Posted online April 10, 2008
Detection and partial characterization of three RNA viruses using improved membrane-based technologiesP. S. CHANG (1), S. A. Tolin (1)(1) Virginia Polytechnic Institute and State University, Blacksburg
A major constraint in examining viral distribution and diversity is the collection, storage of leaf samples, and maintaining sample integrity until testing can be done. Membrane-based technologies for testing plants for viruses, such as tissue blot immunoassay (TBIA) for binding viral antigens to nitrocellulose membranes and Whatman FTA® Plant Cards for nucleic acid binding, have the advantage of requiring no special storage conditions. We modified both the TBIA and FTA® Plant Card protocols and combined them to give one complete method for detecting and partially characterizing three RNA viruses – Cucumber mosaic virus (CMV) and potyviruses Soybean mosaic virus (SMV) and Turnip mosaic virus (TuMV). Cucumoviruses and potyviruses are economically important virus genera that infect over 1,000 plant species in several families worldwide, and are often found co-infecting the same plant in nature. Both viral genera have single-stranded, positive-sense RNA and are aphid transmitted. Isolates of nine CMV, one SMV and one TuMV were positively identified by TBIA. Primers designed from the coat protein of each virus were used for reverse transcription – polymerase chain reactions (RT-PCR) with RNA direct from membranes. All PCR amplifications gave the expected size and sequences from cleaned products, and assigned isolates of CMV to subgroup 1A. These methods alleviate the need for leaf tissue storage or RNA extraction when examining diverse RNA viruses.
Sensitivity of grape downy and powdery mildew to commonly used fungicidesJ. F. COLCOL (1), A. Baudoin (1)(1) Virginia Tech, Blacksburg, VA, USA
Downy mildew (DM, Plasmopara viticola) and powdery mildew (PM, Erysiphe necator) are two important grape diseases that can cause up to 100% crop loss. At present, the application of fungicides is the most practical means to control DM and PM. Unfortunately, many commonly used fungicides (strobilurins, mefenoxam, boscalid, and quinoxyfen) are at risk of resistance development. The ability to quickly detect resistant pathogen populations would allow growers to adjust disease management methods to effectively control DM and PM and minimize the chance of further resistance development. One hundred four PM isolates from 31 mid-Atlantic vineyards were tested by bioassay for resistance to azoxystrobin, boscalid, quinoxyfen, and several ergosterol biosynthesis inhibitors, and 141 DM isolates from 28 vineyards were tested for resistance to azoxystrobin and mefenoxam. The majority of DM and PM isolates were strobilurin (azoxystrobin)-resistant, all DM isolates were mefenoxam-sensitive, all PM isolates were boscalid- and quinoxyfen-sensitive, and a number of PM isolates showed various degrees of reduced sensitivity to ergosterol biosynthesis inhibitors. A point mutation from glycine to alanine at position 143 (G143A) of the cytochrome b gene has been identified as the main cause of high levels of strobilurin resistance in many pathogens. SYBR-green real-time PCR was used to quantify G143A. The majority of the strobilurin-resistant DM and PM isolates contained 94–100% of the 143A allele. Strobilurin-sensitive DM and PM isolates contained less than 1% of 143A. Interestingly, about 1.5% and 19% of strobilurin-resistant DM and PM isolates, respectively, contained less than 1% 143A, indicating that other mutations may be responsible for their resistance.
Clustered genes common to both Aspergillus fumigatus and ergot fungi control early steps within the ergot alkaloid pathwayC. M. COYLE (1), K. E. Goetz (1), D. G. Panaccione (1)(1) Division of Plant and Soil Sciences, West Virginia University, Morgantown, WV 26506-6108, USA
Ergot alkaloids are mycotoxins that affect humans and animals through their interactions with multiple classes of monoamine receptors. They were discovered and have been studied extensively in the ergot fungus, Claviceps purpurea. Ergot alkaloids also have been reported in some closely related fungi, such as Neotyphodium spp. endophytes of grasses. Surprisingly, a different set of ergot alkaloids are produced by the distantly related fungus Aspergillus fumigatus, a common saprophyte and opportunistic pathogen of humans. We hypothesize that the ergot pathways of A. fumigatus and the ergot fungi share early steps and then diverge after the biosynthesis of the important intermediate compound, chanoclavine. A homologue of the gene (dmaW) encoding dimethylallyltryptophan synthase, which catalyzes the first step in the ergot alkaloid pathway of the Neotyphodium spp., was found in the A. fumigatus genome. Knockout of this gene via homologous recombination rendered a mutant that lacked ergot alkaloids. Aspergillus fumigatus dmaW is part of a cluster of genes that resemble genes clustered with dmaW in the ergot fungi. These genes may encode early, shared steps in the ergot alkaloid pathway. Four of these genes, easA, easC, easE, and easF, were knocked out; each mutation altered the profile of ergot alkaloids in A. fumigatus. Three of the mutants were blocked prior to chanoclavine, whereas one accumulated chanoclavine but not ergot alkaloids from the latter part of the pathway. The data indicate that the ergot alkaloid pathways of A. fumigatus and the ergot fungi share common early steps before diverging to produce different end products. Knockout mutants lacking ergot alkaloids will be valuable for testing the role of these toxins in animal pathogenesis and toxicoses. Characterization of the ergot alkaloid pathway and the ability to control the spectrum of alkaloids produced may lead to advances in the agricultural and medical fields.
Protocol for maintenance of Synchytrium solstitiale, cause of false rust on yellow starthistle, under greenhouse conditionsF. M. Eskandari (1), W. L. BRUCKART (1), T. L. Widmer (1)(1) USDA, ARS
An isolate of Synchytrium solstitiale was collected in France and sent to the FDWSRU to evaluate as a candidate for biological control of yellow starthistle (YST, Centaurea solstitialis). Procedure was needed for maintenance of the pathogen in the greenhouse before other research was possible, since inoculum is available from France only a few months of the year. Leaf pieces, 1-cm(^2), with galls were placed at the center of YST rosettes to provide inoculum in this study. YST growing either in pots or whole plants growing in flasks of water (soil was washed from roots) were inoculated as described and placed either in a Percival growth/dew chamber (10°C, night and 15°C, day; 8 hr photoperiod) or in a 13°C conventional dew chamber with continuous light (40 watt incandescent light bulb). Plants put into the Percival were enclosed in plastic bags and misted daily to maintain free moisture on leaves; moisture in the other dew chamber came in the form of dew. After 10 days, plants were removed from the chambers and placed in a 20°C greenhouse with shading. Plants were rated weekly for gall formation, development of resting spores, proportion of infected leaves, and disease severity (a general visual rating of disease). Regardless of variable, whole plants in flasks that were in the Percival chamber developed significantly more infection than those in pots or in the conventional dew chamber. These differences may be due to the fact that rosette leaf bases are submerged in the water and susceptible tissue may be more easily accessed by zoospores. Regardless, it has now been possible to maintain S. solstitiale artificially in the greenhouse for over a year following these protocols.
An improved assay for detection of Acidovorax avenae subsp. citrulli in watermelon and melon seedsJ. FENG (2), T. Zhao (3), A. Sechler (4), P. Randhawa (1), J. Li (2), N. Schaad (4)(1) Calif. Seed Pl. Lab., Elverta, CA, USA; (2) China Agric. Univ., Beijing, China; (3) Chinese Acad. Agric. Sci., Beijing, China; (4) USDA/ARS, FDWSRU, Ft. Detrick, MD, USA
Acidovorax avenae subsp. citrulli (Aac), the causal agent of watermelon fruit blotch, is a serious seedborne pathogen. Although attempts have been made to develop a routine seed assay to detect Aac in seeds, none is routinely used. We describe a agar plating, real-time PCR assay for detection of Aac. Bacteria were extracted by soaking seeds in buffer containing vancomycin and assayed by dilution plating onto ethanol bromothymol blue/brilliant blue R (EBB) agar and EBB agar with ampicillin (EBBA), direct real-time PCR, and real-time BIO-PCR (enrichment PCR). Using extracts of 1000 healthy seeds spiked with cells of Aac, the BIO-PCR assay using EBBA agar detected Aac in extracts containing as few as one cell per ml. Replicates, when spiked with a single naturally infested seed containing as few as 1040 colony forming units of Aac/seed, were positive by all assays. This combined agar plating, real-time PCR protocol is a useful routine assay for detection of Aac in watermelon and melon seeds.
Microbial biofungicides for disease control and enhanced crop yields in organic and sustainable agricultureS. S. GNANAMANICKAM (1), M. Christopher (1), S. Inman (1), L. West (1), S. Semones (1)(1) Novozymes Biologicals, Salem, VA 24153
It is known that the prospect of manipulating crop rhizosphere microbial populations by inoculation of beneficial bacteria to increase plant growth has shown considerable promise in laboratory and greenhouse studies, but responses have been variable in the field. Growth-promotion and pathogen suppression are the two well known mechanisms exhibited by PGPR. Our bio-innovation research with PGPR and biocontrol strains has led to the development of bacterial inoculants that consistently promise higher crop productivity. In this presentation, we describe results from our laboratory profiling work and field tests conducted in India and US during 2006 and 2007 for two of our products whose active ingredients are Bacillus strains. EcoGuardGN is an EPA-registered biofungicide whose active ingredient is Bacillus licheniformis 3086. In a field test conducted during 2006 at Coimbatore, India weekly applications of EcoGuard GN at 260 l/ha led to yields of 30.5 t/ha while the untreated control crop produced 12.6 t/ha (58% increase). The biofungicide used at this concentration also reduced the incidence of downy mildew (caused by Plasmopara viticola). The percent disease index (PDI) in treated plots was reduced by 38.2% compared to untreated control. TAEGRO, also an EPA-registered biofungicide formulated in cornstarch contains 24.5% of freeze-dried spores of Bacillus subtilis var. amyloliquefaciens FZB24. In California tomato trials carried out in 2007, TAEGRO afforded substantial control of Pseudomonas syringae pv. tomato better than Kocide 2000. In addition to its known fungicidal properties against Rhizoctonia, Fusarium, and Phytophthora, TAEGRO has also shown a broad-spectrum of activity in the laboratory against field strains of bacterial plant pathogens such as Ralstonia solanacearum, Erwinia amylovora, and pathovars of Pseudomonas syringae and Xanthomonas campestris. In recent greenhouse tests, the incorporation of TAEGRO in potting mix at 0.613 g/gallon pot suppressed Ralstonia solanacearum (bacterial wilt of tomato) by 75% over that of the untreated control. TAEGRO treatments in the field also consistently afford 7–15% enhancement of crop yields in crops such as tomato, potato, cucumber and several ornamental crops. We believe that TAEGRO has the potential of a biobactericide that can control important diseases such as bacterial wilt and fire blight and afford growth enhancement/crop productivity in the organic and global agricultural market segments worldwide.
Multilocus sequence typing system for Candidatus Liberibacter asiaticus, a causal agent of HuanglongbingH. HU (1), E. L. Schuenzel (2), A. Sechler (2), Z. Wang (1), N. W. Schaad (2)(1) Gene Engineering Research Center, Chongqing University, Chongqing 400045, China; (2) USDA-ARS, FDWSRU, Ft. Detrick, MD 21702
Huanglongbing (HLB) is one of the most devastating citrus diseases worldwide affecting all varieties of citrus. The suspect causal agents have been identified by 16s rDNA sequencing as phloem-limited bacteria belonging to the alpha-subdivision of proteobacteria. Three species have been identified, Candidatus Liberibacter asiaticus (Las), L. americanus, and L. africanus, with Las being the most widespread. Because the organisms can not be cultivated, the evolutionary relationship and geographic diversity are poorly understood. Multilocus sequence typing (MLST) is a nucleotide-based method for identifying strains of bacteria based on sequence differences across 5–10 genes. MLST has proven to be reliable for determining genetic diversity and fingerprinting of strains. We devised an MLST scheme for Las based on 3 loci, tufB, rpoB and dnaA, for about 20 strains collected from China, Thailand, Brazil and Florida. A phylogenetic tree of the results showed little genetic diversity or geographic variation among the strains.
Measuring gene flow from transgenic peanuts to nontransgenics in the same fieldJ. HU (2), P. Phipps (2), D. Partridge (2), S. Chriscoe (1), E. Grabau (1)(1) Plant Path, Phys & Weed Sci, Virginia Tech, Blacksburg, VA 24060; (2) Tidewater Agricultural Research and Extension Center, Virginia Tech, Suffolk, VA 23437
Three transgenic lines of Virginia-type peanut cultivars (Wilson, Perry and NC 7) with a barley oxalate oxidase gene for resistance to Sclerotinia blight were evaluated to determine the frequency and distance of transgene flow through natural hybridization under field conditions. Rows were 3 m long and space 0.9 m apart. One row of a non-transformed variety was planted in the center of each plot and bordered on each side by a single transformed line of the same variety. Additional 24 rows of the corresponding non-transformed variety were planted on each side of the plot. Bumble bees (Bombus terrestris L.) were observed collecting pollen from flowers during the growing season. Whole pods of peanuts were harvested from each row, shelled to remove kernels and 285 seed embryos per row were tested for oxalate oxidase expression by a colormetric measure of hydrogen peroxide released from oxalic acid substrate. The out-crossing rate was 1.1% in Wilson and Perry in the nontransgenic row bordered on each side by a transgenic row. NC7 showed an out-crossing of 1.4% at 1.8 m (2 rows) to the right of the closest transgenic row. Out-crossing rate decreased as distance increased. The out-crossing rate reached zero at 3.7 m from transgenic rows of Wilson and Perry, while outcrossing was detected up to 17.4 m (19 rows) in NC7. These results indicate that transgene flow occurs at low but detectable frequencies and usually over short distances. Physical separation of transgenic and nontransgenic peanuts by greater than 20 m (22 rows) should be sufficient to minimize transgene contamination in seed production.
Phytophthora database: A model for developing a global human and knowledge network against plant pathogenS. KANG (1)(1) Dept. of Plant Pathology, Penn State, University Park, PA, USA
The rapid expansion of global commerce and human travel has greatly accelerated the introduction of non-indigenous pathogens and exotic variants of indigenous ones. This expansion, coupled with global climate change, is reshaping North American pathogen communities. As diagnosis systems have become more sophisticated, detection of diseases caused by novel pathogens also is on the rise. If history is a guide, these forces will lead to the appearance of new diseases and reemergence of quiescent ones. Pathogen dynamics and disease epidemics have biological, environmental, socioeconomical, and geospatial dimensions and thus require integrated analyses of heterogeneous knowledge via the cooperation from diverse stakeholders in order to properly understand and respond to them. Given the pathogen movements across political boundaries and the interconnections among national economies, our response should also be coordinated with international partners. However, to date efforts to study and manage this threat have been fragmented, mostly regional, and limited to coping with immediate crises. I will use the Phytophthora Database (http://www.phytophthoradb.org), an online, forensic database that was established to support rapid detection and diagnosis of Phytophthora species, as an example to outline a blueprint for establishing a global human and knowledge network against plant pathogens.
Infectivity of virulent and transgenic hypovirulent strains of Cryphonectria parasitica, and the influence of inoculation methodology on canker developmentS. C. KENALEY (1), M. L. Double (1), W. L. MacDonald (1)(1) Division of Plant and Soil Sciences, West Virginia University, Morgantown, WV 26505-6108
The deployment of transgenic (TG) hypovirulent strains for the biological control of the chestnut blight fungus (Cryphonectria parasitica) in North America requires a more thorough understanding of the differences in the infectivity of TG and virulent (V) inoculum as well as the influence of inoculation method on canker development. The objectives of this study were to: (1) compare the infectivity of TG and V ascospores, conidia, and a mixture of mycelium and conidia; and, (2) examine the effect(s) of strain, inoculum type, wound size (diameter: 2.0 mm and 9.0 mm), and the delivery medium (potato dextrose and water agar) on the development of artificially initiated cankers. Strain, inoculum, wound size were the most significant factors contributing to infection. In addition, a significant interaction existed among strain, inoculum, and wound size (strain × inoculum × wound size). For all inoculation methods, those performed with V inoculum resulted in the greatest percent infection (55.6%) and was significantly greater than inoculations utilizing TG inoculum (24.3%). The infectivity of V inoculum types was not significantly different regardless of delivery method. Percent infection for V ascospore, conidia, and mycelium and conidia inoculum types was 47.9%, 58.3%, and 60.4%, respectively. In comparison, percent infection for the TG mycelium and conidia inoculum type (41.7%) was significantly greater than TG ascospores (12.5%) and conidia (18.8%). For all treatment combinations, 9.0-mm wounds resulted in infection 66.1% of the time in contrast to 33.9% of the 2.0-mm wounds. Inoculations with V conidia and a wound size of 9.0 mm resulted in the greatest number of infections. Linear growth of cankers that resulted from infection was most significantly influenced by strain, delivery medium, and wound size. Unlike the model for infection, delivery medium contributed significantly to linear growth but inoculum type did not. The mean linear growth of the V cankers (mean = 5.7 cm) was significantly greater than that of TG cankers (mean = 2.6 cm). The growth of cankers associated with 9.0-mm wounds was significantly greater than those initiated with 2.0-mm wounds, and wounds filled with potato dextrose agar resulted in larger cankers than those filled with water agar. Strain, delivery medium, and wound size were the most important effects between or among their associated interactions. Although this experiment did not attempt to examine the natural conditions for infection, it clearly identified the limitations of TG inoculum in initiating cankers and the subsequent growth that occurs. These limitations may dramatically affect the performance of TG strains in forest settings thus influencing biological control strategies.
Potential for some common tank-mix field treatments to control bacterial contaminationM. MAHOVIC (1), S. Rideout (1)(1) Virginia Tech
Water used for pesticide application has been suggested as a potential vehicle for several bacterial diseases to contaminate commercially produced, fresh-market field tomatoes. Several commonly applied pesticides are bactericidal, and often presumed to control any contamination suspended in application waters. Some fungicides have been investigated for such efficacy, but many of the products commonly used on the Eastern Shore of Virginia (ESV) have not been investigated. Here, the causal agents of bacterial soft rot, E. carotovora subsp. carotovora, a common postharvest disease, and the human pathogen Salmonella enterica, which has been linked to enteritis contracted from tomato consumption, were tested in an aqueous environment for susceptibility to 12 common pesticides in use on the ESV. Treatments included copper compounds, oxidants, bio-control agents, antibiotics, and other traditional compounds. Observations and questioning of commercial growers suggests that, beginning at tank-mixing and ending at the initial application in-field, water and pesticides can have as little as 15 min of contact time prior to encountering the biosphere. This would thus be the shortest contact time a treatment would have to act upon any suspended bacteria, and thus was the time chosen for this study. Bacteria were grown in culture and standardized for each treatment. Aqueous suspensions of the bacteria + treatment were then incubated at room temperature for 15 min. Samples of 1 ml each were drawn from each suspension, serial diluted up to 3 log(10), and pour-plated with molten agar and stored for observation. Plates were then observed for colony formation over 72 hrs. Of those materials tested, the only oxidative agent currently labeled for field application, peroxyacetic acid, had significant log reductions compared to control.
Within-season distribution of myclobutanil resistance in populations of Venturia inaequalis in VirginiaS. C. MARINE (1), D. G. Schmale (1), K. S. Yoder (2)(1) Virginia Polytechnic Institute and State University, Blacksburg, VA 24060; (2) Virginia Tech Agricultural Research and Extension Center, Winchester, VA 22602
Apple scab, caused by Venturia inaequalis (Vi), is a devastating disease of apple worldwide. Myclobutanil, a sterol-inhibiting (SI) fungicide, is commonly used to control apple scab in the eastern U.S. Populations of Vi in commercial apple orchards in Virginia have demonstrated resistance to myclobutanil and other SI fungicides, yet little is known about the nature and distribution of fungicide resistance in these populations. The objective of this study was to examine the within-season distribution of myclobutanil resistance in populations of Vi in Virginia. Shoot tips were banded on several branches of apple trees representing four cultivars in four different research blocks. Leaves with new apple scab lesions were collected in May, June, July and August of 2007, and over 400 isolates of Vi were cultured from these lesions. Fungicide resistance was evaluated in 82 of these isolates by monitoring their growth on agar plates containing 0 ppm, 0.1 ppm, 0.5 ppm, or 1 ppm myclobutanil (Nova 40W). Radial colony growth was measured weekly for 4 weeks. A similar range of fungicide resistance was observed for isolates collected at each sampling interval. After 4 weeks, agar treatments were significantly different (P < 0.01), but we did not observe a difference in fungicide resistance between isolates collected from treated or non-treated trees (P = 0.63). Sensitive and moderately resistant isolates were collected from both treated and non-treated trees. Repeated treatments of myclobutanil to a mixed population of Vi may result in a range of fungicide resistance in these populations, but this selective pressure may act over multiple growing seasons.
Global gene expression analysis of the rice blast pathogen Magnaporthe oryzae under stress conditionsS. M. MATHIONI (2), C. Rizzo (1), N. M. Donofrio (2)(1) Agilent Technologies; (2) University of Delaware
Magnaporthe oryzae, the causal agent of the most devastating disease of rice, rice blast, potentially encounters a variety of stresses during its life cycle, such as oxidative environment and temperature shift. The study of the M. oryzae transcriptome can lead to new insights about what molecular mechanisms are used by the pathogen in response to stress. We performed a global gene expression analysis of M. oryzae under various stresses in vitro including oxidative induced by methyl viologen and heat-shock. These conditions were compared to M. oryzae growing in planta in both rice and barley detached leaves. All conditions were compared to the fungus under ideal laboratory growth conditions. Microarrays were used to measure the transcription of all M. oryzae genes and compare them to the control. Results showed that 287 genes are differentially expressed in the treatments in relation to the control. They are involved in electron transport, heavy metal ion transport, oxidoreductase activity, signal transduction, lipid metabolism, and cell organization. Ongoing experiments of validation of the differential gene expression will indicate which of those genes regulate the fungus response to stress conditions and further study of the same genes might help understanding their specific functions and mechanisms of response to stress.
Effects of induced resistance on penetration and development of tobacco cyst nematode in susceptible and resistant flue-cured tobaccoV. PARKUNAN (1), C. S. Johnson (1), J. D. Eisenback (2)(1) Virginia Tech Southern Piedmont AREC, Blackstone, VA, USA; (2) Virginia Tech, Dept. Plant Pathology, Physiology, and Weed Science, Blacksburg, VA, USA
The efficacy of systemic acquired resistance (SAR) and induced systemic resistance (ISR) were evaluated against tobacco cyst nematode (Globodera tabacum solanacearum-GTS) root penetration and development. Nematode penetration and development were evaluated in two experiments on GTS susceptible (cv. K326) and resistant (cv. NC71) flue-cured tobacco cultivars arranged in a randomized complete block design with 5 replications. SAR was activated through a single foliar application of acibenzolar-S-methyl (ASM), whereas ISR was activated through soil incorporation of a combination of two Bacillus spp., B. amyloliquefaciens IN937a and B. subtilis A13 (BioYield™). Pots were infested with 7,000 eggs of GTS 1-week after SAR and ISR treatments had been applied to 6-week-old plants. All roots were stained and counted for preparasitic and parasitic stages of GTS 3 weeks later. Numbers of preparasitic nematodes in roots were similar for both ASM and BioYield™ treatments compared to the untreated control for both susceptible and resistant cultivars, except in one test where BioYield™ reduced the numbers of preparasitic nematodes, but not ASM. Additionally, BioYield™ reduced parasitic stages of GTS more consistently than ASM on both the susceptible and the resistant cultivar. ASM reduced the parasitic stages of GTS in the susceptible cultivar in both tests, but in only one of the two tests for the resistant cultivar. Overall, BioYield™ reduced the numbers of parasitic stages of GTS more than ASM on both the susceptible and the resistant cultivar.
Soybean rust development and disease incidence in the Mid-Atlantic Region in 2006 and 2007P. M. PHIPPS (1), E. L. Stromberg (2), S. L. Rideout (3), D. L. Holshouser (4)(1) Virginia Tech; (2) Virginia Tech, Blacksburg, VA, USA; (3) Virginia Tech, Painter, VA, USA; (4) Virginia Tech, Suffolk, VA, USA
Soybean rust, caused by Phakopsora pachyrhizi, was detected in 2006 on soybean in Georgia (GA) on August 4, South Carolina (SC) on August 14, North Carolina (NC) on September 13, and Virginia (VA) on October 9. High temperatures and below normal rainfall in July and August delayed disease development in 2006. However, soybean rust regained momentum following heavy rainfall (avg. = 8.04 in.) from Tropical Storm Ernesto between August 31 and September 2. By the end of 2006, soybean rust had been detected in 42 counties of NC and 19 counties of VA. In 2007, disease spread was first detected on soybean in GA on August 13, SC on September 10, NC on October 11, and VA on October 19. By season end, soybean rust had been detected in 6 counties of NC and 9 counties in VA. Most of eastern VA in 2007 experienced severe drought, except for above normal rainfall in October in the Tidewater Area and near normal rainfall in August. In 2006 and 2007, disease detection began in states bordering the Gulf of Mexico where P. pachyrhizi overwinters on kudzu. Thereafter, disease spread followed two primary pathways northward that coincided with USDA reports of high soybean acreage by counties in the United States: 1) states bordering the Mississippi River and 2) states in the Mid-Atlantic Region. Efforts for early detection of soybean rust in VA included regular sampling of 10 sentinel plots and multiple commercial fields. Sentinel plots were planted to maturity group III, V and VI cultivars at each location. A total of 363 and 430 samples of leaflets in 2006 and 2007, respectively, were processed from sentinel plots and commercial fields. All samples were placed in moist chambers for 3 to 5 days at room temperature and leaflets were scanned with a dissecting scope for pustules of soybean rust. Positive samples were confirmed by an ELISA test. Spore traps for collection of airborne spores in VA were placed at five locations of sentinel plots in 2006 and 2007, and wet deposition traps were placed at these locations in 2007. A total of 4, 11 and 4 spores with morphological characteristics of P. pachyrhizi were collected in July, August and September, respectively, in 2006 and none were detected in 2007. Wet deposition traps used PCR for rust detection and were positive for P. pachyrhizi during collections from August 6 to 14 at two locations and one location from August 27 to September 11. Soybean growers and county agents in VA rely on weekly to bi-weekly reports on detection of soybean rust for managing disease risk by application of fungicide sprays. These reports and control recommendations are posted throughout the year at http://www.sbrusa.net. To date, applications of fungicides for control of soybean rust have not been recommended in Virginia because disease outbreaks have occurred after growth stage R6 (full seed) when plants are no longer vulnerable to yield loss by soybean rust.
A comparison of CombiMatrix CustomArray and ElectraSense Prunus pathogen detection arraysD. J. SHERMAN (1)(1) USDA-ARS-FDWSRU
A comprehensive pathogen array was developed for the detection of viral, bacterial, fungal, stramenopile and phytoplasma pathogens of many major crops in the Prunus genus. The two detection systems, CombiMatrix’s CustomArray™ and ElectraSense™, were compared to see if there was a difference in specificity, sensitivity and/or background. The CustomArray™ uses a fluorescent labeling system that directly labels the DNA fragments prior to hybridization. The ElectraSense™ platform uses electrochemical detection which includes biotinylating the DNA fragments and a post-hybridization labeling step. Three different samples were tested using each platform: healthy Prunus tissue, cultured Phytophthora cactorum, and an artificial mix of cultured Xylella fastidiosa, cultured Phytophthora syringae and Plum pox virus infected Prunus tissue. For both formats, the samples were amplified by reverse transcription-whole genome amplification and fragmented. After fragmentation the samples were split, and the two halves were labeled separately for the two formats. The CustomArray™ had more positive signals for both specific and non-specific probes. The ElectraSense™ arrays had fewer positive signals suggesting that the platform has lower sensitivity. Both platforms had issues with non-specific hybridization. The ElectraSense™ arrays had reduced sensitivity, but no increase in specificity. Therefore, the CustomArray™ platform was chosen to be more suitable.
Characterization of the functional domains of AvrRxo1 expressed by Xanthomonas oryzae pv. oryzicolaB. ZHAO (1)(1) Virginia Tech
Rice bacterial leaf streak (BLS) disease caused by Xanthomonas oryzae pv. oryzicola (Xoc) is a serious bacterial disease in the tropical rice production areas of Asia. Thus far, no resistance (R) gene that can control BLS has been identified from rice germplasm. We previously isolated a maize gene Rxo1 that conditions a defense response to Xoc carrying the effector gene avrRxo1. Transgenic rice plants expressing Rxo1 are highly resistant to BLS. We recently demonstrated that Rxo1 maintains its functional specificity in the dicot plant Nicotiana benthamiana. This is the first monocot NB-LRR gene that maintains its function in a dicot plant species. Characterization of the molecular basis of AvrRxo1/Rxo1 interaction could help us understand the mechanism of restricted taxonomic functionality and enable us to transfer NB-LRR genes from model species into important crop plants to achieve durable disease resistance. Bioinformatics analysis on AvrRxo1 leads us to identify several function domains including a conserved ATP binding site. Point mutation in the ATP binding site of AvrRxo1 suppressed its ability to trigger an Rxo1 dependent HR in N. benthamiana. Transient expression of avrRxo1 itself in another plant species N. tabaccum results in a strong HR. This newly developed transient assay system provides us a powerful tool to analyze the functional domains of AvrRxo1. The biological and biochemical functions of different domains including the ATP binding sites of AvrRxo1 will be discussed.