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2004 Potomac Division Meeting Abstracts

April 6-7, 2004 - Shepherdstown, West Virginia

Posted online August 9, 2004

New association of Phytophthora species in oak ecosystems in central Appalachian forests. Y. BALCI (1), W. L. MacDonald (1), and K. W. Gottschalk (2). (1) West Virginia University, Division of Plant and Soil Sciences, 401 Brooks Hall, Morgantown, WV 26506-6058; (2) USDA Forest Service, Northeastern Research Station, 180 Canfield St., Morgantown, WV 26505-3180. Publication no. P-2005-0001-PTA.

Recent investigations have shown that there are a variety of Phytophthora species associated with oak trees in Europe and adjacent countries. A preliminary survey has been conducted in oak forests of northern West Virginia and western Maryland to investigate the Phytophthoras present in forest soil. Soil samples taken from around the base of healthy and declining oak trees were baited with 3-7 day old oak leaflets and then plated on PARPNH- medium. P. cinnamomi was the most frequently encountered species. Two other species were recovered including P. europaea and a yet undescribed Phytophthora. These results suggest that there is a diverse population of Phytophthora species in eastern forest soils including natural, exotic and undescribed species. This is the first report of P. cinnamomi in central Appalachian oak forests and P. europaea in North America.

Comparison of chestnut canker treatment procedures for hypovirus introduction.
B. C. Bell and M. L. DOUBLE. Division of Plant and Soil Sciences, West Virginia University, Morgantown, WV 26506. Publication no. P-2005-0002-PTA.

Hypovirulent inoculum of Cryphonectria parasitica historically has been introduced into cankers on American chestnut by inoculating wounds made to the canker margin. This study examined other techniques to deliver hypoviruses, including a non-invasive painting treatment and invasive treatments that employed either blade cuts or punch-wounds made to the canker. The effects of canker coverings and vegetative compatibility also were examined. Hypovirus transmission was evaluated 3 and 9 months after treatment. Hypovirus transmission was greatest in punch-wounded cankers and occurred most often in areas of the canker that formed after treatment. Canker coverings promoted the survival of treatment inoculum but did not enhance hypovirus transmission. Canker expansion was most restricted in cankers that were treated with compatible inoculum, regardless of the application method. Similar results were obtained at the two evaluation dates.

What are the implications of a foreign hemibiotrophic fungus for biological control of weeds?
C. A. CAVIN and W. L. Bruckart. USDA-ARS, Ft. Detrick, MD 21702. Publication no. P-2005-0003-PTA.

An isolate of Colletotrichum gloeosporioides (Cg), which kills Russian thistle (RT, Salsola tragus), is being evaluated for use in biological control. Host range tests indicate minor infection of closely related Salsola spp., and there was limited symptom development on senescent spinach. No symptoms developed on young (non-flowering) spinach, while inoculations with C. dematium, a U.S. spinach pathogen, under the same conditions clearly damaged the plants. This suggests a possible hemibiotrophic disease response in spinach (i.e., symptom development after an extended latent period), something not uncommon among species of Colletotrichum. In research with other Colletotrichum species, latent or “symptomless” infections have been reported after induction of artificial senescence following treatment of plants with paraquat. Similar “symptomless” responses have been noted for at least three crop species in the current host range determination of Cg from RT. Truly latent infections are considered “no risk” regarding evaluation of this candidate, but the hemibiotrophic response complicates the host range determination and it raises issues concerning risk.

Evaluation and identification of a Cladosporium sp. as a biological control agent of yellow starthistle in the USA.
E. L. CRUNKLETON, D. K. Berner, and M. B. McMahon. USDA-ARS-FDWSRU, 1301 Ditto Avenue, Ft. Detrick, MD 21702. Publication no. P-2005-0004-PTA.

Yellow starthistle (Centaurea solstitialis L., YST), an invasive weed in California and the western U.S., is targeted for biological control. In 2003, an epidemic of dying YST plants was found near Kolzani, Greece. Diseased YST plants were sent to the Foreign Disease Weed Science Research Unit, USDA/ARS, Ft. Detrick, MD where the causal organism of the disease was isolated. Based on culture characteristics, fungal morphology and ITS sequence the organism was identified as Cladosporium herbarum. Rosettes and bolted YST plants were inoculated with spores of the fungus and placed in a chamber with 8 hr dew and 12 hr light daily. Plants in the rosette stage were resistant, but the fungus was very aggressive on bolted plants. Within 4-6 days of inoculation necrosis developed on leaves and stems and then spread to capitula, often resulting in plant death. The fungus was also aggressive on developing flowers. The fungus was reisolated consistently from plants in two separate tests. Results of host range tests will establish if this isolate of C. herbarum has potential as a biological control agent of YST in the USA.

Fungicide efficacy and timing trials for the control of downy mildew on baby lima bean.
J. F. DAVEY, R. P. Mulrooney, T. A. Evans, and R. B. Carroll. University of Delaware, Newark, DE. Publication no. P-2005-0005-PTA.

Downy mildew of baby lima bean caused by Phytophthora phaseoli is the number one disease on lima beans in Delaware. In 2003, field experiments tested the timing of applications and efficacy of fungicides for downy mildew control. Baby lima beans were grown and artificially inoculated with a sporangial suspension of Phytophthora phaseoli race E in Newark, DE. Ridomil Gold/Copper WP 2.0 lb applied three times every seven days and Ridomil Gold/Copper WP 2.0 lb followed by two applications of Champ DP 2.0 lb every seven days increased yield and decreased disease severity in the protectant fungicide trial. In the curative fungicide trial Ridomil Gold/Copper WP 2.0 lb applied twice every seven days and Ridomil Gold/Copper WP 2.0 lb followed by two applications of Champ DP 2.0 lb every seven days also performed significantly better than the control. Phostrol 4.0 pt and Ridomil Gold/Copper WP 2.0 lb applied four times every seven days significantly increased yield and decreased disease severity in the fungicide efficacy trial. These experiments have identified both effective and affordable control options for downy mildew of lima bean.

Highbush blueberry flower buds as a winter reservoir of Colletotrichum acutatum. A. DeMARSAY and P. V. Oudemans. Dept. of Plant Biology and Pathology, Rutgers University Blueberry and Cranberry Research and Extension Center, Chatsworth, NJ 08019. Publication no. P-2005-0006-PTA.

Colletotrichum acutatum, a destructive pathogen of many fruit and nut crops, is the causal agent of blueberry anthracnose. Understanding how the fungus overwinters on blueberry is vital to improving disease management, because overwintering infections produce the primary inoculum for fruit infection. In a three-year study of dormant twigs, we found that flower (inflorescence) buds serve as the primary winter reservoir of C. acutatum, harboring more than twice as many infections as blighted wood in both a susceptible and a resistant cultivar. We have also shown that the pathogen overwinters inside the bud rather than on its surface. Infections in dormant buds are concentrated in the outer bud scales. Samples from inoculated and naturally infested bushes indicate that C. acutatum infects buds early in their development in the summer, close to floral initiation. Postharvest fungicide applications might prevent new infections in buds, which would reduce disease pressure in succeeding years. Field trials to assess the effects of several classes of fungicides are underway.

Biological control of Canada thistle (Cirsium arvense) using the rust Puccinia punctiformis. A. M. DEMERS (1), P. A. Backman (1), and D. K. Berner (2). (1) Dept. Plant Pathology, Penn State Univ., University Park, PA 16802; (2) USDA-ARS-FDWSRU, 1301 Ditto Ave., Ft. Detrick, MD 21702. Publication no. P-2005-0007-PTA.

Canada thistle (CT) is an introduced invasive weed in the U.S. and Canada and a target of biological control efforts. Puccinia punctiformis is an endemic, autoecious rust that limits flowering and vegetative growth of CT. Systemic infections of CT root buds by rust basidiospores, give rise to spindly, pale shoots that usually die after producing infective spores. The goal of this study was to determine the density of systemically infected plants per unit patch area needed to initiate an epidemic and achieve biological control. Systemically infected CT seedlings were planted in healthy thistle patches to serve as dynamic sources of infective inoculum. Newly developed systemically infected shoots in each patch were counted regularly over the course of the season and compared to the proportion of healthy plants. A mathematical model, based on field data, is being developed to understand the spread of P. punctiformis in CT patches and determine the density of systemically infected plants necessary to achieve CT control.

and Cougarblight: Is there a difference?
M. M. DEWDNEY (1), A. R. Biggs (2), and W. W. Turechek (1). (1) Cornell University, Geneva, NY; (2) West Virginia University, Kearneysville, WV. Publication no. P-2005-0008-PTA.

MARYBLYT and Cougarblight are fire blight forecasters used to identify infection periods for Erwinia amylovora on apple and pear. MARYBLYT uses flowering, bacterial population (EIP), wetting, and average daily temperature, whereas Cougarblight uses flowering, wetting, a 4-day temperature window, and orchard fire blight history as parameters for predicting blossom infection. All possible thresholds of both models were evaluated with receiver operating characteristic (ROC) curve analysis using historical weather and disease incidence data collected from major apple producing regions in England and North America. Areas under the ROC curves were equivalent based on the Mann-Whitney U statistic signifying that the two forecasters performed similarly in their ability to predict blossom blight. However, the analyses indicated that there are regional differences in predictive ability with best performance of both forecasters occurring in England. Further analysis is needed to determine the reasons for the differences.

Two flies suspected in greenhouse spread of Phoma exigua on Acroptilon repens.
F. ESKANDARI, W. L. Bruckart, and D. K. Berner. USDA-ARS, Ft. Detrick, MD 21702. Publication no. P-2005-0009-PTA.

Wilting and death of noninoculated (healthy) Russian knapweed (RK, Acroptilon repens) have occurred in recent greenhouse experiments at the USDA containment facility in Frederick, MD. Investigations were initiated to identify the pathogen, its source, and mode of dissemination in the greenhouse. A Phoma sp. pathogenic to RK was isolated from diseased plants, and DNA analyses confirmed that the pathogen was P. exigua. It may be that the current infections originated from remnants of Isolate 02-059 from Turkey, evaluated recently for biological control. Abundant pycnidia on diseased plants are most likely the source of inoculum. The fungus gnat (FG, Bradysia coprophila) and the shore fly (SF, Scatella stagnalis) may be involved in transmission of P. exigua. The fungus was easily isolated from adults and larvae of both insect species (47%, n = 147), unless they were surface disinfested with bleach (20% for 10 min.). Conidia were observed clearly on insect wings, and a preliminary study involving caged insects (n = 248 insects of both species) resulted in diseased plants, except in the “no insect” control cage.

Genomic “iceberg”, gene cluster characterized by pseudogenes and horizontally acquired genetic elements in a phytoplasma genome. R. JOMANTIENE (1,2) and R. E. Davis (2). (1) Institute of Botany, Vilnius, Lithuania; (2) USDA-Agricultural Research Service, Beltsville, MD. Publication no. P-2005-0010-PTA.

We cloned and analyzed an 11 kbp clover phyllody (CPh) phytoplasma DNA segment containing a gene cluster that includes pseudogenes, an insertion sequence (IS)-like remnant, and an ORF encoding a putative phage-related protein, and that is flanked by fliA and by a tmk pseudogene and malK. Features indicate the segment represents a genomic “iceberg” of clustered sequences acquired by horizontal transfer. This cluster, and a related one present as variable iterations in onion yellows (OY) phytoplasma genome (GB NC_005303), probably represent variations of an ancestral mosaic composed of sequences acquired and altered in multiple events of horizontal transfer, recombination, and rearrangement. Gene deterioration, seen in presumably non-functional pseudogenes, indicates that repeats of the “iceberg” are not essential, suggesting that the minimal set of genes required for phytoplasma survival is much smaller than present in extant genomes, and that evolutionary genome size reduction is on-going in continued host adaptation of phytoplasmal parasites.

Recovery of hypovirulent isolates from cultures and cankers following hypovirus introduction.
W. E. JONES and W. L. MacDonald. Division of Plant and Soil Sciences, West Virginia University, Morgantown, WV 26506. Publication no. P-2005-0011-PTA.

Previous studies demonstrated that repeated sampling of chestnut blight cankers exposed to hypovirulent (HV) inoculum results in variable recovery of virulent (V) and HV isolates. This study evaluated the influence of mycelial age on hypovirus acquisition by inoculating Cryphonectria parasitica cultures and cankers at different developmental stages with HV inoculum. One- to six-week-old cultures did not acquire hypovirus. When V and HV isolates were coinoculated, almost 100% of the subsequent hyphal growth was HV. In cankers, the greatest recovery of HV isolates was from mycelium that developed after introduction of HV inoculum (44.2%) and the least (4-6%) from samples removed from the oldest part of the canker thallus. When individual bark plugs were divided into three sections from inner to outer bark and cultured, recovery of V and HV isolates occurred variably from the different bark layers. Successful recovery of HV isolates does not appear to represent “conversion” of pre-existing V mycelium. Instead, hypovirus acquisition results from mycelial growth that occurs after anastomoses.

The characterization of genes required for tagetitoxin production by Pseudomonas syringae pv. tagetis.
H. KONG, C. D. Patterson, and J. Lydon. U.S. Department of Agriculture, Agricultural Research Service, Sustainable Agricultural Systems Laboratory, Beltsville, MD 20705. Publication no. P-2005-0012-PTA.

Tagetitoxin is a phytotoxin composed of two six-membered rings, one composed of five carbons and one sulfur atom and the other composed of five carbons and one oxygen atom. Both rings have a nitrogen moiety attached. Tn5 mutagenesis was used to identify genes required for tagetitoxin production by Pseudomonas syringae pv. tagetis EB037, a strain originally isolated from common ragweed (Ambrosia artemisiifolia L.). Mutants with Tn5 inserts in DNA regions that have homology with genes that encode for asparagine synthetase, iron transport systems, sulfate adenylate transferase, and global regulators were identified from the 17 nontoxigenic mutants produced. The asparagine synthetase mutant was not an asparagine auxotroph nor was toxin restored with the addition of free amino acids to the culture media. The sulfate adenylate transferase mutant did not grow in minimal media, however normal growth and toxin production were restored with the addition of cysteine to the growth media. Interestingly, the addition of sulfite or sulfide as a sulfur source restored normal growth in minimal media but did not restore toxin production. Further characterization of these mutants is underway.

A Witch that haunts chocolate lovers: Development of a model system to study the interactions of Crinipellis perniciosa with Theobroma cacao. J.-P. MARELLI (1), S. Kang (1), M. J. Guiltinan (2), and P. A. Backman (1). (1) Department of Plant Pathology, The Pennsylvania State University, Buckhout Lab 16802 University Park; (2) Department of Horticulture, The Pennsylvania State University, 306 Wartik Lab, 16802 University Park. Publication no. P-2005-0013-PTA.

Witches’ broom of Theobroma cacao L. caused by Crinipellis perniciosa, causes abnormal growth of the infected branches, producing stem swelling and a proliferation of axillary buds. There are different biotypes of C. perniciosa. The C-biotype is pathogenic on Theobroma cacao L. and is widespread in cultivated areas in South America. The S-biotype, found on wild solanaceae in the Amazon forest, causes similar symptoms to the C-biotype. This characteristic allowed us to develop a pathogenicity assay on pepper and tomato. Different spore concentrations of the pathogen were evaluated on several varieties of pepper and tomato. The progression of the disease over time was recorded and the disease symptoms were assessed based on macroscopic changes. Electron microscopic observations of the symptoms were also performed.

Evaluating bacterial endophytes as biological control agents for cacao diseases. R. L. MELNICK (1), P. A. Backman (1), B. Bailey (3), and M. Guiltinan (2). Penn State Univ, (1) Dept. Plant Pathology and (2) Dept. of Hort., University Park, PA 16802; (3) USDA-ARS ACSL, Beltsville, MD 20705. Publication no. P-2005-0014-PTA.

The cacao diseases witches’ broom, caused by Crinipellis perniciosa, and black pod rot, caused by Phytophthora spp., reduce pod yield and quality. Phytosanitation and chemical controls are often ineffective and costly. We hypothesize that bacterial endophytes are more suited to control these diseases, since they are ubiquitous colonizers known to induce resistance. Previous research showed that X. c. pv. malvacearum (Xcm) was capable of endophytically inhabiting cacao for up to 28 days when applied with a silicon adjuvant (unpublished). In doing so, Xcm induced plant defense genes without causing disease. This research involves the development of a bioassay using detached leaves to test effectiveness of bacterial endophytes in reducing cacao diseases. Detached leaves of cacao plants are inoculated with a test endophyte to induce plant defense products, then challenged some days later with the cacao pathogen P. palmivora and subsequently evaluated for disease progress and severity in comparison to non-inoculated controls.

The beta-tubulin gene as a means to discriminate species of arbuscular mycorrhizal fungi. Z. MSISKA and J. B. Morton. Division of Plant and Soil Sciences, West Virginia University, Morgantown, WV 26506. Publication no. P-2005-0015-PTA.

The intron region of the beta-tubulin gene is being investigated as a marker to differentiate species of arbuscular mycorrhizal fungi. Polymerase Chain Reaction-Single Strand Conformation Polymorphisms (PCR-SSCP) in polyacrylamide gels under non-denaturing conditions differentiated 10 of 13 species tested in Glomus, Paraglomus, Scutellospora, Acaulospora, and Gigaspora. The three species tested within the genus Gigaspora gave identical SSCP patterns. Isolates of species from different geographical areas showed identical SSCP pattern(s). Sequence differences accounting for variation in SSCP patterns were confirmed by direct sequencing. In addition to sequence differences amongst species, intron length also varied considerably. PCR-SSCP results indicate sufficient evolutionary rates of change in the beta-tubulin gene intron region to differentiate species in all genera except Gigaspora, a genus which also has low variation in other genes (ITS, 18S rDNA) and in morphology.

Actigard and Admire for tomato spotted wilt virus control for flue-cured tobacco in Virginia in 2003.
T. D. Reed (1), P. J. Semtner (1), C. S. JOHNSON (1,2), and M. Parrish (3). (1) So. Piedmont Agric. Res. and Ext. Ctr., Blackstone, VA 23824; (2) Dept. of Plant Pathology, Physiology, and Weed Science, Virginia Polytechnic Institute and State University; (3) Virginia Cooperative Extension, Dinwiddie, VA 23831. Publication no. P-2005-0016-PTA.

Tomato spotted wilt virus (TSWV) is a potentially important disease of tobacco in Virginia. Two field experiments were conducted in 2003 to evaluate use of Admire and Actigard to suppress TSWV incidence. Tests were arranged in a split-plot design with four replications. Main plots involved broadcast- or strip-kill of the cover crop. Subplots included an untreated control; application of Admire, Actigard, or Admire + Actigard to tobacco seedlings before transplanting; or use of Actigard or Admire + Actigard before transplanting and foliar sprays of Actigard 1 and 2 wk after transplanting. Final TSWV incidence peaked at 4% to 5% in untreated control plots. No significant differences were observed among any of the treatments in cumulative incidence of the virus (P < 0.05). However, reductions in plant stand from use of Admire in the greenhouse were significant on 13 August at the Barnes farm compared to the untreated control. Any use of Actigard in the greenhouse reduced plant stand at the Everette farm (P < 0.05). No differences in yield, economic value, grade index, or average price were associated with incidence of TSWV or with the effects of Admire or Actigard application on plant stand. The lack of such differences may be attributable to counteracting influences of low TSWV incidence and negative effects of Actigard and/or Admire on tobacco growth and development.

Evolution of the prokaryotic protein NusA: Comparison of NusA in clover phyllody phytoplasma and other firmicutes. J. SHAO (1), R. E. Davis (1), R. Jomantiene (2), and E. L. Dally (1). (1) USDA-Agricultural Research Service, Beltsville, MD 20705; (2) Institute of Botany, 2021-Vilnius, Lithuania. Publication no. P-2005-0017-PTA.

Clover phyllody (CPh) phytoplasma is a phytopathogenic, cell-wall less prokaryote thought to have descended from walled firmicutes, low G+C Gram-positive bacteria. Because of inability to culture phytoplasmas in artificial media, recent research has emphasized the study of genes and their products to gain greater understanding of these microbes. We cloned and analyzed a CPh phytoplasma DNA locus containing three full-length potential coding sequences (open reading frames, ORFs) encoding NusA and two hypothetical proteins, respectively, and two partial ORFs encoding Small protein B and If2, respectively. The present work focused on NusA, a protein involved in transcription elongation, termination, and anti-termination. The NusA protein from CPh phytoplasma contains three conserved RNA-binding domains (S1, Kh1, and Kh2) typical of NusA from other organisms. We carried out a phylogenetic analysis of each domain separately. The topologies of the phylogenetic trees indicated the evolutionary history of each domain among firmicutes.

Susceptibility of camellia to Phytophthora ramorum, the sudden oak death pathogen.
NINA SHISHKOFF. USDA-ARS, 1301 Ditto Ave., Frederick, MD 21702. Publication no. P-2005-0018-PTA.

Six species of Camellia were tested for susceptibility to Phytophthora ramorum, including two cultivars of C. sasanqua and three cultivars of C. japonica. Plants were inoculated with sporangial suspensions (2000-6800 sporangia/mL) from a P. ramorum isolate originally from camellia. Inoculated plants were placed in a dew chamber at 20 C for 4-5 days, then incubated in the greenhouse for up to a week at 20 C and rated for defoliation and lesions on leaves. Camellias differed in susceptibility to the pathogen, with C. sasanqua ‘Midnight Lover’ and C. oleifera the most susceptible, exhibiting defoliation up to 41.4% and lesions covering up to 9.8% of area of inoculated foliage; C. japonicum and C. sinensis were intermediate, while C. brevistyla and C. crapnelliana showed no symptoms. Defoliation of camellias occurred even when leaves showed no visible lesions, although the pathogen could often be recovered from the petiole of the fallen leaf. Rhododendrons inoculated at the same time showed little defoliation but significant lesion development, up to 72% of inoculated leaf area.

Impacts of host shifting and mode of transmission on PPV microevolution.
C. M. WALLIS (1), F. E. Gildow (1), and W. L. Schneider (2). (1) Dept. of Plant Pathology, Penn State University, Buckhout Laboratory, University Park, PA 16802; (2) USDA-ARS Foreign Disease-Weed Science Research Unit, 1301 Ditto Ave, Ft. Detrick, MD 21702-5023. Publication no. P-2005-0019-PTA.

Plum pox potyvirus (PPV, Family Potyviridae) remains a serious threat to the ornamental and commercial Prunus industries in the United States since its detection in Pennsylvania in 1999. We examined the microevolution of PPV following a host shift from peach (Prunus persicae) to pea (Pisum sativa) during serial transmissions using either aphid-vectored or mechanical inoculation. Herbaceous host adaptation of PPV was characterized by a reduction in time to symptom development and an improvement in transmission efficiency. Viral genomic RNA sequence analyses revealed pea host-specific mutations and mode of transmission-associated mutations. Pea-adapted strains of PPV at every passage were also tested for their ability to infect the original host, peach. Regardless of the number of previous passages, all pea-adapted PPV strains consistently infected peach at 50% efficiency or greater using aphid inoculation. This indicated that herbaceous-adapted PPV strains remain capable of infecting peach, which could undermine eradication efforts if PPV became established in non-Prunus alternate host reservoirs.

Factors affecting the Heterodera glycines suppressiveness of N-Viro Soil.
I. A. ZASADA. USDA, ARS Nematology Laboratory, Beltsville, MD, U.S.A. 20705. Publication no. P-2005-0020-PTA.

N-Viro Soil (NVS) is an alkaline stabilized municipal biosolid that has been used as a soil amendment. Previous laboratory research demonstrated that NVS suppressed Heterodera glycines. This study aimed to identify factors that could potentially influence the efficacy of NVS as a H. glycines management tool. NVS was applied to soil microcosms, and nematode survival and changes in sand solution pH and ammonia were measured. Microbes associated with NVS appeared not to be responsible for the nematode suppressiveness of the amendment; there was no difference in nematode suppression between sterilized and unsterilized NVS. Alkaline-stabilization of biosolids was necessary to achieve nematode suppression. Biosolids applied at rates ranging from 1 to 3% dry w/w did not suppress H. glycines to the same level as equivalent amounts of NVS. Sand solution pH levels after biosolid application ranged from 7.7 to 8.6. The ability of NVS from different geographical sources to suppress nematodes was related to sand solution pH levels. NVS sources applied at 2% dry w/w resulting in sand solution pH levels above 10.0 reduced H. glycines survival 94% or more.