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2002 Northeastern Division Meeting Abstracts

October 1-3, 2002 - Bromont, Quebec, Canada

Posted online November 6, 2002

PCR-based detection of Pseudomonas spp. capable of producing antibiotics in the rhizosphere of conifers. M. ALLAIRE (1,2), L. Bernier (2), V. Vujanovic (3), M. St-Arnaud (3), M. Filion (3), and R. C. Hamelin (1). (1) Natural Resources Canada, Laurentian Forestry Center, QC, Canada; (2) Université Laval Québec Canada; (3) Université de Montréal, Québec, Canada. Publication no. P-2003-0001-NEA.

Rhizobacteria of the genus Pseudomonas have been largely characterized for their antagonism against fungal root pathogens in many agricultural stands around the world. Production of antibiotics like phenazines and phloroglucinol by strains of Pseudomonas spp. is a major determinant in the protection of roots against soil born pathogens. To better characterize the diversity among Pseudomonads in tree rhizosphere, and to identify new antibiotic-producing strains adapted to tree nurseries, we are developing a method to screen populations of Pseudomonas in the rhizosphere of conifers. We use a PCR-based technique with primers designed to amplify genes involved in the biosynthesis of antibiotics production. We have collected samples of rhizosphere from two conifer nurseries, and isolated Pseudomonas strains. Sequencing of the positive amplification products has confirmed the presence of genes involved in phenazine biosynthesis in some of these strains.

Timing of fungicide applications to control Fusarium patch (pink snow mold) and Typhula blight (gray snow mold) on turfgrass.
R. A. ANAIR JR. and G. L. Schumann. Dept. Microbiology, Univ. of Massachusetts, Amherst, MA 01003. Publication no. P-2003-0002-NEA.

Different fungicides and combinations of fungicides were applied to plots of a mixture of bentgrass and annual bluegrass to determine the optimal fungicide timing for snow mold control. Fungicides were applied on 12, 18, and 28 Nov and 4 Dec (1999), 12 and 25 Nov (2000), and 12 and 18 Nov (2001). After the final applications each year, separate experimental areas were inoculated with Microdochium nivale or Typhula incarnata grown on sterilized rye seeds (50 cc / 1 sq m plot). The plots were covered with high density polyethylene greens covers until snowmelt when the plots were rated for percent disease. In the 1999 experiment, 11 of the 19 treatments had no significant differences in disease between application dates. In the 2000 experiment, there were no significant differences in disease between application dates for any of the 35 treatments, and in 2001, only one of the 39 treatments was significantly different. This suggests that there is a significant window for timing of fungicide applications between the end of foliar growth and snowfall for successful control of snow mold with a single fungicide application.

Management of grape powdery mildew by the use of aerobiological data.
R. BACON (1,2), B. G. Talbot (2), and O. Carisse (1). (1) Horticultural Research and Development Centre, Agriculture and Agri-Food Canada, 430 Gouin Blvd, St-Jean-sur-Richelieu, Qc J3B 3E6, Canada; (2) Department of Biology, Sherbrooke University, 2500 Université, Sherbrooke, Qc J1K 2R1, Canada. Publication no. P-2003-0003-NEA.

Grape powdery mildew (Uncinula necator) is among the most important grape diseases in Eastern Canada. The production of secondary inoculum is a key factor in disease development. Consequently, the objective of the present work was to evaluate the potential of initiating treatments against powdery mildew based on inoculum concentration. In 2000 and 2001, experimental plots were established on two commercial sites located in Iberville and Dunham. Spore concentrations (conidia m(^-3)) were measured 3 times a week at heights of 45 cm and 150 cm. Disease incidence, vine phenology and weather were monitored for both years. In 2002, one plot was established in Dunham and treatments were initiated based on spore concentration thresholds of 10 conidia m(^-3) and 15 conidia m(^-3). For both sites in 2000 and 2001, airborne conidia were detected 21 to 35 days prior to the observation of the first sporulating lesions and spore concentrations corresponded to disease incidence. Furthermore, in 2002, measuring spore concentrations allowed the fungicide program to be delayed by 20 days. These results indicate that it may be possible to use airborne conidia concentration measurements to initiate the fungicide applications.

species associated with cavity spot lesions on carrots produced in Québec city area.
N. A.-BOULÉ (1), R. J. Tweddell (1), C. Martinez (1), R. R. Bélanger (1), and C. A. Lévesque (2). (1) Centre de Recherche en Horticulture, Université Laval, Québec, Canada, G1K 7P4; (2) ECORC, Agriculture and Agri-Food Canada, Ottawa, Canada, K1A 0C6. Publication no. P-2003-0004-NEA.

Cavity spot is an important disease affecting carrots worldwide and is readily recognized by the black elliptic lesions on the roots. Studies from France and other European countries have shown that Pythium violae is the most important pathogen associated with cavity spot lesions but other reports from other carrot-producing areas of the world have attributed the disease to different Pythium spp. (e.g. P. sulcatum and P. coloratum). The objective of this study was to identify Pythium species associated with cavity spot lesions on carrots produced in Québec city area. Two hundred and fifty Pythium isolates were recovered from cavity spot lesions. Among them, several pathogenic isolates belonging to different species including P. sulcatum, P. sylvaticum and P. macrosporum, P. sulcatum being the most frequently isolated species. In addition, it was shown that several isolates were resistant to metalaxyl. A new species closely related to P. intermedium was also found.

Molecular epidemiology of poplar leaf rust - temporal and geographic variation.
M. BOURASSA (1), L. Bernier (2), B. G. Milligan (3), and R. C. Hamelin (1). (1) Natural Resources Canada, Laurentian Forestry Centre, Ste-Foy, QC; (2) Centre de recherche en biologie forestière, Université Laval, Ste-Foy, QC; (3) New Mexico State University, Las Cruces, NM. Publication no. P-2003-0005-NEA.

A major threat to plantations of Populus deltoides and its hybrids in North America is the poplar leaf rust caused by Melampsora medusae f. sp. deltoidae. The North American pathosystem is characterized by sympatry of telial and aecial hosts (poplar and larch), where the rust completes its life cycle, and allopatry, where only the telial host is found. To test the hypothesis that epidemics originate from sympatry, populations in sympatry and allopatry were surveyed in 1997 and 1998 using two Polymerase Chain Reaction - Single-Strand Conformational Polymorphism markers. For both years, more alleles were found in sympatry than in allopatry, and most alleles found in allopatry were also present in sympatry. Values of genetic diversity were also higher in sympatry than in allopatry. These results suggest that sympatry represents a center for genetic diversity where epidemics arise. Considering allelic composition, there seems to be sporadic gene flow from sympatry to allopatry.

The lesion-causing efficiency of Venturia inaequalis (Cke.) Wint. on six apple cultivars.
J. M. CLIFFORD (1) and W. E. MacHardy (1). (1) Dept. of Plant Biology, University of New Hampshire, Durham, NH 03824. Publication no. P-2003-0006-NEA.

The lesion-causing efficiency (LCE) of a natural population of Venturia inaequalis was determined on potted trees of Delicious, Golden Delicious, McIntosh, Mutsu, Paula Red, and Rome and evaluated to determine if LCE could account for the relative field resistances of the six cultivars in a mixed planting. The LCE was determined by inoculating the four youngest leaves on extension shoots with a conidial suspension. The 9.8% LCE on McIntosh was significantly greater than the LCE on the other cultivars. The 5.4% LCE on Rome was significantly greater than the 3.0, 2.2, and 2.1% on ‘Delicious’, ‘Golden Delicious’, and ‘Mutsu’, respectively. The 0.2% LCE on Paula Red was significantly less than the LCE on all other cultivars. LCE was correlated with the rankings of relative field resistance of the six cultivars reported in an earlier study. It was concluded that, of all the components of the apple scab disease cycle evaluated to date, LCE of V. inaequalis is the one most correlated with a cultivar’s relative field resistance.

Influence of temperature and moisture on Gibberella zeae perithecial development on corn residue.
N. S. DUFAULT (1), E. D. De Wolf (1), P. E. Lipps (2), and L. V. Madden (2). (1) Dept. Plant Pathology, Pennsylvania State University, University Park, PA 16801; (2) Dept. Plant Pathology, Ohio State University/OARDC, Wooster, OH 44691. Publication no. P-2003-0007-NEA.

The effect of environment on ascospore release from Gibberella zeae perithecia impacts the development of Fusarium head blight epidemics. The influence of temperature and moisture on perithecial production was evaluated by monitoring inoculated corn stalks in a growth-chamber environment. Perithecial development was assessed at combinations of three temperatures (15, 25, and 30 C) and three relative stalk moisture levels (low, moderate, and high). After 11 to 16 days of incubation, the number of perithecia produced at 15 or 25 C was significantly (p < 0.01) greater than numbers produced at 30 C, but no significant difference in the number of perithecia was detected between 15 and 25 C. The number of perithecia was significantly increased at high or moderate compared to low moisture treatments. Significant interactions were identified for treatments that included 30 C, or low moisture. Perithecial development by G. zeae may be limited by extended periods of residue dryness or temperatures above 30 C.

Comparison of pathogenic and nonpathogenic isolates of Fusarium oxysporum for genomic sequences of translation elongation factor 1-alpha.
W. H. ELMER and C. R. Vossbrinck. The CT Agr. Exp. Sta., P. O. Box 1106, New Haven, CT 06504. Publication no. P-2003-0008-NEA.

Genetic variation in Fusarium oxysporum was examined among 18 isolates. Seven isolates represented pathogens on asparagus, banana, basil, cyclamen, lisianthus, and Persian violet. Five isolates were nonpathogenic isolates selected by laboratories in Europe and the US as biocontrol agents, and six isolates were subcultured from soil dilution plates of soil from a field in Hamden, CT and presumed to be nonpathogenic. The protein-coding gene, the translation elongation factor 1-alpha was amplified by PCR and sequenced from the isolates of F. oxysporum. A phylogenetic analysis was performed with F. inflexum serving as the outgroup. There was no pattern among pathogenic, nonpathogenic and soil isolates. Less variation was found among isolates of F. oxysporum cultured from soil than from among pathogens and nonpathogenic strains. Pathogenicity in F. oxysporum may have developed many times among different groups. These findings are in support of other studies that show pathogenicity in F. oxysporum is polyphyletic.

Sensitivity of New Jersey Phytophthora capsici isolates to mefenoxam.
M. L. FOGG and S. A. Johnston. Rutgers Ag Research & Extension Center, Bridgeton, NJ 08302. Publication no. P-2003-0009-NEA.

Phytophthora blight of pepper, caused by Phytophthora capsici, is the most devastating disease of pepper in New Jersey. Mefenoxam is the primary fungicide used to control the crown rot phase of blight. In recent years, incidence of blight has increased to high levels despite the use of mefenoxam. Several states have reported isolates of P. capsici that are insensitive to mefenoxam. To determine if insensitivity to mefenoxam is responsible for blight epidemics in New Jersey, P. capsici populations were sampled from diseased plants within 14 fields located in four counties during the summer of 2001. Sampling locations were logged using global positioning equipment. Single zoospore isolates were screened on clarified V8 juice agar amended with mefenoxam (Ridomil Gold 4E) at 0, 5, and 100 ppm. Isolates were designated as sensitive, insensitive or intermediate based upon linear growth on amended agar compared to that on unamended agar. Isolates were found in all three categories, with a high percentage of isolates determined to be sensitive or intermediately sensitive to mefenoxam in vitro.

The role(s) of silicon amendments in plant-microbe interactions.
D. GHANMI (1), F. Fauteux (1), J.-S. Cloutier (1), J. G. Menzies (2), and R. R. Bélanger (1). (1) Centre de Recherche en Horticulture, Département de Phytologie, Université Laval, Québec, Canada G1K 7P4; (2) Agriculture and Agri-Food Canada, 195 Dafoe Road, Winnipeg, Manitoba, R3T 2M9. Publication no. P-2003-0010-NEA.

Exogenous application of silicon (Si) is known to reduce disease severity in several plant-pathogen interactions. Although the mode of action of Si is unclear, some evidence suggests that insoluble Si deposited in the apoplast of epidermal cells reinforces mechanical resistance against pathogen penetration. However, other observations indicate that soluble Si plays an active role by mediating plant defense reactions. Using Arabidopsis thaliana, a common model plant in host-pathogen interaction studies, and rice (Oryza sativa), a plant species known as a strong Si accumulator, we investigated the effect of Si feeding on the outcome of specific microbial interactions. In A. thaliana, treatment with Si resulted in intense absorption of Si by the plants which in turn conferred them protection against Erysiphe cichoracearum. Microscopic observations indicated a limited development of E. cichoracearum in Si+ plants in spite of successful haustorium formation. In the case of rice, Si feeding resulted in intensification of symbiosis with the endophytic diazotroph Herbaspirillum seropedicae strain Z67 as evidenced by enhanced development of plants under this treatment. These preliminary results indicate that the physiological role of Si in planta may be more complex and diverse than originally thought.

Comparison of butternut curculio wounds and artificial wounds as infection courts for Sirococcus clavigignenti-juglandacearum on butternut seedlings.
S. HALIK, J. E. Stewart, and D. R. Bergdahl. Dept. of Forestry, University of Vermont, Burlington, VT 05405. Publication no. P-2003-0011-NEA.

Butternut curculios (Conotrachelus juglandis) occasionally carry conidia of Sirococcus clavigignenti-juglandacearum (Scj) and on healthy butternut (Juglans cinerea), they create feeding and oviposition wounds that may act as infection courts for the fungus. In 2000 and 2001, we inoculated butternut seedlings with Scj to compare susceptibility of curculio wounds to that of artificial wounds. We also determined if infested curculios deposit conidia in their wounds, resulting in infection. Wound treatments included scalpel scrape, needle punctures, curculio-feeding, or no wound, each of which was inoculated with sterile water or conidia from a culture of Scj in a complete factorial design with 12 seedlings in each treatment combination. Curculio wounds were achieved by placing seedlings in screen cages with curculios. An additional set of 12 seedlings was wounded and inoculated by Scj-infested curculios that had crawled on sporulating cultures. All wounded seedlings and nearly all non-wounded seedlings that were artificially inoculated with conidia became infected with Scj. Of curculio-inoculated seedlings, 67 and 75% became infected with Scj in 2000 and 2001, respectively. Therefore, the butternut curculio may be acting as an occasional vector of Scj in the field.

Population levels and pathogenicity of Burkholderia cepacia isolated from organic soils.
J. S. HAUDENSHIELD and J. W. Lorbeer. Dept. Plant Pathology, Cornell University, Ithaca, NY 14853. Publication no. P-2003-0012-NEA.

The most important bacterial pathogen of onions in New York is currently B. cepacia, cause of the bacterial canker/sour skin disease. We have assayed the soil population levels of B. cepacia in fields historically planted to onions, non-onion crops, and of onion fields rotated with other crops to explore the possibility that crop rotation may reduce the inoculum levels and subsequent disease incidence. We have utilized a laboratory cultivation method, "miniculture," to further test the influence of various crops on rhizosphere B. cepacia levels. It is known that there are several phenotypic sub-groups within the B. cepacia species, and that some are pathogenic in an onion slice assay, while others are not. Many bacterial pathogens are known to exhibit an hypersensitive response (HR, a plant defense mechanism) when infiltrated into the leaves of non-host plants. We hypothesize that an HR response may be elicited by B. cepacia in tobacco, and may correlate with onion slice pathogenicity data, thus providing a more rapid screening technique. Our data on the HR elicited in tobacco by organic soil isolates of B. cepacia support this hypothesis.

Genomic diversity among pine rusts.
D. L. JOLY (1,2), L. Bernier (2), S. F. Covert (3), and R. C. Hamelin (1). (1) Natural Resources Canada, Laurentian Forestry Centre, Ste-Foy, QC, Canada; (2) Centre de recherche en biologie forestière, Université Laval, QC, Canada; (3) Warnell School of Forest Resources, University of Georgia, Athens, GA, USA. Publication no. P-2003-0013-NEA.

Based on cDNA sequences from Cronartium quercuum f. sp. fusiforme, PCR primers have been developed to amplify coding regions of pine rusts to study and compare genomic diversity. Genetic markers generated from this method were sequenced, aligned and compared. Most of the markers studied could be assigned to a function by sequence comparison using BLAST. Strong inter-specific diversity was observed. Most mutations were silent or located in intronic regions but some changes in amino acids were observed. Intra-specific variability was detected, consisting mostly of SNPs (Single Nucleotide Polymorphisms) within C. comptoniae, C. quercuum f. sp. banksianae and C. ribicola. Analysis using SSCP (Single-Strand Conformation Polymorphism) and direct sequencing revealed the presence of heterozygotes as well as homozygotes. So far, on 3.1 kb, 320 positions were different at least for one rust, 15 deletions and 3 insertions were found. Ten sites showed intra-specific polymorphism.

Isolation, cloning and characterization of eleven symbiosis-related plant and fungal genes from arbuscular mycorrhizae.
J. M. LERNER, R. L. Wick, and S. Ciufo. Department of Microbiology, University of Massachusetts, Amherst, MA 01003. Publication no. P-2003-0014-NEA.

Plant and fungal genes expressed during the mycorrhizal symbiosis would presumably be involved in fundamental mycorrhizal processes such as cell wall or membrane synthesis, protein modification, physiological and structural accommodation, induced or suppressed defense responses, nutrient exchange and transport, and communication between the symbionts. In this investigation, eleven (seven plant and four fungal) symbiosis-related cDNAs were isolated and cloned using subtractive hybridization of cDNA libraries from in vitro mycorrhizal and non-mycorrhizal carrot root tissue. Plant cDNAs include a novel disease resistance protein, a polygalacturonase inhibitor, and a precursor of a cell wall polysaccharide. Several plant cDNAs encoded transmembrane proteins, including a zinc and nitrate transporter and a receptor-like protein kinase. Fungal cDNAs include a polyketide synthase, a phosphoglycerate kinase, an ammonium transporter, and an anaphase-promoting protein. These proteins will be characterized and their possible role in the mycorrhizal symbiosis will be discussed.

Organic crop production in Australia.
M. T. MCGRATH. Dept. Plant Pathology, Cornell Univ., Riverhead, NY 11901. Publication no. P-2003-0015-NEA.

Information on organic and biodynamic production of vegetables and other crops in Australia was obtained by visiting growers, researchers, certifiers, wholesalers, and sellers of organic products. Diseases are managed generally by having good soil health and thus healthy, unstressed plants. Growers strive to increase organic matter content and microbial activity in typically infertile Australian soils plus balance levels of minerals and nutrients. Specific control practices include crop rotation and applications of compost tea, liquid worm castings, molasses, milk, oil, copper, sulfur, bicarbonate, and biocontrol agents. Industry concerns include changing certification rules, requirement to use organically-produced seed starting June 2003, packaging needs in supermarkets to prevent contamination, lack of domestic legislation, planned phase-out of copper pesticides, lack of consumer understanding about benefits of organic production, and low demand outside cities. Most organic produce is sold through certified organic wholesalers to health-food shops and home delivery and internet direct buying services. Some goes to supermarkets. The rest is exported. Some cities have farmer's markets.

Genetic variability of Canadian populations of the sapstain fungus Ceratocystis resinifera.
C. MORIN (1), P. Loppnau (2), L. Bernier (1), C. Breuil (2), and P. Tanguay (2). (1) Centre de Recherche en Biologie Forestière, Université Laval, Québec, Canada G1K 7P4; (2) Department of Wood Science, University of British Columbia, Vancouver, Canada V6T 1Z4. Publication no. P-2003-0016-NEA.

DNA was extracted from 129 isolates of C. resinifera and RAPD markers were used to study the population genetic structure of this fungus. The analysis showed an overall low genetic diversity among isolates (H(S) = 0.107). A notable genetic differentiation was nevertheless observed among the 9 geographical populations studied. The genetic differences were especially striking when populations from Eastern Canada were compared to populations from Western Canada, suggesting that geographic reproductive barriers occur within Canada. These barriers may result from intensive agriculture and a lower abundance of hosts across the Great Plains. The occurrence of two distinct biotypes in Canada is questionable, however, since Nei’s genetic distances among populations were very small (0.009 to 0.096). A recent introduction of C. resinifera, probably from one or several sources, and a subsequent clonal reproduction may explain the low level of genetic diversity observed.

Evaluation of chemical seed treatments to eliminate Xanthomonas cucurbitae from naturally infected pumpkin seeds.
Z. ÖZDEMIR and T. A. Zitter. Cornell University, Ithaca, NY 14853. Publication no. P-2003-0017-NEA.

Xanthomonas cucurbitae
is a seedborne pathogen of cucurbits responsible for leaf spot and fruit infection. Economic losses in pumpkin and winter squash occur when bacteria infect the rind leading to fruit rot. Several chemical seed treatments were tested to eliminate bacteria from naturally infected pumpkin (Cucurbita pepo cv. ‘New Rocket’) seeds. Treatments of 3% hydrogen peroxide, 1% peroxyacetic acid, 1% sodium hypochlorite, copper hydroxide plus mancozeb (0.36 g + 0.27 g/100ml water) and water control were tested for 15 min by sampling ca. 500 seeds. Evaluations were made by plating seed washings of treated seeds onto semi-selective XCS agar. Effects of the seed treatments on seed germination were recorded. Sodium hypochlorite, peroxyacetic acid, and copper hydroxide plus mancozeb were effective in eliminating the pathogen on seeds. Hydrogen peroxide treatment was less effective, with bacteria detected in one experiment. None of the treatments adversely affected seed germination. Growing-out tests might be required to observe seed transmission of bacteria and to evaluate the seedling vigor.

Effect of temperature on urediniospore germination and development of uredinia of Thekopsora minima.
S. E. PFISTER, S. Halik, and D. R. Bergdahl. Dept. of Forestry, University of Vermont, Burlington, VT 05405. Publication no. P-2003-0018-NEA.

Thekopsora minima
is a heteroecious rust on needles of hemlock (Tsuga spp.) and leaves of ericaceous plants. Percent germination and germ tube growth of urediniospores were assessed at 10, 15, 20, 25, and 30°C after 3 hours on 1.5% water agar. Polynomial regression analysis revealed a significant effect of temperature on germination (P < 0.001, R(^2)(adj) = 93.6%) and germ tube growth (P < 0.001, R(^2)(adj) = 93.3%), with predicted optimum temperatures of 21.5 and 22.0°C, respectively. Germination and germ tube growth were greatly reduced at 15 and 30°C. Temperature was also found to have a significant effect on infection efficiency, as measured by incubation period (P < 0.001, R(^2)(adj) = 80.8%) and uredinia produced (P < 0.001, R(^2)(adj) = 86.6%). On excised leaf disks of Rhododendron ‘White Lights’ maintained under a 14-hour photoperiod, the shortest mean incubation periods of 10.7 and 10.0 days were at 20 and 25°C, respectively, with a predicted optimum of 23°C. The number of uredinia produced (26-32 uredinia per leaf disk) was similar at 15, 20, and 25°C, but was reduced 6-fold at 30°C. The predicted optimum temperature for production of uredinia was 19.5°C. The optimum temperatures found for urediniospore germination and development of uredinia should be used in future studies of T. minima.

Infection process of Thekopsora minima urediniospores on Rhododendron
White Lights’. S. E. PFISTER, S. Halik, and D. R. Bergdahl. Dept. of Forestry, University of Vermont, Burlington, VT 05405. Publication no. P-2003-0019-NEA.

Two studies on Rhododendron ‘White Lights’ were conducted to better understand the infection process of Thekopsora minima urediniospores. On whole plants, effect of leaf wetness duration on infection efficiency was assessed at 5, 7.5, 10, 15, and 20 hours. After 13 days, numbers of uredinia/cm(^2) were determined and data subjected to polynomial regression analysis. A significant effect of leaf wetness period on infection efficiency was found (P < 0.001, R(^2)(adj) = 71.7.%.). No uredinia developed after 5 hours of leaf wetness; however, after 7.5 hours, trace amounts of infection occurred. Infection efficiency improved after 10 hours and reached a maximum after 15 hours of leaf wetness. A second study examined excised leaf disks inoculated with urediniospores and maintained at 100% relative humidity. Uredinia first began to develop 8 days post-inoculation on abaxial surface-inoculated disks. Microscopic observation found that stomata only occurred on the abaxial leaf surface and appressoria formation appeared to be associated only with stomata. Germ tube penetration into the substomatal chamber was only observed to occur beneath appressoria. These observations of the infection process of T. minima urediniospores may help in the study of possible host resistance mechanisms.

REMI transformation, a tool for gene discovery in Ophiostoma.
K. PLOURDE and L. Bernier. Centre de recherche en biologie forestière, Université Laval, Québec (Qc), Canada G1K 7P4. Publication no. P-2003-0020-NEA.

Dutch elm disease caused two pandemics that modified the landscape of North America and Europe. After many years of research on Ophiostoma novo-ulmi, we still know little on the mechanism of pathogenicity of this fungus. The main goal of this project is to find and characterize pathogenicity genes of O. novo-ulmi. Since natural populations of this fungus are genetically homogeneous, insertional mutagenesis was used to create a bank of mutants, including strains with reduced capacity to induce symptoms in elms. The latter were subjected to phenotypic tests and were crossed to a wild-type strain in order to verify if pathogenicity genes had been interrupted. The other goal of the project is to optimize transformation of O. novo-ulmi by the REMI method, which consists in random integration of a plasmid into the fungal genome in presence of a restriction enzyme. The advantage of REMI is that it facilitates recovery of the sequence mutated by plasmid insertion. We have determined that a concentration of 40U of HindIII with 10(^8) fungal cells and 5 µg plasmid was the most efficient for generating many mutants carrying a unique plasmid insert in their genome.

Effects of fertilizer, pH and biological control on Pythium root rot.
M. P. RAMON and G. Moorman. Dept. Plant Pathology, The Pennsylvania State University, University Park, PA 16801. Publication no. P-2003-0021-NEA.

Production of floriculture crops grown in greenhouses is frequently limited by Pythium spp. that cause seed damping-off and root rot. Biological control agents (BCAs) represent a potential means to suppress pathogenic Pythium spp. whether in the potting soil or within hydroponic systems. However, the success of the BCAs under commercial conditions has often been inconsistent. This variability has been attributed in part to physical and environmental factors in the soil. Our objective in this research is to examine the influence of fertilizer concentrations and pH on fungal and bacterial BCA efficacy. Greenhouse and in-vitro experiments were conducted to evaluate the effects of pH levels ranging from 6.5 to 7.5 and two fertilizer (20-20-20; N-P-K) concentrations, optimum (100ppm) and excessive (400ppm) on five agents (Companion™, Mycostop™, Plant Shield™, Soilgard™ and mefenoxam) for protecting geraniums from Pythium root rot.

Immunolocalization of abscisic acid in poplar following water stress and during compartmentalization processes.
D. RIOUX and M. Simard. Natural Resources Canada, Canadian Forest Service, Laurentian Forestry Centre, P.O. Box 3800, Sainte-Foy, Québec, Canada G1V 4C7. Publication no. P-2003-0022-NEA.

Abscisic acid (ABA) is known to increase rapidly in water-stressed plants. ABA was immunolocalized in light and transmission electron microscopy in Populus balsamifera x deltoids growing under normal and water stress conditions. In water-stressed trees, ABA was detected in the xylem and the phloem while in controls it was only found in the phloem. Under water stress, ABA seemed to be produced within centimetres of the leaves. Following inoculation with Ophiostoma ulmi, a non-pathogenic agent for poplar, compartmentalization processes were obvious around the invaded area. Compartmentalization would primarily be a response to impede air movement within the tree. The labelling for ABA increased during compartmentalization and it was frequently intense in the cambium, within xylem ray cells and around tyloses occluding vessel elements. ABA was also closely associated with barrier zones formed by the cambium in response to inoculation. In electron microscopy, ABA was frequently localized within nuclei and opaque masses of cytoplasm in most living cells.

Relationship of Pasteuria to root-knot and stunt nematodes in golf greens.
W. RUNGRASSAMEE, R. L. Wick, and B. Dicklow. Dept. of Microbiology, U. of Massachusetts, Amherst, 01003. Publication no. P-2003-0023-NEA.

The relationship of Pasteuria penetrans to Meloidogyne graminis was investigated during 2002 on four golf greens. Two greens had a high density of M. graminis and a low encumbrance of M. graminis by Pasteuria (HMLP); two greens had a lower M. graminis density and a higher rate of encumbrance (LMHP). The distribution pattern of M. graminis was highly clumped in all greens. In the HMLP greens, 67 and 70 soil cores were necessary to estimate M. graminis population, and 16 soil cores were needed to estimate the percentage of Pasteuria-encumbered juveniles in both greens. In the LMHP greens, 102 and 110 soil cores were necessary to estimate the nematode population; 29 and 43 soil cores to estimate the percentage of Pasteuria-encumbered juveniles (95% C.I.). A similar study was done with Tylenchorhynchus spp. on three greens; 12, 13 and 15 soil cores were needed to estimate the Tylenchorhynchus population; 5, 8 and 10 soil cores were necessary to estimate encumbrance of Tylenchorhynchus by Pasteuria (95% C.I.). The distribution of Tylenchorhynchus was less clumped than M. graminis, but in both cases described by negative binomial distribution.

Sensitivity to propamocarb and mefenoxam in Pythium isolated from within flower crop greenhouses.
N. SHISHKOFF (1), J. Knoedler (1), Gary W. Moorman (2), and M. Daughtrey (1). (1) Cornell Univ., LIHREC, Riverhead, NY, 11901; (2) Pennsylvania State Univ., University Park, PA 16802. Publication no. P-2003-0024-NEA.

Oomycetes from plants, greenhouse surfaces and soil were collected from greenhouses in NY, and seven other states. When 227 isolates of Pythium were grown on corn-meal agar plates containing a range of concentratrions of the fungicide propamocarb (Banol), 61% were able to grow at 10 mg/L or lower, while 10%, particularly isolates of P. spinosum, were able to grow at 1000 mg/L. When grown on a range of concentrations of mefenoxam (Subdue MAXX), isolates showed a bimodal distribution, either unable to grow at concentrations of 1 mg/L or higher (36%) or else able to grow at the highest concentration tested, 100 mg/L (39%). There was no discernable difference in sensitivity pattern of isolates collected from symptomatic plants and isolates collected from greenhouse surfaces, although isolates from plants were more frequently found to be pathogenic in disease assays. Subdue MAXX has been used widely to control oomycetes in greenhouses, while Banol is only infrequently used in greenhouses.

In vitro
sensitivity of Alternaria solani to chlorothalonil.
C. J. SICA and B. J. Christ. Dept of Plant Pathology, The Pennsylvania State University, University Park, PA 16802. Publication no. P-2003-0025-NEA.

Alternaria solani
the causal agent of early blight of potato, is a highly destructive pathogen that can cause significant economic losses when not controlled by fungicides. In recent years some reports of increased early blight severity by Pennsylvania growers has raised questions as to the efficacy of existing chemical controls, particularly chlorothalonil. As a part of a study to answer these questions, technical grade chlorothalonil was incorporated into water agar medium at 0.2, 0.4, 0.6 and 0.8 mg/L. Isolates of Alternaria solani collected from two locations collected during 1987-2000, were examined by spotting spore suspensions of onto the treated medium. After 24 hours the number of germinated spores was recorded. In most cases, ED 50s were between 0.2 and 0.8 mg/L. There was nearly 100 percent mortality at the highest concentration. Some isolates from one location had ED 50 values between 0.8 and 1.5. It is unlikely that the reported increase in early blight severity is due to a decrease in the efficacy of chlorothalonil.

Involvement of phenols, pectin and callose in the resistance process of Pinus banksiana and P. contorta to the European race of Gremmeniella abietina.
M. SIMARD, D. Rioux, and G. Laflamme. Natural Resources Canada, Canadian Forest Service, Laurentian Forestry Centre, 1055 du P.E.P.S., Sainte-Foy, Quebec, Canada G1V 4C7. Publication no. P-2003-0026-NEA.

In North America, scleroderris canker is caused by the indigenous North American race and the introduced European race (EU) of Gremmeniella abietina. Recently, a high level of resistance to the EU race was observed in jack pine and lodgepole pine; it was suggested that the formation of ligno-suberized boundaries was significant in the defense system of the two pine species. Using naturally infected samples, further cytochemical and immunochemical characterizations of phenols, pectin and callose were carried out to clarify their involvement in the resistance to this disease. Reacting parenchyma cells, including hyperplastic cells, were rich in polyphenols, particularly catechins and condensed tannins. A strong reaction was obtained for pectin in the affected tissue, except for necrotic cells. Enrichment in polyphenols and pectin is considered to be an expression of resistance. Callose does not seem to play a major role in this process.

Conidiospores of the butternut canker fungus carried on the exoskeletons of three different potential beetle vectors.
J. E. STEWART, S. Halik, and D. R. Bergdahl. Dept. of Forestry, University of Vermont, Burlington, VT 05405. Publication no. P-2003-0027-NEA.

Butternut canker, caused by the fungus Sirococcus clavigignenti-juglandacearum (Scj) is threatening the survival of butternut (Julgans cinerea). This study examined the vectoring potential of three beetle species (Astylopsis macula, Eubulus parochus, and Glischrochilus sanguinolentus) that have been found to carry viable conidia of Scj. In 2001 and 2002, beetles were collected, rinsed, and artificially infested with conidia of Scj. The length of time beetles carried viable conidia (0, 6, 12 hours and 1, 2, 4, 8, and 16 days) and the total number carried were determined. Field collected beetles were also freeze-dried and examined using scanning electron microscopy to determine where conidia were on the exoskeleton. All three species carried viable conidia for up to 16 days. Both A. macula and E. parochus carried up to 14 million conidia per beetle. At 16 days, A. macula carried the largest mean number of conidia per beetle (7,600). At each time interval, G. sanguinolentus carried the least mean number of conidia (214,000 and 150 at 0 hour and day 16, respectively). Conidia were observed on the thorax, abdomen, and legs of A. macula and E. parochus. These observations suggest all three beetle species have the potential to vector conidia of Scj; however, A. macula and E. parochus may be more effective vectors.

Differentiation of the pine rusts Peridermium harknessii and Cronartium comptoniae with a PCR-RFLP marker and identification of a rust hyperparasite in North America.
E. ST-MICHEL (1), R. C. Hamelin (2), and L. Bernier (1). (1) CRBF, Université Laval, Québec, Canada, G1K 7P4; (2) Canadian Forest Service, Laurentian Forestry Centre, Quebec, Canada, G1V 4C7. Publication no. P-2003-0028-NEA.

We used PCR-RFLP to differentiate the heteroecious rust Cronartium comptoniae from the autoecious rust Peridermium harknessii which both attack Jack pine (Pinus banksiana). A Hpa I restriction site was observed in the ITS 1 portion of the rDNA in C. comptoniae but not in P. harknessii. This restriction site can thus be used for differentiating the two rusts species. In the course of this work, an additional product was obtained following PCR amplification of the ITS of rust aeciospore DNA samples. Extraction and sequencing of this product indicated that another fungus was present in our rust samples. After molecular and morphological examination, this fungus was identified as Cladosporium tenuissimum, a species reported to be hyperparasite on other rust fungi in Europe. This is the first time this mycoparasite is identified in rust samples in North America.

Systemic alteration of glucanase transcript levels in mycorrizal bean plants infected with Rhizoctonia solani.
K. WEN (1), M. St. Arnaud (2), and S. Jabaji-Hare (1). (1) Dept. of Plant Science, McGill University, Ste.-Anne de Bellevue, (2) Botanical Garden of Montreal and IRBV, 4101 Sherbrooke St. East, Montreal, Quebec, Canada. Publication no. P-2003-0029-NEA.

Arbuscular mycorrhizal (AM) fungus Glomus intraradices was used as a potential bio-control agent to elicit bean plant defense response against Rhizoctonia solani. A temporal and spatial accumulation of glucanase mRNA transcripts was analyzed in G. intraradices colonized bean plants post-infected with R. solani AG4. Real-time RT-PCR analysis revealed that glucanase transcript levels significantly changed with different sampling time. In leaves, stems and roots, R. solani-infected had higher glucanase transcript levels than in non-infected plants. The presence of the AM fungus showed no effect or suppression of glucanase transcription, depending on the tissue analyzed. However, when both fungi were present, glucanase transcript levels from all tissues were lower than in R. solani-infected plants but still higher than in non-infected plants. This may indicate that G. intraradices suppress the expression of glucanase systemically, especially in response to the infection of pathogen.