## Specific TaqMan qPCR and a PvABC qPCR assays for monitoring airborne sporangia of *Plasmopara viticola* f. sp. *riparia*, *aestivalis* and *vinifera*

O. CARISSE (1), M. Tremblay (2), A. Lefebvre (3)

(1) Agriculture and AgriFood Canada, Canada; (2) Agriculture and Agroalimentaire Canada, Canada; (3) Agriculture and AgriFood Canada, Canada

Grape downy mildew (*Plasmopara viticola*) is among the most important grapevine diseases in eastern Canada. Recently, five new cryptic formae species of *P. viticola* from wild and cultivated *Vitis* species were described. In Eastern Canada, only *P. viticola* f.sp. *riparia* (clade A) and *P. viticola* f.sp. *aestivalis* (clade B) have been detected. As part of a biovigilance network aiming at tracking new pathogens and new genotypes, molecular assays for simultaneous quantification of sporangia of clade A and B and detection of clade C (*P. viticola* f. sp. *vinifera*) from air samples were developed. The assays are a specific triplex TaqMan qPCR and a PvABC qPCR developed from sequence alignments of internal transcribed spacer 1 and 2 (ITS1-2) DNA regions for *P. viticola* clade A, B, C and other closely related species. These essays were used to study the temporal and spatial occurrence of these genotypes in two vineyards. At one site, almost exclusively (99%) clade A was found while at the other site it was mostly (83%) clade B. Hence the genotype A was found at both sites, however, it was observed for the first time in early July at one site and in early June at the other site. The impact of these genotypes on grape downy mildew development is under study. Using these new tools, it will be possible to acquire knowledge on aerobiology and spatio-temporal distribution of genotypes A and B and to monitor clade C not present in Eastern Canada, but detected in north-east of USA.

## First report of the Asian citrus psyllid, vector of *Candidatus* Liberibacter asiaticus in Tanzania and their potential further spread in Africa

M. SHIMWELA (1), H. Narouei-Khandan (1), S. Halbert (2), M. Keremane (3), G. Minsavage (1), S. Timilsina (1), D. Massawe (4), J. Jones (1), A. van Bruggen (1)

(1) University of Florida, Gainesville, FL, U.S.A.; (2) Division of Plant Industry, Gainesville, FL, U.S.A.; (3) USDA, Riverside, CA, U.S.A.; (4) Sokoine University of Agriculture, Morogoro, Tanzania

Citrus surveys were conducted at high (>700m), medium (300-600m) and low (<200m) altitudes in Tanzania. Adults and nymphs of *Trioza erytreae* were abundant in the highlands and less abundant at medium altitudes. Unexpectedly, adults and nymphs of *Diaphorina citri*, the Asian citrus psyllid, were found at medium altitudes, around Morogoro. No psyllids were observed at low altitudes. Severe huanglongbing symptoms and tree decline were evident at high altitudes, while mild symptoms were observed at intermediate and low altitudes. DNA was extracted from leaf and psyllid samples and subjected to conventional PCR with seven different primer sets and qPCR with two primer sets for *Candidatus* Liberibacter spp. PCR products were sequenced and subjected to phylogenetic analysis. C. Liberibacter africanus (Laf) was detected in leaf and *T. erytreae* samples from high and medium altitudes by all methods. Sequences of C. Liberibacter DNA from leaves and psyllids were similar to those of Laf from South Africa. Even though Las primers gave a positive reaction in qPCR with medium altitude leaf samples, cPCR and sequencing results were negative for Las, suggesting cross reactivity of Las primers with Laf. Neither Laf nor Las were detected in *D. citri* samples. This is the first report of *D. citri* in Africa. Further surveys are needed to detect Las. The potential distribution of *D. citri* and Las in Africa was predicted using the correlative models MAXENT and Multi-Model Framework.

## Genetic diversity of pathogenic *Clavibacter michiganensis* subsp. *michiganensis* strains from Central Chile

M. VALENZUELA (1), S. Fuentes (2), F. Claverias (2), X. Besoain (3), M. Seeger (4)

(1) Centro de Biotecnología DAL, Universidad Tecnica Federico Santa Maria, Valparaíso, Chile; (2) Centro de Biotecnología DAL, Universidad Técnica Federico Santa Maria, Valparaíso, Chile; (3) Escuela de Agronomía, Pontificia Universidad Católica de Valparaíso, Valparaíso, Chile; (4) Laboratorio de Microbiología Molecular y Biotecnología Ambiental, Universidad Federico Santa Maria, Valparaíso, Chile

The bacterial canker of tomato caused by *Clavibacter michiganensis* subsp. *michiganensis* (*Cmm*) is the most important bacterial disease in tomato. The genetic diversity of *Cmm* strains isolated during 20 years from tomato plants in different Chilean tomato regions was studied. Twenty five isolates were characterized by growth on different media, Gram staining and enzymatic activities. For bacterial identification PSA8/R PCR amplification and 16S rRNA gene sequencing were used. In addition, the housekeeping *gyrB* and *kdpA* genes and the pathogenicity *tomA* and *chpC* genes were sequenced. All genes were polymorphic in these isolates. The *kdpA* gene showed the highest variability. Except for the *kdpA* gene, Chilean isolates genes showed high identity with genes from reference strain NCPPB382. Phylogenetic trees of *gyrB* and pathogenicity genes showed close relationship between Chilean stains and those from New York and Serbia. Analyses of concatenated *kdpA-gyrB* genes yield 7 Chilean haplotypes. Geographical distribution of the isolates was analyzed. Most of the Chilean isolates clustered separately from the Serbian and New Yorker strains, suggesting that Chilean bacteria have a different origin. Additional housekeeping genes and a higher number of isolates will be studied.

## Spatiotemporal analysis of wheat blast epidemiology (*Magnaporthe oryzae* Triticum pathotype) under natural field conditions

K. MILLS (1), L. Madden (1), P. Paul (1), D. Salgado (1)

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

Wheat blast is an emerging fungal disease of wheat caused by the Triticum pathotype of *Magnaporthe oryzae*. In years when weather is conducive for disease development, growers can suffer total crop losses. In order to understand the epidemiology of wheat blast, it is important to characterize the pattern of disease spread within a field. This pattern provides clues about the characteristics of the pathogen that affect the spatiotemporal dynamics of the disease. In this study, inoculum build-up and temporal and spatial spread from naturally occurring in-field disease foci (hotspots) were investigated in six 27 × 27 m² plots at three locations in Bolivia during the 2015 growing season. Plots were assessed for spike blast incidence and severity every 3 days. Assessments were made on a grid, with twenty spikes rated every 2.7 m. Hotspots increased in size over time and were associated with the highest disease ratings. Initially, plants adjacent to the hotspots had higher disease levels than plants located farther from the hotspots. As the disease progressed the steepness of the disease gradient decreased away from hotspots, and high levels of incidence and severity were observed throughout the plots. This trend was observed across the three locations. Preliminary spatial analyses suggest that hotspots are important in-field sources of inoculum.

## A Snapshot of Citrus Black Spot in Southwest Florida: Spatial Distribution in Two ‘Valencia’ Citrus Groves

K. HENDRICKS (1), P. Roberts (1)

(1) University of Florida - IFAS

Citrus black spot (CBS) an emerging disease of citrus in Florida caused by the fungus *Guignardia citricarpa* Kiely (anamorph *Phyllosticta citricarpa* (McAlpine) van der Aa) was identified in March 2010. The source of the introduction into the United States is still unknown. Disease inoculum, conidia and ascospores are dispersed by water and wind respectively. Due to quarantine restrictions and the potential for severe climatic events – hurricanes, it is imperative that the spatial distribution of the disease be monitored within the state and within infected groves. This study investigates the spatial distribution in two commercial ‘Valencia’ citrus groves located within the quarantine zones mandated by the USDA. Trees were mapped for the presence or absence of CBS in groves. Positive trees were defined as having fruit with at least a single hard or shot-hold spot lesion. The hard or shot-hole spot lesion was chosen due to its ease of recognition as a symptom of CBS. Preliminary spatial analysis of disease intensity and clustering were analyzed in R using the binomial dispersion index (D), and Ripley’s K function (K). The incidence of CBS in Grove 1 and 2 were 7.1% and 2.6% respectively in the 2013-2014 citrus season. The binomial dispersion index (D), revealed clustering of symptomatic trees for both groves in all quadrant sizes tested. Monte Carlo simulations for the L function confirmed CBS aggregation in both groves. Monitoring is ongoing.
## Sensitivity survey of *Alternaria* species to SDHI fungicides reveals regional and temporal resistance changes in pistachio orchards of California

P. LICHTEMBERG (1), R. Puckett (1), C. Cunningham (2), V. Leschenne (3), T. Michailides (1)

(1) University of California - Davis, U.S.A.; (2) Fresno State University, U.S.A.; (3) Swiss Federal Institute of Technology, Switzerland

In California, resistance of *Alternaria* spp. to succinate dehydrogenase inhibitor (SDHI) fungicides is common in pistachio orchards and results in difficulties in managing Alternaria late blight (ALB). The objective of this study was to monitor the development and spread of SDHI resistant *Alternaria* phenotypes and evaluate the cross-resistance among fluopyram (Fp), penthiopyrad (Pe), fluxapyroxad (Fd) and boscalid (Bo). In total, 798 isolates of *Alternaria* spp. were collected from 35 pistachio orchards where SDHI were used. *In vitro* assays were used to identify the frequency of SDHI resistant phenotypes and to compare the sensitivity distribution of isolates from previous years. The results revealed 85.8, 83, and 80% phenotypes in the current population resistant to Bo, Fd, and Pe, respectively. The Fp showed the smallest EC₅₀ value 16.74 µg/ml and the greatest frequency of sensitive phenotypes, at 45.8%. For the first time, highly resistant phenotypes for Fp were observed in California pistachio orchards, showing a moderate cross-resistance with Bo, Fd and Pe. Finally, the current population of *Alternaria* isolates (2015) showed sensitivity shifts toward resistance for Fd and Fp in comparison to those of the 2010 survey. Regional and temporal changes in the resistance of *Alternaria* spp. population will be discussed as well as the impact of future studies in order to delay SDHI resistance in California.

## Teratosphaeria pseudoeucalypti: An emerging pathogen with uncertain impact on *Eucalyptus* plantations

C. PEREZ (1), G. Balmelli (2), S. Simeto (2), E. a (3), M. Codina (3), R. Garcia (3), N. Ramirez (3), O. Bentancur (3), M. Wingfield (4)

(1) EEMAC, Universidad de la Republica, Paysandu, Uruguay; (2) INIA Tacuarembo, Uruguay; (3) EEMAC, Universidad de la Republica, Uruguay; (4)

FABI, University of Pretoria, Uruguay

*Teratosphaeria pseudoeucalypti* was first discovered in Queensland, Australia in 2010 causing a serious leaf blight disease known as Kirramyces Leaf Blight (KLB) on *Eucalyptus* commercial plantations. The pathogen was subsequently reported in 2014 in Argentina, Brazil and Uruguay, representing the first occurrence outside Australia. During 2015, a survey was conducted to assess prevalence, incidence and severity of KLB on *Eucalyptus camaldulensis* and *E. tereticornis* in Uruguay. A total of 80 plantations were visited, including these species and their hybrids. In each plantation, one transect of 20 trees in a row was scored for crown damage, based on percentage of defoliation and overall disease severity. The disease was found in all regions of the country. There was no clear pattern of distribution or severity of the disease with a distribution appearing to be generalized. Classification and regression tree analysis indicated that host species was the most important segregating factor based on severity, were *E. tereticornis* plantations showed a significantly lower severity than *E. camaldulensis* and their hybrids. KLB is widespread and serious on susceptible *Eucalyptus* spp., posing a threat to commercial plantation forestry in Uruguay. Future studies will concentrate on understanding the genetic diversity of a large collection of isolates that have emerged from this study and on selecting planting stock resistant to KLB.

## The occurrence pattern of plant viruses and their vectors in pepper fields in Korea

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

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

Pepper viruses usually cause severe symptoms on pepper resulting in a significant loss of the yield. While many viruses have been reported to infect pepper, some of them, including Cucumber mosaic virus (CMV), Broad bean wilt virus 2 (BBWV2), Pepper mottle virus (PepMoV), Pepper mild mottle virus, Tomato spotted wilt virus (TSWV), Beet western yellows virus (BWYV), and Potato virus Y, have already caused epidemics in pepper in Korea. In this study, we performed nationwide surveys from May to October 2015 to examine the occurrence of pepper viruses. Interestingly, the incidence of TSWV was highest in May but decreased dramatically month by month, whereas the occurrence of BWYV and PepMoV was detected from July and increased gradually until October. In addition, CMV and BBWV2 are the most prevalent in July and August and their incidences were maintained at high levels until October. In our surveys, we also investigated the incidences of thrips and aphids. Interestingly, the rate of population of thrips was greatest in May but decreased dramatically from July. On the other hand, the population of aphids gradually increased from May to August and the rate of alate aphids in the population was increased in September in pepper fields. Taken together, our survey results on the occurrence of viruses and their insect vectors suggested that the incidence of the pepper viruses is highly correlated with the population dynamic and life-cycle of their insect vectors.

## Field Survey of Tospoviruses Infecting Tomato in South Florida

O. Abdalla (1), S. Zhang (1)

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

Several viruses are known to infect tomato plants in south Florida, leading to serious losses to growers. *Tomato chlorotic spot virus* (TCSV), *Groundnut ringspot virus* (GRSV) and *Tomato spotted wilt virus* (TSWV), are three tospoviruses currently known to infect tomato in this area. These tospoviruses can cause losses in tomato ranging from a reduction in fruit quality to a complete destruction of the crop. TCSV was first detected from tomato plants in Homestead, FL in 2012, and it has caused significant losses to commercial tomato growers since the fall of 2014. During 2015-2016 growing season, a survey on tospoviruses infecting tomato in Homestead, Florida was conducted to determine the disease incidence and distribution of the tospoviruses in commercial tomato production fields. The percentage of tomato plants showing tospovirus-like symptoms in Homestead tomato fields ranged from 4 - 80% with an average of 35%. Two hundreds and seventy two tomato samples showing the symptoms were collected from over forty tomato fields. RNAs were extracted from the samples and tested by Reverse Transcription-Polymerase Chain Reaction (RT-PCR) using specific primers against TCSV, GRSV and TSWV to determine the incidence of both single and mixed infection of these viruses. RT-PCR results confirmed the infection of tomato plants with tospoviruses in Homestead. The results of the RT-PCR tests will be presented and more details will be discussed.

## Spatial dynamics of a begomovirus disease in processing tomato in central Brazil

A. BERGAMIN FILHO (1), A. Bergamin Filho (1)

(1) University of Sao Paulo, Brazil

The begomovirus disease is one of the most important viral diseases in the tomato crop in Brazil. The objective of this work was to study the spatial aggregation of begomovirus disease in two central-pivot irrigated areas for processing tomatoes in central Brazil. A total of 24 plots (15 × 15 plants) was weekly evaluated by visual inspection for identification of begomovirus symptomatic plants (BSP). The spatial distribution of the disease was analyzed by: binominal index of dispersion, Taylor's power law, and ordinary run. *Tomato severe rugose virus* was the predominant begomovirus species in tomato. It was also detected in a few non-tomato plants. The apatial pattern of symptomatic plants was slightly aggregated. A relevant difference was observed between plots located at the center (PC) and at the edge (PE) of the pivots. The distribution of BSP in PC was more aggregated than in PE. It is most likely that at PE the primary spread is the major component of virus epidemics in the field. These results suggest that the distribution of BSP in PC and PE is ruled out by at least three different dissemination mechanisms: a random (primary spread), and two aggregation components, a real secondary spread, and a "false" secondary spread. However, it is clear that the primary spread is the most important component in begomovirus epidemics. Hence, management of begomovirus diseases focusing only on the target fields is certainly not enough for an efficient control.

## Composition of leaf fibrous ring mediates *Diaphorina citri* preference of *Citrus sinensis* over *Citrus aurantium*

H. SHUGART (1), M. Rogers (1)

(1) University of Florida- Citrus Research & Education Center, U.S.A.
*Candidatus* Liberibacter asiaticus, causal agent of citrus greening disease, is transmitted by *Diaphorina citri*, and together they pose the greatest risk to *Citrus* production worldwide. Currently research into host plant resistance to the vector and bacterial pathogen is ongoing, with the aim of developing resistant *Citrus* selections. Our objective was to test the influence of leaf anatomy on *D. citri* probing behaviors that regulate bacterial transmission. Leaf morphometrics might inhibit bacterial transmission, and thus could be candidates for resistance breeding. We performed an electropenetrographic study and histological examination of leaves of two *Citrus* species differently preferred by the psyllid, *C. sinensis* (var. Valencia) and *C. aurantium* (var. Sour orange). We quantified durations, frequencies, and order of probing behaviors associated with bacterial transmission, and correlated those behaviors with leaf anatomy. Young Sour Orange leaves were less preferred and exhibited a thicker and more complete fibrous ring than did same-aged Valencia leaves. This more developed barrier impacted probing behaviors such that *D. citri* took longer to reach the phloem and ingested from it for shorter durations. Delay and reduction of phloem associated behaviors influences the success of bacterial transmission by *D. citri*. Future resistant varieties could use fibrous ring morphometrics as an anatomical marker for resistance to pathogen transmission.

## Transmission of a 16SrIII-L phytoplasma by leafhoppers (Scaphytopius marginelineatus) to cassava (Manihot esculenta Crantz) in Colombia

E. ALVAREZ (1), C. BETANCOURTH (1)

(1) CIAT, Colombia

As much as 30% of cassava production in Colombia, is affected by cassava frogskin disease (CFSD). The disease is associated with a 16SrIII-L phytoplasma that is transmitted between plants through vegetative propagation or by sap-sucking insect vectors. We collected 250 insect samples in Palmira and Santander de Quilichao; analyzed them for the presence of phytoplasmas during 2009 and 2012–2014; and detected a 16SrIII-L phytoplasma. Greenhouse experiments with symptomatic plants showed that potential insect vectors for cassava were hemipterans, particularly the Cicadellidae and Cixiidae families, and include the cicadellid leafhopper *Scaphytopius marginelineatus*. Colonies of this leafhopper were mass-reared on cassava (‘SM 909-25’) and bean (‘ICA Pijao’) plants. To determine the acquisition period, 50 individual insects were selected and exposed to phytoplasma-infected cassava plants for 2, 4, 6, and 8 days. The insects then underwent an additional 2 weeks’ incubation period, after which surviving insects were allowed to infest healthy cassava plants over a 5-to-7-day inoculation period. Molecular studies of plant tissues, based on PCR amplification and DNA sequencing, confirmed both the acquisition (6 days after inoculation) and transmission (3 weeks) of a phytoplasma. Sequences KP75989, and KP759898-KP759902 were reported to GenBank. Previous studies indicated that potential infestations of cassava crops by leafhoppers can be controlled by organic neem oil spray.

## Cladosporium interactions with the introduced insect spotted wing drosophila (Drosophila suzukii) may alter raspberry fruit rot epidemiology

C. SWETT (1), C. Carignan (1), K. Hamby (1), E. Koivunen (1)

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

In fruit crops, the interaction between frugivorous insects and fungi can alter pest and disease epidemiology, reducing marketable crop yields. In 2015 field surveys of fall red raspberries in Maryland, *Cladosporium* was recovered from fruit-rot affected berries at three farms, and 30% of *Cladosporium*-infested berries were co-infested with SWD larvae. Analyses of larval frass microbes recovered *Cladosporium* from 45% of larvae, with 3.3 colony forming units/ larvae on average. Sequence analysis identified both berry and larval frass isolates as either *Cladosporium cladosporioides* or *C. pseudocladosporioides*. All three tested isolates of each species were pathogenic on raspberries in detached berry assays, and one tested isolate could infect both ripe and unripe fruit in attached-berry assays. Disease incidence was significantly enhanced (*P* < 0.05) both when inoculated detached berries were mechanically wounded and when naturally infested attached berries were exposed to lab-reared SWD adults (ovipositor wounds), compared toounwounded controls. Many endophytic *Cladosporium* species are entomopathogens, so species causing fruit rot may be deleterious to SWD fitness; alternately, this may represent a commensal or mutually beneficial interaction. Overall, these results are among the first to suggest that SWD may interact with *Cladosporium* to significantly alter pre- and post-harvest fruit rot epidemiology.

## The ability of *Olpidium brassicae* to vector blueberry mosaic virus in southern highbush blueberries

T. MILES (1), A. Shands (1)

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

Blueberry mosaic associated Virus (BIMaV) is a recently described ophiovirus that is of concern in blueberry propagation systems. Members of the fungal genus *Olpidium* commonly found in plant roots, have the ability to move in these production systems using flagella, and possess the ability to vector Ophioviruses. In 2015, BIMaV affected blueberry plants were identified in a propagation nursery and a series of trap plants were utilized to determine potential vectors. Trap plant experiments were conducted with varying soil treatments (n=5), and hosts (carrot, lettuce, broccoli, cucumber) to determine the presence of *Olpidium spp.* Olpidium was observed in only lettuce trap plants (average of 6 resting spores/cm of root) and absent in the remaining trap plants. The ability of *Olpidium* species to vector BIMaV was tested through a series of concurrent inoculation experiments consisting of inoculated groups and control groups, treated with lettuce roots containing *Olpidium* resting spores or water, respectively. Each series of inoculation experiments varied in exposure time (10, 8, and 6 months) to root inoculum containing *Olpidium* resting spores and water, respectively. Transmission experiments showed *Olpidium* to be readily observed in the roots of inoculated southern highbush blueberry. By determining the ability of *Olpidium* spp. to vector BIMaV will aid in the management of blueberry propagation and increase our understanding of the basic biology of BIMaV.

## Olive Escudete, caused by *Botryosphaeria dothidea*, as Result of the Interaction fly-mosquito-fungus

J. MORAL (1), I. Eldesouki-Arafat (2), F. López-Escudero (3), E. Vargas-Osuna (2), T. Antonio (4), H. Aldebis (2)

(1) University of California, Davis, Kearney Agricultural Research and Extension Center, Parlier, CA, U.S.A.; (2) Dpto. Ciencias y Recursos Agrícolas y Forestales, Universidad de Córdoba. Campus de Rabanales, Edificio C4, 14071, Cordoba, Spain; (3) Dpto. Agronomía, ETSIAM, Universidad de Córdoba, Campus de Rabanales, Edificio C4, 14071, Cordoba, Spain; (4) Dpto. Agronomía, ETSIAM, Universidad de Córdoba, Campus de Rabanales, Edificio C4, 14071, Cordoba, Spain, Saint Kitts Nevis

The Dalmatian disease or escudete, caused by *Botryosphaeria dothidea*, is the most important disease of table olive fruit. The typical escudete (small shield) symptom is a sunken, necrotic, and circular (≈8mm) lesion that appears in green fruits. The olive fly (*Bactrocera oleae*) and mosquito (*Prolasioptera berlesiana*) whose larvae prey on fly eggs, are disputed as vectors of the pathogen. Here, we study the interaction among these three agents: fly-mosquito-fungus. In the field, we periodically sampled for the presence of the mosquito in fruits, which were naturally punctured by the olive fly or artificially by a needle for three seasons. Under lab conditions, the presence of the fungus in the mosquito and the fly was studied by microscopy, isolation on media, and DNA sequencing. According to our results, the mosquito is attracted by ovipositor punctures on the olive surface made by the olive fly. When the mosquito deposits its own egg adjacent to the fly egg, it also inoculates the fungus in the puncture. This allows the mosquito larvae to feed on *B. dothidea* mycelium when the fly egg is absent; a need that arises due to frequent false punctures (no oviposition) made by the fly to avoid predation by the mosquito larvae. Microscopic studies show that the mosquito female has a special structure (mycangia) to carry *B. dothidea* spores in the last abdominal segments close to the ovipositor, which suggests a mutualistic association between *B. dothidea* and *P. berlesiana*.

## First report of laurel wilt on sassafras in Arkansas

R. OLATINWO (1), C. Barton (2), J. Hwang (3), W. Johnson (3)

(1) USDA Forest Service, Southern Research Station, U.S.A.; (2) Arkansas Forestry Commission, U.S.A.; (3) USDA Forest Service, Forest Health Protection, U.S.A.
Laurel wilt, caused by the fungus *Raffaelea lauricola* T.C. Harrin., Aghayeva & Fraedrich, is a lethal disease of redbay (*Persea borbonia* (L.) Spreng.), sassafras (*Sassafras albidum* (Nutt.) Nees), and other species in the laurel family (Lauraceae). The fungus is transmitted by an exotic ambrosia beetle (*Xyleborus glabratus* Eichh.) native to Asia introduced into the United States in 2002. It was first reported at Port Wentworth, Georgia and rapidly spread across the south causing extensive redbay mortality. On December 2, 2015, visit to a landowner in Bradley County near Warren, Arkansas (33.64511°N, 92.05134°W) led to the first reported mortality of sassafras caused by laurel wilt in Arkansas. Moderate degrees of staining and beetle galleries were found from symptomatic trees at this location. Suspect beetles collected from sapwood of affected trees were later identified as *X. glabratus*. The presence of *R. lauricola* was confirmed by morphological characterization and molecular identification of cultures obtained from stained sapwood tissues. This is the first report of laurel wilt on sassafras or any other Lauraceae species in Arkansas. In 2014, laurel wilt was reported on sassafras in northern Louisiana (83 miles southwest). This new report in Arkansas may indicate northward movement of the disease into natural habitat of sassafras. However, the lack of redbay (a more attractive host) in this region may slow the northward spread of laurel wilt in Arkansas.

## Determination of vector population trends, species composition, and the role they play in the barley yellow dwarf complex in Northeastern Kansas

M. RAMOS (1), D. Rotenberg (1), A. Laney (1), A. Kieffaber (1), A. Whitfield (1)

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

Barley yellow dwarf is a complex disease that includes grass hosts, aphid vectors, and yellow dwarf viruses (YDVs) in the family *Luteoviridae*. BYD is responsible for crop losses worldwide, and is an especially important disease of KS wheat. The goals of this project were to characterize aphid vector species composition in native grasses and crops, and to document associated YDVs in NE KS. Aphid populations were monitored during fall, spring, and summer using yellow sticky cards that were placed in fields of sorghum, corn, wheat, and big bluestem, and aphids were identified via morphological features. *Rhopalosiphum padi* was found to be the most prevalent aphid species. Other species of importance were *Sitobion avenae*, *R. rufiabdominalis*, and *Schizaphis graminum*. Aphid species overlapped between crops and native grasses, supporting the hypothesis that perennial grasses can serve as reservoirs for YDVs. Multiplex RT-PCR was used for virus detection in plants and aphids, and both tested positive for several YDVs. We found barley yellow dwarf virus (BYDV)-PAS to be the virus species most common in both aphids and plants, followed by BYDV-PAV and BYDV-RPV. Aphids and plants were either singly- or co-infected with YDV species. Ultimately, this will lead to determination of the most efficient vectors of YDVs, their association with YDV-species composition in croplands, and the temporal influx of viruliferous aphid populations across agroecosystems in KS.

## Variation in transmission of *Tomato spotted wilt virus* (TSWV) from *Thrips tabaci* is not solely influenced by virus titer within individual thrips

J. BROWN (1), A. Jacobson (2), G. Kennedy (1), S. Lommel (1), T. Sit (1)

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

*Thrips tabaci*, a vector of TSWV, has been shown to vary greatly in the ability to transmit different isolates of TSWV. The objective of this study is to determine if the virus titer in individual *T. tabaci* influences the variation in transmission of TSWV from established *T. tabaci* isofemale lines collected from several locations in North Carolina. The isofemale lines were previously shown to differ in their ability to transmit various TSWV isolates collected from the same series of locations as the thrips. The virus-thrips line combinations were grouped in sympatric and allopatric pairings and the groups classified as low, moderate, or high transmitters based on the percent transmission of TSWV to *Emilia sonchifolia* leaf discs. Quantitative real-time PCR was done on *T. tabaci* from 12 selected virus-thrips line combinations to quantify virus titer in transmitting and nontransmitting individuals to determine if the variation in transmission was correlated with virus titer. The results show that variation in transmission efficiency of the different virus-thrips pairings are not explained by differences in virus titer within the thrips. This is in contrast to findings with other TSWV thrips vectors where virus titer was directly correlated with transmission efficiency. This suggests that the differences in transmission among these thrips-virus isolate pairings may be determined by the genetics of both the virus and the thrips, and their interaction.

## Turnip aphid preference for turnip plants infected with different strains of *Cauliflower mosaic virus*

M. ADHAB (1), J. Schoelz (1), D. Finke (1)

(1) University of Missouri - Columbia, MO, U.S.A.

*Cauliflower mosaic virus* (CaMV) is transmitted in a semi-persistent manner by several species of aphids. In this study, we investigated whether the turnip aphid (*Lipaphis erysimi*) exhibits a preference for healthy turnip plants (*Brassica rapa* L. var. rapa) or plants infected with CaMV. Two strains of CaMV, W260 and NY8153, as well as one recombinant isolate, H12, were used in this study. Preference assays were conducted with detached leaves in petri dishes or with whole plants. When given a choice between infected or healthy plants, aphids chose W260-infected and H12-infected plants significantly more often, regardless of the test. Interestingly, aphids did not exhibit a preference for turnips infected with CaMV strain NY8153 over healthy plants. The recombinant isolate H12 was chosen because it does not elicit symptoms in turnips; the aphid's preference of H12-infected plants over healthy turnips indicated that aphids were not attracted to plants based on differences in color or a pattern associated with disease. We are now focused on identifying differences in volatile profiles between healthy, W260- and NY8153-infected turnips that might influence the preference of an aphid for a specific host. These results suggest that CaMV infection affects the aphid's choice of host and this in turn may have implications for the spread of different strains of CaMV.

## Aphid-mediated potyvirus transmission: Host plant chemistry, vector biology and component interactions

K. GADHAVE (1), B. Dutta (2), J. Schmidt (2), R. Srinivasan (2)

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

Potyviruses pose a significant threat to cucurbit production in Georgia and in the Southeastern United States. Aphids transmit these viruses in a non-circulative manner, causing extensive yield losses worth millions of dollars annually. The present virus and aphid management strategy involves the use of insecticides and transgenic cucurbit cultivars. However, none of these approaches have proven to be sustainable, especially due to the development of vector resistance to insecticides, and the narrow spectrum of virus resistance in transgenic cultivars. It is therefore critical to explore the novel avenues for reducing vector populations and viral incidences. To attain this, we have undertaken a holistic approach that explores the fundamental mechanisms underlying aphid-potyvirus interactions in cucurbit systems. Through an array of lab and field experiments, we have been studying the virus transmission, component interactions in the pathosystem and their impacts on vector behavior, fitness and transcriptome. The impact of these results in the context of virus epidemics and management will be discussed.

## Transovarial and Sexual Transmission of *Tomato yellow leaf curl virus* in Whiteflies

W. MARCHANT (1), R. Srinivasan (1)

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

*Tomato yellow leaf curl virus* (TYLCV) is whitefly-transmitted Geminivirus that causes yield losses in tomato crops. The virus is persistently transmitted by the sweetpotato whitefly, *Bemisia tabaci*. The virus is considered non-propagative and non-transmissible transovarially or through mating. Feeding on infected plant tissue is the only method of acquisition according to most of the literature. However, a few recent studies from Israel demonstrate transovarial and sexual transmission of TYLCV as well as propagation within the whitefly. We have tested to see if transovarial and sexual transmission occur with the whiteflies and TYLCV present in Georgia using both molecular methods and plant transmission experiments. We found a very low rate of transovarial and sexual transmission suggesting negligible impact on virus epidemics. COI sequences from our local *Bemisia tabaci* and full-length TYLCV
genomes have been compared with sequences from Israel available on GenBank. Our *B. tabaci* COI haplotype is present in Israel, but differences in the TYLCV genome were found at the amino acid level. These differences could potentially explain the differences seen in insect-virus interactions.

## Molecular interactions between citrus tristeza virus and its aphid vector, *Toxoptera citricida*

N. KILLINY (1), S. Harper (2), C. C. El Mohtar (2), B. Dawson (2)

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

Citrus tristeza virus is transmitted by several aphid species, including *Aphis gossypii*, *Aphis spiraecola*, *Toxoptera aurantii*, and *Toxoptera citricida*. Considerable variations in the efficacy of transmission both among different vector species, and among CTV isolates are recorded. *T. citricida*, the brown citrus aphid, is the most efficient and specialized vector. It has a host range limited primarily to rutaceous species and transmits most CTV isolates at higher rates than other aphids. We demonstrated that CTV binds specifically to the lining of the cibarium of the aphid using fluorescently-labelled virions. *In vitro* competitive-binding assays between fluorescent virions and free viral proteins determined that the minor coat protein and two heat-shock-like proteins (p61 and p65) are involved in vector interaction. Treating the dissected mouthparts with proteases prior to the binding assay did not affect the binding of CTV virions while, chitinase treatment reduced the CTV binding to foregut. These findings suggest that CTV binds to sugar moieties of the cuticular surface of the aphid cibarium and not to embedded proteins. Additionally, the specific binding involves the concerted activity of three virally-encoded proteins.

## A strategy to study population variation of *Xanthomonas axonopodis* pv. *manihotis* using a new set of multiple-locus variable number tandem repeats

L. RACHE CARDENAL (1), C. Flores (2), C. López (3), B. Szurek (2), C. Vernièr (2), R. Koebnik (2), S. Restrepo (4), A. Bernal (4)

(1) Universidad de los Andes, BOGOTA, Colombia; (2) UMR Interactions Plantes Microorganismes Environnement, IRD-Cirad-UM, Montpellier, France, France; (3) Universidad Nacional de Colombia, Colombia; (4) Universidad de los Andes, Colombia

Multiple-Locus Variable Number Tandem Repeat Analysis (MLVA) is being used to analyze genetic diversity in phytopathogenic microorganisms. One of the most important pathogens affecting Cassava crops is *Xanthomonas axonopodis* pv. *manihotis* (*Xam*) as its infection relates to high losses in crop productivity. Several studies have reported that *Xam* populations change over time. It is important to assess population diversity in this pathogen as one of the basis to develop cassava resistant varieties. Studies to typify *Xam* strains performed by our group showed that using Variable Number of Tandem Repeats (VNTRs) have advantages over Amplified Fragment Length Polymorphisms (AFLPs), such as low costs and high discriminatory power using small sets of markers. Consequently, in this study we standardized and tested a new scheme of VNTR loci to analyze *Xam* populations. First, by analyzing 12 of 65 previously sequenced genomes of *Xam*, which represent the worldwide variability, we selected 16 VNTR loci. We standardized four multiplex PCR to amplify four microsatellite loci in each multiplex. Finally, the 16-VNTR scheme was used to assess population diversity of *Xam* strains collected in four Colombian Caribbean-region localities from 2008 to 2010, 2013 and 2014. Our results show that most of *Xam* genetic diversity is found within populations, and that the 16-VNTR scheme is a useful tool to discriminate *Xam* populations up to a local scale.

## Diversity of *Aspergillus flavus* on important agricultural crops produced in Bangladesh

M. ISLAM (1), P. Cotty (2)

(1) USDA ARS, U.S.A.; (2) USDA-ARS, School of Plant Sciences, the University of Arizona, U.S.A.

The hot, humid climate of Bangladesh is ideal for aflatoxin producing fungi. As a result, rice, maize, peanut, wheat and other crops grown in Bangladesh may become infected by these fungi and contaminated with dangerous concentrations of aflatoxins. Aflatoxins are toxic fungal metabolites produced by *A. flavus*, *A. parasiticus*, and closely related species. Aflatoxins cause increased risk of liver cancer, stunting, immune suppression, and in cases of acute exposure, liver necrosis and death. However, frequencies of aflatoxins and diversity of aflatoxin-producing fungi in crops of Bangladesh are poorly understood. To assess the magnitude of aflatoxin contamination and diversity of *A. flavus* in crops produced in Bangladesh, maize, rice, wheat and peanut samples were collected from 12 districts covering the major agro-ecological regions of Bangladesh. Aflatoxins were quantified in crop samples using immunochromatographic assays. Variable levels of aflatoxins were detected across the samples with a maximum concentration of 122.8 µg/kg total aflatoxins. Fungi associated with the maize and belonging to the L-strain morphotype were genotyped using 16 SSR markers. Genetic diversity of the L morphotype was high with 5 to 24 alleles per SSR locus, and 46 haplotypes among 50 isolates. Atoxigenic strains of *A. flavus* of potential value in aflatoxin prevention and native to Bangladesh were characterized.

## The genetic structure, virulence, and fungicide sensitivity of *Fusarium fujikuroi* in Taiwan

C. CHUNG (1), Y. Chen (2), M. Lai (3), C. Wu (2), A. Cheng (2), T. Lin (3), A. Cheng (4), C. Yang (2), H. Wu (5), S. Chu (6), C. Kuo (7), Y. Wu (4), G. Lin (4), M. Tseng (8), Y. Tsai (9), C. Lin (10), C. Chen (11), J. Huang (11), H. Lin (2)

(1) National Taiwan University, Taipei City, Taiwan; (2) National Taiwan University, Taiwan; (3) Taiwan Agricultural Research Institute, Taiwan; (4) Tainan District Agricultural Research and Extension Station, Taiwan; (5) Taoyuan District Agricultural Research and Extension Station, Taiwan; (6) Miaoli District Agricultural Research and Extension Station, Taiwan; (7) Taichung District Agricultural Research and Extension Station, Taiwan; (8) Kaohsiung District Agricultural Research and Extension Station, Taiwan; (9) Hualien District Agricultural Research and Extension Station, Taiwan; (10) Taitung District Agricultural Research and Extension Station, Taiwan; (11) National Chung Hsing University, Taiwan

*Fusarium fujikuroi* is the causal agent of bakanae disease, a common rice disease causing abnormal growth of seedlings and poorly filled grains. In Taiwan, the disease has long been well managed, but a disease outbreak was found in eastern Taiwan in 2009. To better understand the population genetics of *F. fujikuroi*, 16 polymorphic simple sequence repeat markers were newly developed and used to analyze 637 *F. fujikuroi* isolates collected in 14 rice-growing areas around Taiwan from 1996 to 2013. The isolates were classified into four highly differentiated clusters by Bayesian clustering analysis. The highly diversified vegetative compatibility groups and good numbers of *MAT1-1* and *MAT1-2* isolates sampled from all around Taiwan supported sexual reproduction. However, the biased mating type ratios, reduced effective population numbers, and linkage disequilibrium suggest nonrandom mating between individuals. Analysis of isolation by distance revealed a significant relationship between spatial distance and genetic similarity, which implies a geographical restricted gene flow and low dissemination ability of *F. fujikuroi*. Evaluation of 24 representative isolates on 8 rice varieties revealed differential levels of virulence, however no clear pattern of specific variety x isolate interaction was observed. Investigations of 60 representative isolates from early and recent years indicate that *F. fujikuroi* in Taiwan has developed increased resistance to prochloraz.

## Exploring genetic diversity in *Macrophomina phaseolina*, the causal agent of charcoal rot on soybean

V. ORTIZ LONDONO (1), M. Chilvers (1), K. Wise (2)

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

*Macrophomina phaseolina* is a soil and seed-borne fungal pathogen infecting more than 500 plant species and causing charcoal rot disease on soybean (*Glycine max*). Recently, a set of *Macrophomina* isolates collected from three plant species in Senegal, morphologically similar and phylogenetically closely related to *M. phaseolina*, were described as a new species, *M. pseudophaseolina*. However, occurrence of *M. pseudophaseolina* is unknown outside of Senegal. In this study, 36 putative *M. phaseolina* isolates, collected from soybean grown in the northern and the southern US were identified to the species level. ITS, EF-1α and ACT loci were sequenced and analyzed. Additional sequences were obtained from GenBank. The Incongruence Length Difference (ILD) test was not significant (p=0.95) and supported a decision to combine the sequences. Phylogenetic relationships of the isolates were explored using Maximum Parsimony (MP), Neighbor-Joining (NJ), Maximum Likelihood (ML) and Bayesian Inference (BI). The MP, ML, NJ and BI
phylogenies resulted in two separate clades corresponding to *M. phaseolina* and *M. pseudophaseolina* and placed all 36 isolates from soybeans in the U.S. in *M. phaseolina*. The Shimodaira-Hasegawa test indicates the ML tree as the best, but there were not significant differences among any of them. The ML tree reconstructed the *M. phaseolina* and the *M. pseudophaseolina* clades with a bootstrap support value of 100%.

## Population structure of the wheat pathogen *Zymoseptoria tritici* in France at different scales

A. SIAH (1), L. EL CHARTOUNI (2), B. TISSERANT (2), P. HALAMA (1), P. REIGNAULT (3)

(1) Institut Charles Viollette (EA 7394), Institut Supérieur d'Agriculture, Lille, France; (2) Unité de Chimie Environnementale et Interactions sur le Vivant (EA 4492), Université du Littoral Côte d'Opale, Calais, France; (3) Unité de Chimie Environnementale et Interactions sur le Vivant (EA 4492), Université du Littoral Côte d'Opale, Lille, France

*Zymoseptoria tritici* is currently the most damaging disease on wheat crops in Europe, especially in France, where it causes each year 1.7 t/ha yield loss in average. In order to better understand its genetic features, we characterized several *Z. tritici* populations (more than 1500 isolates), isolated in France over a ten-year period (2005-2015) and at different spatial scales (whole country, Nord-Pas de Calais region, field, 30 cm² square, plant, leaf and lesion) using SSR markers and mating type idiomorphs. Results revealed high and similar levels of genic and genotypic diversities at the country, region and field scales, but the diversity values gradually decreased from the field to finer scales such as plant, leaf and lesion. Bayesian and non-Bayesian statistical analyses revealed significant structuration of the global French population into three genetic clusters, and the Nord-Pas de Calais population into three sub-clusters, all distributed according to their geographical origin. However, no differentiation was found at finer scales. Both mating types were found at equal proportions at the country, region and field scales and co-occurred at all other investigated scales. This study highlighted for *Z. tritici* in France a high potential for sexual reproduction and genetic diversification, as well as a structuration of its populations at largest scales, agreeing with its high fitness degree and its ability of adaptation to local agro-climatic conditions.

## Stripe rust epidemics of wheat and barley and races of *Puccinia striiformis* identified in the United States in 2015

X. CHEN (1), A. Wan (2)

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

In 2015, stripe rust of wheat, caused by *Puccinia striiformis* f. sp. *tritici* (*Pst*), occurred throughout the United States and caused severe yield losses in the Great Plains. Stripe rust of barley, caused by *P. striiformis* f. sp. *hordei* (*Psh*), occurred in California, Montana, Oregon, Utah, and Washington. For monitoring virulence changes of the pathogens, stripe rust samples collected from 21 states were tested on 18 wheat and 12 barley differentials to identify *Pst* and *Psh* races, respectively. From 32 *Psh* isolates, three previously existing races were detected, of which PSH-48 (virulent only on Topper among the 12 barley differentials) and PSH-33 (virulent on differentials Topper and Abed Binder 12) were the major races with frequencies of 53% and 54%, respectively, similar to 2014. From 310 *Pst* isolates, 32 races were detected including 5 new races (PSTv-140, PSTv-141, PSTv-142, PSTv-143, and PSTv-144). The top five frequent *Pst* races were PSTv-37 (36%), PSTv-52 (33%), PSTv-18 (5%), PSTv-53 (4%), and PSTv-48 (3%). PSTv-37 and PSTv-52 were detected in every epidemiological regions, while PSTv-18, PSTv-53, and PSTv-48 were detected only in the western U.S. High virulence frequencies (>50%) were detected for *Yr6*, *Yr7*, *Yr8*, *Yr9*, *Yr17*, *Yr27*, *Yr43*, *Yr44*, and *YrExp2*; moderate (10-50%) for *Yr1*, *YrTr1*, and *YrTye*; low (<10%) for *Yr10*, *Yr24*, *Yr32*, and *YrSP*; and none for *Yr5* and *Yr15*. The information is useful for breeding for resistance and management of stripe rust.

## Preliminary screening of genetic markers for population analyses of *Magnaporthe oryzae* Triticum pathotype

J. STACK (1), C. Cruz (1), S. Dobham (1)

(1) Department of Plant Pathology, Kansas State University, Manhattan, KS, U.S.A.

Wheat blast, caused by *Magnaporthe oryzae* Triticum pathotype (MoT), is a devastating disease of wheat in South America; primarily in Brazil, Bolivia and Paraguay, and recently in a small area of northeast Argentina. It is a potential threat to wheat production worldwide with implications for food security. Very little is known about the population biology of the MoT. Therefore, understanding the population biology of wheat blast is important. Screening of current populations from Bolivia, Brazil, and Paraguay was performed using 30 inter-simple sequence repeat (ISSR) and retrotransposon microsatellite amplified polymorphism (REMAP) primers. Genes for MLST were also screened using primer sets targeting conserved genes and genes involved in different cellular functions. Combined phylogenetic analyses using ISSR and REMAP loci was able to differentiate populations from the three different regions based on 151 total loci, including 119 polymorphic and 32 monomorphic loci; showed 78.80% of percent polymorphism. The MLST based on genes involved in cellular function was found to be informative for the population genetics study. The screening of more markers for the population study is underway. This study will provide valuable information about genetic variation within and among current MoT populations of value for disease management and for the development of reliable and accurate detection tools for this emerging disease.

## Population structure and genetic diversity of *Fusarium effusum* in the USA

C. BOCK (1), M. Hotchkiss (2), K. Stevenson (3), B. Wood (4)

(1) USDA/ARS SE Fruit & Tree Nut Research Laboratory, 21 Dunbar Rd., Byron, GA, U.S.A.; (2) USDA-ARS SE Fruit and Tree Nut Research Laboratory, Byron, GA, U.S.A.; (3) Department of Plant Pathology, University of Georgia, Tifton, GA, U.S.A.; (4) USDA/ARS SE Fruit & Tree Nut Research Laboratory, Byron, GA, U.S.A.

Scab (*Fusarium effusum*) is the most destructive disease of pecan in the Southeast US. Infection is thought to occur solely through asexually produced conidia. To explore the population structure and genetic diversity of *F. effusum*, populations were hierarchically sampled from 11 orchards in Alabama, Florida, Georgia, Illinois, Kansas, Louisiana, Mississippi, Oklahoma and Texas. A total of 734 isolates was collected from up to 20 leaves from each of 3 to 6 trees in each orchard. Isolates were screened against 30 microsatellite markers. Polymorphic loci per population were ≥96.7%. Populations were genetically diverse (Nei's measure of genetic diversity = 0.505-0.692). An analysis of molecular variance showed 81% of variation occurred in the populations of *F. effusum* within trees, 3% in populations between trees within orchards, and 16% between orchards. Population differentiation was low (G<sub>ST</sub> = 0.17) and gene flow relatively high (Nm=2.5). However, there was some association between population genetic and log physical distance (r=0.471, P=0.0003). Ten of the 11 populations showed evidence of gametic disequilibrium (r²<sub>a</sub> = 0.024-0.057), which might be explained by a predominance of within-season asexual reproduction, or possibly by founder effects or migration rates. The results show that *F. effusum* is a genetically diverse pathogen, with the degree and distribution of genetic diversity often associated with sexual recombination.

## Spatial and temporal structure of *Sclerotinia sclerotiorum* in New York State

A. DUNN (1), J. Kikkert (2), S. Pethybridge (3)

(1) Hobart and William Smith Colleges, U.S.A.; (2) Cornell Cooperative Extension, Cornell Vegetable Program, U.S.A.; (3) Cornell University, U.S.A.

*Sclerotinia sclerotiorum* causes white mold on many vegetable and field crops in New York State, but the population structure has not been thoroughly studied. To assess spatial and temporal variation of *S. sclerotiorum* in New York State, 200 isolates were sampled from 10 bean fields in 2014, and 36 isolates were obtained from a historical collection (sampled from various hosts between 1982 and 2008). Isolates were genotyped using eight microsatellite loci. Mycelial compatibility group (MCG), in vitro growth rate, and aggressiveness on snap bean plants were also assessed for 22 selected isolates. Twenty-eight unique multilocus genotypes (MLGs) were identified (24 in 2014). One MLG represented 44 and 47 percent of isolates sampled in 2014 and prior to 2014, respectively. In 2014, genotypic diversity was low (3 to 8 MLGs per field) and Nei's genetic diversity varied among fields (0.42 to 0.90). Analysis of molecular variance indicated no significant spatial structure within or between fields, and the index of association differed significantly from zero in all fields. Isolates of the same MCG almost always shared the same MLG. Growth rate and aggressiveness varied slightly but
significantly among some isolates, but no significant association was found between the two measurements. Thus, the *S. sclerotiorum* population in New York State is highly clonal, and a small number of representative isolates would likely be sufficient to test new disease management strategies.

## Sub-lethal fungicides induce microsatellite mutation in *Sclerotinia sclerotiorum*

B. Amaradasa (1), S. Everhart (1)

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

Stress-induced mutations from sub-lethal fungicide exposure may be an important source of genetic variation for clonal plant pathogens. We used *Sclerotinia sclerotiorum* to assess the role of sub-lethal fungicide exposure in mutation at microsatellite loci. Nine sensitive isolates were exposed to long-term, sub-lethal doses of fungicides with different modes of action: boscalid, iprodione, thiophanate methyl, azoxystrobin and pyraclostrobin. Fungicides were plated in a logarithmic gradient and each isolate sub-cultured from the 50-100% inhibition zone for 12 consecutive generations and experiment repeated. Isolates at the first and twelfth generation from exposed and non-exposed lines were genotyped at six microsatellite loci. Control isolates showed no difference after 12 generations. Five of nine and two of nine fungicide-exposed isolates showed microsatellite mutations in both experiments. One locus did not mutate; all others range from 1 to 34 mutations per locus, with an average of 4.58 stepwise mutations. Most mutations were less than 20 bp, with one 80 bp mutation. The average mutation rate per locus among fungicide-exposed isolates ranged from 4-29 fold greater than the expected mutation rate of $1.3 \times 10^{-5}$ estimated for microsatellites in *Saccharomyces*. All fungicides resulted in mutations, with iprodione- and azoxystrobin-exposed isolates showing the highest frequency. Results suggest long-term sub-lethal fungicide exposure increase mutation rates.

## Molecular characterization and genetic diversity of *Mycosphaerella fijiensis* in Costa Rica using sequence based nuclear markers

J. RISTAINO (1), A. Saville (1), M. Charles (1), M. Wyatt (1), S. Chavan (1)

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

*Mycosphaerella fijiensis* is the causal agent of black sigatoka, a devastating disease of banana that can cause 20-80% yield loss in the absence of fungicides. *M. fijiensis* first appeared in Honduras in 1972 and spread to the rest of the western hemisphere, including major banana production regions such as Costa Rica. In order to characterize the genetic diversity of *M. fijiensis* in Costa Rica, one hundred and eighteen isolates, collected from Costa Rica and Honduras between 2010 and 2014, were examined using multilocus genotyping of six loci and compared to a previously published global dataset. A total of 13 haplotypes were identified from concatenated sequences of Honduran and Costa Rican populations, six of which were unique. Outside of the Americas, Costa Rican and Honduran populations shared haplotype diversity with populations in both Oceania and Southeast Asia for all loci, in particular Australia, Futuna, Tonga, and Papua New Guinea, the latter of which has been proposed as the center of origin for both banana and *M. fijiensis*. The genetic similarity to both regions suggests *M. fijiensis* may have been introduced into Honduras and Costa Rica multiple times. Given a relatively recent introduction, subsequent admixture events are expected to increase diversity of Costa Rican populations over time.

## Genetic and phenotypic diversity of *Puccinia kuehnii* of sugarcane from Brazil

T. MISTURA (1), A. Urashima (1), L. Porto (1), C. Sakuno (2), R. Arias (3)

(1) Federal University of Sao Carlos, Brazil; (2) Syngenta Crop Protection, Brazil; (3) USDA, ARS, South Atlantic Area Nat Peanut Res Lab, U.S.A.

Resistant genotypes are the most employed method to control orange rust of sugarcane (*Puccinia kuehnii*). This approach has been adopted in Brazil; however, recent outbreaks on previously unreported varieties show that the efficacy of this method is dependent and requires better knowledge of pathogen diversity. This can be achieved by applying genetic and phenotypic tools. Therefore, the present work aimed to examine the genetic distance and pathotype variability among isolates of *P. kuehnii* from São Paulo, the most important growing state of the Country. Thirty six rust isolates were genotyped by eight SSR markers and their genetic distances verified by the UPGMA clustering analysis using the software NTSYSpc v.2.2. Phenotypic data employed 22 isolates inoculated on five sugarcane varieties and evaluated at 21 days by examining percent diseased leaf area with Assess 2.0 and rating resistant/susceptible based on Santos et al. (2004). Moreover, an assay of disease progress over time was carried out with three isolates against five sugarcane cultivars examining disease score (Tai et al., 1981) at days 21, 28, 35 and 42. Our results showed two genetically distinct subpopulations of the pathogen with 79% of diversity, phenotypic data identified nine physiological races. In addition, disease progress over time showed that one cultivar considered resistant at day 21 became susceptible at day 42. Part of MSc dissertation by the second author at PPGPVBA, CAPES Scholarship.

## Short-term Host Selection Pressure Has Little Effect on the Evolution of *Verticillium dahliae*

K. PURI (1), S. Gurung (1), D. Short (2), G. Sandoya (3), R. Hayes (4), K. Subbarao (1)

(1) University of California, Salinas, CA, U.S.A.; (2) Division of Plant and Soil Sciences, West Virginia University, Morgantown, WV, U.S.A.; (3) UC Davis Genome Center, Davis, CA, U.S.A.; (4) United States Department of Agriculture, Agricultural Research Service, Salinas, CA, U.S.A.

Understanding pathogen evolution over time is vital for breeding and deployment of host resistance. To study this in the context of a soilborne pathogen, within-field population structure from three major hosts (spinach, tomato and lettuce) and the potential of host-directed evolution of *V. dahliae* race 1 were determined. A total of 408 representing 192, 44, and 172 isolates from spinach, tomato and lettuce, respectively, were collected from WA and CA. An additional 411 isolates were recovered over five years from an experimental screening plot infested with a race 1 strain in Salinas, CA. Isolates were genotyped using 13 microsatellite loci, race and mating type-specific primers. Results indicated no significant variation among the within-field populations from the three hosts. Isolates recovered from the screening plot over five years revealed no demonstrable changes in the race, species composition and mating type structure, except for six SSR locus variants. Replicated virulence assays of these six variants with the wild type race 1 strain on susceptible and resistant lettuce differentials suggested no significant virulence differences between them. This suggests that the deployed host resistance will remain stable against the native *V. dahliae* population, unless novel pathogen genotypes are introduced into the system.

## Population structure of *Sclerotinia subarctica* in England, Scotland and Norway

J. CLARKSON (1), R. Warmington (2), B. Nordskog (3)

(1) Warwick Crop Centre, University of Warwick, United Kingdom; (2) The Eden Project, United Kingdom; (3) Norwegian Institute of Bioeconomy Research, Norway

*Sclerotinia subarctica* is closely related to *S. sclerotiorum* which is a major pathogen of a wide range of dicotyledonous crop plants in many countries across the world. In contrast to *S. sclerotiorum*, *S. subarctica* is little studied as it was only identified relatively recently. However, preliminary evidence suggests that it is usually found sympatrically with *S. sclerotiorum* on the same host plants, but may be confined geographically to more northern latitudes. Using molecular approaches, we established that *S. subarctica* was rarely found in crop plants or wild buttercup in England (4.7% of *Sclerotinia* sp. isolates) and was more prevalent in Scotland (18.3%) and Norway (48.0%). Microsatellite data analysis of 157 *S. subarctica* isolates revealed a multiclonal population structure (75 haplotypes) with one or a few haplotypes more common than the rest. Eight haplotypes were shared between populations from Scotland and Norway while none were shared with England. *S. subarctica* isolates from England were also less diverse and confined to one location suggesting geographic isolation. Preliminary data on the effect of temperature on mycelial growth and sclerotial germination *S. subarctica* is discussed in relation to the pathogen's distribution.

## Tools for analysis of clonal population genetic data in R

Z. KAMVAR (1), J. Brooks (2), N. Grünwald (3)

(1) Oregon State University, U.S.A.; (2) Oregon State University, U.S.A.; (3) USDA-ARS, U.S.A.
Knowledge of the population dynamics and evolution of plant pathogens allows inferences on evolutionary processes involved in their adaptation to hosts, pesticides, and other environmental pressures. These microbial pathogens often require different tools for analysis due to the fact that they can be clonal or partially clonal. With the advent of high-throughput sequencing technologies, obtaining genome-wide data has become easier and cheaper than ever before with techniques such as genotyping-by-sequencing. In 2013, we created the widely used R package poppr for analysis of clonal populations. In 2015, we published several additional extensions to poppr for use with genomic data including the ability to define clones based on a genetic distance threshold, minimum spanning networks with reticulation, and sliding window analysis of the index of association. We present here an overview of poppr as it pertains to traditional and high-throughput population genetic data with select applications.

**Is host specificity influencing the population structure of *Phytophthora* spp. collected from potato and tree tomato in central and southern Colombia?**

C. Chaves (1), M. Mideros (1), G. Danies (1), S. Restrepo (1)

(1) Universidad de los Andes, Colombia

Late blight, caused by *Phytophthora infestans*, has been a major threat to global food security. In Colombia, late blight is considered one of the most limiting diseases on potato and tomato production. Additionally to potato and tomato crops, the pathogen has been associated with outbreaks on semi-domesticated plants such as tree tomato (Solanum betaceum) and other Solanaceous plants. Recently, a new *Phytophthora* species has been described infecting tree tomato crops in Southern Colombia (State of Nariño). Nonetheless, the distribution range of this new emerging pathogen in the country is unknown. Thus, the aim of this study was to investigate: i) if populations of *Phytophthora* associated to tree tomato in the central region of Colombia (States of Antioquia and Cundinamarca), the largest potato producing regions in the country, are genetically different from populations found on potato crops in this region; ii) if host preference exists in populations of *Phytophthora* associated to tree tomato and those associated to potato crops in this region; and iii) if populations of *Phytophthora* associated to tree tomato in the central region of Colombia are genetically distinct from those found in the southern region of the country. Preliminary results indicate that all isolates are of the A1 mating type and that host preference seems to exist between populations of *Phytophthora* associated to tree tomato and those associated to potato crops in the central regions of Colombia.

**Thirty-three years of lettuce downy mildew and counting: A phenotypic and genotypic narrative**

C. TSUCHIDA (1), F. Martin (2), R. Gil (1), O. Ochoa (3), I. Simon (3), R. Michelmore (3)

(1) Department of Plant Pathology, University of California, Davis, U.S.A.; (2) USDA-ARS, Salinas, U.S.A.; (3) Genome Center, University of California, Davis, U.S.A.

Downy mildew, caused by *Bremia lactucae*, is the number one foliar lettuce disease in California and worldwide. This heterothallic oomycete pathogen is highly variable and can rapidly change to overcome resistant cultivars. We are characterizing the pathogen population to determine which sources of wild germplasm will provide effective resistance in California that minimizes dependency on chemical protectants and aids organic farmers. We have been monitoring and archiving isolates of *B. lactucae* in California since 1982, mostly on an opportunistic basis. In December 2013, we began monthly surveys to provide a more comprehensive data set on pathogen variability. We report the patterns in virulence phenotypes observed in the Californian *B. lactucae* population over the past 33 years and an increase in the B₁ mating type. The Californian population has predominantly been the B₂ mating type and reproduces asexually. The occurrence of the B₁ mating type indicates the potential for sexual reproduction with prevalent B₂ isolates. Sexual progeny between recent B₁ and B₂ isolates are fit and highly variable and may explain the recent rise in novel virulence phenotypes. This finding necessitates a change in strategy to achieve more durable disease resistance. We have also sequenced the genomes of representative isolates collected throughout the three decades as a tool for understanding variation of *B. lactucae* in California and the world.

**Population structure of *Pseudoperonospora cubensis* isolates in Michigan and Ontario, Canada**

J. BELLO (1), Y. Guo (1), M. Hausbeck (1)

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

*Pseudoperonospora cubensis* is a highly destructive obligate oomycete pathogen that incites downy mildew disease in cucurbits worldwide. The pathogen populations in Michigan and Ontario are primarily affected by the movement of sporangia annually from the southern U.S. and Mexico or northern greenhouses where the pathogen can overwinter. The objective of our research was to monitor the population structure change over time in Michigan and Ontario, Canada. Sporangia were collected from cucurbit downy mildew (CMD) lesions using leaves of field grown cucurbits from 2007 to 2010, and sporangia collected in 2015. Population structure and genetic diversity were characterized using three marker types: (1) Selected genomic regions from three mitochondrial and nine nuclear genes previously used in oomycete genetic diversity and phylogenetic studies; (2) Selection from 942 simple sequencing repeat (SSR) markers derived from *P. cubensis* reference genome that were inside or partially overlapping with the annotated transcripts; (3) Genomic regions containing higher frequency of host and/or species specific single nucleotide polymorphisms (SNPs) adapted from previous studies. This research provides insight into changes of CMD populations in Michigan and Ontario over time which may contribute to overall management strategies.

**A shift in the population of *Phytophthora infestans* on Egyptian potato crops**

S. EL-GANAINY (1), Y. Ahmed (1), M. Soliman (1), A. Ismail (1), A. Tohamy (1), E. Randall (2), D. Cooke (2)

(1) Plant Pathology Research Institute, Egypt; (2) The James Hutton Institute, Scotland

Potato is an economically important crop in Egypt that is cultivated over three growing seasons, summer, fall (Nili) and winter. The winter crop that is grown from September to the end of February is particularly prone to late blight caused by *Phytophthora infestans* due to weather conditions that are conducive from the start of November and many crops suffer severe yield losses. We obtained 145 purified *P. infestans* isolates from the 2013 and 2014 winter crops which were genotyped using 12 SSR markers to generate data that would inform disease management. In contrast to previous work (3-year period 2010-12) in which the 13_A2 lineage was found, all isolates belonged to the 23_A1 lineage. There was no evidence for the existence of the A2 mating type or 13_A2 lineage even in the destroyed field crops of some cultivars (Cara, Bellini and Valor) that had been reported as resistant to 23_A1. This suggests that the previously reported virulence profile of 23_A1 has changed. Unlike 13_A2 the 23_A1 genotype is reported as sensitive to intermediate in response to metalaxyl. Polymorphisms in mainly 3 loci (SSR3, G11 and D13) discriminated 14 sub-clonal lineages of 23_A1 in Egypt suggesting the population has been present in Egypt for several years. The data have entered into the Euroblight database to allow temporal and spatial genetic diversity to be examined in comparison with other regional *P. infestans* populations.

**Evaluation of the microbial quality of cantaloupe fruit produced on raised or flat beds following a flooding event**

I. KIKWAY (1), M. Lewis Ivey (1), I. Kikway (1)

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

Floodwaters can harbor human and plant pathogens and present a major risk of contamination to fresh fruit and vegetable production in Louisiana. This study evaluated the effect of flooding on the microbial quality and safety of cantaloupe in an agricultural setting. Fields (raised or flat bed) containing mature cantaloupe fruit were flooded with surface water spiked with generic *Escherichia coli* ATCC strains 23716, 25922, and 11775. Generic *E. coli* levels on the surface of cantaloupe were enumerated at 24, 48 and 72 hr after flooding. Incidence of *Sclerotium rolfsii* and *Phytophthora capsici* was measured weekly beginning 7 days prior to flooding. A significant reduction of generic *E. coli* was observed on the surface of cantaloupe 24, 48 and 72
hr post flooding on fruit harvested from flat beds but only after 72 hr on the fruit from raised beds. Coliform levels remained the same over the 72 hr on fruit from both bed types. After 72 hr *E. coli* populations were reduced by an average of 1.8 and 2.5log MPN on fruit harvested from raised beds and flat beds respectively. No differences in *S. rolfsii* or *P. capsci* incidence on fruit from flat compared to raise beds were observed. However, disease incidence was significantly higher in flooded plots compared to non-flooded plots. These results provide science-based data that can be used to develop good agricultural practices programs to minimize food safety and plant disease hazards in flood stricken production regions.

## Weather patterns associated with Gibberella Ear Rot of Corn in Ohio

F. DALLA LANA (1), R. Minyo (1), P. Thimison (1), L. V. Madden (1), P. A. Paul (1)

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

Gibberella ear rot (GER), caused by *Fusarium graminearum* is an important disease of corn. Since outbreaks of GER are sporadic, understanding the weather patterns associated with this disease is fundamental for GER risk assessment. The objective of this study was to determine the association between weather conditions and GER intensity. During 2015, 15 hybrids were tested at 10 locations in Ohio. At the R1 growth stage, ten arbitrarily-selected primary ears per hybrid were inoculated in the silk channel with 2 ml of a spore suspension containing 2.5 × 10⁷ macroconidia of *F. graminearum* per mL. Air temperature, relative humidity (RH), surface wetness, and rainfall data were collected at 15-min intervals using local weather stations. At R6 all inoculated ears plus five non-inoculated ears were hand-harvested to estimate the incidence and severity of GER. Symptomatic infection was not observed on non-inoculated ears of any hybrid at any location, indicating that natural infection likely did not occur. Two locations had relatively high GER incidence (18 to 25%), four had low incidence (0.7 to 1.4%), while incidence was zero at the other locations. A preliminary descriptive analysis of the data indicated that GER was associated with 3 to 5 rainy days during the 7 days before R1, moderate temperatures (approximately 20°C) during R1, and RH above 80% during the week after R1.

## Evaluating soybean production in fields infested with *Heterodera glycines* and *Macrophomina phaseolina* with spatial regression analyses

H. LOPEZ-NICORA (1), J. Carr (1), A. Dorrance (2), T. Niblack (1)

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

*Heterodera glycines*, the soybean cyst nematode, and *Macrophomina phaseolina*, causal agent of charcoal rot, are economically important soybean pathogens. The spatial distribution of both pathogens was previously described as aggregated. The objective of this study was to use and compare ordinary least squares (OLS) and spatial regression models to evaluate soybean production in fields co-infested with these pathogens and to assess their role in soybean performance. Six trials were conducted in fields naturally infested with *H. glycines* and *M. phaseolina*. Within 30 days after planting, a 10 × 10 grid of contiguous quadrats (7.62 × 7.62 m) was marked in each field. Fifteen to 20 soil cores were collected at each grid for initial (Pi) and final (Pf) *H. glycines* egg and *M. phaseolina* colony-forming unit (CFU) counts, respectively. Twenty-five plants were collected and hand-threshed from every grid for yield analysis. Moran's *I* test indicated that yields from five fields were spatially correlated (P < 0.05). In these fields, spatial error and spatial lag regression models were fitted and compared with the OLS model, which does not account for spatial correlation. Spatial regression analyses revealed a significant interaction effect between *H. glycines* and *M. phaseolina* on soybean yield for fields with high initial Pi of both pathogens. Results from this study will aid in long-term disease management and evaluation of pathogen interaction in reducing crop production.

## A model for predicting onset of Stagonospora nodorum blotch in winter wheat based on pre-planting and weather factors

P. OJIAMBO (1), L. Mehra (1), C. Cowger (1)

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

Stagonospora nodorum blotch (SNB), caused by *Parastagonospora nodorum*, is a serious disease of wheat. Its management relies mainly on fungicide application after flag leaf emergence. The disease can occur prior to flag leaf emergence but the impact of onset time on yield has not been determined. Using 390 disease cases collected across 11 counties in North Carolina from 2012 to 2014, the effect of SNB onset on yield was analyzed to identify a disease onset threshold. A model was then developed to predict the time of SNB onset using pre-planting factors and cumulative daily infection values (cDIV) starting 1 to 3 weeks prior to the disease onset threshold. Time of disease onset explained 32% of the variation in yield (P < 0.0001), and day of year (DOY) 102 was identified as the disease onset threshold. Below-average yield occurred in 87% of the disease cases when onset occurred before DOY 102, but only in 28% of those cases where onset occurred on or after DOY 102. A binary logistic regression model indicated that cDIV accumulated until two weeks prior to DOY 102 and wheat residue were significant (P < 0.0001) predictors of SNB onset. The model had a correct classification rate of 0.94, and specificity and sensitivity rates were ≥ 0.91. Internal model validation based on two cross-validation techniques was also high. The model could serve as a useful decision support tool for fungicide application for SNB management in wheat.

## Incidence and severity of Phytophthora disease and assessment of inoculum levels in Texas citrus orchards

S. CHAUDHARY (1), M. Setamou (1), O. Alabi (3), J. daGraca (1), M. Kunta (1), V. Ancona (1)

(1) Texas A&M Kingsville Citrus Center, U.S.A.; (2) Texas A&M Kingsville Citrus Center, Weslaco, TX, U.S.A.; (3) Texas A&M AgriLife Research and Extension Center, U.S.A.; (4) Texas A&M Kingsville Citrus Center, TX, U.S.A.

*Phytophthora* foot and root rot disease, caused primarily by *P. nicotianae*, is present in Texas citrus orchards but there is a lack of information on the incidence and severity of the disease in the State. In addition, Texas citrus growers lack information on relative levels of *P. nicotianae* inoculum which are needed to make treatment decisions based on established thresholds. To address these knowledge gaps, a survey was conducted from February to June of 2015 across the three-county of Rio Grande Valley region where commercial citrus orchards in Texas are located. Twenty-four grapefruit, two tangerines and four sweet orange orchards were surveyed. 20 trees per orchard were rated for foot rot and gummosis lesion severity on a scale of 0 (no lesion) to 5 (entire trunk covered with lesion and gummosis). The survey revealed that 96% of Texas citrus groves are affected by foot rot and gummosis with the disease severity index ranging from 1 to 57%. Further analysis revealed that 20% of the surveyed citrus orchards had more than 50% disease incidence with an average disease severity of 23.6%. *Phytophthora* propagule counts in the soil ranged from less than, five to 118 cfu/cm³ with 46% of orchards having more than the reported damaging levels of 20 cfu/cm³. The data will be useful to growers for making treatment decisions and in predicting yield losses in the region due to foot rot and gummosis.

## Potential global distribution of blueberry twig blight (Diaporthe vaccinii) predicted by two species distribution modeling approaches

A. VAN BRUGGEN (1), H. Narouei-Khandan (1), C. Harmon (1), P. Harmon (1), C. Gardi (2), I. Koufakis (2), W. van der Werf (3), J. West (4)

(1) University of Florida, Gainesville, FL, U.S.A.; (2) European Food Safety Authority, Parma, Italy; (3) Wageningen University, Wageningen, Netherlands; (4) Rothamsted Research, Harpenden, United Kingdom

Blueberry twig blight (Diaporthe vaccinii) is an endemic disease in parts of the USA and Canada. It has been detected in limited locations in Belarus, Chile, China, Europe and Russia, but was reported to be eradicated in Chile and most of Europe, where it is a quarantine organism. Models on the potential spread of *D. vaccinii* do not exist. Published data on its worldwide occurrence were inventoried and supplemented with data from the National Plant Diagnostic Network. Occurrence and long-term climate data were entered in the species distribution models MaxEnt and Multi-Model Framework. The models correctly predicted that the climate in central-eastern USA, the US and Canadian west coasts, Latvia, Belarus and western Russia would be conducive to blueberry twig blight. Large areas in Europe, Eastern Australia, New Zealand, and smaller areas in South America and East Asia also were predicted to be conducive. Precipitation in the driest quarter contributed most to the prediction, followed by mean annual temperature, with an optimum of 10°C. The potential impact of *D. vaccinii* in Europe and its potential spread to native *Vaccinium* species is unknown.
The pathogen can be spread by natural and human-assisted means, and the risk of introduction, establishment and spread of *D. vaccinii* along different pathways, as well as potential impacts and the efficacy of risk reduction measures, will be assessed by the Plant Health Panel of the European Food Safety Authority.

**Symptomatology and epidemiology of Exobasidium leaf and fruit spot of blueberry**

R. INGRAM (1), H. Scherm (1), R. Allen (1)

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

Over the past decade, Georgia has become one of the largest producers of cultivated blueberries in the United States. As production has expanded, several new blueberry diseases have emerged, with Exobasidium leaf and fruit spot caused by the fungal pathogen *Exobasidium maculosum* being the most recent. The disease causes early-season leaf spotting and green spots on fruit. The pathogen produces both basidiospores and yeast cells, but its life cycle, including its overwintering biology and primary and secondary inocula, is unknown. Epidemiological field studies initiated in 2014 included the use of trap plants, a leaf spot demography study, spore trapping, and epiphytic population enumeration. Monitoring of surface populations showed that *E. maculosum* is capable of overwintering epiphytically on all blueberry tissues tested during dormancy. Demography and trap plant data collected in 2015 suggested that dispersal of primary inoculum occurs shortly after leaf/flower emergence, and that infection of primarily young and tender tissue is favored by prolonged rainy periods (>3 days/week). Disease progress in 2015 indicated that the disease is active from March through late May, and that initial fruit and leaf infection occur simultaneously. Additional observations revealed a novel symptom, lesions on emerging shoots that cause girdling and blighting. Although initial results suggest that the disease is monocyclic, further confirmatory studies are needed.

**Effect of soybean canopy closure on *Sclerotinia sclerotiorum* apothecia production, ascospore release, and primary plant infection of soybean**

M. FALL (1), M. Chilvers (1), A. Byrne (1), J. Wilbur (2), D. Smith (2)

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

Identifying the best timing for fungicide applications to get the optimum white mold control poses a major challenge to soybean producers. This study investigated the impact of canopy closure on apothecia development and ascospore release, to optimize fungicide application timing. In 2015, 0.355 m and 0.762 m row spacing replicated soybean plots were established at the MSU Montcalm Research Farm. The foliar fungicide Endura (boscalid) was applied to treated plots at the beginning of flowering, R1. The number of apothecia, ascospores, and the disease severity were monitored two times/week. Apothecia were first observed in the 0.355 m plots at full flower, R2, 8 d before they were observed in the 0.762 m plots, at beginning of pod development, R3. The number of apothecia was 50 times higher in the 0.355 m plots than in the 0.762 m plots. Incubated plants were symptomatic up to 13 and 18 days prior to symptom development in the 0.355 m and 0.762 m plots, respectively. There were no significant differences between non-treated and Endura treated plots in terms of final disease intensity and yield. However, there was a significant delay in disease severity (P<0.000) between treated and non-treated fungicide plots for the first 5 and 10 d after fungicide application in the 0.355 m and 0.762 m plots, respectively. There was a quantitative relationship between the number of apothecia and the soil temperature and between the number of apothecia and canopy closure.

**Modeling regulatory decisions in response to exotic pest detections: Pest case studies for decision makers**

L. BROWN (1), L. Brown (1)

(1) APHIS Plant Protection & Quarantine, Science & Technology, U.S.A.

Detections of exotic pest require responses from decision makers. Response options include management actions such as: eradication, containment, localized control, or the decision to not implement a program. When uncertainty is high, situational context can drive decision-making, leading to responses that are counter intuitive to epidemiological principles of infectivity, symptom expression, and pathogen spread. Spatially explicit models like Webidemics or spatially implicit models like ExPAT are used to model outcomes of decisions, reducing the reliance on highly uncertain information. These tools are applied based on the situation presented by the particular case study. Based on multiple case studies of all pathogen groups, we will present a matrix highlighting for decision-makers which tools are best suited for different exotic pest detections. In the future, the selection and parameterization of proper tools will supply decision makers with quantitative data to evaluate exotic pest detections.

**An impact network analysis for management of Xanthomonas wilt of banana: Adoption thresholds for regional success of single diseased stem removal**

K. GARRETT (1), C. Ocimati (2), J. Ntamwira (3), G. Blomme (4)

(1) University of Florida, U.S.A.; (2) Bioversity, Uganda; (3) Bioversity, DR Congo, DR Congo; (4) Bioversity, Ethiopia

Xanthomonas wilt of banana (BXW) is a major challenge to production for smallholder farmers of Central and Eastern Africa. Lack of resistant varieties and the often high rate of disease spread necessitate timely management. Single diseased stem removal (SDSR; removal at soil level of all visibly infected plants) is a promising approach to management requiring lower labor input compared to the previously advocated whole mat removal. We evaluate SDSR in a regional context using a new framework, Impact Network Analysis (INA). An INA is an evaluation of how technologies can have system-level impact through linked socioeconomic and biophysical networks. Estimates of short-term success rates for on-farm application of SDSR are available for several locations. INA includes projection of long-term regional outcomes based on rates of SDSR adoption. The risk of spread by garden tools is a given, but successful BXW management strategies vary by region. In most highland areas of eastern DR Congo, there is lower insect transmission risk associated with AAA type highland cooking/beer bananas. There, a regional strategy for community-level management does not depend on collective action; individual farmers can effectively manage BXW using SDSR, even if their neighbors do not manage effectively. By contrast, in most lowland areas, collective action is necessary for management, especially in zones planted to ABB type cooking/beer bananas highly susceptible to insect transmission.

**Seedborne inoculum thresholds of *Pseudomonas syringae* pv. *aptata*, causal agent of bacterial leaf spot, in 'baby leaf' Swiss chard crops**

L. DU TOIT (1), M. Derie (1), B. Holmes (1), I. Safni (2), C. Bull (2)

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

Most US table beet and Swiss chard seed production (~90%) occurs in western Washington and Oregon. *Pseudomonas syringae* pv. *aptata* (Psa), the cause of bacterial leaf spot, has been identified in beet and chard seed from this region, but seedborne inoculum thresholds have not been established. Chard seed naturally infested with Psa at 8.6 × 10³ CFU/g was mixed with seed of the same cultivar infested at <10 CFU/g to generate a log₁₀ series of infestation. Five replicate plots/infestation level were planted in a field in Mount Vernon, WA at ~10.7 × 10⁶ seeds/ha in May 2015 in a randomized complete block design. Bacterial leaf spot incidence was 52 ± 18% in plots planted with seed infested at 8.6 × 10³ CFU/g vs. 15 to 21% in plots sown with seed infested at <10³ CFU/g. Seed infestation level did not affect plant stand or amount of Psa recovered from leaves onto KBC medium. The trial was repeated in the fall, when moist, cool conditions resulted in a leaf spot incidence of 93 ± 2% across all plots, and Psa was recovered from every plot (9.97 × 10¹¹ ± 1.82 × 10¹¹ CFU/g dry leaf tissue), with no differences among seed infestation levels. Plots sown with seed infested at 8.6 × 10³ CFU/g had 33% fewer plants, 29% less leaf dry weight, and 126% more severe leaf spot than plots planted with seed infested at <10³ CFU/g. Psa was transmitted from infested chard seed, even at <10 CFU/g. Environmental conditions in the two trials affected seed transmission and leaf spot severity.
## Multilayer Networks Supporting Healthy Seed Systems for Smallholder Potato Producers in Tungurahua, Ecuador

J. HERNANDEZ NOPSA (1), J. Andrade-Piedra (2), G. Forbes (3), P. Kromann (4), S. Lei (5), J. Brisbane (5), K. Garrett (5)
(1) University of Florida, Gainesville, FL, U.S.A.; (2) International Potato Center, CGIAR Research Program on Roots Tubers and Bananas (RTB), Lima, Peru; (3) International Potato Center, CGIAR Research Program on Roots Tubers and Bananas (RTB), Beijing, China; (4) International Potato Center, CGIAR Research Program on Roots Tubers and Bananas (RTB), Quito, Ecuador; (5) Plant Pathology Department, Emerging Pathogens Institute and Institute for Sustainable Food Systems, University of Florida, Gainesville, FL, U.S.A.

The potato seed system is a multilayer network that includes social, economic, and agroecological components. Producers, breeding institutions, markets, technical support, and diseases are components of this complex system that interact and modify each other. We studied the structure of the networks of seed sources, markets, and IPM information sources, to understand impacts on the potato seed system of the Consortium of Small Potato Producers (CONPAPA) in Tungurahua, Ecuador. Using information about the structure of the seed system, we can consider how resilient the system is likely to be to stressors such as the introduction of a new pathogen. Which types of farmers may be more vulnerable to losses to such new stressors? One development goal is for there to be good access to quality seed and quality management recommendations, regardless of gender or farm size. Female farmers reported fewer diseases causing seed degeneration than male farmers, and fewer sources of IPM recommendations among the sources less likely to have commercial biases. Identification of key nodes that influence the success of seed systems (e.g., farmers, information, and seed sources) supports enhancement of the system, for improvements such as maximizing the distribution of good varieties, managing outbreaks, and targeted improvement of communication and infrastructure.

## Genetic diversity of *Pseudomonas syringae* causing bacterial leaf spot on table beet (*Beta vulgaris*) and Swiss chard (*Beta vulgaris* subsp. *cicla*)

I. SAFNI (1), L. Ramos-Sepulveda (2), P. Goldman (3), M. Derie (4), L. du Toit (4), S. Koike (5), V. Stockwell (6), C. Bull (2)
(1) Department of Agroecotechnology, Faculty of Agriculture, University of Sumatera Utara, Utara, Indonesia; (2) Department of Plant Pathology and Environmental Microbiology, Penn State University, University Park, PA, U.S.A.; (3) USDA/ARS, Salinas, CA, U.S.A.; (4) Washington State University, Mount Vernon, WA, U.S.A.; (5) Univ of California Cooperative Extension, Salinas, CA, U.S.A.; (6) USDA/ARS, Corvallis, OR, U.S.A.

Table beet and Swiss chard are grown throughout the US, with a significant proportion of baby leaf production in California and 90% of US seed production in Oregon and Washington. Bacterial leaf spot on beet and Swiss chard, caused by *Pseudomonas syringae* pv. *aptata* (Psa), has been reported from California and Oregon, but not yet from Washington. Since 2001, pseudomonads were isolated from surface-disinfested, symptomatic beet and chard plants grown in the western US, including Washington. Strains were variable for fluorescence on KMB agar medium, indicating populations may be diverse. Genetic diversity of the 99 strains was evaluated by repetitive extragenic palindromic PCR (rep-PCR) assay using the BOX AIR primer. Strains from a single Washington outbreak from a chard crop planted with infested seed had DNA fragment-banding patterns identical to the pathotype strain of Psa. The majority of the other isolates were identical, with a pattern similar to but distinct from the pattern of the pathotype. Three additional DNA fragment-banding patterns were observed. Strains from single outbreaks were represented in each of the alternative banding patterns. Preliminary multilocus sequence analysis and pathogenicity tests suggest a majority of bacterial leaf spot outbreaks on chard and table beet in California, Oregon, and Washington are caused by haplotypes distinct from Psa.

## Diversity and phylogeny of moko disease-causing *Ralstonia solanacearum* strains in Colombia

M. RAMIREZ (1), R. Jackson (2), V. Villegas-Escobar (3), J. Correa-Álvarez (3), C. Ramirez (1)
(1) Universidad de Antioquia, Colombia; (2) Reading University, England; (3) Universidad Eafit, Colombia

*Ralstonia solanacearum* is a soilborne plant pathogen that causes bacterial wilt on a wide range of hosts of economic importance. This disease is particularly devastating in *Musa* spp., where it is known as Moko disease. A major challenge in controlling this disease is to understand the diversity of the pathogen and how it adapts over time. With such knowledge, it may be possible to identify drivers of adaptation and inform changes in management practices. We therefore sought to characterize the genotypic and phenotypic diversity of *R. solanacearum* isolates obtained from plantations of *Musa* spp. in Colombia, and infer their phylogenetic relationship. Sixty-six isolates were collected. The genetic diversity and phylogenetic relationship among all the isolates were assessed by multiplex PCR and sequence analyses (*mutS*, *rplB*, and *egl*). Multiplex PCR assigned all the strains to phylotype II/sequevars 4 and 6. Similarly, the phylogenetic analysis of the gene sequences confirmed the classification of the strains into phylotype II; but, in addition, identified three separate clusters: IIA/6 (banana), IIB/4 (banana), and IIB/4 (plantain). Pathogenicity tests showed symptoms of wilt on banana plants after one month, but only strains isolated from plantain infected tomato. This study shows that plantain and banana plantations in Colombia harbor genetically diverse strains of *R. solanacearum*, belonging to at least three separate clades, and with differences in pathogenicity.

## Phylogenomic analysis supports polyphyly in *Pythium sensu lato*

E. GOSS (1), M. Ascunce (2), J. Huguet-Tapia (2), E. Braun (2), A. Ortiz-Urquiza (2), N. Keyhani (2)
(1) University of Florida, U.S.A.; (2) University of Florida, U.S.A.

*Pythium* is a large and morphologically diverse genus that includes economically costly root pathogens. Molecular phylogenies support the morphological clustering of *Pythium* into three monophyletic groups: Clades A-D are characterized by filamentous sporangia, clades E-J by globose sporangia, and clade K has been reassigned to the new genus *Phytopthium*. However, different markers have supported different relationships among these clades and to the genus *Phytophthora*. The absence of a robust phylogeny has prevented advances in *Pythium* systematics and evolutionary genomics. In the last six years, whole genome sequences have been generated for seven *Pythium* species and ten other oomycetes. We conducted a phylogenomic analysis using the nucleotide sequences of 277 orthologous genes found in the 17 analyzed oomycete genomes to examine the evolutionary relationships among the major clades of *Pythium*. Our results supported the position of *Phytophthium vexans* as a sister clade to *Phytophthora*. As expected, the remaining *Pythium* taxa formed two monophyletic groups. One group was composed of three taxa that correspond to *Pythium* clades A, B and C, and the other group contained taxa representing clades F, G and I, which was consistent with previous *Pythium* phylogenies. However, the group containing *Pythium* clades F, G and I formed a sister group to the *Phytophthora-Phytophthium* clade and thus *Pythium* clades F-J are not monophyletic.

## Identification of essential protein machinery involved in the plant disease resistance-signaling node in *Arabidopsis*, NDR1

A. CORRION (1), B. Day (2)
(1) Michigan State University, East Lansing, MI, U.S.A.; (2) Michigan State University, U.S.A.

Plant disease epidemics result in an estimated $200 billion in crop losses globally each year. To understand how plants and pathogens interact, and moreover, to develop a fundamental understanding of the mechanisms that drive pathogen virulence and adaptation, research in our group is focused on the molecular-genetic processes underpinning host resistance. To do this, we use the *Arabidopsis thaliana* - *Pseudomonas syringae* pv. *tomato* (Pst DC3000) pathosystem to study the role of non-host disease resistance-1 (NDR1), a key regulator of the disease signaling network of CC-NB-LRR (coiled-coil nucleotide binding leucine rich repeat) resistance (R) proteins. In the current study, we present a comprehensive analysis, utilizing a combination of genetic-, biochemical- and cell biology-based approaches to identify and define the NDR1 interactome. In brief, we utilized immunoprecipitation methods, coupled with liquid chromatography mass spectroscopy, to identify a list of candidate interacting proteins. Through this approach, we have identified a family of proteins that interact with NDR1 directly in planta, and have begun to characterize the role of these interactions in the NDR1-dependent resistance-signaling pathway. Data will be presented that provides support for a model that posits NDR1's association with key, Rho-related GTPases from plants (ROPs), plasma membrane-localized signaling proteins involved in the resistance to Pst DC3000.
### Examining putative motility genes in *Clavibacter michiganensis* subsp. *michiganensis*

C. PERITORE (1), M. Tancos (2), C. Smart (2)

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

The Gram-positive bacterium *Clavibacter michiganensis* subsp. *michiganensis* (*Cmm*) is the causative agent of bacterial canker of tomato (*Solanum lycopersicum*). *Cmm* historically has been characterized as a non-motile pathogen, however, it is known to move acropetally and basipetally in the xylem of infected plants. Under artificial xylem conditions in microfluidic chambers, non-flagellated *Cmm* actively moved against the media flow at ~6 µm/minute; these speeds were similar to those observed in * planta*. We have hypothesized that *Cmm* uses a type IV pilus (Tfp) mediated motility system similar to that of *Xylella fastidiosa*. Tfp are diverse in that biosynthesis can be mediated by *pil*, *com*, and/or *tad* operons in both Gram-negative and positive bacteria. Two tight adhesion (*tad*) operons have been identified through sequence similarity in the annotated genome of *Cmm*, and literature suggests they may play a crucial role in biosynthesis of Tfp. *Cmm* transformants were generated by disrupting the *tad* operon promoter. Microfluidic chambers will be utilized to mimic *in planta* conditions of xylem flow to assay and compare motility between mutants and wildtype. Genetic and microscopic studies will be complemented with *in planta* virulence assays to investigate the role of *tad* genes in the *Cmm*-tomato pathosystem. Understanding how the pathogen infects and moves in the plant will strengthen our biological knowledge of *Cmm*, possibly leading to novel disease control strategies.

### Differential effects of *Fusarium thapsinum* on host nitric oxide (NO) synthesis among stalk rot resistant and susceptible sorghum lines

A. BANDARA (1), C. Little (1)

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

*Fusarium thapsinum* (FT) is a hemibiotrophic fungus causing stalk rot in sorghum (*Sorghum bicolor* (L.) Moench). NO is a powerful oxidant involved in programmed cell death and FT-mediated NO synthesis was investigated in host stalk tissues. Two greenhouse-grown stalk rot resistant (SC599, SC35) and susceptible (Tx7000, BTx3042) sorghum lines were inoculated with FT and phosphate-buffered saline (control). At 7 and 30 d post-inoculation (d.p.i.), FT- and mock-inoculated stem cross sections were treated with 4-amino-5-methylamino-2',7?-difluorofluorescein diacetate, and observed under a confocal microscope (excitation 488; emission 515 nm). The fluorescence generated by controls was used as the baseline. At 7 d.p.i, strong green fluorescence was observed in FT-inoculated resistant lines, confirming the presence of NO, while no (or weak) signal was evident at 30 d.p.i. The susceptible lines revealed the opposite phenomenon, except for the signal detected in BTx3042 at 7 d.p.i. These patterns suggested that FT switches from biotrophy to necrotrophy by 30 d.p.i. While the presence of NO in SC599 and SC35 at 7 d.p.i contributes to resistance against the biotrophic phase, its absence at 30 d.p.i may decrease vulnerability to the necrotrophic phase. Tx7000 showed the opposite response and is susceptible to both phases of the pathogen's lifestyle. Based on NO response, BTx3042 appeared to be moderately resistant to the biotrophic phase but prone to the necrotrophic phase of FT.

### Stalk sugar content and charcoal rot disease reaction in grain sorghum

A. BANDARA (1), D. Weerasooriya (1), S. Liu (1), C. Little (1)

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

Charcoal rot, caused by *Macrophomina phaseolina* (MP), is an important fungal disease in grain sorghum (*Sorghum bicolor* (L.) Moench). An RNA-Seq experiment was conducted to explore the molecular basis of the sorghum-MP interaction. Stalk RNA extracted from MP- and mock-inoculated resistant (SC599) and susceptible (Tx7000) sorghum lines at 2, 7, and 30 d post-inoculation (d.p.i.) was subjected to RNA-Seq. Genes involved in UDP-glucose conversion, sucrose degradation to ethanol and lactate, and fructose degradation to pyruvate and lactate metabolism were significantly up-regulated in Tx7000 at 7 d.p.i., while those of SC599 were not differentially expressed. To confirm the results, stalk juice was extracted at 7 d.p.i from two MP- and mock-inoculated resistant (SC599, SC35) and susceptible (Tx7000, BTx3042) lines and subjected to HPLC. Concentrations (mg/mL) of sucrose, glucose, and fructose in stalk juice of SC599 (95.3, 23.9, 20.7, respectively) and SC35 (66.2, 31.2, 23.4) were significantly greater than those of Tx7000 (2.7, 6.5, 4.1) and BTx3042 (9.1, 10.9, 6.9) across MP- and mock-inoculations (P < 0.0001). Moreover, the reduction of sucrose, glucose, and fructose in MP-inoculated susceptible lines was 86.4, 26.5, and 19.8%, respectively, while those of the resistant lines were 17.0, 21.5, and 7.3%. Therefore, sorghum genotypes with higher stalk sugar content along with lesser sugar reduction under MP infection appeared to be more resistant to charcoal rot disease.

### Spectral signatures of chemical changes in the wheat cell-wall resulting from stripe rust infection in compatible and incompatible near isogenic lines

R. LAHLALI (1), G. Brar (2), D. Qutob (3), C. Karunakaran (1), H. Kutcher (2)

(1) Canadian Light Source Inc., Saskatoon, SK, Canada; (2) Department of Plant Sciences/Crop Development Centre, University of Saskatchewan, Saskatoon, Canada; (3) Canadian National Research Council- Plant Biotechnology Institute, Saskatoon, SK, Canada; (4) Department of Plant Sciences/Crop Development Centre, University of Saskatchewan, Saskatoon, SK, Canada

Wheat stripe rust, caused by *Puccinia striiformis* f. sp. *tritici*, is one of the most devastating diseases in North America. The biotrophic fungus produces uredia, consisting asexual urediospores, on wheat leaves along the veins and obtains nutrition from host through haustoria. In a compatible interaction, the fungus damages the cell-wall in a susceptible cultivar, however in a resistant cultivar the incompatible interaction occurs as a result of hypersensitive cell death in the host. The purpose of this study was to understand the biomolecular changes in cell-wall compounds in each of the interactions. Two Avocet lines: one carrying *Yr10* and other susceptible (no resistance allele) were inoculated with two isolates (one virulent on both and the other virulent only on *Yr10*). Infected and freeze dried leaves collected at 1 day and 15 days post inoculation were assessed by Fourier transform infrared (IR) spectroscopy. A significant difference in disease severity was observed between the two wheat lines. Principal component analysis of the IR spectra indicated substantial differences in the cell-wall components of the two wheat lines before and after pathogenic infection. There was a significant difference in the biomolecular changes between compatible and incompatible interactions. The most important IR peaks in the leaf cell wall that contributed to such differences were assigned to phenolic and aromatic rings, and proteins.

### Small RNA processing in *Sclerotinia sclerotiorum*

S. MARZANO (1), L. Domier (2)

(1) South Dakota State University, U.S.A.; (2) USDA/ARS, University of Illinois, Urbana-Champaign, U.S.A.

RNA silencing pathways are under-studied in plant pathogenic fungi. Once elucidated, they can be utilized to improve plant health by more effectively silencing fungal genes from host plants or viral vectors. In this study, we characterized changes in gene expression and small RNA accumulation in *Sclerotinia sclerotiorum*, a damaging and widely distributed fungal plant pathogen, in response to hypovirus infection. Our results indicated that the fungus produced small RNAs of predominantly 22 nt. The *S. sclerotiorum* genome was predicted to contain 59 families of microRNA-like (miRNA) genes, which similar to other recently characterized fungal species, were poorly conserved with other eukaryotes. In degradome analyses, 39 of the miRNAs sequences were predicted to mediate cleavage of 47 *S. sclerotiorum* mRNAs. Consistent with the role of RNA silencing in defense against mobile and infectious genetic elements, most of the small RNA sequences in virus-free *S. sclerotiorum* were derived from retrotransposons. Similarly, large amounts of predominantly 22 nt sequences were detected from the hypovirus genome, indicating that it also was targeted for degradation by host RNA-mediated antiviral defenses. These findings suggest that virus-induced hypovirulence could result from disruption of RNA silencing and that a robust RNA silencing pathway exists in *S. sclerotiorum* that can be exploited for RNA-based pathogen resistance strategies.
Investigation on the role of molecular composition and lignification of canola plant cell wall in resistance mechanisms associated with CR gene Rcr1

R. LAHLALI (1), T. Song (2), M. Chu (2), F. Yu (3), L. McGregor (2), B. Gossen (2), S. Kumar (2), C. Karunakaran (1), G. Peng (2)

(1) Canadian Light Source Inc., Saskatoon, SK, Canada; (2) Agriculture and Agri-Food Canada, Saskatoon, SK, Canada; (3) Agriculture and Agri-Food Canada, Saskatoon, Canada

Clubroot disease, caused by *Plasmodiophora brassicae Woronin*, is a serious soil-borne disease of crucifer crops worldwide. It poses a serious threat to canola production in western Canada. *Rcr1* is a new clubroot resistance gene identified in *Brassica rapa*. A multifaceted approach was employed to understand the molecular changes in canola plant cell wall associated with *Rcr1*-mediated clubroot resistance and the expression of defense-related genes involved in the lignin pathway. Fourier Transform mid Infrared (FTIR) spectral analysis showed that non-inoculated canola roots from both resistant (R) and susceptible (S) populations were grouped together and distinctly separated from those inoculated with *P. brassicae*. R and S plants responded differentially to the infection; it appeared that the cell-wall components of roots were changed for plants carrying *Rcr1*, including lipids, proteins, lignin, aromatic and phenolics ring, polysaccharides, and carbohydrates. qPCR identified the upregulation of nine defense-related genes involved in the lignin pathway, as well as in chitinase and xyloglucan endo-transglycosylate activities in roots carrying *Rcr1* relative to those of non-inoculated, but the level of increase was more pronounced with plants carrying *Rcr1*. The changes cell-wall components may reflect differential regulation of genes involved in host defense responses, in particular, the lignin pathways and chitinase synthesis mediated by *Rcr1* during clubroot resistance.

A sensitive method for quantifying cercosporin, a fungal-derived secondary metabolite, in plant tissue

J. SMITH (1), J. Lay (2), B. Bluhm (2)

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

*Cercospora* species infect a wide range of host plants, including soybean and maize. Many species of *Cercospora* produce the phytotoxic secondary metabolite cercosporin. The role of cercosporin as a virulence factor has been demonstrated in several *Cercospora* species. However, it is not clear whether cercosporin is important for virulence in all species of *Cercospora*. To gain a more complete understanding of the role of cercosporin in plant pathogenesis, sensitive methods for its detection and quantification are required. Therefore, the objective of this research was to develop a mass spectrometry based method for quantification of cercosporin from plant tissues with high sensitivity. To this end, extraction, sample cleanup, chromatographic separation, and mass spectrometry based detection methods were developed and optimized. The use of liquid chromatography coupled with a triple quadrupole mass spectrometer allowed detection of cercosporin at concentrations as low as 1 ng/ml. Currently, the feasibility of using this method for quantifying cercosporin in soybean leaves and seed infected with *C. kikuchii* and maize leaves infected with *C. zeae-maydis* is being examined. A sensitive method for detecting and quantifying cercosporin will provide a valuable tool for determining the role of this compound in virulence among *Cercospora* species.

Role of Cryphonectria parasitica Cdc48-like gene, CpCdc48 in regulation of growth & cell division

D. KIM (1)

(1) Chonbuk National University, South Korea

Functional analysis of a cell division cycle 48 (CDC48) ortholog, CpCdc48, from Cryphonectria parasitica was performed via construction of a CpCdc48-null mutant. Genotype analysis revealed that the putative CpCdc48-null mutant was a heterokaryotic transformant containing two different types of nuclei (i.e., one with the wild-type CpCdc48 allele and the other with the CpCdc48-null mutant allele). Although stable mycelial growth of the heterokaryotic transformant was observed on media containing hygromycin B, neither germination nor growth of the resulting spores was observed on selection media, suggesting that the CpCdc48 gene is essential. Microscopic analysis of germinated conidia from the heterokaryon demonstrated that although there were normal germinating spores due to the wild-type conidia, there were many residual conidia that did not germinate. However, with prolonged incubation, non-germinating conidia began to swell into gigantic globose spores. DAPI staining and FACS analysis of the gigantic spores revealed the presence of multiple nuclei. These gigantic conidia did not show any signs of polarized growth and underwent autolysis with further incubation. These findings indicate that the CpCdc48 gene is responsible for delayed cell cycle during spore germination, resulting in some karyokinesis, but not following spore cytokinesis. Thus, CpCdc48 is essential for cell division and polarized growth.

Wheat streak mosaic virus-encoded NIa-Pro and coat protein are involved in virus superinfection exclusion

S. TATINENI (1)

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

Cross protection or superinfection exclusion (SE) is defined as the phenomenon whereby initial infection by one virus prevents subsequent infection by closely related viruses. The mechanisms of SE are just beginning to be understood. Wheat streak mosaic virus (WSMV; genus: Tritimovirus; family: Potyviridae) is an economically important wheat virus. Proteins encoded by WSMV were individually expressed in wheat using an unrelated Triticum mosaic virus (TriMV) gene expression vector, followed by challenge inoculation with GFP-tagged WSMV (WSMV-GFP). Wheat plants infected with TriMV expressing WSMV P1, HC-Pro, P3, 6K1, CI, 6K2, NIa-VPg or NIb cistrons did not prevent superinfection by WSMV-GFP. However, expression of WSMV NIa-Pro or CP greatly reduced superinfection by WSMV-GFP, suggesting that NIa-Pro and CP are involved in SE. Expression of WSMV NIa-Pro or CP cistron with frameshift mutations did not exhibit SE against WSMV-GFP, indicating that the NIa-Pro and CP proteins but not the RNA sequences are involved in SE. A series of deletions comprising 60 amino acid (aa) sets covering the entire NIa-Pro cistron revealed that the complete NIa-Pro protein is required for SE activity. In contrast, WSMV CP aa 3-100 and 301-349 were dispensable for SE but not aa 101-300. Expression of WSMV CP aa 101 to 300 in TriMV exhibited the same SE activity as full-length CP. In reciprocal experiments, wheat infected with WSMV expressing TriMV CP prevented superinfection by TriMV-GFP.

In planta interaction between *Tobacco mosaic virus* 126 kDa protein and a host membrane protein (SNARE) and its influence on virus RNA accumulation

R. NELSON (1), A. Ibrahim (1), J. Schoelz (2)

(1) Samuel Roberts Noble Foundation, Inc., U.S.A.; (2) University of Missouri, U.S.A.

*Tobacco mosaic virus* (TMV) 126 kDa protein aids virus accumulation and is necessary for virus intercellular movement. This protein also co-localizes or associates with the endoplasmic reticulum and vacuole membrane (reviewed in Liu et al. 2013 Front. Plant Sci. 4:12. doi: 10.3389/fpls.2013.00012). The importance of the 126 kDa protein interaction with different host membranes as it relates to virus accumulation is not understood. As shown through yeast two hybrid analysis the 126 kDa protein interacts with two host proteins: one putatively involved in membrane fusion with vacuoles and the second with intra-vacuolar protease activity. Here we will show the co-localization of the membrane fusion protein, a plant SNARE (soluble N-ethylmaleimide sensitive attachment protein receptor) protein, in planta with the 126 kDa protein. Additionally, we are analyzing the influence of this and other SNARE proteins on tobamovirus [TMV and Turnip vein clearing virus (TVCV)] RNA accumulation using Arabidopsis T-DNA mutants. TVCV is being studied because of the different characteristics of its 126 kDa protein homolog, the 125 kDa protein (e.g. different aggregation states displayed during their ectopic expression). The importance of this SNARE protein for subcellular localization of the TMV 126 kDa protein and virus RNA accumulation will be summarized.
Overexpression of a modified plant thionin enhances disease resistance to citrus canker and Huanglongbing (HLB, citrus greening)

G. HAO (1), E. Stover (1)

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

Huanglongbing (HLB or citrus greening disease) caused by *Candidatus* Liberibacter asiaticus (Las) is a great threat to the US citrus industry. Citrus canker is also an economically important disease associated with a bacterial pathogen (*Xanthomonas citri*). In this study, we characterized endogenous citrus thionins and investigated their expression in different citrus tissues. We aim to develop citrus resistant to both HLB and citrus canker through expression of modified plant thionin. In order to improve effective for disease resistance, we modified and synthesized the sequence encoding plant thionin and cloned into binary vector pBinPlus/ARS. The construct was introduced into *Agrobacterium* strain EHA105 and used for citrus transformation. Transgenic Carrizo plants expressing the modified plant thionin were successfully generated through *Agrobacterium*-mediated transformation. Transgenic Carrizo plants expressing the modified thionin gene were challenged with *X. citri* 3213 at various concentrations, and showed a significant reduction in canker symptoms and a decrease in bacterial growth compared to nontransgenic plants. Furthermore the transgenic citrus plants were challenged with HLB via graft inoculation. The results showed significant decrease in the Las titer in transgenic Carrizo compared with nontransgenic plants. These data, thus, provide a promising approach to engineering citrus disease resistance against HLB and canker.

Transgenic Carrizo expressing 'Candidatus Liberibacter asiaticus' effector P235 displays Huanglongbing-like symptom and alters some gene expression

G. HAO (1)

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

*Candidatus* Liberibacter asiaticus' (Las) is the agent causing destructive citrus huanglongbing worldwide. A putative protein (P235) was identified in Las genome prophage region, which encodes a 123 amino-acid protein with a molecular weight of 14.19 kD. Green fluorescence protein (GFP)-fused with P235 appeared to accumulate in *Nicotiana benthamiana* cell nuclei by *Agrobacterium*-mediated transient expression, however, P235-GFP fusion protein in transgenic citrus targets the citrus chloroplast. P235 alone was constructed into a binary vector for citrus transformation. Over 40 independent transgenic Carrizo plants were obtained. Gene integration and expression were confirmed by molecular analysis. We observed that some P235-expressing Carrizo plants displayed HLB-like symptoms with leaf chlorosis and plant growth retardation. P235 expression levels, determined by RT-qPCR, correlated with HLB-like symptoms. Transcriptome analysis revealed that carbohydrate metabolism and biotic stress pathways were significantly affected in transgenic citrus expressing p235 effector. Higher transcript levels were determined for several stress-related genes involved in hormone metabolism and zinc ion binding. This is the first report that *Candidatus* Liberibacter asiaticus' encodes a protein that targets the chloroplast of plant cells and alters plant-host phenotypes.

Bioluminescent imaging as a sensitive tool for evaluation of *Ralstonia solanacearum* infection dynamics in resistant and susceptible pepper lines

H. DU (1), B. Chen (1), X. Zhang (1), S. Miller (2), G. Rajashekara (3), X. Xu (1), S. Geng (1)

(1) Beijing Vegetable Research Center (BVRC) of BAAFS, China; (2) Department of Plant Pathology, Ohio Agricultural Research Development Center, Ohio State University, Wooster, U.S.A.; (3) Food Animal Health Research Program, Ohio Agricultural Research Development Center, Ohio State University, Wooster, U.S.A.; (4) Beijing Vegetable Research Center (BVRC) of BAAFS, Beijing, China

Bacterial wilt, caused by *Ralstonia solanacearum*, is a major disease affecting pepper (*Capsicum annuum*) production worldwide. In this study, we evaluated the resistance of 100 pepper lines using *R. solanacearum* strain Rs-SY1 (phylotype I, isolated from sweet pepper in South China) and identified one resistant pepper line, BVRC 1. In order to study the bacterial spatial-temporal distribution in planta in real time, we generated a strongly bioluminescent *R. solanacearum* strain (BL-Rs7) using pXX3 carrying *luxCDABE* genes. BL-Rs7 was similar to its parent strain Rs-SY1 in morphology and pathogenicity. Furthermore, luminescence intensity of BL-Rs7 strongly correlated with bacterial population in planta (R² = 0.88). The utility of bioluminescence assay was validated by real-time in vivo monitoring of *R. solanacearum* infection dynamics in resistant and susceptible pepper lines BVRC 1 and BVRC 25, respectively, following root inoculation. The luminescence signals were detected in susceptible pepper plants from 3 days post inoculation (dpi), while little signal was detected in roots or stem of the resistant pepper line over 10 dpi and only a very low titer of bacteria was detected in the root. The results suggested that pepper line BVRC 1 exhibits resistance by interfering with translocation and multiplication of *R. solanacearum* in the stem.

Transgenic approaches for the genetic improvement of cowpea (Vigna unguiculata(L.)Walp) and soybean (Glycine max(L.) Merrill)

V. RAMACHANDRAN (1)

(1) BHARAT UNIVERSITY, India

Herbicide-resistant transgenic improvement of Cotyledone explants excised from 3 day old in vitro of *Vigna unguiculata* and *Glycine max* were infected with *Agrobacterium tumefaciens* (LBA4404) harboring chitinase (*chi II*) and bar gene. Co-cultivated cotyledon explants were inoculated on MS medium containing B5 vitamins BA, TDZ, L-glutamine, adenine sulphate, basta and cefotaxime. Shoots formed at the proximal end of cotyledons were transferred to MS medium containing B5 vitamins, GA3, basta and cefotaxime for elongation. Elongated shoots rooted in hormone free half strength MS medium. Rooted plants were subsequently established in soil. Transformed plants were conformed by herbicide resistance and PCR analysis. These ready systems enable us to improve seed qualities and agronomic characteristics by transgenic approaches. In addition, with the accumulation of cowpea and soybean genomic resources, convenient or promising approaches will be requisite for the determination and use of gene function in cowpea and soybean. In this article, we describe recent advances in and problems of cowpea and soybean transformation, and survey the current transgenic approaches for applied and basic research in India.

Co-expression of proteins by two virus vectors in the same cells of infected plants

M. Mendoza (1), H. Scholthof (1)

(1) Texas A&M University, U.S.A.

Plant viral vectors represent a technology that allows expression of valuable proteins at high levels in a relatively short time. For many purposes it may be desirable to express more than one protein in a single cell but that is often not feasible when using a single virus vector. Such a co-expression strategy requires the simultaneous delivery by two non-competitive viruses to express proteins. Here we report on the use of two agro-launchable virus vector systems based on *Tomato bushy stunt virus* (TBSV) and *Tobacco mosaic virus* (TMV) that both express suppressors to minimize the effects of Argonaute2-mediated silencing. To co-express proteins we used TBSV and TMV each expressing green fluorescent protein (GFP) of different sizes, in addition to co-expressing TBSV-GFP with TMV expressing red fluorescent protein (RFP). The results in *Nicotiana benthamiana* and tomato demonstrated that these two vectors accumulated in the same plant, same leaves, and in the same cells. Therefore, co-expression by these two vectors provides a biotechnological platform for fast and high level expression of potentially valuable proteins that need to form oligomers for activity.

Displaying foreign proteins and peptides on the surface of *Pepino mosaic virus* virions

C. LI (1), S. Shi (1), N. Yu (1), Z. Xiong (1)

(1) University of Arizona, U.S.A.
*Pepino mosaic virus* (PepMV), a member of *Potexvirus* genus, infects tomatoes and causes only mild symptoms under high intensity light conditions. However, its infection is highly productive and yields a large amount of virions in tomatoes. The flexuous PepMV virions (510 nm x ~13 nm in dimensions) are made of 1290 identical copies of a 25 kDa capsid protein (CP) with exposed N-terminus at the virion surface. Therefore, PepMV virions are ideal nanoparticles to display useful antigens and antibodies fused at the N-terminus of the CP. Using a highly infectious cDNA clone as the backbone, we have developed a virus-based expression system that is capable of displaying foreign proteins and peptides on the surface of PepMV virions. Two unique restrictions sites have been engineered immediately downstream the AUG translation initiation codon of PepMV CP to allow convenient and directional cloning of any foreign proteins and peptides into the expression vector. Several 2A self-cleavage peptides from picornaviruses have also been inserted between the restriction sites and PepMV CP to modulate variable ratios of CP fusion proteins and mature CP. mG-capped *in vitro* transcripts synthesized from the vectors are highly infectious, yielding high level expressions of foreign proteins and peptides in inoculated plants. The PepMV-based protein display system is expected to have a wide range of applications in pharmaceuticals and diagnosis.

## A single nucleotide polymorphism at the right terminal region of Mexican papita viroid is a virulent determinant factor on tomato

K. LING (1), R. Li (1)

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

*Mexican papita viroid* (MPVd), a pospiviroid, causes serious disease outbreaks on greenhouse tomato in North America. Two dominant genotypes (MPV-S and MPV-M), sharing 93.8% sequence identity, incited striking different symptom expression (severe and mild) on tomato 'Rutgers'. To determine genetic factor that is responsible for differential pathogenicity, using synthetic DNA approach, a series of chimeric infectious clones were generated through progressive replacement of each secondary structural domain in MPVd-M with their corresponding domain of MPVd-S. In plant inoculation assays, a chimaera containing the Terminal Right domain of MPVd-S incited severe symptom on tomato similar to that induced by MPVd-S. The other three chimeras incited only mild symptoms similar to that of MPVd-M. A single nucleotide polymorphism in the Terminal Right region between MPVd-M (176U:A185) and MPVd-S (174G:C183) was identified as the virulent determinant factor for MPVd. This result supports variable pathogenicity functions in different pospiviroids, which may lead us to design a better strategy in managing this emerging viroid disease.

## Two complete genome sequences of a new pathovar of *Xanthomonas oryzae* infecting wild grasses provide insight into evolution of pathogenicity

J. LANG (1), R. Koebnik (2), A. Pérez-Quintero (2), E. DuCharme (1), B. Szurek (2), J. Leach (1), V. Verdier (2)

(1) Colorado State University, U.S.A.; (2) Institut de recherche pour le développement (IRD), France

*Xanthomonas oryzae* (*Xo*) is a group of plant pathogenic bacteria composed of weak and highly virulent members that are genetically distinct from one another. Strains belonging to different virulent lineages from Africa and Asia and less virulent strains from the US have been used for comparative studies of pathogenicity and establishing effector repertoires. A close relative of *Xo*, *X. campestris* pv. *leersiae* (*Xcl*) was first described in 1957, and causes bacterial streak on the perennial grass, *Leersia hexandra*. *L. hexandra* is closely related to rice, globally ubiquitous around rice paddies, and is a reservoir of pathogenic *Xo*. We used draft and long read, single molecule, real time (SMRT) genome sequences of two strains of *Xcl* from China and Burkina Faso to determine the genetic relatedness of this organism with *Xo*. Novel transcription activator like (TAL) effectors and repeat variable domains (RVD) were discovered. Predicted TAL effector target sequences were identified in the available *L. perrieri* genome. Rice susceptibility gene homologs and unique TAL effector targets were also uncovered. Pathogenicity screening on *L. hexandra* and diverse rice cultivars confirmed that *Xcl* can colonize rice and produces weak not progressive symptoms. Overall, based on average nucleotide identity, effector repertoires and phenotype, we propose to rename *Xcl* to *X. oryzae* pv. *leersiae* and use this parallel system to better understand the evolution of pathogenicity in grasses.

## Phytopathogenic interaction between *Herbaspirillum rubrisubalbicas* and different genotypes of *Sorghum*

T. TULESKI (1), T. Tuleski (1), C. Espinoza (2), F. Plucani do Amaral (2), T. Pereira (2), R. Monteiro (4), E. Maltempi de Souza (4), F. Pedrosa (4), G. Stacey (2)

(1) University of Missouri, Columbia, MO, U.S.A.; (2) University of Missouri, U.S.A.; (3) University of Missouri, Columbia, U.S.A.; (4) Federal University of Parana, Brazil

*Herbaspirillum rubrisubalbicans* is a nitrogen fixing and plant growth promoting bacterium found in association with important grasses, such as maize and wheat. In contrast, this bacterium is a pathogen on some genotypes of *Sorghum bicolor*, being responsible for red stripe disease. The pathogen infects sorghum leaves leading to tissue necrosis, a reduction in photosynthesis and impaired leaf development. Twenty five genotypes of sorghum were inoculated with *H. rubrisubalbicans* with the great majority showing symptoms after 7 days. Bacteria were recovered from portions of the leaf increasingly distal from the site of inoculation. The results showed the spread of both bacteria and symptoms along the expanding leaf. Plants have the ability to mount a defense response to invading pathogens due to recognition of conserved motifs, termed microbe-associated molecular patterns (MAMPs). We tested the ability of MAMP-triggered immunity to protect sorghum plants from *H. rubrisubalbicans* infection. Sorghum leaves were pre-treated with chitin and flg22 (a conserved 22 amino acid peptide derived from bacterial flagellin) and then challenged with the pathogen. Evaluation of disease symptoms 5 days after inoculation showed strong plant protection in those leaves pretreated with chitin and flg22. The data suggest that *H. rubrisubalbicans* will be a useful pathogen in which to study the innate immunity response of sorghum, an important grain, forage and bioenergy crop.

## Expression of *FLS2s* from ‘Nagami’ kumquat and ‘Sun Chu Sha’ mandarin enhance citrus canker resistance in ‘Duncan’ grapefruit

V. FEBRES (1), Q. Shi (1), G. Moore (1)

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

In citrus, flagellin 22 (flg22), a pathogen-associated molecular pattern (PAMP) from *Xanthomonas citri* ssp. citri (Xflg22), triggers stronger defense responses in canker resistant genotypes than susceptible ones (Shi et al. Mol. Plant Pathol. 2025, 16:507). We have now identified the citrus genes encoding putative bacterial flagellin/flg22 receptors (*FLS2*). We isolated two genes each from ‘Sun Chu Sha’ mandarin (*Citrus reticulata*, *CrFLS2-1* and *CrFLS2-2*) and ‘Duncan’ grapefruit (*C. paradisi*, *CpFLS2-1* and *CpFLS2-2*), a canker resistant and a highly susceptible citrus species, respectively. Only one *FLS2* gene was obtained from the highly canker-resistant ‘Nagami’ kumquat (*Fortunella margarita*, *FmFLS2-1*). Flanking sequences suggest a rearrangement event resulted in the deletion of *FLS2-2* from the genome of kumquat. Transcriptional analysis indicated that the *FLS2-2s* reached higher expression levels in response to Xflg22 than the *FLS2-1s*, and the induction levels positively correlated with the level of canker resistance. Kumquat showed the highest *FLS2-1* pre-treatment expression level among the citrus species tested. We also transiently expressed *FmFLS2-1*, *CrFLS2-2* and *CpFLS2-2* in leaves of susceptible grapefruit. *FmFLS2-1* and *CrFLS2-2* conferred stronger responses to Xflg22 and reduced canker symptom development when compared to *CpFLS2-2*. We conclude that these two genes are valuable for engineering enhanced canker resistance and possibly other bacterial pathogens.

## Homologs of *CsLOB1* can function as disease susceptibility genes in citrus canker

J. ZHANG (1), J. Tapia (1), Y. Hu (2), J. Jones (1), N. Wang (1), S. Liu (3), W. Frank (1)

(1) University of Florida, Gainesville, FL, U.S.A.; (2) Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, P. R. China, Gainesville, FL, U.S.A.; (3) Kansas State University, Gainesville, FL, U.S.A.

The *lateral organ boundary domain (LBD)* genes is a plant-specific family of proteins that function as transcription factors for plant growth and development. *Citrus sinensis* *lateral organ boundary 1* (*CsLOB1*) functions as a disease susceptibility (S) gene in citrus bacterial canker (CBC). The *Citrus sinensis* genome contains thirty-four LBD members. We assessed the potential for additional LBD genes in citrus to function as surrogates for
*CsLOB1* and compared host gene expression upon induction of different genes. Using custom designed TAL effectors (dTALes), *CsLOB2* and *CsLOB3* functioned similarly to *CsLOB1* in CBC. Quantitative RT-PCR of mRNA during infection revealed a set of cell wall metabolic genes that are associated with *CsLOB1*, *CsLOB2* and *CsLOB3* expression. The genes may represent downstream genes involved in CBC.

### Ca. Liberibacter asiaticus carries a highly conserved, chromosomal CI repressor that binds an early gene promoter in its prophage

L. FLEITES (1), M. Jain (1), S. Zhang (1), D. Gabriel (1)

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

*Candidatus* Liberibacter asiaticus (Las) is one of three species of bacteria associated with Huanglongbing, arguably the most devastating disease of citrus. The majority of Las strains harbor bacteriophage that are maintained as stable lysogens in psyllids, but begin to enter a lytic cycle in planta. Las carries a CI phage repressor (LCI) with internal direct repeats that readily recombine and can create truncated translation products. LCI has been used as an epidemiological marker to follow the movement of Las populations; however, LCI has not been functionally characterized. Las strain UF506 has been maintained in isolation in a containment facility and transmitted from citrus to citrus and to periwinkle via dodder. All five LCI sequences isolated from curated Las strain UF506 in infected citrus encoded a full-length protein. By contrast, all seven UF506 LCI sequences derived from artificially infected periwinkle encoded a truncated version. Mobility shift assays demonstrated that LCI binds its own promoter and an early gene promoter on SC1, indicating potential involvement in lytic cycle activation. Promoter reporters are being evaluated to quantify repression in the presence of the full-length and truncated proteins, and additional potential binding sites are being investigated. If the LCI does function as a phage repressor, it would become a promising target for HLB control, since interfering with this function could trigger the lytic cycle in Las.

### The Type II-dependent secretion of virulence factors is necessary for systemic colonization by the xylem-limited pathogen, *Xylella fastidiosa*

B. INGEL (1), P. Wang (2)

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

*Xylella fastidiosa* (*Xf*) is a xylem-limited, gram-negative bacterium that causes Pierce's disease (PD) in grapevines. PD symptom progression is highly correlated with systemic colonization of the xylem, which is achieved by bacterial movement via the degradation of pit membranes separating adjacent xylem vessels. Pit membranes are readily dismantled by *Xf* cell wall-degrading enzymes (CWDEs), three of which have been implicated as virulence factors: a polygalacturonase (PG), and two endoglucanases (EngXCA1 and EngXCA2). As pathogenicity was nullified in a PG-deficient mutant, the PG has been a focal point for *Xf* inhibition studies. However, PG expression has proved to be difficult, and we have sought alternative CWDE inhibition targets. A knockout mutation of the Type II secretion system (T2SS) ATPase gene (*ΔxpsE*) resulted in a complete loss of pathogenicity similar to that displayed by the PG-deficient mutant. As *Xf* lacks a Type III secretion system, it is predicted that virulence factors such as CWDEs are secreted via the T2SS. Secretome analyses of both the *Xf* wild-type and *ΔxpsE* mutant strains had distinct differences in protein composition, and an antibody targeting EngXCA2 revealed that secretion of this endoglucanase is Type II-dependent. These results provide clear evidence that *Xf* relies on the T2SS for secretion of the CWDEs necessary for systemic colonization, and inhibition of this secretion system will significantly reduce *Xf* pathogenicity in grapevines.

### The chemotaxis regulator *pilG* of *Xylella fastidiosa* is required for virulence in *Vitis vinifera* grapevines

H. LIN (1), X. Shi (2)

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

*Xylella fastidiosa* is a Gram-negative, xylem-limited pathogenic bacterium that causes Pierce's disease of grapevines. Type IV pili of *X. fastidiosa* are regulated by *pilG*, a chemotaxis regulator in Pil-Chp operon involving signal transduction pathways. To elucidate the role of *pilG* in twitching motility and pathogenicity, phenotypes of wild type, *pilG*-mutant and a complementary strain were characterized. While *in vitro* studies showed that all tested strains had similar growth curves, mutant *XfApilG* exhibited a twitching defective phenotype and significant reduction in biofilm formation and cell-cell aggregation. Greenhouse *in planta* experiment further confirmed that grapevines infected with *XfApilG* showed very mild PD symptoms whereas grapevines inoculated with *X. fastidiosa* wild type and *XfApilG-C* developed typical PD symptoms. These results demonstrate that *pilG* of *X. fastidiosa* is required for twitching motility and full virulence.

### Biological functional Analysis of *tatB* in *Acidovorax citrulli*

T. ZHAO (1)

(1) Chinese academy of agricultrual sciences, institute of plant protection, China

*Acidovorax citrulli* causes bacterial fruit blotch of cucubits world wide. In order to explore the relationship between Twin-arginine Translocation system (Tat) and pathogenicity of *A. citrulli*, a *tatB* disruption mutant (?tatB) and its complement strains were constructed by homologous recombination. The virulence, motility, growth ability, extracellular polysaccharide and cellulase producing ability and biofilm formation of the wild type, mutant and the complement strains were tested to illustrate the influence of Tat system on *A. citrulli*. By comparing the expression of *trbC*, *hrcN*, *Aave_1810*, *Aave_0034* and *hrpE* gene with real-time fluorescent quantitative PCR, we explored the relationship between *tatB* genes associated with type III secretion system, Tat system related genes and cell toxicity related genes. The results showed the deficiency of *tatB* weakened the virulence, motility and growth ability of *A. citrulli*, while had no effect on the production of extracellular polysaccharide (EPS)?extracellular cellulase and biofilm formation. Real-time fluorescent quantitative analysis showed that the deficiency of *tatB* made the expressions of *trbC* and *hrcN* genes in mutant strain significantly increase, while the expressions of other genes notably decreased. Consequently, the deficiency of functional gene *tatB* in Tat affects some biological characteristics of *A. citrulli*, and ultimately weakens pathogenic ability.

### Secretome annotation in the citrus greening bacterial pathogen

L. Cano (1), M. Pitino (2), Y. Duan (2)

(1) University of Florida, U.S.A.; (2) US Horticultural Research Laboratory, U.S.A.

Citrus greening or also known as Huanglongbing (HLB) is one of most deadly citrus disease in Florida and in the world. The causal agent is the bacterium *Candidatus liberibacter asiaticus* (*CLas*). Plant pathogens secrete molecules that manipulate host physiology to achieve colonization. The main goal of our study was to identify and make available to the citrus community a set of candidate pathogen secreted proteins from *CLas* using genome sequences and *in planta* gene expression. Identified secreted protein candidates can be employed in high throughput functional genomics screens, commonly referred to as effectoromics, to test citrus germplasm for specific pathogen recognition by resistance *R* proteins. Our findings highlight about 10 candidate secreted proteins for pathogen-assisted breeding in citrus for resistance to HLB.

### The bifunctional catalase/peroxidase *KatG* is required for pathogenicity of *Xanthomonas albilineans* on sugarcane

M. JAIN (1), L. Fleites (2), S. Zhang (2), D. Gabriel (2)

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

Survival and multiplication of phytopathogenic bacteria, and disease progression are essentially contingent upon the pathogen's ability to detoxify reactive oxygen species (primarily $H_2O_2$) generated either endogenously through aerobic respiration or by the oxidative burst of plant cells during plant-pathogen interactions. *Xanthomonas albilineans* (*Xal*) is a gram-negative, xylem-invading pathogen that causes sugarcane leaf scald disease. Two
putative catalase genes XALc_0265 (*srpA*) and XALc_2991 (*katE*), and a bifunctional catalase/peroxidase XALc_0959 (*katG*) have been annotated in *silico* in the genome of Xal FL07-1. Xal *katG* was replaced by an enhanced Green Florescent Protein marker gene (*gfp*) via splice overlap PCR. Challenge inoculations of decapitated sugarcane plants by Xal ÄkatG::gfp exhibited a significant reduction in pathogenic symptoms on the newly emerged leaf blades. In addition bacterial viability in planta was significantly reduced compared to the wild type Xal FL07-1. Full-length *katG* cloned in the broad host range vector pUFR047 complemented the ÄkatG::gfp mutant strain for both pathogenicity and endophytic survival of the bacterium. Given that Xal *katG* is predicted to have a non-classical secretion potential, its role is also being investigated in dampening the apoplastic, non-specific low amplitude systemic wave of H₂O₂ elicited by the host cell that is essential for establishment of innate plant immunity.

## The role of the tomato genotype in susceptibility to *Salmonella enterica*

M. TEPLITSKI (1), M. Teplitski (1), M. Marvasi (2), J. Giovannoni (3)

(1) University of Florida, U.S.A.; (2) Middlesex University, United Kingdom; (3) Boyce Thompson Institute, Cornell University, U.S.A.

Tomatoes were linked to multiple outbreaks of salmonellosis. A hypothesis that some tomato genotypes are less conducive to *Salmonella* growth was tested. A screen of 30 cultivars revealed 10-1,000 fold differences in the ability of this human pathogen to multiply within fruits. Expression of *Salmonella* genes involved in the interactions was tomato cultivar- and ripening-dependent. *Salmonella* reached lower numbers within immature fruit, however, pigment accumulation *per se* did not have an effect on its growth within tomatoes. *Salmonella* reached similar population densities in tomatoes carrying green flesh or Hyper pigmented mutations. Growth of *Salmonella* was reduced in the tomato genotypes with defects in ethylene synthesis, perception and signal transduction. While mutation in the ripening-related ethylene receptor *Nr* resulted only in a modest reduction in *Salmonella* numbers within tomatoes, strong reduction of the *Salmonella* growth was observed in *rin* and *nor* tomato mutants. A commercial cultivar Sebring heterozygous for *rin* was less susceptible to *Salmonella* under the greenhouse conditions than other large-fruited varieties. No significant differences in growth of *Salmonella* within green and red tomatoes of cv. Sebring were observed in the greenhouse experiments, consistent with the hypothesis that ethylene signaling is responsible for the phenotype of green tomatoes. However, in the field, the phenotype of the cv. Sebring was masked by environmental conditions.

## Determining the genetic basis for stomatal aperture modulation by *Salmonella enterica* serovar Typhimurium strain 14028

J. MONTANO (1), S. Porwollik (2), M. McClelland (2), M. Melotto (1)

(1) Department of Plant Sciences, University of California, Davis, U.S.A.; (2) Department of Microbiology and Molecular Genetics, University of California, Irvine, U.S.A.

Pathogens that cause foodborne illness pose a challenge to food safety and security, as crops that are vectors for these human diseases may appear healthy, resulting in their integration to marketplaces throughout the world. Although some human pathogens that are introduced to the phyllosphere elicit an immune response within the plant, *Salmonella enterica* serovar Typhimurium strain SL1344 has been shown to disrupt plant innate immune signaling (including stomatal immunity) and survive for long periods of time inside the leaf. We have determined that, like SL1344, *S. enterica* serovar Typhimurium strain 14028 (14028) also disrupts stomatal immunity after 4 hours post-inoculation (hpi). We set up a genetic screen to elucidate the mechanism(s) by which 14028 modulates stomatal movement using a mutant library with high-coverage and multi-gene deletions created with lambda-red mediated gene-replacement. Leaf sections of lettuce (*Lactuca sativa* cultivar Salinas) were floated on bacterial inoculum (10⁵ CFU/mL) or a water control under light and stomatal aperture widths were measured via microscopy at 4 hpi. A total of 535 mutants were analyzed for their inability to stimulate stomatal opening at 4 hpi and compared to the wild type and water control. We observed that mutant strains lacking operons involved in secretion could not open stomata. Further characterization of identified mutants will aim to determine the individual genes that are pertinent for stomatal opening.

## In vivo and in vitro investigation of the mobilization of pathogenicity islands of *Streptomyces*

Y. ZHANG (1), D. Bignell (2), R. Zuo (3), Q. Fan (4), J. Huguet-Tapia (1), Y. Ding (3), R. Loria (1)

(1) Department of Plant Pathology, University of Florida, Gainesville, FL, U.S.A.; (2) Department of Biology, Memorial University of Newfoundland, St. John's, Newfoundland and Labrador, Canada; (3) Department of Medicinal Chemistry, University of Florida, Gainesville, FL, U.S.A.; (4) College of Plant Protection, Fujian Agriculture and Forestry University, Fuzhou, China

Approximately ten *Streptomyces* species including the most widely distributed *Streptomyces scabiei* cause the potato scab disease. The main pathogenicity determinant of scab-causing *Streptomyces* species is the phytotoxin thaxtomin A (ThxA). In *S. turgidiscabies*, ThxA biosynthetic genes are localized on a mobile pathogenicity island (PAI). However, the PAI mobilization in other *Streptomyces* species remains uncharacterized. We investigated the mobilization of the PAI of *S. scabiei* 87-22 both *in vivo* and *in vitro*. We sequenced genomes of ten pathogenic *Streptomyces* isolates from different locations, inferred the evolutionary relationships of pathogenic *Streptomyces* species, and identified a novel pathogenic species (*S. sp. 96-12*) that was isolated from Egypt. The PAI in *S. sp. 96-12* is identical to the PAI in *S. scabiei* 87-22 despite differences in their whole genome sequences. This strongly suggested *in vivo* mobilization of this PAI in these two strains. To test whether the PAI in *S. scabiei* 87-22 could indeed be mobilized, *S. scabiei* 87-22 deletion mutants with antibiotic resistance markers in the PAI, were mated with *S. diastatochromogenes*, a nonpathogenic species. The PAI of *S. scabiei* was found to be site-specifically inserted into the *aviX1* gene of *S. diastatochromogenes* and conferred pathogenicity. Our results demonstrated that the mobilization of the *S. scabiei* PAI could be responsible for the emergence of novel pathogenic species.

## Characterization of the T6SS secretion system of *Ralstonia solanacearum* and its role in virulence at low temperatures

A. BOCSANCZY (1), J. Yuen (1), A. Mangravita-Novo (1), D. Norman (1)

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

*Ralstonia solanacearum* is the causal agent of Bacterial Wilt, the worldwide devastating disease of hundreds of plant species. *R. solanacearum* is a 'complex' species due to its genetic diversity and wide host range; however all strains possess conserved secretion systems. The type VI secretion system (T6SS) has been associated with virulence, symbiosis and bacterial competition, however its function is not known in *R. solanacearum*. Previously, we discovered a functional T6SS in several strains of *R. solanacearum*, through a proteomic comparison. We created a T6SS related (Vip-B like) gene defective mutant in strain P673. In this work we confirmed that this mutant renders the T6SS non-functional. We show that the T6SS contributes to P673 virulence at low temperatures, only when soil inoculated. We hypothesized that the T6SS contributes indirectly to virulence, either by competition with other bacterial species, or with populations of the same species. To test this hypothesis we performed bacterial competition assays with other bacterial species commonly found in soil, however we did not find a difference. Currently, we are developing intra-species competition assays at 30°C and 18°C in order to determine the T6SS function and its dependency on temperature. We also present a comparative genomics of the T6SS system for all the strains sequenced to date. Characterization of the T6SS system in *R. solanacearum* will help elucidate its role in virulence at low temperatures.

## Global regulatory changes allow for the increased growth of *Salmonella* in soft rotted tomatoes

A. GEORGE (1), A. George (2)

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

While little is known about the mechanisms that allow *Salmonella* to multiply in tomatoes, even less is understood about how interactions of this human pathogen with phytobacteria contribute to its survival in alternate hosts such as plants. It is now known that bacterial soft rot contributes to an increased fitness of *Salmonella enterica* sv. Typhimurium in tomato fruits. The goal of this research is to understand the underlying mechanisms involved in this
phenomenon. The hypothesis is that a change in the tomato environment caused by *Pectobacterium* provides an overall benefit for *Salmonella*. In order to determine the full complement of functions that may be vital for the success of *Salmonella* in soft rot, we employed high throughput transposon sequencing (TN-seq). Libraries of mutants made via transposon mutagenesis were seeded into tomatoes inoculated with fully virulent *Pectobaterium*, less virulent *Pectobacterium*, and no *Pectobacterium*. As a result, pathways involved in nitrogen metabolism, motility, and other regulatory pathways have been identified as potentially playing a role in these interactions. Deletion mutants of genes identified by the TN-seq were then constructed via Wanner-Datsenko mutagenesis for individual testing in tomatoes. These individual mutations have confirmed the results of the TN-seq. We conclude that a variety of metabolic changes do in fact contribute to the increased growth of *Salmonella* in tomatoes.

## Expression of the FST1 from *Fusarium verticillioides* in a yeast strain lacking a major myo-inositol transporter gene

C. NIU (1), G. Payne (2), C. Woloshuk (3)

(1) Purdue University, U.S.A.; (2) North Carolina State University, U.S.A.; (3) Purdue University, U.S.A.

*Fusarium verticillioides* causes an important seed disease on maize and produces fumonisin B1 (FB1), a mycotoxin that is detrimental to human and animal health. Previous studies discovered that expression of *FST1* is required for FB1 production. In addition, strains lacking a functional *FST1* are less virulence on maize seeds and more inhibited by hydrogen peroxide than the wild-type strain. The mutation also causes reduced hydrophobicity of hyphae and macroconidia production. *FST1* encodes a putative protein with 12 transmembrane domains with sequence similarity to hexose transporters. However previous studies have failed to prove its ability to transport glucose, fructose or mannose. In this research, we used an *Saccharomyces cerevisiae* strain lacking a functional myo-inositol transporter (*ITR1*). This strain grows poorly in myo-inositol medium and is not inhibited by increasing concentrations of FB1. Expression of *FST1* in the *itr1* mutant restored growth on myo-inositol medium and the inhibitory effects of FB1 to levels observed in the wild-type yeast strain. These results indicate that *FST1* can function as a myo-inositol transporter. The evidence also suggests that *FST1* can transport FB1 into the fungal cell.

## Evaluation of Rice Varieties for Resistance for Autumn Decline in Arkansas

H. YAN (1), H. Zhang (2), M. Kim (3), B. Yoon (4), W. Shim (4)

(1) Texas A&M University, Plant Pathology & Microbiology, Texas A&M University, College Station, TX, 77843, College Station, TX, U.S.A.; (2) Texas A&M University, Plant Pathology & Microbiology, Texas A&M University, College Station, TX, 77843, College Station, TX, U.S.A.; (3) Texas A&M University, U.S.A.; (4) Texas A&M University Electrical & Computer Engineering, Texas A&M University, College Station, TX, 77843, U.S.A.; (5) Texas A&M University Electrical & Computer Engineering, Texas A&M University, College Station, TX, 77843, TX, U.S.A.

Symptoms of autumn decline include black root rotting with stunted and yellowish rice foliage starting as early as two weeks following permanent flood establishment. The problem is often most severe where cold well water first enters a rice field and may spread throughout the field, except on levees. The phenomenon was reported in Arkansas in a limited number of fields in 2004. However, there were several reports of autumn decline across the state of Arkansas in 2012 to 2015. Observations have shown fields having a clay loam soil texture as more prone to the autumn decline phenomenon than others commonly cropped to rice. The root rotting symptoms often start a few weeks after flood establishment and become progressively worse throughout the season if unmitigated. In situations where root rotting is severe, fungi grow into the crown which limits function of the whole root system and prevents translocation of water and nutrients from the soil to the plant. In moderate to severe cases, tillers break off easily and plant death may occur rapidly leading to significant yield losses. Twenty commercial rice varieties were evaluated in the field in 2015 and based on their reactions a rating scale was developed. The nature of autumn decline problem, reactions of the varieties and the rating scale will be presented.

## Global Analysis of Horizontal Gene Transfer in *Fusarium verticillioides*

S. GAO (1), J. Wisecaver (2), Y. Zhang (3), L. Ma (3), A. Rokas (2), S. Gold (4), A. Glenn (4)

(1) Department of Plant Pathology, University of Georgia, Athens, GA, U.S.A.; (2) Department of Biological Sciences, Vanderbilt University, Nashville, TN, U.S.A.; (3) Department of Biochemistry and Molecular Biology, University of Massachusetts, Amherst, MA, U.S.A.; (4) USDA ARS, Richard B. Russell Research Center, Toxicology & Mycotoxin Research Unit, Athens, GA, U.S.A.

The co-occurrence of microbes within plants and other specialized niches may facilitate horizontal gene transfer (HGT) affecting host-pathogen interactions. We recently identified fungal-to-fungal HGTs involving metabolic gene clusters. For a global analysis of HGTs in the maize pathogen *Fusarium verticillioides* (*Fv*), we used a phylogenomic pipeline to identify potential candidates. From an initial output of 1801 genes, manual curation revealed 117 strong HGT candidates acquired mostly from bacteria. Preliminary evaluation suggested several enriched metabolic pathways (e.g. lysine biosynthesis and nitrogen metabolism) compared to the frequency of such genes within the *Fv* genome. Interestingly, among the five candidates that were categorized as being involved in lysine biosynthesis, FVEG_09873 appears only in *Fv* and has no orthologs in other fungi. This gene was acquired from plant-associated Proteobacteria and putatively encodes a diaminopimelate epimerase in the diaminopimelic acid pathway employed by most bacteria but not commonly found in fungi. Another promising HGT candidate acquired from bacteria is FVEG_10494 that encodes a putative class-III aminotransferase, functioning in nitrogen metabolism. It is highly expressed when *Fv* is exposed to nitric oxide. Additional analyses will focus on other HGT candidates having limited fungal distribution. Functional characterization is underway to determine the significance of select HGT candidates in fungal adaptation.

## A Battle in a Kernel: Molecular Exploration of Antagonisms between Two Maize Endophytes, *Fusarium verticillioides* and *Acremonium zeae*

M. GAO (1), A. Glenn (2), S. Gold (2)

(1) The University of Georgia, U.S.A.; (2) United States Department of Agriculture Toxicology and Mycotoxin Research Unit, U.S.A.

*Fusarium verticillioides* (*Fv*) is a prevalent seed-borne maize endophyte capable of causing severe kernel rot and fumonisin mycotoxin contamination. Within maize kernels, *Fv* is primarily confined to the pedicel, while another seed-borne fungal endophyte, *Acremonium zeae* (*Az*), is observed in embryos. *Az* produces two lactam-containing antibiotics, named pyrrocidine A (PA) and B (PB), with PA exhibiting greater inhibitory activity against *Fv* and other fungi. PA differs from PB only by having a double bond in the lactam ring, suggesting the importance of the lactam moiety for toxicity. Preliminary experiments indicated the fungus is able to metabolize PB. In the *Fv* genome, 38 out of the 46 identified lactamase domain-containing proteins are predicted to carry out lactam hydrolysis. We recently characterized one lactamase gene (FVEG_08291) essential for the hydrolysis of 2-benzoxazolinone, a maize phytoanticipin, that significantly induced the expression of this lactamase and adjacent clustered genes. Likewise, we predict that pyrrocidines will induce one or more lactamase gene(s) and that the encoded enzyme(s) will function to hydrolyze these *Az* metabolites. RNA-seq experiments are underway to assess the *Fv* transcriptional response to pyrrocidines and to *Az* co-inoculation. RNAseq data should allow us to identify the interacting lactamase gene(s) and help us better understand the molecular mechanisms underlying the metabolic antagonism between these endophytes.

## A dynamic regulatory network model underlying stomatal infection in *Cercospora zeae-maydis*

J. RIDENOUR (1), B. Bluhm (1)

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

*Cercospora zeae-maydis* causes gray leaf spot of maize, which is considered to be one of the most important foliar diseases of maize worldwide. Intriguingly, *C. zeae-maydis* has evolved an infection strategy that exploits host stomata. Prior to entering the host, the fungus undergoes a series of defined morphogenetic transitions collectively termed pre-infectious development. Despite the importance of pre-infectious development, the underlying genetics are poorly understood. Genetic regulatory network (GRN) modeling offers a novel approach to understand the complexity underlying plant-
pathogen interactions. The overarching goal of this project is to utilize GRN modeling to identify central regulatory hubs that control pre-infectious development in *C. zeae-maydis*. To this end, we first developed a methodology to collect fungal tissue from inoculated maize during pre-infectious development and extract RNA of sufficient quality for RNA sequencing (RNA-seq). RNA-seq data is being generated via the Ion Torrent platform, and will be used to generate a GRN model. Preliminary transcriptome analysis has revealed that genes involved in transcriptional regulation, signaling, and stress and defense are enriched during pre-infectious development. Defining pre-infectious development at the systems level will provide mechanistic insight into the developmental program employed by *C. zeae-maydis* and augment efforts to identify antifungal targets and improve disease resistance.

## Arabidopsis thaliana: A model host plant to study plant-pathogen interaction using rice false smut isolates of *Ustilaginoidea virens*

J. LI (1), M. Andargie (2)

(1) South China Botanical Garden, Chinese Academy of Sciences, China; (2) South China Botanical Garden, Chinese Academy of Sciences, China

Rice false smut fungus which is a biotrophic fungal pathogen causes an important rice disease and brings a severe damage where rice is cultivated. We established a new fungal-plant pathosystem where *Ustilaginoidea virens* was able to interact compatibly with the model plant *Arabidopsis thaliana*. Disease symptoms were apparent on the leaves of the plants after 6 days of post inoculation in the form of chlorosis. Cytological studies showed that *U. virens* caused a heavy infestation inside the cells of the chlorotic tissues. Development and colonization of aerial mycelia in association with floral organ, particularly on anther and stigma of the flowers after 3 weeks of post inoculation was evident which finally caused infection on the developing seeds and pod tissues. The fungus adopts a uniquely biotrophic infection strategy in roots and spreads without causing a loss of host cell viability. We have also demonstrated that *U. virens* isolates infect Arabidopsis and the plant subsequently activates different defense response mechanisms which are witnessed by the expression of pathogenesis-related genes, *PR-1*, *PR-2*, *PR-5*, *PDF1.1* and *PDF1.2*. The established *A. thaliana*-*U. virens* pathosystem will now permit various follow-up molecular genetics and gene expression experiments to be performed to identify the defense signals and responses that restrict fungal hyphae colonization in planta and also provide initial evidence for tissue-adapted fungal infection strategies.

## Macrophomina phaseolina promotes charcoal rot susceptibility in sorghum through induced host nitric oxide (NO) production

A. BANDARA (1), D. Weerasooriya (1), S. Liu (1), C. Little (1)

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

*Macrophomina phaseolina* (MP) is an important necrotrophic pathogen causing charcoal rot disease in sorghum (*Sorghum bicolor* (L.) Moench). Exploring the host-pathogen interaction is a key to understanding the molecular basis of resistance. An RNA-Seq experiment was conducted to discover differentially expressed genes (DEG) in response to MP infection. SC599 (resistant) and Tx7000 (susceptible) sorghum lines were grown in the greenhouse and inoculated with MP and phosphate-buffered saline (mock-inoculated control). RNA was extracted from three biological replicates at 2, 7, and 30 d post-inoculation (d.p.i.) from stem tissues adjacent to the inoculation point and subjected to RNA-Seq. Among the DEG, three genes (*Sb08g011530*, *Sb05g000680*, and *Sb04g027860*) involved in NO biosynthesis were significantly upregulated in Tx7000 at 7 d.p.i., while those of SC599 were not differentially expressed. To further confirm the findings, confocal microscopy was used. At 7 d.p.i, in situ NO production in MP- and mock-inoculated stem cross sections of two resistant (SC599, SC35) and two susceptible (Tx7000, BTx3042) lines was analyzed using 4-amino-5-methylamino-2',7?,difluorofluorescein diacetate (DAF-FM DA). Strong green fluorescence was observed in both MP-inoculated susceptible lines confirming the presence of NO while no signal was detected in the resistant lines. We believe that NO-mediated cell death contributes to the rapid proliferation of MP within susceptible hosts.

## Induced rhamnose biosynthesis in sorghum by *Macrophomina phaseolina* contributes to charcoal rot susceptibility

A. BANDARA (1), D. Weerasooriya (1), S. Liu (1), C. Little (1)

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

*Macrophomina phaseolina* (MP) is a necrotrophic pathogen that causes charcoal rot disease in sorghum (*Sorghum bicolor* (L.) Moench). To detect differentially expressed genes (DEG) in response to MP infection, an RNA-Seq experiment was conducted. SC599 (resistant) and Tx7000 (susceptible) sorghum lines were stalk-inoculated with MP hyphal fragments and phosphate-buffered saline (control). Stalk RNA extracted at 2, 7, and 30 d post-inoculation (d.p.i.) was subjected to RNA-Seq. Among the DEG, 7 genes involved in the rhamnose biosynthetic pathway (*Sb01g038050*, *Sb01g039220*, *Sb01g043370*, *Sb02g029130*, *Sb08g022850*, *Sb09g018070*, and *Sb10g024490*) were significantly upregulated in Tx7000 at 7 d.p.i., while those of SC599 showed no differential expression. To confirm the results, stem juice extracted from two MP- and mock-inoculated resistant (SC599, SC35) and susceptible (Tx7000, Btx3042) sorghum lines were subjected to HPLC. Compared to control, the MP-inoculated charcoal rot susceptible lines had significantly higher (P < 0.005) stalk rhamnose concentrations (mg/mL) (Tx7000, 7.2; BTx3042, 5.8), while no such effect was evident in the resistant lines (SC599, 1.6; SC35, 1.4). To test the effect of rhamnose on MP growth, two isolates were grown on minimal medium supplemented with rhamnose at 0, 25, 50, 75, 100, and 125 mM concentrations. MP grew on rhamnose in a concentration-independent manner. Results suggested the potential role of host rhamnose as a virulence factor for MP.

## Macrophomina phaseolina induces stalk senescence in charcoal rot susceptible sorghum genotypes through up-regulated host *chlorophyllase-2* biosynthesis

A. BANDARA (1), D. Weerasooriya (1), S. Liu (1), C. Little (1)

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

Charcoal rot (*Macrophomina phaseolina*, MP) and Fusarium stalk rot (*Fusarium thapsinum*, FT) are important sorghum diseases worldwide. An RNA-Seq experiment was conducted to explore the interaction of MP and FT with known resistant and susceptible sorghum genotypes. SC599 (R) and TX7000 (S) sorghum lines were grown in the greenhouse and inoculated with FT, MP, and phosphate-buffered saline (control). RNA extracted from stem tissues at 2, 7, and 30 days post-inoculation (d.p.i.) was subjected to RNA-Seq. At 7 d.p.i., a gene that encodes *chlorophyllase-2* (*Sb02g012300*) showed two and 200-fold up-regulation in FT- and MP-inoculated Tx7000, respectively. SC599 did not show a significant differential expression. To confirm the effect of up-regulated *chlorophyllase-2*, stalk chlorophyll was extracted with acetone from two pathogen- and mock-inoculated resistant (SC599, SC35) and susceptible (Tx7000, BTx3042) sorghum lines at 7 d.p.i. Total chlorophyll content (mg/g stalk) was computed using spectrophotometer readings. Compared to control, MP significantly reduced total chlorophyll of Tx7000 (control, 1.56; MP, 0.40) and BTx3042 (control, 1.45; MP, 0.63) (P < 0.016). No such effect was evident with the two resistant genotypes. FT did not significantly affect the chlorophyll content of any genotype. Results revealed the ability of MP to induce stalk senescence through increased chlorophyll degradation, which contributes to the enhanced vulnerability to this necrotrophic fungus.

## Genome-wide study on cultivar-specific and stripe rust responsive miRNAs 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 grain production 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.

## Genotype and chemotype profiles of endophytes associated with wild barley

M. YI (1), J. Kaste (2), N. Charlton (1), W. Hendricks (1), N. Krom (1), P. Nagabhyru (3), D. Panaccione (4), C. Young (1)
(1) The Samuel Roberts Noble Foundation, Ardmore, OK, U.S.A.; (2) Cornell University, Ithaca, NY, U.S.A.; (3) University of Kentucky, Lexington, KY, U.S.A.; (4) West Virginia University, Morgantown, WV, U.S.A.

The genetic and chemotypic diversity of *Epichloë* species (Clavicipitaceae, Ascomycota) isolated from wild barley (*Hordeum* spp.) was explored to provide potential valuable resources for forage improvement. We screened 56 wild barley PI lines from the US Department of Agriculture's National Plant Germplasm System (USDA-NPGS), and identified 16 PI lines with endophyte incidence. Five distinct endophyte genotypes representing three nonhybrid and two hybrid *Epichloë* species were identified among the isolated endophytes from nine viable PI lines. Genotypes were identified based on the presence of alkaloid biosynthesis and mating type genes. Molecular phylogenetic analyses indicated that all nonhybrid genotypes are *E. bromicola*, whereas the two hybrid genotypes are *E. bromicola* by either *E. typhina* or an ancestor of *E. amarillans* or *E. baconii*. One of the hybrid genotypes was identified as a new taxonomic group currently referred to as HboTG-3. Alkaloid profiles were predicted from the genotyping data and the chemical profiles of distinct isolates were confirmed by HPLS and mass spectrometry with endophyte-infected plants, which confirmed the predicted chemotypes. Whole genome sequencing of four isolates provided insights for the complete alkaloid biosynthesis clusters in the wild barley endophytes and the unique features of the *Epichloë* species genomes. The beneficial effects each endophyte may provide the host will be tested under various biotic and abiotic stresses.

## Exploring the *Monilinia vaccinii-corymbosi*-blueberry pathosystem for the discovery of genes governing host-specificity

K. BANSAL (1), J. Rollins (1)

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

*Monilinia vaccinii-corymbosi*, a Sclerotiniaceae member, causes mummy berry disease on blueberries. Primary infection of vegetative tissues is caused by ascospores and secondary infection of flowers is caused by conidia. Fruit infection is highly specific as conidial germ tubes enter the host through flowers. Conidia germinate on stigma, traverse the stylar canal to colonize developing ovules, converting fruit into a mummy. To understand the underlying genetics of this specificity, we are developing standardized tools for generating inoculum, visualizing the course of infection, and collecting infected materials for transcriptomic analysis. A comparison of culture media and temperature conditions demonstrated that four day old cultures of *M. vaccinii-corymbosi* isolate RL1 (Mvc-RL1) grown on oatmeal agar medium, in the dark, at 22°C followed by a low temperature shift (15°C) induced sporulation most abundantly after 24 hours. For characterization of the gynoecium infection process, a protoplast-mediated genetic transformation system was utilized to produce constitutive GFP-expressing Mvc-RL1 strains. Floral inoculation of Southern High Bush variety Legacy with conidia of GFP-Mvc-RL1 documented germinating conidia and the course of hyphae traversing the stylar canal. Using these tools, floral infections will be utilized for comparison with uninoculated flowers for transcriptomic analysis and identification of genes underlying host specificity in *M. vaccinii-corymbosi*.

## Fungal transcriptome analysis of the *Sclerotinia sclerotiorum* and *Pisum sativum* interaction

M. CHILVERS (1), J. Rojas (1), P. Santos (2), J. Wang (1)

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

*Sclerotinia sclerotiorum* is a broad host range necrotrophic pathogen that causes white mold on pea (*Pisum sativum*). Gene expression profiling during infection has been characterized for *S. sclerotiorum* on several model plants. However, the *S. sclerotiorum-P. sativum* interaction is still poorly understood. In this study, fungal transcriptomic data was collected at three time points: 12 h, 24 h, and 48 h post inoculation (hpi), from susceptible and partially resistant pea cultivars inoculated with *S. sclerotiorum*. As a control, transcriptomic data was also collected from mycelia grown on PDA medium used in the inoculations. Four gene clusters were identified using expression profile analysis, one cluster was exclusively up-regulated on PDA, while the remaining three clusters were either associated with the host or sampling time. Gene ontology enrichment analysis demonstrated that 83% of the up-regulated cluster 1 genes were categorized as oxidoreductases at 12 hpi. Whereas, 43% of the up-regulated cluster 2 genes were involved in hydrolysis and transport functions at 24 and 48 hpi. The temporal gene expression profile change may indicate the lifestyle transition of the pathogen from biotroph to necrotroph. In addition, three out of 486 predicted effector candidate genes were up-regulated in planta at 24 and 48 hpi. The identification of key genes involved in the *S. sclerotiorum-P. sativum* interaction will facilitate effector assisted breeding for disease management.

## Aspartyl protease mediated cleavage of AtBAG6 triggers autophagy and fungal resistance in plants

Y. LI (1), M. Kabbage (2), M. Dickman (1)

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

The Bcl-2-associated-athanogene (BAG) family is an evolutionarily conserved group of co-chaperones that modulate numerous cellular processes. Previously we found that *Arabidopsis* BAG6 (AtBAG6) is required for basal immunity against the fungal phytopathogen *Botrytis cinerea*. However, the mechanism(s) by which AtBAG6 controls immunity are obscure. Here, we address this important question by determining the molecular mechanisms responsible for AtBAG6 mediated basal resistance. We show that AtBAG6 is cleaved *in vivo* in a caspase-1 like dependent manner, and via a combination of pull-downs, mass spectrometry, yeast-two-hybrid and chemical genomics, we demonstrate that AtBAG6 interacts with a C2-GRAM domain protein (AtBAGP1) and an aspartyl protease (AtAPCB1), both of which are required for AtBAG6 processing. Furthermore, fluorescence and transmission electron microscopy established that AtBAG6 cleavage triggers autophagy in the host that coincides with disease resistance. Targeted inactivation of *AtBAGP1* or *AtAPCB1* results in the blocking of AtBAG6 processing and loss of resistance. Mutation of the cleavage site blocks cleavage, inhibits autophagy and disease resistance is compromised. Taken together, these results couple an aspartyl protease with a molecular co-chaperone to trigger autophagy and plant defense, providing a key link between fungal recognition and the induction of cell death and resistance.

## The MAP kinase kinase gene, *CpBck1*, regulates sectorization and pathogenicity of the phytopathogenic fungus *Cryptonectria parasitica*

D. KIM (1)

(1) Chonbuk National University, Korea

*CpBck1*, an ortholog of the cell-wall integrity mitogen-activated protein kinase kinase kinase of *Saccharomyces cerevisiae*, was cloned and characterized from the chestnut blight fungus *Cryptonectria parasitica*. The *CpBck1*-null mutant displayed cell wall integrity-related phenotypic changes such as abnormal cell morphology and wall formation and hypersensitivity to cell wall-disrupting agents. In addition, the mutant showed severely retarded growth without any sign of normal development, such as hyphal differentiation, conidiation, or pigmentation. As the culture proceeded, the mutant colony showed sporadic sectorization. Once sectored, the sectored phenotype of robust mycelial growth without differentiation was stably inherited. Compared with the wild type, both the parental *CpBck1*-null mutant and the sectored progeny exhibited marked impaired virulence. The present study
revealed that a mutation in a signaling pathway component related to cell-wall integrity resulted in sporadic sectorization and these sectored phenotypes
were stably inherited, suggesting that this signal transduction pathway is implicated in adaptive genetic changes for sectorization.

A band of misfits: Role of unexpected proteins in the plant symbiotic signaling pathway

M. VENKATESHWARAN (1), A. Wiley-Kalil (2), D. Jayaraman (3), M. Banba (4), A. Binder (5), S. Bernard (6), J. Maeda (6), M. Otegui (7), H. Imaizumi-Anraku (8), M. Parniske (5), J. Ané (9)

(1) University of Wisconsin-Platteville, Platteville, WI, U.S.A.; (2) NDSU Williston Research Extension Center, U.S.A.; (3) University of Wisconsin-Madison, U.S.A.; (4) Division of Plant Sciences, National Institute of Agrobiological Sciences, Japan; (5) Ludwig-Maximilians-Universität München, Germany; (6) Department of Agronomy, University of Wisconsin, Madison, U.S.A.; (7) Department of Botany, University of Wisconsin-Madison, U.S.A.; (8) Division of Plant Sciences, National Institute of Agrobiological Sciences, U.S.A.; (9) Departments of Bacteriology and Agronomy, University of Wisconsin, Madison, Madison, WI, U.S.A.

Rhizobia and arbuscular mycorrhizal fungi produce lipo-chito-oligosaccharidic signals that are perceived by legume hosts at the plasma membrane level. These signals are then transduced to the nucleus where they trigger oscillations of the nuclear calcium concentration (calcium spiking) that, in turn, regulates symbiotic gene expression. Genetic and protein interaction studies identified some relatively expected components in such a signaling pathway: receptor-like kinases at the plasma membrane that perceive the microbial signals, ion channels and ion pumps on the nuclear envelope that allow calcium spiking, and nuclear transcription factors that regulate gene expression. We will present new data on more unexpected members of this signaling pathway. (1) An HMGR (3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase) that interacts with and is regulated by the symbiotic receptor kinases. In particular, we will show that the activity of this HMGR is not only necessary but also sufficient to trigger calcium spiking. (2) Nucleoporins of the NUP107-160 sub-complex that have been identified through forward genetic approaches. We will show that these nucleoporins are required for the proper localization of symbiotic ion channels to the inner nuclear membrane. Finally, we will compare results between two legumes, *Medicago truncatula* and *Lotus japonicus*, and show the value of comparing carefully these two genetic systems.

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

D. FERNANDEZ (1), D. Fernandez (2), M. Grossi de Sá (3), I. Mezzalira (4), M. Beneventi (4), M. Lisei de Sá (5), D. Amora (4), H. Baimey (6), A. Petitot (7), J. de Almeida Engler (8), É. Saliba Albuquerque (4), M. Grossi de Sá (4)

(1) IRD - French Research Institute for Development, Montpellier, cedex 5, France; (2) IRD- French Research Institute for Development, Montpellier, France; (3) IRD, Montpellier, France; (4) EMBRAPA CENARGEN, Brazil; (5) Epamig and Embrapa Cenargen, Brazil; (6) Parakou University, Benin; (7) IRD- French Research Institute for Development, France; (8) INRA, France

Root-knot nematodes are endo-parasites with a wide host range, encompassing mono- and dicotyledonous plant crops. *Meloidogyne javanica* is responsible for rice (*Oryza sativa*) production losses in Brazil and Africa (Benin). Successful infection is likely achieved by effector proteins produced in the nematode esophageal gland cells and released in the host plant cells. The aim of this study was to assess the functional role of three esophageal gland cell proteins of *M. javanica* in rice - nematode interactions. RT-qPCR assays showed that *Mj-SP2*, *Mj-SP18* and *Mj-SP19* genes are over-expressed all along the infection cycle in rice roots. Immunodetection experiments using specific antibodies raised against synthetic peptides showed that the nematode proteins are localized in circular granules structures within the nematode body. Transgenic rice (*O. sativa* *Nipponbare*) plants expressing the candidate proteins or artificial micro-RNAs (*amiRNAs*) able to silence the cognate genes in the nematode were produced. Assessment of nematode reproduction on homozygous transgenic lines allowed the identification of rice lineages with altered susceptibility, indicating that these proteins may be involved in *M. javanica* virulence. Data obtained significantly widen our knowledge of molecular players contributing to nematode pathogenicity, and open new avenues for nematode control strategies in rice and other crops of interest.

MetacodeR: An R package for comparative analysis and visualization of microbial communities and evaluation of metabarcoding primer specificity

Z. FOSTER (1), N. Grunwald (1)

(1) USDA Agricultural Research Service / Oregon State University, U.S.A.

Metabarcoding, the sequencing of a locus amplified from environmental DNA, is revealing the unculturable majority of microbes for the first time, but new methods present new challenges. Complex hierarchical data are difficult to plot and the commonly used stacked bar charts are ineffective when plotting multiple taxonomic ranks or treatments. In addition, the choices of loci and barcode primers are a source of underexplored bias. MetacodeR is a new tool kit for metabarcoding and community ecology, focusing on visualization of taxonomic data and *in silico* primer/barcode evaluation. It provides tools to integrate quantitative data from thousands of taxa and multiple communities into intuitive tree-based plots. Data from one or more communities are mapped to tree element size or color, as in a heat map. This allows for rapid exploration of data and information-rich, publication-quality graphics. In addition, metacodeR provides critical tools for evaluating potential primers and barcode loci. *In silico* PCR evaluates primer specificity and barcode gap analysis estimates error rates and resolution. MetacodeR is currently being applied to evaluating the diversity of fungi and oomycetes in the rhizosphere of nursery-grown rhododendron. The ability to easily test new primers/barcodes will accelerate the adoption of metabarcoding to emerging or understudied pathogen groups, such as oomycetes, and intuitive plotting will make subtle patterns in complex data more apparent.

Molecular dissection of resistance signaling in watermelon fruit through transcriptomic approach

M. MANDAL (1), J. Ikerd (2), A. Soorni (3), C. Kousik (2)

(1) ORISE Participant sponsored by the U.S. Vegetable Laboratory, USDA, ARS., Charleston, SC, U.S.A.; (2) U.S. department of Agriculture, Agricultural Research Service, U.S. Vegetable Laboratory, Charleston, SC, U.S.A.; (3) Department of Horticulture, Virginia Tech, Blacksburg, WV, U.S.A.

Plant defense signaling involves a complex array of events initiated by direct or indirect interactions between pathogen effectors and host resistance
proteins. Our study involves developing watermelon (Citrullus lanatus) lines resistant to various pests and pathogens through conventional breeding
techniques. One of the most common and widely spread diseases in watermelon is Phytophthora fruit rot caused by *Phytophthora capsici*, which is
especially severe in the southeastern US due to prevalence of favorable weather conditions. In order to elucidate the molecular mechanism of signaling
events that occur during watermelon - *Phytophthora* interactions we are utilizing the available watermelon and *P. capsici* genome sequence data along
with our RNA-seq data to identify the differentially regulated transcriptome between resistant and susceptible watermelon lines. Our preliminary data
indicates that most sample sequence reads matched to watermelon genome (70-90%) and the pathogen transcript reads were higher in susceptible cultivar
'Sugar Baby' (5-30%) when compared to resistant germplasm line 'USVL020-PFR' (0.5-1%). The genotypic data correlates well with the phenotypic
data. The overall goal is to identify plant defense markers contributing to host resistance against *P. capsici* that will prove useful in watermelon breeding
programs.

Identification of silencing suppressor proteins in Maize chlorotic mottle virus

N. BACHELLER (1), H. Garcia-Ruiz (1)

(1) University of Nebraska-Lincoln, U.S.A.

Maize Lethal Necrosis (MLN) is a devastating disease of maize caused by a synergistic interaction between *Maize chlorotic mottle virus* (MCMV) and *Sugarcane mosaic virus* (SCMV), *Wheat streak mosaic virus* (WSMV) or *Triticum mosaic virus* (TriMV). RNA silencing is a prominent antiviral
defense system in plants. In single and double infections, MCMV and SCMV activate maize antiviral RNA silencing machinery, resulting in the accumulation of virus-derived small RNAs. Most plant viruses encode silencing suppressor proteins to inactivate RNA silencing and overcome the host defense system. No silencing suppressor has been identified in MCMV. To identify silencing suppressors in MCMV, individual proteins were cloned into binary vectors for transient expression in *Nicotiana benthamiana*. Proteins with silencing suppression activity were identified by the ability to complement the pathogenicity defect in two different suppressor-deficient viruses. Identification and characterization of silencing in MCMV and SCMV establishes the foundation to further study the molecular mechanisms involved in Maize Lethal Necrosis Disease.

## Resistance of cultivated soybean to *Clover yellow vein virus* apparently originated during domestication from wild soybean

K. NAKAHARA (1), J. Abe (1), T. Yamada, (1) Y. Wang, (2), M. Hajimorad (2)

(1) Hokkaido University, Japan, (2) University of Tennessee, Knoxville, TN, U.S.A.

*Clover yellow vein virus* (CIYVV) (genus *Potyvirus*, family *Potyviridae*), is pathogenic to legume plants, including broad bean (*Vicia faba*) and pea (*Pisum sativum*). However, inoculation of dozen cultivated soybean (*Glycine max*) genotypes with CIYVV strain no30 (CIYVV-no30) did not result in systemic infection in any of the inoculated plants. In contrast, inoculation of 24 accession lines of wild soybean (*G. soja*) with the green fluorescent protein (GFP)-tagged CIYVV-no30 (CIYVV-no30-GFP) resulted in systemic infection in all, except one line. This observation led us to hypothesize that resistance to CIYVV in cultivated soybean originated from wild soybean during domestication. It seems likely that during soybean domestication, parental plants with natural resistance to CIYVV were selected. To test this hypothesis, and to elucidate the genetic basis of the resistance, recombinant inbred lines (RILs) were generated from a cross between cultivated soybean TK780 and wild soybean line B01167. Analysis of responses of 64 derivative RILs inoculated with CIYVV-no30-GFP resulted in identification of one major and one minor quantitative trait loci (QTL). The role of each of these loci in conferring resistance to CIYVV in cultivated soybean is being investigated.

## Examination of the mechanism of superinfection exclusion by *Citrus tristeza virus*

S. FOLIMONOVA (1), O. Atallah (1), S. Kang (2)

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

Superinfection exclusion (SIE), referred to as cross-protection when used as an agricultural practice, is a phenomenon in which a primary virus infection prevents a secondary infection with the same or closely related virus. Earlier we demonstrated that SIE by *Citrus tristeza virus* requires viral p33 protein, yet p33 alone is not sufficient for virus exclusion. In this study we show that a 5'-proximal genomic region encoding leader proteases L1 and L2 is involved in SIE. Substitution of this region with the cognate sequences from a heterologous strain affected virus ability to exclude the parental virus. As the next step, we are interested to assess whether SIE is conferred by the L1/L2 proteins or by their coding sequences. We created four mutant viruses having alterations in the RNA sequences of the L1 and L2 N-terminal (NTD) and protease domains by changing the wobble base of each possible codon to avoid changing the amino acid sequences of these proteins. All mutant viruses were able to replicate in *Nicotiana benthamiana* at the levels comparable to the wild type virus, and three of them except L1 NTD mutant were able to establish systemic infection in *N. benthamiana* and citrus plants. Additional constructs containing mutations within the L1 NTD domain are being made with the goal to produce virus variants capable of systemic infection of both plant hosts. The effect of the introduced mutations in the L1-L2 domains on SIE ability will be assessed.

## Exchange of HC-Pro cistron between *Soybean mosaic virus* and *Clover yellow vein virus*: Impact on pathogenicity

M.R. HAJIMORAD (1), Y. Wang (1,2), J. Abe (3), K. Nakahara (3)

(1) University of Tennessee, Knoxville, TN, U.S.A., (2) Jilin Academy of Agricultural Sciences, Jilin, China, (3) Hokkaido University, Japan

*Soybean mosaic virus* (SMV)-N and *Clover yellow vein virus* (CIYVV)-no30, members of *Potyvirus* genus within *Potyviridae* family, have narrow host range. SMV-N is well adapted to cultivated soybean (*Glycine max*) while infects systemically wild-type soybean (*G. soja*) as well. However, SMV-N is incapable of systemic infection in broad bean (*Vicia faba*). In contrast, CIYVV-no30 is capable of systemic infection in both broad bean and wild-type soybean; however, incapable of systemic movement in cultivated soybean. Because SMV-N and CIYVV-no30 both establish local infection in all these plants, we hypothesized that lack of systemic movement by SMV-N in broad bean and by CIYVV-no30 in cultivated soybean is due to incompatibility of respective HC-Pro, a key long-distance movement potyviral protein, in these two hosts. To test this hypothesis, chimeras were constructed with precise exchanges of HC-Pro cistrons between SMV-N and CIYVV-no30 and inoculated onto cultivated soybean "Williams", wild-type soybean "B01167" and broad bean "Windsor". Analyses showed that pathogenicity of recombinant viruses did not differ from those of the parental viruses. This observation suggests that i) HC-Pro of SMV-N and CIYVV-no30 are functionally equivalent, and ii) the determinant(s) for systemic infection in cultivated soybean by SMV-N and by CIYVV-no30 in broad bean reside elsewhere.

## Devising a custom 13K unigene-based *Nicotiana benthamiana* microarray to study host-pathogen interactions at the transcriptome level

P. THAMMARAT (1), P. Thammarat (1), T. Sit (2), S. Lommel (2)

(1) Functional Genomics Program, North Carolina State University, U.S.A.; (2) Department of Entomology and Plant Pathology, North Carolina State University, U.S.A.

Selecting a model plant to study host/pathogen interactions at the transcriptome level is a crucial part of experimental design. Although *Arabidopsis thaliana* is a preferred model based on the availability of comprehensive genomic information, it is only susceptible to a restricted number of pathogens. Due to this limitation, exploring alternative plant models is necessary. *Nicotiana benthamiana* (Nb) is highly susceptible to a wide range of pathogens. In order to enable the use of Nb in high-throughput studies, we created a genomic tool for this plant. We developed the first Nb array from 13,014 unigenes (average 752 bp) which were originally derived from 40,000 expressed sequence tags (ESTs; ~70% of publicly available Nb ESTs representing ~38% of the transcriptome). Homology searches against GenBank, KEGG, and GO databases (BLASTX, E-value < 1 × 10⁻⁵) indicated that 66% of the unigenes have > one hit, with the most frequent hit to *Solanaceae* plants. Our unigene-based array has broad coverage of plant pathways: metabolism, transportation, transcription, translation, signal transduction and defense. 261 unigenes were involved in transcription with the most abundant ones belonging to the AP2/ERF, WRKY and GRAS families. The Nb array consists of 9 probes per unigene. We conducted an array performance test to study interactions between Nb and *Red clover necrotic mosaic virus*. The array successfully yielded 100% hybridization capacity to Nb mock and infected samples.

## Reimagining the *Cauliflower mosaic virus* genome as an interactome between host and virus proteins

J. SCHOELZ (1), R. Nelson (2), S. Leisner (3), C. Angel (4)

(1) University of Missouri, U.S.A.; (2) The Samuel Roberts Noble Foundation, U.S.A.; (3) University of Toledo, U.S.A.; (4) Cenicafe, Colombia

The genome of *Cauliflower mosaic virus* (CaMV) was the first plant-associated organism to be completely sequenced, in 1980. The CaMV nucleotide sequence revealed that its genome was composed of seven substantial open reading frames (ORFs), and six proteins were subsequently matched up with their respective ORFs; the seventh putative protein has never been found in CaMV-infected plants. Since the initial CaMV sequence was finalized, numerous studies have been completed to ascertain the functions of the six CaMV proteins, as well as their subcellular localization. Furthermore, many of the CaMV proteins have been the subject of yeast two-hybrid and co-immunoprecipitation screens to characterize the interactions of host and virus proteins. For example, the P6 protein of CaMV is a multifunctional protein with distinct roles in translation, intracellular movement, elicitation of defenses in resistant hosts or symptoms in susceptible hosts, and modulation of plant defenses. The P6 protein has been shown to interact with at least 14