The Rise and Management Challenges of Multi-Fungicide-Resistant PathogensEvolution of multi-fungicide resistance in cereal pathogens and impact on disease controlBart FraaijeRothamsted Research
<div>NW-Europe is always under high disease pressure of fungal pathogens due to favourable weather conditions and a high intensity of cereal production. Host resistance breeding has provided good control of powdery mildews and rusts, albeit subjected to boom-and-bust cycles of major single resistance genes, but it has proved more difficult to control diseases such as Septoria, net blotch and Ramularia. Control of these diseases has been heavily dependent on chemical control. Unfortunately, pathogens like <i>Zymoseptoria tritici</i>, <i>Pyrenophora teres</i> and <i>Ramularia collo-cygni</i> have been able to shown a remarkable adaptation to fungicides over the years and current UK field populations are resistant or less sensitive to different groups of systemic fungicides, including benzimidazoles, quinone outside inhibitors and azoles. As a consequence, farmers have only a few options available to control these diseases. Further threats for disease control are the first reports of resistance development to the most effective group of fungicides, the succinate dehydrogenase inhibitors (SDHIs), in different pathogen field populations, and loss of current actives and drying up of the fungicide pipeline due to tighter legislation. DNA diagnostics able to measure the dynamics of SDHI-resistant alleles in <i>Z. tritici</i>populations are now being used to establish which strategies are most effective for resistance management and can help to extend the shelf life of current and future fungicides.</div>
Biology and Disease ManagementOralPaper3446Paper3446.mp4
The Rise and Management Challenges of Multi-Fungicide-Resistant PathogensHealthy vegetables at the cost of multi-fungicide-resistant pathogensMargaret McGrathCornell University
<div>A commonly recommended and implemented resistance management strategy is applying in alternation fungicides with different modes of action (different chemical groups based on FRAC code). Premise is that cross resistance occurs among related but not unrelated fungicides, because mode of resistance usually is the result of changes in different target-sites, and also that pathogen strains with resistance to multiple chemistries are unlikely to develop or be fit to survive. Unfortunately, resistance has developed to multiple chemistries in single strains of several pathogens causing key diseases in vegetables, including powdery mildew of cucurbits (FRAC Code 1, 7, 11, and 13), gummy stem blight of cucurbits (1, 7, and 11), early blight of potato (7 and 11), Fusarium dry rot of potato (1 and 12), and likely downy mildew of cucurbits (4 and 11 plus 28 and/or 43). Stepwise accumulation of resistance to fungicides with successive exposure is the assumed reason. When the first fungicides at risk for resistance development were registered there was not other chemistry to use in alternation except multi-site contact fungicides, which typically are less effective. However, recent cases of resistance (cucurbit powdery mildew pathogen developing resistance to FRAC Code 13 fungicide in addition to Codes 1, 7, and 11) occurred when there were ample chemistries to alternate with (3, U6, and U8). Premixes can contribute to the problem when resistance has developed to one ingredient.</div>
Biology and Disease ManagementOralPaper3448Paper3448.mp4
The Rise and Management Challenges of Multi-Fungicide-Resistant PathogensMultiple fungicide resistances in <em>Cercospora beticola</em> of sugar beetGary SecorNorth Dakota State University
Viviana Rivera-Varas: North Dakota State University; Melvin Bolton: USDA ARS NCSL
<div>Cercospora leaf spot (CLS), caused by the fungus <i>Cercospora beticola</i>, is the most damaging disease of sugar beet worldwide. It is endemic in the midwestern region of the US causing yield losses and reduced sugar quality. Disease is managed by a combination of crop rotation, resistant varieties and timely fungicide applications. This integrated management strategy has minimized disease losses with no major disease outbreaks for many years. Six fungicides from four classes of fungicides are applied one to six times during the growing season. The fungicides used are triphenyl tin hydroxide, thiophanate methyl, tetraconazole, difenoconazole, prothioconazole and pyraclostrobin. Since 1998, sensitivity to these fungicides has been monitored in over 1000 samples/year from commercial fields collected by sugar company agronomists throughout Minnesota and North Dakota. Changes in sensitivity to all fungicide classes have been documented in all factory districts, and isolates with resistance to multiple fungicide have steadily increased since 2013. In 2016, 14.4% of the isolates tested (n=1326) were resistant to all fungicide classes used for CLS management. The combination of decreased fungicide sensitivity, resistance to multiple fungicide classes and increased disease pressure in recent years may have an impact on future disease management and an accelerated need for resistant varieties.</div>
Biology and Disease ManagementOralPaper3449Paper3449.mp4
The Socioeconomic Impact of New and Re-emerging Bacterial Diseases: A National PerspectiveEpidemiology and distribution of bacterial leaf streak of corn caused by <em>Xanthomonas vasicola</em>Terra HartmanUniversity of Nebraska-Lincoln
Jim Harbour: University of Nebraska-Lincoln; Brad Tharnish: University of Nebraska-Lincoln; Julie Van Meter: Nebraska Department of Agriculture; Tamra Jackson-Ziems: University of Nebraska-Lincoln
<div>In 2016, <i>Xanthomonas vasicola</i>, causal agent of bacterial leaf streak, was confirmed in Nebraska corn fields, the first detection in the United States. In 23 countries around the world, <i>X. vasicola</i> is known to cause disease on a wide range of hosts, most notably gumming disease of sugarcane. In 2016, a survey was conducted, primarily in Nebraska, to determine the distribution of the pathogen and to learn about agricultural practices that may favor disease development. To gather survey data, farmers, crop consultants, and other agribusiness cooperators were queried about their cropping practices. They were also asked to submit symptomatic leaf samples for pathogen isolation. A total of 409 samples and surveys were returned for analyses. <i>X. vasicola</i> was confirmed by PCR on bacterial colonies isolated from samples submitted from 51 Nebraska counties. Survey responses are being analyzed with classification and regression tree (CART) and the random forest algorithm. Results of virulence tests conducted on additional plant species will also be presented.</div>
Diseases of PlantsOralPaper3459Paper3459.mp4
The Socioeconomic Impact of New and Re-emerging Bacterial Diseases: A National PerspectiveUnderstanding the Recent Outbreak of <i>Dickeya dianthicola</i> Causing Potato Soft Rot and Blackleg in the United StatesJianjun HaoUniversity of Maine
Amy Charkowski: Colorado State University; Steve Johnson: Univ of Maine Coop Ext; Gary Secor: North Dakota State University
<div>An outbreak of <i>Dickeya</i> caused disease occurred in 2015 and 2016 in the Eastern US, and <i>D. dianthicola</i> has been determined to be the major causal agent. This problem continues to be a threat since then and <i>D. dianthicola</i> has been found in at least 23 states in the US. Furthermore, multiple <i>Pectobacterium</i> spp. are frequently found in blackleg and soft rot samples along with <i>D. dianthicola</i>. This pathogen complex causes significant economic losses throughout the season due to low emergence and necrotic stems during the growing season and rotted tubers in storage. Contaminated seed potato tubers are a major source of inoculum, but the bacteria can survive and spread on crops other than potatoes, and can survive in the environment, particularly in surface water. Pathogen detection by PCR from tuber stem ends or periderm is a primary tool to evaluate seed potato lots for Dickeya and test results are used to eliminate seed lots with high pathogen incidence from production. Unfortunately, most published detection primers failed when we attempted to validate them with field samples. Sanitation and proper handling of tubers at planting and storage can reduce disease spread. Five antibacterial products were tested to identify additional chemicals farmers could use for sanitation. Elite breeding lines were screened to identify resistant or tolerant lines. To better understand the outbreak, researchers are focused on strain identification, the effect of climate, as well as pathogen distribution, spread and biology. Findings such as where <i>D. dianthicola</i> resides on potato tubers and how it goes to dormant state will improve the efficiency of detection and elimination of pathogens.</div>
Biology and Disease ManagementOralPaper3460Paper3460.mp4
The Socioeconomic Impact of New and Re-emerging Bacterial Diseases: A National PerspectiveZebra chip, what we know and where are we headedElizabeth PiersonTexas A and M University
<div>Outbreaks of Zebra Chip (ZC) on potatoes in the Americas and New Zealand arising in the 1990’s and the rapid and devastating proliferation of Huanglongbing (HLB) on citrus elevated the recognition of '<i>Candidatus</i> Liberibacter' pathogens. ‘<i>Ca</i>. L. solanacearum’ (CLso) is the causative agent of ZC and diseases on major solanaceous crops as well as carrot and celery and is transmitted by psyllids. The epidemiology and management of CLso-associated diseases is complicated by different haplotypes of the pathogen and multiple, genetically variable psyllid vectors with different host ranges. Progress in understanding disease epidemiology and developing management strategies was facilitated by active interaction between researchers, industry, and producers. Management of potato production depends on aggressive psyllid control, albeit with few chemicals and developing resistance. Worldwide proliferation of CLso-associated diseases on current and new hosts will continue with vector range expansion. No host resistance to CLso has been discovered, though breeding for disease tolerance has met with some success. Biological control of insects, microbial agents with plant growth-promoting capabilities, and phage-based therapies targeting the pathogen are promising approaches to augment disease management. Gene silencing and CRISPR/Cas gene editing may yield insights and strategies to control insect populations and modulate host symptoms. Despite progress there is much to do.</div>
Biology and Disease ManagementOralPaper3461Paper3461.mp4
The Socioeconomic Impact of New and Re-emerging Bacterial Diseases: A National PerspectiveDetection and characterization of <i>Xanthomonas</i> <i>vasicola</i> pv. <i>vasculorum</i> pv. nov. causing bacterial leaf streak of corn in the United StatesJillian LangColorado State University
Elysa DuCharme: Colorado State University; Jorge Ibarra: Colorado State University; Emily Luna: Colorado State University; Terra Hartman: University of Nebraska-Lincoln; Kevin Korus: Alachua County Extension, University of Florida; John Rascoe: USDA-APHIS-PPQ-S&T-CPHST; Mary Carmen Ortiz: Colorado State University; Tamra Jackson-Ziems: University of Nebraska-Lincoln; Kirk Broders: Colorado State University; Jan Leach: Colorado State University
<div>Bacterial leaf streak of corn (<i>Zea mays)</i> recently reached epidemic levels in three corn-growing states, and was detected in another six states in the central United States. <i>Xanthomonas vasicola</i> was identified as the causal agent of this disease, however, the biology and taxonomy of this pathogen are not well understood. Multi-locus sequence alignment (MLSA) of six housekeeping genes and comparison of average nucleotide identity (ANI) from draft genome sequence confirmed phylogenetic relationships and classification of this bacterium relative to other <i>Xanthomonas vasicola </i>strains. <i>X. vasicola</i> strains from Nebraska and South Africa were highly virulent on corn and less virulent on sorghum or sugarcane, but caused water-soaking symptoms that are typical of <i>X. vasicola </i>infection on the leaves of all three hosts. Based on host range and phylogenetic comparison, we propose the taxonomic designation of this organism to <i>X. vasicola</i> pv. <i>vasculorum </i>pv. nov. Robust molecular diagnostic assays were developed that distinguish <i>X. vasicola</i> pv. <i>vasculorum</i> pv. nov. and <i>X. vasicola</i> pv. <i>holcicola</i> from each other and from other <i>Xanthomonas</i> spp.</div>
Diseases of PlantsOralPaper3462Paper3462.mp4
The Socioeconomic Impact of New and Re-emerging Bacterial Diseases: A National PerspectiveThe socioeconomic impact of emerging and re-emerging disease epidemicsAmy CharkowskiColorado State University
<div>The fundamentals of on-farm bacterial disease management in plants have not changed for most crops since the 1930s. Exclusion and sanitation are used in almost every crop. Plant resistance is sometime available and when appropriate, insect vector management can reduce disease spread. Since the 1930s, phytosanitary regulations on production and import of plant propagules have been used to manage the spread of bacterial plant pathogens via planting material. The impact in disease reduction of these regulations can take decades to detect and long term datasets that can be correlated with regulatory changes are only available for a few crops, such as seed potato. With these datasets, the cost of implementing these regulations can be estimated and compared to potential disease losses. Examples of the impact of regulation on management of <i>Clavibacter</i>, <i>Ralstonia</i>, <i>Liberibacter</i>, and <i>Dickeya</i> in potato production over the past 80 years will be detailed. The socioeconomic impacts of these disease in potato, a vegetatively propagated crop, will be compared to the impacts of diseases caused by these same bacterial genera in true seed crops. Potential unintended negative socioeconomic effects of regulations targeted to disease management, particularly as they impact small farms, will also be presented.  </div>
Biology and Disease ManagementOralPaper3463Paper3463.mp4
Genomics-based Approaches Facilitate Diagnostic and Population Genetic Marker Development for Plant PathogensSearching for the genetic basis of phenotypic traits of interest of the causal agent of late blight disease through a genome-wide association studyGiovanna DaniesUniversidad de los Andes
Laura Gonzalez-Garcia: Universidad de los Andes; Kevin Myers: Cornell University; Howard Judelson: University of California; William Fry: Cornell University; Silvia Restrepo: Universidad de los Andes
<div>Management of <i>Phytophthora infestans</i>, the causal agent of late blight of potatoes and tomatoes has proven to be challenging. This is mainly due to the lack of durable resistance genes and the capability of the pathogen of evolving resistance to certain highly effective fungicides. An understanding of the genetic basis of fungicide resistance would be of value in managing late blight because rapid analysis using molecular markers could inform the selection of the most effective mitigation tactic. In this study, we conducted a genome-wide association study to identify genetic markers associated with sensitivity to mefenoxam in a diverse panel of <i>P. infestans </i>isolates. To reduce the number of contigs in the <i>P. infestans </i>genome, we reassembled it using Pacific Biosciences sequencing data. The closest genes within a 1000 kb flanking region, upstream and downstream, of each significant SNP marker associated with sensitivity to mefenoxam in <i>P. infestans </i>were carefully annotated. Further analyses are needed to confirm these associations.</div>
Biology and Disease ManagementOralPaper3474Paper3474.mp4
Genomics-based Approaches Facilitate Diagnostic and Population Genetic Marker Development for Plant PathogensMarker development for <em>Puccinia striiformis </em>f. sp.<em> tritici</em>Xiaoping HuNorthwest A&F University, China
<div>Strip rust, caused by <i>Puccinia striiformis</i><i> </i>Westend. f. sp. <i>tritici</i> Eriks. (<i>Pst</i>), results in severe economic losses for wheat growers in many countries. The current <i>Pst</i> race identification mainly depends on the virulence phenotype of the pathogen on a set of wheat differential genotypes. Usually, the approach needs increasing enough urediniospores and testing a set of differential genotypes in a conditioned greenhouse. Such testing can be limited by space and temperature conditions, and is also time-consuming and labor-intensive. Usually, one person can only identify ca. 400 <i>Pst</i> samples per year. <p>In 1990s, the moderately repetitive sequences from <i>Pst</i> were developed to identify 96 genotypes among 160 isolates in the Chinese <i>Pst</i> population. In 2000s, RAPD-SCAR markers of CYR17, CYR29, CRY31, CYR32, CYR33 and V26 were developed to identify races from large scale field samples. In 2009, we identified 20 new polymorphic microsatellite loci derived from the expressed sequence tag (EST) data of Pst. Since 2015, we developed 5741 SNPs using 15 RNA-seq data set of <i>Pst</i> race CYR32 compared to PST78, among which 67 SNPs associated with 13 SP-SNP (secreted protein SNP) were identified. Now, we are developing SNPs related to virulence variation of<i> Pst</i> based on 600 whole-genome re-sequence data set. <p>In summary, these RAPD-SCARs, EST-derived SSRs, and SNPs will be particularly valuable for virulence-related gene discovery as well as for studies on population evolution, population diversity, and dispersal route of <i>Pst</i>.</div>
Genomics-based Approaches Facilitate Diagnostic and Population Genetic Marker Development for Plant PathogensComparative genomics informed detection of <i>Pseudomonas syringae</i> associated with bacterial leaf spot of watermelon and squashEric NewberryUniversity of Florida
Dave Mol: Syngenta Seeds; J. Jones: Department of Plant Pathology, University of Florida; Mathews Paret: North Florida Research and Education Center, University of Florida; Roland Willmann: Syngenta Seeds; Bert Woudt: Syngenta Seeds
<div>Seed infestation is an important factor contributing to the dissemination of <i>Pseudomonas syringae</i> in vegetable crops. In recent years, disease reports identifying the bacterial species as a leaf spot pathogen of watermelon and squash have increased in frequency in the United States and Europe, indicating a need for improved diagnostic methods. Previous studies have shown the strains associated with disease outbreaks to be members of <i>P. syringae</i> phylogroup 2, however genotypic heterogeneity coupled with an unresolved taxonomy pose a serious problem for the precise identification of pathogenic strains. We used comparative genomic approaches to design several molecular markers and screened them against an international collection of over 300 <i>P. syringae</i> strains isolated from contaminated seeds and diseased fields over various years. A phylogenetic analysis of <i>syl</i>C involved in the biosynthesis of the virulence factor syringolin A revealed two divergent groups (<i>syl</i>C<sub>a</sub> and <i>syl</i>C<sub>b</sub>) within <i>P. syringae</i> phylogroup 2, and a TaqMan probe showed 93% of the collected strains to possess <i>syl</i>C<sub>b</sub>. Preliminary pathogenicity testing indicated that the <i>syl</i>C<sub>a</sub> strains were weakly virulent to non-pathogenic on squash (cv. Spineless Beauty) whereas all <i>syl</i>C<sub>b </sub>strains tested were moderately to highly virulent.<i> In-silico</i> analysis suggests <i>syl</i>C<sub>b</sub> to be semi-specific for <i>P. syringae</i> strains isolated as pathogens of watermelon and squash, and therefore increased specificity can be achieved with additional strain specific markers.</div>
Diseases of PlantsOralPaper3476Paper3476.mp4
New Insights into NLR on Plant Immunity: Pathogen Recognitions, Molecular Interactions, and Novel Disease Control StrategiesDistinct E3 ligases regulate the turnover of individual components of paired typical NLR immune receptorsXin LiUniversity of British Columbia
Oliver Dong: University of British Columbia; Fang Xu: University of British Columbia; Kevin Ao: University of British Columbia
<div>In plants, immunity mediated by nucleotide-binding leucine-rich repeat (NLR) immune receptors often requires the formation of NLR hetero-pairs. As the partner has not yet been identified for the Arabidopsis NLR SUPRESSOR OF NPR1, CONSTITUTIVE 1 (SNC1), a reverse genetic screen was undertaken and the NLRs SIDEKICK SNC1 1 (SIKIC1), SIKIC2, and SIKIC3 were found to be redundantly required for SNC1-mediated defense. IP-MS analyses indicated that the SIKICs indeed interact with SNC1 as typical NLR pairs. Furthermore, the protein levels of the SIKICs are regulated by either MUTANT, <i>SNC1</i>-ENHANCING 1 (MUSE1) or MUSE2, two previously uncharacterized redundant E3 ubiquitin ligases that were identified from a genetic screen for <i>snc1</i> enhancers. As SNC1 accumulation is regulated by the E3 ligase SCF<sup>CPR1</sup>, this report provides the first evidence that the homeostasis of individual components of a typical NLR pair are subject to differential regulation via ubiquitin-mediated protein degradation.</div>
Molecular and Cellular Plant-Microbe InteractionsOralPaper3480Paper3480.mp4
New Insights into NLR on Plant Immunity: Pathogen Recognitions, Molecular Interactions, and Novel Disease Control StrategiesBarley Bait-and-Switch: a receptor kinase bait switches on programmed cell death in response to <i>Puccinia graminis</i>Robert BrueggemanNorth Dakota State University
Shyam Solanki: North Dakota State University; Roshan Sharma Poudel: North Dakota State University; Jonathan Richards: North Dakota State University; Gazala Ameen: North Dakota State University
<div>The barley NLR genes, <i>Rpg5</i> and <i>HvRga1</i>, are required together for <i>rpg4</i>-mediated resistance to <i>P. graminis</i> f. sp. <i>tritici </i>(<i>Pgt</i>)<i>,</i> wheat stem rust, and have the head-to-head genome architecture of integrated decoy (ID) resistance mechanisms. The ID hypothesis explains why one NLR partner contains an atypical ID domain that mimics a pathogen’s virulence effector target. Once these host effector targets are fused to NLR receptors they function as IDs that recognize the presence of the pathogen switching on defense responses. Alleles of <i>Rpg5 </i>contain unique C-terminal IDs, with the resistance allele having a protein kinase (PK) ID and susceptible alleles containing a protein phosphatase 2C (PP2C) ID. The <i>Rpg5</i>-PK ID progenitor, HvApk1b, is the <i>Arabidopsis</i> AtAPK1b ortholog that is involved in stomatal aperture opening. Laser Capture Microdissection followed by qPCR determined that HvApk1b is also highly expressed in barley stomata. Thus, we hypothesize that the <i>Pgt</i> effector, Avr-4/5, manipulates HvApk1b during the infection process facilitating pathogen entry through otherwise closed stomata. Barley counter evolved the <i>Rpg5-PK</i> ID immunity receptor via duplication and translocation of the HvAPK1b target protein. Thus, <i>Pgt</i> evolved a virulence effector that facilitates stomata entry, yet, barley counter evolved a “bait” that betrays the pathogen switching on defense responses resulting in resistance to diverse <i>Puccinia graminis</i> formae speciales and races including TTKSK.</div>
Molecular and Cellular Plant-Microbe InteractionsOralPaper3482Paper3482.mp4
New Insights into NLR on Plant Immunity: Pathogen Recognitions, Molecular Interactions, and Novel Disease Control StrategiesTransfer of an NLR gene from pigeonpea into soybean confers resistance to Asian soybean rustPeter van EsseThe Sainsbury Laboratory
Cintia Kawashima: The Sainsbury Laboratory; Jonathan Jones: The Sainsbury Laboratory; Sérgio Brommonschenkel: Universidade Federal de Viçosa
<div>The fungus Phakopsora pachyrhizi is the causal agent of Asian soybean rust (ASR). In Brazil, the second largest producer of soybean in the world, the direct and indirect losses incurred by this pathogen are estimated at approximately 2 Billion US$ per year. Since there are currently no commercial soybean cultivars that have durable resistance against P. pachyrhizi, frequent fungicide applications are the way to control the disease. Genetic resistance is highly desirable given the high cost to growers and the environmental impact of current ASR control strategies. Eight major loci for resistance against the disease have been identified, however none of the causal genes have been cloned. In addition, many of these loci have been introduced into commercial soybean cultivars individually. As a consequence, P. pachyrhizi isolates that have overcome these introduced loci can be readily identified in the field. We have cloned a P. pachyrhizi resistance gene from pigeonpea (Cajanus cajan) and named it CcRpp1 for Cajanus cajan Resistance against Phakopsora pachyrhizi 1. The CcRpp1 gene confers full resistance to P. pachyrhizi when introduced into soybean. Our findings show that legume species related to soybean such as pigeonpea, cowpea, common bean and others could provide a valuable and diverse pool of resistance traits for crop improvement.</div>
Molecular and Cellular Plant-Microbe InteractionsOralPaper3485Paper3485.mp4
Best Practices in Diagnostic Test Development and DeploymentThe need for accurate repeatable diagnostic assays and the pitfalls of testing anomalies: Case study examples from the seed industryScott HeuchelinDuPont Pioneer
<div>Diagnostic assays are only as good as the rigor of the validation process. Too often the validation process prior to publication is limited and doesn’t always show the true limitations of the assay. These issues are often identified when the assay is used in different environs or geographies and false positives or false negatives are observed. These errors in diagnosis for phytosanitary certification can cause commercial or research seed to be rejected entry into a country where it is needed for research and development or to meet market needs. National Plant Protection Organizations (NPPOs) often employ diagnostic assays that they find published in science journals. When these assays are published without extensive inclusivity and exclusivity validation testing, problems often arise from related species or subspecies that exist in the given nation’s environs. Both false positives and false negatives can have major repercussions when the test is used for phytosanitary purposes. These direct losses of seed from impoundment, or the inability to get the seed to its destination due to assay error, can cost companies large sums of money or market competitiveness. Many of these issues could be avoided by better assay development and validation and international standardization of diagnostic assays for pathogen species or subspecies. This presentation will illustrate some of these issues and their resolution via real-world examples from the seed industry.</div>
Diseases of PlantsOralPaper3492Paper3492.mp4
Best Practices in Diagnostic Test Development and DeploymentObtaining DNA template of adequate purity for amplification and avoiding compounds present that inhibit amplification: A <em>Verticillium dahliae</em> exampleGuillaume J. BilodeauCanadian Food Inspection Agency
<div><span>Obtaining a good quality and yield from a DNA extraction is the key in direct detection samples for looking at plant pathogens and organism. In best practices in diagnostic test development, the quality of the DNA is the first step to optimize in order to have success. Over the years, in different projects, we evaluated different DNA extraction methods with optimization in order to obtain the best DNA for Real-Time PCR detection and some metagenomics experiments. Real-time PCR was employed as a tool to determine the best extraction method. Internal controls and specific qPCR assays for <i>Verticillium</i><i> dahliae</i> and other target organisms were used to determine PCR inhibition (quality) and DNA yield. Soil and water samples were collected and spiked with the target organisms to evaluate the best DNA extraction procedure. From the DNA extraction methods tested, some additional steps with magnetic bead purification and chemical flocculation to remove PCR inhibitors were evaluated. Clearly, the methods giving the highest DNA concentration do not always provide the best quality of DNA, and the ones with the best quality do not provide the highest yield. Removal of PCR inhibitors such as humic acids usually requires addition of a chemical flocculant during the extraction, or a post-extraction step such as magnetic bead-based purification. Further using qPCR to directly targeting organisms, some comparison of kits and methods have also been done for metagenomic analysis using Next generation sequencing (NGS).</span></div>
Diseases of PlantsOralPaper3494Paper3494.mp4
Best Practices in Diagnostic Test Development and DeploymentTransferring PCR assays into isothermal platforms: How to make sure it works in real world settings.Timothy MilesCalifornia State University-Monterey Bay
<div>PCR technologies have been the backbone of molecular diagnostics in plant pathology for almost 30 years. Utilizing these tools researchers have been able to detect various species and genotypes in a variety of environmental samples (e.g. soil, air, and plant tissue). While PCR is powerful it is not without limitations including; 1) the requirement of very clean template DNA, 2) the amount of time required to extract this DNA from environmental samples, and 3) a requirement of a high level of skill to run the assays. Isothermal assays offer significant advantages over traditional PCR assays. Many approaches exist but the two main techniques include loop mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA). Like any molecular technology there are advantages and disadvantages to either technique but both approaches are tolerant of PCR inhibitors, can be completed within 30 minutes and can utilize more crude template DNA. Another plus is these assays require significantly less training to perform and have been successfully deployed in field settings to many different groups of plant pathogens. We will discuss examples used in transferring a mitochondrial based PCR marker system for <i>Phytophthora</i> species to LAMP and RPA as well as other examples and compare these approaches in terms of sensitivity, specificity and overall accuracy of the platforms.</div>
Diseases of PlantsOralPaper3495Paper3495.mp4
Best Practices in Diagnostic Test Development and DeploymentUSDA APHIS NPPLAP Proficiency Testing and planned methods deviation as a part of the methods’ validation process for a network of laboratoriesVessela MavrodievaUSDA-APHIS-PPQ-S&T-CPHST
<div><span>The National Plant Protection Laboratory Accreditation Program (NPPLAP) mission is to expand USDA Plant Protection and Quarantine laboratory capacity and promote continuous process improvement for diagnostics of regulated plant pathogens by accrediting network laboratories, use of validated diagnostic protocols and Proficiency Testing (PT). A type of inter-laboratory comparison, PT is where a blind randomized set of samples that mimic field samples is sent to network laboratories for analysis using approved methods. Therefore, in addition to testing labs’ competency, the PT provides data on methods’ performance by different diagnosticians from different laboratory settings and using different equipment. The NPPLAP PT programs for <i>Phytophthora ramorum</i>, citrus greening and plum pox virus have collected multiyear data on the PPQ approved diagnostic methods from approx. 30 laboratories. Analysis of the PT results has provided invaluable data on critical test parameters such as detection limit, precision and range, and methods’ fitness for intended use. This and participant feedback have allowed NPPLAP to identify needs for improvement of these methods. The PT data supports the NPPLAP planned methods deviation process. This process is applied to demonstrate comparability of modified methods, used by a particular laboratory, with approved PPQ methods. This allows network laboratories to implement updated technology and higher throughput to fit their needs. </span><p> </div>
Biology and Disease ManagementOralPaper3496Paper3496.mp4
Adapt, Change, and Improvise: How to Control Diseases as the Climate Is ChangingAdapting disease forecasting models to climate change scenariosKaren GarrettUniversity of Florida
Robin Choudhury: University of Florida Plant Pathology Department; Kelsey Andersen: Plant Pathology Department
<div>Disease forecasting models can be an important component of climate change adaptation. However, climate change offers challenges for the construction and maintenance of disease forecasting models, which must not only address weather variability, but also underlying trends in weather. We developed a framework for evaluating the climate scenarios where forecasting models will become more or less useful. In general, forecasting models may be of little use in environments where the disease in question is extremely common or rare. Optimizing forecasting models may involve tracking where climate change is predicted to shift disease prevalence. The quality (skill) of models used for constructing weather indices for yield loss is also an important factor, along with the spatial heterogeneity of the environment in which the models are applied. Where climate change results in increased weather variability, this may also reduce the success of forecasting models. One aspect of this effect is the potential for more frequent weather conditions that fall outside those used to parameterize the model. The extent to which a forecasting model is mechanistic will help to determine whether it can be applied effectively in novel weather conditions. We discuss the types of ongoing modifications that can help to make forecasting models resilient under climate change, so that they can successfully contribute to climate change adaptation strategies.</div>
Adapt, Change, and Improvise: How to Control Diseases as the Climate Is ChangingAdapting to water insecurity: Balancing reduced water use with root disease riskJohanna Del Castillo MuneraUniversity of California, Davis
Cassandra Swett: Department of Plant Pathology, University of California - Davis; Bruk Belayneh: University of Maryland; John Lea-Cox: University of Maryland
<div>The agricultural sector consumes the highest percentage of available fresh water and, with climate change driving increased in water insecurity, agricultural demands are becoming unsustainable. Sensor-driven deficit irrigation networks use less water by allowing a mild crop stress, but may pose disease risks. The objectives of this study were to evaluate (i) effects of deficit irrigation (DI) on <i>Phytophthora</i> root disease in tomato and (ii) effects of DI on root disease in commercial greenhouse crops. When tomatoes inoculated with <i>Phytophthora</i> <i>capsici</i> were placed under saturated, mild deficit and severe deficit irrigation treatments, disease severity was significantly lower under the saturated and mild deficit treatment compared to severe deficit (<i>P</i> = 0.025). The mild deficit deployed 18% less water than the saturated treatment; irrigation treatment did not affect growth of non-inoculated plants. There was no effect of minor deficit on Pythium root rot in commercially grown poinsettias. When poinsettias were inoculated with<i> Pythium aphanidermatum</i>, disease<i> </i>incidence was significantly lower under saturated and mild deficit conditions, compared to the severe deficit treatment (<i>P </i>= 0.05). These studies indicate that, although severe deficit increases risk of disease losses and should be avoided, minor deficit may not increase root disease risk and can reduce water usage, improving water security in agricultural systems.</div>
Biology and Disease ManagementOralPaper3521Paper3521.mp4
Adapt, Change, and Improvise: How to Control Diseases as the Climate Is ChangingHow changes of annual soil temperature and moisture affect rhizosphere oomycete communitiesMarty ChilversMichigan State University
Alejandro Rojas: Duke University; John Rupe: University of Arkansas; Alison Robertson: Iowa State University, Department of Plant Pathology; Keiddy Urrea: University of Arkansas; Rashelle Matthiesen: Iowa State University
<div>Yield loss in the US alone due to seedling and root rot diseases caused by oomycete and fungal pathogens are estimated to be in the order of 100 million bushels for soybean and corn, respectively. Soil temperature fluctuations and precipitation pulses affect the severity of oomycete disease and the species responsible. Using multiple approaches, we demonstrate the impact of temperature on the oomycete species profile that cause disease and the interaction between these pathogens and fungicides as influenced by temperature. A culture-based survey of oomycetes from soybean across the US soybean belt demonstrated a significant shift in oomycete species between years with differing weather conditions. A follow up study using an amplicon sequencing approach, demonstrated that the oomycete “seed bank” differed by latitude/longitude but not by year. The influence of temperature on the recovered oomycete community profile was confirmed using controlled temperature baiting experiment and controlled temperature pathogenicity and virulence assays. In addition to the effect of temperature on species composition, fungicide sensitivity was also demonstrated to be affected by temperature regimes. The knowledge generated through these studies will aid in improved modelling of oomycete diseases and management through breeding resistance to appropriate species and improved targeting of chemical and biological seed treatments.</div>
Biology and Disease ManagementOralPaper3522Paper3522.mp4
Adapt, Change, and Improvise: How to Control Diseases as the Climate Is ChangingPresence of <i>Ralstonia solanacearum</i> cold virulent strains in the U.S. and mechanisms of virulence at low temperatureAna Maria BocsanczyUniversity of Florida MREC
David Norman: Univ of Florida MREC; Jose Huguet-Tapia: University of Florida; Arianna Mangravita-Novo: University of Florida MREC
<div>Climate change has a very diverse effect on different agricultural areas of the world. In southern United States, the consistent rise in temperature, longer planting periods, and extreme weather events are already favoring the introduction of tropical climate related diseases such as Bacterial Wilt caused by the bacterium <i>Ralstonia solanacearum</i>. We are documenting the increasing presence of strains introduced from the Caribbean that are capable of causing disease at lower temperatures than the usual tropical temperature range with the potential to further spread to northern states. The increase in temperature is also favoring the movement of tropical strains towards higher latitudes. This presence not only can increase the potential for epidemics in economical important crops such as tomato, and tobacco but also can increase the risk of acquiring low temperature virulence and of extending host ranges to include novel temperate climate hosts such as blueberry. It is very important to understand virulence at low temperature and its mechanisms. In our lab we used comparative proteomics, and genomics approaches to understand at molecular level this capability which until recently was assigned to only a monophyletic group of <i>R. solanacearum</i> [R3B2] which is not present in the United States. We are also studying Type 3 effectors that can be horizontally transferred between diverse strains extending their host range to include temperate climate plant crops.</div>
Molecular and Cellular Plant-Microbe InteractionsOralPaper3523Paper3523.mp4
Adapt, Change, and Improvise: How to Control Diseases as the Climate Is ChangingHow to use outreach to help farmers adapt to climate changeAlison RobertsonIowa State University
J. Arbuckle, Jr.: Iowa State University
<div>Outreach shares information with communities to enable them to adapt or mitigate challenging situations. Understanding the community with whom the information is shared is important to ensure the outreach is successful. Within the past few years, considerable research has been done in the Corn Belt through surveys of farmers, extension educators, and private crop advisors to understand their beliefs and attitudes regarding climate change and potential adaptive management strategies. The data suggest that due to political sensitivity surrounding climate change and its causes, direct outreach to farmers and advisors should focus on the impacts of climate change: weather extremes and associated problems such as pests and disease. Most farmers have experienced extreme weather events that impacted their production, thus outreach that addresses adaptation to extreme weather events and related impacts resonate with farmers. Moreover, research suggested that farmers are generally confident in their adaptation skills and are more responsive to outreach focused on ways to adapt to variable weather rather than discussion of climate change. The research also showed that extension is the most trusted source of information on climate change and dealing with extreme weather, among both farmers and advisors, and this should be capitalized on. Today, farmers are likely to go to private advisors, since there are fewer extension educators. Private advisors reported they depend on extension for reliable information on climate change. Thus, a potentially effective way to help farmers adapt to climate change is to foster relationships between extension and private industry and “train the trainer”.</div>
A Bridge over Troubled Ecosystems: How Host Cultivation Creates Novel PathogensRapid Ohia Death: The fast track from houseplants to Hawaii's native forests?Lisa KeithUSDA-ARS
<div>Ohia, <i>Metrosideros</i> <i>polymorpha</i>, is Hawaii’s most common and widespread native tree, ranging from sea level to 2,500 m elevation in both dry and wet forests. It is the most ecologically important native tree, defining ecosystem function and providing critical habitat to many endemic and endangered flora and fauna. Rapid Ohia Death (ROD) is a newly discovered phenomenon causing widespread mortality of ohia on Hawaii Island. Crowns of infected, mature ohia trees turn yellow, then brown in a matter of days to weeks. Fungal growth occurs within ohia wood with characteristic staining patterns. Greenhouse and growth chamber inoculation experiments on ohia seedlings and saplings have proven that <i>Ceratocystis</i> <i>fimbriata</i> causes ROD. Several genotypes of <i>C</i>. <i>fimbriata</i> were already present in Hawaii, affecting sweet potato, taro, and Syngonium, a common ornamental plant in the family Araceae. The possibility that <i>C</i>. <i>fimbriata</i> from these hosts were the culprit of ROD was examined using pathogenicity tests and phylogenetic analysis and results indicate that no direct link exists. ROD poses a serious threat to Hawaii’s flagship native tree species whose loss would be catastrophic for the diversity, structure, and function of Hawaii’s remaining native forests and the critical services they provide.</div>
Diseases of PlantsOralPaper3526Paper3526.mp4
A Bridge over Troubled Ecosystems: How Host Cultivation Creates Novel PathogensPathways and populations in <em>Phytophthora</em>: A legacy of destructionErica GossDepartment of Plant Pathology, University of Florida
Jianan Wang: Kansas State University
<div>The genus <i>Phytophthora</i> contains incredibly successful pathogens in both forest and agricultural ecosystems. <i>Phytophthora</i> species have long taken advantage of cultivated hosts to expand their geographic range. For example, genetic data indicate that early explorers may have spread <i>P. palmivora</i> around the world with coconuts. More recently, nursery stock has been implicated in the movement of multiple <i>Phytophthora</i> species, most notably the multiple introductions of <i>P. ramorum</i> leading to disease epidemics in wild and cultivated forests. Evolutionary changes in these pathogens are also likely to be responsible for their success upon introduction to new hosts and environments. Several <i>Phytophthora</i> pathogens exhibit high heterozygosity within genomes, which may contribute to their adaptive potential. Some of this intra-individual variation is due to polyploidy. We have found substantial triploidy or aneuploidy even in sexual populations of <i>P. infestans</i>. <i>Phytophthora </i>genomes may also exhibit high heterozygosity because they are interspecific hybrids. The tree pathogens <i>P. andina</i>, <i>P. alni</i>, and <i>P. palmivora</i> all exhibit high allelic variation within genomes due to interspecific hybridization. The relative stability of cultivated systems may provide the opportunity for the eventual success of otherwise high risk evolutionary genetic strategies in <i>Phytophthora</i> pathogens.</div>
A Bridge over Troubled Ecosystems: How Host Cultivation Creates Novel PathogensTree domestication and host jump trigger the making of a novel tree diseaseNicolas FeauDepartment of Forest and Conservation Sciences, University of British Columbia
Richard Hamelin: Department of Forest and Conservation Sciences, University of British Columbia; Richard Hamelin: Institut de Biologie Intégrative des Systèmes, Université Laval
<div><span>Tree cultivation is a large contributor to the success in establishment of pathogens, as the use of a few productive clones in intensively managed plantations may encourage susceptibility to diseases. This has been observed in poplars where severe epidemics of native pathogens that were innocuous in their natural pathosystems occurred following host domestication. The North American fungus <i>Sphaerulina musiva</i> is responsible for leaf spots on the eastern cottonwood <i>Populus deltoides. </i>Following introduction of exotic poplar species 100 years ago, a canker disease caused by this pathogen emerged. This disease is severe, causing stem breakage and resulting in plantation failure. By comparing the genome of this fungus to the related poplar leaf pathogen <i>S. populicola</i> we found that the acquisition of a gene arsenal required for growth in woody tissues was at the origin of the adaptation of <i>S. musiva</i> to colonize trees. Recently, an outbreak of cankers induced by <i>S. musiva</i> was observed on <i>P. balsamifera</i> in Alberta. This suggests a host shift since this is not a known host. Analysis of individuals from this outbreak revealed an unusual pattern of diversity, with islands of diversity separated by fixed genome regions. This new population was likely the result of the hybridization of two individuals followed by limited number of recombination events. The population infecting <i>P. balsamifera</i> has strong clonality: we observed a transposon proliferation as well as fixation of the Mat1.1 allele. The shift from a sexual to a clonal population may constitute one rapid way to fix beneficial mutations. The development and expansion of poplar cultivation triggered changes in a native pathogen, resulting in a specialized population with higher fitness.</span><p> </div>
Biology and Disease ManagementOralPaper3528Paper3528.mp4
A Bridge over Troubled Ecosystems: How Host Cultivation Creates Novel PathogensFly without wings: Genetic structure and adaptation of fungal pathogen and associates with bark beetle outbreak in the western Canada and USAClement TsuiDepartment of Forest and Conservation Sciences, University of British Columbia
Nicolas Feau: Department of Forest and Conservation Sciences, University of British Columbia; Dario Alayon: Department of Forest and Conservation Sciences, University of British Columbia; Arnaud Capron: Department of Forest and Conservation Sciences, University of British Columbia; Braham Dhillon: University of Arkansas; Richard Hamelin: Department of Forest and Conservation Sciences, University of British Columbia; Yiyuan Zhang: Department of Forest and Conservation Sciences, University of British Columbia
<div>Over 18 million hectares of forests have been destroyed in the past two decades in Canada by the mountain pine beetle (MPB) and its fungal symbionts <i>Grosmannia clavigera</i> (<i>Gc</i>), <i>Leptographium longiclavatum</i> (<i>Ll</i>) and <i>Ophiostoma montium </i>(<i>Om</i>) (Ophiostomatales, Ascomycota). These fungal symbionts are crucial in the establishment and reproductive success of MPB by aiding in nutritional acquisition and overcoming host-tree defenses. Understanding their population structure and adaptations are important to predict their expansion pattern and to improving modeling of beetle epidemics. We investigated their genetic structures using single nucleotide polymorphisms (SNPs) and we monitored fungal growth rates at different temperatures. We found a strong north-south differentiation in their population structure that is strongly correlated with geographical distance. Genetic variation within each species are best explained by distinct spatial and environmental variables. SNP genotyping coupled with genotype-environment association analysis and phenotypic characterization of growth rate, suggested that the coexistence of three MPB fungal symbionts is the result of niche partitioning. We observed that both common (temperature seasonality and host species) and distinct (drought/cold stress, precipitation) environmental and spatial factors shaped these fungal genomes resulting in contrasting outcomes. Intraspecific phenotypic variations in <i>Gc </i>and <i>Ll</i> suggests potential for adaptive selection in these two species. By contrast, <i>Om</i> displayed narrower intraspecific variation but greater tolerance to high temperatures. Our study highlights the unique genotypic and phenotypic characteristics in these symbionts.</div>
A Bridge over Troubled Ecosystems: How Host Cultivation Creates Novel Pathogens<em>Sphaerulina</em> through history in North America: Consequences of human-aided disseminationMonique SakalidisMichigan State University
<div>Commercial planting practices and strategic breeding for traits that are desirable for commercial production (fast growth, high fiber content) may inadvertently shape the evolution of pathogens that exist on these trees. Domestication of wild pathogens alongside their host can generate new diseases that can have devastating consequences. The Dothideomycete fungus, <i>Sphaerulina musiva </i>is an endemic fungus that occurs naturally on wild poplar in north-eastern and north-central North America where it causes innocuous leaf infections. In domesticated poplars this fungus causes a new disease that results in wounds (cankers) on the stem of the trees- in the most extreme cases trees literally snap in half. In order to manage disease spread, prevent further incursions and identify genes involved in plant attack, a detailed review of historical records and the genomes of 83 strains of the pathogen were decoded. Genes involved in adaptation across all populations and regional adaptation generating local geographic populations were found. These geographic populations also displayed significant difference in virulence profiles on different genotypes of <i>Poplar trichocarpa</i>. Modeling using genomic profiles shows that the pathogen originated in the US, a center of diversity and has repeatedly spread or been introduced into Canada. The most likely vector enabling these repeated introductions is symptomless poplar material exchanged for commercial plantations or breeding programs.</div>
Diseases of PlantsOralPaper3530Paper3530.mp4
Beautiful Efficiency: The Multifunctional Nature of Virus ProteinsThe P6 Effector Protein of <em>Cauliflower mosaic virus</em>: A Masterswitch in the Virus Infection CycleJames SchoelzUniversity if Missouri
Mustafa Adhab: University of Missouri - Columbia; Carlos Angel: Cenicafe - Colombia; Richard Nelson: Samuel Roberts Noble Foundation, Inc.
<div>The P6 protein of <i>Cauliflower mosaic virus</i> (CaMV) is a 66 kDa protein that forms the matrix for the electron dense, amorphous inclusion bodies (IBs) that accumulate in the cytoplasm. Early electron micrographs revealed that host ribosomes were found in association with the P6 IBs, and nearly all of the icosahedral CaMV virions accumulate and are retained within the P6 IBs. Subsequent studies provided an explanation for the association of P6 IBs and host ribosomes; the P6 protein physically interacts with host ribosomes to reprogram them for expression of all CaMV proteins on the polycistronic 35S mRNA, a process called translational transactivation. For many years, translational transactivation was thought to be the primary function of the P6 protein. However, several recent studies have expanded our understanding of the true impact of the P6 protein on CaMV infections, as the P6 protein has been shown to mediate intracellular movement, elicit defenses in hypersensitive hosts or symptoms in susceptible hosts, modulate SA- and JA-mediated host defenses, and suppress antiviral gene silencing. Furthermore, the P6 protein interacts with at least 14 host and virus proteins, of which distinct subsets are associated with individual functions. This presentation will illustrate how newly identified virus-host protein interactome maps complemented with subcellular localization studies can be used to further define the role of the P6 protein in the CaMV disease cycle.</div>
Molecular and Cellular Plant-Microbe InteractionsOralPaper3531Paper3531.mp4
Beautiful Efficiency: The Multifunctional Nature of Virus ProteinsThe p33 protein of <em>Citrus tristeza virus</em>: A conundrum of multiple functionsSvetlana FolimonovaUniversity of Florida
<div><em>Citrus tristeza virus</em> (CTV), the most economically important viral pathogen of citrus, encodes a unique protein, p33. Research conducted over the recent years showed that this protein plays multiple important roles in viral pathogenesis. It was found that while p33 is dispensable for infection of a number of citrus varieties, it is required for systemic infection of a few others. Furthermore, p33 is one of the key viral factors mediating virus ability to exclude superinfection with the same or closely related virus. The characterization of p33 demonstrated that it is a self-interacting integral membrane protein, and its membrane localization is important for virus ability to infect an extended host range. In the infected cells, p33 shows plasmodesmata localization and forms extracellular tubules, which are the characteristic features found for a number of movement proteins of other plants viruses. Although p33 is not conserved among other closteroviruses, a few members of the genus <i>Closterovirus</i> encode proteins of ~30 kDa whose corresponding genes are situated at the same genomic position as that of the CTV p33 gene. While primary sequences of those proteins do not share significant homology, some of their characteristics along with their secondary structures show some similarities. Further research is required for a better understanding of the functions that these proteins play in virus infection and the overall genome complexity of closteroviruses.</div>
Molecular and Cellular Plant-Microbe InteractionsOralPaper3533Paper3533.mp4
Beautiful Efficiency: The Multifunctional Nature of Virus ProteinsFunctional variation in cognate proteins of nucleotrophic negative-strand RNA viruses of plantsMichael GoodinUniversity of Kentucky
<div>Rhabdoviruses infect a wide range of hosts, and members of this group include viruses that infect humans, terrestrial animals/vertebrates, fish, arthropods, and plants. The genomes of the plant-adapted viruses are organized generally into seven open reading frames with the gene order 3’-N-X-P-Y-M-G-L-5’, which encodes the nucleocapsid, phospho, movement, matrix, glyco, and RNA-dependent RNA polymerase proteins, respectively, except for X, which is of unknown function. In addition to its structural role in virion formation, the M protein of <i>Potato yellow dwarf virus </i>(PYDV) is capable of inducing the intranuclear accumulation of the inner nuclear membranes (INM) in transfected cells. The M protein also interacts with the nuclear import and export receptors Importin-alpha and Exportin 1, suggesting a role for M in transport of condensed nucleocapsids from the nucleus. Interestingly, the ability to remodel the INM is conferred by the P, but not M, protein of coffee ringspot dichorhavirus, demonstrating that functional domains within rhabdoviral proteins are portable. This variation likely contributes to the finding that protein interaction and localization maps (PILMs) for each virus are unique. How these variations in protein sequence contribute to the manner by which these viruses remodel the architecture of plant nuclei will be discussed. <i></i></div>
Molecular and Cellular Plant-Microbe InteractionsOralPaper3534Paper3534.mp4
Phytobiomes 2.0: Functional Approaches in Forest EcosystemsMovement of bacteria between plant species drive assembly of epiphytic bacterial communitiesSteven LindowUniversity of California Berkeley
<div>Aerial plant surfaces often support large population sizes (> 10<sup>6</sup> cells/cm<sup>2</sup>) of a variety of bacterial colonists. The large majority of these colonists occur in relatively large cellular assemblages, apparently driven by localized abundance of limiting carbon resources and more conducive environmental conditions at such sites. Bacterial growth and survival is strongly favored in cellular aggregates, which also are sites of preferential recruitment of immigrant inoculum. Emigration of bacteria from plants is quite efficient and thus the abundance and composition of microbes in air near plants is strongly influenced by the amount and type of vegetation nearby. While some host plant species selection for particular epiphytic bacterial colonists occurs, phyllosphere microbial communities appear to be assembled from a metacommunity contributed and shared by nearby plants in a process that likely involves microhabitat modification at sites of microbial aggregation by initial plant colonists. The selection appears to occur from bacterial strains adapted for life on foliar plant surfaces rather than from other habitats. Leaf surface microbial communities therefore are quite context-dependent and can be managed either by direct inoculation or by changing the agroecological context in which crops are grown.</div>
Phytobiomes 2.0: Functional Approaches in Forest EcosystemsMicrobial communities of bristlecone needles and resistance in white pine blister rustAlyssa AlbertsonColorado State University
Zaid Abdo: Colorado State University; Anna Schoettle: USDA Forest Service; Jane Stewart: Colorado State University; Ken Kassenbrock: Colorado State University
<div><i>Cronartium ribicola</i>, the causal agent of white pine blister rust (WPBR), threatens susceptible North American pine species, including Rocky Mountain bristlecone pine (<i>Pinus aristata</i>). Since fungal endophytes have been identified to alter a hosts’ response to pathogens this research examined the endophytic fungal communities of healthy Rocky Mountain bristlecone pine trees previously inferred to vary in susceptibility to WPBR (using artificial inoculation progeny testing). Three sites were visited in Colorado, where six trees were sampled at each site. Two techniques were used to assay the fungal endophyte community of each tree: culturing from surface-sterilized needles, and of PCR-amplified ITS1 fungal ribosomal gene. Culturing recovered 255 fungal isolates in 33 morphological groups. The Next Generation data yielded over 42,000 usable sequences that were clustered into 789 operational taxonomic units. There was good overlap between both methods; detecting small variations between the fungal communities of individual trees, resistant and susceptible trees, and sites.</div>
Molecular and Cellular Plant-Microbe InteractionsOralPaper3539Paper3539.mp4
Phytobiomes 2.0: Functional Approaches in Forest EcosystemsThe <em>Populus</em> microbiome: Progress and limitations of our understanding of phytobiome communitiesDan JacobsonOak Ridge National Laboratory
Piet Jones: University of Tennessee; Gerald Tuskan: Oak Ridge National Laboratory; Wellington Muchero: Oak Ridge National Laboratory; Jay Chen: Oak Ridge National Laboratory
<div>The phytobiome (here defined as the combination of the microbiome, mycobiome and viriome) not only interacts with the host, and thus may elicit or suppress a defense response, but there may be interactions amongst the constituents of the phyotbiome. To better understand these influences we have applied modified genome-wide association (GWAS) methods in order to associate host genotypic variation to the putative viral, bacterial, fungal and archaeal community composition. Metatranscriptome samples extracted from <i>Populus trichocarpa</i> xylem and leaf tissue were used to identify the different taxa present via several computational approaches. The resultant members of the phytobiome then served as phenotypes in a GWAS analysis involving 444 genotypes of <i>Populus trichocarpa</i> (against over 28 million single-nucleotide polymorphisms). We view the results as a network and network find shared associations between phytobiome phenotypes and receptor kinases, signal transduction genes, transcription factors, stress and cell organization among other functions. The resultant network provides a rich framework for biologically driven hypothesis generation. Furthermore, we have used the plant gene expression data from these genotypes as expression phenotypes for eQTN analysis. In so doing we are able to identify a significant portion of the regulatory network surrounding the genes that map to the phytobiome phenotypes described above. This unified model is a powerful tool for understanding host-phytobiome associations and the regulatory circuits that control them.</div>
Molecular and Cellular Plant-Microbe InteractionsOralPaper3541Paper3541.mp4
Translation of Basic Biological Control Research into Effective Grower Products and PracticesThe phytobiome and biological control: What does the future hold?Jan LeachColorado State University
<div>Meeting demands for food, feed and fiber will require doubling production in less than 35 years, and doing so in the face of increasing resource constraints, extreme weather events, uncertain water availability, and increasingly limited arable land. The Phytobiomes Roadmap, published in 2016, offered a vision for sustainable crop productivity obtained through a systems-level understanding of diverse interacting components. One component of the phytobiome that is being actively targeted for improving healthy crop production is the plant microbiome.<span> </span>Strategies involve the incorporation of beneficial microbiomes into agricultural systems by managing, selecting, or engineering microbiomes that enhance plant growth, nutrient use efficiency, tolerance to abiotic stresses, or disease resistance, including biological control. However, there are many knowledge gaps that need to be closed to successfully and sustainably translate this information to the field; these gaps and strategies to approach them will be discussed in this presentation. </div>
Biology and Disease ManagementOralPaper3547Paper3547.mp4
Translation of Basic Biological Control Research into Effective Grower Products and PracticesWhat happens to good results? From the academic research lab to labeled product for growersBarry JacobsenMontana State University
Michael Dimock: Certis USA, LLC
<div>This presentation will discuss the pathways and pitfalls in developing a commercial biological control. Focus will be on initial discovery, laboratory research, taxonomic identification, deposit of reference samples with national or international culture collections, development of provisional and final patents, publication, field testing and working with companies who will develop a product and regulatory agencies who will label a product. Precautions and suggestions on sharing samples early in the discovery and development phases, field testing and testing for key commercial markets will be discussed. The 20 year development of Bacillus mycoides isolate J into the product LifeGard <p> </div>
Biology and Disease ManagementOralPaper3548Paper3548.mp4
Translation of Basic Biological Control Research into Effective Grower Products and PracticesNonbiological biological controlGary HarmanAdvanced biological marketing
Andrea Moreno: Advanced Biological Marketing; Walid Nosir: Advanced Biological Marketing; Molly Cadle-Davidson: Advanced Biological Marketing, Inc
<div>Biocontrol and plant growth promotive microbes, especially endophytic ones, interact strongly with plants. They frequently do so via chemical communication with plants. These chemical communicants are, by necessity, active at very low concentrations and may provide many of the same benefits as the microbial agents that produce them. One advantage of use of these biorational compounds is that it may be possible to use these in environments, e.g., in the presence of chemical pesticides and surfactants, that would be lethal to the microbial agents that produce them. An example of such microbial metabolites are the lipopolysaccharides from <i>Bacillus </i>spp. In our studies with <i>Trichoderma </i>strains, we evaluated 1-octen-3-ol (1o3) and 6-penyl pyrone (6PP). In field trials, seed treatments with 1 ul/seed or less of 1o3 induced many of the same effects in corn as did the strains from which they were produced. Plant growth and yields were enhanced and root growth was remarkably stimulated. Similarly, resistance to abiotic stresses such as flooding also was enhanced. In greenhouse tests, both 1o3 and 6PP had similar effects on both corn and soybeans. The effects in the field lasted for the life of at least an annual crop. Clearly, the chemicals must induce changes in the plant, perhaps through epigenetic changes in the nucleic acid and/or through long-term alterations in the plant microbiome. Formulation and delivery systems with these chemicals are being developed.</div>
Biology and Disease ManagementOralPaper3550Paper3550.mp4
Pursuit of Solutions to Mycotoxin Risks by Next-Generation Plant PathologistsComputational prediction of time-course subnetwork modules associated with histidine kinase activities in maize pathogen <i>Fusarium verticillioides</i>Man KimTexas A&M University
Won-Bo Shim: Texas A&M University
<div><i>Fusarium verticillioides</i> is a fungal pathogen causing maize ear rots worldwide, and maize contaminated with fumonisins poses human and animal health risks. There is a critical need to improve our understanding of the disease to develop effective control strategies. Recently, we developed computational methods to analyze large-scale RNA-Seq datasets and identify <i>F. verticillioides</i> genes as potential molecular targets for disrupting pathogenesis and reducing fumonisin contamination. This study demonstrates our effort to systematically investigate the role of histidine kinase (HK) genes during Fusarium ear rot. We performed network-based comparative analysis in a time-course manner to monitor differential HK gene activities in <i>F. verticillioides</i> on maize kernels compared to vegetative growth. We found that majority of HK genes are significantly activated with other genes at certain time points only. Meanwhile selected HK genes are significantly activated at multiple time points, but with closely correlated genes drastically changing over time. We identified HK genes associated with the osmotic-stress response signaling, a well-known function of HK, and show that the transcriptional subnetwork modules between HK two-component system and Hog MAP kinase cascade are correlated in a complex manner. This computational method allows us to advance the discovery of time-specific co-expression subnetwork modules associated with HK gene regulation and Fusarium ear rot pathogenesis.</div>
Molecular and Cellular Plant-Microbe InteractionsOralPaper3554Paper3554.mp4
Pursuit of Solutions to Mycotoxin Risks by Next-Generation Plant PathologistsModeling complex associations among weather, deoxynivalenol contamination, and Fusarium head blight in wheatWanderson Bucker MoraesThe Ohio State University
Pierce Paul: Ohio State Univ, Ohio Agricultural Research and Development Center, Dept of Plant Pathology; Felipe Dalla Lana: Ohio State University; Byung-Kee Baik: United States Department of Agriculture; Paul B. Schwarz: North Dakota State University; Laurence Madden: Ohio State Univ, Ohio Agricultural Research and Development Center, Dept of Plant Pathology
<div>Fusarium head blight (FHB), caused by <i>Fusarium graminearum</i>, is associated with wheat grain contamination with harmful mycotoxins such as deoxynivalenol (DON). Although FHB is a good indicator of DON, this relationship breaks down under certain weather conditions. One possible explanation for this could be the conversion of DON to DON-3-Glucoside (D3G), which is often missed by common DON testing methods. An experiment was conducted to investigate the effects of rainfall patterns on DON-D3G conversion in wheat grains. Separate replicate plots with different levels of FHB were subjected to one of six rainfall treatments: rainfall every day (Rain1); every other day (Rain2); two days with, two days without rainfall (Rain3); three days with, three days without rainfall (Rain4); four days with, five days without rainfall (Rain5); and no supplemental rainfall (check). There was a significant positive relationship between DON and D3G. Rain1 resulted in significantly higher mean D3G than the other treatments. Rain4 resulted in higher mean D3G than Rain2, Rain3, and Rain5. All rainfall treatments induced sprouting, as indicating by low falling number (FN). Rain1 consistently had the lowest mean FN, whereas Rain4 resulted in significantly lower mean FN than the other intermittent rainfall treatments. There was a significant positive relationship between DON-D3G conversion (based on D3G/DON ratios) and sprouting. This study is the first to associate DON-D3G conversion to pre-harvest rainfall patterns and sprouting, and provides new information that is invaluable for understanding this complex disease-toxin system.</div>
Recruiting Next Gen Scientists: Strategies for Inclusive OutreachFostering aGIRLculture: STEM camp solving the grand challenges of the 21st centuryDenita HadziabdicUniversity of Tennessee
Lori Duncan: University of Tennessee; Andrea Ludwig: University of Tennessee; Annette Wszelaki: University of Tennessee; Bonnie Ownley: University of Tennessee, Department of Entomology and Plant Pathology; Bonnie Ownley: University of Tennessee; Christine Buschermohle: University of Tennessee; Kimberly Gwinn: University of Tennessee, Department of Entomology and Plant Pathology; Natalie Bumgarner: University of Tennessee; Faith Critzer: University of Tennessee; Lannett Edwards: University of Tennessee; Lisa Muller: University of Tennessee; Carrie Stephens: University of Tennessee; Margarita Velandia: University of Tennessee; Jennifer DeBruyn: University of Tennessee; Elizabeth Gall: University of Tennessee; Joanne Logan: University of Tennessee; Rebecca Trout Fryxell: University of Tennessee; Heather Kelly: University of Tennessee; Karen Lewis: University of Tennessee; Sharon Jean-Philippe: University of Tennessee; Karen Jones: University of Tennessee; Doris D'Souza: University of Tennessee, Food Science Dep
<div>To meet the demands of increased world population and expectations of feeding approximately 9.7 billion people by 2050, there is a critical need for expanding post-secondary education focused on science, technology, mathematics and engineering<i> </i>(STEM) disciplines. Current college participation rates for rural Tennessee, U.S., are low compared to national standards and students are unaware of professional career opportunities in STEM areas. Although the majority of students that enroll in colleges are female, they are not pursuing STEM disciplines. Recent findings indicated that having a female professor as a guide and role model from a female student’s perspective was linked to increased implicit science identity and decreased implicit gender stereotyping. Hence, a team of 24 female faculty members from the University of Tennessee developed and implemented a 3-day summer camp titled ‘Cultivating aGIRLculture’. The project objectives were 1)<b> </b>to<b> </b>recruit high school female students from rural communities and expose them to higher education and careers in STEM disciplines, 2) to<b> </b>cultivate a climate that is free of gender biases and stereotypes in STEM careers, and 3)<b> </b>to<b> </b>enhance female faculty collaboration across different disciplines to further improve educational, research, and outreach efforts. Data included pre- and post-surveys to evaluate attitudes, perceptions, and knowledge of camp participants, undergraduate chaperones, and faculty collaborators. <p><b> </b></div>
Recruiting Next Gen Scientists: Strategies for Inclusive OutreachTeach the Teachers: The Florida ExperienceMonica ElliottUniversity of Florida
<div><span>During summer 2016, seven of the University of Florida-IFAS research and education centers located across the state served as hosts for a Plant Pathology Teachers Workshop, using themes from APS: “Healthy Plants=Healthy World” and “Don’t Get Caught With Your Plants Down”). The target audience was middle school and high school teachers and 83 people participated. Hard copies of all materials were provided, but the materials were also available, and continue to be available, on-line at <a href=""></a></span><span>. Much of the material used was obtained from APS resources. The overall increase in plant pathology knowledge was 50%, and the overall evaluation for the workshop was 4.5, on a 5-point scale with 1=low and 5=high. Of particular note was the general view that the concepts would not be easy to incorporate into their current science curriculum, but the teachers enjoyed gaining new knowledge. Hands-on activities were included, and teachers stated these were an important aspect of the workshop, especially if designed so teachers can use them in the classroom. However, the main challenge public school teachers face in Florida is the restriction on using microbes in the classroom. There are even restrictions on flowering plants and foods that could be used for experiments. The workshop was also useful as a tool to engage graduate students in teaching and outreach.</span><p> </div>
Recruiting Next Gen Scientists: Strategies for Inclusive OutreachIntegrating plant diseases into the K-12 STEM classroomRobert HirschUniversity of Kentucky
<div>Despite being ideal systems that allow STEM teachers to integrate critical content areas and address relevant teaching standards, plant diseases are largely absent from K-12 STEM curricula. Two case studies present models of partnership that capitalize on the respective strengths of research scientists and STEM educators by fulfilling content standards, meaningfully involving students in the research process, and increasing the broader impact of competitive grant proposals. The first example explores the population genetics of the tall fescue endophyte <i>Epichlo</i><i>ë coenophiala</i>. A research field trial was established on public school property, and was utilized as a living laboratory for high school students to learn the process of plot design and sample collection. Research scientists assisted STEM teachers with developing grade-band appropriate curricula modules and assessments based on the ecological implications of fescue/fungi symbioses. In the second case study, APS scientists and STEM teachers utilized bacterial soft rot as a model disease and drafted a middle school laboratory curriculum module. Utilizing these materials, over 200 students manipulated variables of the disease triangle and explored complex host/pathogen interactions. Both case studies offer tangible examples of successful outreach collaborations that benefited the STEM community, broadened the impact of scientific research, and introduced the next generation of scientists to plant pathology.</div>
Recruiting Next Gen Scientists: Strategies for Inclusive OutreachNurturing agricultural migrant workforceLuisa SantamariaOregon State University
<div>There is a growing demand for appropriate training opportunities for the U.S. agricultural workforce. Immigrants make up the majority of this workforce and about 67% of them are Spanish-speakers. Traditional, face-to-face training is the most common approach in extension, but work schedules make this method a limited option for consistent, ongoing trainings. We are constantly evaluating the educational needs to help this workforce be more efficient in their daily activities. Steps have been taken in this direction to train workers in different aspects of plant health to prevent plant diseases in Oregon's nursery and Christmas tree production systems. One of our new approaches was to develop a hybrid teaching class (blend of online as well a face-to-face instruction) and to assess the reception of this teaching method by immigrant workers. The delivery method was very well received by the participants and the possibility to reach more workers with this approach is promising. Printed educational material is also an important tool to support trainings for this adult audience. Some bilingual materials (pocket booklets, fact sheets, posters, and web responsive and mobile friendly materials) have been developed to create plant disease awareness. This presentation will provide an overview of the challenges and opportunities of creating an active and inclusive educational program geared towards Spanish speaking migrant workers.</div>
An Ever-changing Extension Environment: Keeping a Foot in the Furrow and a Hand in CyberspaceBlippity BLOPs: Goals, information, uncertainty, and trust in Extension adoption problemsNeil McRobertsUniversity of California-Davis
<div>Extension problems in agriculture are Bi-level Optimization Problems (BLOPs) in which actors (i.e. extension agents and farmers) operating at two different scales have overlapping but not identical goals. Standard economic approaches for optimizing goals routinely fail to work for BLOPs resulting in economic inefficiency and social costs. Newer methods for solving BLOPs point to the key roles that information flow, uncertainty, and trust among actors within and between scales play in the likelihood of satisfactory outcomes. Even without formal quantitative analysis, framing extension problems as BLOPs helps us to see these points and develop ways to incorporate them into extension practice. Many of the issues involved in achieving satisfactory outcomes can also be considered from the social network perspective, and by considering the ways in which the study of BLOPs might be undertaken we begin to characterize the analytical toolkit for future extension scientists and the means by which extension can be revitalized and given higher priority by policy makers.</div>
Show Me the Money! Assessing the Value of Disease Control in a Changing LandscapeImpact Network Analysis: A framework for evaluating the effects of technologies through linked socioeconomic and biophysical networksKaren GarrettUniversity of Florida
<div>Decision-making about disease management is ultimately driven by money. Farmers decide what management strategies to adopt, and sponsors decide what research and development projects to support, based on their financial constraints. Understanding how to optimize the effects of research and data collection typically requires integration across three general types of system components: (a) the type and quality of information and other technologies, (b) socioeconomic networks that determine communication and influence about technology use, such as networks of farmers, and (c) biophysical networks where decisions about use of technologies determine epidemiological outcomes. Impact Network Analysis is a framework to integrate these three components, providing a systems perspective to evaluate potential outcomes from research and development investments. Impact Network Analyses can also be used to assess the resilience of a system to new perturbations, such as climate change or the introduction of a new pathogen. Network analysis, as compared to more aggregated models, allows for consideration of the role of geographic and social structures on the likelihood of success of technological innovations. Applications of Impact Network Analysis will be illustrated for disease management in the field, in seed systems, and in postharvest food chains. Experimental choice games will also be introduced, in the context of evaluating willingness to pay for new technologies.</div>
Show Me the Money! Assessing the Value of Disease Control in a Changing LandscapeDisease Control: A Seed Company PerspectiveJohn PitkinMonsanto
<div><span>There are three main modes of delivery for disease control solutions by private agricultural companies: in the seed (disease traits) on the seed (seed treatments) or over the top (fungicides). The desire to combat diseases must be balanced by the overall profitability of any disease control system for the farmer customer. The most important disease to our customers when making seed choices is often the disease the farmer has struggled to control over the last season or two on his or her farm. Given that disease pressure is regional and periodic, all of these factors must be considered when determining the value of “in the bag” seed solutions for disease control in row crops. </span><p> </div>
Biology and Disease ManagementOralPaper3587Paper3587.mp4
Phyllosphere Microbial Assemblages: Friends, Foes, and StrangersAn unusual ménage à trois in the phyllosphereRichard BélangerUniversité Laval
Joan Laur: Université Laval; Caroline Labbe: Université Laval; Gowsica Bojarajan Ramakrishnan: Université Laval; Pietro Spanu: Imperial College of Sci Tech & Medicine
<div>The phyllosphere harbors a complex microbial community in which fungi occupy a predominant space. In the course of evolution, all leaf surface fungi have acquired specific properties that enable them to compete and survive in this restricted ecological niche in spite of a scarcity of resources. While we, as scientists, have been trying to ascribe a certain hierarchy among the fungi inhabiting the phylloplane, it is nonetheless important to remember that in a balanced environment, each of these fungi manages successfully to acquire the resources necessary for its establishment and reproduction on the leaf surface. In the case of tritrophic interactions involving a biocontrol agent, a fungal pathogen and a plant, they have historically been analyzed solely from the perspective of the mode of action of the biocontrol agent. However, the tripartite association between Pseudozyma flocculosa, powdery mildews and the host plant has seemingly evolved from the conjugated action of the three protagonists. Recent evidence indicates that P. flocculosa can in fact modulate the virulence of powdery mildew fungi and divert the resources that the pathogen extracts from the plant for its own benefit. This intricate mode of action, described as hyperbiotrophy, causes a rapid collapse of the powdery mildew haustorium, which, in turn, leads to a premature death of the pathogen, and brings an end to the interaction.</div>
Molecular and Cellular Plant-Microbe InteractionsOralPaper3592Paper3592.mp4
Phyllosphere Microbial Assemblages: Friends, Foes, and StrangersGet me out of here: Modeling in-canopy turbulence and pathogen dispersionWalter MahaffeeUSDA
Brain Bailey: University of California, Davis; Nate Miller: University of Utah; Lucas Ulmer: University of Utah; Eric Pardyjak: University of Utah; Rob Stoll: University of Utah
<div>Understanding and predicting how microbes disseminate through the environment requires a nuanced understanding of the factors that influence microbial dispersion beyond data correlations. To this end, we are developing and evaluating the uncertainty in improved modeling tools to predict and examine spatially explicit three-dimensional (3D) canopy microclimate and turbulence. Advances in computational sciences (e.g., ray tracing algorithms, graphics processing unit computing), and biophysical modeling are used to estimate microclimate heterogeneity and its effect on microbial development, while physics-based models are used to estimate in- and above-canopy turbulence and its impacts on dispersion and deposition. These tools are being integrated into a 3D simulation environment that will be used to test hypotheses or to predict temporally evolving dispersion plumes and the probability of infection risk. Our research is showing that the complex architecture of trellised agricultural systems requires an improved understanding of how canopy heterogeneity and local orography interact with meso and microscale events to influence air turbulence and dispersion at field and regional scales.</div>
Labs, A Mechanism to Enhance Learning in the Changing World of Plant PathologyDesign and teaching of plant pathology and biology labsDoug RouseUniversity of Wisconsin-Madison
<div>Hands-on experiences that can be achieved in a teaching laboratory are an important means of learning in the biological sciences. Different kinds of laboratory experiences illustrate the range of possibilities and the kind of learning on Blume’s taxonomy that may be achieved in the laboratory. There are activities that simply allow students to see the pathogens and diseases discussed in lecture first hand, for example make a wet mount and see an oospore inside an infected root. Students may also conduct experiments designed to illustrate a concept, such as inoculate viruses onto indicator plants. Students may engage in completely open-ended inquiry activities where they get to practice the process of science and develop their own ideas about plant pathology concepts. Ultimately there is the classroom undergraduate research experience (CURE) model. All of these have their place, and a mix of different kinds of activities may be best. Nevertheless, among education researchers there is much discussion about what is best, and there is a strong theme currently to drive as far toward inquiry based activities as possible. The importance of the teaching laboratory for experiencing and learning how to work in small groups should also not be underestimated.</div>
Labs, A Mechanism to Enhance Learning in the Changing World of Plant PathologyLess Watching, More Doing: Hands-on Activities to Enhance Undergraduate CoursesBrantlee Spakes RichterUniversity of Florida
<div>Educational research has consistently reinforced the concept that we learn best by doing, rather than by watching, and results of a recent APS research survey indicate that employer satisfaction with BS-degreed employees is marred by frustration with deficiencies in hands-on skills. Yet the lecture format remains the most common mode for teaching. Lectures are efficient for delivering information to large groups, they are familiar and comfortable for both professors and students, and they require less preparation than most experiential learning modes. Classroom design and time periods often favor the lecture format, and many of us struggle to provide meaningful content interactions in short time frames, with no lab facilities, to students whose lectern-facing chairs are literally bolted to the floor. Yet, even in the most inconducive of settings, there are ways to incorporate hands-on activities. This presentation will share results of employer surveys, identifying particular skills that hiring managers and supervisors would like to see better developed among undergraduate students, and will explore ways to address these deficits and incorporate hands-on learning in non-laboratory classes.</div>
Labs, A Mechanism to Enhance Learning in the Changing World of Plant PathologyDesign and delivery of immersive online labsDavid ShewNorth Carolina State University
Alyssa Koehler: North Carolina State University; Arlene Mendoza-Moran: North Carolina State University
<div>Labs and lab-based activities are vitally important to teaching an effective undergraduate Plant Pathology course. Well designed labs build the connections between conceptual/fact-based learning and application of that knowledge to real-world scenarios. Interactive labs, whether face-to-face or online, provide needed opportunities to complete activities that allow students to achieve outcome-based learning objectives that cannot be met solely in lecture-based classrooms. To be effective, online labs must employ methods that effectively reach a diverse student population, with the goal of creating a learning experience that is as similar as possible to in-class labs. In this presentation, we will discuss how a variety of presentation styles and technologies such as interactive web platforms and gaming can be used to enrich the online lab experience for undergraduates in an Introductory Plant Pathology course. Emphasis is placed on how we help students achieve an enhanced understanding of the fundamental skills of plant pathology and experimental design.</div>
Labs, A Mechanism to Enhance Learning in the Changing World of Plant PathologyMolecular biology-based lab methods using plant nematodesKris LambertUniversity of Illinois
<div>The identification of plant parasitic nematodes in a laboratory class setting can be challenging. Often high-resolution microscopic equipment required for careful nematode observations are not available. While counter intuitive, the use of molecular biological techniques in nematode identification is often simpler, more accurate and offers students the opportunity to combine classical nematology techniques with more modern laboratory methods. By mixing a combination of “hands-on” nematode wrangling, micro-scale DNA extraction and computational analysis of nematode DNA sequences, the often tedious task of plant nematode identification can become a highly interactive experience for undergraduate plant pathology students. To successfully use molecular methods in identifying plant nematodes, access to a thermocycler and basic agarose gel electrophoresis equipment, common in many research laboratories, is necessary. Having DNA sequence files for student practice is also useful. For molecular methods to function smoothly in a plant pathology laboratory, it is critical that all methods are as streamlined as possible. Simple single-tube DNA extractions and PCR reactions are conducted by the students and then, while the thermocyclers are running, a discussion on how the data is analyzed is conducted. Students are provided with a list of web sites that host free bioinformatics tools so they can analyze sequence data and conduct homework assignments. The use of locally extracted nematodes from student-collected samples can provide a useful survey of common plant nematodes in the area and raise awareness of nematode ecology, which can feedback in to the lecture portion of the class.</div>
Biology and Disease ManagementOralPaper3597Paper3597.mp4
Unfriendly and Beneficial Plant-Parasite InteractionsInfluence of root exudates and soil on attachment of <em>Pasteuria penetrans</em> to root-knot nematode <em>Meloidogyne arenaria.</em>Chang LiuUniversity of Georgia
Patricia Timper: USDA ARS; P. Ji: University of Georgia
<div><i>Pasteuria penetrans </i>is a parasite of root-knot nematode (<i>Meloidogyne </i>spp.). Spores of <i>P. penetrans </i>attach to the cuticle of second-stage juvenile (J2) and subsequently sterilize infected females. This study looked at different factors that influence spore attachment of <i>P. penetrans</i> to <i>M. arenaria</i>. Incubating J2 with root exudates reduced spore attachment compared to incubation with phosphate-buffered saline (PBS), suggesting that root exudates altered or blocked spore recognition domains on the nematode surface. Spore attachment was equally reduced following exposure to root exudates from both host and non-host of <i>M. arenaria</i>, indicating there is common signal that affects spore attachment. Root exudates reduced spore attachment more in sterilized soil than in natural soil. Sterilization may have eliminated microbes that consume root exudates. The effect of root exudates on spore attachment was greater in sand than in clay. Clay is likely to adsorb more components from root exudates which may affect spore attachment. Results in this study provide valuable information on the interaction between <i>P. penetrans </i>and its host nematode in the root zone of plants and may help improve biological control.</div>
Biology and Disease ManagementOralPaper3631Paper3631.mp4
Addressing Nematode Parasitic Tactics Through BiologyGlobal efforts to control potato cyst nematodesLouise-Marie DandurandUniversity of Idaho
<div><span>In today’s globalized world, intensified international trade has increased the risk of an introduction of noxious pests, including the economically important potato cyst nematodes (PCN). Native to South America, PCN are some of the most specialized nematode pests in agriculture. PCN are limited in host range to potato and a few other solanaceous crops, and are well adapted to survive in soil for many years. The cyst, the remnants of the body wall of the female, contains 200-500 eggs which remain dormant until stimulated to hatch by root exudates from its host. Infestation retards plant growth, and as a result yield can be decreased by up to 80%. In the US, stringent adherence to phytosanitary programs have contained <i>Globodera rostochiensis</i> to eight counties in New York, fewer than 6,000 acres, despite its documented presence since 1941. The infestation of <i>Globodera pallida</i>, first found only in Idaho in 2006, continues to be contained to fewer than 3,000 acres which is less than 1% of the total acreage planted to potato in Idaho. Trade of potato from the US and from Idaho, originally interrupted by the detection of <i>G. pallida</i>, has resumed with Canada, Mexico and South Korea, whereas negotiations to resume export of potato from Idaho to Japan are ongoing. The use of containment and quarantine methods, plant resistance, soil fumigation, and alternatives to fumigation in the United States, and elsewhere in the world against potato cyst nematodes will be discussed. </span><p> </div>
Biology and Disease ManagementOralPaper3635Paper3635.mp4
Addressing Nematode Parasitic Tactics Through BiologyNematode parasitism genes and their manipulation of host plantsCynthia GleasonWashington State University
Lei Zhang: Washington State University; Natthanon Leelarasamee: Georg August University; Jan Utermark: Georg August University; Samer Habash: Bonn University; Abdelnaser Elashry: Bonn University
<div>In the Pacific Northwest, root-knot nematodes (<i>Meloidogyne spp</i>.) are a serious problem on potatoes. There is a critical need to reduce the use of toxic chemicals in nematode management. In order to develop the tools for engineering nematode resistance in potato, we must first understand the molecular components of plant defense and the nematode strategies used to overcome these defenses. During infection, the nematode secretes molecules called effectors. The effectors suppress plant defenses and/or alter the host physiology to enable successful nematode infections. Our lab studies root-knot nematode effectors, and we identified a novel effector called Mh265. Plants that ectopically express Mh265 were more susceptible to nematode infection. Interestingly, Arabidopsis that expressed Mh265 exhibited a suppression of elicitor-induced callose deposition. We concluded that Mh265 is secreted by the nematode in order to modulate basal plant immunity. In addition to effectors, our group is also interested in plant immune responses during nematode infections, and in particular, we have focused on the role of the defense hormone jasmonic acid (JA). Using several mutants in JA biosynthesis/perception, we were the first to find that a jasmonic acid precursor called OPDA acts as a defense-signaling molecule in the plant-nematode interaction. This result has given us insights into the mechanisms regulating plant defenses against nematodes. <p> </div>
Molecular and Cellular Plant-Microbe InteractionsOralPaper3637Paper3637.mp4
Addressing Nematode Parasitic Tactics Through Biology<span>The soybean cyst nematode: Managing a chronic disease using sustainable approaches</span>Kris LambertUniversity of Illinois
<div>The soybean cyst nematode (SCN), <i>Heterodera glycines</i>, is a damaging pest of soybean that has been spreading within the U.S. for over 60 years. The nematode is now found in all soybean producing states and causes significant yield losses each year. SCN owes its persistence to cysts that protect its eggs for many years. The economics of growing soybean do not support the use of soil-applied nematicides, but many natural SCN resistant germplasm accessions have been discovered. A diverse set of SCN resistant soybeans have been developed in academic laboratories, but the majority of commercially available SCN resistant varieties are derived from one source of resistance, PI88788. The over use of one SCN resistance mechanism has applied a strong selection pressure on SCN populations, causing an increase in nematodes that can reproduce on resistant plants, or virulent SCN. In theory, SCN could be managed more effectively if soybean with different mechanisms of resistance could be rotated. However, for rotation strategies to be effective the virulence profiles of nematode populations must be known and plants with the most effective SCN resistance must be grown. For this kind of precision agriculture to be implemented, rapid methods of measuring SCN virulence need to be deployed and new mechanisms of SCN resistance must be bred into commercial soybean varieties. New insights into understanding SCN resistance in soybean, and virulence in the nematode, promise to produce diagnostic technologies that can be used for sustainable SCN management.</div>
Biology and Disease ManagementOralPaper3638Paper3638.mp4
Phyllosphere Microbial Assemblages: Friends, Foes, and StrangersStrangers with benefits, remaking the phyllosphere through diseaseJeri BarakUniversity of Wisconsin-Madison
<div>Among the complex microbiome membership of the phyllosphere is the periodic inclusion of human enteric pathogens, such as <i>Salmonella enterica. </i>Despite the reoccurrence of <i>S. enterica</i> in the phyllosphere heralded by the numerous outbreaks of salmonellosis due to consumption of raw produce, little is known about the mechanisms which influence its persistence. <i>S. enterica</i> is non-pathogenic on plants, and populations normally decline in the phyllosphere, indicating that it is less fit on healthy plants. Bacteria frequently form alliances in the phyllosphere, resulting in enhanced fitness success. Plant pathogen infection has been shown to be a critical factor for epiphytic persistence of <i>S. enterica</i> on plants. Virulent <i>Xanthomonas perforans </i>(Xp) suppress pathogen associated molecular patterns-triggered immunity resulting in effector-triggered susceptibility which in turn increases persistence of <i>S. enterica </i>on infected leaves. Furthermore, we found that <i>S. enterica </i>could replicate on plants infected with <i>X. euvesicatoria </i>or<i> X. gardneri. </i>Thus, phytobacterial infections and the disease caused by these pathogens directly increase the risk that produce will maintain viable enteric human pathogen populations. Since our previous work ruled out any physical interaction between <i>S. enterica </i>and <i>Xanthomonas</i>, we hypothesize that physiological change to the phyllosphere during disease development influences <i>S. enterica </i>populations. Specific changes in the phyllosphere caused by a discrete set of the xanthomonads lead to <i>S. enterica </i>replication and changes in colonization sites of the human pathogen revealing an important mechanism that influences this plant pathogen – human pathogen interaction.</div>
Molecular and Cellular Plant-Microbe InteractionsOralPaper4146Paper4146.mp4
The Rise and Management Challenges of Multi-Fungicide-Resistant PathogensMultiple resistance in fungal plant pathogens, its selection schemes and impact on disease control management strategiesStefano TorrianiSyngenta Crop Protection
<div><span style="font-family:'Calibri';"><span size="3"> </span></span></p> <p><span style="font-family:'Arial';">Several fungicide classes are used to control a fungal disease. Fungicide classes inhibit independent biochemical targets and are often applied in mixtures or in spray program during the season. By sexual recombination or accumulation of independent mutations, multiple resistant individuals can be selected. Each resistance is characterized by different resistance factors, fitness, frequency and impact on field performance. Multiple resistances have been observed in various pathogens including Zymoseptoria tritici, Plasmopara viticola and Botrytis cinerea. Z. tritici evolved resistance to MBC, DMI, QoI and SDHI fungicides, but also might express weak multidrug characteristics. P. viticola developed resistance to PA, QoI, CAA and cymoxanil. B. cinerea showed targeted and multidrug resistance towards a range of fungicide classes. Whereas frequency of single resistance is high, the combined resistance might remain at lower frequencies and can be managed through appropriate spray applications and good agronomic practices. Multiple fungicide resistance can follow different evolutionary directions in response to the selection imposed by the spray strategies (alternation/mixtures), the number of registered products, the characteristics of each fungicide and the putative contribution of multidrug resistance. A better understanding of how multiple resistances evolve is beneficial to frame the impact to disease control and to propose sound new anti-resistance strategies.</span></p> <p><span style="font-family:'Calibri';"><span size="3"> </span></span></p> <p><span size="3"> </span></div>
Biology and Disease ManagementOralPaper4153Paper4153.mp4
Biological ControlBioplastic seed coating formulations combining pesticides with biocontrol isolates to control agricultural pestsHamed AbbasUSDA ARS BCPRU
Cesare Accinelli: University of Bologna; W. Thomas Shier: University of Minnesota, College of Pharmacy
<div>Film-coating of agronomic seeds is a rapidly developing technology that generally uses petroleum-derived polymers and synthetic pesticides. However, serious consideration has recently been given to using renewable source polymers and reducing use of synthetic pesticides. We evaluated the feasibility of film-coating agronomic seeds with a starch-based bioplastic formulation, and combining conventional pesticides with beneficial microorganisms. Corn and canola seeds were film-coated with bioplastic formulations containing the insecticide imidacloprid, the fungicide metalaxyl-M, the growth promoting fungus <i>Trichoderma harzianum</i> or a non-aflatoxigenic biocontrol isolate of the fungus <i>Aspergillus flavus</i>. Bioplastic coating did not affect germination of either seed type <i>in vitro</i> or in soil. Bioplastic film-coating reduced dust-off from coated seeds by up to 95% compared to coating with a commercial polymer. This is important, particularly when neonicotinoid insecticides are included. When spores of the biocontrol fungus, <i>T. harzianum</i>, were incorporated into bioplastic coatings, growth of corn and canola seedlings was significantly stimulated (i.e., shoot and root lengths of corn seedlings were 29% and 44% longer, than in uncoated seeds, respectively; similar results were observed with canola). No growth stimulatory effects were observed with bioplastic coatings containing the biocontrol <i>A. flavus</i> isolate. Preliminary field studies indicated that coating corn seeds with bioplastic and spores of non-aflatoxigenic <i>A. flavus</i> biocontrol isolate significantly reduced aflatoxin contamination in harvested seed. More extensive studies are in progress in corn growing areas of USA and Southern Europe.</div>
Biology and Disease ManagementOralPaper4214Paper4214.mp4
New Insights into NLR on Plant Immunity: Pathogen Recognitions, Molecular Interactions, and Novel Disease Control StrategiesUsing decoys to expand the recognition specificity of a plant disease resistance proteinMatthew HelmIndiana University
Roger Innes: Indiana University
<div>Genetic-based disease resistance is the most effective and environmentally sustainable approach to protecting crops from disease. Although significant progress has been made with respect to understanding the mechanistic basis of plant-pathogen interactions, a remaining challenge is to expand the recognition specificity of plant resistance (<i>R</i>) proteins to confer entirely new specificities. We recently reported a novel approach for expanding the recognition specificity of an <i>Arabidopsis R </i>protein by generating ‘decoy’ proteins that function as substrates for pathogen proteases. This strategy is based on the observation that modifying the protease cleavage site within <i>Arabidopsis</i> PBS1, a substrate of the AvrPphB cysteine protease, expands the recognition specificity of the <i>Arabidopsis</i> RPS5 immune response pathway. Thus, the specificity of RPS5 can be changed simply by altering the protease cleavage sequence within PBS1. We are now using this technology to engineer resistance in soybean (<i>Glycine max</i>) to <i>Soybean Mosaic Virus</i> (SMV) based on the recognition of the viral protease by a PBS1 ‘decoy’ protein. Soybean encodes three co-orthologs of PBS1, and all three can be cleaved by AvrPphB. Furthermore, we show soybean recognizes AvrPphB, thus soybean likely contains an <i>R</i> protein that detects PBS1 cleavage. We hypothesize that we will be able to engineer resistance to SMV by introducing a cleavage site recognized by the SMV protease into a soybean PBS1 ortholog.</div>
Molecular and Cellular Plant-Microbe InteractionsOralPaper4228Paper4228.mp4
Floral and Fruit DiseasesDeciphering blueberry and cranberry floral involvement in the disease cycle of <i>Colletotrichum fioriniae</i>Timothy WallerRutgers University
Joshua Gager: Rutgers University; Peter Oudemans: Rutgers University; Thomas Gianfagna: Rutgers University
<div><i>Colletotrichum fioriniae</i> is a hemibiotrophic, fruit-rotting pathogen that limits blueberry (BB) and cranberry (CB) production. Since fungicide applications during bloom are most effective, the link between floral tissue and infection structure formation was investigated. Floral components influence both production of inoculum (secondary conidiation) and formation of infection structures (appressoria). Effects of flower tissue bioactivity on <i>C. fioriniae </i>were tested using two distinct extraction methods: water (W), to simulate mobilization of floral compounds in the field, and chloroform (C), to evaluate nonpolar cuticle compounds. W and C floral extracts significantly increased the rate (200%) and magnitude (500%) of appressorium formation and W extracts increased secondary conidiation (500%) over controls. Extracts of susceptible BB cultivars significantly increased appressorial stimulation compared to resistant cultivars, however, this was not observed in CB. Floral rainwater runoff stimulated significantly more secondary conidiation and appressorium formation compared to controls, demonstrating the presence of this phenomenon in the field. Analysis of CH extract of CB flowers and pure chemical assays showed methyl ester of hexadecanoic acid (HEX) as the most stimulatory compound characterized. Utilizing this, HEX was field-tested with fungicides as a ‘trap’ spray, synchronizing fungal germination to fungicide applications with moderate early season control.</div>
Biology and Disease ManagementOralPaper4334Paper4334.mp4
Pathogen Detection and DiversityExtracting Genomic DNA from Root-Knot Nematode Infested Soil using Fe<sub>3</sub>O<sub>4</sub> Super Paramagnetic NanoparticlesAdrienne GornyCornell University, Plant Pathology & Plant Microbe Biology Section
Xiaohong Wang: USDA-ARS, Robert W. Holley Center for Agriculture and Health; Xiaohong Wang: Cornell University, School of Integrative Plant Science; Sarah Pethybridge: Cornell University, Plant Pathology & Plant Microbe Biology Section
<div>Quantifying the level of plant-parasitic nematodes in field soil prior to planting using a DNA-based soil test could result in a more accurate estimation of crop damage or yield loss observed at harvest. Incorporation of this information into management decisions may promote conservative use or targeted application of pesticides, reducing the environmental impact and cost of production. The isolation of DNA from soil is a critical first step in molecular quantification, and standard techniques for extracting DNA from soil include phenol-chloroform based methods and commercial kits. However, these are often time consuming, produce hazardous waste, are cost prohibitive, and may only be designed for isolating DNA from small volumes of soil. The variability in pathogen population density estimations may be greatly reduced when larger volumes of soil are analyzed. Recently, the use of super paramagnetic nanoparticles has been successful in genomic DNA isolation, providing a quicker, easier, and more economical protocol than traditional kit or phenol-chloroform based methods. Here, a method using Fe<sub>3</sub>O<sub>4</sub> super paramagnetic nanoparticles for the isolation of genomic DNA from soil was investigated for its utility in quantification of plant-parasitic nematodes directly from soil. DNA was isolated from 100 g of soil inoculated with a known quantity of <i>Meloidogyne hapla</i> (root-knot nematode). The method was optimized, and several permutations assaying the volume of nanoparticles used and number of nematodes in the soil were trialed. Resultant DNA was assessed for quantity and quality using spectroscopy, fluorometry, and PCR technologies. Trade-offs between parameter optimization and DNA quality and quantity are discussed.</div>
Show Me the Money! Assessing the Value of Disease Control in a Changing LandscapeLosses, regrets and expectations: an overview of attaching value to disease management decisionsNeil McRobertsUniversity of California-Davis
<div>While not being entirely neglected by plant pathologists, economics has not so far been incorporated into our discipline as comprehensively as it has in cognate disciplines such as weed science and entomology; there is no equivalent in plant pathology of the Journal of Economic Entomology. At an applied level there is, of course, a long-standing interest in comparing different crop mangement approaches using cot-benefit and cost effectiveness approaches. However, if we consider the concept of ecnomics in its wider sense as the study of decision making under constraints it is clear that a more general economic theory of plant disease management has been slow to develop. We consider recent interest in decision theory, information theory and game theory within plant pathology and review the prospects for these concepts to contribute to the development of a more clearly defined economic framework for plant disease management.</div>
Show Me the Money! Assessing the Value of Disease Control in a Changing LandscapeRisk analysis and economic optimization of late blight management tacticsIan SmallUniversity of Florida
Yangxuan Liu: Dept. of Agriculture, Eastern Kentucky University; Michael Langemeier: Dept. of Ag. Economics, Purdue University; Laura Joseph: Cornell University; William Fry: Cornell University
<div>Calendar-based fungicide application schedules are often employed to manage late blight regardless of existing disease severity, disease-resistance level of the potato cultivar, or prevailing weather. Such strategies may not be economically or environmentally efficient. BlightPro decision support system (DSS)-based fungicide application schedules are influenced by prevailing weather and host resistance. Objectives of this study were to assess the economic value of information created by the DSS and to optimize forecasting rules in the DSS for disease suppression and profitability. Three fungicide scheduling strategies were evaluated: calendar-based strategy, DSS-based strategy, and unsprayed. Using results from simulation experiments for several locations in the United States, we constructed distributions of the net return to all costs excluding fungicide cost and application cost (net return per acre) for calendar-based and DSS-based strategies at each location. These distributions were then compared using risk management methods: stochastic dominance with respect to a function and stochastic efficiency with respect to a function. The DSS-based strategy was identified as the most effective approach in terms of disease suppression, net return per acre, and risk-adjusted net return. The value of the information created by the DSS varied by cultivar resistance, producers’ risk-aversion level, and production location.</div>
Phenotyping and Population DynamicsDifferences in <i>Fusarium </i>species in <i>brown midrib </i>sorghum and in air populations in production fieldsDeanna Funnell-HarrisUSDA-ARS
Erin Scully: USDA-ARS; Scott Sattler: USDA-ARS; Patrick O'Neill: USDA-ARS
<div>Several <i>Fusarium </i>species cause sorghum [<i>Sorghum bicolor </i>(L.) Moench] grain mold, resulting in deterioration and mycotoxin production in the field and during storage. Fungal isolates from air (2005-2006), and from leaves and grain from wild-type and <i>brown midrib </i>(<i>bmr</i>)-<i>6 </i>and <i>12 </i>sorghum plants (2002-2003) at two locations were collected. Plants containing <i>bmr </i>have reduced lignin content, altered cell wall composition and different levels of phenolic intermediates, compared with wild-type. Multilocus maximum likelihood analysis identified two <i>Fusarium thapsinum</i> operational taxonomic units (OTUs). One OTU was identified at greater frequency in grain and leaves of <i>bmr </i>and wild-type plants, but was infrequently detected in air. Nine <i>Fusarium graminearum/Gibberella zeae</i> species complex OTUs were identified: one was detected at low levels in grain and leaves while the rest were only detected in air. Wright’s <i>F-</i>statistic (<i>F<sub>ST</sub></i>) indicated that fungal air populations differentiated between locations during crop anthesis, but did not differ during vegetative growth, grain development and maturity. <i>F<sub>ST </sub></i>also indicated that the <i>Fusarium</i> population from wild-type grain was differentiated from those in either <i>bmr6</i> or <i>bmr12</i> grain at the first location but at the second location, populations from wild-type and <i>bmr6</i> grain were more similar. Thus, impairing monolignol biosynthesis in <i>bmr </i>plants substantially effected <i>Fusarium </i>populations but the environment also had a strong influence.</div>
Virology Detection and DescriptionsInvestigating the longevity and host range of <i>Cucumber green mottle mosaic virus</i> in the Northern Territory, AustraliaDavid LovelockDepartment of Primary Industry and Resources
Nadine Kurz: Department of Primary Industry and Resources; Sharl Mintoff: Department of Primary Industry and Resources; Merran Neilsen: Department of Primary Industry and Resources; Lucy Tran-Nguyen: Department of Primary Industry and Resources
<div><i>Cucumber green mottle mosaic virus</i> (CGMMV) is a tobamovirus that is able to infect a number of economically important cucurbits crops. The first detection of CGMMV in Australia was reported in the Northern Territory (NT) in September 2014; 26 properties in the NT have now been identified as CGMMV positive. As of February 2017, CGMMV has also been detected in Queensland and Western Australia. Initially all infested properties (IP) in the NT were placed under quarantine for a period of two years, where all host plants including cucurbitaceae weeds were destroyed. Previous research had indicated CGMMV was viable for up to 9 months in host free soils. Four IP with varying soil types and climates in the NT were chosen to determine the longevity of the virus starting at 12 months into quarantine, using watermelon and cucumber. At 12, 15 and 18 months of the quarantine period, 80 soil samples were collected from each IP, with plant bioassays conducted to determine the viability of any remaining virus. A field trial was also conducted on all four IP at the 12 month soil sampling point. At the initial test period of 12 months, 2 IP from the bioassay and 1 IP from the field trial tested positive for CGMMV, while all 4 IP tested positive in the 15 month bioassays. Because of this discovery, the status of other non-cucurbit commercial crops and selected weeds is being tested to determine the risk of transmission of CGMMV in a wider range of plant host.</div>
Diseases of PlantsOralPaper4469Paper4469.mp4
OomycetesGrowth medium affects physiology and virulence of <em>Phytophthora infestans</em> sporangia.<em> </em>William FryCornell University
Sean Patev: Clark University
<div>Our lab has observed that the medium on which <em>P. infestans</em> is grown can strongly influence the results of experiments with sporangia produced on those media. The first observation was that sporangia produced from pure cultures (culture sporangia) on agar (Rye B or Pea) medium were more sensitive to solar radiation than were sporangia produced on leaflet lesions (leaflet sporangia). The second observation was that arachidonic acid induced resistance in potatoes to culture sporangia, but not to leaflet sporangia. To quantify the effect of “source” of sporangia on the virulence of those sporangia, we conducted inoculation studies in the field and in moist chambers. Leaflet sporangia produced three- to twenty-times as much disease per sporangium as did culture sporangia. In order to develop hypotheses concerning mechanism, gene expression in both types of sporangia was assessed. The ratio of expression of an RxLR effector to expression of INF1 differed more than 50-fold in the two types of sporangia. The ratio declined gradually as an isolate was obtained from leaflets and then passaged on agar medium. Thus, we conclude that for <em>P. infestans,</em> leaflet sporangia are physiologically and pathogenically different from culture sporangia, and leaflet sporangia should be used for the most accurate assessment of host-pathogen interactions under natural/agricultural conditions.</div>
Molecular and Cellular Plant-Microbe InteractionsOralPaper4489Paper4489.mp4
Plant Pathologists of the Future: Showcasing the Top Graduate Students from APS Division MeetingsEffects of fluopyram on soybean cyst nematode resistance management under greenhouse conditionsKyle BroderickUniversity of Nebraska-Lincoln
Carol Picinini: Univeristy of Sao Paulo - Escola Superior de Agricultura Luiz de Queiroz; Loren Giesler: Univ of Nebraska-Lincoln
<div>Soybean cyst nematode (SCN, <i>Heterodera glycines</i>)<i> </i>is the most economically important soybean pathogen in the United States. Host resistance effectively controls SCN, however populations virulent to PI 88788 resistance are evolving, warranting additional controls options. The objectives of this study were: 1) to determine the effects of nematode seed treatments on SCN reproduction on different SCN resistance sources, and 2) to determine if seed treatments combined with host resistance could slow development of virulence to host resistance sources. Four seed treatments (clothianidin + <i>Bacillus firmus</i>, fluopyram, clothianidin + <i>B. firmus</i> with fluopyram, and a nontreated control) were compared on two resistant sources, PI 88788 and Peking. Treatments received a base seed treatment of penflufen, prothioconazole, and metalaxyl. Using a population virulent to PI 88788, eggs were serially passed for 3 generations on the same treatment and then a modified SCN-type tests was performed on each SCN population. Fluopyram significantly decreased SCN reproduction but there was not a significant difference between resistant sources (p=0.05). Fluopyram treatments significantly slowed development of virulence to PI 88788 (p=0.10), but this was not observed with virulence to Peking. These results suggest that seed treatments containing fluopyram could have additional benefits in the management of SCN resistance when paired with resistant varieties. <p> </div>
Biology and Disease ManagementOralPaper4492Paper4492.mp4
Plant Pathologists of the Future: Showcasing the Top Graduate Students from APS Division Meetings<span>Etiology and Management of Sour Rot in Grapes</span>Megan HallCornell University
Gregory Loeb: Cornell Univ; Wayne Wilcox: Cornell Univ
<div><span face="Calibri" style="font-family:Calibri;"><span size="3">Sour rot is a disease complex affecting grape growers and winemakers worldwide, characterized by the oxidation of the berry skin, internal development of acetic acid and partnered with the presence of <i>Drosophila </i></span><span size="3">fruit flies. We have shown that acetic acid production requires yeast to produce ethanol and bacteria to subsequently convert the ethanol to acetic acid through an oxidation reaction. We have characterized the microorganisms involved, determining that several yeast species (</span><i><span size="3">Saccharomyces </span></i><span size="3">spp., </span><i><span size="3">Hanseniaspora </span></i><span size="3">spp., and </span><i><span size="3">Pichia </span></i><span size="3">spp.) partnered with acetic acid bacteria (</span><i><span size="3">Gluconobacter </span></i><span size="3">spp. and </span><i><span size="3">Acetobacter </span></i><span size="3">spp.) can cause this reaction, but only in the presence of </span><i><span size="3">Drosophila </span></i><span size="3">spp. To investigate potential non-microbial contributions of </span><i><span size="3">Drosophila </span></i><span size="3">spp. to the complex, we produced axenic </span><i><span size="3">D. melanogaster </span></i><span size="3">eggs and reared larvae until axenic conditions, yielding adults devoid of gut or surface microbiota. Wounded berries exposed to the wild type flies for 8 days produced typical sour rot symptoms, but in order for symptom development to occur in those in the presence of axenic flies, co-inoculation with a yeast and acetic acid bacteria was required. In field trials conducted on cv. 'Vignoles' in 2013-16, both insecticide and antimicrobial treatments significantly reduced sour rot development. In 2015, untreated vines averaged 20.5% sour rot severity, and this was reduced by 73-81% on vines treated prophylactically post-veraison with weekly sprays containing a combination of the insecticide zeta-cypermethrin plus the antimicrobial potassium metabisulfite or hydrogen dioxide; severity was reduced by 49% on vines receiving only insecticide sprays. These trials further support the hypothesis that sour rot results from a complex of yeast, bacteria, and </span><i><span size="3">Drosophila</span></i><span size="3"> spp. and that targeting these organisms can provide significant levels of control.</span></span></div>
Diseases of PlantsOralPaper4493Paper4493.mp4
Pursuit of Solutions to Mycotoxin Risks by Next-Generation Plant Pathologists<em>Aspergillus flavus</em> functional genomics: Toward enhancing host resistance to aflatoxin contamination under drought using biotechnologyJake FountainUniversity of Georgia
Gaurav Agarwal: University of Georgia; Prasad Bajaj: International Crop Research Institute for the Semi-Arid Tropics; Manish Pandey: International Crop Research Institute for the Semi-Arid Tropics; Spurthi Nayak: University of Agricultural Sciences, Dharwad; Robert Kemerait: University of Georgia - Department of Plant Pathology; Rajeev Varshney: International Crop Research Institute for the Semi-Arid Tropics; Baozhu Guo: USDA ARS CPMRU
<div>The contamination of maize and peanut with aflatoxin during <i>Aspergillus flavus</i> infection is exacerbated by drought stress. This is correlated with the accumulation of reactive oxygen species (ROS) in host tissues. These ROS also stimulate the production of aflatoxin by <i>A. flavus</i>, which is postulated to provide fringe antioxidant benefits. In order to investigate the functional causes for isolate-to-isolate variation in oxidative stress responses and to characterize components pertinent to host resistance, 10 field isolates of <i>A. flavus</i> and <i>A. parasiticus</i> were used for whole genome re-sequencing (WGRS). Sequencing reads were aligned to the NRRL3357 reference genome with an average of 86.6X coverage for each isolate. Variant calling between the re-sequenced isolates and the reference genome found that toxigenic isolates exhibited fewer non-synonymous single nucleotide polymorphisms (SNPs) than atoxigenic isolates with averages of 22,601 and 34,294, respectively. Also, greater numbers of non-synonymous SNPs were observed than synonymous SNPs suggesting possible niche specialization in progress proportional to observed stress tolerance. Gene family variant enrichment analysis and variant influences on gene expression are under investigation. Understanding the factors influencing <i>A. flavus</i> stress responses and aflatoxin production will allow for targeted enhancement of host resistance through breeding, and the application of novel biotechnologies.</div>
Molecular and Cellular Plant-Microbe InteractionsOralPaper4575Paper4575.mp4
Fungicide Resitance and FitnessFitness, Competitive Ability and Mutation Stability of Strawberry <em>Colletotrichum acutatum</em> Isolates Resistant to Quinone-outside Inhibitor FungicidesBruna ForceliniUniversity of Florida
Natalia Peres: Gulf Coast Research and Education Center; University of Florida; Carolina Rebello: Universidade Estadual Paulista
<div>Quinone-outside Inhibitor (QoI) fungicides, are used to manage anthracnose of strawberry, caused by <i>Colletotrichum acutatum</i>. However, selection for <i>C. acutatum</i> resistance to QoI fungicides was first reported in 2013 in Florida, and subsequently in strawberry nurseries and production areas across the USA and Canada. <i>C. acutatum</i> resistance is due to the G143A point mutation in the cytochrome <i>b</i> gene. This mutation is known to cause complete resistance even at high rates of QoIs. In this study, we investigated the relative fitness and competitive ability of QoI-resistant and sensitive <i>C. acutatum</i> isolates. A fitness comparison did not indicate any difference between resistant and sensitive isolates in mycelial growth at different temperatures or on hypertonic media. However, when different ratios of resistant and sensitive isolates were inoculated on detached strawberry fruit, production of conidia by the resistant isolates increased in relation to sensitive isolates. Cultivation of QoI-resistant and QoI-sensitive isolates for four culture cycles <i>in vitro</i> in the absence of azoxystrobin showed that QoI resistance was stable. The observed competitive advantage and stability of the G143A mutation in QoI-resistant <i>C. acutatum</i> populations suggest that the interruption and further reintroduction of QoI fungicides might not be an option for strawberry nurseries and fruit production areas.</div>
Biology and Disease ManagementOralPaper4590Paper4590.mp4
Fungicide Resitance and FitnessSensitivity and resistant management for the SDHI fungicide fluxapyroxad in the apple scab pathogen <i>Venturia inaequalis</i>Katrin AyerCornell University
Kerik Cox: Cornell University; Sara Villani: North Carolina State University
<div>The recent registration of the SDHI fungicide fluxapyroxad for apple scab management prompted investigations into the sensitivity of <i>Venturia</i> <i>inaequalis </i>and fungicide application practices. Baseline isolates of <i>V. inaequalis </i>were used to determine the effective concentration of fluxapyroxad at which conidial germination and mycelial growth was inhibited by 50% (EC<sub>50</sub>). Mean EC<sub>50</sub> values for fluxapyroxad conidial and mycelial growth were 0.028 and 0.25 μg ml<sup>-1</sup>, respectively. Using a draft genome of <i>V. inaequalis</i>, the <i>sdhC</i> and <i>sdhD </i>genes were identified and characterized to compliment the previously characterized <i>sdhB </i>gene. Translated proteins had > 60% identity with <i>sdhC</i> and <i>sdhD </i>genes from other fungi. This baseline sensitivity and genetic information will serve as the foundation for phenotyping and genotyping SDHI sensitivity in populations<i> </i>of <i>V. inaequalis</i>. To evaluate fungicide application practices for resistance management, field experiments at two research orchards using commercial fluxapyroxad products (Sercadis® and Merivon®) were performed using both low and high application rates as well as a combination with single-site with multi-site fungicides. In the first year, few significant differences were observed between treatments at both locations. However, plots left untreated and those treated with Merivon had populations with the highest sensitivity to fluxapyroxad. Repeated experiments in subsequent years may lead to an improved evaluation of application practices for resistance management.</div>
Biology and Disease ManagementOralPaper4600Paper4600.mp4
Fungal DiseasesEmerging plant pathogens on ornamental crops in south FloridaGeorgina Sanahuja SolsonaTropical REC
Patricia Lopez: University of Florida; Stephanie Suarez: University of Florida; E. Vanessa Campoverde: University of Florida Extension Miami-Dade; Edward Evans: University of Florida - Tropical Research and Education Center; Aaron Palmateer: Bayer Environmental Science
<div>The state of Florida is geographically at risk for the introduction of new plant pathogens and insect pests due to it’s location, subtropical climate, and agro tourism industry. Florida’s hot and humid climate is highly favorable for newly introduced plant pathogens to become established and the movement of plant material through the ornamental industry creates an effective means for long distance dispersal. The University of Florida’s Extension Plant Diagnostic Clinic has reported six new ornamental diseases in 2016. Accurate identification and characterization of new plant pathogen introductions and the diseases they cause is the first and most important step in successful and effective disease management. This presentation provides examples of several newly emerging diseases affecting plants in the Florida landscape and in commercial production. One fungal plant pathogen, <i>Bipolaris oryzae</i> first reported causing brown spot disease on rice leaves in the United States has recently been reported on two new ornamental hosts plants including a popular bromeliad hybrid, <i>Aechmea tayoensis </i>and a tropical foliage plant, <i>Strelitzia nicolai</i>.<i> </i>Notably, glyceraldehyde-3-phosphate dehydrogenase (GPD) sequences extracted from <i>B. oryzae </i>isolates causing disease on <i>A. tayoensis </i>and <i>S. nicolai </i>were a 99% match with an isolate of <i>B. oryzae </i>previously reported causing disease on <i>Panicum virgatum </i>in New York. This is an excellent example of the potential for fungal pathogens to infect new plant host species.</div>
Diseases of PlantsOralPaper4656Paper4656.mp4
Fungicide ResitanceManaging an oomycete community: fungicide sensitivity and evolution of resistance to ethaboxamZachary NoelMichigan State University
Alejandro Rojas: Duke University; Janette Jacobs: Michigan State University; Martin Chilvers: Michigan State University
<div>Seedling diseases caused by oomycetes pose a significant threat to soybean production. In 2011 and 2012, over 80 oomycete species were found to be associated with soybean seedlings. Over half of those species were found to be pathogenic. Evaluation of these species for fungicide sensitivity is important for management. Fungicide amended medium assays are slow, labor intensive and expensive. A high-throughput assay to evaluate fungicide sensitivity of many oomycete isolates at once was developed using optical density measurements of macerated mycelial fragments. Z’-factor was used as a quality control statistic. The assay was utilized to evaluate the sensitivity of 81 oomycete species to mefenoxam and ethaboxam. Of the isolates tested, 87.5% had an EC<sub>50 </sub>< 1 μg ml<sup>-1 </sup>and only one <i>Phytopythium </i>isolate had an EC<sub>50 </sub>> 10 μg ml<sup>-</sup>1 mefenoxam. For ethaboxam, 61.7% of isolates tested had an EC<sub>50 </sub>< 1 μg ml<sup>-1</sup>, whereas, species within <i>Pythium</i> clades A, B and E had EC<sub>50</sub> ≥ 20 μg ml<sup>-1</sup> ethaboxam. This suggested that reduced sensitivity to ethaboxam may be inherent and possibly related phylogenetically. Therefore, we investigated the evolutionary history and mechanism of resistance to ethaboxam. Phylogenies indicated that species with reduced sensitivity to ethaboxam followed a convergent evolutionary pattern and had evolved three separate times. Two different transversion mutations lead to the same amino acid change in the target gene of lineages with reduced sensitivity to ethaboxam.</div>
Biology and Disease ManagementOralPaper4668Paper4668.mp4
MycotoxinsDescription of a novel aflatoxin-producing <i>Aspergillus </i>species from a region of the United States with perennial aflatoxin contaminationPummi SinghUniversity of Arizona
Peter Cotty: USDA-ARS, University of Arizona
<div>Aflatoxins are potent carcinogens that contaminate a wide range of crops in warm regions, and are produced by several species in <i>Aspergillus </i>section <i>Flavi. </i>To completely describe the etiology of aflatoxin contamination<i>, </i>aflatoxin-producing species must be identified and characterized. During the course of molecular phylogenetic analysis of aflatoxin-producing fungi native to North America, a novel aflatoxin-producing species was identified. The new taxon was assigned the name <i>Aspergillus texensis</i>, P. Singh and P.J. Cotty, species novum. The new taxon has sclerotia production not differentiable from the <i>A. flavus </i>S strain morphotype<i>. </i>However<i>, A. texensis</i> produces both B and G aflatoxins whereas <i>A. flavus</i> produces only B aflatoxins. Total aflatoxins produced by isolates<i> of A. texensis</i> on maize did not differ (p > 0.05; mean = 172 ± 9.5 mg/kg) from quantities produced by the <i>A. flavus </i>S strain. <i>Aspergillus texensis</i> formed a highly supported monophyletic clade in phylogenies based on concatenated partial gene sequences of the beta-tubulin (0.5 kb), calmodulin (1.1 kb), and nitrate reductase (2.1 kb) genes. <i>Aspergillus texensis</i> is known from eleven isolates, nine from soils cropped to maize in Texas, where aflatoxin contamination of crops is a perennial issue, and one isolate each from maize grown in Arkansas and Louisiana. This is the first report of the occurrence in the United States of an S morphotype <i>Aspergillus </i>with ability to produce both B and G aflatoxins.</div>
Biology and Disease ManagementOralPaper4670Paper4670.mp4
OomycetesIdentification and characterization of <i>Phytophthora </i>isolates from citrus orchards of South TexasShima ChaudharyTexas A&M University
Veronica Ancona: Texas A&M Kingsville Citrus Center; Christopher Barbola: Texas A&M University-Kingsville, Citrus Center
<div>Foot rot and root rot caused by <i>Phytophthora</i> species are prevalent diseases in commercial citrus orchards of South Texas. There is a need for comprehensive study to identify and characterize <i>Phytophthora</i> isolates in the orchards. Ninety four isolates, were collected from soil and infected roots from 30 citrus orchards located across the three citrus producing counties in the Lower Rio Grande Valley of Texas. ITS sequence generated based on primers ITS4 and ITS6, exhibit 98 to 100% identity to <i>Phytophthora nicotianae </i>for all the isolates tested. This was also supported by morphological characteristics such as colony motif, sporangium and oogonium shape and size. Thirty Isolates were tested for mating type with known A1 and A2, <i>P.nicotianae</i> testers. Twenty two and eight isolates were identified as A2 and A1 mating type respectively. Thirty isolates of <i>P. nicotianae</i> were also screened for sensitivity to mefenoxam on 10% clarified V8 agar at 1 a.i.µg mL <sup>–1</sup>. Six isolates were inhibited less than 50%, twenty-two isolates were inhibited 50% to 90% and two isolates were inhibited more than 90% and were marked as insensitive, intermediate sensitive and sensitive respectively. We conclude from this study that prevalent species in the commercial citrus groves is <i>P.nicotianae</i> and most of the isolates tested, are intermediate or insensitive to mefenoxam at 1 a.i.µg mL <sup>–1</sup>. Occurrnce of both mating types in the orchards, can have an implication for the management of the <i>Phytophthora </i>diseases in the region.</div>
Biology and Disease ManagementOralPaper4709Paper4709.mp4
Fungicide Resitance and FitnessHigh levels of fludioxonil resistance in <i>Botrytis fragariae </i>and investigation of potential resistance mechanismsMengjun HuClemson University
Guido Schnabel: Clemson University
<div>Fludioxonil, a phenylpyrrole, is a fungicide commonly used for control of gray mold of strawberry caused by <i>Botrytis spp</i>., including <i>B. cinerea</i> and <i>B. fragariae</i>. In contrast to other site-specific fungicides, resistance frequencies have been comparably low in strawberry fields in the eastern United States. Therefore, the fungicide has been used extensively in rotational programs in recent years. In 2016, <i>B. fragariae</i> isolates were collected from strawberry fields in the east coast. They displayed MIC values greater than 100 µg/ml. In contrast, previously reported MIC values of <i>B. fragariae </i>from Germany ranged from 0.5 to 3.3 µg/ml. Resistance to fludioxonil in <i>B. cinerea</i> from eastern strawberry fields is conferred by mutations in the <i>mrr1</i> gene, leading to enhanced expression of the ATP-binding cassette (ABC) transporter gene <i>atrB</i>. Analyses of <i>mrr1</i> sequences indicated no variation in nucleotide sequence compared to sensitive isolates, but real-time gene expression analysis revealed the <i>atrB</i> gene was constitutional higher expressed in resistant <i>B. fragariae </i>isolates compared to sensitive isolates. Relative <i>atrB</i> copy numbers were identical in resistant and sensitive isolates indicating a lack of gene duplication. Nucleotide sequence analysis of <i>Bos1</i>, the osmosensing histidine kinase, did not reveal variations between sensitive and resistant isolates as well. In lab mutants and more recently in field isolates from China, mutations in this gene have been reported to be associated with resistance to dicarboxamides and phenylpyrroles. The high levels of fludioxonil resistance in <i>B. fragariae</i> isolates may pose a selective advantage over <i>B. cinerea </i>in commercial strawberry fields. <p> </div>
Biology and Disease ManagementOralPaper4711Paper4711.mp4
Biological ControlIntegration of biological control and transgenic insect protection for mitigation of mycotoxins in cornMark WeaverUSDA ARS
Hamed Abbas: USDA ARS BCPRU; Michael Brewer: Texas AgriLife Research and Extension Center-Corpus Christi; Luke Pruter: Texas A&M AgriLife Research and Department of Entomology; Nathan Little: Southern Insect Management Research Unit USDA-ARS
<div>Field trials were conducted over two years in two states (MS and TX) to examine the efficacy of biological control of aflatoxin in corn and the interactions with other mycotoxins and transgenic insect protection. Corn hybrid N78N3111, expressing the Cry 1Ab, Cry3A and Vip3Aa20 insecticidal proteins, was nearly 100 percent free from corn earworm damage and generally≥ 50% as much fumonisin contamination as compared to N78NGT, a near isogenic corn hybrid without insect protection. This insect protection, however, did not significantly prevent aflatoxin contamination. Soil application of non-aflatoxigenic biocontrol strains of <i>Aspergillus flavus</i> significantly reduced aflatoxin concentrations in corn. Biocontrol strain 21882 of <i>A. flavus</i> was especially effective, reducing aflatoxin contamination by about 90 percent over the seven field trials. There was no significant interaction between the insect protection and biocontrol treatments. Although no synergies were detected, the reduction of mycotoxins by both strategies supports application of both strategies in tandem. Economic factors external to the cost of the technologies will be a major determinant if the mycotoxin mitigation attained by use of these technologies will have a positive economic benefit.</div>
Biology and Disease ManagementOralPaper4771Paper4771.mp4
Bacterial Virulence and EffectorsDynamic expression of T3SS genes in single cells of <i>Dickeya dadantii</i> during the interaction with potatoZhouqi CuiDepartment of Plant Pathology & Ecology, The Connecticut Agricultural Experiment Station
Quan Zeng: Department of Plant Pathology & Ecology, The Connecticut Agricultural Experiment Station; Ching-Hong Yang: Department of Biological Sciences, University of Wisconsin-Milwaukee; Xiaochen Yuan: Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences
<div><i>Dickeya dadantii </i>is a bacterial pathogen and causal agent of soft rot on potato and other tuber producing plants. The type III secretion system (T3SS), which induces plant cell death, is an important virulence factor in <i>D. dadantii</i>. The expression of T3SS in is induced in plant apoplast or in Hrp-inducing minimal medium (Hrp-MM). Despite the understanding of induction conditions, how individual cells in a bacterial<i> </i>population respond to the induction signal is not well understood. Our previous studies revealed that when cultured in Hrp-MM, <i>D. dadantii </i>express the T3SS genes in a bi-stable manner: only a small proportion of cells highly expresses T3SS genes, whereas the rest of the population does not express these genes. In this study, we further characterized the T3SS gene expression in single cells of <i>D. dadantii </i>under <i>in vivo </i>conditions, using potato as a model host. Using a dual fluorescence reporter and single-cell techniques, we proved that the expression of T3SS genes in <i>D. dadantii</i> is also bistable in potato apoplast. Furthermore, we characterized the dynamics of “T3SS On” and “T3SS Off” populations at different infection stages and locations. More cells are in the “T3SS On” population at the initial infection stage than in the late infection stage in potato leaves, and more cells are in the “T3SS On” population when inoculated in stems than in leaves. No “T3SS On” cells were observed in tuber. These results implicate that the T3SS bistability might be important for <i>D. dadantii </i>to adapt the hostile host environment.</div>
Molecular and Cellular Plant-Microbe InteractionsOralPaper4834Paper4834.mp4
Viral-Host InteractionsCharacterization of a symptom determinant of grapevine fanleaf virus<i> </i>suggests a novel function for its RNA-dependent RNA polymeraseLarissa OsterbaanCornell University
Marc Fuchs: Cornell University
<div>Fanleaf degeneration is one the most devastating viral diseases of grapevine. It is caused by a number of viruses of the genus <i>Nepovirus </i>(family<i> Secoviridae)</i>, including <i>Grapevine fanleaf virus </i>(GFLV). These viruses have a bipartite (+) sense RNA genome encapsidated in isometric particles and are transmitted by dagger nematodes. Grapevines infected with GFLV exhibit a range of symptoms such as fan-like leaf morphology, mosaics, vein yellowing, shortened internodes and premature death. Though GFLV is one of the most well characterized grapevine viruses, the mechanisms underlying symptom development remain poorly understood. Previous research revealed that symptom determinant(s) of GFLV strain GHu on <i>Nicotiana benthamiana</i>, a systemic herbaceous host, map to the 3’-end of the RNA-dependent RNA polymerase (1E<sup>Pol</sup>) coding region. Mutagenesis of amino acids within this region of protein 1E<sup>Pol</sup> followed by infectivity studies <i>in planta </i>ruled out several residues unique to strain GHu as being individually responsible for symptom development. Unexpectedly, some of the corresponding chimeric viruses failed to establish systemic infections in <i>N. benthamiana</i>,<i> </i>suggesting that this region may also be involved in systemic movement, a function not previously ascribed to the 1E<sup>Pol</sup> protein. These results highlight the multi-functional nature of this GFLV protein and advance our understanding of molecular interactions that enable infection and symptom development by GFLV.</div>
Molecular and Cellular Plant-Microbe InteractionsOralPaper4841Paper4841.mp4
Floral and Fruit DiseasesHow does <i>Botrytis cinerea</i> infect red raspberry?Olga KozharWashington State University
Tobin Peever: Washington State University
<div><i>Botrytis cinerea</i>, causal agent of gray mold, is the most important pathogen of raspberry in the US Pacific Northwest and worldwide. Despite intensive fungicide application programs used to control the disease, fruit losses often exceed 25% due to incomplete disease control. Fungicides currently are applied on a calendar basis rather than based on inoculum pressure and infection risk because the disease cycle on red raspberry is poorly understood. The objectives of this study were to determine the dynamics of flower and fruit infection in association with environmental conditions. Recovery of <i>B. cinerea</i> from seven developmental stages of raspberry flowers and fruit in northwestern Washington was recorded in 2015-2016 in a field not sprayed with fungicides. Colonization of raspberry flowers by <i>B. cinerea</i> was limited (15%), increased as fruit developed, and peaked on mature fruit (67%). In early stages of flower development, the floral part with greatest <i>B. cinerea</i> recovery was the carpel (70%). As fruit matured, additional floral parts were colonized by <i>B. cinerea</i>, which could increase the risk of secondary infections of mature fruit. <i>B. cinerea</i> colonization of fruit was significantly associated with air temperature and rainfall. The results suggest that fungicide applications at flowering may be less effective than those at fruit development and maturation, and monitoring environmental conditions may help time fungicide applications effectively to control gray mold.</div>
Biology and Disease ManagementOralPaper4861Paper4861.mp4
Plant Pathologists of the Future: Showcasing the Top Graduate Students from APS Division MeetingsA phylogenetic network of the soilborne fungal pathogen Sclerotium rolfsii in the Southeastern USPatricia SoriaUniversity of Florida
Nicholas Dufault: Univ of Florida
<div><i>To avoid publishing twice, the original abstract will be published with the other “<b>Southern Division 2017</b>” abstracts in Phytopathology.</i><p>Stem rot is one of the most important soilborne diseases in the Southeastern US, causing significant yield loss in tomato, pepper, and peanut. Population structure and genetic variation of the causal agent, <i>Sclerotium rolfsii</i>, is yet to be explored for Southeastern isolates, despite significant variability in colony morphology and host virulence. Mycelial compatibility groups have indicated the possibility of genetic diversity and previous phylogenetic analysis of the non-coding internal transcribed spacer (ITS) region indicate sequence variation among Florida isolates. The objective of this study was to determine the number of genotypes in the Southeastern US using sequence analysis of a protein coding gene. Thirty-five isolates infecting peanut in Florida, Georgia, and Alabama were collected and whole genomic DNA was extracted. RNA polymerase II subunit I (Rpb1) protein was amplified and sequenced. Sequences were analyzed using the Templeton, Crandall, Sing (TCS) algorithm for determining phylogenetic networks, where each node in the network is a genotype and links between nodes represent mutational steps. This study reports the first phylogenetic network for <i>S. rolfsii</i> in the Southeastern US using a protein coding gene sequence. Genotypes in the Southeastern US were distinct from previously characterized Mediterranean genotypes. Knowledge of gene flow among populations of <i>S. rolfsii</i> is needed for durable host resistance and can improve disease management.</div>
Fungicide Resitance and FitnessAssessing fitness of <i>Pythium aphanidermatum</i> isolates with dual resistance to mefenoxam and fenamidoneEmma LookabaughNorth Carolina State University
Barbara Shew: NC State University
<div><i>Pythium aphanidermatum</i> is the predominant species causing Pythium root rot on commercially grown poinsettias in NC. Mefenoxam (M) and fenamidone (F) are among the fungicides labeled for control of Pythium root rot on poinsettia. Widespread resistance to mefenoxam (MR) has been documented in <i>P. aphanidermatum</i>, but resistance to fenamidone and other QoIs has not been reported. <i>In-vitro</i> sensitivity (S) or insensitivity (R) to mefenoxam (17.6 μl a.i/ml) and fenamidone (488 μl a.i./ml) was tested on 96 isolates and the isolates were assigned to four fungicide resistance groups: MSFS, MRFS, MSFR, MRFR. 56% of isolates were insensitive to one (MRFS=35%; MSFR=15%) or both fungicides (MRFR = 6%). A single point mutation in the cytochrome-b gene (G143A) was identified in fenamidone-insensitive (FR) isolates. Mycelial growth rate, oospore production, and aggressiveness were evaluated to assess fitness of fungicide-resistant isolates. MRFR isolates had reduced growth rates at 30°C and were less aggressive on inoculated poinsettias than other isolate groups. MSFS isolates produced significantly more oospores <i>in-vitro</i> than isolates resistant to one or both fungicides. Fungicides failed to prevent disease on plants inoculated with resistant isolates, demonstrating the practical implications of resistance. This is the first report of QoI resistance in <i>P. aphanidermatum</i> from greenhouse floriculture crops and the first report of dual resistance to mefenoxam and QoIs.</div>
Biology and Disease ManagementOralPaper4886Paper4886.mp4
Translation of Basic Biological Control Research into Effective Grower Products and PracticesThe new biofungicide natamycin as a preplant dip treatment against QoI-resistant populations of <i>Colletotrichum acutatum </i>in strawberryStacey HaackDepartment of Plant Pathology and Microbiology, University of California
Kelly Ivors: Strawberry Center, California Polytechnic State University; Gerald Holmes: Strawberry Center, California Polytechnic State University; Helga Förster: Department of Plant Pathology and Microbiology, University of California; James Adaskaveg: Department of Plant Pathology and Microbiology, University of California
<div>Anthracnose crown rot of strawberry, caused by <i>Colletotrichum acutatum</i> (<i>Ca</i>), is an important disease in California nursery transplant and fruit production. In nursery plant multiplication, the disease can increase in wet years or with overhead irrigation and may reach epidemic levels when infected plants are moved to fields for fruit production. Cyprodinil/fludioxonil and azoxystrobin are currently registered as pre-plant dips of bare-root transplants. Additional management options are needed because of phytotoxicity concerns with cyprodinil/fludioxonil if roots are over-exposed to the treatment and presence of QoI resistance in <i>Ca</i> populations. The broad-spectrum biofungicide natamycin, never reported to develop resistance in filamentous fungi, was evaluated for its in vitro toxicity and as a dip treatment. Mean EC<sub>50</sub> values for mycelial growth of 45 QoI-sensitive and 29 -resistant <i>Ca</i> isolates were 0.996 and 0.902 mg/liter, respectively. Dipping transplants inoculated with QoI-sensitive or –resistant <i>Ca</i> for 5 minutes in 500 or 1000 mg/liter natamycin reduced plant mortality in the field by 58 to 95% as compared to the control (42% or 72% mean mortality for cultivars Fronteras and Monterey, respectively). Fludioxonil/cyprodinil reduced mortality by 86 to 100%. Azoxystrobin was only effective against QoI-sensitive isolates, reducing mortality by 80 to 92%. Based on this work, registration of natamycin as a dip treatment of strawberry transplants is in progress.</div>
Biology and Disease ManagementOralPaper4901Paper4901.mp4
Resistance IdentificationNew sources of soybean cyst nematode resistance in ‘Forrest’ soybean identified through forward and reverse geneticsZhou ZhouSouthern Illinois University
Naoufal Lakhssassi: Southern Illinois University; Shiming Liu: Southern Illinois University; Sadia Bekal: Southern Illinois University; Vincent Colantonio: Southern Illinois University; Kris Lambert: University of Illinois; Abdelali Barakat: University of South Dakota; Khalid Meksem: Southern Illinois University
<div>Soybean cyst nematode (SCN, <i>Heterodera glycines</i> Ichinohe) is the most economically destructive pathogen for soybean production worldwide. Although planting resistant cultivars is the primary management practice to control this pathogen, the mechanism of soybean [<i>Glycine ma</i>x (L.) Merr.] resistance to SCN remains unknown. In Peking-type soybean cultivar (cv.) ‘Forrest’, <i>rhg1</i> and <i>Rhg4</i> are two major quantitative trait loci (QTL) controlling resistance to SCN. Ethyl methanesulfonate (EMS) mutagenized ‘Forrest’ populations have been extensively and successfully used in both forward and reverse genetics approaches to study the function of SCN resistance genes in soybean. In this study, a total of 193 ‘Forrest’ mutant families was screened for their SCN female index (FI), from which 18 mutants were found susceptible to SCN (FI>10%). Five of the 18 mutants carried mutations in the <i>GmSHMT08</i>, a gene at <i>Rhg4</i> locus contributing SCN resistance, while four of them carried no mutations in both <i>GmSHMT08</i> and <i>GmSNAP18</i>, a gene conferring SCN resistance at <i>rhg1</i> locus. Our findings suggest that other gene(s) may play a role of SCN resistance in ‘Forrest’. The next-generation sequencing (NGS) is considered to identify EMS-induced mutations in novel candidate SCN resistance genes. Using a recombinant inbred line (RIL) to conduct SCN phenotype screening, <i>GmSNAP11</i>, a member of <i>GmSNAP</i> gene family sharing highly structural similarity with <i>GmSNAP18</i>, were identified to contribute to an additive resistance to SCN. Combined with the expression profiles and structural analyses, it is confirmed as a novel minor SCN resistance gene.</div>
Molecular and Cellular Plant-Microbe InteractionsOralPaper4905Paper4905.mp4
MycotoxinsApple post-harvest infection by <em>Paecilomyces niveus</em> causes spoilage of thermally processed productsMegan DanielsCornell University
Abigail Synder: Cornell University; Randy Worobo: Cornell University; Kathie Hodge: Cornell University
<div><i>Paecilomyces niveus</i> is known in the food industry as an important mycotoxigenic mold that spoils heat-processed fruit products. Though regarded as a soil-borne contaminant, our research shows it can be an apple pathogen like its relative <i>Penicillium expansum</i>. Both fungi synthesize patulin, which acts as a virulence factor in apple infection. Our goals were to assess the ability of <i>P. niveus </i>to infect apples and test whether infected apples<i> </i>can be a source of<i> </i>spoilage fungi in the finished apple juice concentrate, a heat-processed product often made from low quality apples. Two varieties of apples were inoculated with <i>P. niveus </i>to study pathogenicity and complete Koch’s postulates. To determine whether <i>P</i>. <i>niveus</i> can survive heat-processing, juice concentrate was made from infected apples. We confirmed <i>P</i>.<i> niveus</i> can infect apples through wounds, causing lesions similar to Bull’s-Eye Fruit Rot. Apple varieties differed significantly in lesion size (p=0.003). Juice concentrate made from infected apples contained viable <i>P</i>.<i> niveus</i> propagules, which were reduced by 4-log CFU/L but not eliminated across five stages of processing. We conclude <i>P</i>.<i> niveus</i> is an overlooked post-harvest apple pathogen that can survive thermal processing and produce patulin, a mycotoxin limited to < 50 ppb in apple products. The link between post-harvest apple disease and food spoilage is novel and may explain the episodic nature of <i>P</i>.<i> niveus </i>food spoilage.</div>
Diseases of PlantsOralPaper4947Paper4947.mp4
MycotoxinsAtoxigenic <i>Aspergillus flavus</i>: A hidden mechanism of biocontrolLourena AroneUniversity of Arizona
Peter Cotty: USDA-ARS, University of Arizona
<div>Aflatoxins are potent mycotoxins produced by several <i>Aspergillus</i> species. These toxins contaminate foods and feeds worldwide impacting health and trade. Research on managing aflatoxin contamination has focused on breeding, agronomic practices, and biocontrol. Biocontrol with atoxigenic genotypes of <i>A. flavus</i> may result in aflatoxin reductions of over 90%. Applications change the structure of <i>Aspergillus</i> communities so that aflatoxin producers are less common and atoxigenics dominate. In co-infected crop components, contamination is reduced both through competitive exclusion and a second, minor, undefined mechanism. To improve understanding of mechanisms through which atoxigenic <i>A. flavus</i> reduce aflatoxins, degradation of aflatoxins by atoxigenic strains of <i>Aspergillus flavus</i> was evaluated. Two commercial biocontrol agents (Aflaguard and AF36) and one biocontrol candidate MZM221-5 were inoculated onto both maize and liquid media containing aflatoxins. The three atoxigenic <i>A. flavus</i> genotypes degraded aflatoxins over the 4 week period. The current study reports for the first time the ability of atoxigenic genotypes of <i>A. flavus</i> currently used in biocontrol products to degrade aflatoxins in maize. Although all the evaluated atoxigenic genotypes degraded aflatoxin B<sub>1, </sub>there was variation<sub> </sub>among the genotypes in the speed of degradation. Optimal selection of atoxigenic biocontrol agents may include assessment of both competitively ability and ability to degrade aflatoxins.</div>
Biology and Disease ManagementOralPaper4952Paper4952.mp4
Phenotyping and Population DynamicsThree complete genome sequences of novel<i> Xanthomonas citri</i> strains from Texas carry atypical PthA alleles and unusual large plasmidsAlejandra Munoz BodnarUniversity of Florida
Vessela Mavrodieva: USDA-APHIS-PPQ-S&T-CPHST; Dean Gabriel: University of Florida; Zhaowei Liu: USDA APHIS PPQ; Mark Nakhla: USDA-APHIS-PPQ-S&T-CPHST; Gem Santillana: APHIS
<div>In 2015, backyard lime (<i>Citrus </i>spp.) trees in Texas showing typical citrus canker symptoms were confirmed positive for <i>Xanthomonas citri </i>subsp. <i>citri</i>. This confirmation triggered a regulatory response since the disease has been absent from the state since 1940s. The complete genomes of three strains from Texas were determined by PacBio Single Molecule, Real-Time (SMRT) sequencing. The genomes were 5,330,822 bp, 5,341,733 bp and 5,337,252 bp in size with an average read length of 15,756 bp, 15,261 bp and 16,590 bp, respectively. Bioinformatic analyses indicated that the new strains were related to the Florida A<sup>w</sup> strain. However, all three strains carried nearly identical and unusual large plasmids (123557bp, 123557bp, and 129139bp, respectively), that matched contigs in the NCBI Whole Genome Shotgun (WGS) database from some <i>X. citri subsp citri, X. fuscans</i> pv. fuscans and <i>X. axonopodis</i> pv. manihotis strains. These plasmids contain a region of ca. 39 kB similar to Type 4 conjugational transfer genes, indicating recent horizontal transfer into <i>X. citri</i>. All three Texas strains carried predicted functional <i>pthA</i> homologs with 18.5 tandem repeats each instead of the typical 17.5 repeats and a second homolog predicted to be nonfunctional. Atypically for citrus canker causing strains, two of the Texas strains carried both <i>pthA</i> homologs on the chromosome. Specific primers to distinguish the Texas strains from other canker-causing strains were designed and evaluated.</div>
Diseases of PlantsOralPaper4961Paper4961.mp4
Integrated Pest ManagementEffect of tillage and cultivar on sudden death syndrome and yield of soybean in IowaYuba KandelIowa State University
Daren Mueller: Iowa State University; Leonor Leandro: Iowa State University
<div>Reduced-tillage has become a common practice of soybean farming in the Midwestern U.S. Field trials were established in a field with a history of sudden death syndrome (SDS; caused by <i>Fusarium virguliforme</i>) in Iowa in 2011, and evaluated for five consecutive years, to determine the impact of long-term tillage on SDS and yield. The study site contains mainly webster clay loam, bemis moraine, and nicollet loam soil, with 1 to 3% slopes that are poorly drained. The experiment was laid out in a split split-plot design with four replicates. The main plot factor was tillage (no till both crops, no till corn and chisel plow soybean, and disc corn and chisel plow soybean, respectively), and each main plot was divided into subplots of corn or soybeans (in a 2-year rotation). Each subplot was again divided into sub-subplots where two soybean cultivars, susceptible and moderately resistant to SDS, were planted each year. Root rot and SDS disease index (FDX) differed among years, as some years were more favorable for the disease than the others. However, tillage did not affect any parameters, including yield, in any year (P > 0.05). Cultivar effect was significant for each parameter occasionally. Compared to the susceptible cultivar, the moderately resistant cultivar had 61% less root rot in 2015; 61 and 62% less FDX in 2014 and 2015, respectively, and 21% greater yield in 2015. These data suggest planting resistant cultivars can be an effective management tactic but tillage is not an effective option for SDS management.</div>
Biology and Disease ManagementOralPaper5016Paper5016.mp4
17th I. E. Melhus Graduate Student Symposium: Today's Students Addressing Tomorrows Challenges Concerning Plant Diseases and PhytobiomesOptimization of fungicides for disease management and enhanced overwintering of steviaAlyssa KoehlerNorth Carolina State University
<div>There are many challenges facing the establishment of stevia (<i>Stevia rebaudiana) </i>as a perennial crop in the US. Southern blight (caused by <i>Sclerotium rolfsii)</i> and Septoria leaf spot (caused by <i>Septoria sp.</i>) are the most economically important diseases in the southeast. Fungicide efficacy trials for management of these diseases were conducted between 2014 and 2017 and multiple products for management of <i>S. rolfsii </i>and <i>Septoria</i> were identified<i>.</i> Crowns from fungicide trials in 2014 were left in the field to overwinter and stand counts were taken in spring 2015 to determine percent emergence compared to final stand counts in fall 2014. Treatments that received one or more applications of the QoI (strobilurin) fungicide azoxystrobin had enhanced overwintering survival (mean survival rate, 78%) compared to other fungicides (38%), and the non-treated control (38%). In spring 2016, plots receiving QoI treatments in 2015 had higher overwintering emergence compared to controls at two locations. Roots and crowns were destructively sampled in spring 2016 and QoI treated plants had higher mean weights (62g) compared to non-treated controls (31g). Trials planted in 2016 were destructively sampled monthly for 12 months to record root weights and document fungi associated with roots throughout the growing season and overwintering. This study may open a new avenue of investigation into the role of fungicides and the root phytobiome in the vigor and survival of perennial plants.</div>
Biology and Disease ManagementOralPaper5029Paper5029.mp4
17th I. E. Melhus Graduate Student Symposium: Today's Students Addressing Tomorrows Challenges Concerning Plant Diseases and PhytobiomesEffects of three chemical compounds on <i>Ralstonia solanacearum</i> physiological functions and disease developmentHsien-Tzer TsengNorth Carolina State University
Asimina Mila: North Carolina State University
<div>The effects of three chemical compounds, known to have anti-microbial activities, were examined against <i>Ralstonia solanacearum</i> (Rs). The compounds were 3-indolyacetonitrile (IAN), p-benzoquinone (pBQ), and 6,7-dihydroxycoumarin (6-7-D). The minimal inhibition concentration against Rs was 640μM, 25μM, and 125μM for IAN, pBQ and 6-7-D, respectively. Sub-inhibitory concentrations, 160μM for IAN, 12.5μM for pBQ, and 50μM for 6-7-D, were used to determine bacterial growth rates. IAN was significantly more effective in inhibiting growth compared to pBQ and 6-7-D. Fluorescent microscopy with GFP-labeled W7 strain demonstrated that IAN limited bacterial colonization of plant roots the most. Tobacco cultivars K326 and Speight168 were treated with the compounds 48-hours before, at the same time or 48-hours after inoculation. IAN was the most effective compound in limiting disease and treating at inoculation suppressed the disease the most regardless of the compound. Quantitative real-time PCR revealed that pBQ significantly inhibited the expression of the three pathogenicity-related genes, <i>phcA</i>,<i> xpsR</i>,<i> and pehR</i>, compared to the untreated control whereas 6-7-D did not have any significant effect. With IAN, expression of <i>phcA</i> and <i>xpsR</i> was repressed, but the expression of <i>pehR</i> was increased. Understanding the unique Rs physiological traits and their responses to stimuli may provide insights for future phytobiome research.</div>
Biology and Disease ManagementOralPaper5031Paper5031.mp4
Bacterial Virulence and EffectorsA change in perspective: PAMP triggered immunity as told by <i>Pseudomonas syringae</i> pv. tomatoAmelia LovelaceUniversity of Georgia
Brian Kvitko: University of Georgia
<div>The recognition of conserved Pathogen-Associated Molecular Patterns (PAMPs), such as bacterial flagellin, by Pattern Recognition Receptor (PRRs) membrane proteins activates an immune response that confers broad defense against many microbes. When pre-induced, PAMP triggered immunity (PTI) restricts the growth of pathogenic bacteria such as <i>Pseudomonas syingae</i> pv. <i>tomato</i> (<i>Pto</i>) in the apoplastic space. Although the plant cellular processes involved in flagellin/FLS2 signaling have been well studied, the mechanisms by which PTI suppresses further colonization of the apoplastic space by <i>Pto</i> are poorly understood. The objective of this study is to use <i>Pto</i> as a probe to better understand the mechanism(s) of PTI. <i> Pto </i>was exposed to pre-induced immune activated (Flg22) and naïve (Mock) <i>Arabidopsis</i> leaves for 1, 3, and 5 hours before extraction using a newly developed method to access the <i>Pto </i>transcriptome <i>in planta</i>. Principal component analysis of transcriptome profiles between treatments show separation of Flg22 and Mock samples after one hour of exposure. Differential expression of genes in <i>Pto</i> exposed to these two conditions reveal that virulence genes and sugar metabolism are downregulated whereas chemotaxis, motility, and nutrient uptake are upregulated by exposure to immune defenses. Expression profile of <i>Pto</i> exposed to immune defenses is suggestive of entering stationary phase with consistent levels of expression for major cellular processes.</div>
Molecular and Cellular Plant-Microbe InteractionsOralPaper5108Paper5108.mp4
Virology Detection and DescriptionsField status and dynamics of cassava mosaic disease in ZambiaRabson MulengaZambia Agriculture Research Institute
Patrick C. Chikoti: Zambia Agriculture Research Institute; James Legg: International Institute of Tropical Agriculture; Olufemi Alabi: Dept. of Plant Pathology & Microbiology, Texas A&M University
<div>The status of cassava mosaic disease in Zambia was determined during a country-wide survey conducted in 2014 in farmers’ fields in six major cassava-growing provinces (Luapula, Lusaka, Northern, Eastern, Western and Northwestern). A symptoms-based assessment of CMD was performed on 30 plants of a predominant variety counted along two diagonals to determine disease incidence and severity. Incidence was calculated as a percentage of symptomatic plants out of 30 counted plants while severity was rated using the standard 1 to 5 scale. Assessment of infection type (cutting- or whitefly-borne) was made and values for whitefly-borne infections were transformed into multiple infection units. The results indicated that mean CMD incidence was lowest in Northern (44%) and highest in Lusaka province (81%); averaged 57% across all six provinces. Mean CMD severity varied significantly (χ<sup>2</sup>= 249.8, df = 10, P < 0.001) among provinces ranging from 2.8 in Eastern to 3.0 in Northwestern province. Mean CMD incidence was higher for cassava landraces (~25 to 86%) than improved varieties (~32 to 60 %). Disease severity also varied between landraces (2.2 to 3.0) and improved varieties (2.7 to 2.8). Cutting-borne infections accounted for ~92% of CMD-affected plants relative to 8% for whitefly-borne infections. DNA fingerprinting of field survey samples revealed the occurrence of <i>African cassava mosaic virus</i>, <i>East African cassava mosaic virus</i> and <i>East African cassava mosaic Malawi virus</i> as single or mixed infections of different proportions. The results underscore the importance of clean plant materials for sustainable management of CMD in Zambia.</div>
Pathogen Virulence and EffectorsLocalization and functional study of <i>Ustilago hordei</i> avirulence effector UhAVR1 using barley and <i>Nicotiana benthamiana</i> plant systemsAna Priscilla Montenegro AlonsoUniversity of British Columbia
Guus Bakkeren: Agric & Agi Food Canada
<div>UhAVR1 is a 190-aa protein of the barley smut fungal pathogen <i>Ustilago hordei</i> (<i>Uh</i>). It has a predicted 19-aa signal peptide (SP) and this candidate effector was identified as an avirulence factor triggering an HR within 48 hrs after infection (hpi) of cv. Hannchen having resistance gene<i> Ruh1</i>. To decipher its virulence function, host target and host response triggered upon its recognition, transcriptomic analyses and localization studies were performed. Barley seedlings from cvs. Odessa (<i>ruh1</i>) and Hannchen (<i>Ruh1</i>), representing compatible and incompatible interactions respectively, were inoculated with teliospores from wild type and an <i>UhAVR1</i> deletion mutant. <i>UhAvr1</i> expression was quantified via ddPCR during infection from 24 hpi till heading showing expression only at the early biotrophic stage and peaking between 72 and 96 hpi. Global transcriptome analysis via RNA-seq at 48 hpi revealed 151 differentially expressed genes (DEGs) in Odessa including kinases, transcription factors, chloroplast genes among others, whereas in Hannchen 173 DEGs were detected including peroxidases, <i>R</i> genes, and chitinases, among others. To identify putative interactors, localization experiments were performed. In heterologous <i>Nicotiana benthamiana </i>(<i>Nb</i>),<i> </i>chimeric UhAVR1-SP, C-terminally tagged with eGFP, was delivered by <i>Agrobacterium tumefaciens</i> into infiltrated leaves. Confocal microscopy revealed localization to cytosolic bodies at 24 and 48 hpi, compared to free eGFP control which was found in the nucleoplasm and cytosol. Protein analysis from leaf samples at the same time points by Western blots using anti-GFP and anti-UhAVR1 antibodies, confirmed the expression of full length chimeric mature UhAVR1 protein of 46 kDa.</div>
Molecular and Cellular Plant-Microbe InteractionsOralPaper5133Paper5133.mp4
Viral-Host InteractionsMapping Loci that Control Tuber and Foliar Symptoms Caused by PVY in Autotetraploid Potato (<i>Solanum tuberosum </i>L.)Washington Da SilvaCornell University
Gary Stewart: USDA, Agricultural Research Service; Gary Stewart: Cornell University/USDA-ARS; Walter De Jong: Cornell Univ
<div>Potato tuber necrotic ringspot disease (PTNRD) is a tuber deformity associated with infection by the tuber necrotic strain of potato virus Y (PVY<sup>NTN</sup>). PTNRD negatively impacts tuber quality, marketability, and poses a serious threat to seed and commercial potato production worldwide. PVY<sup>NTN</sup> symptoms differ in the cultivars Waneta and Pike; Waneta expresses severe PTNRD and foliar mosaic with vein and leaf necrosis, whereas Pike does not express PTNRD and mosaic is the only foliar symptom. To map loci that influence tuber and foliar symptoms, 244 F<sub>1</sub> progeny of a cross between Waneta and Pike were inoculated with isolate PVY<sup>NTN</sup>-NY090029 and also genotyped using 12808 Potato SNP markers. Foliar symptom type and severity were monitored for 10 weeks, while tubers were evaluated for PTNRD expression at harvest and again after 60 days in storage. Pairwise correlation analyses indicate a strong association between PTNRD and veinal necrosis (τ = 0.4195). QTL analyses revealed major-effect QTLs on chromosomes IV and V for mosaic, IV for PTNRD, and V for foliar-necrosis symptoms. Locating QTLs associated with PVY-related symptoms provides a foundation for breeders to develop markers that can be used to screen out potato clones with undesirable phenotypes, e.g., those likely to develop PTNRD or to be symptomless carriers of PVY.</div>
Biology and Disease ManagementOralPaper5136Paper5136.mp4
Virus BiologyStrain specificity of helper components encoded by<i> Potato virus Y</i>Shaonpius MondalCornell University
Gary Stewart: Cornell University
<div>In recent years, the recombinant strains of <i>Potato virus Y</i> (PVY<sup>NTN</sup> and PVY<sup>N:O</sup>) have steadily replaced their nonrecombinant parent, PVY<sup>O</sup>, in the U.S. potato crop. Previous studies showed a transmission bias for recombinant strains when acquired sequentially with PVY<sup>O</sup>, regardless of acquisition order. To understand this bias, the nonstructural helper component (HC) and infectious virus particles were separated, mixed in various combinations, and fed to green peach aphids using Parafilm® sachets. Following a 20 min acquisition access period, groups of 10-12 aphids were transferred to potato seedlings for a 24 h inoculation access period. Treatments included all possible combinations of HC and purified virus (PV) from PVY<sup>O</sup>, PVY<sup>N:O</sup>, and PVY<sup>NTN</sup>. Plants were assayed 21-28 days post inoculation by TAS-ELISA. When heterologous or homologous combinations of PV and HC of PVY<sup>N:O</sup> and PVY<sup>NTN </sup>were mixed and fed immediately to aphids, transmission from any treatment was <20%, except the PVY<sup>NTN</sup> PV - PVY<sup>N:O </sup>HC combination (>60%). Following a 3-4 h incubation of PV with HC at 4°C, transmission efficiency of the all combinations increased to >70% except the PVY<sup>N:O</sup> PV - PVY<sup>NTN </sup>HC combination, which remained <20%. These data indicate a strain specific function of HC, and suggest that formation of a HC-PV protein complex prior to aphid acquisition facilitates transmission. Ongoing research is determining transmission of HC-PV combinations of PVY<sup>O</sup>-PVY<sup>N:O </sup>and PVY<sup>O</sup>-PVY<sup>NTN</sup>.</div>
Biology and Disease ManagementOralPaper5139Paper5139.mp4
Pathogen Detection and DiversityExpanding the scope of a single pathogen survey to solve the mystery of the ailing red raspberriesJerry WeilandUSDA ARS
Chris Benedict: WSU; Inga Zasada: USDA ARS; Bryan Beck: USDA ARS; Anne Davis: USDA ARS; Amy Peetz: USDA ARS HCRU; Kim Graham: USDA ARS; Robert Martin: USDA ARS; Jeremiah Dung: Oregon State University; Andres Reyes Gaige: Oregon State University; Lindsey Thiessen: North Carolina State University
<div>Washington state produces almost 60% of the processed raspberries ($79 M value) in the U.S. Production is severely limited by Raspberry Bushy Dwarf Virus (RBDV) and the soilborne pathogens <i>Phytophthora rubi</i> and <i>Pratylenchus penetrans</i>. However, in 2012, growers began noticing plants with unusual symptoms that were not attributed to any of these pathogens. Starting in late summer, individual primocanes or entire plants would suddenly wilt and die. Adjacent plants were often chlorotic and stunted. Early diagnostic work indicated that <i>Verticillium dahliae</i> was the culprit at three locations, but analyses of soil populations often produced conflicting results depending on the lab and detection method. Growers became concerned that this pathogen was a previously undiagnosed problem for the industry. Therefore, a survey was conducted to determine if <i>V. dahliae</i> was common in production fields, to determine if <i>V. dahliae </i>was associated with disease, and to compare the sensitivity of two detection methods (selective media versus qPCR) for quantifying <i>V. dahliae </i>in the soil. The presence of <i>P. rubi</i>, <i>P. penetrans</i>, and RBDV was also assessed. In 2013 to 2014, primocanes, roots, and soil were collected from 51 disease sites and 20 healthy sites within 24 production fields. Results show that <i>V. dahliae</i> is common and that qPCR is more sensitive than plating for detection. But, the main question remains, which pathogen(s) are causing the disease? Come find out!</div>
Diseases of PlantsOralPaper5167Paper5167.mp4
Pathogen Virulence and Effectors<em> Phytophthora</em> hijacks host BAG7 and bZIP28 resulting in sequestration in endoplasmic reticulum for compatibility via effector-mediated BiP accumulationMaofeng JingTexas A&M University
Yurong Li: Texas A&M University; Yuanchao Wang: Nanjing Agric Univ; Martin Dickman: Texas A&M University
<div>The endoplasmic reticulum (ER) is a central hub for stress responses to pathogens as well environmental<i> </i><i>Phytophthora</i> species secrete a large array of specific effector proteins to manipulate host innate immunity.<i> Phytophthora </i>uses an essential effector, Avh262 to stabilize Binding immunoglobulin Proteins (BiPs) in the ER, that act as negative regulators of plant resistance to <i>Phytophthora</i> and ER stress-induced cell death (ERCD), can result in attenuated plant defense responses. However, little is known about how <i>Phytophthora</i> hijack the host BiPs to regulate the ER machinery for successful infection. Here we show Avh262 interacts with both AtBAG7, an ER-localized cochaperone that helps maintain for the maintenance of the unfolded protein response, and bZIP28, an ER membrane-tethered transcription factor. The translocation of AtBAG7 and bZIP28 from the ER to the nucleus is required for activating the downstream pathways. We found AtBAG7 negatively regulates plant resistance to <i>Phytophthora</i>, and bZIP28 plays a positive role. The culture filtrate of <i>Phytophthora</i><i> </i>triggers the translocation of AtBAG7 and bZIP28 from the ER to the nucleus, which can be prevented by the Avh262-mediated accumulation of BiPs. In conclusion, <i>Phytophthora</i> pathogens secrete effectors to accumulate high level of BiPs, which retains AtBAG7 and bZIP28 in the ER lumen, resulting in suppressing the ERCD pathway and blocking expression of downstream defense genes, beneficial to infection.</div>
Molecular and Cellular Plant-Microbe InteractionsOralPaper5182Paper5182.mp4
Virus BiologyCurrent status of <em>Tomato chlorotic spot virus</em> in Florida and the CaribbeanScott AdkinsUSDA ARS USHRL
Samuel Hutton: University of Florida; Consuelo Estevez de Jensen: University of Puerto Rico, Agro-environmental Sciences; Joe Funderburk: University of Florida; William Turechek: USDA ARS SAA SPP
<div>Damaging outbreaks of <i>Tomato chlorotic spot virus</i> (TCSV), an emerging thrips-vectored tospovirus, and several invasive species of thrips are significantly impacting vegetable and other crops in Florida and the Caribbean. Host and geographic ranges of TCSV are continuing to expand in this region. Development of effective strategies requires a multi-pronged approach against TCSV and its key vectors, including western flower thrips and common blossom thrips. The <i>Sw-5</i> gene for resistance to <i>Tomato spotted wilt virus </i>(TSWV), a related tospovirus, also confers resistance to TCSV. New hybrids containing this gene are currently being evaluated in south Florida field trials. Epidemic development is being characterized through intensive sampling of commercial pepper fields. Viral incidence data is being used to characterize the spatial and temporal progress of the virus. Corresponding flower samples are being collected for thrips identification and to characterize the relationship between thrips density and disease incidence. New qRT-PCR protocols have been developed for rapid, quantitative and specific identification of TCSV. Symptomatic plant samples tested to date have been infected primarily by TCSV although TSWV is also occasionally detected. Collectively, this information will lead to development of management strategies that are better focused on disrupting virus and/or thrips biology.</div>
Biology and Disease ManagementOralPaper5196Paper5196.mp4
Phyllosphere Microbial Assemblages: Friends, Foes, and StrangersLight as a driver of phyllosphere microbial behaviorGwyn BeattieIowa State University
<div>Light is a cue that influences the behavior of foliar bacterial pathogens. Whereas plants exploit light signals to enhance defense against pathogens, we have shown that <i>Pseudomonas syringae </i>exploits light signals to influence movement and colonization, and this may enable it to evade circadian-based plant defenses. Far-red light is enriched relative to blue and red light in plant tissues due to blue/red absorption, enabling far-red light to be abundant within tissues and conducted over long distances through stems and roots. We discovered a much stronger response of <i>P. syringae </i>to far-red than to red or blue light, and a significant role of a bacteriophytochrome, a far-red light-sensing photosensory protein, in global gene expression patterns. We have also found that <i>P. syringae </i>integrates responses to far-red light, blue light and and water limitation, which are environmental signals that co-occur on leaves. The impact of far-red light and a bacteriophytochrome on >25% of the genes in the <i>P. syringae </i>genome illustrates a major role for light-sensing in <i>P. syringae </i>biology, consistent with the possibility that this pathogen and others exploit far-red light gradients as spatial and temporal cues within plant tissues.</div>
Molecular and Cellular Plant-Microbe InteractionsOralPaper5197Paper5197.mp4
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