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2006 Potomac Division Meeting Abstracts

March 15-17, 2006 - Rehoboth Beach, Delaware

Posted online April 12, 2006

First report of QoI fungicide resistance of grape downy mildew in North America. A. BAUDOIN and M. Baldwin. Dept. of Plant Pathology, Physiology, and Weed Science, Virginia Tech, Blacksburg, VA 24061.

QoI fungicides, also known as strobilurins, have been commonly used for control of grape downy mildew (Plasmopara viticola) since the first one was registered for use on USA grapes in 1997. QoI resistance of P. viticola has been reported in several European countries and in Brazil, but not previously in North America. In 2005, P. viticola samples from six locations in Virginia and North Carolina were tested by bioassay, using methods patterned after those of Wong and Wilcox (2000, Plant Dis. 84:275-281). Five of 22 isolates had an ED(50) for azoxystrobin (disease incidence) near the baseline (below 1 mg/L) whereas the other 17 isolates had ED(50) values near or over 100 mg/L. The ED(50) of these 17 isolates for pyraclostrobin was approximately 25 mg/L. Potted Vitis labrusca ‘Catawba’ plants were treated with the highest labeled rate (concentration based on 100 gallons per acre) of pyraclostrobin (0.1 g a.i./L, Cabrio) or azoxystrobin (0.3 g a.i./L, Abound), and inoculated the following day with resistant downy mildew isolates at 2 × 10(^4) sporangia per ml. Disease control was poor to nil. Resistant isolates were from four widely separated locations in the two states.

Host range determination of Colletotrichum gloeosporioides from Russian thistle.
C. A. CAVIN and W. L. Bruckart. USDA-ARS-FDWSRU, Ft. Detrick, MD 21702.

Salsola tragus
(Russian thistle) is a major weed pest in the western United States. An isolate of Colletotrichum gloeosporioides (CG) from Hungary currently is a candidate for biological control of S. tragus. Risk assessment to determine potential non-target susceptibility involved inoculation of 64 species with 10(^6) conidia from two-week-old CG cultures grown on V-8 Juice Agar at room temperature and lighting. Over half (36) of the test species were from the Chenopodiaceae, including seven cv of Beta vulgaris (Beet, Chard) and six cv of Spinacia oleracea (Spinach). Inoculated plants were incubated in a dark dew chamber at 25°C for 18 hr and then placed on a greenhouse bench (21–25°C, natural light supplemented to give 16 hr photoperiod) for observation of symptom development. Non-target infections (macroscopic symptoms) were noted on 15 non-target species in the Chenopodiaceae, including (genus, [number of symptomatic species]) Bassia (1) Chenopodium (4), Nitrophila (2), Salicornia (2), Salsola (4), Spinacia (6 cv) and Suaeda (1). All S. tragus were killed, and a native, Suaeda taxifolia, was clearly infected. The other species had only minor symptoms.

Access to web-based information about fungal geographic distribution, host range, and scientific names using the USDA-ARS Systematic Botany and Mycology (SBML) databases: What can SBML do for you?
E. T. CLINE and D. F. Farr. USDA-ARS SBML, Beltsville, MD 20705.

Nomenclature is the science of naming organisms. Stable and descriptive names allow us to comprehend the diversity of the natural world and are the foundation for accurate communication. Multiple names may have been used to refer to the same organism, especially for fungi. Scientific information may be lost if these names are not linked to the current name. Our web-based database provides these links for pathogenic fungi (, which allows users to find the accepted name and all synonyms that have been used in the literature. We will demonstrate how the linking of synonyms is essential in determining for a fungal pathogen: i) the reported host range, ii) the geographic distribution, and iii) whether or not it has ever been reported in the United States. This information is vital for plant quarantine policy makers in deciding when to restrict imports to protect the U.S.A. from introduction of exotic and potentially invasive fungal pathogens.

Detection of Phytophthora ramorum chlamydospores in soil.
G. C. COLBURN and N. Shishkoff. USDA-ARS Foreign Disease Weed Science Research Unit, 1301 Ditto Ave., Fort Detrick, MD 21702.

Phytophthora ramorum
is the causal agent of sudden oak death and is present in Oregon and California. P. ramorum produces abundant chlamydospores but their significance in the disease cycle is unknown. Techniques for the detection of P. ramorum spores in the soil are needed to give regulators a tool to prevent the spread of infested soils. Chlamydospores of an A2 mating type U.S. isolate of P. ramorum were produced in culture to infest soils at 10 and 50 chlamydospores/cm(^3) substrate in sand and natural forest soil from Frederick, MD. Two techniques, direct plating and leaf bating, were used for detecting P. ramorum. Direct plating of soils is conducted by diluting a soil sample in 0.2% water agar solution which is placed on a Phytophthora selective medium. Leaf baiting of soils is conducted by floating 5-mm leaf disks in a flooded soil sample. After a 3 day incubation period, the leaf disks are placed on a Phytophthora selective medium. Direct plating allowed for the quantification of viable chlamydospores and recovered 100% of the spores from both soil types. After an incubation time of 72 h, P. ramorum was detected in 100% of the leaf baits in sand at both concentrations but detection fell to 27% in the forest soil at the higher concentration. Soil samples were collected near symptomatic plants at nine locations in California. Phytophthora spp. were not detected using direct plating or the leaf bait methods. However, these techniques did detect P. ramorum in the California soils after they were infested with 10 chlamydospores/cm(^3).

An Acholeplasma genome encodes enzymes with the potential to degrade and utilize plant cell wall components.
R. E. Davis (1), R. Jomantiene (1,2), I.-M. Lee (1), Y. Zhao (1), R. W. Hammond (1), J. Shao (1), E. L. Dally (1), A. Dawe (3), K. Davis (4), and D. Nuss (4). (1) USDA-Agricultural Research Service, Beltsville, MD USA 20705; (2) Institute of Botany, Vilnius LT-08406, Lithuania; (3) New Mexico State University, Las Cruces, NM USA 88003; (4) University of Maryland Biotechnology Institute, College Park, MD USA 20742.

Phytoplasmas and Acholeplasma spp. are wall-less bacteria classified in class Mollicutes. Phylogenetic analyses based on ribosomal RNA have indicated that phytoplasmas descended from acholeplasma-like ancestors. Yet, little is known concerning genes that were lost and genes that were retained during the divergence of phytoplasmas and acholeplasmas from their last common ancestor (LCA). Whereas, phytoplasmas are obligately parasitic phytopathogens, known Acholeplasma spp. are apparently not obligate parasites in nature and have not been described as phytopathogenic. In silico reconstruction of the genome of their LCA could aid understanding of both Acholeplasma and phytoplasma evolution. Acholeplasma palmae was originally isolated from rotting tissues of a coconut palm (Cocos nucifera), but the precise nature of the association of this or other Acholeplasma spp. with plants has remained undetermined. We now report that the A. palmae genome contains nucleotide sequences potentially encoding putative enzymes involved in plant cell wall degradation and product assimilation, genes not previously found in the genomes of other members of class Mollicutes, including phytoplasmas. The results suggest that such genes were present in the phytoplasma-acholeplasma LCA genome and were lost in phytoplasma evolution. They further point to utilization of plant products by A. palmae and support the hypothesis that this acholeplasma may be capable of penetrating plant tissues as a secondary invader.

Discovery of Cercosporella leaf spot on Russian knapweed from Montana.
F. M. Eskandari (1), W. L. Bruckart (1), J. Littlefield (2), M. B. McMahon (1), and D. G. Luster (1). (1) USDA-ARS-FDWSRU, Ft. Detrick, MD 21702; (2) Dept. Entomology, Montana State Univ., Bozeman, MT 59717.

Acroptilon repens
(Russian knapweed) is a major weed pest in the western United States. A Turkish isolate of Cercosporella acroptili has been a candidate for biological control of A. repens, having undergone evaluations in the containment greenhouse at the FDWSRU. In August, 2005, leaf spot disease symptoms were noted on samples of A. repens collected in Montana that were infected by Puccinia acroptili. A Cercosporella sp. was isolated that was pathogenic on inoculated A. repens in greenhouse tests. The pathogen was identified as C. acroptili on the basis of fungal morphology and symptomatology on A. repens. This isolate was identical to both the type specimen and the candidate isolate from Turkey (FDWSRU #98-001) on the basis of morphological and molecular characteristics. Discovery of C. acroptili in Montana has caused reconsideration of program objectives involving continued evaluation of the Turkish isolate for biological control. Development of the U.S. isolate for biological control of A. repens may now be a possibility.

Genes clustered in sequence-variable mosaics: A unique phenomenon in the genomes of phylogenetically diverse phytoplasmas.
R. Jomantiene (1,2) and R. E. Davis (2). (1) Phytovirus Laboratory, Institute of Botany, Vilnius LT-08406, Lithuania; (2) Molecular Plant Pathology Laboratory, USDA-Agricultural Research Service, Beltsville, MD USA 20705.

Clover phyllody (CPh) and onion yellows (OY-M) phytoplasmas are ‘Candidatus Phytoplasma asteris’-related strains belonging to group16SrI. Recent work showed that the genomes of these strains contain similar gene clusters that are present in multiple, sequence-variable mosaics (Jomantiene and Davis. 2006. FEMS Microbiol Letters 255:59-65). The clusters include full length or partial transposase, ATP-dependent Zn protease, thymidylate kinase, MalK, SMC, FliA, and hypothetical protein genes. To learn whether other phytoplasmas contain similar mosaics of clustered genes, we designed primers to amplify partial sequence-variable mosaics from additional group 16SrI strains and from phytoplasmas belonging to groups 16SrV, 16SrVI, and 16SrXII. The results provided evidence that similar gene clusters were present in all phytoplasmas tested, suggesting that the genomic sequence-variable mosaics (GSVMs) are a common feature of phytoplasma genome architecture.

Expression analysis of genes involved in Rpp1-mediated resistance to soybean rust.
K. T. Schneider (1), J. J. Choi (1), N. W. Alkharouf (3), B. F. Matthews (2), and R. D. Frederick (1). (1) Foreign Disease-Weed Science Research Unit, USDA-ARS, Fort Detrick, MD 21702; (2) Soybean Genomics and Improvement Laboratory, USDA-ARS, Beltsville, MD 20705; (3) Genome Sciences Centre, BC Cancer Agency, Vancouver, BC V5Z 4S6.

Asian soybean rust, P. pachyrhizi, is endemic to southern Asia, Australia, Africa and South America. In 2004, soybean rust was identified for the first time in the continental U.S. in 9 states, and last year the disease was found in more than 100 counties. There is no known rust resistance in commercial U.S. soybean cultivars. Four resistance genes (Rpp1-Rpp4) have been described, but isolates have been identified that overcome each of these resistance genes. A soybean-rust cDNA library enriched for resistance-related transcripts was constructed using suppressive subtractive hybridization (SSH) to identify soybean genes that are expressed in the Rpp1 resistant reaction. Gene expression was compared between Rpp1 resistant and susceptible interactions using a 7883 soybean cDNA microarray that included clones from the SSH library. Differentially expressed cDNAs in the Rpp1 interactions revealed by the microarrays will be discussed.

Detection of Phytophthora ramorum chlamydospores in soil using real-time PCR. K. E. SECHLER, G. C. Colburn, and N. Shishkoff. United States Department of Agriculture, Agricultural Research Service, Foreign Disease-Weed Science Research Unit, 1301 Ditto Avenue, Fort Detrick, MD 21702.

Phytophthora ramorum is the quarantined plant pathogen responsible for the rapid decline of thousands of oak trees in California and Oregon. Similar to other Phytophthora spp., this pathogen produces multiple spore types including chlamydospores, which are capable of persisting in soil. Using real-time PCR with specific primers and probe based on the mitochondrial gene region, detection limits of P. ramorum chlamydospores were established in potting soil and natural forest soil. When artificially infested, a single chlamydospore could be detected in 250 mg of either soil type. P. ramorum was not detected in any of the natural soil samples collected in California from areas known to have infected trees. However, artificial infestation of these soils did result in positive detection.

Development of an interactive web-based resource center for phytoplasma research. J. Shao (1), I.-M. Lee (1), Y. Zhao (1), W. Wei (1), R. Jomantiene (1,2), and R. E. Davis (1). (1) USDA-Agricultural Research Service, Beltsville, MD USA 20705; (2) Institute of Botany, Vilnius LT-08406, Lithuania.

Phytoplasmas are cell-wall-less bacteria that specifically inhabit phloem sieve cells of plants and are transmitted from plant to plant by insect vectors, primarily phloem-feeding leafhoppers. Along with Mycoplasma and other genera of wall-less bacteria, phytoplasmas are classified in class Mollicutes, but unlike many other members of the class, phytoplasmas cannot be isolated in culture. Currently, no less than 15 groups, each representing a distinct species, and over 40 subgroups have been delineated based on restriction fragment length polymorphisms (RFLP) of 16S rDNA, tuf gene, and ribosomal protein gene sequences. New phytoplasmas are discovered each year, and work in phytoplasma classification, phylogeny, genomics is accelerating. To keep pace with this rapid progress, we are constructing a web-based resource at that will include up-to-date information on phytoplasma strains, group and subgroup affiliations, described ‘Candidatus Phytoplasma species’, plant hosts, insect vectors, countries of origin, sources of gene sequence data, and published references. The site will also provide interactive tools for phytoplasma identification, classification, and genomics to further aid the field of phytoplasma research.

Direct observation of infection of Viburnum roots by Phytophthora ramorum using a Petri dish rhizotron.
N. Shishkoff. USDA-ARS FDWSRU, 1301 Ditto Ave., Fort Detrick, MD 21702.

To study root infection by P. ramorum chlamydospores, Viburnum tinus ‘Spring Bouquet’ was rooted in Turface MVP (a fired clay potting medium) in “rhizotrons” made from 9 cm diam Petri dishes stood on edge. 10-15 chlamydospores from a US isolate stored a year in sterile water culture were placed on 8 × 8 mm squares of cellophane and stuck to the side of the Petri dish at four sites. The behavior of chlamydospores and roots was then observed with a dissecting microscope. Petri dishes were also filled with turface, vermiculite, or perlite to observe spore behavior in the absence of roots. Over the course of a month, chlamydospores continuously produced sporangia in all treatments. Some plant roots became visibly infected, with sporangia produced on the root surface. Not every cutting became infected, but on average 22-30% of root tips gave rise to P. ramorum colonies when, at the end of the experiment, roots were plated on selective media. These results suggest chlamydospores present a risk not only to a plant in infested potting mix, but to neighboring plants that might be exposed to irrigation runoff from that pot.

Genetic transformation of pear cultivar ‘Old Home’ and regeneration of transgenic plants for potential disease resistance.
Q. SUN (1,2), W. Wei (1), R. W. Hammond (1), R. E. Davis (1), and Y. Zhao (1). (1) U.S. Department of Agriculture, Agricultural Research Service, Beltsville, MD; (2) Shandong Institute of Pomology, Taian, China.

Pear (Pyrus communis L.) is a nutrient-dense fruit with strong consumer demand and high commercial value. However, most cultivated pear varieties are often susceptible to diseases caused by fungi, bacteria, and viruses. Since pear is highly heterozygotic and has a long juvenile period, conventional breeding for disease resistance is difficult. With the recent advances in molecular biology, artificially engineered resistance has become a new approach to plant disease control. Such genetic engineering requires transformation of parent tissues to introduce foreign genes and subsequent regeneration of transgenic plants with desired characters. Major factors that influence transformation and regeneration of pear cultivars were examined, and optimal conditions were established for efficient transformation and regeneration from leaves of a popular pear cultivar, ‘Old Home’. High transformation efficiency was achieved due to an improved induction stage following initial Agrobacterium infection. In the induction stage, Agrobacterium cells and parent leaf segments were co-cultivated on a liquid induction medium, which yielded a five-fold increase of transformation frequency over conventional co-cultivation on a solid medium. Transgenic shoots were regenerated from transformed cells via an indirect regeneration pathway, which involves a callus proliferation / shoot primordium induction phase and a shoot elongation phase using different hormone combinations. With the new protocol, independent transgenic pear lines carrying antimicrobial peptide genes were regenerated. The transgenic pear plants are being analyzed for foreign gene expression and for their potential disease resistance.

In planta
functional screening of phytoplasma genes: Toward identification of effector proteins involved in pathogenesis.
W. WEI (1), Y. Zhao (1), R. E. Davis (1), C.-J. Chang (2), and Xiurong Shi (1). (1) U.S. Department of Agriculture, Agricultural Research Service, Beltsville, MD; (2) Department of Plant Pathology, University of Georgia, Griffin, GA.

Candidatus Phytoplasma asteris’ and related phytoplasmas are a group of phloem-restricted, cell wall-less bacteria that cause numerous plant diseases worldwide. Despite the recent accomplishments in genome sequencing of two aster yellows phytoplasma strains, knowledge about genes involved in phytoplasma pathogenicity and about molecular interactions between phytoplasmas and their host plants is still lacking. To gain insights into what genes and gene products may play roles in disease induction and/or symptom expression, we launched a genome-scale in planta functional screening of aster yellows phytoplasma effector proteins. A complete set of protein-encoding genes from ‘Candidatus Phytoplasma asteris’–related strain AY1 were amplified by polymerase chain reactions and cloned into Potato virus X (PVX)-based binary expression vector pGR107. Plasmid constructs harboring individual phytoplasma genes were transformed into Agrobacterium tumefaciens strain GV3101. In planta functional screening was carried out by infiltration inoculation of Agrobacterium cells into leaves of Nicotiana benthamiana seedlings. While most of the inoculated plants showed systemic symptoms typical of PVX infection, some exhibited unusual phenotypes including stunting, shoot proliferation, and chlorosis or yellowing of the foliage tissues. Studies are being focused on those plants showing unusual symptoms. Findings of this study may lead to identification of phytoplasma genes and gene products that are involved in phytoplasma pathogenesis and symptom expression.

Development of a computer-simulated 16SrDNA restriction fragment length polymorphism analysis system for classification of phytoplasmas.
W. WEI, Y. Zhao, I.-M. Lee, R. E. Davis, and Q. Sun. U.S. Department of Agriculture, Agricultural Research Service, Beltsville, MD.

Phytoplasmas are cell wall-less pathogenic bacteria that cause numerous plant diseases. Due to the inability to culture phytoplasmas in vitro, it is impossible to differentiate and classify phytoplasmas by the traditional methods that are applied to cultured prokaryotes. To date, restriction fragment length polymorphism (RFLP) analysis of highly conserved 16S rRNA gene sequences has served as a primary method for differentiation and classification of phytoplasmas into groups and subgroups according to similarity coefficients. However, RFLP analysis requires expensive multiple restriction enzyme digestions and subsequent polyacrylamide gel electrophoresis of DNA fragments. To facilitate the implementation and expansion of the existing 16S rRNA gene sequence-based phytoplasma classification scheme, we developed a computer-simulated RFLP analysis method, which allows rapid determination of the classification status of any phytoplasma under investigation. Over 700 publicly available phytoplasma 16S rRNA gene sequences were used in this study. The sequences were aligned using the ClustalW algorithm, and the aligned 1.2 kb fragments were exported to software pDRAW32 for in silico restriction analysis and virtual gel plotting. Applying the previously established classification criteria [Lee et al., IJSB (1998) 48, 1153-1169], the phytoplasma strains were classified into 22 major groups and 77 subgroups based on the simulated RFLP patterns. This study also determined the classification status of more than 250 previously unclassified phytoplasmas, and expanded the number of phytoplasma groups and subgroups.

Integrated management of foliar diseases of watermelon by green manure and biofungicides alternated with chlorothalonil.
X. G. ZHOU (1) and K. L. Everts (1,2). (1) University of Maryland, Salisbury, 21801; (2) University of Delaware, Georgetown, 19947.

Gummy stem blight (GSB), anthracnose and Cercospora leaf spot (CLS) are common foliar diseases of watermelon in the Mid-Atlantic. We evaluated the effect of 1) hairy vetch green manure and 2) biofungicides alternated with chlorothalonil (Chl) applied according to a weather-based forecasting program, Melcast, alone and in combination, on disease severity in 2 years. The hairy vetch winter cover crop that was soil-incorporated in spring suppressed GSB on foliage and anthracnose on fruit. The alternation of Chl with Bacillus subtilis (Serenade), Reynoutria sachalinensis (Milsana), chitosan (Elexa) or harpin (Messenger) on a Melcast schedule reduced GSB and CLS to the levels comparable to those obtained with Chl alone or in rotation with EBDC. The green manure combined with Melcast-scheduled sprays of B. subtilis rotated with Chl resulted in an average of 34% fewer sprays per season and led to control of GSB and CLS comparable to Chl alone applied weekly. The integrated approach developed here could be useful to effectively manage these diseases in the Mid-Atlantic.