host and virus proteins. This presentation will illustrate how interactome maps coupled with subcellular localization studies can be used to describe the CaMV disease cycle.

## Two eIF4E proteins in Yellow Lantern chili pepper interact with Pepper veinal mottle virus VPg

Z. ZHANG (1), N. Yu (1), X. Zhang (2), Y. Zhang (1), J. Wang (1), Z. Liu (3), Z. Xiong (4)

(1) Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou, China; (2) Institute of Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou, China; (3) Chinese Academy of Tropical Agricultural Sciences, Haikou, China; (4) University of Arizona, Tucson, AZ, U.S.A.

Pepper veinal mottle virus (PVMV) causes a major disease on the Yellow Lantern chili peppers (Capsicum chinense), an economically important specialty crop on Hainan Island, China. PVMV is a *Potyvirus* with a genome-linked protein (VPg) attached at the 5' end of the (+)-strand RNA genome. Interaction between PVMV VPg and pepper eIF4E is necessary for the PVMV to infect peppers. As there is no natural resistance against PVMV in the Yellow Lantern chili pepper, disruption of the VPg-eIF4E interaction by genome editing appears to be a feasible approach to engineer PVMV resistance. Four eIF4E genes were identified from bioinformatic analysis of pepper genomes, cloned by RT-PCR with specific primers, and analyzed by sequencing. The pepper eIF4E family consists of two eIF4E genes (Cc-eIF4E and Cc-eIF4E-Xm), one eIF(iso)4E gene (Cc-eIF(iso)4E), and one novel cap binding protein gene (Cc-nCPB9090). Only Cc-eIF4E and Cc-eIF(iso)4E interacted with PVMV VPg in the LexA yeast two-hybrid system. The specific interaction between these two pepper eIF4E proteins and PVMV VPg was further studied *in vivo* with bimolecular fluorescence complementation (BiFC) tests in infiltrated tobacco cells. Cc-eIF4E showed a strong and consistent interaction while Cc-eIF(iso)4E displayed a weak and inconsistent interaction with PVMV VPg by BiFC. These two eIF4E genes will be further evaluated as potential targets for genome editing in our quest for PVMV resistance in the chili pepper.

## QTL Analysis of flg22-triggered Basal Resistance in Maize

X. ZHANG (1), P. Balint-Kurti (1), X. Stacey (2), Y. Cao (2)

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

Plants are constantly under attack by different pathogens. Molecules released or generated during microbial entry can be perceived by the plant and produce activate defense, including early responses such as the oxidative burst. An important component of this defense system is based upon the recognition of microbe-associated molecular patterns (MAMPs) also known as pathogen-associated molecular patterns or PAMPs). Once MAMPs are detected by the plant, a defense response, termed MAMP-triggered immunity (MTI, also known as PAMP-triggered immunity or PTI) happens. In a few cases, a direct link has been established between MTI and speci?c quantitative trait loci (QTLs) for partial resistance. The MTI response has been widely studied, mainly in the model plant Arabidopsis and a few crops, such as Brassica and soybean. Few studies have examined MTI in maize. There is signi?cant potential in harnessing maize MTI to improve cultivars to withstand a variety of pathogens. In this study, we evaluated the MTI responses in 26 different maize genotypes by challenging with one well known MAMP (flg22), and then assessed a recombinant inbred line mapping population (CML228×B73) derived from parent lines with differential flg22 responses. We identified one major QTL on chromosome 2 associated with variation in the flg22 MAMP response. We will describe our progress in characterizing this QTL and in identifying QTL associated with other MAMP responses.

## Physiological and Molecular-Genetic Characterization of Basal Resistance in Sorghum

J. KIMBALL (1), D. Chen (2), G. Stacey (2), P. Balint-Kurti (1)

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

Early detection of pathogens is a critical component of plant immunity. Basal resistance, aka innate immunity, is the first line of defense to protect plants against a range of pathogens. Triggered by the recognition of microbe-associated molecular patterns (MAMPs) by pathogen recognition receptors (PRRs), the induction of basal resistance has been shown to vary quantitatively within and across species. In sorghum (Sorghum bicolor (L.) Moench), preliminary results supports this hypothesis. The goal of this project is to investigate the genetic architecture and transcriptional response associated with the basal defense response in sorghum. Specific objectives in this project are to 1. Develop robust assays to measure disease resistance and the MAMP response in sorghum, and screen a set of diverse sorghum germplasm for variation in these traits, 2. Identify genes differentially regulated during the MAMP response in high and low responding sorghum genotypes, and 3. Examine the activity of fungal extracts of sorghum pathogens in inducing the MAMP response, 4. Measure the effect of the MAMP response on disease resistance in controlled assays, and 5. Determine whether control of variation in the MAMP response and variation in disease resistance is under shared genetic control.

## The Role of Ethylene in PAMP-Triggered Immunity in Fusarium Crown Rot (FCR) Disease Resistance in Wheat

S. ALLEN (1), X. Xiao (1), K. Bhide (1), S. Scofield (2)

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

Fusarium Crown Rot (FCR) is a devastating cereal crop disease cause by necrotrophic fungal pathogens *Fusarium graminearum* and *Fusarium pseudograminearum*. Plants detect conserved pathogen-associated molecular patterns (PAMPs) through pattern-recognition receptors. Then, a signal cascade is triggered resulting in PAMP-Triggered Immunity (PTI) response such as expression of defense response genes and production of plant hormones jasmonic acid, salicylic acid, and ethylene. A model proposed by Zipfel in 2013 suggests that ethylene functions to prime additional signaling pathways for sustained PTI response. Our laboratory has shown that disease resistance to FCR can be enhanced in susceptible wheat cultivars by stimulating the ethylene-signaling pathway with ethylene biosynthetic precursor 1-aminocyclopropene-1-carboxylic acid (ACC). Additionally, resistant cultivars become more susceptible when ethylene-signaling is inhibited by treatment with the competitive inhibitor 1-methylcylopropene (1-MCP). RNA-Seq analysis of FCR-susceptible wheat cv 'Kennedy' in response to ACC or 1-MCP application and *F. graminearum* inoculation demonstrates significant difference in Differentially Expressed Genes (DEGs) within several disease-response categories such as peroxidases, pathogenesis-related proteins, and WRKY transcription factors. Currently, we are examining the transcriptome of FCR-resistant cv 'Bobwhite' to identify additional candidate genes involved in FCR resistance.

## Use of synchrotron Fourier transform spectroscopy to shed light on the chemical composition of cell wall appositions papillae on winter wheat leaf

R. LAHLALI (1), T. Song (2), C. Karunakaran (1), G. Peng (2), Y. Wei (3)

(1) Canadian Light Source Inc., Canada; (2) Agriculture and Agri-Food Canada, Canada; (3) Department of Biology, University of Saskatchewan, Canada

Papillae formation is known as one of the most important plant immunity mechanisms. Its major function is to prevent the entry of fungal pathogens into the host plant cells. In this study, a synchrotron based Fourier transform spectroscopy (sFTIR) was used to elucidate the chemical composition of the cell wall of forming papillae and non-forming papillae in response to powdery mildew infection. Spectroscopic results indicated an increase in peaks intensity of asymmetric and symmetric stretching vibrations of CH₃ and CH₂ that correspond to fatty acid groups, phenolics groups, lignification, cellulose, hemicellulose, and glucans following pathogenic infection. Principal Component Analysis (PCA) applied to sFTIR spectra confirmed these findings and distinguished two different groups with significant differences in cell wall composition including lignin, phenolics, polysaccharides and carbohydrates. This study highlighted, for the first time, the potential application of synchrotron-based infrared technique to study the *in situ* composition of lignified papillae and plant cell in the plant disease resistance, which remained to be a challenge with biochemical approaches. Subsequently, further
studies with more cultivars, as well as other synchrotron imaging techniques will be undertaken to confirm these preliminary findings and explore other
defense response mechanisms.

miR858 functions as a negative regulator of plant susceptibility to the beet cyst nematode *Heterodera schachtii*

S. PIYA (1), T. Baum (2), T. Hewezi (1)

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

Recent studies have indicated that microRNA genes play key roles in shaping the compatibility of the interaction between plant-parasitic cyst nematodes and host plants. In this study, we report a novel regulatory role of the Arabidopsis miR858 and its MYB83 transcription factor during plant-cyst nematode interactions. Promoter-GUS fusion assays pointed to a role of miR858 in post-transcriptional regulation of MYB83 in the syncytium induced by the beet cyst nematode *Heterodera schachtii*. qPCR quantification further supported the negative association between miR858 and MYB83 expression levels at different time points post *H. schachtii* infection. Overexpression of miR858 in Arabidopsis resulted in significant decreases of plant susceptibility to infection by *H. schachtii*, whereas a *mir858* mutant showed the opposite effect of increased susceptibility. In addition, we found that T-DNA insertion mutation of MYB83 was less susceptible to nematode infection. In contrast, overexpression of miR858-resistant *myb83* produced hyper-susceptibility phenotypes. Taken together, these data revealed the importance of tight regulation of MYB83 transcription factor expression by miR858 in the syncytium, apparently to control downstream target genes that encode components that may be needed for syncytium formation/function. Identification of target genes that are directly or indirectly regulated by MYB83 is currently underway.

Agroinfiltration-based screening to discover new potato germplasm with resistance against *Globodera* nematode pests

S. Chen (1), R. Cui (1), X. Wang (2)

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

Potato cyst nematodes (PCN) (*Globodera rostochiensis* and *G. pallida*) are of worldwide regulatory concern and considered to the most economically important pests of potato, causing > 80% yield loss. The U.S. potato industry has been adversely affected due to the emergence of *G. pallida* in Idaho and a virulent *G. rostochiensis* pathotype in New York and the lack of resistant potato cultivars. Using host resistance is the most effective and sustainable means for nematode control. Like other plant pathogens, nematodes secrete effector proteins into root cells to suppress host plant defenses, leading to successful infection. However, specific effectors can be recognized by host resistance proteins and trigger plant defense, which often results in a hypersensitive response (HR) believed to halt pathogen infection. "Effectoromics" is a high-throughput, functional genomics approach that uses effectors to potentially detect R genes in plants. Wild potato species offer an extremely rich source of resistance against *Globodera* nematodes. We have established an agroinfiltration assay in wild potato species. Our initial screening of forty-two wild potato accessions with a group of PCN-secreted effectors showed the occurrence of an HR phenotype when specific effectors were transiently expressed in potato leaves. The results indicate that this "Effectoromics" approach may be a valuable tool that helps to accelerate resistance breeding against *Globodera* nematode pests.

Transcriptome analysis of resistance against *Phytophthora cinnamomi* and the role of β-cinnamomin eliciting in pathogenicity

M. ISLAM (1), H. Hussain (1), J. Rookes (2), A. Chambery (3), A. Schallmey (4), W. Oßwald (5), D. Cahill (2)

(1) Deakin University, Waurn Ponds, Australia; (2) Deakin University, Waurn Ponds, Australia; (3) Università degli Studi di Napoli, Italy; (4) RWTH Aachen University, Aachen, Germany; (5) Technische Universität München, Freising, Germany

Resistance to the destructive oomycete pathogen *Phytophthora cinnamomi* is rare. We have explored resistance in roots, following infection, of the Australian native monocot *Lomandra longifolia*. Inoculated plants developed restricted lesions and analysis of lignin, callose and hydrogen peroxide indicated their involvement in resistance. We then used a transcriptomic approach to further examine resistance in this system. As the genome of *L. longifolia* is not sequenced, we performed RNA-seq analysis and the data showed the elevated expression of a range of resistance-related genes including those involved in jasmonic acid synthesis, lignin biosynthesis and in disease resistance signalling. Blast2GO analysis of contigs revealed the presence of elicitin and elicitin-like proteins. We then investigated the role of elicitins in pathogenicity of *P. cinnamomi*. Elicitins were isolated from liquid culture and different isoforms were identified by isoelectric focusing analysis. A β-cinnamomin elicitin was purified, an antibody produced and its specificity was shown by Western blot analysis. β-cinnamomin production at different life stages of the pathogen was confirmed by confocal microscopy. Elicitins were also detected in inoculated roots of the susceptible plant species, *Lupinus angustifolius*. Elucidation of resistance-related mechanisms and pathogenicity factors provides opportunities for modification of susceptible plants and identification of targets to prevent infection.

A synergistic effect of two plant antimicrobial peptides from defensin and lipid-transfer protein families towards *Phytophthora infestans*

E. ROGOZHIN (1), D. Zaytsev (2)

(1) Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry Russian Academy of Sciences, Russia; (2) Timiryazev Russian State Agrarian University, Russia

Late blight caused by *Phytophthora infestans* (Mont.) de Bary is one of the most important diseases afflicting potatoes and tomatoes worldwide. It results in economically meaningful yield losses and decreases in the quality of harvested produce. In the last two decades, researchers have focused on developing chemical and biological pesticides to treat against the diseases following plant tissue necrotization and dying off. Antimicrobial peptides are one of the primary components of a plant innate immunity to biotic stress factors involving diseases and pests. Most of these peptides can act directly against plant pathogenic microorganisms (fungi, oomycetes and bacteria) based on disruption of cell wall and plasma membrane integrity. In our study, we investigated two antimicrobial peptides (plant defensin NsD2 isolated from *Nigella sativa* seeds and lipid-transfer protein EcLTP purified from *Echinochloa crusgalli* kernels) belonging to different plant defense families. We showed that these peptides exhibited synergistic action against *Phytophthora infestans* on potato tuber disks assays when used in tandem. An application of plant antimicrobial peptides that demonstrates a diverse mode of action towards *Phytophthora infestans* would enable increases in the inhibition effect up to complete elimination of the pathogen.

Wheat streak mosaic virus P1: Defining the minimal region required for the suppression of RNA silencing

A. KASAMSETTY (1), G. Hein (2), S. Tatineni (1)

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

Wheat streak mosaic virus (WSMV) is the most economically important wheat virus in the Great Plains region of the USA. WSMV is the type species of the genus *Tritimovirus*, family Potyviridae, and is transmitted by the wheat curl mite, *Aceria tosichella* Keifer. Previously, we reported that WSMV P1 functions as the viral suppressor of RNA silencing (VSR) and pathogenicity enhancer. In this study, we examined the minimal region of P1 required for VSR function. Twenty one deletion mutants of WSMV P1 were generated by progressively deleting from the N- and C-terminal regions. VSR assays were conducted by agro-infiltration of deletion mutants in pCASS4 together with the 35S-GFP into *Nicotiana benthamiana* leaves. WSMV P1 with deletions comprising up to 23 amino acids at the N-terminal region exhibited VSR activity, while deletion of N-terminal 25 amino acids completely abolished the VSR activity. In contrast, even a single amino acid at the C-terminus is indispensable for VSR activity of P1, suggesting that the C-terminal region of WSMV P1 is crucial for VSR activity. Mechanistic studies of WSMV P1 in VSR activity will be discussed.

Development of HRM markers tightly linked to extreme resistance to *Potato virus Y* using next-generation sequencing

X. NIE (1), D. De Koeyer (1), A. Murphy (1), V. Dickison (1), M. Singh (2)

(1) Fredericton Research and Development Centre, Agriculture and Agri-Food Canada, Canada; (2) Agricultural Certification Services, Canada
Tetraploid potato breeding clone F87084 possesses a resistance gene (*Ry*) that confers extreme resistance (ER) to *Potato virus Y* (PVY). However, markers reported to be highly linked to different *Ry* genes were not detected in F87084. Next generation sequencing of genomic DNA from F87084 (*Rrrr*), F02010 (*rrrr*) and the respective bulk DNA of resistant and susceptible progenies of F87084 x F02010 was carried out by Illumina HiSeq for identification of single nucleotide polymorphisms (SNP). Polymorphic positions matching the expected pattern in parents and progeny bulk samples were determined. The genomic regions flanking the target SNP were extracted and used to design primers and probes for high-resolution DNA melting (HRM) assays. Of the 10 primer/probe sets designed and tested, five produced amplicons that exhibit different HRM profiles between the resistant parent/bulk and the susceptible parent/bulk. Further analysis of the HRM primer/probe sets on a segregating population comprised of 300 progenies revealed near-perfect matches between the HRM assays and the phenotyping results, demonstrating high linkage between these newly developed HRM markers and *Ry* in F87084. The efficacy of the HRM markers for marker-assisted selection of ER to PVY is validated with different populations involving F87084.

## The virulence function and regulation of the metalloprotease gene *prtA* in the bacterial plant pathogen, *Burkholderia glumae*

T. DE PAULA LELIS (1), J. Peng (1), S. Osti (1), J. Hyun Ham (1)

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

Bacterial panicle blight caused by *Burkholderia glumae* is a major bacterial disease of rice in the United States. Our preliminary RNA-seq study showed that a serine metalloprotease gene, named *prtA*, is positively regulated by the Tof1/TofR-mediated quorum sensing system like other genes for producing important virulence factors of *B. glumae*, such as toxoflavin. To study the function of *prtA* in *B. glumae* 336rg-1, a null mutant of *prtA* was generated through an insertional mutagenesis approach. Extracellular protease activity was not detected when the *prtA* mutant was grown on LB-agar plates amended with 1% skim milk, indicating that *prtA* is solely responsible for the extracellular protease activity of *B. glumae* 336gr-1. In addition, inoculation of rice panicles with the *prtA* mutant resulted in reduction of disease severity compared to the wild type strain, showing the requirement of *ptrA* for full virulence. This study also revealed that the regulation pattern of extracellular protease activity is similar to that of toxoflavin production. Particularly, it was newly found that *qsmR*, known as the quorum-sensing master regulator gene, is essential for both extracellular protease activity and toxoflavin production by *B. glumae* 336gr-1. Furthermore, additional genetic elements that control extracellular protease activity were newly identified through screening of random mini-Tn5 mutants, which provide useful information to understand the mechanism of *prtA* regulation.

## The genome of the endophytic *Curtobacterium* strain ER 1.4/2 and its potential as a biocontrol of several plant diseases

L. CURSINO (1), K. Pawlak (2), G. Li (2), R. Worobo (3)

(1) Keuka College, U.S.A.; (2) Hobart and William Smith Colleges, U.S.A.; (3) Cornell University, U.S.A.

*Curtobacterium* are Gram-positive bacteria that can live inside of plants as endophytes and also have the ability to cause disease such as wilting disease in dry beans. *Curtobacterium* sp. strain ER1.4/2 (ER1.4/2) was isolated from branches of healthy sweet-orange (*Citrus sinensis*). The genomic DNA of this bacterium was extracted and the whole DNA sequencing was performed using next-generation DNA sequencing by the Illumina HiSeq 2000 method. The genome annotation of *Curtobacterium* strain ER1.4/2 was initially performed using a RAST (Rapid Annotation using Subsystem Technology) server. The draft genome contains 3,692,034 bases with a GC content of 71.6%. Important genes such as a bacteriocin and siderophores were found in the genome. This bacteria showed antibiosis *in vitro* against several important phytopathogens such as *Xylella fastidiosa* CVC, *Xanthomonas campestris* pv. *Glycines*; *Pseudomonas syringae* pv. *syringae* and *Agrobacterium tumefaciens*. We are currently identifying ER1.4/2 at the species level by FAME to confirm its 16S rDNA identification. In addition, studies on its interaction with several cultivars of *Phaseoulus vulgaris* and *Glycine max* are underway. The ability of ER1.4/2 to colonize multiple plants, with its wide-spectrum antibiosis and non-pathogenicity to all plants tested make this bacterium an excellent biocontrol candidate against bacterial diseases.

## The *dlt* locus contributes to virulence in the necrotrophic pathogen *Pectobacterium carotovorum*

I. RUBIO (1), C. Guerin (1), R. Schaub (1), A. Charkowski (1)

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

In Gram-positive bacteria, the *dlt* locus contributes to antimicrobial peptide resistance through D-alanylation of cell wall teichoic acids. Homologs of *dlt* are present in the Gram-negative, soft rot pathogens *Pectobacterium* and *Dickeya*. Gram-negative bacteria lack teichoic acids, so the function of the *dlt* operon is unclear. We mutated the *Pectobacterium carotovorum* *dltB* and *dltD* genes and assayed for virulence attenuation in *Solanum tuberosum* (potato) stems and tubers. Red Norland plant stems were stab-inoculated and evaluated with a rating scale for wilt and stem maceration symptoms. Stem colonization by the different strains was determined by slicing and plating 1cm stem sections. Russet tubers were inoculated with serial dilutions of bacteria, and evaluated for the presence or absence of maceration. None of the mutants assayed were reduced in ability to cause symptoms on stems. However, *ΔdltD* was reduced in ability to spread inside stems. In tubers, both mutants were reduced in virulence compared to the wild type strain. Therefore, the *dlt* appears to contribute to *P. carotovorum* virulence in tubers and spread in stems. Investigating possible targets of gene products using HPLC and biolog analysis are on-going and will provide insight into *dlt* function in *P. carotovorum*.

## Systematic computational network-based analysis to predict subnetwork modules associated with pathogenicity and fumonisin in *Fusarium verticillioides*

M. KIM (1), B. Yoon (1), W. Shim (1)

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

*Fusarium verticillioides* is a notorious pathogen of maize causing ear rot and fumonisin contamination. In order to develop innovative preharvest control strategies, such as new biocontrol agents and resistant hybrids, to minimize the entry of fumonisins into our food supply, there is a need to identify new molecular targets for disrupting *F. verticillioides* pathogenicity. In this study, we performed a systematic computational network-based comparative analysis of two distinct *F. verticillioides* - maize kernel RNA-seq datasets (one on moderately resistant inbred and the other on susceptible hybrid). For the systematic analysis of the pathogenicity mechanism, we first inferred *F. verticillioides* co-expression networks. Subsequently, we identified functional subnetwork modules on the co-expression networks consisting of interacting genes that display harmoniously coordinated behavior in the respective datasets. A computationally efficient branch-out technique applied with an adopted probabilistic pathway activity inference method was used to identify functional subnetwork modules likely involved in *F. verticillioides* pathogenicity. Here we exhibit key potential subnetwork modules, where the modules contain several enriched GO terms as well as potential pathogenicity genes from other pathogenic fungi. Putative hub genes in each subnetwork will be functionally characterized to test their candidacy as new targets for ear rot and fumonisin control strategies.

## Computational and functional analyses of MADS-box transcription factor-mediated regulation of fumonisin biosynthesis in *Fusarium verticillioides*

A. HILTON (1), A. Hilton (1), M. Kim (1), C. Ortiz (2), C. Woloshuk (3), B. Yoon (2), W. Shim (2)

(1) Texas A&M University, U.S.A.; (2) Texas A&M University, U.S.A.; (3) Purdue University, U.S.A.

*Fusarium verticillioides* is an important maize pathogen, leading to Fumonisin B1 (FB1) in infested kernels. MADS-box transcription factors (TF) were found to modulate polyketide synthase (PKS) gene expression and FB1 production in *F. verticillioides*. With next-generation sequencing of *F. verticillioides* wild type and MADS-box TF mutant cultures, we used a suite of computational network-based tools, e.g. partial correlation, log-likelihood
ratio matrix and seed-and-extend approach, to perform a system-oriented transcriptome analysis to predict downstream genetic subnetwork modules associated with FB1 production. Our aim was to identify and characterize system-level changes across correlated genes rather than simply focusing on individual gene expression. The resulting subnetwork modules are predicted to contain hub genes, which likely play a crucial regulatory role within functional modules. Two predicted subnetwork modules were analyzed *in silico*, and five putative hub genes were subjected to functional characterization. Deletion of RAS GTPase (*FvRSR1*) and methyltransferase (*FvEFM3*) led to a significant reduction in FB1 levels. A comparative qPCR was performed to investigate the impact of FvRsr1 and FvEfm3 on the expression of neighboring genes in two subnetworks, which did show significant changes. Collectively, these results support our prediction that *FvRSR1* and *FvEFM3* serve as a key hub gene in each subnetwork and regulate FB1 biosynthesis.

## Independent amplification of a housekeeping gene and its evolutionary significance in the Dothideomycetes

B. DHILLON (1), G. Kema (2), S. Goodwin (3), B. Bluhm (1)

(1) University of Arkansas, U.S.A.; (2) Plant Research International, Wageningen University and Research Centre, Netherlands; (3) USDA/ARS Purdue University, U.S.A.

Transposable elements (TEs) are key drivers of genome evolution, but the exact mechanisms of how they modulate gene and genome function are largely unknown. We identified an event where a housekeeping gene (histone H3) was captured and amplified by a hAT DNA transposon in the genome of the banana pathogen *Pseudocercospora fijiensis*. Comparative genome analysis in related Capnodiales revealed a similar event occurred in the corn pathogens *Cercospora zeae-maydis* and *C. zeina*, but was lacking in the soybean pathogens *C. sojina* and *C. flagellaris*. Histone H3 amplification had been described previously in Pleosporales (*Pyrenophora tritici-repentis*). Our analysis showed key differences in the amplification events between the Pleosporales and Capnodiales, namely, 1) extent of histone H3 amplification, 2) difference in H3 nucleotide composition and 3) number of functional H3 genes present. In the Capnodiales, all amplified H3 copies were inactivated by RIP, which suggests the original H3 gene was under strong negative selection pressure. The interplay of TEs and RIP can result in various outcomes for gene and genome evolution. For instance, when TEs act as breeding grounds for pathogenicity genes, such interactions can influence gene function and/or regulation, and ultimately affect host-pathogen interactions.

## Transcriptomic analysis identifies specificity in *Fusarium verticillioides* metabolic response to *Bacillus mojavensis* lipopeptides

A. BLACUTT (1), S. Gold (2)

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

*Fusarium verticillioides* is a mycotoxigenic fungus capable of both pathogenic and asymptomatic endophytic lifestyles in maize; such intimate association renders efficient chemical control cost-prohibitive. *Bacillus mojavensis* RRC101 is a maize endophyte demonstrating both *in vitro* antagonism of *F. verticillioides* and *in planta* reductions of disease and mycotoxin accumulation, the former attributed to production of lipopeptide antibiotics. Although both RRC101 surfactins and fengycins induce increase mycotoxin accumulation in plate assays, only fengycins are antagonistic and induce hyphal distortion, pigment accumulation indicative of a stress response, and violent lysis. Preliminary analysis of RNA sequencing data has identified common functional groups in transcripts enriched in *F. verticillioides* under fengycin antagonism, particularly structural proteins and hydrolytic enzymes. Genes responsible for secondary metabolism, specifically antibiotic production, are also upregulated under lipopeptide challenge. These data suggest that differential lipopeptide responses previously observed in *F. verticillioides* reflect an underlying cross-kingdom "conversation" of secondary metabolites between maize endophytes.

## Temporal succession of an *Aspergillus flavus* biocontrol agent in commercial cornfields in Texas

M. SEXTON (1), I. Carbone (1), J. White (1), T. Isakeit (2)

(1) Center for Integrated Fungal Research, North Carolina State University, U.S.A.; (2) Texas A&M University, U.S.A.

*Aspergillus flavus* contaminates corn by producing carcinogenic aflatoxins (AF). This results in devastating economic losses worldwide and poses serious health risks. The most effective control strategy for reducing AF contamination is the application of non-aflatoxigenic strains of *A. flavus*, such as NRRL 21882 (=Afla-Guard®) or NRRL 18543 (=AF36). However, we have a limited understanding of the succession of biocontrol agents in cornfields. From a genomic perspective, we are examining the influence of NRRL 21882 on the populations of *A. flavus* from Texas commercial cornfields. In 2014, *A. flavus* strains were isolated from kernels from fields that were not treated or treated with NRRL 21882 in 2011. To obtain fine resolution of genetic variation, we conducted genome-wide sequencing, which allowed us to detect changes in the genomes of NRRL 21882-derived strains and introgression of NRRL 21882 into native strains over a three year period. From an analysis of 168 *A. flavus* strains, 32 strains are missing their entire AF gene cluster, a marker of NRRL 21882; 14 of these were in untreated fields. Eight of the strains missing the cluster are putative recombinants, phylogenetically grouping with full cluster strains. Ongoing sexual reproduction is indicated by approximately equal distributions of both mating types in treated and untreated fields.

## Comparative genomics of S and L morphotypes of *Aspergillus flavus*

M. OHKURA (1), M. Ohkura (1), P. Cotty (2), M. Orbach (1)

(1) School of Plant Sciences, University of Arizona, U.S.A.; (2) Agricultural Research Services USDA, University of Arizona, U.S.A.

Strains of *Aspergillus flavus* vary greatly in their level of aflatoxin production and atoxigenic strains are not uncommon in nature despite the fungus' notoriety for producing the carcinogenic mycotoxin. *Aspergillus flavus* strains are of two morphotypes; the S morphotype that produces numerous small sclerotia and limited conidiospores and the L morphotype that produces relatively fewer large sclerotia and abundant conidiospores. S strains are consistently toxigenic, whereas L strains vary greatly in toxin production ranging from atoxigenic to highly toxigenic. This suggests the two morphotypes have adapted to different niches where selection pressure to maintain aflatoxin production is high in the environment the S strains inhabit, and low in which the L strains inhabit. To develop hypotheses on differential niche adaption, we compared the genomes of three S strains and three L strains to identify genes unique to each morphotype. A notable difference in secondary metabolite genes was observed and the results from the comparative genomics analyses will be presented.

## Functional characterization of candidate effector proteins identified from the wheat scab fungus *Fusarium graminearum*

S. Lu (1), M. Edwards (1), S. Lu (1)

(1) USDA-ARS Cereal Crops Research Unit, U.S.A.

Fungal pathogens often produce certain small secreted cysteine-rich proteins (SSCPs) during pathogenesis that may function in triggering resistance or susceptibility in specific host plants. We have recently identified a total of 190 SSCP s encoded in the genome of the wheat scab fungus *Fusarium graminearum* and found at least 23 SSCP s to be true extracellular proteins that are expressed *in planta*. To facilitate functional characterization, we compared the expression patterns between compatible and incompatible interactions by transcriptional analysis. A majority of SSCP s examined were found to be expressed in identical or similar patterns when the fungus attacked susceptible wheat cultivars such as Grandin and Wheaton. However, the expression patterns of at least 13 SSCP s were altered when the same pathogen infected the resistant cultivar Sumai 3: the expression of 11 SSCP s appeared to be suppressed or "down-regulated" at certain stages of infection while two others were apparently "up-regulated" at 4-8 days post inoculation. Gene knock-out experiments are underway with the priority given to these differentially expressed SSCP s. The resultant deletion mutants will be subjected to pathogenicity assays on both susceptible and resistant wheat cultivars to determine the roles of these candidate effector proteins in *F*.
*graminearum*-wheat interactions. This study may provide new molecular insights into Fusarium head scab, a devastating disease of wheat crops worldwide.

## Analysis of a ketide synthase from fungal endophytes of toxic locoweeds

A. NOOR (1), D. Baucom (2), D. Cook (3), R. Creamer (2)

(1) Molecular Biology, New Mexico State University, U.S.A.; (2) New Mexico State University, U.S.A.; (3) USDA, ARS, U.S.A.; (4) New Mexico State University, U.S.A.

Toxic *Astragalus* and *Oxtropis* locoweeds are legumes in which toxicity is due to fungal endophytes of *Alternaria* section *Undifilum* sp. (A. sec. *U.* sp.). The fungi produce swainsonine, an alkaloid α-mannoside inhibitor that causes locosim disease when consumed by grazing animals. Polyketide synthase (PKSs) is a key enzyme in swainsonine biosynthesis. KS is the most highly conserved and informative of the multi-domain enzymes for predicting pathway associations. The objective of this study was to analyze the KS sequences from *A.* sec. *U.* sp. isolated from locoweed plants of *Astragalus lentiginosus* var. *diphysus*, *lentiginosus*, *araneosus*, and *wahweapensis*, and *A. mollissimus* var. *biglovii*, *earleii*, *wootoni* and *mollissimus* that were collected from the western USA. The KS from these endophytic fungi were compared to *A.* sec. *U.* *oxytropis* isolated from *Oxytropis lamberteii* and *O. sericea* using PCR. A 741 bp amplicon was sequenced and analyzed to produce maximum parsimony trees. Fungi isolated from different varieties of locoweed and locations had different morphologies and colors in culture. The KS sequences were highly conserved across *A.* sec. *U.* sp., but differed slightly between *A.* sec. *U.* *oxytropis* and *A.* sec. *U.* *fulvum* and *A.* sec. *U.* *cinereum*. Comparing the KS from additional locoweed endophytes and *A.* sec. *U.* sp. will allow the further examination of phylogenetic relationships among these fungi.

## A metabolomic approach to investigate the putative role of drought stress in Esca disease of grapevine

M. LIMA (1), A. Machado (1), W. Gubler (1)

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

Esca is a destructive disease of grapevine known to be associated with several xylem inhabiting fungi, namely *Phaeomoniella chlamysdospora* (*Pch*) and *Phaeoacremonium alephilum* (*Pal*). However, the disease mechanism(s) remains unclear. These fungi seem capable of living for several years as endophytes without apparently causing the disease. It is unknown why these fungi become pathogenic, but symptom appearance has been associated with abiotic factors, particularly water stress. We hypothesize that changes induced by drought on the components of xylem sap contribute to symptom appearance. H¹-Nuclear Magnetic Resonance spectroscopy coupled with multivariate statistical techniques was used to investigate the interaction of drought stress and Esca-associated pathogen infection. Young, greenhouse-grown, *Vitis vinifera* cv. Chardonnay cuttings were inoculated with *Pch* or *Pal* six weeks before inducing drought stress. Xylem sap was collected thirteen weeks after inoculation. Drought stress induced major changes in several compound classes, e.g. amino acids, carbohydrates and aromatic compounds. The modulation of these metabolic changes by fungal inoculation is being considered. This “omic” approach allowed to gain better insight into the biotic-abiotic interaction, and suggested candidates that could be tested in the control of esca-associated fungi, thus leading *Science* closer to *Practice*.

## PiCS, a predicted protein-protein interactome of the frog eye leaf spot pathogen *Cercospora sojina*

A. SUBEDI (1), B. Musungu (1), M. Geisler (2), B. Bluhm (3), A. Fakhoury (1)

(1) Southern Illinois University Carbondale, U.S.A.; (2) Southern Illinois University Carbondale, U.S.A.; (3) University of Arkansas Fayetteville, U.S.A.

Frog eye leaf spot (FLS) is a foliar disease of soybean caused by the fungus *Cercospora sojina* Hara. FLS is a severe problem for soybean production, particularly in the mid-southern United States causing millions of dollars in losses every year. A predicted protein-protein interaction (PPI) map provides an overall snapshot of all the protein interactions occurring in an organism. Therefore, the development of PiCS, a predicted protein-protein interactome of *C. sojina*, can decipher many of the primary metabolic processes, biological processes and developmental processes in the fungus. This will help further our understanding of the mechanisms governing pathogenesis in the *C. sojina* - soybean pathosystem. PPI maps have been generated for some model organisms such as human, fruit fly, and arabidopsis using high throughput experimental methods. In this study, we used PPI data from 11 model organisms to predict the PPI map in *C. sojina* via the interlog method using the INPARANOID and ENSEMBL algorithms. PiCS is comprised of 4,092 proteins (nodes) and 144,488 interactions (edges). It covers 33% of the total *C. sojina* proteome. Subnetworks were also built for secreted proteins in *C. sojina* as well as for putative pathogenicity related genes. This is a first attempt to build a PPI map for a member of the *Cercospora* genus. It is a new tool that could be used to understand the interaction between soybean and *C. sojina*.

## Comparative analysis of predicted proteomes from forma speciales of *Fusarium oxysporum* causing palm wilt

S. PONUKUMATI (1), S. Ponukumati (2), J. Huguet (2), J. Rollins (2), M. Elliott (1)

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

*Fusarium oxysporum* f. sp. *palmarum* [FOP] and *Fusarium oxysporum* f. sp. *canariensis* [FOC] cause lethal fusarium wilt of palms in Florida. While FOC and FOP cause similar diseases, FOP has a wider host range and appears to be more aggressive than FOC. The focus of the current study is to compare the predicted FOC and FOP proteomes with various formae speciales of *F. oxysporum* to identify common and unique gene families. To date, the FOP and FOC genomes were sequenced, assembled and annotated using multiple bioinformatic approaches. Genome sizes of FOP and FOC are 46.9 Mbp and 46 Mbp, respectively, and the predicted proteomes consist of 15528 (FOC) and 15974 (FOP) proteins. CAZy analysis indicate FOP and FOC share similar copies of sugar transporters, cellulase, and secretory lipase, whereas FOP has additional copies of necrosis inducing proteins, cysteine rich secretory proteins and cell degrading enzymes, such as cutinase and pectate lyase. OrthoMCL was used to cluster related proteins among *Fusarium* genomes, resulting in 396 (FOC) and 511 (FOP) unique clusters. Secondary metabolite genes were present in 22 OrthoMCL clusters. Two gene clusters related to nonribosomal peptide synthases have increased copy number in FOP, indicating the possible role of toxins in pathogenicity. Further annotation is underway to identify putative functions of unique and common genes and to develop hypotheses concerning differences in pathogenic specificity.

## Fungal endophyte community analysis of green coffee beans: A comparison across growing regions and qualities

R. CAPOUYA (1), T. Mitchell (1), V. Devi Ganeshan (1)

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

Coffee is considered a global dietary staple due to its distinct flavor, longstanding cultural significance, and stimulating caffeine content. During the production process, defective green coffee beans are sorted out so the batch contains below a certain percentage of them, as a non-homogenous batch can negatively impact the roasting process. A reduction of this percentage of defects would result in higher yields and better quality coffee. We hypothesize that there are notable differences in the endophyte communities between good and defective green coffee beans, and one or more fungal taxa can be associated with bean health or with particular economically significant defects. Through next-generation amplicon sequencing, we analyzed the endophytes present within beans from six defect categories across three coffee growing regions and made comparisons to identify the fungal taxa associated with these categories. We identified a variety of fungal communities present within the samples and were able to make statistical associations between both healthy and defective beans and certain endophytic taxa. Particularly, our studies indicate that multiple species from the *Pichia* genus may be responsible for promoting healthy bean development. Our findings will open the door to new research regarding the manipulation of endophytic fungi to improve overall coffee production and quality.
### Transcriptomic response of *Fusarium verticillioides* associated with nitrogen availability and development of sugarcane pokahh boeng disease

M. ZHANG (1), Z. Lin (2), J. Wang (3), Y. Bao (3), Q. Guo (3), C. Powell (1), S. Xu (3), B. Chen (3)

(1) IRREC-IFAS, University of Florida, U.S.A.; (2) Guangxi University, China; (3) Guangxi University, China

Pokahh boeng, caused by *Fusarium verticillioides*, is a serious disease in sugarcane industry. The impact of nitrogen source (ammonium sulfate, urea or sodium nitrate) on sugarcane pokahh boeng disease and its pathogen was investigated in *planta* and fungal growth and sporulation production was measured *in vitro*. The results showed that ammonium and nitrate were beneficial to fungal mycelium growth, cell densities and sporulation and enhanced the disease symptomology of sugarcane pokahh boeng in comparison to urea fertilization. Total 1,779 transcripts out of 13,999 annotated genes identified from global transcriptomic analysis by RNA sequencing were differentially expressed in *F. verticillioides* CNO-1 grown in the different sources of nitrogen. A subset of these genes were associated with organic nitrogen assimilation and reproductive processes of *F. verticillioides*. The identified DEGs suggested the modes of nitrogen uptake and assimilation for *F. verticillioides* during different nitrogen regimes. Our results further demonstrated that nitrogen availability might play an important role in disease development by increasing fungal cell growth as well as influencing the expression of genes required for successful pathogenesis. These genes included those associated with phenotypic alteration, reproduction, nitrogen and carbon metabolism as well as several transcription factors responsible for these metabolisms.

### Metagenomic analysis of oomycete communities in rhizosphere soil from field pea on the Canadian prairies using Illumina Miseq

B. GOSSEN (1), S. Chatterton (2), N. Foroud (3), A. Esmaeili Taheri (3), D. McLaren (3)

(1) AAFC, Canada; (2) AAFC, Canada; (3) AAFC, Canada

Oomycetes include several important soil-borne pathogens that cause damping-off and root rot in many crops including pea. Samples of soil from the rhizosphere of pea plants were collected from patches of healthy or diseased plants (127 samples from 26 commercial fields) on the Canadian prairies in 2013 and 2014. Oomycete communities were characterized using metagenomic analysis of the ITS1 region on Illumina Miseq. Roughly 4 million high quality sequences were obtained. Clustering took place at 85% coverage and 97% similarity. A representative sequence of each operational taxonomic unit (OTU) was compared to NCBI sequences. Species were identified at (minimum) 90% coverage and 97% similarity, and genera at 80% coverage and similarity. Clusters below 80% coverage or identity were labelled as unidentified. From 102 identified OTUs, 43 species and 16 oomycete genera were identified. *Pythium* was the dominant genus and *P. heterothalicum* was the most prevalent species. *Aphanomyces euteiches*, a very important pea root rot pathogen, was detected in 71 sites, but in very low abundance (≈ 0.2%). Multivariate analysis revealed difference in oomycete communities of healthy and diseased sites, and among years and provinces. This study shows that deep amplicon sequencing on Miseq can reveal unknown diversity of oomycete communities in agricultural soil, and that primer optimization for higher detection power of economically important oomycetes such as *A. euteiches* is warranted.

### Apple replant disease and the -omics: Interaction of apple rootstock metabolome and the soil microbiome

R. Leisso (1), R. Leisso (2), M. Mazzola (2)

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

Apple replant disease (ARD) negatively impacts tree health and reduces crop yield in new orchard plantings. Use of tolerant rootstock cultivars can diminish the growth limiting effects of ARD; however specific rootstock attributes enabling ARD tolerance are not understood. Systems biology tools were used to contrast root exudate biochemical profiles and corresponding soil microbial ecology between ARD tolerant and susceptible rootstock genotypes. A metabolomic approach utilizing LC-MS/MS QTOF was used to characterize water-soluble root exudate metabolites collected periodically from water percolated through rootstock roots planted in pasteurized quartz sand over 12 weeks. Soil microbial community profiling was conducted by terminal restriction fragment length polymorphism (TRFLP) or next-generation sequencing (NGS) on samples collected concurrently from soil with a history of ARD treated daily with the flow through exudates from rootstocks cultivated in quartz sand. Results indicate rootstock cultivars differ in both root exudate composition and quantity, and correspondingly soil microbial communities are altered in a rootstock genotype-dependent manner. Differences in exudate metabolome associated with ARD cultivar tolerance, and temporal dynamics of root exudate production and microbial populations during early stages of rootstock growth following dormancy, may offer insight into ARD tolerance and the early stages of disease development.

### Differential metabolome analysis of field-grown maize kernels in response to drought stress

L. YANG (1), J. Fountain (1), X. Ni (2), R. Lee (1), S. Chen (3), B. Scully (2), R. Kemerait (1), B. Guo (2)

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

Drought stress constrains maize kernel development and can exacerbate aflatoxin contamination. In order to identify drought responsive metabolites and explore pathways involved in kernel responses, a metabolomics analysis was conducted on kernels from a drought tolerant line, Lo964, and a sensitive line, B73, with and without drought stress treatment for 7 and 14 days after drought induction beginning 7 days after pollination. Using ultra-performance liquid chromatography coupled with MS/MS, we profiled 445 metabolites covering 42 pathways. Drought stress induced higher accumulation of glycerolipid and phospholipid metabolites, and decreased content of galactolipid, amines and polyamines, amino sugar, and nucleotide sugar metabolites in both lines. However, B73 was found to accumulate fewer metabolites involved in dipeptide and pyrimidine metabolism under drought stress than Lo964. Lo964 also exhibited increases in sphingolipid, sterol, and purine metabolism in comparison to B73. Collectively, higher sugar accumulation, a lower rate of energy metabolism, and elevated lipid and nucleic acid turnover was observed in B73. Conversely, higher amino acid and dipeptide accumulation, a lower TCA cycle rate, and elevated lipid and nucleic acid turnover was observed in Lo964 in response to drought stress. These results suggest that drought sensitivity is associated with differential metabolite accumulation, and diverse metabolic pathways in maize kernels.

### Comparison of *Fusarium graminearum* transcription factors affecting Fusarium graminearum virus 1 accumulation based on phenome analysis

J. Yu (1), Y. Lee (1), K. Kim (1)

(1) Seoul National University, Korea

The Fusarium graminearum virus 1 (FgV1) causes hypovirulent phenotypic changes in *Fusarium graminearum*. It has been reported that the numerous *F. graminearum* genes were differentially expressed upon FgV1 infection including genes encoding regulatory factors. Using gene-deletion mutant library of 657 putative transcription factors (TFs) in *F. graminearum*, we transferred FgV1 by hyphal anastomosis to screen transcription factors that might be associated with viral replication or symptom induction. FgV1-infected TF mutants were divided into three groups according to the mycelial growth phenotype compare to the wild-type FgV1-infected strain (WT-VI). The FgV1-infected TF mutants in Group 1 exhibited slow or weak mycelial growth compare to that of WT-VI on CM media at 5-dpi. In contrast, Group 3 consists of virus-infected TF mutants showing faster mycelial growth and mild symptom compared to that of WT-VI. The hyphal growth of FgV1-infected TF mutants in Group 2 was not significantly different from that of WT-VI. These growth differences among the FgV1-infected TF mutant groups might be related with FgV1 RNA accumulations in infected host fungi. To confirm this, we tested FgV1 RNA accumulation of some of FgV1-infected TF mutants in each group. Taken together, our analysis will help us identifying regulator(s) of FgV1-triggered signaling and antiviral responses and thus expand our understanding on complex regulatory networks between FgV1 and *F. graminearum* interaction.
## Root transcriptome analysis reveals viral diversity in two species of blueberry

N. SAAD (2), J. Olmstead (1), R. Alcalá-Briseño (2), A. Varsani (3), J. Polston (2)

(1) Horticultural Sciences Department, University of Florida, U.S.A.; (2) Department of Plant Pathology, University of Florida, U.S.A.; (3) School of Biological Sciences, University of Canterbury, Christchurch, New Zealand

A growing number of metagenomics based approaches have been used for the elucidation of viral diversity in insects, cultivated plants, and water in agricultural production systems. However, while the generation of plant transcriptomes has greatly increased over the last few years, their use for data mining viral sequences has been largely unexplored. In this study, blueberry root transcriptomes (8 libraries each of Vaccinium arboreum and V. corymbosum x V. darrowi 'Emerald') were analyzed using bioinformatic analyses for plant viral sequences. *De novo* assembly of 'Emerald' libraries yielded a complete viral genome of the *Blueberry red ringspot virus* (BRRV), genus *Soymovirus*, family *Caulimoviridae*, a plant virus with a dsDNA genome. BRRV was detected by PCR in 100% and 25% of the 'Emerald' and *V. arboreum* samples, respectively. Since sequences of species in the *Caulimoviridae* are known to be present as integrants in their hosts, back to back primers were designed to detect the episomal form of BRRV genome. The episomal form of BRRV was detected in root tissue of 'Emerald' but not *V. arboreum*, which indicated that the BRRV sequences in *V. arboreum* are most likely those of integrants. In addition, BLASTx analysis of 16 blueberry libraries revealed contigs that match sequences in 20 virus genera and 10 families. Overall, this study demonstrates the feasibility of using available host transcriptome data for mining viral sequences.

## Replication and transcription are independently modulated for each *Banana bunchy top virus* DNA component

N. YU (1), H. Xie (1), J. Wang (1), X. Zhang (1), Z. Liu (1), Z. Xiong (2)

(1) Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, China; (2) University of Arizona, U.S.A.

*Banana bunchy top virus* (BBTV) causes a severe disease in banana world-wide. BBTV is a single-stranded DNA virus with a genome consisting of six circular molecules of ~1kb each. Some BBTV isolates also carry satellite DNA molecules that are not essential for BBTV infection. To understand the coordinated actions of the multiple genomic components during BBTV infection, we measured the relative and absolute amounts of each DNA component of isolate B2 and its transcript levels by real-time PCR (qPCR) and reverse transcription (RT)-qPCR, respectively. Significant differences in the absolute amounts and expression levels were found among BBTV genomic components. The most abundant DNA molecules are those of DNA-U3, DNA-M, and DNA-N, 4 to 6 times more than those of DNA-R. The least abundant DNA molecules are those of DNA-C, 12 times less than that of DNA-U3. Interestingly, two satellite DNA components are also present at high levels, with amounts similar to that of DNA-U3. Transcript levels determined by RT-qPCR did not correspond to the absolute amount of each DNA component. The most abundant transcripts came from DNA-N, followed by DNA-U3, and DNA-S, and the least abundant transcripts came from DNA-C, 110-fold less than those from DNA-N. The ratios of transcript copies over DNA copies ranged from 0.15 to 2.63. These data suggest that both replication and expression levels are independently modulated for each DNA component, possibly by cis-acting DNA sequence elements.

## Identification of defense-related genes associated with tomato Sw-7 line against Tomato spotted wilt virus in tomato through transcriptome analysis

C. PADMABHAN (1), Y. Zheng (2), R. Shekaste-band (3), K. Stewart (1), J. Scott (3), Z. Fei (2), K. Ling (1)

(1) USDA-ARS, U.S. Vegetable Laboratory, Charleston, SC, U.S.A.; (2) Boyce Thompson Institute for Plant Research, Ithaca, NY, U.S.A.; (3) University of Florida, IFAS, Gulf Coast Research and Education Center, Wimauma, FL, U.S.A.

Tomato spotted wilt virus (TSWV) is a highly infectious tospovirus, and one of the most damaging plant viruses infecting tomatoes worldwide. Developing a tomato cultivar with TSWV-resistance would be the most effective approach for disease management. Comparative analysis of differential expression of genes in plant functional pathways between highly isogenic tomato breeding lines with striking difference in resistance and susceptibility to TSWV may lead to understanding of the mechanism of disease resistance. In the present study, RNA-seq was performed on samples from a near-isogenic breeding line containing Sw-7 and its recurrent TSWV-susceptible parent at 0, 4, 7, 14, 21 and 35 days post inoculation (dpi). Comparative transcriptome analysis revealed a large number of genes that are differentially expressed (DE) by TSWV infection over time, including pathogenesis related 1 (PR1) and 5 (PR5) (Osmotic-like protein), Pto-like, Serine/threonine kinase protein, N-methyltransferase-like protein, auxin, glycine/proline rich protein, MADS box transcription factors, histidine kinase, histone proteins, as well as the RNA silencing pathway genes, argonaute, DCL3 and PoIV and microRNA target genes. Genome-wide transcriptome analysis offers us an opportunity to understand the mechanism of disease resistance, which may lead us to an effective management of TSWV in tomato and other crops.

## Transcriptome profiling to discover defense-related genes associated with resistance line ty-5 against Tomato yellow leaf curl virus in tomato

C. PADMABHAN (1), Y. Zheng (2), R. Shekaste-band (3), K. Stewart (1), D. Hasegawa (1), J. Scott (3), Z. Fei (4), K. Ling (1)

(1) USDA-ARS, U.S. Vegetable Laboratory, Charleston, SC, U.S.A.; (2) Boyce Thompson Institute for Plant Research, Ithaca, NY, U.S.A.; (3) University of Florida, IFAS, Gulf Coast Research and Education Center, Wimauma, FL, U.S.A.; (4) zf25@cornell.edu, Ithaca, NY, U.S.A.

Tomato yellow leaf curl virus (TYLCV), a whitefly-transmitted begomovirus, has caused serious economic losses to tomato crops in the U.S. and around the world. The most effective management would be the use of a TYLCV-resistant tomato cultivar. Several sources of TYLCV resistance genes have been identified and incorporated in tomato breeding. The ty-5 gene recently discovered in 'Tyking' was a recessive gene. Although its function has not been well-characterized, near-isogenic breeding materials with TYLCV resistance have been generated. In the present study, after feeding by viruliferous whiteflies (*Bemisia tabaci*, MEAM1) at 0, 4, 7, 14 and 21 days post inoculation (dpi), leaf materials were collected on the test tomato plants for RNA-seq. Differentially expressed (DE) genes based on genome wide transcriptome profiling were analyzed and categorized based on their functions. Those defense-related genes included pathogenesis related 1 (PR1) and 4b (PR4b), CC-NBS-LRR, defensin protein, and heat shock protein were differentially expressed in the ty-5 line. Furthermore, RNA silencing and transcriptional silencing pathway genes, such as RdRp family protein, DNA (Cytosine-5)-methyltransferase 3 and microRNA target genes were also identified. To conclude, the genome wide transcriptome analysis allows us to have a global view on the differential gene expression, which may lead us to an effective management of TYLCV through breeding for durable disease resistance cultivars.

## National Seed Health System: Standardizing seed health practices in the United States for seed exports

N. GONZALEZ (1), T. Bruns (1), G. Munkvold (2)

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

The U.S. National Seed Health System (NSHS) is a program authorized by USDA-APHIS and administered by the Iowa State University Seed Science Center in Ames, Iowa. NSHS provides accreditation to non-government entities to perform certain activities needed to support phytosanitary certification for the international movement of seed, and conducts peer review of protocols for approval as standard phytosanitary seed health testing methods. NSHS is governed by a Policy and Procedures Advisory Board in collaboration with the USDA-APHIS Seed Health Accreditation Manager and the National Plant Board Council/PPQ Strategy Team. Currently, there are 19 entities accredited by NSHS to perform field inspections, seed sampling and/or the seed health testing necessary to obtain phytosanitary certificates for exporting seeds. These organizations include private seed companies, state crop improvement associations and private testing labs. NSHS has been operating since 2001 and has approved 86 methods as standards for testing seeds for 66 different pathogens within a range of vegetable and field crops. NSHS works closely with international organizations to harmonize seed health testing methods that are accepted around the globe for phytosanitary certification. The standardization of seed health methods and practices increases confidence in the health status of US seed exports and facilitates safe and efficient international movement of seed for research and commercial purposes.
# Implementation of a Quality Management System in an ISO 17025 Accredited Plant Pathogen Diagnostic Laboratory

D. PICTON (1), A. Barth (1), K. Burch (2), P. Shiel (2)

(1) USDA-APHIS-PPQ-S&T-CPHST, U.S.A.; (2) USDA-APHIS-PPQ-S&T-CPHST, U.S.A.

In 2015, the USDA-APHIS-PPQ-S&T Beltsville lab (Bldg 580) became the first ISO 17025:2005 accredited regulatory plant pathogen diagnostic lab in the United States. The ISO/IEC 17025 Standard is used world-wide by testing labs in the human clinical realm to the Federal Bureau of Investigation criminal analysis labs to ensure accuracy and reliability of test results. As a result of incorporating a system for technical and quality management, the diagnostics and results generated by the lab are internationally recognized to be technically competent, traceable, repeatable and statistically valid. The implementation of a Quality Management System (QMS) required the establishment of a systematic approach to control all laboratory processes as well as well-defined work instructions that were developed using statistically sound validation and verification data. The newly implemented Laboratory Information Management System (LIMS) has been integrated into the Laboratory QMS to provide complete sample, equipment, reagent and report tracking. This provides an added level of confidence that all data is sufficiently documented, reproducible, and traceable. An annual assessment by an accreditation body ensures that our laboratory continues to meet the requirements of technical competence. Continual improvements to the lab and its commitment to stakeholders through use of the QMS are now an operational component of the USDA-APHIS-PPQ-S&T Beltsville Lab.

# eFile and the USDA Animal Plant Health Inspection Service's (APHIS) PPQ Pest Permitting Process

J. ABAD (1)

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

USDA APHIS safeguards U.S. agriculture against the entry, establishment, and spread of economically and environmentally significant pests. APHIS PPQ uses a permit type called the "PPQ 526 permit" to regulate the interstate movement and importation of plant pathogens (such as bacteria, fungi), pests (such as insects and nematodes), and biocontrols. We currently issue permits through ePermits, a web-based system that allows users to submit and track applications. PPQ receives over 6,000 of these applications per year. The average processing time is 80 days. Depending on the risk of the organism(s) requested, we may require that the work to be done in a USDA APHIS- inspected containment facility to adequately contain the organisms. This may increase processing time as PPQ determines if the facility is adequate. In most cases, this applies for the receipt of foreign organisms, federal quarantine pests, and diagnostic facilities. APHIS is developing a new permitting system known as "eFile" which we will deploy soon. We expect that this new system could substantially reduce the processing time for certain permits.

# National Clean Plant Network for Roses

K. ONG (1), D. Byrne (2), D. Golino (3), N. Anderson (2), S. Sim (3), B. Pemberton (4)

(1) Texas Plant Disease Diagnostic Lab, Texas A&M AgriLife Extension Service, College Station, TX, U.S.A.; (2) Department of Horticultural Sciences, Texas A&M University, College Station, TX, U.S.A.; (3) Foundation Plant Services, University of California-Davis, Davis, CA, U.S.A.; (4) Texas A&M AgriLife Research, Overton, TX, U.S.A.

At the urging of the grape and fruit tree industries, the National Clean Plant Network (NCPN) was established to provide high quality asexually propagated plant material free of targeted plant pathogens and pests for growers in the United States. In 2008, the NCPN was initiated with grapes and fruit tree crops. This was followed by the addition of citrus, berries and hops in 2010 and roses and sweet potatoes in 2015. Each specialty crop has its own commodity network composed of representatives from industry, academia, extension, state government and other interested experts. This program supports translational research, education and extension initiatives to maintain the network's high quality collections and strengthen its services. Since being established in 2015, the National Clean Plant Network for Roses (NCPNR) has been working to augment, renovate and improve the current rose collection at the Foundation Plant Services (Davis, CA), to assess the frequency of viruses in roses in the USA, to develop educational materials, and to establish the best protocols for determining the pathogen status of a rose.

# Master in Plant Health Management: Education and training to meet 21st century trends and challenges in Extension and industry

M. LEWANDOWSKI (1), A. Dorrance (2), L. Canas (3), B. Kleinke (4), E. Roche (5), J. Schoenhals (6), S. Williams (1), F. Peduto Hand (1), J. Jasinksi (7), M. Gardiner (3), P. Paul (2), A. Londo (6)

(1) The Ohio State University, Department of Plant Pathology, Columbus, OH, U.S.A.; (2) The Ohio State University, Ohio Agricultural Research and Development Center, Department of Plant Pathology, Wooster, OH, U.S.A.; (3) The Ohio State University, Ohio Agricultural Research and Development Center, Department of Entomology, Wooster, OH, U.S.A.; (4) Ohio State University Extension, Xenia, OH, U.S.A.; (5) Ohio State University Extension, Cleveland, OH, U.S.A.; (6) Ohio State University Extension, Columbus, OH, U.S.A.; (7) Ohio State University Extension, Urbana, OH, U.S.A.

In response to the growing demand for qualified professionals to address emerging trends and challenges in plant health, the Master in Plant Health Management (MPHM) was established by the Department of Plant Pathology and the Department of Entomology at The Ohio State University. The MPHM program, which is affiliated with the Professional Science Master's Association, is distinguished by an interdisciplinary curriculum that integrates agricultural science coursework with professional development and workforce skills in the College of Food, Agricultural, and Environmental Sciences. An online option was added in 2014. The program has attracted a diverse population of students, many of whom are working professionals and non-traditional students. A new graduate associateship program was established with OSU Extension to recruit, educate and support future Extension educators to work in the field of agriculture and natural resources. Students work directly with Extension teams and mentors on projects that include agronomic field trials, ornamental pathology, and citizen science programs. The MPHM program's placement rate is very strong, with graduates working in Extension, industry and research.

# Virtual disease management using Blightpro DSS

M. MCKELLAR (1), I. Small (1), W. Fry (1)

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

Experiential learning activities can support student understanding of material in an introductory plant pathology course where student base knowledge related to content can be limited. Unfortunately not all course content can be successfully translated into authentic hands-on activities. Computer-based activities such as simulations can be successful in meeting this challenge. We developed a laboratory exercise using a practice version of BlightPro DSS (Decision Support System), which models the progress and control of potato late blight epidemics. The major learning outcome for students is to synthesize and apply their knowledge of disease management to the "real-time" management of late blight in relation to weather, cultivar resistance, and fungicide application. Working in small groups, students developed fungicide schedules to protect their potato crop from late blight while minimizing economic costs and environmental impact. A class discussion on the outcome of student group data concluded the exercise. Students demonstrated a strong cognitive presence throughout the exercise, thinking critically about plant disease management. They sought information from instructors and electronic resources during the exercise, beyond what was required for completion. Student collaboration in small groups promoted cooperative learning opportunities with students sharing previous experiences and knowledge applicable to the exercise outside of content presented in lecture.

# Seed treatment with multiple rhizobacterial strains promotes soybean growth and suppresses reproduction of soybean cyst nematode in the field

L. CHEN (1), Y. Zhou (2), Y. Wang (2), X. Zhu (2), R. Liu (2), P. Xiang (3), J. Chen (4), X. Liu (2), Y. Duan (2)
(1) Shenyang Agricultural University, Ithaca, NY, China; (2) Shenyang Agricultural University, Shenyang, China; (3) Heihe Branch, Heilongjiang Academy of Agricultural Sciences, Heihe, China; (4) Daqing Branch, Heilongjiang Academy of Agricultural Sciences, Daqing, China

The soybean cyst nematode (*Heterodera glycines*, SCN) is the most damaging pathogen of soybean (*Glycine max*) worldwide. Seed coating with nematicides or biological control agents represent a valuable method for controlling nematode pests. Here, we presented the study that evaluated the effects of combining three rhizobacterial strains, including *Sinarhizobium fredii*, *Bacillus megaterium*, and *Bacillus simplex*, as a seed treatment on SCN control and soybean yield enhancement under greenhouse and field conditions in northeast China. An optimal formulation consisting of different proportion of the above three rhizobacterial strains was determined based on the results of the germination vigor of the coated soybean seeds and subsequently designated as SN101. SN101 coated soybean seeds were then used in greenhouse studies and field trials conducted in two field locations for a period of three years. After each growing season, SCN reproduction was determined by nematode egg and cyst numbers and soybean growth and yield were also evaluated. Our results showed that SN101 treatment greatly inhibited SCN reproduction and significantly promoted plant growth and yield production in both greenhouse and field trials, suggesting that SN101 is a promising seed-coating agent that may be used as an alternative tool for controlling SCN in soybean fields.

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

N. KAUR (1), R. Cating (1)

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

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

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

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

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

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

## Frequent isolation of *Colletotrichum* species complex from citrus tissues infected with leaf and fruit spot disease in Ethiopia

A. D. Moges (1,3), D. Belew (2), B. Admassu (1), M. Yesuf (1), S. Maina (3), S. R. GHIMIRE (3)

(1) Ethiopian Institute of Agricultural Research, Ethiopia; (2) Jimma University, Ethiopia; (3) Biosciences eastern and central Africa Hub-International Livestock Research Institute (BecA-ILRI) Hub, Box 30709, Nairobi 00100, Kenya; Email: s.ghimire@cgiar.org

Citrus are economically important fruit crops in Ethiopia with estimated acreage of 7,040 hectares and annual production of 72,459 tons. The citrus leaf and fruit spot is among the major diseases contributing to a low productivity (10.3 t/ha) of citrus in Ethiopia. Therefore, a study was conducted to study diversity and relationships of the pathogens associated with leaf and fruit spot disease in major citrus growing regions of the country. Citrus leaves and fruits samples with distinct leaf and fruit spot disease symptoms were collected from 16 districts of Ethiopia and a total of 167 fungal isolates were recovered and monosporic or hyphal tip cultures were developed for each isolates. The genomic DNAs of all 167 isolates were amplified and sequenced for internal transcribed spacers (ITS), long subunit (LSU) and actin (ACT) genes. The sequences obtained were compared with the GenBank database sequences. The multilocus sequences based phylogenetic analysis revealed that 167 isolates were from either *C. gloeosporioides* or *C. boninense* species complexes. Surprisingly, this study did not recover any *Pseudocercospora angolensis* isolate, the causal agent reported for citrus leaf and fruit spot diseases. The detached leaf assay revealed the ability of both *C. gloeosporioides* and *C. boninense* isolates to cause foliar symptoms on multiple citrus species.