Posted online May 7, 2007
Characterizing conserved effector proteins from Hyaloperonospora parasitica. R. ANDERSON (1), R. Jiang (2,3), D. Dou (2), X. Wang (2), B. Tyler (2), and J. McDowell (1). (1) Dept. Plant Pathology, Physiology, and Weed Science, Virginia Tech, Blacksburg, VA 24061; (2) VBI, Virginia Tech, VA 24061; (3) Laboratory of Phytopathology, Wageningen University, The Netherlands NL-6709 PD and Broad Institute, Cambridge, MA 02141.
Many plant pathogens utilize targeted effector proteins to promote disease. Oomycete effectors carry a host targeting (HT) sequence used for translocation into the host cell. We are using the model interaction between Arabidopsis and Hyaloperonospora parasitica (Hpa) to study how oomycete effectors manipulate host cells. Bioinformatics analyses of the Hpa genome has revealed over 180 candidate effector genes with a HT sequence. We are focusing on candidate effectors that have conserved homologs in Phytophthora sojae (Ps). Conserved effectors between Hpa and Phytophthora may have important functions in oomycete pathogenicity. We have determined in planta expression for each candidate effector during the Hpa interaction with Arabidopsis. We have shown that the HT motifs from an Hpa effector and the Ps effector, Avr1b, are functionally equivalent. Several effectors contain a functional nuclear localization sequence suggesting that they modulate host gene expression.
Characterizing isolates of cucumber mosaic virus from heirloom species at a Virginia historic site. P. G. CHANG, R. Loveday, and S. A. Tolin. Dept. of Plant Pathology, Physiology and Weed Science, Virginia Tech, Blacksburg, VA 24061.
Cucumber mosaic virus (CMV) is one of the most destructive viral diseases worldwide, with a host range that includes more than 1,200 plant species in over 100 families and transmissibility through seed and by aphid vectors. The virus, whose genome of four single-stranded RNAs is packaged in three isometric capsids, is highly diverse. Discovered in 1916, CMV is considered endemic in most states of the United States. However, little recent work has been done to characterize U. S. isolates from crop or naturalized hosts. High yield losses in beans in New York since 2000, attributed to CMV, has renewed interest in this virus. In August 2005 and May 2006, several plants in a demonstration garden at a Virginia historical site in Blacksburg were observed with virus-like symptoms of mosaic, leaf curling and stunting. Leaves from five species were serologically positive for CMV. Efforts were focused on characterizing isolates mechanically transmitted from flowering tobacco, gourd, and Vinca sp. to tobacco. Host range and symptomatology varied suggesting biological diversity among the isolates. Primers designed from published sequences were used to amplify the coat protein gene, using Reverse-Transcription Polymerase Chain Reaction (RT-PCR), for sequence analysis. Comparison of the molecular diversity of these isolates to different CMVs isolated in Virginia is in progress.
Gaining governmental approval for the release of disease resistant transgenic peanuts expressing oxalate oxidase. S. M. CHRISCOE (1), D. E. Partridge (2), P. M. Phipps (2), and E. A. Grabau (1). (1) Dept. Plant Pathology, Physiology, and Weed Science, Virginia Tech, Blacksburg, VA 24061; (2) Tidewater Ag. Research and Extension Center, Suffolk, VA 23437.
Sclerotinia minor is a devastating fungal disease of peanut (Arachis hypogaea). Transgenic plants of three Virginia-type peanut cultivars have been engineered to express an oxalate oxidase enzyme from barley. Oxalate oxidase degrades oxalic acid, a major pathogenicity factor of S. minor, thereby suppressing fungal infection and increasing disease resistance. Three years of field trials have been conducted and six transgenic lines have been identified to submit for governmental regulatory review. In 2006, these lines had 85% less disease and yielded 537 to 2491 kg/ha more than the parental lines, giving an added value of $222 to 1043/ha. Before these peanuts can be marketed, they must be evaluated by the US Department of Agriculture, the Food and Drug Administration and the Environmental Protection Agency. Petitions will be submitted to each of the three agencies to address various data requirements including allergenicity, outcrossing, genetic characterization and transgene expression.
Development of an autonomous unmanned aerial vehicle for aerobiological sampling. B. R. DINGUS (1,2), D. G. Schmale III (1), and C. Reinholtz (2). (1) Dept. Plant Pathology, Physiology, and Weed Science, Virginia Tech, Blacksburg, VA 24061; (2) Dept. Mechanical Engineering, Virginia Tech, Blacksburg, VA 24061.
The ability to detect, monitor, and forecast the movement of airborne plant pathogens in agricultural ecosystems is essential for developing rational approaches to managing these habitats. We developed an autonomous (self-controlling) unmanned aerial vehicle (UAV) platform for aerobiological sampling tens to hundreds of meters above agricultural fields. We equipped a Senior Telemaster model airplane with two spore-sampling devices and a MicroPilot autonomous system, and we have conducted over 60 autonomous microbe-sampling flights at Virginia Tech’s Kentland Farm. To determine the most appropriate sampling path for aerobiological sampling, we have explored a variety of different sampling patterns for our autonomous UAVs including multiple GPS waypoints plotted over a variety of spatial scales. Autonomous UAVs have the potential to extend the range of aerobiological sampling, improve positional accuracy of sampling paths, and enable coordinated flight with multiple aircraft at different altitudes.
Crosses between pycnia of Puccinia acroptili from the United States, Russia, and Turkey. F. M. ESKANDARI and W. L. Bruckart, III. USDA, ARS, FDWSRU, Ft. Detrick, MD 21702.
Russian knapweed (Acroptilon repens) is infected both in the United States (U.S.) and its native range of Eurasia by Puccinia acroptili, a candidate for biological control in the U.S. Small, consistent differences in teliospore length and ITS sequences were found between a Turkish and three U.S. isolates in earlier studies. Research to clarify taxonomic relationships of these isolates included artificial crosses. When flecks developed after foliar inoculation with teliospores, leaves were detached and grown in sterile tap water in insect exclusion cages. Pycniospores from a single pycnium were suspended in sterile distilled water and a 1 microliter drop placed on each of the remaining pycnia. Successful reciprocal crosses have been achieved between the Turkish isolate (02-048) and one isolate each from the U.S. (05-055) and Russia (05-085). One-way crosses between a U.S. isolate (05-056) and isolates from Turkey and Russia also have been successful. These results suggest that isolates are the same species, despite differences noted earlier. Classical and molecular characterization of isolates from these and subsequent crosses is in progress.
Architecture or the signaling network activated by Arabidopsis RPP7 for resistance to Hyaloperonospora parasitica. T. HOFF and J. McDowell. Dept. Plant Pathology, Physiology and Weed Science, Virginia Tech, Blacksburg, VA 24060.
The RPP7 gene activates race-specific resistance to the downy mildew pathogen Hyaloperonospora parasitica. RPP7 is not suppressed by mutations in a variety of putative signal transducers that are required by various other NBS-LRR resistance genes (e.g. pad4-1, ndr1-1, npr1-1, pbs2-1). In an effort to better understand the genetic requirements for signal transduction for RPP7, we have constructed a series of double mutants to test for additive of functionally redundant contributions by known defense signaling components. Most of these combinations display a slightly enhanced level of asexual sporulation, with the ndr1/pad4 combination having the strongest effect. Trypan blue staining revealed that all of the double mutants are capable of inducing the HR, but this response is delayed to varying degrees. The effect of the double mutants on the kinetics of ROI accumulation is similar to what is seen in the trypan blue staining. These results suggest that RPP7 activates resistance through multiple signaling pathways that collectively regulate the kinetics of the HR.
Apple scab sensitivity to myclobutanil in Virginia. S. C. MARINE (1), D. G. Schmale III (1), and K. S. Yoder (2). (1) Dept. Plant Pathology, Physiology, and Weed Science, Virginia Tech, Blacksburg, VA 24061; (2) Virginia Tech AREC, Winchester, VA 22602.
Apple scab, caused by Venturia inaequalis, is an economic threat to commercial apple production in the eastern U.S. Populations of V. inaequalis in VA orchards may be developing resistance to myclobutanil and other DMI fungicides. Little is known about the frequency, timing, and mechanisms of fungicide resistance in apple scab populations in VA. We evaluated fungicide resistance in a total of 71 single-spored V. inaequalis isolates collected in Winchester, VA in 2006. Percent growth suppression on agar containing 0 ppm, 0.1 ppm, 0.5 ppm, or 1 ppm myclobutanil showed that 22 of the isolates were sensitive, 16 were resistant, and 33 were moderately resistant to myclobutanil. The growth of isolates from treated trees was significantly greater than those from non-treated trees for all myclobutanil treatments in agar. High levels of fungicide resistance in populations of V. inaequalis suggest that replacement programs should be considered. Future research may rely on DNA-based methodologies to determine fungicide resistance and employ appropriate disease management strategies.
Functional genomic analysis of apple (Malus) ESTs associated with fire blight (Erwinia amylovora). J. L. NORELLI (1), E. Borejsza-Wysocka (2), A. M. Baldo (3), H. S. Aldwinckle (2), C. L. Bassett (1), R. E. Farrell, Jr. (4), M. Malnoy (2), D. A. Lalli (1), S. S. Korban (5), K. Gasic (5), and M. E. Wisniewski (1). (1) USDA-ARS, Appalachian Fruit Research Station, Kearneysville, WV 25430; (2) Cornell University, Department of Plant Pathology, Geneva, NY 14456; (3) USDA-ARS, Plant Genetic Resources Unit, Geneva, NY 14456; (4) Pennsylvania State University, York, PA 17401; (5) University of Illinois, Urbana, IL 61802.
The goal of this project is to use a functional genomic analysis to characterize the response of apple to fire blight disease and thereby identify new opportunities for improving fire blight resistance. Expressed sequence tags (ESTs) derived from mRNA isolated from a specific tissue and treatment provide a crude "inventory" of genes that are being expressed in that tissue. Bioinformatics was used to identify publicly available apple ESTs uniquely associated with Erwinia amylovora infected apple or similar to Arabidopsis ESTs associated with Pseudomonas syringae pv. tomato infection. To determine the function of 19 apple ESTs in fire blight resistance and susceptibility, RNA interference (RNAi) is being used to silence the expression of specific candidate genes. To select apple RNAi mutants, an efficient high-throughput system of transformation for apple was developed in which only one EST-silencing gene was inserted per transgenic line. The system uses a multi-vector transformation approach and PCR primers developed for pHellsgate8-derived vectors that can: 1) detect the presence of either single or multiple EST-silencing genes in RNAi transgenic lines and 2) provide sequencing template to determine the EST contained in the silencing insert. Additional ESTs are currently being selected based upon on-going cDNA suppression subtractive hybridization and cDNA-AFLP analyses.
Induced resistance to Globodera tabacum solanacearum in tobacco. V. PARKUNAN (1,2), C. S. Johnson (1,2), and J. D. Eisenback (2). (1) Southern Piedmont AREC, Virginia Tech, Blackstone, VA; (2) Dept. of Plant Pathology, Physiology, and Weed Science, Virginia Tech, Blacksburg, VA 24060.
Reproduction of Globodera tabacum solanacearum (tobacco cyst nematode – Gts) on Xanthi NN and flue-cured tobacco cultivar K 326 was compared in greenhouse trials that included an untreated control, 7 foliar applications of acibenzolar-S-methyl (ASM) on a 10-day interval, and preplant soil incorporation of 4 combinations of Bacillus subtilis A13 with B. pumilis INR-7, B. pumilis SE34, B. licheniformis IN937b, or B. amyloliquifaciens IN937a. Reproduction was evaluated in 2 experiments on each tobacco type arranged in a randomized complete block design with 6 replications. Pots were infested with 5,000 eggs of Gts 5 weeks after seeding, and Gts cysts were extracted from 250 cm(^3) of soil 11 weeks later. Among the four rhizobacterial combinations, B. subtilis A13 + B. amyloliquifaciens IN937a exhibited the most consistent reduction in Gts cysts. Use of B. subtilis A13 + B. pumilis INR7 reduced Gts reproduction on K 326, but not on Xanthi NN. Application of ASM reduced final numbers of Gts cysts, but also resulted in chlorosis, stunting, and reduced plant fresh weight.
Development and field evaluation of genetically modified peanuts with high levels of resistance to Sclerotinia blight. D. E. Partridge (1), P. M. Phipps (1), S. M. Chriscoe (2), and E. A. Grabau (2). (1) Tidewater Agricultural Research and Extension Center, Virginia Tech, Suffolk, VA 23437; (2) Dept. Plant Pathology, Physiology, and Weed Science, Virginia Tech, Blacksburg, VA 24061.
Sclerotinia blight, cause by Sclerotinia minor, is a major limiting factor for Virginia-type peanut production. High levels of resistance have been achieved through the development of transgenic peanut transformed with a barley oxalate oxidase gene. T(3) and T(4) transformed lines and non-transformed controls of three Virginia cultivars were evaluated in fields naturally infested with S. minor. Bioengineered peanuts averaged 76.9% and 85.8% less disease compared to their non-transformed parent in 2005 and 2006, respectively. All transformed lines produced yields that were equal to or greater than their non-transformed parent, with a number of lines increasing yield 488 to 2490 kg/ha and value $304 to 623/ha. These studies demonstrate that the oxalate oxidase gene will provide stable resistance to Sclerotinia blight in peanut and allow maintenance of yield and quality through selection.
Asian soybean rust confirmed in 18 counties of Eastern Virginia in 2006. P. M. PHIPPS (1), D. L. Holshouser (1), S. L. Rideout (2), E. L. Stromberg (3), E. A. Bush (3), M. A. Hansen (3), and M. E. Palm (4). (1) Virginia Tech, Tidewater AREC, Suffolk, VA 23437; (2) Virginia Tech, Eastern Shore AREC, Painter, VA 23420; (3) Dept. Plant Pathology, Physiology and Weed Science, Virginia Tech, Blacksburg, VA 24061; (4) USDA/ARS, Systematic Botany and Mycology Lab, Beltsville, MD 20705.
Sentinel plots of three cultivars (early-, mid-, late-maturing) were planted in May at 10 locations in Virginia for early detection of soybean rust (SBR). A sample of 100 leaflets from the lower canopy was collected biweekly prior to flowering and weekly thereafter from sentinel plots and less frequently from 70 commercial fields. All leaflets were examined under a stereoscope for pustules of Phakopsora pachyrhizi. A total of 363 samples were processed from mid June through early November. SBR was detected on soybeans in Virginia on 9 October, which was 98, 49 and 26 days after detection on commercial soybeans in Georgia, South Carolina and North Carolina, respectively. By 7 November, SBR had been confirmed in 18 counties in Virginia. No significant losses of yield occurred, since SBR appeared after the crop had full to nearly mature pods.
Monitoring the development of downy mildew (Phytophthora phaseoli) of lima bean (Phaseolus lunatus) under field and controlled conditions. L. SANTAMARIA (1), T. A. Evans (1), K. L. Everts (2), A. P. Grybauskas (2), and R. P. Mulrooney (1). (1) Department of Plant and Soil Sciences, University of Delaware, Newark, DE 19716; (2) Plant Science and Landscape Architecture, University of Maryland, College Park, MD 20742 and Salisbury, MD 21801.
Downy mildew of lima bean, caused by Phytophthora phaseoli Thaxt., is one of the most devastating diseases associated with lima bean production in Delaware and the mid-Atlantic region. The objective of this research was to determine the effect of leaf wetness duration (LWD) on the development of downy mildew in the field and under controlled conditions. Field experiments were conducted for three growing seasons during the summers of 2003, 2004 and 2005. Three LWD treatments were evaluated, one with natural conditions and two with different misting treatments during the afternoon. Experiments under controlled conditions consisted in different LW periods inside of a dew chamber. Disease evaluation in the field began when typical symptoms of downy mildew on pins, pods and racemes first appeared, and every three days thereafter. LW from 16:00 – 24:00 resulted in a significant increase in disease incidence over the no mist treatment. Under controlled conditions, a 2 hr LWD was enough to develop 100% incidence of downy mildew with a severity about 30%. A 4 hr LWD produced a 100% incidence and increased severity from 30 to 60%. LWD has a direct effect in the severity of the disease. Release of sporangia occurs most commonly between 9:00 – 17:00 with the highest peak between 13:00 – 15:00.
Using synthetic RPP8 gene clusters to model R gene evolution by meiotic unequal crossing-over. S. SIMON, B. Woffenden, C. Gilbert, J. Jelesko, and J. McDowell. Dept. Plant Pathology, Physiology, and Weed Science, Virginia Tech, Blacksburg, VA 24061.
Unequal crossing-over between different linked genes of a cluster can create new combinations of R genes as well as chimeric genes. The Arabidopsis RPP8 gene belongs to a two-gene cluster, and sequence comparisons suggest that unequal crossing-over has significantly affected the evolution of allelic diversity at RPP8. We are utilizing a genetic screen to model both the frequency and character of unequal crossing-over within a synthetic RPP8 transgenic cluster. We will identify rare meiotic unequal crossover events by coupling chimeric gene formation to the activation of the Firefly Luciferase gene. The recombination breakpoints will be mapped and the pathogen resistance specificities of the chimeric RPP8 genes will be tested. We will also address whether the frequency of meiotic recombination is affected by abiotic and biotic stress. This study will provide general insights into the frequency and character of meiotic unequal crossing-over and its impact on the evolution of functional diversity within R gene clusters.
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