*Phytophthora infestans*, causal agent of the Irish potato famine, is a threat to food security globally and an important pathogen in the US on tomato and potato. In 2009, late blight epidemics in the eastern US were the worst in modern history due to widespread inoculum distribution from infected tomato transplants and favorable weather. That year the US-22 lineage was identified as the dominant lineage. USABlight (www.usablight.org) was launched in 2011 to improve disease detection and communication between researchers, growers, the industry, and the public about late blight. A total of 1094 late blight reports were recorded between 2011-2015. Of these, 64% were from tomato and a third primarily in the NE US. A shift in the dominant clonal lineage from mefenoxam sensitive US-22 to US-23 occurred and 82% of genotyped strains were US-23. Disease outbreaks begin each year in Florida winter tomatoes. Spread through movement of tomato fruit is suspected. We developed a loop-mediated isothermal amplification (LAMP) assay specific for detection of *P. infestans*. The assay can be run in the field at 65°C, needs no thermocycling, and can detect 100-10 pg/ul of pathogen DNA. A prototype mobile reader developed by Mobile Assay has been used for in-field detection using a SYBR green stain and hand-held tablet. This mobile reader and LAMP assay is being tested in both the US and East Africa to improve pathogen detection and will be used to develop an Afriblight tracking database.

## LAMP lights the way: Early detection of airborne inoculum of *Magnaporthe oryzae* in turfgrass fields

F. PEDUTO HAND (1), F. Peduto Hand (1), C. Villari (1), W. Mahaffee (2), T. Mitchell (1)

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

A host-specialized form of *Magnaporthe oryzae* (Lolium pathotype, MoL) is the causal agent of Grey Leaf Spot (GLS), a detrimental disease of perennial ryegrass. Early diagnosis of this disease is crucial for making timely management decisions. A pathogen-specific quantitative loop-mediated isothermal amplification (LAMP) assay, that is able to detect as low as 25 spores of the pathogen, was developed in our laboratory to monitor GLS airborne inoculum. To assess the suitability of the method for field use, two perennial ryegrass plots were artificially inoculated with pathogen-infested dried grass clippings at The Ohio State University Turfgrass Research Center in the summer of 2015, and five continuously running custom impaction spore traps were placed in each plot. Sampling units were replaced daily and tested with the developed qLAMP assay while plots were monitored for symptom development. Results confirmed that the qLAMP assay-trap system was able to detect the pathogen's inoculum up to two weeks before symptoms developed in the field. The implementation of this assay by practitioners has the potential to be a useful decision support tool to guide initiation and timing of fungicide applications for GLS management.

## Detection of the select agent *Rathayibacter toxicus* using recombinase polymerase amplification coupled with a lateral flow device

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

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

Department of Economic Development, Jobs, Transport and Resources, Latrobe University, Bundoora, Australia; (5) Department of Plant Pathology, Kansas State University, Australia

*Rathayibacter toxicus* is a gram-positive plant pathogen responsible for a lethal disease of livestock in Australia. An easy, sensitive in-field detection method would enhance capabilities for port of entry inspections and biosecurity surveillance surveys of annual ryegrass (*Lolium rigidum*) hay and seed. Recombinase polymerase amplification (RPA) is a fast and sensitive isothermal DNA amplification and detection technology. Long forward and reverse primers (32 bp) and a long probe (49 bp) labelled with FAM were designed using gene involved in toxin production. TwistDX kit was used to amplify the target DNA (at ~37°C) and a lateral flow device used to visualize the amplified products. RPA primers and probes targeting the ITS region of the host genomes were designed for use as internal controls, to enhance the reliability and accuracy of the assays. RPA assays were tested against inclusivity and exclusivity panels for high specificity and accuracy. In-field validation in South Australia confirmed the efficiency and sensitivity of the RPA assays; detected the *R. toxicus* from infected samples. The proposed RPA method will enhance diagnostics, surveillance, disease management, and in-field biosecurity decisions.

## Development of a rapid Amplify® RP Acceler8 test for grapevine red blotch-associated virus in grapevines

R. LI (1), M. Fuchs (2), K. Perry (3), T. Mekuria (4), S. Zhang (1)

(1) Agdia Inc., U.S.A.; (2) Cornell University, U.S.A.; (3) Cornell University, U.S.A.; (4) Vintage Nurseries, U.S.A.

Grapevine red blotch-associated virus (GRBaV) is of unprecedented concern to the grape industry since its discovery in 2011. A rapid, reliable, sensitive, and user-friendly test is needed to quickly identify GRBaV-infected grapevines and facilitate their timely removal from vineyards. An Amplify® RP Acceler8 detection kit was developed for GRBaV for use both in laboratories and vineyards. The test is performed in a disposable detection chamber for 20 minutes using crude grapevine extracts. The test specifically detects up to 11 copies of GRBaV genomic DNA in a matrix of healthy crude extract. The test has no cross reactivity to host plant tissues and grapevine-infecting pathogens including arabis mosaic virus (ArMV), grapevine fanleaf virus (GFLV), grapevine leafroll-associated virus 1 (GLRaV-1), grapevine leafroll-associated virus 2 (GLRaV-2), grapevine leafroll-associated virus 3 (GLRaV-3), grapevine leafroll-associated virus 4 strain 5 (GLRaV-4 strain 5), grapevine fleck virus (GFkV), tomato ringspot virus (ToRSV), tobacco ringspot virus (TRSV), *Xylella fastidiosa*, and *Botrytis cinerea*. The pellets contain all the necessary reaction components and are stable for a long period of storage even at 37°C. The test has been validated using both viral DNA and crude plant extracts as templates.

## An Integrated Microfluidic Device for Rapid Plum Pox Virus Enrichment and Detection

S. ZHENG (1), Y. Yeh (1), V. Mavrodieva (2), W. Zhang (1), Z. Liu (2)

(1) The Pennsylvania State University, U.S.A.; (2) USDA, APHIS, PPQ, CPHST-Beltsville Laboratory, U.S.A.

Rapid and sensitive detection is highly desirable for plant viral pathogens. However, plant samples with low virus concentrations pose significant challenges for early detection. We present a miniature disposable device, carbon nanotube size tunable enrichment microdevice (CNT-STEM), that is capable of rapidly concentrating and purifying plum pox virus (PPV) from leaf tissues. This microdevice enriches PPV in the sample by employing physical size-based exclusion. The device consists of patterned carbon nanotube (CNT) arrays and a microfluidics channel made of polydimethylsiloxane (PDMS). The microdevice captures PPV from homogenized leaf samples inside the CNT nanostructure while most of the host contaminants are removed. After elution, the PPV enriched sample is subjected to real-time RT-PCR. The procedure takes less than 2 hours, significantly faster than standard immunocapture virus enrichment procedure. The results showed that the microdevice increased analytical sensitivity of PPV real-time RT-PCR by up to 100 fold compared with the current USDA standard immunocapture sample preparation method. The microdevice could enable early detection of PPV by enriching virus particles directly from plant samples. The technology can also serve as a platform for enrichment of other plant viral pathogens.

## Aspergillus communities contaminating red chilies with aflatoxins

P. SINGH (1), P. Cotty (1)

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

Aflatoxins are carcinogenic, immunosuppressive metabolites that cause health concerns across the globe. Several *Aspergilli* have been implicated in aflatoxin contamination events with some aflatoxin-producers associated with specific crops. Red chilies (*Capsicum spp.*) are produced in warm regions that favor aflatoxin producers. Aflatoxin in red chilies may result in market loss and even crop destruction. In order to gain insight into causal agents of aflatoxin contamination of red chilies, naturally contaminated chilies from markets in Nigeria and the US were examined. The *A. flavus* L morphotype
was the most frequently isolated member of *Aspergillus* section *Flavi* (89%). Fungi with S strain morphology were also detected in chilies from both countries (10%). Multi-gene phylogenetic analyses of partial gene sequences for nitrate reductase (*niaD*, 2.1 kb) and aflatoxin pathway transcription factor (*aflR*, 1.8 kb) resolved S morphotype fungi into three distinct clades. Fungi that produced only B aflatoxins formed a single clade with reference *A. flavus* regardless of L or S morphology. All S morphotype fungi from chilies purchased in Nigeria produced both B and G aflatoxins and were assigned to either *A. minisclerotigenes* or the previously described unnamed taxon S<sub>BG</sub> on the basis of phylogenetic affinities; over 60% of these fungi were assigned to *A. minisclerotigenes*. The results suggest the etiology of aflatoxin contamination of chili is complex and may vary with region.

## Diversity of the *Aspergillus* section *Flavi* S morphotype in Mozambique

L. ARONE (1), J. Augusto (2), R. Bandyopadhyay (3), P. Cotty (4)

(1) University of Arizona, Tucson, AZ, U.S.A.; (2) International Institute of Tropical Agriculture (IITA), Nampula, Mozambique; (3) International Institute of Tropical Agriculture (IITA), Ibadan, Nigeria; (4) Agricultural Research Service, USDA and School of Plant Sciences, University of Arizona, Tucson, AZ, U.S.A.

Aflatoxins are potent mycotoxins that contaminate foods and feeds worldwide. These cancer causing toxins also impair development and suppress immune systems. In Mozambique rejection of groundnuts from export markets and high prevalence of liver cancer are linked to aflatoxins. To improve understanding of contamination in Mozambique, aflatoxin producers were compared with known causal agents from other regions. Since, *Aspergillus* section *Flavi* isolates with S morphology are linked to severe contamination in North America and East Africa, relationships of S morphology aflatoxin producers to previously described fungi were characterized. Partial sequences of aflatoxin transcription factor (*aflR*, 2.1 kb) and nitrate reductase (*niaD*, 2.1 kb) genes were compared among S isolates from Mozambique, Kenya, Benin, and USA. Phylogenetic analyses sorted 200 S isolates from Mozambique into four clades. Isolates in the first clade produced only B aflatoxins and were closely related to fungi previously associated with lethal aflatoxicosis in Kenya. The other three clades produce both B&G aflatoxins. One of these contained *A. minisclerotigenes* and another was sister to the Kenya isolates. The third B&G clade is divergent from but most closely related to both the NRRL isolate A-11612 and fungi from West Africa belonging to an unnamed taxon referred to as S<sub>BG</sub>. Fungi in all four clades produce high aflatoxin concentrations and are potentially important causes of crop contamination.

## RNAseq analysis reveals oxidative stress responses of *Aspergillus flavus* are related to stress tolerance and aflatoxin production

J. FOUNTAIN (1), P. Bajaj (2), L. Yang (3), M. Pandey (2), S. Nayak (2), V. Kumar (2), A. Jayale (2), A. Chitikineni (2), S. Chen (4), R. Lee (3), B. Scully (5), R. Kemerait (3), R. Varshney (2), B. Guo (5)

(1) University of Georgia, Tifton, GA, U.S.A.; (2) International Crop Research Institute for the Semi-Arid Tropics (ICRISAT), India; (3) University of Georgia, U.S.A.; (4) University of Florida, U.S.A.; (5) USDA-ARS, U.S.A.

Aflatoxin contamination by *Aspergillus flavus* is exacerbated by drought stress in the field. Given that reactive oxygen species (ROS) both accumulate in plant tissues during drought and can stimulate aflatoxin production *in vitro*, we examined the responses of toxigenic isolates of *A. flavus* to oxidative stress focusing on secondary metabolite production using whole transcriptome sequencing. We examined two high toxin producing isolates, Tox4 and AF13, and one moderate producer, NRRL3357, in aflatoxin conducive yeast extract sucrose medium amended with various levels of H₂O₂. The high producers exhibited fewer differentially expressed genes (DEGs) than the moderate producer in response to increasing stress. Altered aminobenzoate degradation expression along with altered conidiation and growth also suggests reduced developmental rates in the isolates caused by oxidative stress. Aflatoxin genes were upregulated in response to increasing stress along with genes encoding aflatrem and kojic acid biosynthesis. Additional changes in primary metabolic genes also indicates adaptations to increasing stress. Given that the number and diversity of DEGs observed correlated with previously observed aflatoxin production and oxidative stress tolerance for each isolate, these data suggest that secondary metabolite production is involved in *A. flavus* oxidative responses. Continuing proteomic analyses using gel and iTRAQ methods are underway to confirm the transcriptome results.

## Hypoxia tolerance as a factor in *Aspergillus flavus* invasion and toxin contamination of developing maize seeds

S. Chalivendra (1), C. DeRobertis (2), K. Damann (1)

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

*Aspergillus flavus* predominantly colonizes the embryo and peripheral tissues (e.g., pericarp and the aleurone layer) of maize seeds but shows limited or late invasion of the endosperm. This pattern correlates with the O₂ levels in seed tissues and is suggestive of a role for hypoxia tolerance in the successful invasion of the fungus in seeds. This proposal was tested by comparing *A. flavus* isolate responses to O₂ deprivation *in vitro* with those in field inoculated seeds. Hypoxia (3 to 5% O₂) allowed conidial germination but retarded radial growth and inhibited conidial and sclerotial development. Anoxia imposed suspended animation in most isolates. Growth differences among isolates correlated with biomass differences in infected kernels. Gene expression studies showed that canonical hypoxia genes (e.g., *pdc1*) were induced, while key development genes (e.g., *brlA*) were strongly suppressed by O₂ deprivation. Aflatoxin (AF) levels were relatively high in inoculated kernels, although O₂ deprivation *in vitro* decreased AF content in most isolates. Oleic acid addition to the growth medium enhanced toxin levels under hypoxia, consistent with high AF levels observed in seed. Isolate differences in growth, sporulation, gene expression and AF accumulation patterns correlated with their responses during seed colonization, implicating hypoxia tolerance as a key virulence factor in *A. flavus*.

## Relationship between invasive brown marmorated stink bug and fumonisin contamination of field corn in the Mid-Atlantic

J. OPOKU (1), H. Mehl (2), N. Kleczewski (3), K. Hamby (4)

(1) Virginia Tech TAREC, U.S.A.; (2) Virginia Tech Tidewater AREC, U.S.A.; (3) University of Delaware, U.S.A., (4) University of Maryland, College Park, MD, U.S.A.

Invasive brown marmorated stink bug (BMSB) feeding damages a variety of crops including field corn. In 2013, a survey of BMSB-infested corn fields in VA indicated BMSB damage is correlated with fumonisin contamination. The objective of the current study was to quantify the relationship between BMSB injury and fumonisin contamination in field corn. Field trials were conducted in DE, MD, and VA in 2015. Ears were covered with mesh bags at tasseling to prevent insect damage. BMSB treatments were 0 or 4 adults applied to bagged ears, and *Fusarium* treatments were water or a spore suspension of *F. verticillioides* applied to ears at the R3 growth stage. Treatments were arranged in a randomized complete block factorial design. At maturity, ears were harvested and stink bug damage, *Fusarium* infection, and fumonisin concentrations were assessed. Stink bug damage and *Fusarium* infection were significantly different among locations, but there was not a location by treatment interaction. Treatments with BMSB had more damaged kernels compared to those without BMSB. Both BMSB and *Fusarium* treatments increased *Fusarium* infection of kernels. However, only the BMSB treatment influenced fumonisin concentrations. Results suggest BMSB increases *Fusarium* infection and fumonisin contamination in field corn. Further studies are needed to understand mechanisms by which BMSB increases fumonisins, and to develop management strategies to mitigate impacts of BMSB on field corn in the region.

## Exploration of biofilm formation and its potential link to virulence in the Goss's wilt pathogen *Clavibacter michiganensis* subspecies nebraskensis

M. BOTTI-MARINO (1), M. Botti-Marino (1), J. Jacobs (1), M. Chilvers (1), G. Sundin (1)

(1) Michigan State University, U.S.A.
Goss's wilt is a bacterial disease of corn that has re-emerged in the Corn Belt of the United States since 2006 and can cause up to 50% yield loss in extreme cases. Management of the disease is difficult, and little is known about the interaction of the causal bacterium *Clavibacter michiganensis* subsp. *nebraskensis* (*Cmn*) with its host. Bacterial cells are known to form simple to complex aggregations, termed biofilms, on biotic and abiotic surfaces, and biofilm formation contributes to the virulence of several plant pathogenic bacteria. We examined the occurrence of biofilm formation in *Cmn*, and the potential linkage of this phenotype to virulence. A total of 19 *Cmn* strains were screened for biofilm formation using two in vitro methods, quantification of biofilm formation on glass coverslips and scanning electron microscopy evaluation of biofilms produced on gold mesh grids. These strains were screened for virulence using stab and cut inoculation techniques on Goss's wilt-susceptible corn hybrids and rated for disease symptoms 10 dpi. Results from these assays indicated variation in biofilm formation ability among strains. There did not appear to be a correlation between biofilm formation and virulence of the pathogen, suggesting that biofilm formation may play other roles in *Cmn* ecology.

## The negative regulatory function of tepR for the virulence of *Burkholderia glumae* in rice is exerted via the quorum-sensing master regulator gene qsmR

J. HAM (1), J. Peng (1)

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

*Burkholderia glumae* causes bacterial panicle blight of rice, which is a globally emerging rice disease. The quorum-sensing system mediated by *tofl* and *tofR* plays a key role in the regulation of major virulence factors, such as toxoflavin (a phytotoxin), lipase and flagella. *tepR*, a newly found regulatory gene of *B. glumae*, negatively regulates multiple virulence factors including toxoflavin, lipase and exoprotease. In this study, a comparative RNA-seq analysis and a series of genetic research were performed to characterize the global regulatory function of *tepR* in *B. glumae*. The RNA-seq analysis revealed that none of the known regulatory genes required for toxoflavin production (*tofl*, *tofM*, *tofR*, *toxJ*, and *toxR*) was differentially expressed between the wild type (336gr-1) and its *ΔtepR* derivative (LSUPB401), while the structural genes for the toxoflavin biosynthesis and transport were more highly expressed in the *ΔtepR* background. Similar results were also obtained from quantitative reverse transcription PCR experiments, which validated the RNA-seq analysis. Likewise, expression of *tepR* was not affected by the *tofl/tofR*-mediated quorum sensing system. Interestingly, *qsmR*, a key regulatory factor for flagellum biogenesis, was found to be upregulated in the *ΔtepR* strain and essential for both toxoflavin production and exoprotease activity. These results suggest that *tepR* exerts its regulatory functions on toxoflavin and exoprotease through *qsmR* in this pathogen.

## Research on Bacterial Endophytes at the Institute for Advanced Learning and Research

C. MEI (1), R. Chretien (2), J. Carey (2), Y. He (2), S. Kadali (2), S. Lowman (2)

(1) Institute for Advanced Learning and Research, U.S.A.; (2) Institute for Advanced Learning and Research, U.S.A.

Bacterial endophytes live inside plants and represent a largely unexplored resource for enhancing sustainable agricultural production. They are also recognized as an untapped reservoir of novel natural products. We have demonstrated significant growth promotion of the bioenergy crop switchgrass by bacterial endophytes (*Burkholderia phytofirmans* PsJN and *Panteoagglomerans* PaKM) under *in vitro*, growth chamber and greenhouse, as well as field conditions, particularly in low fertility soil. Currently, we have established a library of 225 bacterial endophytes isolated from various environments and characterized their abilities in N fixation, P solubilization and auxin production, as well as ACC deaminase activities. Many endophytes exhibited one or several of these plant beneficial traits. In the agricultural sector, researchers are exploring growth enhancement and disease resistance of crops important to the region such as tobacco, grapevine and tall fescue and testing both individual microbes and consortia for possible applications. In the environmental sector, scientists are using switchgrass and bacterial endophytes able to breakdown toxic organic compounds to remediate polychlorinated biphenyls (PCBs) in a contaminated waste water overflow pond and also to reduce PCBs in storm water. In the natural product sector, researchers are searching for unique natural products with wide-ranging applications as antibiotics, antivirals and anticancer agents.

## Genome analysis of *Rathayibacter toxicus* strain WAC3373 from Western Australia: Sequencing, assembly and annotation

M. ARIF (1), G. Busot (1), R. Mann (2), B. Rodoni (2), S. Liu (1), J. Stack (1)

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

Annual ryegrass toxicity (ARGT) is an often fatal disease of livestock caused by *Rathayibacter toxicus*, a toxin producing, nematode-vectored (*Anguina funesta*) gram positive bacterium. *R. toxicus* is believed indigenous to Australia (mainly in Western Australia and South Australia) but is also present in South Africa. Complete genome information increases our understanding of the biology of *R. toxicus*. A complete genome of *R. toxicus* isolate WAC3373 (population RT-III from Western Australia) was obtained through *de novo* assembly (HGAP3 assembly pipeline: Hierarchical Genome Assembly Process) using PacBio sequences. The finished assembly revealed one chromosome of 2,346,032 bp length (no plasmid) with a mean coverage of 99X. The GC content was 61.5%. Annotation projected a total of 2277 genes, including 2221 coding sequences (CDS) of which 1601 genes were assigned a putative function and 620 designated as hypothetical proteins. A total of 1331 genes were assigned to COG function categories. The total number of RNA genes were 51 including, 45 tRNA CDS and 6 rRNA CDS (LSU rRNA, SSU rRNA and 5S rRNA were 3117-, 1486- and 121-bp in length, respectively). Genome analyses will provide a better understanding of the genetic constitution of this high consequence bacterium. Unique regions of the genomes were identified to develop effective diagnostic tools for plant biosecurity applications.

## Effect of biofertilizer applications on bacterial communities in the rhizosphere of citrus affected by huanglongbing

J. LI (1), Y. Zhang (2), L. Li (3), U. Handique (2), N. Wang (3)

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

Biofertilizer is increasingly used to maintain the health and productivity of citrus trees affected by huanglongbing (HLB) in Florida. Although the practice is of controversy regarding effectiveness, little is known about its non-target effects in the environment. We investigated the impact of soil application of bacterial biofertilizer agents (BFA) on rhizosphere microbial communities of HLB diseased citrus trees. Soil samples were collected at one week before applying BFA and 14 months after the first application respectively. Bacterial communities were profiled by high-throughput sequencing of 16S rDNA V3-V4 hypervariable region. Comparison of taxonomic communities only revealed marginal changes after application of BFA. The phyla Proteobacteria, Bacteroidetes Firmicutes, Actinobacteria, and Acidobacteria were most abundant in all samples, representing 95.0 to 97.0% of all assigned sequences. Analysis of the most prominent genera revealed that *Pseudomonas*, *Chitinophaga*, *Bacillus*, *Pedobacter*, *Flavobacterium*, *Bradyrhizobium*, and *Rhizobium* represented between 5% and 25% of the microbial communities in all the samples that where either treated or not treated. A general shift within the composition of the microbial communities that was independent of the application of BFA was observed after application. The results indicate that BFA application has limited effect on citrus rhizosphere bacterial communities; however, its lasting effects need to be determined.

## Impacts of abundance of *Candidatus Liberibacter* on the citrus phyto-microbiome

R. BLAUSTEIN (1), J. Meyer (1), A. Conesa (2), G. Lorca (2), M. Teplitski (1)

(1) Soil and Water Science Department, University of Florida, U.S.A.; (2) Microbiology and Cell Science Department, University of Florida, U.S.A.

Understanding how the citrus microbiome is affected by (and impacts) Huanglongbing (HLB) may be critical to disease management. To test the hypothesis that phyto-microbiome composition and diversity are related to the prevalence of *Candidatus Liberibacter*, the HLB-causative agent, leaves were collected from HLB-infected Valencia trees that differed in the extent of disease symptoms and the leaf-associated microbial communities were
characterized by sequencing the V4 region of 16S rDNA. Approximately 1,900 OTUs were detected, representing 215 genera. The relative abundance of *Candidatus Liberibacter*, which ranged from 0.7 - 71.3%, induced PCA clustering and negatively correlated with alpha diversity, as indicated by Shannon (*p*<0.001) and Chao1 (*p*=0.001) indices. Positive interactions or co-occurence (*p*<0.05) of *Candidatus Liberibacter* with other genera were detected only with *Pseudomonas* and an unclassified genus of Oxylobacteraceae. These two, in turn, had negative interactions (*p*<0.05) with 5 genera that formed a complex network of positive interactions with 39 other genera, which likely represent the “healthy” phyto-microbiome. These interactions suggest that (1) the impacts of *Candidatus Liberibacter* on the microbiome are likely mediated through its key interactions with pseudomonads and oxylobacteria and/or (2) antagonistic effects of the bacteria from the core citrus microbiome on these two groups may lessen the pathogen titer and mitigate disease progression.

## Quick genome sequencing of "Candidatus Liberibacter" strains by use of Enrichment-Enlargement-Next generation sequencing (EEN)

J. CHEN (1), Z. Zheng (2), F. Wu (3), X. Deng (3)

(1) USDA-ARS, U.S.A.; (2) South China Agricultural University, China; (3) South China Agricultural University, China

Members of "Candidatus Liberibacter" are associated with several important plant diseases such as citrus Huanglongbing (HLB) and potato zebra chip (ZC) disease. The inability to culture and the low titers in infected hosts of the bacteria have been major obstacles for research. Whole genome sequence analysis could be an effective means to study the bacteria. Taking the advancement of Next Generation Sequencing (NGS) technology, a procedure, called EEN (enrichment-enlargement-NGS), has been developed for quick genome sequencing of liberibacteria. Enrichment includes increasing the DNA ratio of liberibacter to its host using alternative hosts with higher titer capacity, removal of the highly methylated plant DNA and selection of low Ct values samples; Enlargement includes increasing the enriched DNA quantity through whole DNA amplification to meet the requirement of a sequencing platform; And the NGS step includes selection of sequencing platform and development of liberibacter-specific pipelines. With EEN, >10 G of sequence data was generated from MiSeq, followed by collection of liberibacter reads using Perl scripts, de novo or/and referenced genome assembling, and sequence annotation. Thus far, seven draft genome sequences have been generated, five HLB liberibacteria (China, California, and Florida) and two ZC liberibacteria (California) from plant or psyllid hosts. The EEN procedure could also benefit genome research in other fastidious prokaryotes.

## Phenotypic effects of anti-Candidatus Liberibacter asiaticus antibody expression in grapefruit

T. MCNELLIS (1), T. Gottwald (2), J. Sinn (1), V. Orbovic (3)

(1) Penn State University, U.S.A.; (2) USDA ARS, U.S.A.; (3) CREC-University of Florida, U.S.A.

Citrus greening, or huanglongbing (HLB), is a globally-important, fatal citrus disease caused by the gram-negative, phloem-limited bacterium "Candidatus Liberibacter asiaticus" (CLas). HLB-resistant citrus germplasm is not available. This project aims to develop HLB-resistant grapefruit. Based on homology to *Xylella fastidiosa* and *E. coli*, the CLas NodT outer membrane transporter might export factors required for virulence. The goal of this project is to block NodT-mediated virulence factor export, potentially disrupting CLas virulence. A single-chain antibody (scFv) directed at the major predicted extracellular loop of CLas NodT was expressed in stably transformed 'Duncan' grapefruit. To promote protein expression, stability and mobility in the phloem, the antibody was fused to the phloem-mobile Flowering Locus T (FT) protein. Full-length, stable FT-scFv protein was expressed in multiple transgenic lines, most of which had no phenotypic abnormalities. Thus, FT-antibody fusion protein can be expressed in citrus without deleterious effects on plant development. However, the highest expressing line exhibited precocious flowering and fruit set, presumably due to the bloom-inducing activity of the FT domain. This suggests that sufficiently high FT-scFv expression triggers flowering, which could be useful in developing rapid-flowering citrus lines to expedite breeding. The HLB resistance status of the FT-scFv grapefruit lines is now being determined.

## Modification of chemically defined medium XF-26 for in-vitro cultivation of *Xylella fastidiosa* and *Xylella taiwanensis* isolated in Taiwan

W. DENG (1), N. Sharma (1), S. Hsu (1), C. Su (2), C. Chang (3), Y. Tseng (1), F. Jan (1)

(1) National Chung Hsing University, Taiwan; (2) Taiwan Agricultural Chemicals and Toxic Substances Research Institute, Taiwan; (3) University of Georgia, U.S.A.

Strains of *Xylella fastidiosa* (PD) and *X. taiwanensis* (PLS) are plant pathogenic bacteria that were respectively isolated from grapevine and pear tree in Taiwan. The bacteria are nutritionally fastidious and require a complex nutrient for ex-planta growth. The Taiwan *Xylella* PD and PLS strains can grow on PD2 but not on the chemically defined XF-26 medium that was developed for in-vitro cultivation of *X. fastidiosa* strains isolated in North America. To develop a defined medium that supports growth of the Taiwan *Xylella* strains, different medium formulations modified from the original XF-26 were tested. A XF basal medium containing 20 amino acids resulted in good growth of Taiwan PD and PLS strains. By omitting amino acids one by one or in combination from the basal medium, tyrosine was found to be an essential amino acid for cultivating Taiwan PD and PLS strains, and the bacteria can grow in the XF-26 medium supplemented with tyrosine (XF-Tyr). Moreover, replacing asparagine with aspartic acid in the XF-Tyr medium also supported bacterial growth of purified PD and PLS strains or the ones isolated from diseased tissues during primary cultivation. To simplify the preparation of the chemically defined medium, the 17 amino acids in the XF-26 medium were substituted by 0.4% (w/v) casamino acids containing tyrosine, aspartic acid, and 16 other amino acids, and the modified XF-26 (mXF-26) was demonstrated to support good growth of the Taiwan-isolated *Xylella* strains.

## Virulence traits and disease development by *Xylella fastidiosa* are impaired in a mutant on the outer membrane protein MopB

H. CHEN (1), P. Kandel (1), L. Cruz (1), L. De La Fuente (1)

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

Outer membrane (OM) proteins are key elements to maintain the integrity of the outer membrane, and play an important role in bacterial behavior. MopB is a major OM protein in the plant pathogen *X. fastidiosa*, which causes major losses on many economically important crops due to occlusion of the xylem network in plants. Based on DNA sequence analysis of the gene encoding MopB, the C-terminal part is a homologue of the OmpA family, and includes a conserved calcium binding motif. However the function of MopB protein in *X. fastidiosa* remains poorly understood. Here, MopB function has been studied by site-directed mutagenesis. Taking advantage of the natural competence of *X. fastidiosa*, *mopB* mutants were obtained in two different *X. fastidiosa* strains, the type strain 'Temecula' and the more aggressive 'WM1-1'. *mopB* mutants in both background strains were impaired in surface attachment, biofilm formation, twitching motility and natural competence. Further analysis of the bacterial surface showed that the *mopB* mutants were impaired in pilus formation as observed by electron microscopy. Additionally, *mopB* mutants in both backgrounds showed reduced virulence as compared to WT when tested on tobacco (*Nicotiana tabacum*) plants as a host under greenhouse condition. These results suggest that MopB has an effect on pilus biogenesis and is important for the virulence of *X. fastidiosa*.

## Insights from comparative analyses between the genomes of two model strains of the cucurbit pathogenic bacterium *Acidovorax citrulli*

S. BURDMAN (1), N. Eckshtain-Levi (2), D. Shkedy (3), M. Gershovits (3), G. Mateus da Silva (4), D. Tamir-Ariel (1), R. Walcott (4), T. Pupko (3)

(1) The Hebrew University of Jerusalem, Rehovot, Israel; (2) The Hebrew University of Jerusalem, Rehovot, Israel; (3) Tel Aviv University, Tel Aviv, Israel; (4) University of Georgia, Athens, GA, U.S.A.

Bacterial fruit blotch (BFB) of cucurbits, caused by *Acidovorax citrulli* (Ac), has been responsible for serious economic losses in watermelon and melon production worldwide. Ac strains are divided into two main groups based on genetic traits. While group I strains have been mainly isolated from melon, most group II strains were isolated from watermelon. The genome sequence of the group II model strain AAC00-1 is available since 2007. We recently sequenced the genome of the group I model strain M6. The M6 genome is 4.85 Mb in length, which is approximately 500 Kb shorter than that of
AAC00-1. Comparative analysis revealed this size difference is mainly explained by eight AAC00-1 genomic regions (FAs; for fragments of AAC00-1), ranging from ~35 to 120 Kb and distributed throughout the AAC00-1 genome. The FAs are absent in the M6 genome. While AAC00-1 possesses over 500 open reading frames (ORFs) that are absent in M6, only about 120 M6 ORFs are absent in AAC00-1. Coverage analyses with draft genomes of other strains support the notion that significant portions of the FAs are discriminatory between groups I and II. Moreover, GC content, effective number of codon values' and COG analyses support these FAs were introduced into group II strains by horizontal gene transfer. This study provides the first comprehensive genome comparison between the two major groups of Ac. Further investigation is needed to sharpen the distinctive genetic markers of the two groups.

## Evaluation of assembling methods on determination of whole genome sequence of Xylella fastidiosa blueberry bacterial leaf scorch strain

J. CHEN (1), C. Wallis (1), C. Chang (2)

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

Blueberry bacterial leaf scorch (BBLS) disease, a threat to blueberry production in the Southern USA and elsewhere, is caused by Xylella fastidiosa. Efficient control of BBLS requires knowledge of the pathogen. Research is challenging because Xylella fastidiosa is difficult to culture. This study was to study BBLS strain based on whole genome sequence analyses. A total of 13,739,924 sequence paired reads (mean = 251 bp) were generated from a BBLS strain isolated from Georgia, USA. Draft whole genome sequences (minimum contig size = 1,000 bp) were generated by both de novo assembling (DA) and referenced assembling (RA) using CLC Genomic Workbench. The DA draft genome was 2,537K (95 contigs). Sizes (bp) of RAs varied depending on reference genome sequences: 2,449K (39 contigs) with NC_010513 (M12, subsp. multiplex), 2,461K (70 contigs) with NC_010577 (M23, subsp. fastidiosa), 2,467K (96 contigs) with NC_004556 (Temecula1, subsp. fastidiosa), and 2,457K (180 contigs) with NC_002488 (9a5c, subsp. pauca). Percentage coverages were 99.0% (M12), 97.1% (M23), 97.1% (Temecula1) and 91.7% (9a5c). ANI (average nucleotide identity) values were 99.9 (M12), 97.5 (M23), 97.6 (Temecula1) and 96.2 (9a5c). These data suggest that 1) DA alone was promising yet contig number remained high; 2) For RA, reference genome sequences had significant influence on the BBLS genome size; And 3) BBLS strain is a member of subsp. multiplex based on its similarity to strain M12.

## Bacterial Communities in Cultivated and Wild Cranberry Bogs

S. SOBY (1), G. Ebadzad (1), E. Batory (1)

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

Transitions in microbial population structure between fallow and cultivated soils may be an important early determinant of disease-suppression of the cultivated soil. Cranberry bogs are an ideal model system for comparing native and cultivated phytophages because of the genetic similarity between wild and domesticated cranberry plants, and their location in the same geographic region of southeastern Massachusetts. Soil samples were collected from wild and cultivated cranberry bogs over the course of several growing seasons to determine the composition of phytophages in native and cultivated cranberry bogs. Population structures were determined by pyro-sequencing of the V4 region of 16S rRNA genes. Paired-end reads were assembled using PANDAseq, and analyzed using the QIIME software package to call operational taxonomic units and to calculate richness and diversity indices. Bacterial communities in both cultivated and wild bogs had different relative abundances, but were composed of five dominant phyla: *Proteobacteria*, *Acidobacteria*, *Bacteriodetes*, *Verrucomicrobia* and *Actinobacteria*. Principal Components Analysis based on soil bacterial populations among sampled sites indicate that sampling location or soil type are more important than cultivation effects in determining bacterial community structure. These results suggest that a descriptive population structure is associated with each bog type, independent of cultivation status, season, or sampling year.

## Host range potential of bacterial soft rot pathogens isolated from *Phalaenopsis* and *Oncidium* orchids

R. CATING (1), B. Robinson (1), A. Palmateer (2), K. Frost (3)

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

Bacteria from the genera *Pectobacterium* and *Dickeya* (formerly *Erwinia chrysanthemi*) are known to cause soft rot disease in many plant species. Since 2008, bacterial soft rot caused by a *Dickeya* sp. has been reported on several orchid genera in South Florida. Ten isolates (5 from *Phalaenopsis*, 5 from *Oncidium*) were identified as belonging to the *Dickeya* or *Pectobacterium* genera based on phenotypic and 16S sequence data and were cultured on nutrient agar for 24 h at 28°C. Cultures were suspended in sterile water and adjusted to a concentration of 1 × 10⁶. Carrots, onions, potatoes (R. Burbank), and *Phalaenopsis* orchids were inoculated by placing a 10 µL drop of bacterial suspension directly over a wound made with a sterile pipette tip. Inoculated plant material was incubated at 28°C and evaluated for symptoms at 24, 48, 72, and 96 h. Bacteria were re-isolated from symptomatic tissue and identified based on the 16S and *Pel* genes. Orchid isolates caused soft rot symptoms in onion, potato, carrot, and *Phalaenopsis* orchids. Recently, *Dickeya* spp. and a newly proposed species, *D. solani*, have been reported to cause soft rot disease of potato tubers in other parts of the world, and there is concern that new species will end up in the US. We are continuing work on the orchid strains to determine their appropriate species designation.

## Bacteriophages: The over-looked component of the phyllosphere of *Xanthomonas*-diseased plants

D. RITCHIE (1), M. Munster (2), S. Butler (2)

(1) North Carolina State University, U.S.A.; (2) North Carolina State University and Plant Disease & Insect Clinic, U.S.A.

Bacteriophages specifically infect and replicate within bacteria. Phages are the most numerous, diverse, and ubiquitous entities on earth, but little is known about their existence in the phytobiome, particularly the phyllosphere. Bacteria in the genus *Xanthomonas* cause foliar diseases in many plants. Our objective was to determine occurrence of phages on plants infected with *Xanthomonas*. Foliar samples from diseased *Brassica* spp., crape myrtle, *Corylus* sp., Nandina, pepper, poinsettia, *Prunus* spp., and Verbena were assayed. Diseased tissue was chopped/soaked in 1-3 ml sterile deionized water (SDW). Non-enrichment phage extraction used either filtering (0.22 um) and/or treating with 10% chloroform. Indicator/host bacteria were isolated from the same disease sample or bacteria that were isolated previously from similar samples. Bacteria isolated from samples were identified using Biolog, ITS region, and elicitation of HR in a non-host plant. Bacteria lysed by phages represented 4 *Xanthomonas* spp. and 5 pathovars (pv). Phage titers greater than 10³ pfu/ml extraction SDW were detected representing all 8 plant genera. Plaque morphologies were hazy Xanthomonas-disease microbiota.

## Comparative genomic analyses of 15 *Acidovorax* pathogens provide insights into the emergence of a new turfgrass disease and the host specificity

Q. ZENG (1), J. Wang (2), P. Giordano (3), F. Bertels (4), M. Chilvers (5), J. Vargas (5), G. Sundin (5), N. Mitkowski (6)

(1) The Connecticut Agricultural Experiment Station, New Haven, CT, U.S.A.; (2) Michigan State Univ, U.S.A.; (3) Bayer Corporation, Canada; (4) University of Basel, Switzerland; (5) Michigan State University, U.S.A.; (6) URI, U.S.A.

Bacterial etiolation and decline (BED), caused by *Acidovorax avenae* subsp. *avenae* (*Aaa*), is an emerging disease of creeping bentgrass on golf course putting greens. The causal agent of BED belongs to the genus *Acidovorax*, members of which can infect various hosts with strong host specificity. In this research, we aim to understand the emergence of BED, and identify genes in turf *Aaa* that define its host specificity. Eighteen *Acidovorax* draft genomes (14 turf, 3 maize, and 1 orchid pathogenic strains) were generated. Using whole genome SNP analysis, the 14 turf-pathogenic *Aaa* isolates were grouped into three clades. High levels of single nucleotide polymorphisms, gene deletions, insertions, and inversions were detected within the turf-pathogenic *Aaa* isolates, suggesting that the 2009-2011 BED outbreak was caused by a genetically diverse population. Population genomic analyses identified genes
whose sequences are conserved in turfgrass pathogenic *Aaa* in comparison to the closely related maize pathogenic *Aaa* isolates. Interestingly, many of these genes identified are located within the type III secretion system pathogenicity island, an important virulence factor of phytopathogenic bacteria. We identified homologous recombination as the potential cause of the gene conservation observed in the T3SS pathogenicity island. Our results suggested a strong host selection during the evolution of the turf-pathogenic *Aaa*.

## Epiphytic survival of *Pantoea ananatis* on Florida Pusley (*Richardia scabra* L.)

B. DUTTA (1), R. Gitaitis (1), F. Anderson (1)

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

*Pantoea ananatis*, the causal organism of center rot of onion (*Allium cepa* L.), can survive on different weeds, but in a previous survey it was most commonly found on Florida pusley (*Richardia scabra* L.). The epiphytic survival of *P. ananatis* on Florida pusley under different temperature and moisture regimes was investigated. Weed seedlings were spray-inoculated with $10^3$ colony forming units (CFU)/ml of rifampicin-resistant strain Pan 97-1<sup>rif</sup> and incubated in a growth chamber at 15.5° or 21.1°C at 65% RH for 96 h, which represented mean environmental conditions during mid-March to mid-May in Vidalia, GA. *P. ananatis* survived for 96 h when incubated at 21.1°C with a mean population of $3.2 \times 10^1$ CFU/g of leaf tissue. In contrast, no viable bacteria were detected after 96 h at 15.5°C. Survival of *P. ananatis* on Florida pusley was also monitored during alternating 12-h wet and dry periods, or continuous wet or dry periods for 96 h at 15.5° or 21.1°C. Compared to initial or continuous dry periods, *P. ananatis* survived significantly better with a 12/12-h wet/dry cycle or a continuous 24-h wet period at both 15.5° and 21.1°C. Unlike at 15.5 °C, *P. ananatis* populations ($1.8 \times 10^1$ CFU/g) survived for 96 hours post inoculation at 21.1 °C under a 12-h of dry/12-h wet cycle. These results demonstrate that *P. ananatis* can survive on Florida pusley leaves under conditions of 21.1°C and prolonged leaf wetness and may potentially serve as a source of inoculum to onion.

## Further evidence for host preference among *Acidovorax citrulli* strains based on a detached melon fruit assay

L. YAN (1), R. Walcott (2), B. Hu (1)

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

Bacterial fruit blotch (BFB), caused by Acidovorax citrulli, is a serious threat to cucurbit crop production. A. citrulli strains can be divided into two genetically distinct groups with group I strains being associated with a range of cucurbits and group II strains being associated with watermelon. Despite this, seedling pathogenicity assays for A. citrulli lack the sensitivity to distinguish the groups. In this study, we developed an immature, detached melon (cv. Joaquin Gold) fruit assay that clearly distinguished the two A. citrulli groups. We observed that four group I strains induced typical water-soaked lesions on melon fruit rind tissue 7 to 10 days after pin-prick inoculation. In contrast, four group II strains did not induce symptoms on detached melon fruits. Additionally, a type III secretion (T3S) system mutant of the group I strain, M6 (M6?hrcV), failed to induce watersoaking in the same assay. Based on these observations, we conclude that group I A. citrulli strains have the capacity to infect immature melon cv. Joaquin Gold fruit, while group II strains do not. The difference in the ability of group I and group II A. citrulli strains to induce watersoaking on immature melon fruit is determined by T3S effectors. Further investigation using this detached melon fruit assay will help to identify the A. citrulli T3S effectors responsible for this differential pathogenicity phenotype.

## Characterization and Management of *Ralstonia solanacearum* Causing Bacterial Wilt of Tomato in Louisiana

A. JIMENEZ MADRID (1), A. Jimenez (1), M. Lewis Ivey (1)

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

*Ralstonia solanacearum* (Rs), the causal agent of bacterial wilt of tomato can cause severe economic losses to tomato growers in Louisiana. Traditional management tactics such as soil fumigation and crop rotation are ineffective. Resistant cultivars lack durability and have undesirable horticultural traits. For these reasons, many producers in Louisiana have abandoned their fields for tomato production. Although *R. solanacearum* is endemic in Louisiana, pathogen populations have not been characterized. Tomato samples with bacterial wilt symptoms were collected from three parishes in 2015. Bacterial wilt was confirmed using commercial Rs-specific immunostrips and the bacterial streaming test. Fifteen isolates were recovered from four fields and a greenhouse and all belonged to phylotype II. Thirty-three percent of the strains were characterized as biovar 1 and 20% as biovar 3. The remaining strains did not conform to a published biovar pattern. None of the isolates were Race 3 biovar 2 based on results from R3bv2-specific PCR. Grafting with resistant rootstock varieties is being explored as a sustainable management strategy. Louisiana tomato growers were surveyed for their willingness to use grafted plants with Rs resistance. Sixty-six percent of the growers indicated that they would be willing to use Rs-resistant grafted plants but ranked not knowing how to graft and the cost of grafted plants as the top two factors that would hinder their adoption of this strategy.

## Development of green fluorescent protein-expressing *Xanthomonas gardneri* strains to study tomato infection processes

F. ROTONDO (1)

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

Bacterial leaf spot (BLS) is a serious disease of tomato, causing millions of dollars in losses worldwide. While BLS is associated with several species of *Xanthomonas*, *X. gardneri* is the predominant species in Ohio where it causes severe damage to processing and fresh market tomatoes. To improve our understanding of BLS disease development, two green fluorescent protein (GFP)-labeled *X. gardneri* strains were developed to study colonization in *plantae*. Wild-type *X. gardneri* strains SM605-2011 and SM775-2012 were isolated from tomatoes produced in Ohio. Each strain was transformed by electroporation utilizing a GFP-expressing kanamycin/tetracycline-resistant plasmid, CGT46339. Overall, transformation efficiency for this system was low, approaching $10^2$ CFU/ug DNA. Two GFP-expressing transformants, SM605-2011gfp and SM775-2012gfp, were evaluated against their wild-type counterparts for growth *in vitro*. No significant differences were observed based on optical density measurements (Permutation test, p>0.05), and both reached the stationary phase at 1.8 OD600 and $10^9$ CFU/ml. Stability of the GFP marker and virulence of the transformed strains will be determined. These strains will be used to study leaf, fruit and seed infection using confocal microscopy (Leica TCS SP5). The transformed strains will be a powerful tool to visualize infection processes and provide critical information for disease management.

## Improvement of the environmental DNA extraction method for profiling the culturable microbiome of bacterial wilt-conducive and non-conducive soils

R. GICANA (1), W. Deng (1), J. Wu (1)

(1) National Chung Hsing University, Taiwan

The culturable microbiome profile of the bacterial wilt-conducive and nonconducive soils were compared using the PCR-DGGE profile of the V4-V5 hypervariable region of the 16S rRNA, and using the high throughput sequencing (HTS) analysis of the V1-V3 region of the 16S rRNA. PCR-DGGE results showed DNA fingerprints unique only to either the conducive or the nonconducive soils, which was confirmed by HTS analysis with 63 culturable taxa unique only to the nonconducive soil compared to 94 in the conducive soil. Exclusively cultured taxa in the nonconducive soil belong to the Phylum Planctomycete, Nitrospirae and Gemmatimonadetes while those enriched in the conducive soil belong to the Phylum Verrucomicrobia, Alphaproteobacteria and Gammaproteobacteria. While studying the culturable microbiome of the two soil types, we have improved a previous DNA extraction method to generate high quality DNA used in this study. The modified method needs no further purification steps, yields more DNA which are unsheared and with high molecular weight, with low humic acid content, and can extract DNA from bacteria of different cell wall compositions. The microbiome was cultured using low nutrient medium and with an improved DNA isolation technique we were able to capture some taxa previously
reported uncultured or were recalcitrant to culturing under traditional laboratory methods such as *Nitrospira calida*, *Planctomyces* sp., and several genera belonging to the classes Gemm-3 and Gemm-5.

**Introduction and seed transmission of *Fusarium proliferatum* in the field**

A. REYES GAIGE (1), W. Yue (2), C. Toomajian (2), J. Stack (2)

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

*Fusarium proliferatum* (Fp) is a seed-borne fungus in maize seed. It has a broad host range with worldwide distribution, and it is a prolific producer of mycotoxins. The introduction of this seed-borne organism into new environments can result in geographic range expansion of undesirable populations. The potential introduction of Fp via infected seed planted into a new environment and subsequent dispersal from that site via seed transmission was studied. Genotyping-by-sequencing (GBS) was used to identify unique polymorphisms in Fusarium species isolated from two maize genotypes. The polymorphisms were used to design primer sets to accurately detect specific isolates of Fp by end-point PCR. Extensive exclusion panels were performed to validate the accuracy of the primer sets and two Fp isolates with unique GBS loci from two maize hybrids were identified. In field experiments, 2 plots each containing 6 subplots (3 of each hybrid) were established (completely randomized design). At harvest, 20 random maize ears were collected from each subplot, and 6 ears/subplot were used. 50 kernels/ear were sampled, 1855 isolates collected, and DNA extracted; 817 isolates were identified as Fp and 751 as *F. verticillioides*. 23 isolates were identified as the unique GBS locus isolates thus indicating that transmission from the planted seed to the next generation of seed occurred. AFLP fingerprinting will confirm their identity as the original isolates that were characterized with GBS.

**Genetic mapping of pathogenesis in the northern corn leaf blight fungus**

S. MIDEROS (1), C. Chung (2), J. Poland (3), G. Turgeon (4), R. Nelson (4)

(1) University of Illinois at Urbana Champaign, U.S.A.; (2) National Taiwan University, Taiwan; (3) Kansas State University, U.S.A.; (4) Cornell University, U.S.A.

*Setosphaeria turcica* (=Exserohilum turcicum) causes northern leaf blight (NLB) of maize (*Zea mays*). Identification of genes associated with pathogenesis/virulence suggests possible targets for control, generates molecular tools to characterize populations of the pathogen, and provides valuable information to breeders and maize geneticists. In this study we used a forward genetics approach to i) identify fungal genes that determine race-specific reactions with two maize resistance genes, and ii) determine the genetic regions for other virulence mechanisms in the pathogen. We generated a cross between a race 1 and a race 23N strain and collected 220 single-ascosporic progeny. The ability of each progeny to cause disease on maize lines with or without the *Ht1* and *Ht2* genes was evaluated in greenhouse experiments. Mycelial abundance, radial growth, sporulation and melanin content were visually evaluated on artificial media. Genotyping-by-sequencing generated 2,078 single nucleotide polymorphic markers (SNPs). Using 310 high-quality SNPs we calculated a genetic map composed of 22 linkage groups spanning 1981.1 cM. Genetic mapping pinpointed regions of interest for the *avrHt1*, *avrHt2*, and the melanin biosynthetic locus, as well as quantitative trait loci for mycelial abundance, speed of growth, and sporulation. Molecular markers for the identification of fungal strains capable of causing disease in maize plants with *Ht1* and *Ht2* genes are being validated.

**Characterizing *Fusarium* species infecting corn roots in South Dakota**

A. ADHIKARI (1), P. Okello (1), B. Kontz (1), M. Dunbar (1), A. Varenhorst (2), F. Mathew (2)

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

There are multiple *Fusarium* spp. that are associated with root diseases on corn, *Zea mays*, in the United States. However, the importance of *Fusarium* associated with root disease of corn in South Dakota, is still unknown. In 2015, a survey of 50 corn fields from 23 counties in South Dakota, was conducted to characterize the *Fusarium* spp. associated with root diseases of corn. A total of 250 samples (5 plants per field) were collected between V1 (first leaf) to V3 (third leaf) growth stages. Root pieces from each sample were surface sterilized and placed on potato dextrose agar (PDA) for 7 to 10 days at 25°C under 12 hour fluorescent light/dark conditions. Isolates of *Fusarium* were hyphal-tipped, cultured, and then identified microscopically based on colony growth and spores produced on PDA. Representative *Fusarium* isolates were confirmed by sequencing of the translation elongation factor (EF1-α) gene region. At least seven *Fusarium* spp. were identified. *Fusarium graminearum* and *F. oxysporum* were the most frequently recovered (≥20%) *Fusarium* spp. from the corn roots. In the aggressiveness study to compare *Fusarium* isolates in the greenhouse, significant differences in aggressiveness (*P* ≤ 0.05) was observed among the *Fusarium* spp. with the most aggressive isolates being *F. graminearum* (FG13) and *F. acuminatum* (FA8). Results from this research will help us develop disease management strategies for corn farmers in South Dakota to maximize their yield.

**Understanding sexual fertility in *Aspergillus flavus* through analysis of F1 progeny**

R. GELL (1), I. Carbone (1)

(1) Center for Integrated Fungal Research, North Carolina State University, U.S.A.

*Aspergillus flavus* produces aflatoxin, which is a constant threat and economic burden to corn and oil seed crops worldwide. In order to manage this problem more cost effectively, a greater understanding of *A. flavus* biology and genetics is required. One important question is how genetic information moves between strains. *A. flavus*, previously thought of as only asexual, has recently been found to undergo sexual reproduction both in laboratory crosses and in the field. During the mating process, the sclerotium, a survival structure, of one strain acts as the female parent providing both the mitochondria and a matrix for the ascocarps and progeny to grow, while a spore or propagule from a second strain fertilizes as the male. The fertility of mating pairs is highly variable and influenced by the directionality of the cross. We are examining crosses that exhibit high fertility with one direction, but low fertility when male and female parents are reversed. Genome wide polymorphism screening using double digest Restriction Associated DNA sequencing (ddRADseq) was performed for 36 progeny from each direction of two biased crosses. These data are being used to provide an understanding of genetic inheritance and recombination in *A. flavus* and serve as genetic markers for mapping genomic regions that may bias fertility. By understanding these aspects of *A. flavus* genetics, we create opportunities to utilize strain fertility in the selection of biological control agents.

**Progress on genomic analysis and taxonomy of the rice blast fungus (*Pyricularia oryzae*) and other Magnaporthales species**

N. ZHANG (1), G. Cai (2), D. Bhattacharya (3), J. Luo (3), H. Qiu (3)

(1) Rutgers University, U.S.A.; (2) USDA, ARS, U.S.A.; (3) Rutgers University, U.S.A.

The order Magnaporthales includes the rice blast fungus *Pyricularia oryzae* (=Magnaporthe oryzae), the take-all pathogen *Gaeumannomyces graminis*, and saprotrophs. Recent phylogenomic analyses strongly support the idea that the sexual genus *Magnaporthe* is polyphyletic and that the placement of the rice blast fungus in this genus was based on an incorrect morphological identification in the 1970's. The asexual genus *Pyricularia* is the older (1880s) and correct name for the blast fungus, which corresponds well with pathogenicity, and ecological and evolutionary features. Following the new "One Fungus One Name" nomenclature, we propose to use *Pyricularia oryzae* for the rice blast fungus, and treat *Magnaporthe oryzae* as a synonym that can continue to be included in publications as *Pyricularia oryzae* (=Magnaporthe oryzae). This will help to bridge the literature and knowledge gap for this important species. In January 2016, a workshop entitled "Comparative genomic approaches to understanding the evolution of Magnaporthales" was held at Rutgers University. About 30 working group members are currently analyzing genome data from selected Magnaporthales lineages to understand the evolution of pathogenicity and life-style adaptation. Specifically, the following research topics are being addressed: OrthoMCL analysis of gene families, the secretome, positive selection acting on orthologous genes, MAT, secondary metabolism, and the molecular clock for Magnaporthales.
## Anything But Barren: Fungal Diversity and Functions in the Pine Barrens

N. ZHANG (1), J. Luo (2), S. Miller (2), E. Walsh (2), S. Bonos (2), Z. Helsel (2)

(1) Rutgers University, U.S.A.; (2) Rutgers Univ, U.S.A.

Pine barrens ecosystem has acidic and nutrient-poor soils, where switchgrass and other stress tolerant species are dominant understory plants. Our results indicate that grass (Poaceae) roots in the pine barrens are one of the major reservoirs of novel fungi with 47% being undescribed species. Importantly, we observed that *Acidomelania panicicola* (Leotiomycetes), a new genus and species we described from switchgrass in the New Jersey Pine Barrens increases root hair growth of switchgrass and rice in low nutrient conditions. We also described another new genus *Pseudophialophora* (Magnaporthales, Sordariomycetes) that contains several pathogenic species. Naming and description of a number of other new fungal taxa are in progress. We compared the root fungal community between the wild switchgrass from the Pine Barrens and cultivated switchgrass in managed farms using both culture and metagenomic methods. Both methods suggest that Leotiomycetes are dominant fungi in the switchgrass roots from pine barrens soils, while Sordariomycetes are dominant in the roots growing in the rich farm soils. More experiments are being conducted to test our hypothesis that *Acidomelania* and other Leotiomycetes play a role in increasing plant tolerance to abiotic stresses (e.g., low pH, low nutrients, drought) and contribute to improved establishment in poor soils. Results from this work will facilitate ecological and evolutionary studies on root-associated fungi.

## Improved plant-based culture media for growth and sporulation of *Cercospora janseana*

S. UPPALA (1), B. Liu (2), M. Wu (2), L. Zhou (3), X. Zhou (4)

(1) Texas A & M AgriLife Research Center, U.S.A.; (2) Hunan Agricultural University, China; (3) Texas A&M AgriLife Research, U.S.A.; (4) Texas A&M AgriLife Research Center, U.S.A.

*Cercospora janseana* is the fungus causing narrow brown leaf spot, a disease that has increased its importance in recent years in the U. S. *In vitro* studies are essential to better understand the biology of this pathogen and to develop effective strategies for management of this disease. However, the fungus grows very slowly and produces sparse conidia in common agar media. An *In vitro* study was conducted to evaluate various plant-based agar media in comparison with common agar media for their effects on radial growth and sporulation of *C. janseana*. Included in this study were extracts of dried rice leaves, stems, straw, roots and hulls; fresh rice leaf extract, dried extracts of barnyard grass leaves and sorghum leaves, clarified V8, and potato dextrose agar. All plant-based agar media were tested at four concentrations (25, 50, 100 and 200 g/L). Petri plates with various media were plated with a disc of 1-week-old actively growing fungus at the center and incubated at 28 °C with the 12:12 h (light:dark) cycle. Radial growth was measured at weekly intervals for 2 weeks and sporulation was measured at the end of 2 weeks. Radial growth and sporulation differed among various media and their concentrations. Among the various media tested, dried rice leaf extract, dried barnyard grass, fresh rice leaf extract and V8 media consistently supported highest radial growth of the fungus. V8 medium also supported the maximum production of conidia of this fungus.

## Virulence of *Rhizoctonia solani* anastomosis groups on cotton, corn, rice and soybean

K. URREA (1), C. Rothrock (1), S. Winters (1)

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

*Rhizoctonia solani* is an important pathogen on numerous crops. Isolates of *R. solani* were recovered from major row crops in the southern United States as part of a Rhizoctonia survey, and represented anastomosis groups (AG) 2, 4, 7, and 11. Virulence was evaluated for two isolates of each AG using stand and hypocotyl/coleoptile ratings on soybean, corn, rice and cotton using colonized millet seed inoculum placed 1-cm from seed at the time of planting (seed rot assay) or seedlings 7 days after planting (hypocotyl/coleoptile assay) in controlled environmental experiments at 25°C. Plant stands and discoloration ratings were recorded 7 days after planting or hypocotyl/coleoptile inoculation. In the seed rot assay, isolates of AG2 caused significant stand loss and brown-reddish discoloration of coleoptiles in corn. In cotton, the AG4 isolates were highly virulent causing significant stand loss compared to other AGs. In soybean, there were not significant differences in stands among isolates. In the hypocotyl assay on cotton, AG4 isolates caused significant damping-off. There were no significant differences in number of seedlings for corn, rice or soybeans. In all four crops, AG4 and AG11 isolates caused reddish brown lesions on hypocotyls or coleoptiles. This research supports the results of the soil and crop survey that crop increases the recovery of AGs of *R. solani* that are virulent on that crop.

## Identity, characteristics, and fungicide sensitivity of isolates in the *Diaporthe* species complex associated with soybean stem disease in Minnesota

D. MALVICK (1), C. Floyd (2)

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

Late season stem diseases of soybean with symptoms of browning, canker, and top dieback are common in Minnesota. Characteristics of causal agents and factors that influence disease development are poorly understood. The objectives of this study were to: (i) assess the identity and relatedness among isolates in the *Diaporthe* spp. complex associated with both symptoms and asymptomatic infection, (ii) compare virulence among the isolates, and (iii) assess fungicide sensitivity. Isolates of *D. longicolla* (n=13) and *D. phaseolorum* var. *caulivora* (n=11) were obtained from symptomatic and asymptomatic plants across MN. The ITS and beta-tubulin genes were sequenced for all isolates and a phylogenetic tree with four branches was formed. Seedling stems of three soybean cultivars were inoculated with the isolates, and lesion length was measured to reveal significant differences in virulence among isolates. The groups in the consensus tree were associated with isolate species and virulence. No significant differences in sensitivity to the fungicides Folicure® 3.6F and Headline® were detected among isolates *in vitro*, and the mean EC50 for all isolates was 0.37 to 0.38 ug/ml. These studies reveal genotypic and virulence differences, but not differences in fungicide sensitivity, among isolates in the *Diaporthe* spp. complex from symptomatic and asymptomatic soybean stems, and suggest explanations for inconsistent disease development and avenues for disease management.

## Standardizing water potential of salt-amended growth media at different temperatures for microbiological studies

I. AUJLA (1), T. Paulitz (2)

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

Temperature and moisture profoundly influence the activity of fungi. Water potential (a measure of available water) for optimal growth of a fungi may differ with temperature. Depending on the habitat and life stage of the fungus, different components of the water potential (WP) become important. The matric component of WP dominates in soil but following plant infection, osmotic effects are important. Nutrient media amended with NaCl or KCl and polyethylene glycol (PEG) are used to study the response of fungi to osmotic and matric potentials, respectively. The advent of more precise WP measuring devices such as WP4C (Decagon Devices, Pullman, WA), using the chilled-mirror dew point technique, allowed us to standardize salt-amended potato dextrose broth with or without agar for a range of basal nutrient media from -0.2 to -9 MPa at 20°C, using NaCl and KCl for osmotic potential and PEG-8000 for matric potential. The same salt and nutrient media combinations standardized at 20°C were also measured for WP at 15, 25, 30 and 35°C. WP was extrapolated for 4 and 10°C with linear/curvilinear regression due to the temperature range limits of the instrument between 15 and 40°C. These standardized tables are more accurate than previous literature due to technological advancements and will be useful for studying the response of different life stages of fungi to osmotic and matric potential.

## Ascospore biology of *Parastagonospora nodorum* under Norwegian field conditions

A. FICKE (1), B. Asalf (2), A. Ruud (3)

(1) Norwegian Institute for Bioeconomic Research, Norway; (2) Norwegian Institute of Bioeconomy Research, Norway; (3) Norwegian University of Life Sciences, Norway
*P. nodorum* (syn. *Stagonospora nodorum*) is the dominant pathogen of the Norwegian leaf blotch disease complex on wheat and can cause substantial yield losses. To improve the forecasting model for leaf blotch diseases, available at our web-based pest- and disease forecasting platform (vips-landbruk.no), we studied ascospore biology of *P. nodorum*. Airborne ascospores were monitored with Burkard volumetric spore traps for three years in southeastern Norway. In the laboratory, we conducted wind tunnel studies to examine the effect of simulated rain and light conditions on ascospore discharge. We determined the mating types for 117 *P. nodorum* isolates (MAT1 and MAT2) using MAT-specific primers and inoculated wheat plants with opposite mating types to follow ascocarp maturation under field conditions. In the field, ascospore dispersal was not associated with rain events nor any specific day/night periodicity, but with daily relative humidity above 70%. In the laboratory, over 95% of ascospores were released during simulated rain events at 100% relative humidity, independent of light conditions. Mating type ratios of *P. nodorum* varied with location between 1:1 and 1:2 (M1:M2). Mature ascocarps formed 12 months after inoculation, corresponding to 551 degree-days (base 10°C) for the 2014/2015 season. We conclude that ascocarp formation can take an extended period of time and that daily humidity above 70% is sufficient for ascospore release in the field.

## Evaluation of chemotype, pathogenicity, and aggressiveness of *Fusarium graminearum* isolates of wheat and soybean

B. MUELLER (1), B. Mueller (1), C. Groves (1), D. Mueller (1)

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

*Fusarium graminearum* is the causal agent of head blight (FHB) of wheat and damping off disease of soybean. *F. graminearum* can be classified as one of three chemotypes: 3-acetyldeoxynivalenol (3-ADON), 15-acetyldeoxynivalenol (15-ADON), and nivalenol (NIV). Besides causing yield loss, *F. graminearum* in wheat, reduces seed quality by contaminating grain with deoxynivalenol (DON) and NIV and can threaten animal and human health. Previously, 15-ADON was found to be the predominant *F. graminearum* chemotype in North America. Recently, however, the more aggressive 3-ADON chemotype is increasing in prevalence in Canada and the United States. Twelve total isolates of *F. graminearum* were collected in Wisconsin, Kansas, Iowa and Indiana from soybean and wheat between 2007 and 2015. Characterization of *F. graminearum* isolates were done using polymerase chain reaction (PCR) chemotyping. Thus far, all 12 isolates are the 15-ADON chemotype. Additional isolates will be collected in Wisconsin and further research will evaluate pathogenicity and aggressiveness on soybean and soft red winter wheat in Wisconsin. Results of this study will pave the way for the development of improved management options for Wisconsin farmers.

## Pathogenicity and Virulence of *Alternaria alternata* on 'Alamo' Switchgrass

S. COLLINS (1), B. Ownley (1)

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

*Alternaria alternata* causes foliar disease in switchgrass (*Panicum virgatum*), and was the most frequently isolated fungus from a survey of 30 commercial switchgrass seed lots. The aims of this study were to determine 1) pathogenicity and virulence of *A. alternata* isolates recovered from commercial switchgrass seed, and 2) the impact of isolates on seed germination, plant health, and biomass. Tests were arranged in a completely randomized design and data were analyzed with mixed model ANOVA and F-LSD (SAS 9.4). Six isolates were screened for pathogenicity with a detached leaf assay using 'Alamo' switchgrass. All *A. alternata* isolates were pathogenic on detached leaves (P<0.05), with lesion areas ranging from 8.8% to 23.3%. Isolates of *A. alternata* were inoculated onto surface-sterilized 'Alamo' seed at 10⁵ conidia/ml. Plant stand and health, and biomass yield were measured. Foliar disease ratings of plants from seed inoculated with all isolates was significantly greater than in untreated controls (P<0.0001). Isolates were inoculated (10⁵ conidia/ml) on foliage of whole plants. All isolates caused greater foliar disease severity than untreated controls (P=0.10), and two isolates significantly reduced shoot fresh weight (P=0.05). Although *A. alternata* can cause foliar disease, isolates did not reduce plant stand, suggesting that this fungus may cause less economic losses than fungi that affect stand establishment, such as *Bipolaris* and *Fusarium* spp.

## Genetic characterization of the *Colletotrichum gloeosporioides* complex in apple orchards of North Carolina

B. HOGE (1), M. Cubeta (1), D. Ritchie (1)

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

Glomerella leaf spot caused by fungi in the *Colletotrichum gloeosporioides* (Cg) species complex is an important disease of apple (*Malus domestica*). It is associated with leaf and fruit spots, resulting in yield loss and premature defoliation of trees. While several studies have characterized the genotypic diversity of species complexes within *Colletotrichum*, little information is available on pathogen diversity on apple cultivars in North Carolina (NC). Development of sustainable management strategies requires consideration of both host and pathogen factors and information on their association with disease incidence and severity. The primary objective of this study was to examine the genetic diversity of field populations of Cg sampled from infected fruits and leaves of cultivars Gala, Golden Delicious, Pink Lady, Granny Smith, and Yellow Transparent in 15 orchards in NC. Genomic DNA was extracted from a phenotypically diverse subsample (n=50) of 150 isolates and sequence analysis of four independent genetic loci was performed to identify multi-locus haplotypes (MLHs). Eight distinct MLHs were detected, and three well-supported lineages corresponding to species in the Cg complex were identified. Isolates identified as MLH1 belonged to the Cg clade and were sampled from three of five cultivars. MLH2 isolates were identified as *C. fructicola*, and sampled from four of five cultivars with some overlap. The remaining MLHs were restricted to a single cultivar or isolate.

## Fitness ability and fungicide sensitivity of *Lambertella corni-maris* isolates from apple

A. AMIRI (1), A. Hawkins (2), K. Mulvaney (2)

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

*Lambertella corni-maris* is a newly reported postharvest decay of apple. Additional knowledge is needed to understand disease ecophysiology and develop adequate control strategies. In this study, we investigated fitness ability of three isolates in vitro i.e. growth on different media, sensitivity to pH and osmotic stress and in vivo pathogenicity on nine apple cultivars. All three isolates grew significantly faster on apple juice agar compared to potato dextrose agar whereas growth on V8 agar and malt extract agar was intermediate. Isolates grew equally at pH ranging from 3 to 7 and growth was inhibited at pH ≥8. Significant sensitivity to osmotic stress was only observed at higher KCl concentrations (1M). *L. corni-maris* was pathogenic to all nine test apple cultivars, however, Honey Crisp, Fuji, Pixata, and Gala were significantly more susceptible compared to Cameo, Braeburn and Red Delicious. Furthermore, the sensitivity of 100 *L. corni-maris* isolates to fludioxonil, boscalid, pyraclostrobin, pyrimethanil and thiabendazole was evaluated using a mycelial growth inhibition assay. Mean EC₅₀ values were 0.07, 0.84, 1.32, 2.45 and 3.68 µg/ml, respectively. Fludioxonil and pyrimethanil were highly effective in controlling the disease on detached fruit whereas thiabendazole was the least effective. Our results indicate that *L. corni-maris* may be able to survive extreme conditions and is better controlled by postharvest rather than preharvest fungicides.

## Nutritional and environmental effects on conidial germination and appressorium formation of *Phyllosticta citricarpa*, the citrus black spot pathogen

N. WANG (1), M. Dewdney (2)

(1) Department of Plant Pathology, Citrus Research and Education Center, University of Florida, U.S.A.; (2) mmdewdney@ufl.edu, U.S.A.

Citrus black spot caused by *Phyllosticta citricarpa* has been present in Florida since 2010 and can reduce fruit yield and marketability. Conidial germination and appressorium formation (AF) are essential for successful infection by *P. citricarpa*, although the conditions required for this process are poorly understood. In this study, the effects of citrus juices, concentration, pH, various carbon and nitrogen sources, and environmental conditions were evaluated *in vitro*. All tested juices, especially Valencia (> 85%, P < 0.05), favored germination and AF, whereas sterile water rarely stimulated
germination (< 1%). Juice quality analysis using Pearson's correlation test revealed a moderate relationship between the Brix/Acid ratio and germination (r = 0.783, P < 0.05). The Valencia juice effect was concentration- and pH-dependent, and the maximum rate was reached in 1.5% juice with pH of 3.4. Most carbon, nitrogen, or complex sources did not favor germination or AF with exception of PDB, ammonium nitrate, and yeast extract. An incubation period of 18 to 24 h at 24°C were required for peak germination and AF. Further analysis of critical juice components using synthetic juice revealed sugars, salts, citric acid, and thiamine combined were most important for germination and AF (> 80%, P > 0.05). These results provide a better understanding of fungal biology and a robust and convenient system for further applications such as screening for efficacious fungicides.

## Host colonization and substrate utilization by wood-colonizing Ascomycete fungi in the grapevine trunk disease complex

E. GALARNEAU (1), C. Wallis (2), K. Baumgartner (2)

(1) University of California-Davis, U.S.A.; (2) USDA-Agricultural Research Service, U.S.A.

Grapevine trunk diseases cause chronic wood infections (cankers) in mixed infections within the same vine. To determine the synergistic interactions of trunk-pathogen communities and their impact on the host we are characterizing, on a pathogen-by-pathogen basis, fungal damage to woody cells and tissues. Our focus is on two taxonomically unrelated Ascomycetes: Botryosphaeria dieback pathogen *Neofusicoccum parvum* and Eutypa dieback pathogen *Eutypa lata*. After inoculating plants and identifying the host-defense compounds that accumulated in wood cankers, we tested their effects on fungal growth in vitro. Both fungi significantly degraded and utilized rutin, epicatechin, and piceid, suggesting that these fungi tolerate and possibly subvert the biochemical host-defense response. In contrast, fungal growth was significantly inhibited by lignin and gallic acid. Only *E. lata* discolored solid media amended with gallic acid, signifying phenoloxidase activity, which is consistent with its status as a soft-rot fungus. Previous work showed *E. lata* colonizes the ray parenchyma, xylem vessels and fibers, which we verified are also proliferated by *N. parvum*. The ability of these fungi to degrade wood was evaluated in planta by imaging the microscopic features of soft rot. As both fungi have similar gene families associated with cell-wall degradation and secondary metabolism, it is not surprising that they may share similar strategies for colonization and substrate utilization.

## Virulence of Botrytis prunorum associated with blossom blight on Japanese plum in Chile

E. FERRADA (1), B. Latorre (1), J. Zoffoli (1), G. Diaz (2)

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

Blossom Blight (BB) is an important disease that affects the productivity of stone fruits. Recently, *B. prunorum* was identified causing BB on Japanese plum (*Prunus salicina*) in Chile. Symptoms consisted on a light brown necrosis of flowers. The objective of this study was to compare the virulence exhibited by isolates *B. prunorum* and isolates of *B. cinerea* affecting plums. Virulence tests were conducted with seven isolates of *B. prunorum* on detached plum flowers and mature fruits 'Larry Ann' inoculated with a conidial suspension (10⁶ conidia/mL) and mycelial plugs (4 mm in diameter). Mature fruits of 'Granny Smith' apples and 'Hayward' kiwifruits were similarly inoculated. Flowers and fruits were incubated for at least 48 h at 20°C in humid chambers. Seven *B. cinerea* isolates were included in all virulence tests. In addition, the virulence of *B. prunorum* and *B. cinerea* was studied on detached bean leaves that were inoculated with a mycelial plug (4 mm) incubated for 4 d at 20°C in humid chambers. All isolates of *B. prunorum* and *B. cinerea* were pathogenic on flowers and fruits of plums as well as on apple and kiwifruit and bean leaf. All isolates of *B. prunorum* were significantly (P<0.001) less virulent than *B. cinerea* isolates. Differences in virulence between *B. cinerea* and *B. prunorum* can be attributed to differences in the genetic expression of virulence factors. These results suggest that *B. prunorum* plays a secondary role in the epidemiology of BB of plums.

## Modelling potential inoculum availability of Mycosphaerella nawae in persimmon leaf litter using Bayesian growth curves

A. VICENT (1), J. Martínez-Minaya (2), A. López-Quílez (3), D. Conesa (3)

(1) Instituto Valenciano de Investigaciones Agrarias (IVIA). Moncada 46113, Valencia, Spain., Valencia, Spain; (2) Instituto Valenciano de Investigaciones Agrarias (IVIA), Moncada 46113, Valencia, Spain., Valencia, Spain; (3) Departament d'Estadística i Investigació Operativa, Universitat de València. Burjassot 46100, Valencia, Spain., Valencia, Spain

Circular leaf spot, caused by *Mycosphaerella nawae*, is a serious disease of persimmon (*Diospyros kaki*) inducing necrotic lesions on leaves, defoliation and fruit drop. The disease was initially restricted to humid regions in Japan and South Korea and in 2008 it was detected in arid areas in Spain. The fungus forms pseudothecia in leaf litter during winter and ascospores are released in spring infecting susceptible leaves. Persimmon growers are advised to spray with fungicides during the period of inoculum availability, which was defined based on ascospore counts. In order to develop a model of potential inoculum availability, samples of leaf litter were collected weekly in six affected orchards from 2010 to 2015. Leaves were soaked, placed in a wind tunnel, and released ascospores were counted under the microscope. Hierarchical Bayesian methods were used to fit the dynamics of ascospore production in the leaf litter to Gompertz and logistic growth curves, including temperature and rainfall as nonlinear effects. MCMC methods were used to approximate the posterior distribution of the parameters. Results showed that accumulated degree days best described ascospore dynamics with both Gompertz and logistic curves. The predictive ability of the models was evaluated using ascospore data from two orchards not used in model development. The resulting best model was implemented in a disease warning system to schedule fungicide sprays for the control of circular leaf spot.

## Factors affecting germination of pseudosclerotia of Monilinia vaccinii-corymbosi on lowbush blueberry

T. CASE (1), S. Annis (1)

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

In the lowbush blueberry (*Vaccinium angustifolium*) industry of Maine, substantial yield loss can occur annually from the disease, mummy berry, caused by *Monilinia vaccinii-corymbosi*, if left unmanaged. The primary objective of this study is to better understand the environmental conditions that favor the germination of pseudosclerotia. We have conducted *in vitro* incubation experiments for the past two years, in which the germination of apothecia from pseudosclerotia has been observed in response to chill-hours (time between 0-7°C). To further identify conditions affecting the germination of pseudosclerotia, we have incubated pseudosclerotia over a wide range of chill-hours (800-2380 chill-hours), under three different soil saturations (50, 60 and 75%), as well as three different post-chill temperature regimes (10, 13 and 16°C). Germination of pseudosclerotia was greater under the highest temperature we tested (16°C), and moderate soil moisture conditions (60%). The development of apothecia from pseudosclerotia was affected by chill-hours, but varied substantially between treatments from different fields. Identifying the environmental conditions favoring the development of apothecia in the lab assists in managing this pathogen in the field through the development of a predictive model for germination. We will use this model in continuing our program for forecasting mummy berry, which gives growers an opportunity to effectively manage this disease.

## Relationship between genetic and morphological characters of Botrytis cinerea isolates obtained of blueberry fields in southern Chile

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

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

*Botrytis cinerea* is the most important pathogen in blueberry crops (*Vaccinium* spp.) in southern Chile. The climate conditions of the zone and the presence of some populations fungicide resistant, increase its incidence. Due to the high morphological variability observed on Botrytis populations, the aim of this work was to characterize isolates from eight locations (Lat. 39°06'S to 41°28'S), in order to establish some relationship between morphological and genetic variability. For this, 39 monosporic cultures of *B. cinerea* obtained from mature blueberry fruits were morphologically characterized about mycelia color, sporulation level, growth rate, conidia size and sclerotia production. On the other hand, genetic characterization was done by PCR detection of transposons *Boty* and *Flipper*, to determine their genotype. In addition, coding fragments for the MS457 gene were sequenced and phylogenetic tree was built. The genetic studies revealed the presence of four genotypes, predominating Vacuma in the geographic region.
The results indicate that the presence of transposable elements does not affect the growth rate of the isolates. However, different sizes of conidia are distinguished by genotype, possessing at least one of the transposons conidia size increases, then Transposa presented conidia larger while Vacuma the smaller. On the other hand Boty produced the bigger sclerotia.

## Mining of abiotic and biotic factors for prediction of soybean sudden death syndrome (SDS) symptoms

Z. NOEL (1), M. Roth (1), J. Wang (1), E. Papenfuss (1), D. Kramer (1), M. Chilvers (1)

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

*Fusarium virguliforme* is the causal agent of soybean sudden death syndrome (SDS) in the U.S. The pathogen infects roots initially, but foliar chlorosis and necrosis caused by a phytotoxin, do not usually appear until later in the growing season when it is too late to consider replanting. In this study, parameters measured before planting were used as indicators of SDS development later in the season. Prior to planting, soil nutrients (pH, K, Ca, Mg, Zn, Mn, Cu, Fe, and organic matter) soybean cyst nematode counts (eggs, juveniles, and adults), and *F. virguliforme* inoculum density were used to determine which variables or combination of variables helped explain SDS development throughout the growing season. Principle components analysis allowed for dimensional reduction and indicated which variables were most important for disease prediction. Disease symptoms at R5 and R6 were significantly correlated with soybean cyst nematode data and *F. virguliforme* inoculum density. Other multivariate techniques are currently being performed to allow for a better understanding of the factors that contribute to the disease development of SDS.

## Effects of inoculum substrates on root rot of soybean caused by *Fusarium oxysporum* and *Fusarium graminearum*

D. CRUZ (1), D. Mayfield (2), Y. Meng (1), G. Munkvold (1), L. Leandro (1)

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

*Fusarium oxysporum* (Fo) and *F. graminearum* (Fg) are associated with the Fusarium seedling disease and root rot complex in soybeans. Published soil infestation methods have limitations in the expression of disease. Our objective was to compare the efficacy of infested substrates for generating root rot caused by Fo and Fg and evaluate adverse effects of non-infested substrates on roots. Autoclaved millet and rice hulls infested with Fo and Fg were added to soil at a rate of 5% (by vol.). Susceptible cultivars Jack and MN1805 were evaluated 2 weeks after planting for root length and root rot using the WinRhizo image analysis software; root rot was also estimated visually on a percent scale. Cultivars did not differ in root length and root rot assessed by image analysis, but visually-assessed root rot was greater in Jack (P=0.008). There was a significant interaction between substrate and Fusarium species for root rot assessed by both methods (P<0.0001). Millet infested with Fg produced the greatest root rot severity (37%), followed by millet infested by Fo (24%), whereas infested rice hulls resulted in low root rot (15 and 10% for Fo and Fg, respectively). Root rot for non-inoculated substrates was less than 5%. Infested and non-infested millet reduced root length compared to rice hulls. Pathogen colonization was assessed using qPCR. Our study suggests that millet is more effective for producing seedling root rot but can cause some adverse effects on root growth.

## Preliminary results from Multilocus Sequence Typing of the soilborne fungal pathogen *Sclerotium rolfsii*

P. SORIA (1), M. Smith (1), N. Dufault (1)

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

*Sclerotium rolfsii* is an important soilborne fungal pathogen of hundreds of plant species worldwide, including economically important crops such as tomato and peanut in the southeastern US. Currently, molecular studies are scarce for this pathogen, leading to a lack of an experimental framework to study the biology *S. rolfsii* using genetic methods. There is, however, evidence for significant phenotypic variation among *S. rolfsii* isolates in terms of virulence, growth rate, sclerotial size, and temperature tolerance. Methods of measuring genetic variation are currently limited to ambiguous mycelial compatibility groups, and few sequence analyses using the ITS (internal transcribed spacer) rDNA. To accurately characterize genetic diversity among phenotypically variable *S. rolfsii* populations, a more elaborate and sequence-based experimental framework is required. Multilocus Sequence Typing (MLST) was chosen as a sequence-based method for cryptic species recognition and for identifying genetic diversity of *S. rolfsii* in the Southeastern US. Several housekeeping genes (LSU, RPB1, RPB2, TEF, and MCM7) that are commonly used in MLST analyses were evaluated to choose the most informative loci to characterize the population structure of *S. rolfsii*. The resulting MLST scheme will contribute to establishing a molecular framework for this important soilborne pathogen and will increase the current understanding of the biology and epidemiology of *S. rolfsii*.

## A real-time PCR and digital droplet PCR assay for quantification of *Polymyxa betae* in sugar beet roots

B. SMITH (1), F. Martin (1)

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

*Polymyxa betae* is an obligate pathogen capable of vectoring several viruses of sugar beet that can cause large losses in production. In the past, quantifying *P. betae* infection levels required time-consuming staining and visual examination using light microscopy. A new species-specific quantitative real-time PCR assay that allows for rapid quantification of *P. betae* infection levels in sugar beet roots has been developed. The assay has been designed to target the *P. betae* internal transcribed spacer (ITS) region of the genomic rDNA using sequence data from forty isolates. To achieve species-specificity, the assay primers and probe were designed against nineteen *Polymyxa graminis* ITS sequences from GenBank. In practice, the assay has demonstrated species-specificity in all fifteen *P. betae* isolates tested and has not amplified *P. graminis* isolates. Three internal plant controls, two targeting single copy nuclear genes and one targeting the ITS region of the rDNA, have been multiplexed with the pathogen assay to standardize results across samples. Further, the assay has also been optimized for digital droplet PCR, which allows for more precise quantification of minor differences in infection levels than conventional real time PCR. These tools will allow for more efficient quantification of *P. betae* infection levels in experimentation evaluating host resistance, variation in aggressiveness among isolates and virus transmission studies.

## Ray blight disease (*Stagonosporopsis tanaceti*) development in pyrethrum

M. BHUIYAN (1), T. Groom (2), M. Nicolas (3), P. Taylor (3)

(1) The University of Melbourne, Australia; (2) Botanical Resources Australia, Australia; (3) The University of Melbourne, Australia

Pyrethrum (*Tanacetum cinerariifolium*) is commercially cultivated in Australia for pyrethrins which are used as natural pesticides. Ray blight caused by *S. tanaceti* is considered as the most devastating fungal disease and is recognized as one of the limiting factors of pyrethrum production. The pathogen is known to infect the flowers and leaves however, little is known about the mechanism of infection and process of tissue colonisation. Therefore, this study was undertaken to identify disease development within the whole plant using traditional culturing and histopathology, and elucidate the life cycle of *S. tanaceti*. Ray blight disease development in pyrethrum plants was the result of colonisation of the parenchyma cells in the hypodermis and cortex with no infection of the vascular tissues. Pycnidia developed in severely infected tissues which developed a deep brown discolouration due to necrosis caused by intra- or inter cellular colonization. Secondary infection of upper flower stems, flower buds and flower tissues most likely occurred from pycniospores released from the pycnidia in the infected leaves and petioles and being disseminated through wind, rain or water-splash. The pathogen did not infect roots of plants with infected crown tissue. This process of infection and colonisation of pyrethrum plants provides a better understanding of the complete life/disease cycle which will lead to improved control measures through the strategic use of fungicides.

## Identifying Diversity Within *Corynespora cassiicola*, Cause of Target Spot of Cotton, Tomato, and Soybean in the Southeastern U.S.

L. SUMABAT (1), M. Brewer (1), R. Kemerait (2)

(1) University of Georgia, U.S.A.; (2) University of Georgia, U.S.A.
Target spot, caused by the fungus *Corynespora cassiicola*, is a disease that is emerging on crops in the southeastern U.S., including cotton. Previous studies suggest that populations from cotton, tomato, and soybean in the U.S. are host specific and genetically differentiated based on host of origin. Although variation in aggressiveness to cotton was observed, no genetic variation was detected within populations sampled from cotton. The aim of this study was to develop markers to detect genetic variation in *C. cassiicola*. Variable microsatellite motifs with conserved flanking regions were identified in draft genomes of two isolates from cotton, an isolate from tomato, and an isolate from soybean. Thirty-one sets of primers were designed, and then tested for amplification of these four isolates. The nineteen loci that amplified well were evaluated on a panel of 15 isolates from diverse hosts of origin and geographic regions. Fourteen of the markers were robust and informative. Preliminary results showed diverse genotypes among *C. cassiicola* populations from different hosts, yet there was no genotypic variation detected within tomato or cotton isolates sampled from different cultivars, states, and years, suggesting that populations from these hosts represent distinct clonal genotypes. Determining levels of variation among isolates of *C. cassiicola* from cotton is critical for screening for cultivar resistance and identifying fungicides for management of target spot.

## Oxygen and an aerial state are independently required for asexual development of Aspergillus species

M. CHI (1), K. Craven (2)

(1) The Samuel Roberts Noble Foundation, U.S.A.; (2) The Samuel Roberts Noble Foundation, U.S.A.

Asexual sporulation is one of the primary means for aerial dissemination of many plant pathogenic fungi. The generation of asexual spores usually involves dynamic changes in cellular polarity and morphogenesis (e.g. from filamentous hyphae to oval spores). While sporulation appears to be suppressed in *in planta*, it is seemingly de-repressed outside of plant tissues, where it encounters an aerial environment. In this study, we identify key components that allow fungi to distinguish between *in planta* and aerial environments. We observed conidiophore development of several *Aspergillus* species in an artificial environment that mimicked the *in planta* condition, but where we could enforce either high or low oxygen conditions. This experimental design enabled us to reveal that not only oxygen but also the physical, aerial state are positive environmental factors inducing conidiogenesis in most of the aspergilli tested in this study. Transcriptional analysis combined with direct observation of conidiation-defective mutants revealed that expression of key regulatory genes for conidiophore development is indeed regulated by these environmental cues and that the production of reactive oxygen species is required for polarized cell differentiation. Our findings provide not only novel insights into how fungi respond to an aerial environment but also inform an improved model for the regulatory pathway controlling conidiophore development.

## Current Status and Ongoing Conservation Efforts for the Federally Endangered Species Pityopsis ruthii

T. EDWARDS (1), P. Wadl (2), R. Trigiano (3), E. Hatmaker (3), S. Boggess (3), P. Moore (1), M. Staton (5), W. Klingeman (1), E. Bernard (1), A. Dattilo (6), B. Ownley (1), T. Rinehart (7), M. Pistrang (8), J. Skinner (1), G. Call (9), A. Windham (1), D. Hadziabdic (1)

(1) University of Tennessee, Knoxville, TN, U.S.A.; (2) United States Department of Agriculture, Agricultural Research Service, Charleston, SC, U.S.A.; (3) University of Tennessee, U.S.A.; (4) University of Tennessee, Knoxville, U.S.A.; (5) Universitiy of Tennessee, Knoxville, TN, U.S.A.; (6) Tennessee Valley Authority, Knoxville, TN, U.S.A.; (7) United States Department of Agriculture, Agricultural Research Service, Poplarville, MS, U.S.A.; (8) United States Department of Agriculture, Forest Service, Cleveland, TN, U.S.A.; (9) United States Fish and Wildlife Service, Cookeville, TN, U.S.A.

*Pityopsis ruthii* is an endangered species endemic to the Hiwassee and Ocoee rivers in Tennessee. Due to limited information regarding biology and genetics of *P. ruthii*, our research over the past six years have focused on conservation and recovery efforts. Using applied and molecular techniques, a basic understanding of the biology, propagation, population genetics, and diseases of *P. ruthii* has been gained. Micropropagation protocols using leaf and receptacle tissues have been developed. Clones from these experiments had a 73% survival rate one year after introduction to native habitats. Vegetative propagation of stems is highly successful. Seed germination for long-term stored seeds was low (<1%) and highly variable for freshly collected seeds across different locations. A total of 814 individuals from both rivers were analyzed using chloroplast and nuclear microsatellites and indicated higher genetic diversity than initially hypothesized. Analyses of chloroplast and nuclear microsatellites showed differences between the respective data sets, indicating presence of gene flow and population structure. Two diseases were identified from greenhouse grown plants: aerial blight, caused by *Rhizoctonia solani*, and powdery mildew, caused *Golovinomyces cichorecearum*. Understanding the biology and genetics of *P. ruthii* will facilitate long-term conservation efforts and provide information on reintroduction and preservation of biodiversity for this endangered species.

## Genome sequencing of Synchytrium endobioticum offers insight into its carbohydrate degrading enzymes and effectors

H. NGUYEN (1), B. van de Vossenberg (2), T. van der Lee (2), D. Joly (3), D. Smith (4), M. van Gent-Pelzer (2), P. Bonants (2), C. Levesque (1)

(1) Agriculture and Agri-Food Canada, Ottawa, ON, Canada; (2) Wageningen University and Research Centre, Netherlands; (3) University of Moncton, Moncton, NB, Canada; (4) Canadian Food Inspection Agency, Canada

*Synchytrium endobioticum* is an obligate fungal parasite causing severe and persistent potato wart disease that render potato tubers unmarketable. Therefore, quarantine measures are enforced worldwide to prevent the spread of this economically important plant disease. The first detection of potato wart in North America occurred in 1909 in Newfoundland. More recently in 2000, it was found in a field of Prince Edward Island. We sequenced and assembled the genomes of two different genotypes: *S. endobioticum* MB42 from the Netherlands and *S. endobioticum* DAOM 229326 from Canada. The genome size ranged from 20.6 to 23.5 Mb and between 8000 and 8800 genes were predicted for the respective strains. Carbohydrate degrading enzymes and effectors are being analyzed to help the development of more robust genotyping and pathotyping assays and to understand better the evolution of this early lineage of plant pathogens.

## Origin and consequence of myrtle rust (Puccinia psidii) in the New Caledonian biodiversity hotspot

S. JULIA (1), M. Laurent (2), C. Fabian (1)

(1) Institut Agronomique Néo-Calédonien, New Caledonia; (2) CIRAD (Centre de coopération internationale en recherche agronomique pour le développement), New Caledonia

The myrtle rust disease, caused by the fungus *Puccinia psidii*, is considered as one of the main environmental threat to plants belonging to Myrtaceae family worldwide. In New Caledonia, a biodiversity hotspot renowned for its extraordinary botanical richness and endemicity, the fungus was first reported in 2013. The Myrtaceae, the principal target of *P. psidii*, holds a prominent position in the New Caledonian flora by being the most species-rich family. Taking into account the ecological importance of Myrtaceae in many ecosystems, it was agreed to establish a disease management approach to mitigate the spread and the potential long term impacts of the pathogen. In this perspective, it is essential to refine our understanding of the dynamics of the host-pathogen-environment interactions. In order to reach this goal, we have conducted field-based assessments of the host range and disease monitoring on several species in a semi-controlled environment during two years. A genotyping survey of the rust showed that a unique genotype has been established throughout the whole territory. To date, *P. psidii* was reported on at least 40 host species. The results showed a wide variation in responses to the disease within Myrtaceae and across different habitats, as previously showed in other locations where the rust occurs. Intraspecific variability was also observed among a range of susceptible species.

## Development of a multiplex PCR microsatellite marker set for Raffaelea lauricola, and its potential applications

T. DREADEN (1), M. Hughes (2), J. Smith (2)

(1) USDA FS, U.S.A.; (2) University of Florida, U.S.A.
Laurel wilt is an emerging disease of trees in the family Lauraceae characterized by systemic wilt and rapid mortality. It is caused by the fungus *Raffaelea lauricola* and its vector, the redbay ambrosia beetle (*Xyleborus glabratus*) that was likely introduced to the southeastern United States from Asia. The origin of the US introduction and the population genetics of this devastating pathogen have not yet been characterized. To help facilitate this we developed a panel of microsatellite markers that can be multiplexed, amplify two or more loci in a single reaction, into eight PCRs. The marker set can be used to screen 11 *R. lauricola* isolates per 96 well PCR plate, eliminating the need to combine samples post amplification. The marker set reduces cost by using conventional hot start Taq, 15ul reaction volumes, multiplexing PCRs and fluorescein-12-dUTP to label PCR amplicons instead of fluorescently labeled primers. Future work will utilize the microsatellite marker set to investigate the genetic structure of *R. lauricola* within both the US and Asian populations and generate information regarding the number of introductions, possible origins of the introductions into the US and potential for development of new strains.

## Genome and transcriptome analysis of *Phellinus noxius*, a wood decay fungus

H. LEE (1), C. Chen (2), I. Tsai (3), R. Liou (2), C. Chung (2)

(1) National Taiwan University, Taiwan; (2) National Taiwan University, Taiwan; (3) Academia Sinica, Taiwan

*Phellinus noxius*, which causes brown root rot disease in more than 200 tree species, has recently gained much attention in East Asia. However, little is known about the genome of *P. noxius*. In this study, four mate-pair libraries of a *P. noxius* basidiospore isolate A42 were sequenced by using Illumina platform. Genome assembly and *ab intio* gene prediction resulted in a draft genome of ~32 Mb (N50 = 1.55 Mb) which includes 11,025 coding sequences. Both *A* and *B* mating type loci were identified. Sequence alignment of A42 and a Japanese isolate showed high similarity in *B* locus and one of the two HD1-HD2 pairs in *A* locus. Sequence analysis of the *B* locus of 12 basidiospore isolates from a single basidiocarp showed no polymorphism, which suggests a bipolar mating system. To facilitate gene annotation and to better understand the expression profiles of genes involved in stress responses, transcriptomes under various stress conditions were examined, encompassing temperature shift and nutrient deficiency (minimal medium, carbon deficiency, nitrogen deficiency). Genes involved in nutrient metabolism, secondary metabolites biosynthesis, hydrophobin production, and stress responses were induced. A particularly up-regulated pattern was found in the glyoxylate metabolism pathway which produces oxalate, an organic acid known to facilitate the activity of cell-wall degrading enzymes, as a byproduct. Investigations of the stress-related genes in disease development are underway.

## Ganoderma Species Associated with Declining or Dead Trees in the Southeastern United States

A. LOYD (1), J. Smith (2), R. Blanchette (3), B. Held (3), C. Barnes (4), M. Schink (5)

(1) Bartlett Tree Experts/University of Florida, U.S.A.; (2) University of Florida, U.S.A.; (3) University of Minnesota, U.S.A.; (4) Departamento Nacional de Proteccion Vegetal, INAP, Ecuador; (5) Self, Port Crane, NY, U.S.A.

*Ganoderma* is a large and diverse genus of wood decay fungi that can cause a white rot of the lower bole or roots of hardwood, conifer and palm trees. The pathogenicity and decay ability within the genus is understudied. Some, such as *G. zonatum* are aggressive pathogens, and have been associated with tree failure and mortality of mature palms, while others, such as *G. applanatum s.l.*, are often saprophytic causing decay in old, weakened tress, but can also be facultative pathogens. *Ganoderma* species identification tools are needed to assist arborists with predicting tree failure when fruiting bodies of *Ganoderma* are present. Based on a preliminary survey of *Ganoderma* species found in Southeastern United States, over 100 isolates representing different taxa were collected from living tree species and dead wood across 4 states: Florida, Georgia, South Carolina and North Carolina. Taxa investigated in the survey were the *G. applanatum* complex and related species, *G. curtisii*, *G. meredithae*, *G. sessile*, *G. tsugae*, *G. tuberculosum*, *G. weberianum-subamboinens* species complex, *G. zonatum*, and *Tomophagus colossus* (syn. *G. colossus*). Wood block decay assays are underway to quantify decay ability of select species. Future studies will investigate differences in decay rates and pathogenicity, host specialization and phylogeography of the most commonly observed *Ganoderma* taxa collected in the Southeastern United States.

## Progress of severity of brown rust and orange rust during two sugarcane crop seasons in Florida

S. SANJEL (1), M. Hincapie (1), B. Chaulagain (1), J. Comstock (2), R. Raid (1), P. Rott (1)

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

Brown rust (caused by *Puccinia melanocephala*) and orange rust (caused by *Puccinia kuehnii*) are currently the two most damaging fungal diseases of sugarcane in Florida. To determine progress of these two diseases during the crop season, brown rust susceptible cultivar CL90-4725 and orange rust susceptible cultivar CL85-1040 were planted in field experiments at Belle Glade in November 2013 and in November 2014. Intensity of pathogen sporulation and percent rust affected area were assessed on the top visible dewlap leaf (TVD) and the fourth leaf below TVD every two weeks. Brown rust symptoms were mainly observed from mid-March to mid-July in 2014 and from mid-April to mid-August in 2015. Sporulation intensity and leaf damage was highest in May-June 2014 but this period shifted to June-July in 2015. In contrast, orange rust symptoms were observed all year round, but sporulation and percent diseased leaf area peaked from mid-May to early August. Later on, disease severity decreased until October when another significant increase of orange rust occurred until the end of the year. This pattern of orange rust progress was similar during the 2015 and 2016 crop seasons. Overall, disease severity was higher for orange rust than for brown rust and orange rust impacted sugarcane growth for a longer period of time during the crop season. Conducive environmental conditions for disease progress remain to be determined but appear to be broader for orange rust than for brown rust.

## Host specificity of *Colletotrichum* sp. on tree tomato (*Solanum betaceum*) and mango (*Mangifera indica*) crops

S. RESTREPO (1), L. Cabrera-Villamizar (1), C. pardo (1), S. Rojas (1), P. Rojas (1), G. Danies (1), P. Jiménez (2)

(1) Universidad de los Andes, Colombia; (2) Universidad Militar Nueva Granada, Colombia

*Colletotrichum* species are one of the main problems in crop production in Colombia. Several research efforts have been devoted to characterize the populations of this fungus. However, the phylogenetic relationships among species within this genus are still uncertain. *Colletotrichum* species have been traditionally classified using morphological traits and based on their host, despite the fact that the host range of these species is still unclear. In Colombia, the phylogenetic relationships of *Colletotrichum* species have been determined using molecular markers as well as morphological and physiological characteristics. Preliminary results have suggested host specificity among the species assessed. However, this assays were conducted by directly damaging the plant tissue. The aim of this study was to investigate host-pathogen interactions among isolates collected from tree tomato and mango from the pre-penetrating up to the colonization stages of the pathogen. Isolates were thoroughly characterized molecularly and microscopically and were subsequently inoculated onto both hosts. Strains of *C. gloeosporioides*, *C. asianum*, *C. theobromicola*, and *C. tamarilloi* were inoculated on whole-plants and detached-leaves. Pathogenicity, latent period, aggressiveness, and sporulation were recorded to evaluate the fitness of each species on the different hosts. Preliminary results indicate the presence of host preferences among the different species of *Colletotrichum* assessed.

## Arbuscular mycorrhizal fungal communities associated with organic and conventional onion crops in the Columbia Basin of Washington

L. DU TOIT (1), A. Knerr (2), D. Wheeler (2), D. Schlatter (3)

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

Onion mycorrhizal associations were assessed in conventional vs. organic fields in the Columbia Basin of the Pacific Northwest USA in 2014. Soil was sampled from five sites in each of four conventional and five organic fields prior to planting in March. Arbuscular mycorrhizal fungi (AMF) were baited from each soil by planting seed of the onion cv. Tamara. Plants also were sampled from each field at bulb initiation (mid-June). Onion roots were stained
to quantify mycorrhizal colonization, and DNA was extracted for 454 pyrosequencing of AMF communities using 18S rDNA and Glomerales-specific primers. AMF were prevalent in all fields, with no significant differences in colonization of baited roots in soil samples from organic vs. conventional fields (mean ± SE of 77 ± 4 vs. 75 ± 4%, respectively). Baited AMF communities from spring soil samples differed in community structure in conventional vs. organic fields (PERMANOVA Pseudo-F = 4.08, *P* = 0.02), though AMF richness and evenness did not differ significantly (t-tests and Welch-Satterthwaite t-test *P* ≥ 0.2). AMF communities in mid-summer root samples also differed in community structure (PERMANOVA Pseudo-F = 7.92, *P* = 0.0005), with greater richness (Welch-Satterthwaite t-test *P* = 0.02) but similar evenness in organic vs. conventional fields. Differences in AMF communities between organic and conventional fields were attributed mostly to differences in relative abundance of *Glomus* and *Claroideoglomus* spp.

## Fungicide resistance and host susceptibility of *Colletotrichum orbiculare* infecting cucurbit crops in North Carolina

N. NOËL (1), L. Quesada-Ocampo (1)

(1) NCSU, U.S.A.

Cucurbit anthracnose, caused by the ascomycete Colletotrichum orbiculare syn. lagenarium, is a widespread fruit and foliar disease that results in yield losses across the United States. In North Carolina, favorable environmental conditions, host availability, and long growing seasons offer ample opportunity for disease development, resulting in significant economic losses. Application of effective fungicides and planting of resistant cultivars, along with appropriate cultural practices, have proven effective in reducing anthracnose. However, the appearance of new races of the pathogen and development of fungicide resistance has limited the efficacy of these control strategies. The objective of this study is to characterize C. orbiculare populations infecting cucurbit crops in North Carolina, determine their sensitivity to fungicides, and their virulence in watermelon cultivars. Isolates obtained from cucurbit fields in North Carolina and elsewhere for comparison, where evaluated for fungicide sensitivity in vitro with several fungicides and various concentrations. Local isolates of different races were used to evaluate host susceptibility of 24 popular cultivars of watermelon (Citrullus lanatus) in field and greenhouse trials. Results indicate that an integrated disease management strategy that combines effective fungicides and host tolerance will be required to control cucurbit anthracnose in North Carolina.

## Determining infection courts for *Fusarium oxysporum* f. sp. *niveum* leading to watermelon seed infestation

A. PETKAR (1), P. Ji (1)

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

Fusarium wilt of watermelon caused by *Fusarium oxysporum* f. sp. *niveum* (FON) is a seedborne and seed transmitted disease. Seeds latently infected by the pathogen serve as a significant inoculum source and may result in serious disease in the field. It is unknown how watermelon seeds can get infected by the pathogen. To determine potential infection courts leading to watermelon seed infestation, experiments were conducted in 2013 to 2015 using different methods to inoculate watermelon plants with FON. Watermelon seeds were harvested when mature and seed infestation by FON was determined by isolation on peptone PCNB agar and real-time polymerase chain reaction (PCR) analysis. Swab-inoculation of immature fruit resulted in infestation of 18 to 54% of the seedlots under field conditions and 0.5 to 13% under greenhouse conditions when seeds were not surface sterilized before isolation. Seed infestation was detected on 0 to 15% of the seedlots when seeds were surface sterilized before isolation. Inoculation of stigma of female flowers resulted in infestation of 0 to 14% of the seedlots. Inoculation of peduncle also resulted in infestation of 0 to 10% of the seedlots in the field experiments when seeds were not surface sterilized before isolation. The results suggest that watermelon tissues including immature fruit, flowers and peduncle could be potential infection courts of FON leading to seed infestation by the pathogen.

## Evaluating isolate aggressiveness of *Fusarium oxysporum* f.sp. *niveum* in Florida

T. SANCHEZ (1)

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

Fusarium wilt, caused by *Fusarium oxysporum* f. sp. *niveum*, is a major soil borne disease of watermelon with limited host resistance available. Understanding isolate aggressiveness is important for variety selection and other management strategies. Pathogenicity tests of 72 isolates collected from 10 Florida counties were conducted to examine their aggressiveness on the variety Black Diamond under greenhouse settings. Seedlings were inoculated with a conidial suspension using a root dip method. Aggressiveness was measured by the percentage of wilted plants and AUDPC for incidence, over a 4 week period after inoculation. Significant (P<0.05) differences between isolates were found using ANOVA. Based on wilted plants, isolates were characterized into 4 aggressiveness levels; not aggressive, weak, medium and severe (0%, 1-32%, 33-67% and 68-100% wilt respectively). It was observed that 33.3% of the isolates were not aggressive, 15.3% weak, 25.0% medium, and 26.4% severe. Quantitative diversity indices (Simpson's = 0.27 and Shannon entropy *H*' = 1.35) indicate that these aggressiveness levels are highly diverse in Florida, and that no single level was dominant. Characterizing the aggressiveness is a necessary step for race determination, as well as the assessment of various integrated management strategies. Further research is presently being conducted that evaluates the efficacy of current integrated management strategies on isolates with a severe aggressiveness rating.

## Characterization of fungi associated with tomato leaf mold in the United States

L. GARBER (1), N. LeBlanc (2), A. Orhinsky (1), C. Smart (3)

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

Tomato leaf mold is one of the most prevalent diseases affecting high tunnel-grown tomatoes in the USA. Disease symptoms include chlorotic to necrotic lesions, defoliation and decreased yields. Previous studies focused on the fungus, *Passalora fulva* (syn: *Cladosporium fulvum*, *Fulvia fulva*) as the causal pathogen of leaf mold. Our initial preliminary data suggests that sporulation on lesions is associated with a number of *Cladosporium* spp., primarily *C. cladosporioides* and *C. pseudocladosporioides*. Leaf mold-associated fungi were collected from MN and NY by surface sterilization of symptomatic tissue and by direct plating of spores from lesions. Single spore isolates were generated and PCR amplification of internal transcribed spacer regions and alpha-actin were performed. Amplicons were sequenced and aligned with available reference species sequences obtained from GenBank using Seaview. A majority of leaf mold isolates collected fell within the *C. cladosporioides* complex. Representative isolates from each *Cladosporium* spp. did not appear to be pathogenic. The frequency with which *C. cladosporioides* and *C. pseudocladosporioides* are associated with leaf mold lesions suggests that they may be important in the epidemiology and etiology of the disease. Studies regarding the association between *C. cladosporioides*, *C. pseudocladosporioides* and leaf mold of tomatoes are ongoing and may lead to the development of unique disease management strategies in high-tunnel tomatoes.

## Mitochondrial and nuclear gene sequences to infer the phylogeny of Pezizomycotina (Ascomycota)

E. Franco (1), S. Lopez (1), M. Saparrat (2), P. BALATTI (1)

(1) Facultad de Ciencias Agrarias y Forestales, Argentina; (2) Faculad de Ciencias Agrarias y Forestales, Argentina

Pezizomycotina is the largest subphylum of Ascomycota. It includes filamentous species widespread in nature, where they live as saprotrophs, mutualists and/or parasites of animal, plants and fungi. We evaluated the phylogeny of such an important subphylum not only by analyzing the sequence of nuclear genes but also of the core mitochondrial ones, since this organelle may provide additional information. Hence, we compared the phylogenetic reconstitution provided by the concatenated nucleotide sequences of 12 core mitochondrial genes (*atp6*, *cob*, *cox1-3*, *nad1-6* and *nad4L*) and the individual and concatenated nucleotide sequences of 5 widely used nuclear genes (*β-tub*, *tef-1a*, *gpd*, *rpb1* and *rpb2*) of 28 Pezizomycotina species. Alignments were generated with ClustalW and automatically curated with Gblocks 0.91b. Best-fit model of evolution was selected with jModelTest
2.1.7 and data matrices were analyzed under maximum likelihood and bayesian criteria in PhyML 3.0 and MrBayes 3.2, respectively. We found that only *rph2* and the concatenated nuclear and mitochondrial datasets clustered species in accordance with the current systematic of this subphylum, although incongruences were observed in some internal nodes. Discrepancies between the remaining datasets are indicative of the complex evolutionary histories and confirmed that the genes for an accurate phylogenetic estimation should be chosen with special care.

## In silico screening of genes coding for secondary metabolites in the phytopathogenic fungus *Stemphylium lycopersici*

P. BALATTI (1), E. Franco (2), S. Lpez (2), M. Saparrat (3)

(1) Facultad de Ciencias Agrarias y Forestales, La Plata, Argentina; (2) Centro de Investigaciones de Fitopatopatologia Facultad de Ciencias Agrarias y Forestales UNLP, La Plata, Argentina; (3) Catedra de Microbiologia -INFIVE Facultad de Ciencias Agrarias y Forestales UNLP, La Plata, Argentina

Secondary metabolites (SMs) are a highly diverse set of low molecular weight bioactive compounds that play different biological roles. They are dispensable when microorganism are cultivated *in vitro*, but usually play a key role in nature, providing organisms with adaptive advantages in different niches. Among them, toxins produced by phytopathogenic fungi play crucial roles in virulence and host nutrition. We hypothesized that the genome of *Stemphylium lycopersici* strain CIDEFI-216, an etiological agent of tomato gray leaf spot, contains several gene clusters that code for the synthesis of SMs, such as those involved in the biosynthesis of virulence factors. We searched by means of the bioinformatic tools Secondary Metabolite Unknown Region Finder (SMURF) and Antibiotics and Secondary Metabolite Analysis Shell (antiSMASH) enabling the ClusterFinder algorithm for potential SMs gene clusters within the draft genome sequence of the fungus. This lead to the prediction of 32, 33 and 64 additional SMs gene clusters by SMURF, antiSMASH and ClusterFinder, respectively. They included polyketides synthase (PKS), non-ribosomal peptide synthase (NRPS), terpene synthase, dimethylallyl tryptophan synthase, hybrid PKS-NRPS, lanthipeptide synthase, fatty acid synthase and others gene clusters of unknown nature. This knowledge provides critical information to understand the biological bases of the tomato-*S. lycopersici* interaction.

## Greenhouse Evaluation of Dosage Responses of the Tomato I3 Gene to *Fusarium oxysporum f. sp. lycopersici* race 3

C. LAND (1), C. Land (2), G. Vallad (2), S. Hutton (2), R. Willis (2)

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

*Fusarium oxysporum f. sp. lycopersici* (FOL) causes an aggressive vascular wilt disease on tomato. The goal of this study was to evaluate increasing soil levels of FOL on tomato plants with and without the I3 gene conferring resistance to race 3 FOL isolates. Soil inoculum of a race 3 FOL isolate was produced in a corn meal:sand medium and diluted with a pasteurized field soil to levels of $10^4$ to $10^1$ cfu/g soil. Tomato seedlings with and without the I3 gene were transplanted to pots filled with 6 levels of infested soil, including a non-infested soil, and periodically evaluated for plant height and disease severity over 2 months. Afterwards, plants were uprooted, shoot fresh weights were measured, and roots and hypocotyls were plated onto Komada's medium. A significant difference in total biomass was observed between I3 homozygous and heterozygous plants, with I3 homozygous plants exhibiting 20% greater growth compared to susceptible plants regardless of the initial level of soil inoculum. Plants grown in non-infested soils were symptomless, exhibiting either undetectable or very low levels of FOL recovery from the hypocotyls. Heterozygous I3 plants grown in 10 cfu/g of FOL infested soil exhibited 70% hypocotyl infection, which was the highest rate of vascular infection of all treatments.

## *Fusarium oxysporum f.sp. lycopersici: Small Chromosomes Define Friend vs Foe*

K. FENSTERMACHER (1)

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

*Fusarium* is a genus of filamentous fungi well-known for causing persistent wilt diseases on almost all cultivated crops. The *Fusarium oxysporum* Species Complex has no known sexual stage, yet is diverse and contains over 120 *formae speciales* that cause disease on different plant hosts. In 2010, Ma *et al.* sequenced *F. oxysporum* f.sp. *lycopersici* (Fol) to find virulence loci of differing GC content throughout the genome, including small, conditionally dispensable chromosomes (CDCs) that do not match known *Fusarium* sequences, contain genes with no known homologs, can convey pathogenicity when transferred from a pathogenic strain to a non-pathogenic relative—making them functionally similar to virulence plasmids in bacteria. CDCs could explain the high level of diversity observed in a microbe with no sexual recombination, and the inconsistent number and sizes of chromosomes consistently found in these plant pathogens. This project sampled over 40 sites, with and without known cases of Fol, to examine 1) the prevalence of CDCs where Fol is found, 2) the variation among CDCs and pathogen phenotypes, and 3) the variation within genetic backgrounds harboring different CDCs. This data will help determine the roles of CDCs in the context of the populations and growing systems in which they are found, to analyze the potential of previous or future horizontal chromosome transfer events under various conditions, and analyze the risk factors and effects on agricultural systems.

## Root knot nematode effects on metabolic profiles of susceptible and resistant grapevine rootstocks

C. Wallis (1)

(1) USDA-ARS

Root knot nematodes (*Meloidogyne* spp.) can negatively impact newly planted and stressed vineyards. Nematode infestations also may increase grapevine susceptibility to other stresses such as water deficit or various diseases. However, little is known about direct or indirect effects of nematode feeding on grapevine physiology, or potential underlying mechanisms that impart resistance to certain rootstock cultivars. Therefore, this study assessed metabolite profiles in Cabernet Sauvignon grapevines grafted to either susceptible (O39-16) or resistant (Freedom) rootstocks established in root knot nematode-infested soil or steam-sterilized soil. Grapevines of both cultivars grown in soil infested with nematodes had reduced fructose and glucose levels compared to control grapevines grown in non-infested soil. Phenolic profiles in resistant grapevines did not differ due to exposure to nematodes, and only a few phenolic compounds increased in susceptible grapevines due to nematode feeding. Regardless of the presence of nematodes, root phenolic profiles between resistant and susceptible rootstocks were dissimilar, with different individual compounds more prevalent in one cultivar than the other. Taken together, these results demonstrated that nematode infestations altered root physiology, and that resistant grapevines expressed phenolic profiles different from susceptible grapevines.

## Influence of root exudates and soil on attachment of *Pasteuria penetrans* to *Meloidogyne arenaria*

C. LIU (1), P. Timper (2)

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

*Pasteuria penetrans* is a widely distributed endospore-forming bacterium which is a hyperparasite of *Meloidogyne* spp. Previous research showed that root exudates can reduce attachment of *P. penetrans* to *Meloidogyne arenaria*. The objective of this study was to determine if soil microorganisms modify the effect of root exudates on attachment of *P. penetrans* spores to *M. arenaria*. The following treatments were set up in both sand and clay soil: natural soil with a plant, sterilized soil with a plant, and natural soil without plant. For treatments with a plant, 1-month-old eggplant seedlings were used. All treatments were incubated in greenhouse for 3 days, and soil solutions were obtained by vacuum filtering saturated soil. Controls included phosphate-buffered saline (PBS) as well as root exudates in water. Second stage juveniles (J2) were incubated in 5 ml of different solutions for 6 h, rinsed and incubated in PBS for another 6 h with $10^5$ *P. penetrans* spores. Compared to J2 incubated in PBS, attachment was significantly reduced when root exudates were present, with root exudates having a greater effect on attachment in sand than in clay. Spore attachment was always higher in clay than in
sand solutions. Root exudates reduced spore attachment more in sterilized soil than in natural soil. The presence of microorganism and chemical
composition of different soils may interact with root exudates to affect endospore attachment.

Soybean Cyst Nematodes of Ohio: Deciphering mechanisms of virulence

E. WALSH (1), T. Miller (2), A. Grenell (3), B. Cassone (4), C. Taylor (1)

(1) The Ohio State University, U.S.A.; (2) The Ohio State University, U.S.A.; (3) The College of Wooster, U.S.A.; (4) Brandon University, Canada

Soybean cyst nematodes (SCN; *Heterodera glycines*) are the most yield-reducing pest of soybean in North America. Resistant soybean cultivars are widely utilized for management purposes, however SCN populations are quickly adapting to overused sources of resistance. Counties across Ohio were surveyed for SCN populations and their virulence against the available sources of resistance was tested with HG Type assays. Of the populations tested to date, 96% are infective on PI88788, the most prevalent source of resistance in commercial varieties. The objective of this study was to determine how SCN populations have overcome this source of resistance. Nine SCN populations from across Ohio were selected, which vary in their HG Type and/or level of virulence. These populations were grown on both PI88788 and a susceptible cultivar, Lee 74. Female SCN were harvested 15 days after inoculation with juvenile worms, total RNA was extracted, and submitted for sequencing on the Illumina Hi-Seq 2500. Interesting expression patterns were found common among virulent populations such as putative effectors and genes related to digestion, the immune response, and detoxification. Also, populations avirulent on PI88788 share the expression of an effector not present in virulent populations, indicating that the loss of this effector may play a role in virulence to this resistance source. Further work includes cloning identified candidate genes to determine their role in this interaction.

Investigating the role of maize for influence on soybean cyst nematode

R. MEDINA (1)

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

Soybean cyst nematode (SCN; *Heterodera glycines*) has a significant impact on the soybean industry, costing $90 million dollars annually. SCN has been managed mostly through the use of resistant cultivars and non-host crop rotations. Recently, resistance-breaking populations of SCN have made the use of SCN-resistant cultivars far less effective in controlling this nematode. Reliance on crop rotation has now become significantly more important for SCN control. However, little is known about how widely used non-host crops can impact SCN populations. Previous reports suggest that SCN numbers can be reduced during rotations with maize but the mode of action in this interaction is unknown. This study aimed to identify parental lines from the maize Nested Association Mapping population that are most effective at decreasing SCN populations through the production of allelopathic chemicals in root exudates. Field experiments were established using a simulated crop rotation scheme with maize and soybean. When exposed to developing maize seedlings, nematodes showed altered infection rates on soybean. A controlled experiment to complement field data collected showed significant differences in cyst production after pretreatment with maize lines. Soybean growers will ultimately directly benefit from this research by gaining knowledge on biochemical characteristics that can improve non-host selection and aid in further reducing SCN nematode populations in infested fields.

Comparison of ITS1 versus ITS2 regions for characterizing fungal communities colonizing soybean cyst nematode

W. HU (1), K. Bushley (1), S. Chen (2)

(1) Department of Plant Biology, University of Minnesota, U.S.A.; (2) Southern Research and Outreach Center, University of Minnesota, U.S.A.

The development of next generation sequencing has enabled investigation of microbial communities from a wide range of environmental samples. We examined fungal communities associated with cysts of soybean cyst nematode (SCN) to understand the effects of different crop rotation practices. The primers most frequently used for fungal communities on the Illumina Miseq platform target either the rRNA internal transcribed spacer 1 region (ITS1) or the ITS2 region. In order to assess which primers are better for amplifying fungal communities in SCN cysts, we performed 300 bp paired-end sequencing on two Miseq runs using fungal ITS1f/ITS2r and 5.8SR/ITS4 primers. Cysts of the SCN were collected from a soybean-corn rotation field in Waseca, MN, and washed with 0.2% Tween™ 20. The DNA was extracted from 50 cysts using the CTAB method, and normalized for PCR reactions. After quality control, 361224 (ITS1) and 230404 (ITS2) reads were used for OTU picking in QIIME. OTUs were blasted against the UNITE database with the default setting in QIIME. About 99% of the ITS1 region sequences were classified as fungal sequences, however, 99% of the ITS2 region sequences were not fungal sequences. In addition, significant changes in the relative abundance of some taxa was observed between rotation treatments for only the ITS1 sequences. We conclude that primers targeting the fungal ITS1 region rather than ITS2 region are more suitable for characterizing fungal communities in SCN cysts.

Gossypium arboreum accessions resistant to *Rotylenchulus reniformis* identified

S. STETINA (1), J. Erpelding (1)

(1) USDA, Agricultural Research Service, U.S.A.

Reniform nematode (*Rotylenchulus reniformis*) is a serious pest of upland cotton (*Gossypium hirsutum*) in the southeastern United States. Resistance does not exist naturally in *G. hirsutum* and no resistant cultivars are available. To identify sources of reniform nematode resistance, 225 *G. arboreum* accessions were evaluated in repeated growth chamber experiments. Root infection was measured on 3 plants 4 weeks after inoculation with 1,000 nematodes. The 16 accessions supporting the fewest infections were evaluated again in confirmation tests lasting 8 weeks. The numbers of nematodes extracted from soil and eggs extracted from roots of 5 plants were combined and totals were analyzed (ANOVA and differences of least squares means, *P* ≤ 0.05). Susceptible controls *G. hirsutum* cv Deltapine 16 and *G. arboreum* A₂-101 had 85,999 and 46,902 reniform nematodes, respectively; the resistant control *G. arboreum* A₂-190 had 1,665 reniform nematodes, and only 136 reniform nematodes remained in fallow pots. All 16 accessions tested supported smaller reniform nematode populations than the susceptible controls, ranging from 10,883 to 707 individuals. Nine accessions (A₂-254, A₂-272B, A₂-294, A₂-362, A₂-456, A₂-497, A₂-514, A₂-558, and A₂-665) supported reniform nematode populations comparable to the resistant control, and accession A₂-354 had fewer reniform nematodes than the resistant control.

Movement of Fluopyram in Sandy Soil to Affect *Meloidogyne incognita* Motility

T. FASKE (1), K. Hurd (2)

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

Fluopyram is nematicidal to *Meloidogyne incognita*. It has limited xylem movement, thus direct contact is important for nematode suppression; however, the effective movement of fluopyram in sandy soil is unknown. The objective of this study was to determine the movement of fluopyram from treated seed and a water dilution in sandy soil. Treatments consisted of 0.15 mg abamectin, 0.25 mg fluopyram, and 0.75 mg thiodicarb + imidacloprid treated cotton seed, while water dilutions of 25 µg abamectin and 25 µg fluopyram were used in the second experiment. Each treatment was placed on the surface of the soil column and flushed with 500 µl of water. The soil was removed after 24 h from three, 5-cm long segments and mixed with an equal volume of water. A portion of the supernatant was placed into a well, which contained 30-40 J2 in 500 µl of water. In the first experiment, a higher percentage of immotile J2 were observed from fluopyram collected in the 0-5 and 5-10 cm segments than abamectin. No nematode immotility was observed with thiodicarb or water control. In the second experiment, a higher percentage of immotile J2 were observed in the 5-10 cm segment from the water dilution of abamectin and fluopyram compared to the treated seed. No nematode immotility was observed past the 10-cm depth for either nematicide. The effective movement of fluopyram in sandy soil within 24 h was limited to the upper 10-cm of soil, which was similar to that of abamectin.
## Root Knot Nematode (Meloidogyne incognita) populations on eggplant could be reduced using Marigold extracts

A. HAMEED (1), N. Liaqat (2), K. Riaz (2), M. Alam (2), S. Sarfraz (2), S. Jameel (2)

(1) University of Agriculture, Faisalabad, Pakistan; (2) University of Agriculture Faisalabad, 38040-Pakistan, Pakistan

Root knot nematode diseases are gaining importance in vegetables in Pakistan. Chemical pesticides are often hazardous and are discouraged due to their non-targeted effects. In this study, amongst the several powder plant extracts (5 gram equivalent) of selected indigenous plants such as Marigold (Tagetes erecta), Akk (Calotropis procera), Kanair (Nerium oleander) and Neem (Azadirachta indica), the marigold extracts not only decreased the root galling index (55.1%) and number of knots per plant on root system (47.4%) in the susceptible eggplant variety (cluster king) but also increased the germination (28%) and plant vigor (32%) as compared to control plants harvested. Kanair (42.09 & 32.8%), Neem (37.56 & 28.5%) and Akk (31.51 & 21.69%) were also effective. These extracts were applied as seed treatment followed by drenching at an interval of ten days post emergence upto one month. Data was collected after two months and correlated scientifically.

## Soybean cyst nematode culture collections and field populations from North Carolina and Missouri reveal high incidences of infection by viruses

C. RUARK (1), S. Koenning (2), M. Mitchum (3), E. Davis (2), C. Opperman (2), S. Lommel (2), T. Sit (2)

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

Five viruses were previously discovered within Illinois greenhouse cultures of soybean cyst nematode (SCN; *Heterodera glycines*). In this study, the five viruses have been identified in SCN greenhouse and field populations from North Carolina (NC) and Missouri (MO). The prevalence and titers of viruses in SCN were examined using qRT-PCR in 47 greenhouse cultures and 25 field populations [20 from NC and 5 from MO]. Viral titers within SCN greenhouse cultures were similar throughout juvenile development, and the presence of viral anti-genomic RNAs within egg, second-stage juvenile (J2), and pooled J3 and J4 stages suggests active viral replication within the nematode. Overall, viruses were detected more frequently and at higher titers within greenhouse cultures versus field populations. Five greenhouse cultures harbored all five known viruses whereas in most populations a mix of viruses was detected. In contrast, three greenhouse cultures of similar descent to one another did not possess any detectable viruses and primarily differed in location of the cultures (NC versus MO). Furthermore, several of these SCN viruses were also present in the related nematodes species *Heterodera trifolii* (clover cyst) and *Heterodera schachtii* (beet cyst). If nematode infection with viruses is truly more common than first considered, the potential influence on nematode biology, pathogenicity, ecology, and control warrants continued investigation.

## Potential of trap cropping for managing root-knot nematode

B. WESTERDAHL (1)

(1) University of California Entomology & Nematology Department, U.S.A.

Trap cropping is a nematode management technique that has been tested periodically since the late 1800's. A susceptible host is planted and larvae of a sedentary parasitic nematode such as root-knot are induced to enter and establish a feeding site. Once this has occurred, and the female begins to mature, she is unable to leave the root. The plants are then destroyed before the life cycle of the nematode can be completed, trapping nematodes within the root. By itself, trap cropping is not likely to provide the same level of control as a fumigant nematicide, because not all nematodes are induced to enter the roots. However, the potential for loss of registration of fumigants for various environmental reasons is great enough that an IPM approach using two or more techniques in combination that will each provide partial control of the nematode population is warranted. Several years of field testing of trap crops with and without a biological nematicide for management of root-knot nematode on carrots demonstrated that trap cropping resulted in statistically significant increases in weight and number of marketable carrots. A wet fallow treatment, in which a trap crop was not planted, but the weeds that germinated following irrigation served to trap nematodes within the roots, followed by tillage two weeks later, also produced a statistically greater yield of marketable carrots than the untreated.

## Cover cropping affects plant-parasitic and free-living nematodes in Michigan carrot production

Z. GRABAU (1), Z. Maung (1), D. Noyes (1), D. Brainard (1), D. Baas (2), B. Werling (3), H. Melakeberhan (1)

(1) Michigan State University Department of Horticulture, East Lansing, MI, U.S.A.; (2) Michigan State University Extension-St. Joseph County, Centreville, MI, U.S.A.; (3) Michigan State University Extension-Oceana County, Hart, MI, U.S.A.

Cover crops are often used for reducing soil erosion, retaining soil nutrients, and building organic matter. Free-living nematodes (nematodes that do not feed on plants) are well-established bioindicators and may respond to changes in soil ecology due to cover cropping. Some cover crop cultivars are also marketed for managing plant-parasitic nematodes. This study assessed the impact of common Michigan cover crops on plant-parasitic and free-living nematodes in carrot production systems. Research was conducted at 4 sites in Michigan where cover crops were grown in the fall preceding summer carrot production. Oats, 'Defender' radish, dwarf essex rape, a mixture of oats and 'Defender' radish, and a fallow control were treatments at each site. Black oats and 'Graza' radish were also grown at sites 3 and 4 while 'Image' radish was grown only at site 4. Lesion nematode (*Pratylenchus* sp.) was present at all four sites. At sites 1, 3, and 4, lesion nematode densities were relatively small (less than 20 nematodes/100 cm³ soil) and not significantly (*P* > 0.05) affected by cover crops. At site 2, during carrot production, lesion nematode density was significantly (*P* < 0.05) greater following 'Defender' radish than fallow or other cover crop treatments. Similarly, at site 2 during carrot production, free-living nematode densities were greater following 'Defender' radish or oats than fallow, but were not significantly affected by cover crops at the other sites.

## Mode of action studies on the nematicide fluensulfone

P. NAVIA GINE (1)

(1) 1973, Wellington, FL, U.S.A.

Fluensulfone has proven efficacy in the field against a range of nematode species. The mode of action of fluensulfone is however currently unknown. Fluensulfone was found to have nematicidal activity against C. elegans but at concentrations ≥100-fold greater than those reported to be effective against the PPN Meloidogyne javanica. Fluensulfone affected a number of C. elegans behaviours, including locomotion, pharyngeal pumping, egg laying and development. The pharyngeal effects suggested that fluensulfone might influence feeding behaviour in PPNs. In PPNs, the behaviour of the stylet mouth spear is critical in feeding and host invasion. The action of fluensulfone on the stylet of the PPN Globodera pallida was therefore investigated, to validate observations made with C. elegans. The pharmacology of the stylet was also investigated, using knowledge of the C. elegans pharynx as a guide. Fluensulfone stimulated stylet activity and blocked 5-HT-stimulated activity. If metabolic impairment is identified as the causative agent of fluensulfone nematicidal activity this must be further studied through metabolomics and other techniques.

## Temporal analysis of oomycete communities associated with soybean and corn

M. CHILVERS (1), J. Rojas (1)

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

Corn and soybean rotation is typically practiced in field crop production, with the purported benefit of reducing pathogen inoculum density. This management practice has been studied before with concentration on a single pathogen species. There is limited information on how the associated oomycete community is modulated by using different crops in the rotation. We hypothesized that the genetic background of the hosts and their different growth stages could have an effect on the associated oomycete community. In 2013-14, field trials including two soybean cultivars (cv. Sloan and
Archer) and two corn cultivars (cv. DK5261 and P0255AMXT) were planted in a randomized block design with non- and inoculated treatments. The inoculated treatment consisted of a mixed inoculum of equal parts of *Pythium irregulare*, *Py. sylvaticum*, *Py. ultimum*, *Py. heterothallicum*, *Py. oopapillum* and *Phytophthora sojae* colonized rice. Soil and root samples were collected at different growth stages and subjected to DNA extraction. Samples were sequenced for ITS 1 amplicons. The results showed a variability in community composition between the two crops, however cultivars did not significantly vary in community composition despite different genetic backgrounds. Host could play an important role in filtering the associated oomycete community, however it did not effectively change the composition, suggesting this specific rotation system might not be conducive to pathogen inoculum reduction.

## Survey data reveals potential interactions between soil-borne fungi and oomycetes isolated in the midwest

E. LERCH (1), A. Fakhoury (2), M Chilvers (3), A Robertson (1)

(1) Iowa State University, IA, U.S.A., (2) Southern Illinois University, IL, U.S.A., (3) Michigan State University, MI, U.S.A.

Root rot pathogens, such as Fusarium and Pythium species are frequently isolated together from damped-off soybean seedlings. Consequently, they are hypothesized to form a multi-species complex that infects soybean, however the interaction has not been extensively studied and is not well understood. The objective of this research was to identify potential interactions between fungi and oomycetes. Data from 2012 Midwest survey funded by the USDA-NIFA and USB/NCSRP was used. Data comprised species recovered from seedlings collected by soybean pathologists from fields with seedling disease or a history of disease. Hierarchical clustering was used to identify groups of isolates frequently recovered together from the same field. When data were analyzed including all fungi and oomycetes, regardless of pathogenicity, six distinct clusters were identified. Within each cluster were fungal and oomycete pathogens and non-pathogens. When data were reanalyzed with only pathogenic species, four distinct clusters were present. Cluster 3 contained only Fusarium species, while cluster 2 contained several Python species as well as Phytophthora sojae, Phytophthora sansomeanea and F. verticillioides. The remaining two clusters contained Fusarium and Pythium species. These data should allow soybean pathologists to identify potential interactions between specific species of soil-borne fungi and oomycetes that can be explored and used to enhance our understanding of root rot on soybean.

## Breadth of resistance of Phytophthora fruit rot resistant watermelon germplasm to *Phytophthora capsici* isolates from across United States of America

C. KOUSIK (1), J. Ikerd (1), M. Mandal (2)

(1) U.S. Department of Agriculture, Agricultural Research Service, U.S. Vegetable Laboratory, U.S.A.; (2) ORISE Participant, U.S. Vegetable Laboratory, USDA, ARS., U.S.A.

Phytophthora fruit rot of watermelon caused by *Phytophthora capsici* is particularly severe in southeastern U.S where optimal conditions for disease development are prevalent. We have developed Phytophthora fruit rot resistant (PFR) germplasm lines (USVL020-PFR, USVL203-PFR, USVL489-PFR and USVL782-PFR) for use in breeding programs. The breadth of resistance of these lines to 20 *P. capsici* isolates collected from various states and different crops was evaluated. Mature fruit of PFR lines or susceptible checks 'Mickylee' and 'Sugar Baby' were collected from plants grown in a field. Fruit were inoculated by placing a 7-mm agar plug from a 4-day-old colony of a given *P. capsici* isolate. Inoculated fruit were kept in a walk-in-humid chamber (>95% RH, temperature 26±2 °C) for 5 days. Ratings on lesion diameter and visible pathogen growth were then recorded. Variability in aggressiveness among the 20 isolates on checks and some PFR lines was observed. Check cultivars were susceptible to all isolates whereas PFR lines were significantly more resistant. However, PFR lines were not immune to all isolates. Significant differences in lesion size on PFR lines for some isolates was observed. In most cases very small lesions (0.8mm) formed on PFR lines under the agar plug and on the fruit surface. Our results suggest that watermelon cultivars developed using these PFR lines as resistant sources will be able to provide resistance against Phytophthora fruit rot across many states.

## The Effect of Increased Soil Fertility on Seedling Disease Development of Soybean in Ohio

M. EYRE (1), S. Culman (1), A. Dorrance (1)

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

Low or high soil fertility levels have been shown to have potential secondary impacts on root pathogens, some of which decrease yield. Fertility recommendations for soybean in Ohio are over 20 years old and one proposed scenario is that more phosphorus and potassium may be required to support the increase in yields over the past 2 decades. Therefore, the objective of this study was to evaluate the impact of higher rates of P, K, and P + K on seedling diseases caused by oomycetes in both field and greenhouse trials. Field trials were carried out at two locations in 2014 and three locations in 2015 at fields each with a known history of seedling disease. Stand counts, percent emergence, and yield were not significantly different between treatments at any of the locations. However, a greater number of *Pythium* spp. were recovered from plants with the addition of K. In greenhouse assays using soil collected from the same infested fields, final stand and root weights were significantly (P<0.05) higher in pots without fertility treatments for one of the two locations. Further monitoring of the influence of higher rates of K at the time of planting on disease development of soil borne diseases caused by oomycetes is warranted.

## Cold stress at planting increase susceptibility to damping-off caused by *Pythium sylvaticum*

M. SERRANO (1), A. Robertson (1)

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

*Pythium sylvaticum* is an Oomycete that causes damping-off of soybeans in Iowa, particularly when cool weather occurs at planting. Seed treatments are applied to over 90 percent of soybean seed in Iowa to protect germinating seedlings and consequently plant stand. The objectives of this experiment were to (i) evaluate the effect cold stress at different times after planting, and (ii) the effect of seed treatments on damping off. Soybean seed was treated with Intego Suite, Intego Suite + mandrobin or left untreated and planted into 8 oz cups inoculated with *P. sylvaticum* or non-inoculated. The cups were placed in growth chamber and subjected to 96h of cold stress (4°C) at 0h or 24h after planting. A significant three-way interaction was detected between cold stress, inoculation and seed treatment (P<0.0001). In general, emergence of untreated seeds was reduced more than 70% when subjected to periods of cold stress (P<0.05). Emergence was improved with seed treatments (P<0.0001). These data indicate that periods of cold stress at planting increases the susceptibility of soybean to damping-off caused by *P. sylvaticum* and that seed treatments reduce damping-off under these adverse conditions.

## Pathogenicity and Virulence of Oomycete Species on Common Bean

D. ROSSMAN (1), M. Chilvers (1), A. Rojas (2), J. Jacobs (2)

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

Common bean is often affected by soilborne oomycetes that cause seed and seedling rot. A study was conducted to determine the pathogenicity and virulence of 24 *Pythium* spp. and *Phytophthium* aff. *vexans* on two common bean (Phaseolus vulgaris) cultivars, 'Red Hawk' kidney bean and 'Zorro' black bean from Andean and Meso-American gene pools, respectively. A seedling assay was conducted at 20°C in a growth chamber. Oomycete inocula were added to the root zone at the time of planting, and seedlings were harvested after 12 days. Root length, root area, root dry weight, and emergence were quantified. Seedling assay results indicated that 12 species caused significant root length and root area reductions from the control. Similarly, 11 of the same oomycete species caused significant reductions in root dry weight. Four *Pythium* spp. and *Phytophthium* aff. *vexans* caused significantly lower emergence than the control and were considered to be seed rot pathogens. An in vitro seed assay was conducted at 20°C and 26°C, in which disease
ratings were assessed. In this assay, 13 species caused disease severity that was significantly higher than the control. Moreover, it was observed that the virulence of certain oomycete species varied depending temperature and host gene pool. Findings help elucidate the oomycete species that are most aggressive and identify factors that may influence oomycete species virulence on common bean.

**Putative sucrose esters from *Petunia x hybrida* may contribute to this host's reduced susceptibility to *Phytophthora infestans***

M. BECKTELL (1)

(1) Colorado Mesa University, U.S.A.

Petunias have been established as a less susceptible host of *P. infestans*, but the reasons for this have not been elucidated. We have reported that petunia leaf extracts contain a potent zoosporicide. Previous biochemical analyses of crude leaf extracts suggested the presence of sucrose esters, a class of compounds known to be produced by the trichomes of *Petunia* and *Nicotiana* species. Subsequent experiments with commercially available sucrose esters have revealed that sucrose monolaurate (SM), an amphiphilic sucrose monoester known to interact with cell membranes, exhibits zoospore lysing activity. The goal of our current work is to confirm the presence of and identify the chemical structure of the putative sucrose esters present in petunia leaf extracts. Samples were purified using hydrophilic-lipophilic balanced cartridges and an established protocol for the purification of sucrose esters from *Nicotiana tabacum*. The purified samples were analyzed, along with SM as a control, via LCMS-ELSD, ¹H and ¹³C NMR, and TLC. The data shows a lack of monoesters, excluding SM as the bioactive component, but supports the presence of several known sucrose di- and polyesters. Analyses are ongoing to deduce the specific chemical structures of these sucrose esters. Results from this work will bring us one step closer to understanding an aspect of the petunia-late blight interaction that has received little attention.

**Identification and characterization of potential oomycete pathogens from the University Agricultural Laboratory in Fresno, California**

M. ELLIS (1), H. Deniston-Sheets (1), D. Sieperda (2), J. Bushoven (1)

(1) Plant Science Department, California State University, Fresno, U.S.A.; (2) California State University, Fresno, U.S.A.

The University Agricultural Laboratory (UAL) at California State University, Fresno is a 1000-acre farm that grows a wide range of fruit, nut, vegetable, and field crops. Many of the crops grown on the UAL are negatively impacted by oomycetes; however, identification of pathogenic species has not been determined. Therefore, the goal of this research was to identify and characterize potential sources of oomycete pathogens from soil and irrigation reservoirs on the UAL. Oomycetes were baited over a 5-day period using pears or cucumbers as bait. Symptomatic fruit tissue was excised and plated on the oomycete selective medium PARP. Isolates were then characterized using morphology and DNA sequencing of the internal transcribed spacer region. Forty-four isolates of *Pythium* spp., 6 isolates of *Phytophthora* spp., 4 isolates of *Phytopythium* spp., and 1 isolate of a *Pythiogeton* sp. were identified. *Pythium* spp. (86%), *Phytophthora* spp. (11%), and *Phytopythium* spp. (3%) were identified from the irrigation reservoir. The most common species isolated from irrigation water were *Pythium oopapillum* (54%) and *Pythium dissotocum* (25%). Of the isolates baited from soil 75% were *Pythium* spp., while 13%, 8%, and 4% were *Phytopythium* spp., *Phytophthora* spp., and a *Pythiogeton* sp. respectively. These results suggest that the UAL has a diverse number of potential oomycete pathogens and that irrigation reservoirs may act as a source for potential inoculum in farming operations.

**Development of a split-root technique for canola (Brassica napus L.)**

G. BRUCE (1), J. Robson (2), M. McDonald (2)

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

Canola (*Brassica napus*) is an important oilseed crop in Canada. A split-root system was developed for studies of host-pathogen interaction with root pathogens. Canola seed was germinated on moistened filter paper within pouches. After 7 days, the root tip of each seedling was clipped back to stimulate secondary branching. Each seedling was then carefully planted into soilless mix and allowed to grow for 18 days. When uprooted, each seedling had a pair of secondary roots. One root of each pair was then potted into each side of a specially modified plant pot for subsequent growth and assessment. The modified pots were created by placing two small plastic bags filled with potting mix into a square pot to create two equal but independent sections of soil per pot. A bamboo support was inserted between the bags. The roots were placed into each bag and covered with soil. The young seedling was loosely tied to the support, then sand was added to cover the soil and support the seedling. This resulted in almost 100% survival of the treated seedlings. As an initial proof of concept, seedling roots on one side of each pot were inoculated with resting spores of *Plasmodiophora brassicae* 7 days after potting and visually assessed for clubroot symptoms at 42 days after inoculation. Clubroot symptoms developed on the inoculated side but not on the control.

**Estimates of viable resting spores of *Plasmodiophora brassicae* using propidium monoazide and qPCR**

B. GOSSEN (1), F. Al-Daoud (2), M. McDonald (2)

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

*Plasmodiophora brassicae* [clubroot of *Brassica* spp.] persists in soil as long-lived resting spores. Quantitative polymerase chain reaction (qPCR) estimates of spore concentration do not distinguish between viable and non-viable spores. Propidium monoazide (PMA) penetrates non-viable cells and binds to DNA when exposed to high-intensity light, and so inhibits amplification with qPCR. The impact of pre-treating resting spores with PMA and light prior to qPCR (PMA-PCR) was assessed. Spore suspensions from 6-wk-old (immature) and 9-wk-old (mature) clubs were heat-treated, and concentrations of spores were estimated using qPCR and PMA-PCR. Spore viability was assessed using bioassays on canola seedlings. Prior to heat treatment, comparison of estimates from qPCR and PMA-PCR indicated that all mature resting spores were viable, but ≥ 74% of immature spores were not viable. This was consistent with substantially higher clubroot severity in plants inoculated with mature resting spores than immature spores. Heat treatment produced little or no change in estimates of mature spores with qPCR, but substantially reduced both estimates of spore concentration from PMA-PCR and clubroot severity in bioassays. For immature spores, PMA-PCR did not consistently demonstrate a heat treatment effect. We conclude that PMA allows for estimation of viable resting spores using qPCR.

**Phenotypic variation between two clades of *Phytophthora cinnamomi* isolated from avocado fields in California**

R. BELISLE (1), B. McKee (2), W. Hao (2), P. Manosalva (2)

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

*Phytophthora cinnamomi* (*Pc*) is the causal agent of Phytophthora root rot (PRR), the most serious disease of avocado worldwide. Pagliaccia *et al.* (2013) first assessed the genetic diversity of this pathogen in California and identified 16 genotypes corresponding to two distinct clades of A2 mating type isolates. Additionally, differences in virulence were observed between these two clades after infecting avocado rootstocks with different levels of tolerance to *Pc*. To reveal additional phenotypic differences in these specific genotypes, three representative isolates from clade I and clade II were characterized for: i) mycelial growth rate at different temperatures, ii) zoospore production, iii) zoospore germination, and iv) fungicide sensitivity. More virulent *Pc* isolates of clade II showed slower growth but an earlier release of zoospores. The molecular interaction between these *Pc* isolates and their hosts are being studied using lupin and avocado plants. *Pc* nuclear and mitochondrial genes will be used to study the genetic relationship among isolates from these two clades. Our data suggests that the isolates representing clade II differ in most of the phenotypic traits assessed. Fungicide efficacy among these isolates is being tested under greenhouse conditions using avocado seedlings. This work represents an integrated approach to study the molecular interaction of *P. cinnamomi* with its host and provide practical solutions to manage this pathogen in the field.
## Phylogenetic relationships among Philippine isolates of *Peronosclerospora* based on sequence analysis of multiple mitochondrial loci

F. DELA CUEVA (1), M. Samaco (1), M. Ramon (2), F. Martin (2)

(1) Institute of Plant Breeding, University of the Philippines Los Baños, Philippines; (2) USDA ARS, U.S.A.

Downy mildews cause substantial yield losses of sugarcane and corn in the Philippines with several species of Peronosclerospora (*P. sacchari*, *P. spontanea*, *P. miscanthi*, and *P. philippinensis*) commonly reported. Due to the obligate nature of the pathogen only spore morphology and host genus have been used as the bases of species identification. However, these parameters are highly effected by environmental conditions. Therefore, molecular tools are needed for investigating pathogen diversity and taxonomy. Primers have been developed for amplification of three mitochondrial loci from fifty-four isolates of *Peronosclerospora* recovered primarily from sugarcane representing geographically diverse regions in the Philippines. Limited sequence divergence of the mitochondrial loci was observed among these isolates. Additional isolates recovered from corn are being sequenced and added to the analysis to evaluate the taxonomy and phylogenetic relationships among corn and sugarcane downy mildews. Studies involving a broad sampling of the pathogen from other grasses in the Philippines and neighboring Asian countries are being conducted to assess the diversity and identify species of tropical graminicolous downy mildews. To facilitate rapid pathogen identification, species-specific molecular diagnostic assays targeting mitochondrial loci are under development. This is especially important for detection of the USDA-APHIS Select Agent *P. philippinensis*.

## Identification of conserved transcriptome responses to propagative plant viruses in insect vectors

K. MARTIN (1), K. Barandoc-Alviar (1), D. Rotenberg (1), A. Whitfield (1)

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

The plant *Nucleorhabdovirus*, *Maize mosaic virus* (MMV), is vectored by the corn planthopper, *Peregrinus maidis*. Previously, we conducted an RNA-Seq project to identify differentially expressed (DE) transcripts in adults during infection with MMV. We identified a total of 144 transcripts, with 77 genes upregulated and 67 genes downregulated. To identify conserved transcriptome responses in vectors to propagative viruses, we compared the DE transcripts in our system to two other systems: white backed planthoppers, *Sogatella furcifera*, exposed to *Southern rice black-streaked dwarf virus* (SRBSDV, *Fijivirus*), and the black-faced leafhopper, *Graminella nigrifrons*, infected with *Maize fine streak virus* (MFSV, *Nucleorhabdovirus*). In a tblastx analysis with a cut-off value of 10-3, we identified DE transcripts that are shared between systems. Twenty three transcripts were shared between all three systems. Two transcripts were unique to *P. maidis* and *G. nigrifrons* and 38 transcripts were unique to *P. maidis* and *S. furcifera*. Protein binding was the largest category of the 144 *P. maidis* transcripts (19), 10 were shared in all three systems. Functional characterization is underway on transcripts potentially involved in viral replication, intercellular movement and defense. The responsive genes identified in these vectors of propagative viruses may represent a conserved anti-viral strategy and could be targets for resistance against a diverse range of viruses.

## An optimized multiplex detection protocol captures divergent isolates of yellow dwarf virus species in Kansas wheat

A. LANEY (1), R. Acosta-Leal (1), A. Whitfield (1), D. Rotenberg (1)

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

Barley yellow dwarf (BYD) is a complex aphid-transmitted virus disease that can be caused by multiple yellow dwarf virus (YDV) species classified in the family *Luteoviridae*. Previous research revealed that *Barley yellow dwarf virus-PAS* (PAS) is prevalent in KS. However, currently available multiplex RT-PCR detection assays designed for a subset of YDVs do not include PAS, and primers we designed to the reference sequence of PAS (NC_002160) did not detect all KS isolates. The objectives of this study were to i) sequence two KS field isolates of PAS and one of PAV to enable primer design, and ii) to modify and optimize a multiplex RT-PCR assay to detect six YDV species, including divergent isolates of KS PAS. To test the new assay, 101 archived single-plant leaf samples obtained from a wheat virus survey were tested to determine YDV species composition. We found that PAS was the predominant isolate detected with 59% incidence followed by PAV with 57%; mixed infections of PAS and PAV (30%) were common. Single infections for PAS and PAV (23% each) were also found. The other YDVs were only detected in mixed infections of PAS and/or PAV in 12%, or less, of the samples. These include *Cereal yellow dwarf virus-RPV*, BYDV-SGV, BYDV-MAV, and a variant of *Maize yellow dwarf virus-RMV* first identified in MT. This new protocol will provide a tool for examining the association between BYD disease development and YDV species composition in Kansas wheat fields.

## Impact of downy brome (*Bromus tectorum*) and volunteer wheat on risk of winter wheat infection by *Wheat streak mosaic virus*

N. RANABHAT (1), Z. Miller (1), E. Lehnhoff (2), F. Menalled (1), T. Seipel (1), M. Burrows (1)

(1) Montana State University, U.S.A.; (2) New Mexico State University, U.S.A.

Wheat streak mosaic is caused by *Wheat streak mosaic virus* (WSMV) which is vectored by the wheat curl mite (WCM, *Aceria tosichella* Keifer). The mite and virus can survive on multiple grass species that act as reservoirs for WSMV. Understanding the effect of alternative hosts on risk of WCM infestation and WSMV infection in newly emerged winter wheat crops is crucial for disease management. We assessed WCM immigration onto trap plants and the influence of alternative hosts such as downy brome (*Bromus tectorum* L), and volunteer wheat on WCM infestation and WSMV infection of wheat. Wheat trap plants were monitored weekly in the fall over three years to assess the relationship among patterns of WCM infestation and WSMV infection with cumulative growing degrees days (GDD) of spring wheat, daily mean temperature with respect to alternative hosts. The relationship among GDD on WCM infestation and WSMV infection across alternative hosts was non-linear. Mite infestation and virus infection was positively correlated with daily mean temperature (P < 0.001) in the fall. The risk of WCM infestation and WSMV infection on trap plants from downy brome and volunteer wheat plots was higher during the late fall when compared to the bare ground control plots (P < 0.05). Overall, daily mean temperature is a better predictor of WSMV risk and the Wheat streak mosaic infection on emerging crop increases with the presence of downy brome and volunteer wheat species during the fall.

## A full genome cDNA clone of Cherry rusty mottle associated virus induces disease in sweet cherry

D. VILLAMOR (1), S. Pillai (1), K. Eastwell (1)

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

Cherry rusty mottle-associated virus (CRMaV), a member of the family *Betaflexiviridae*, is associated with cherry rusty mottle disease (CRMD). To date, fulfillment of Koch's postulates for this virus has yet to be demonstrated. A full genome of CRMaV isolate 98CI194 was cloned into a modified pORE-E4 *Agrobacterium* binary vector between the ENT CUP2 promoter and the *Nos-t* terminator. Leaf agroinfiltration with the binary vector carrying ENT CUP2-CRMaV failed to infect *Nicotiana benthamiana*, *N. occidentalis* 37B, and *N. glutinosa* whereas agro-inoculation using either the bark flap technique or root dipping resulted in the induction of CRMD symptoms in Krymsk 6 virus-tested cherry rootstock. Further confirmation of infection was verified by RT-PCR detection of CRMaV amplicon and by positive reaction of symptomatic leaves in ELISA and Western blotting using an antibody against the coat protein of CRMaV 98CI194. Finally, bark patch inoculation using lignified wood from one of the CRMaV positive, agroinoculated, Krymsk 6 trees resulted in CRMD symptoms on virus-tested sweet cherry seedling rootstock *Prunus avium* 'Mazzard'. These results provide conclusive evidence of the etiology of CRMD to be caused by CRMaV. This infectious clone provides a tool for future studies of genome expression and pathogenesis of CRMaV. Moreover, the construction of this infectious clone opens a path forward to pursue etiological studies of other stone fruit virus-like diseases.

## Comparative analyses of world-wide cucumber green mottle mosaic virus (CGMMV) isolates

T. PITMAN (1), B. Falk (1)

(1) UC Davis, U.S.A.
CGMMV is a +ssRNA *Tobamovirus* that infects curcurbit crops. It is capable of contaminating seeds inside the seed coat, making it a problem with the increased globalization of seed trade. 2013 was the first report of this virus in North America, which prompted federal and state responses and quarantine procedures where it was detected. Subsequent finds in 2014 and 2015 occurred in California, suggesting this is a recurring problem with global seed movement. Our efforts focus on better detection methods in seed. Currently, industry standard for detection is ELISA, which relies on antibody recognition of coat protein. Industry participants have described isolates that are not detected with available ELISA kits, but are identified with molecular assays. We are working to improve detection methods for CGMMV by serological and molecular methods. To support those efforts we need a better understanding of the diversity of CGMMV isolates. We previously sequenced two California isolates and found them to share 90% sequence homology. GenBank has 197 accessions for CGMMV, of which 42 are full genome sequences. Of these, the majority are Asian in origin. Given that CGMMV was originally described in England and is endemic to Europe and the Middle East, we worked with collaborators to amass a collection of 35 isolates from regions where CGMMV is endemic. We used enriched virion samples for next generation sequencing, and bioinformatics to analyze the sequence information.

**The virosome of *Rhizoctonia solani* and its effect in fungal pathogenicity**

T. STETINA (1), C. Rothrock (1), T. Spurlock (2), I. Tzanetakis (1)

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

*Rhizoctonia solani* consists of at least 14 genetically distinct anastomosis groups (AG). Mycoviruses have been shown to play an important in fungal epidemiology and pathogenicity. The goals of this research are to understand mycovirus epidemiology in *Rhizoctonia solani* on important row crops in Arkansas and their potential to influence pathogenicity. Mycoviruses have been extracted from 190 geotagged isolates from five different fields using cellulose affinity chromatography. The cDNA from double-stranded RNA enriched material was sequenced and sequences analyzed to determine whether they belong to known or undescribed viruses. Detection primers were designed for each virus and then used in all geotagged isolates. Our results are consistent with previous research indicating that mycoviruses are commonly found in isolates of *R. solani*. More than 20 new viruses, belonging to at least three different virus families have been discovered. Future experiments will determine the effects of mycovirus presence on growth and virulence of the fungus, and to determine rates of horizontal transmission. Spatial analysis will determine whether interactions exist between *Rhizoctonia* spp., AG, and mycovirus distribution in fields, as well as crop and soil properties.

**Studies on Groundnut rosette disease and implications of the newly reported *Groundnut ringspot virus* for groundnut production in Ghana**

A. APPIAH (1), R. Tegg (2), S. Offei (3), C. Wilson (2)

(1) Tasmanian Institute of Agriculture, School of Land and Food, University of Tasmania, Australia, Hobart, Australia; (2) Tasmanian Institute of Agriculture, School of Land and Food, University of Tasmania, Australia, New Town, Australia; (3) School of Agriculture and Consumer Sciences, University of Ghana, Legon, Accra, Ghana, Accra, Ghana

Groundnut rosette is a devastating disease of groundnut (peanut) and presents a major constraint to crop production. Twelve cultivars of groundnut were screened in field trials for resistance to Groundnut rosette disease (GRD), caused by infection with *Groundnut rosette assistor virus* (GRAV), *Groundnut rosette virus* (GRV) and its satellite RNA. GRAV infection rates were higher in the wet (13.9–100%) than the dry (11.8–61.8%) season. Four local varieties were rated as resistant along with three of six improved varieties previously regarded as possessing resistance to GRD. These resistant cultivars could be used in the interim management of the disease in the field and further exploited in breeding programmes for the development of virus-resistance. *Groundnut ringspot virus* (GRSV)-like symptoms were observed within these trials. The identity of the virus was confirmed by ELISA, RT-PCR and amplicon sequencing, and represents a first record of GRSV for Ghana. GRSV was found in most cases in mixed infection with the GRD agents. The identification of GRSV in groundnut fields in Ghana highlights the need for its inclusion in future groundnut-breeding programs. Genetic diversity analysis of the coat protein gene of GRAV revealed more than 97% nucleotide and amino acid identity among the Ghanaian, Nigerian and Malawian isolates, providing a good opportunity for the development of pathogen-derived resistance to combat the disease.

**Detection, Distribution and Effect of Hop stunt viroid on different hop cultivars**

M. KAPPAGANTU (1), J. Bullock (1), S. Kenny (2), K. Eastwell (2)

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

Hop stunt disease caused by Hop stunt viroid (HSVd) is a growing threat to hop cultivation globally. It severely impacts growth and yield of certain hop cultivars. In 2004, HSVd was first detected in hop yards of Washington State, the major hop growing area in the United States. RT-PCR has been the major technique used for HSVd diagnosis; however, it is costly and sample handling is technically challenging. A more robust Reverse Transcription-Recombinant Polymerase Assay (RT-RPA) was developed to facilitate larger sample numbers. To understand the current distribution of HSVd in Washington, a survey was conducted and it revealed that 17% of hops grown in this region are infected with HSVd. Symptom expression is highly variable among different hop cultivars infected with HSVd. To understand the effect of HSVd, a 5 year study was conducted of six cultivars of hop to determine the impact on yield. Over the study period, the average cone yield of infected cultivars Glacier, Cascade and Willamette was reduced by 62%, 14% and 34%, respectively, compared to non-inoculated healthy plants. No significant yield reduction was observed in cultivars Nugget, Columbus and Galena cultivars. In the final year 2013, horticultural parameters of Willamette and Nugget were also measured.

**Discovery of various dsRNA mycoviruses in *Trichoderma* spp. causing green mold disease of shiitake *Lentinula edodes***

D. KIM (1)

(1) Chonbuk National University, South Korea

A total of 315 fungal isolates causing green mold disease were collected from contaminated artificial logs and sawdust bags used for cultivating shiitake mushroom *Lentinula edodes* in Korea and were analyzed for the presence of dsRNA. dsRNA, which was purified using dsRNA-specific chromatography and verified by dsRNA-specific RNaseIII digestion, was detected in 32 isolates. The molecular taxonomy of dsRNA-infected isolates based on the ITS region of the rDNA indicated that all isolates belonged to the Trichoderma spp. The number and size of dsRNAs varied among isolates and the band patterns could be categorized into 15 groups. The most common dsRNA group, groupVI, occurred in 10 isolates encompassing three species of Trichoderma. Partial cDNA clones were obtained from two selected dsRNA groups and the sequence comparison of the cloned fragments of these dsRNAs revealed a high degree of similarity to sequences of a hypothetical protein and polyprotein genes of other mycoviruses, indicating the existence of mycoviruses in *Trichoderma* spp. Northern blot analysis using cloned cDNA showed specific hybridizing patterns in the dsRNA bands for isolates from which the clones were obtained, suggesting that many different mycoviruses, which have not been identified yet, exist in *Trichoderma* spp.

**Mixed Infection of Two Economically Important Tospoviruses in *Nicotiana benthamiana***

K. ZHAO (1), C. Rosa (1)

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

*Tomato spotted wilt virus* (TSWV) and *Impatiens necrotic spot virus* (INSV) are two economically important tospoviruses. Mixed infection of TSWV and INSV was first reported on tomato plants from Italy in 2000; and mixed infection has also been found in tobacco in many states in the US for more than two years. Sialer and Gallitelli suggested that mixed infection in tomato could represent progressive adaption of INSV to vegetable crops. To test this hypothesis, healthy *Nicotiana benthamiana* plants were mechanically inoculated with different ratios of TSWV/INSV and examined for how mixed
infections influence disease development. After two weeks, systemically infected leaves were collected to measure the viral titer by ELISA. Results show that TSWV is a poor competitor compared to INSV, since INSV could establish infection and reach high titer even when inoculated with equal amount of TSWV inoculum, but TSWV was not able to infect *N. benthamiana* efficiently in the presence of INSV. Mixed infections of tospoviruses are commonly found in nature, but the interaction between these two economically important tospoviruses and how mixed infections affect viral host range have not been studied yet. Our results provide insights into the interaction between TSWV and INSV in *N. benthamiana* and basic knowledge for future tospovirus mixed infection studies.

## Complete genome sequence of a *Watermelon mosaic virus* isolate from watermelon after 50 years of its detection in the United States of America

A. ALI (1), N. Rajbanshi (1)

(1) The University of Tulsa, Tulsa, OK, U.S.A.

*Watermelon mosaic virus* (WMV) belong to one of the important plant virus genus *Potyvirus*. This virus has been detected for the first time in 1965 in the Rio Grande Valley of Texas but so far no complete genome of WMV is available from cucurbits in the United States (US). WMV isolate (TX29) was collected from Rio Grande Valley of Texas during a survey in 2010 which showed severe mosaic in a watermelon plant. The complete genome of WMV-TX29 isolate was found to be 10,049 nucleotides excluding the poly-A tail. Nucleotide sequence comparison of TX29 isolate showed more sequence similarities with a WMV-French isolate. This is the first report of the complete genomic sequence of a WMV isolated from cucurbits in the US.

## Interactions between Bell pepper endornavirus and acute viruses

C. ESCALANTE (1), R. Valverde (1)

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

Persistent plant viruses do not cause detectable symptoms in their plant hosts. In contrast, acute viruses cause symptoms, and in most cases, disease. Most bell pepper (*Capsicum annuum*) cultivars are infected with the persistent virus *Bell pepper endornavirus* (BPEV). To study possible interactions between endornaviruses and acute viruses, we developed two near-isogenic lines of the bell pepper cultivar Marengo; one BPEV-infected and the other BPEV-free. The two lines were mechanically inoculated individually, with a mild (M) and a severe (S) isolate of the acute virus *Pepper mild mottle virus* (PMMoV). Symptoms were recorded and relative amounts of PMMoV determined by ELISA. When the two lines were inoculated with PMMoV-M, all plants reacted with mosaic symptoms. In contrast, when PMMoV-S was inoculated, BPEV-free plants reacted with systemic necrosis and mosaic while BPEV-infected plants reacted only with mosaic. Results of the ELISA test showed that plants of the BPEV-infected line yielded consistently less PMMoV than plants of the BPEV-free line; however, the differences were not statistically significant. The two bell pepper lines were also inoculated with *Tomato spotted wilt virus*, *Potato virus Y*, *Cucumber mosaic virus*, and *Tobacco mosaic virus*, and in all cases, plants reacted with similar symptoms. Results obtained in this investigation suggest that BPEV inhibit the systemic necrotic reaction of Marengo bell pepper caused by PMMoV-S.

## Characterization of recombinant Potato virus Y<sup>NTN</sup> (PVY<sup>NTN</sup>) isolates from Sulawesi, Indonesia

A. KARASEV (1), M. Chikh-Ali (1), N. Bosqui-Perez (1), D. Vander Pol (1), D. Sembel (2)

(1) University of Idaho, U.S.A., (2) Universitas Sam Ratulangi, Manado

In the last three decades, Granola, a potato cultivar originally from Germany, has become the most common cultivar for fresh consumption in Indonesia. In August 2014, a survey was conducted in Sulawesi, where potato fields cultivated with Granola and its selection, cultivar Super John were surveyed for *Potato virus Y* (PVY) presence using visual symptoms and serology. PVY was found in Sulawesi for the first time. Samples determined to be positive for PVY were subsequently typed to strain using RT-PCR assays. All PVY isolates sampled were identified as PVY<sup>NTN</sup> recombinants, with three recombination junctions in P3, VPg, and CP regions of the genome. Three local PVY isolates were subjected to whole genome sequencing and subsequent sequence analysis. The whole genomes of the Indonesian PVY<sup>NTN</sup> isolates I-6, I-16 and I-17, were found to be closely related to the European PVY<sup>NTNa</sup>. This recombinant type was shown previously to cause potato tuber necrotic ringspot disease (PTNRD) in susceptible potato cultivars. The dependence of potato farmers on mostly a single cultivar, Granola, may have given a competitive advantage to PVY<sup>NTN</sup> over other PVY strains resulting in the predominance of the PVY<sup>NTN</sup> recombinant. The dominance of PVY<sup>NTN</sup> in Sulawesi, and possibly in Indonesia as a whole, represents a potential risk to any newly introduced potato cultivar to the country, especially cultivars susceptible to PTNRD.

## Variation in aphid abundance and Potato Virus Y incidence in Oregon potato

S. BAG (1), K. Frost (1), S. Rondon (1), B. Charlton (2), D. Walenta (3)

(1) HAREC, OSU, U.S.A.; (2) Klamath Basin Research and Extension Center, OSU, U.S.A.; (3) OSU Extension Service, U.S.A.

*Potato virus Y* (PVY) is an economically important potato pathogen that can induce potato tuber necrotic ringspot disease. More than 25 aphid species are reported to vector PVY in a non-persistent manner. Green peach aphid (GPA, *Myzus persicae*) and bird cherry-oat aphid (*Rhopalosiphum padi* L.) are considered to be the primary PVY vectors. In 2015, aphid abundance and PVY incidence were measured in nine commercial potato fields in Oregon. Aphids were collected weekly, counted and identified based on morphological characteristics. Potato leaf tissue was sampled from each location biweekly and assayed for PVY using ELISA. PVY strain composition of ELISA positive samples was determined using an RT-PCR-based strain-typing methodology. Potato aphid (*Macrosiphum euphorbiae*) and GPA were less abundant than all the other unidentified aphids combined. PVY incidence was highest in Klamath (8.4%), followed by Umatilla (3.3%), Union (1.75%), and Morrow (0%) counties. Strain typing of the ELISA positive samples revealed the PVY<sup>N.O</sup> strain to be most prevalent (56%) followed by PVY<sup>O</sup> (10.34%), PVY<sup>NTN</sup> (5.74%) and a mixed infection of PVY<sup>NTN</sup> and PVY<sup>NO</sup> (1.14%). Potato fields with border crops as sudan grass had lower PVY incidence and fewer aphids captured when compared to fields without border crops. Also, traps placed in fields adjacent to hay crops captured more aphids and had higher PVY incidence when compared to traps placed in fields adjacent to maize, mint, sunflower or wheat.

## Isolation and characterization of FRs551, a bacteriophage associated with the phytobacterium *Ralstonia solanacearum* strain UW551

M. STULBERG (1), J. Mershon (2), A. Ahmad (3), D. Mollov (4), Q. Huang (2)

(1) FNPRU, USDA/ARS & ORISE, Beltsville, MD, U.S.A.; (2) FNPRU, USDA/ARS, U.S.A.; (3) FNPRU, USDA/ARS & Department of Plant Pathology, Faculty of Agriculture, Minia University, El-minia 61519, Egypt, U.S.A.; (4) NGRL, USDA/ARS, Beltsville, MD, U.S.A.; (5) FNPRU, USDA/ARS, Beltsville, MD, U.S.A.

*Ralstonia solanacearum* species complex members are soil-borne, plant pathogenic bacteria that cause devastating diseases in a wide range of economically important crops around the world, with global losses in potato alone exceeding $950 million annually. The race 3 biovar 2 strains of *R. solanacearum* causing potato brown rot are listed as select agent pathogens due to their potential threat to U.S. agriculture. Genome analysis of the select agent strain UW551 revealed the presence of a putative intact prophage sequence with high homology to the family *Inoviridae*. Active virions of this phage, designated *ΦRs551*, were isolated from the supernatant of UW551 cells grown under normal culture conditions and were found to infect other *R. solanacearum* strains. Electron microscopy showed that *ΦRs551* is a filamentous-type phage approximately 1200 nm long and 7 nm wide. *ΦRs551* has a circular DNA genome that is 7929 nt in length, with 100% identity to the putative prophage sequence in the UW551 genome. It is most closely related to *ΦRS603*, another filamentous phage of *Ralstonia* isolated from soil. Our draft *ΦRs551* genome contains open reading frames (ORFs) similar to those found in other *Inoviridae* members, but has a unique overall ORF makeup not found in any other phage relative.