.S.A.; (4) Department of Plant Pathology and Environmental Microbiology, Pennsylvania State University, University Park, PA, U.S.A.; (5) Virginia Polytechnic Institute and State University, Suffolk, VA, U.S.A.; (6) Department of Plant Pathology and Crop Physiology, Louisiana State University, Baton Rouge, LA, U.S.A.; (7) Plant Pathology and Plant-Microbe Biology, SIPS, Cornell University, Riverhead, NY, U.S.A.; (8) Department of Plant Pathology, Ohio State University, Wooster, OH, U.S.A.; (9) Department of Entomology and Plant Pathology, Auburn University, Auburn University, AL, U.S.A.

Cucurbit downy mildew (CDM) caused by *Pseudoperonospora cubensis* is considered the most damaging disease of cucurbitaceous crops worldwide. Sporangia produced by the pathogen are carried by wind currents and deposited on host plants following rain events. The CDM ipmPIPE forecasting system integrates data on current disease outbreaks and trajectories of sporangia transport from source fields to forecast the risk of new disease outbreaks in the eastern United States. The forecasting system was validated by relating the predicted risks of disease outbreak to the presence or absence of sporangia after a rain event and the onset of disease symptoms in the field. Rainwater samples from eight states, AL, GA, LA, NC, NY, OH, PA, and SC were collected from planting to the first report of disease during the 2013 to 2015 seasons. The presence of sporangia was then analyzed using primers specific to the pathogen. The pathogen was detected in 4-30% of the 203 samples collected from 2013 to 2015. Presence of sporangia in rainwater samples correctly classified disease onset in 67%, 81% and 78% of the cases, while the forecasting system correctly predicted disease onset in 75%, 85% and 60% of the cases in 2013, 2014 and 2015, respectively. Predictions from the forecasting system and presence of sporangia in rainwater samples were in agreement in 60-87% of the cases analyzed except in 2015 when presence of sporangia in rainwater samples were detected infrequently.

## Rapid LAMP diagnostics for detection and disease alerts of *Phytophthora infestans*

J. RISTAINO (1), A. Saville (1), R. Guenter (1), C. Lagaly (1), D. Cooper (2)

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