Posted online February 1, 2006
Reproduction of Meloidogyne hapla on potato cultivars and its management with soil or foliar Vydate applications. G. S. ABAWI, J. W. Ludwig, and B. K. Gugino. Dept. Plant Path., Cornell Univ., Geneva, NY 14456.
The northern root-knot nematode (Meloidogyne hapla, Mh) is a major pathogen of fresh and processing vegetables in NY. It has been observed that damage by Mh to carrot, lettuce and onion is most severe when they are grown after potato. Thus, the reproduction of Mh on 12 potato cultivars was evaluated in pasteurized soil infested with 10 eggs of Mh/cc soil in the greenhouse. All cultivars were excellent hosts to Mh (Reproductive Factor = 32 - 95) and exhibited galls on fibrous roots, but not on coarse roots. In field microplots, Vydate C-LV was applied as a broadcast spray incorporated before planting (1.29 gal /A) or as four foliar sprays (total of 2 gal /A). The number of Mh juveniles was reduced and potato yield significantly increased in the Vydate treatments in 2003, but not in 2004 or 2005, probably due to extreme wet and drought conditions, respectively. In 2003, foliar sprays of Vydate C-LV reduced the population of Mh in one grower field, but did not affect yield. In another field infested with 40 juveniles of Mh and 1600 Pratylenchus penetrans/100 cc soil, foliar Vydate sprays greatly reduced nematode populations and increased yield.
Increased resistance to fire blight in apple plants by silencing DspE-interacting proteins. E. E. BOREJSZA-WYSOCKA (1), M. Malnoy (1), S. V. Beer (2), and H. S. Aldwinckle (1). Cornell University, (1) Geneva, NY 14456; (2), Ithaca, NY 14853.
The DspE gene of Erwinia amylovora encodes a pathogenicity effector that is essential for development of the serious disease, fire blight, in apple plants. Genes encoding the four DspE-interacting proteins of Malus (DIPM genes) are conserved in all hosts of E. amylovora tested. Interaction between DIPM’s and DspE is thought to be involved in disease development. The fire blight susceptible cultivar Galaxy was transformed with seven silencing constructs. Transgenic lines with all constructs were recovered. Net expression of the target DIPM’s was determined by RT-PCR for their mRNA’s, resulting in evidence of partial to complete silencing of some of the DIPM’s in some transgenic lines. Resistance to fire blight was evaluated in the growth chamber by inoculation of vigorously growing shoot tips with the virulent strain Ea273 of E. amylovora. Some lines with silencing had increased resistance. Resistance due to silencing of a native apple gene(s) is likely to be more acceptable to regulators, growers and consumers than the addition of genes from other organisms.
Liposomes as a tool for detection of Erwinia amylovora. W. S. BOREJSZA-WYSOCKI (1), H. S. Aldwinckle (2), R. A. Durst (1), T. R. DeCory (1), and R. A. Montagna (3). (1) Dept. Food Science and Technology; (2) Dept. Plant Pathology, Cornell University, Geneva, NY 14456; (3) IBI, Inc., Grand Island, NY 14072.
This bioassay for Erwinia amylovora is based on simultaneous immunomagnetic separation and rapid, highly sensitive signal-amplification strategies using marker-loaded immunoliposomes. Monoclonal antibody Mab3B recognizes only strains of E. amylovora and was used for preparation of the immunomagnetic beads and immunoliposomes. Cells of E. amylovora are captured and concentrated from the sample extract by immunomagnetic beads, and immunoliposomes are simultaneously captured by the target bacterium. The resulting “sandwich” complex, consisting of numerous immunoliposomes bound to the bacterium which, in turn, is captured by the immunomagnetic beads, is then magnetically separated from the sample solution. After lysis of immunoliposomes, the marker level is measured by a photodetector, and the signal is proportional to the number of target bacteria. E. amylovora (Ea 273) cells were separated from a 10(^8) cells/ml suspension by immunomagnetic beads and showed a strong positive signal with immunoliposomes.
Management of bacterial speck in the field using plant activators. M. A. BORSICK, H. W. Lange, and C. D. Smart. Dept. Plant Pathology, Cornell University, Geneva, NY 14456.
Plant activators, which do not directly affect pathogens, are a promising management option in agriculture. These compounds activate plant defense responses and may work via the systemic acquired resistance (SAR) and/or induced systemic resistance (ISR) pathways. Regrettably, most of these products are underutilized due to inconsistent disease control. The goal of this research is to determine when plants in the field are “activated” in order to ascertain a more practical management strategy. Marker gene expression for known defense pathways will be followed using real-time quantitative PCR. We have conducted a preliminary field experiment testing the efficacy of plant activators in managing bacterial speck in New York. The results reveal that the SAR-inducing compound (acibenzolar-S-methyl) controls the pathogen to the same level as a copper-based spray program. The ISR-inducing Bacillus spp. ineffectively managed disease but did increase yield. Interestingly, the combination of both products controlled disease without a yield increase. It will be intriguing to see whether this interaction between SAR and ISR in the field is repeatable.
Exploiting genetic diversity within a germplasm collection to address questions of disease resistance. L. CADLE-DAVIDSON. USDA-ARS, PGRU, Geneva, NY 14456.
Germplasm repositories not only preserve genetic variation but also provide resources for exploiting traits not found among cultivated varieties. My laboratory at the USDA-ARS Grape Genetics and Genomics Group utilizes genetic resources maintained at the Plant Genetic Resources Unit (PGRU) to address fundamental questions in grapevine pathogenesis and to identify novel sources of disease resistance from the Vitis collection. Enzymatic resistance (eR) conferred by a glyoxylate aminotransferase in the breeding line 124111F has recently been proposed as a novel mechanism of resistance to Pseudoperonospora cubensis in Cucumis melo. We assayed 45 genotypes of Cucumis spp. with characterized resistance and susceptibility to P. cubensis, and only 124111F expressed characteristic eR, suggesting that eR is not a general marker of resistance but a specific mechanism of resistance. We then assayed all 1200 Vitis accessions maintained at the PGRU to search for this apparently rare, novel mechanism of disease resistance. I will present some results from this study and emphasize the potential value of germplasm collections when applied to biological research.
Outbreak of anthracnose caused by Elsinoe ampelina in vineyards in Quebec. O. Carisse. Agriculture and Agri-Food Canada, 430 Gouin, St-Jean-sur-Richelieu, Qc, Canada, J3B 3E6.
The first outbreak of anthracnose was observed in 2003 almost exclusively on the cultivar Cliche. On leaves, first symptoms appeared as small lesions, often numerous. As lesions age the center became grey and dries giving a ‘shot hole’ appearance. On shoots, lesions were circular, dark brown and surrounded by a purple margin. On berries, lesions were purple under wet conditions and brown surrounded by a grey margin under dry conditions. In 2005, outbreaks of anthracnose were observed in most vineyards planted with the cultivars Cliche and Prairie-Star. The first lesions on leaves were observed at the end of June following a major rain event. The disease progressed mainly during rainy periods. Optimum culture conditions were tested. The susceptibility of ten cultivars and selections was evaluated by spraying potted plants with a spore suspension of 10 × 5 spores per ml. The plants were covered with clear plastic bags for 48 hrs at 25°C (±3 C). Optimum growth was obtained when E. ampelina was grown on Czapek Dox agar incubated under light at 20°C. The most susceptible cultivars were ES-8-2-43, Cliche, Prairie-Star and ES-5-4-71.
A spray deposition analysis system for accurately quantifying the amount of fungicide or biocontrol agent applied to leaves or fruit of cacao. RONALD T. COLLINS. USDA, ARS Sustainable Perennial Crops Laboratory, 10300 Baltimore Ave-Bldg. 001, Rm. 223, Beltsville, MD 20705.
Various chemical and biocontrol agents have been used to control diseases of cacao. One difficulty in evaluating the effectiveness of these agents is quantifying the accuracy of placement of the control agent at the desired location in the necessary quantity. Due to the remoteness of cacao fields in Central and South America, West Africa and Asia and to the lack of facilities, the commonly used methods of measuring spray depositions are impractical. A new methodology was needed which: (1) Did not require elaborate laboratory equipment. (2) Had low cost per sample analyzed. (3) Did not require refrigeration for shipment (sample was stable). (4) Allowed easy sampling. (5) Was able to accurately and consistently quantify the amount of spray solution deposited on a unit area. After evaluating different dyes and colorants, it was found that spray deposition analysis system utilizing a blue colorant named Bulls Eye met all of the desired characteristics. This system accurately quantifies the spray deposition in all or selected portions of leaves as well as fruit.
Mechanism of biological control of grape crown gall by Agrobacterium vitis strain F2/5. J. E. CREASAP, G. Hao, H. Zhang, and T. J. Burr. Dept. Plant Path, NYSAE Station, Cornell Univ, Geneva, NY 14456.
A. vitis causes crown gall in grapevines, which can be a major problem in grape-growing regions. Via an unknown mechanism, the nontumorigenic strain, F2/5, prevents crown gall in grape tissue. M852, a Tn5 mutant of F2/5, lacks biological control ability, does not induce an HR on tobacco, and induces a reduced on grape shoots. The disrupted gene has been identified as homologous to an ATP-dependent Clp protease, clpA, with similarities to A. tumefaciens (86%) and Sinorhizobium meliloti Rm1021 (83%) clpA genes. Using the genome sequence of A. vitis strain S4, the ORF of clpA was amplified from F2/5 DNA, sequenced, and confirmed similar to clpA in S4 (97%). Site-directed mutagenesis has shown this gene is involved in biological control, and this clpA homolog will be cloned into an expression vector and used to complement M852. Additionally, to determine whether clpA is solely responsible for the lack of biological control, the genes immediately upstream and downstream have been sequenced and identified as a clpS homolog and ORFD, respectively. Site-directed mutants of these genes have been tested for biological control, HR, and necrosis.
Sensitivity to the fungicide quinoxyfen of powdery mildew isolates collected from pumpkin in New York in 2004. J. F. DAVEY and M. T. MCGRATH. Dept. Plant Pathology, Cornell Univ., Riverhead, NY 11901.
Isolates of Podosphaera xanthii were collected from a research field on Long Island in 2004. Of these isolates, 15 were selected for this study based on their sensitivity to quinone-outside-inhibiting (QoI; FRAC group 11) and demethylation inhibiting (DMI; 3) fungicides. Their baseline sensitivity to quinoxyfen (13), active ingredient in Quintec, was determined using a leaf disk bioassay (PD 80:633). Squash seedlings were treated with 5 quinoxyfen concentrations from 0 to 10,000 ppb. All 15 isolates tolerated 10 ppb, exhibiting some decrease in severity on these disks compared to 0 ppb. Highest concentration tolerated ranged from 100-10,000 ppb. No relationship was seen between quinoxyfen sensitivity and QoI sensitivity, DMI sensitivity, collection time, or whether Quintec was applied to the research plot where the isolate was collected. Quintec does not yet have a Section 3 US registration. It was granted a Section 18 registration in NY in 2004 and 2005. Monitoring sensitivity of P. xanthii over time to fungicides at risk for resistance, prior to widespread use, provides critical information for fungicide recommendations.
Evaluation of population estimates of Erwinia amylovora in the MARYBLYT™ program. M. M. DEWDNEY, R. C. Seem, and H. S. Aldwinckle. NYSAES, Cornell University, Geneva, NY 14456.
Fire blight, caused by Erwinia amylovora (Ea), is a catastrophic disease of apples. Blossom infection forecasting and bactericidal sprays are the best available strategy to control blossom blight. MARYBLYT (MB), a fire blight forecaster, estimates apple stigma Ea populations as epiphytic inoculum potential (EIP). To determine how well EIP described presence of Ea, we stigma-printed blossoms on CCT medium in 2004 and 2005, and recorded percent blossoms infested (PBI). Bacterial identification was later confirmed by PCR with Ea specific primers. To calculate EIP, weather data from the period from green tip to petal fall were collected. EIP poorly described the PBI but when EIP and PBI were plotted by date: a lag between PBI and EIP was observed. Correlations between PBI and the lag of several temperature-related variables were calculated. Correlations were greater with a 1- to 4-day lag. When lagged EIP (1 or 2 days) was used to estimate PBI, simple correlations were improved from 0.71 to 0.88 and 0.86, respectively. Since this study has shown that PBI has a delayed response to EIP, the addition of a lag period to EIP may improve MB forecast reliability.
BTH molecular response assessment in petunia, potato and tomato. A. P. DUQUE and W. E. Fry. Department or Plant Pathology, Cornell University.
Induced resistance by chemicals such as benzothiadiazole BTH (Syngenta Inc) mimics the biological activation of Systemic Acquired Resistance (SAR) by necrogenic pathogens, taking the place of salicylic acid (SA) in the SAR signal pathway, inducing the same molecular markers and range of resistance. Previous work in our laboratory found that BTH activates resistance against late blight caused by P. infestans, on petunias and tomatoes while it did not activate resistance against the same pathogen on potatoes, suggesting that the spectra of resistance activated by BTH are very crop and pathogen specific. Our current research seeks to understand the molecular mechanism by which BTH mimics the SAR response and further understanding why BTH works in some plants and not others. To address this question we used microarray technology to identify the genes expressed in response to BTH in petunias, tomatoes and potatoes. Knowledge of the differences of BTH effect on these plants will have important implications to practical disease management and will increase our understanding of disease resistance mechanisms in plants.
Influence of earthworm activity on soilborne diseases. W. H. ELMER. The CT Agr. Exp. Sta., P.O. Box 1106, New Haven, CT 06504.
Earthworms (Lumbricus terrestris) (EW) were raised in pots in the greenhouse and in field microplots and planted with asparagus, tomatoes, or eggplants to assess their influence on specific soilborne diseases caused by Fusarium oxysporum (FO) and Verticillium dahliae (VD). Greenhouse asparagus was grown in pots filled with FO-infested soil with and without EW (control). Compared to controls, asparagus + EW had 60% larger root systems and had 50% fewer root lesions, and the soil had a 10-fold increase in fluorescent pseudomonads (FP), an increase of 1.2-units in rhizosphere pH, and greater fertility. Greenhouse tomatoes grown in soil with FO f. sp. lycopersici and EW were twice as large, had 3X as many fruit, had 50% less stem discoloration, and a 12-fold increase in FP in the soil compared to controls. Asparagus grown in FO-infested microplots and eggplants and tomatoes grown in VD-infested microplots were larger, had more fruit, and less wilt when EW were added than plants grown in no EW microplots. The disease-suppressing mechanism with EW may involve an increase in beneficial microbes.
The effect of girdling and cutting on perithecia production in Nectria cankers on black birch. F. J. FERRANDINO, J. S. Ward, and S. L. Anagnostakis. CT Agric. Expt. Sta., P.O. 1106, New Haven, CT 06504.
Black birch (Betula lenta), prized for use as a veneer, is becoming an increasingly important component of CT forests (~27% of the stems). However, trunk deformations by Nectria cankers (Nectria galligena) can render the lumber valueless. A five-year study was undertaken to understand the impact of culling on disease epidemiology. In late April 2001 seven infected trees out of sixty in a 0.4-ha plot near Lake Saltonstall, CT were cut down and forty cankered logs were left on the ground. In addition, 20 of the infected trees were girdled. Periodic observations of perithecia development were made for over 200 cankers from June 2001 to May 2005. No fruiting bodies were observed from mid June to mid September in any year. For the first two years, cankers on cut and girdled trees produced 2-3 times the number of perithecia as cankers on control trees. After 28 months there was a sharp reduction in the production of fruiting bodies on cankered logs. Sporulation within cankers on girdled trees continued for 36 mo but by 47 mo there was a marked decline in perithecia production as girdled trees died and became infected with other wood rotting fungi.
Alternative methods for genetic transformation of fungal biocontrol agents and related species. E. FORTIER, G. Marchand, F. Belzile, and R. R. Belanger. Dept. Phytologie, Universite Laval, Quebec, Qc, G1K 7P4.
Over the past 30 years, remarkable progress has been achieved in genetic transformation of microorganisms. The availability of methods for genetic transformation has benefited phytopathology by providing new tools for the study of plant-pathogen interactions but reports of such approaches with biocontrol agents have been scarce. In this work, our objectives were to develop alternative transformation methods for Pseudozyma flocculosa, a biocontrol agent of powdery mildew fungi and P. antarctica, a related species. Electroporation and Agrobacterium tumefaciens -mediated-transformation were investigated as alternatives to PEG-mediated transformation of protoplasts. Results have shown that P. antarctica is easily amenable to genetic transformation by either method. In the case of P. flocculosa, it seems that specific and non-standard parameters are required to achieve reproducible transformation. Once optimized, these techniques will facilitate subsequent studies of biological control agents, such as P. flocculosa, and their interactions with plant pathogens.
Consequences of gene flow on the dynamics and survivability of wild x transgenic squash hybrid populations. M. FUCHS. Dept. Plant Pathology, Cornell University, New York State Agricultural Experiment Station, Geneva, NY 14456.
Gene flow is one of the major environmental safety issues over transgenic crops. We recently reported on the movement of transgenes between commercial virus-resistant transgenic squash CZW-3 and its wild relative, Cucurbita pepo spp. ovifera var. texana. Transgene transfer was documented over three generations under conditions of low disease pressure whereas it was not sustained beyond the first generation under conditions of high disease pressure. Wild progeny that acquired transgenes exhibited increased fitness over C. texana and their nontransgenic counterparts under conditions of high disease pressure. In contrast, C. texana outperformed all the other genotypes under conditions of low disease pressure. Limited information is available on factors that provide a selective advantage to C. texana hybrids and affect their dynamics and survivability. Some of these factors are being investigated under field conditions to address consequences of gene flow and determine if risk management strategies should be devised.
Pythium crypto-irregulare, a new species within the Pythium irregulare Buis. complex. C. D. Garzón, J. M. Yánez, and G. W. MOORMAN. Dept. Plant Pathology, The Pennsylvania State University, University Park, PA 16802-4506.
Pythium irregulare Buisman, is an important plant pathogenic species that exhibits great genetic and morphological variability. Both sequences of the internal transcribed spacers (ITS region) and the cytochrome oxidase (cox) II gene and the adjacent spacer, and AFLP banding patterns clearly indicate that P. irregulare is a species complex composed of Pythium irregulare sensu stricto and at least one other species. Based on these genetic analyses and measurements of morphological features of isolates from different host plants and geographical regions, we propose that a new species be recognized, Pythium crypto-irregulare, referring to its hidden identity greatly resembling P. irregulare sensu stricto. Among the isolates used in this study, many members of P. crypto-irregulare are resistant to mefenoxam while none of those belonging to P. irregulare sensu stricto exhibit such resistance.
Variation in ontogenic resistance to Uncinula necator in the USDA-ARS, PGRU Vitis germplasm collection. C. T. GEE (1,2), D. M. Gadoury (2), and L. Cadle-Davidson (1,2). (1) USDA-ARS, PGRU, Geneva, NY 14456; (2) Department of Plant Pathology, Cornell University, Geneva, NY 14456.
Ontogenic resistance (OR) to grape powdery mildew (Uncinula necator) has been shown to confer near-immunity to infection by ca 20 days post anthesis in several cultivars of Vitis vinifera and V. labrusca. We quantified variation in the magnitude and temporal distribution of OR within a diverse collection of Vitis spp. Candidate genotypes were inoculated with U. necator in a time series beginning at bloom for two growing seasons. In year 1, forty genotypes were highly resistant at all phenological stages, 9 exhibited a statistically significant decrease in susceptibility, and 4 showed no change in OR within 30 days after bloom, and remained susceptible. Year 2 results will be discussed. Berries of selected genotypes were harvested for a biochemical time-course study of cuticle development, as it relates to changes in OR. These results will facilitate our subsequent studies on the genetics and biochemistry of OR in grapevine berries.
Comparative efficacy of different silicon treatments on the control of wheat powdery mildew. M.-H. GUEVEL (1), J. G. Menzies (2), and R. R. Belanger (1). (1) Dept. Phytologie, Universite Laval, Quebec, Qc, G1K 7P4; (2) AAFCanada, Winnipeg, Man, R3T 2M9.
The mode of action by which Si protects the plants, its effect on plant growth and the means of application for optimal effect remain a subject of controversy. In this work, our objectives were: 1) to evaluate the efficacy of different silicon-based formulations in the wheat-powdery mildew interaction, and 2) to compare the efficacy of foliar versus root applications. To fulfill our objectives, Si absorption was determined by X-ray microanalysis techniques, and disease incidence and plant growth were measured over time. Results from our experiments indicated that only plants receiving root applications absorbed Si in their tissues. Also root-treated plants had a much lower disease incidence than plants sprayed with Si solutions. Potassium silicate, applied to the root system gave consistently the best results in terms of disease control. Measures of plant height and weight failed to link fertilizing properties with Si applications. These results tend to confirm the concept that systemic distribution of Si within the plant is necessary for optimal prophylactic effects.
Relating soil health management practices to root health and nematode populations. B. K. GUGINO, J. W. Ludwig, and G. S. Abawi. Dept. Plant Pathology, Cornell Univ., Geneva, NY 14456.
As a result of soil degradation and reduced profitability, growers have become interested in soil health (SH) and in implementing sustainable soil management practices. The SH program work team at Cornell is developing a cost-effective SH assessment protocol and is researching the effects of soil management practices (tillage systems, cover and rotational crops, and organic amendments) on soil quality and productivity. Our lab is quantifying the suppressive capacity of soil to root pathogens (root health, RH) using a bioassay with bean as well as enumerating nematode populations. RH is greatly affected by the soil management practices employed. For example, root rot severity (RRS) ratings of beans grown in soils managed using soil building crops and IPM practices was better (RRS 2.1) than conventionally managed (RRS 7.3) based on a scale of 1 (healthy) to 9 (>75% roots decayed). RH was improved with reduced tillage, long rotation with grain crops, certain cover crops and organic amendments. Seasonal and spatial variability of nematode populations within and between sampled fields has made it difficult to clearly assess their relationship to SH.
Viability of Sirococcus clavigignenti-juglandacearum conidia in beetle fecal pellets. S. HALIK, J. E. Stewart, and D. R. Bergdahl. University of Vermont, Burlington, VT 05405.
Conidia of Sirococcus clavigignenti-juglandacearum (SCJ), the fungus causing butternut canker, are carried and consumed by the beetles Astylopsis macula and Eubulus parochus. Conidia remain viable on exoskeletons, but viability is unknown after passage through the beetles’ digestive tracts. In 2004 and 2005, we collected 30-40 each of A. macula and E. parochus and fed all but 10 of each sporulating SCJ on butternut twigs. Fecal pellets were collected up to 96 h after feeding. Pellets were dissolved in sterile distilled water and streaked on malt extract agar to observe growth of SCJ and percent germination of conidia. Remaining pellet solution was stained with aniline blue and conidia counted using a hemacytometer. Numbers of conidia ranged from 0 to 533,750 and 0 to 86,250 in pellets of A. macula and E. parochus, respectively. Viable conidia were deposited by A. macula for up to 48 h and by E. parochus for up to 24 h. Germination of conidia from E. parochus pellets remained above 90% for at least 6 h. Fecal pellets from A. macula were used to inoculate butternut seedlings, resulting in infections and confirming another means of spore dissemination for SCJ.
Novel genes in Agrobacterium vitis affect grape necrosis and tobacco hypersensitive responses. G. HAO, H. Zhang, and T. J. Burr. Dept. Plant Path., NYSAES, Cornell Univ., Geneva, NY 14456.
Quorum-sensing system in A. vitis was associated with regulation of a tobacco hypersensitive response and a grape-specific necrosis (GSN). Two Tn5 mutants of strain F2/5 (M1123 and M901) are both HR-negative and necrosis-reduced. M1123 carries a Tn5 insertion in hypothetical protein (ORF4) that resides directly upstream of disrupted ORF5 in M901. ORF5 shows homology to the lysR family of transcriptional regulators which typically contain an N-terminal DNA binding domain and a C-terminal sensing domain. Mutations in the putative ORF4 and ORF5 were verified by site-directed mutagenesis and changes in HR and GSN phenotypes. ORF2, a homolog of lysE, and ORF3, a homolog of a homoserine kinase located upstream of ORF4, are also essential for HR and GSN. ORF1 and ORF6 are not associated with HR and GSN. A cosmid clone that carries this cluster (ORF4 and ORF5) complements the phenotypes. No differences were observed between profiles of N-acyl-homosering lactone autoinducers that were produced by M1123, M901 and F2/5. Efforts are under way to determine if lysR (ORF5) regulates other genes in this cluster and/or members of the luxR gene family in A. vitis.
Efficacy of treatments to reduce grape cluster compactness and Botrytis bunch rot. B. HED (1) and J. W. Travis (2). Dept. Plant Pathology, Penn State University, (1) North East, PA 16428; (2) Biglerville, PA 17307.
The compactness of clusters of Vitis interspecific hybrid ‘Vignoles’ plays a major role in the development of Botrytis bunch rot (Botrytis cinerea). In 2004, leaf removal (at trace bloom), cluster clipping (after shatter), and two foliar applications of either Ultra-Fine or SprayTech oil (at 1.5 or 2 percent, at pre-bloom plus full bloom or trace bloom plus full bloom) were evaluated for effects on fruit set, cluster compactness, and Botrytis bunch rot. Leaf removal and oil treatments reduced fruit set by 17.5 and 9 to 37 percent, respectively, and reduced compactness by 13 and 2 to 18 percent, respectively. Amending a Botrytis fungicide program with leaf removal, cluster clipping, and oils, improved Botrytis control by 60, 77, and 2 to 77 percent, respectively, over the fungicide program. Cluster clipping significantly reduced the severity of Botrytis and was statistically equivalent to two Botrytis fungicide applications. Cluster clipping was significantly more effective at reducing Botrytis than trace bloom leaf removal. Treatments that reduce the compactness of grape clusters can improve Botrytis control.
Pantoea stewartii subsp. stewartii requires motility for infection. C. M. HERRERA (1), M. D. Koutsoudis (2), D. Tsaltas (2), and S. B. von Bodman (1,2). (1) Plant science; (2) Molecular Cell Biology, University of Connecticut, Storrs, CT.
Pantoea stewartii subsp. stewartii (Pnss) is the causal agent of Stewart’s vascular wilt and leaf blight in maize. The bacterium colonizes primarily the xylem vessels where the production of an exopolysaccharide leads to vascular occlusion and plant wilt. How bacteria colonize and disseminate within the plant host is not well understood, although bacterial motility is an important component of bacterial plant colonization. Thus it seems surprising that Pnss is considered a non-motile, non-flagellated plant pathogenic bacterium. Our interest in studying plant colonization from a developmental perspective led us to re-evaluate the motility of Pnss during defined phases of bacterial population growth. We report that 1) the genome sequence of Pnss contains a number of gene systems consistent with bacterial motility. 2) Pnss exhibits swarming motility. 3) Non-motile mutants are impaired in the xylem colonization and dissemination. We conclude that Pnss is motile bacterium and should be classified as such. In addition, motility in Pnss is an essential aspect of virulence.
Effect of mowing and rolling practices on anthracnose severity of an annual bluegrass putting green. J. C. INGUAGIATO, J. A. Murphy, and B. B. Clarke. Dept. of Plant Biology & Pathology, Rutgers University, New Brunswick, NJ 08901.
Annual bluegrass (Poa annua) putting green turf can be extensively damaged by anthracnose caused by the fungus Colletotrichum graminicola. The objective of this field study was to evaluate anthracnose severity and ball roll distance in response to putting green mowing and rolling practices during 2004 and 2005. Treatments were arranged in a factorial combination using a split-split plot design with four replications. Mowing height (2.8-, 3.2-, and 3.6-mm) was the main plot, mowing frequency (7 or 14 times wk(^-1)) was the subplot, and lightweight vibratory rolling (rolling vs. no rolling) was the sub-subplot. Disease severity was greatest at the 2.8-mm mowing height, while plots mowed at 3.6-mm incurred the lowest levels of disease in both years. A significant interaction between mowing height and frequency on three rating dates in 2004 indicated no difference in anthracnose observed between mowing at 2.8- and 3.2-mm height at 7 times wk(^-1) while 2.8-mm mowing had more disease than 3.2-mm at 14 times wk(^-1) mowing frequency. Increased mowing frequency at 3.2- or 3.6-mm either reduced or had no effect on disease severity in 2004; whereas under 2.8-mm mowing, 14 times wk(^-1) increased disease compared to 7 times wk(^-1). Mowing frequency did not effect disease development in 2005. Rolling lessened anthracnose 9-23% over both years.
Fungicide resistance in Venturia inaequalis in Québec orchards: An overview of the problem. T. Jobin and O. Carisse. Horticultural Research and Development Centre, Agriculture and Agri-Food Canada, 430 Gouin, St-Jean-sur-Richelieu, Qc, Canada, J3B 3E6.
Fungicide resistance in apple scab has been thoroughly investigated in the past decades but only one study has been done in Québec, in 1994, on fenarimol. In the summer of 2003, more than 50 orchards with various levels of scab incidence were sampled and a collection of more than 300 monoconidial isolates was established. Isolates were tested in vitro for their sensitivity to kresoxim-methyl, myclobutanil, dodine and thiophanate-methyl. Baseline populations were constructed for all fungicides with 30 isolates collected in untreated abandoned trees. More than 70 percent of the isolates tested were resistant to myclobutanil, 34 percent to dodine, and 60 percent to thiophanate-methyl. Resistance to kresoxim-methyl imputable to alternative respiration has been identified in 25 percent of the isolates. No significant cross-resistance has been found between the different families. In vivo tests on in-vitro produced apple trees are currently underway to determine the implication of these results in the field.
A pectate lyase homolog associated with the hypersensitive response ability of Xanthomonas axonopodis pv. glycines strains from Thailand. S. KAEWNUM (1), S. Prathuangwong (1), and T. J. Burr (2). (1) Dept. Plant Path, Kasetsart Univ. Bangkok, Thailand 10900; (2) Cornell Univ., NYSAES, Geneva, NY 14456.
Xanthomonas axonopodis pv. glycines (Xag) causes bacterial pustule disease of soybeans. Isolates of Xag (26) from soybean areas in Thailand differed in their induction of a hypersensitive response (HR) on tobacco, pepper, tomato, cucumber, pea and sesame. Isolate KU-P-34017 caused an HR on all the plants tested. The minimal conc. of KU-P-34017 needed to induce HR on tobacco was 5 × 10(^8) CFU/ml. A period of 2.5 h was necessary for HR. Eukaryotic inhibitors, CoCl, LaCl and Na orthovanadate, and cycloheximide (partially) blocked the HR on tobacco, indicating a plant response. HR on tomato was inhibited only by CoCl. A transposon insertional mutant of Xag no longer induced a tobacco HR on most plants. A 1.4 Kb fragment was sequenced and revealed an open reading frame that has 78% amino acid identity with a pel gene from Xag. The mutant had the same growth rate as the wildtype strain, but did not induce soft rot or express polygalacturonase activity. The results demonstrate that the pel homolog is involved in the elicitation of HR on tobacco.
Deletion of the DspA/E and HrpN genes alters the virulence of Erwinia amylovora. W.-S. Kim, S. C. D. Carpenter, J. M. Bonasera, and S. V. BEER. Dept. of Plant Pathology, Cornell University, Ithaca, NY 14853.
The hrp/dsp gene cluster of the fire blight pathogen had been shown via transposon mutagenesis to include several genes that are involved in pathogenesis to host plants. We created in-frame gene deletions to determine the importance of two encoded, secreted proteins, HrpN and DspA/E, to the ability of E. amylovora to cause fire blight. The highly virulent strain, Ea273, isolated from apple in western NY, was the parent strain. DNA fragments upstream and downstream of the gene of interest were amplified by PCR, ligated together, then exchanged for the wild-type gene by homologous recombination. The mutations were confirmed by PCR. The dspA/E mutant lacked virulence in immature pear fruit and in apple shoots on greenhouse-grown trees. Complementation with the dspA/E gene in trans restored virulence to near wild-type levels. The hrpN mutant likewise lacked virulence in immature pear fruit and was greatly reduced in virulence in apple. Interestingly, complementation with the hrpN gene in trans in two vectors restored virulence to different degrees. These mutants are proving useful for addressing molecular genetic questions.
Bacteriophages, potential biological control agents of fire blight and its ecological monitoring by multiplex real-time PCR. W.-S. KIM (1), S. Lehman (1,2), K. E. Schneider (1), E. Barszcz (1), A. J. Castle (2), and A. M. Svircev (1). (1) Agriculture and Agri-Food Canada, SCPFRC, 4902 Victoria Ave. North, P.O. Box 6000 Vineland Station, ON, Canada L0R 2E0; (2) Dep. of Biol. Sci. Brock Univ., St. Catharines, ON, Canada L2S 3A1.
Fire blight caused by Erwinia amylovora (Ea) is a destructive disease on pear and apple in North America. The appearance of streptomycin-resistant Ea strains and the potential loss of streptomycin registration in Canada have driven development of biological control. We have isolated, enriched and screened phages based on their specific lytic activity on Ea. Selected phages were applied to blossoms with a unique phage carrier system in the field. To understand the impact of the phages in nature, we developed a multiplex real-time PCR detection system based on TaqMan® probe. The system was validated for use on target microorganisms isolated directly from blossoms and used to track the populations of those organisms during field trials. Our multiplex real-time system can detect 200 cfu/ml, and as little as 20 cfu/ml. The efficacy of the carrier-phage system was evaluated under the field conditions.
Surface adhesion is an important aspect of Pantoea stewartii subsp. stewartii virulence. M. D. KOUTSOUDIS (1), D. Tsaltas (2), C. M. Herrera (2), and S. B. von Bodman (1,2). (1) Plant science, (2) Molecular Cell Biology, University of Connecticut. Storrs, CT.
Pantoea stewartii subsp. stewartii (Pnss) is a vascular pathogen of maize. The organism has a quorum sensing system that governs the cell density-dependent synthesis of Stewartan exopolysaccharide (EPS). The timing of EPS synthesis is a critical aspect of virulence. An N-acyl-homoserine lactone (AHL) deficient mutant strain is unable to synthesize EPS and appears to be arrested in the adhesion phase of bacterial development. This strain is also highly attachment proficient and localized to the site of infection. Exogenous addition of AHL promotes EPS synthesis and a parallel decrease in attachment. The signal deficient strain is capable of forming tightly packed pillar structured biofilms. The wild type strain exhibits a more developed, less compact three-dimensional biofilm architecture. A mutant lacking the EsaR quorum sensing repressor produces EPS constitutively. This strain is adhesion deficient and forms “floating” biofilms. Both quorum sensing regulatory mutants are significantly compromised in their ability to infect the host.
Systemic acquired resistance and fungicides for management of tobacco blue mold. J. A. LAMONDIA. Valley Laboratory, The Connecticut Agricultural Experiment Station, Box 248, Windsor, CT 06095.
Field experiments were conducted over several years to determine the efficacy of fungicides and the systemic acquired resistance initiator Actigard 50 WG (a.i. acibenzolar-S-methyl) for management of tobacco blue mold, a disease caused by Peronospora tabacina that has caused millions of dollars of losses in Connecticut over the last few years. We evaluated Actigard alone, the standard fungicides Acrobat MZ and Quadris at label rates, and a combination of Actigard at rates ranging from 2.2 to 35.1 g per ha with fungicides for disease management. Applications were made on six dates to 4 replicate plots at 14-day intervals. Actigard was applied only on the last 3 spray dates. Acrobat MZ and Quadris significantly reduced disease. Actigard alone resulted in similar disease control but caused some damage to tobacco at the highest rate. The low rates of Actigard in combination with fungicides resulted in superior disease control.
Weeds as reservoirs of Xanthomonas campestris pv. campestris in New York. H. W. LANGE, G. C. Meeks, and C. D. Smart. Dept of Plant Pathology, Cornell University, Geneva, NY 14456.
The relative role of weeds and soil debris as sources of the pathogen Xanthomonas campestris pv. campestris (Xcc) in New York is unknown. In other locations cruciferous weeds have been shown to harbor Xcc, contributing to field infections of black rot. In 2004, there was a severe outbreak of black rot in NY. The goal of this study was to determine if weeds serve as a pathogen reservoir for subsequent seasons in New York. Five fields that had a significant black rot problem in 2004 were chosen for the study. Individual plant species, including crucifers and other weeds, were collected at multiple times throughout the spring and summer from each site. Xcc was isolated from several species of cruciferous weeds at each site, but non-cruciferous weeds were not found to be hosts of the pathogen. DNA fingerprint analyses using rep-PCR is currently being used to determine if strains isolated from weeds in 2005 are the same as those isolated from cabbage in 2004. This comparison will allow us to determine if there are certain strains that are common in New York every year, or if new strains enter the state each season.
Genes required for twitching motility in Xylella fastidiousa. YAXIN LI, Guixia Hao, Yizhi Meng, Cheryl D. Galvani, Harvey C. Hoch, and Thomas J. Burr. Dept. of Plant Pathology, Cornell University-NYSAES, Geneva, NY 14456.
Xylella fastidiosa Temecula 1 (T1), an important phytopathogen causing Piece’s disease of grapevine, was recently shown to exhibit type IV pili mediated twitching motility on modified PW agar surface and the ability to migrate preferentially against a flowing current. EZ::TN transposome system was used to develop twitching-defective mutants. Cloning and sequencing analysis revealed seven associated genes residing in three pil gene clusters, including pilX cluster (fimT and pilX and pilY1), pilQ cluster (pilQ and pilO) and pilA cluster (pilB and pilR). fimT, pilX, pilQ, pilO, pilB and pilR mutants lack the twitching phenotype, while pilY1 mutant colony exhibited significantly reduced twitching motility. With transmission electron microscopy, we observed the cell morphologies for wild type and two non-twitching mutants. No type IV pili but only type I pili were found in non-twitching mutants whereas both types of pili exist in the wild type. Inoculation experiment indicated that the basipetal movement in planta was inhibited in non-twitching mutants compared as that in the wild type.
An additional copy of the apple gene MpNPR1 in transgenic Malus × domestica induces increased disease resistance. M. MALNOY (1), E. E. Borejsza-Wysocka (1), S. Y. He (2), and H. S. Aldwinckle (1). (1) Cornell Univ., Geneva, NY 14456; (2) Michigan State Univ., East Lansing, MI 48824.
The NPR1 gene is thought to be pivotal in the defense cascade caused by systemic acquired resistance and by R gene resistance in plants. An NPR1 homolog, MpNPR1, was cloned from Malus × domestica and over-expressed in two apple cvs, Galaxy and M26. Over-expression of MpNPR1 mRNA was shown by RT-PCR. Activation of some PR proteins was also demonstrated. Resistance to the serious bacterial disease, fire blight, was evaluated in the growth chamber by inoculation of vigorously growing shoot tips of 30-cm high own-rooted potted plants with the virulent strain Ea273 of Erwinia amylovora. Galaxy clones with an additional copy of MpNPR1 under control of the potato Pin2 promoter had necrotic lesion length of 32-40% compared with 80% in the control Galaxy. M26 clones with an additional copy of MpNPR1 under the control of the CaMV35S promoter showed a significant reduction in lesion length compared to the control M26. Preliminary tests indicate that several MpNPR1 over-expressing Galaxy clones may also have increased resistance to two important fungal diseases of apple.
Spectrum of virulence in the Puccinia sorghi population. D. A. MASSEY, D. A. Shah, and H. R. Dillard. Dept. of Plant Pathology, Cornell University, NYSAES, Geneva, NY 14456.
Eleven genetically pure Puccinia sorghi, isolates were obtained from samples collected in IL, KS, MN and NY in 2004. Five of these were obtained from samples growing on corn of known resistance (Rp) phenotype. The other six were obtained from a sample of infected leaves of the hybrid HiII (no known Rp genes), which was used to serially inoculate near isogenic seedlings homozygous for one of ten Rp alleles in the H95 inbred background: Rp1-A, Rp1-B, Rp1-C, Rp1-D, Rp1-E, Rp1-J, Rp1-M, Rp-G, Rp3-A, or Rp4-A. The virulence of each of the 11 pure isolates was tested on 2-3 week old seedlings of the above set of differentials. Ten isolates exhibited unique virulence phenotypes. Six exhibited intermediate interactions or were virulent against more than one of the following: Rp1-D, Rp1-E, Rp1-J, and Rp-G. None of the Rp genes were completely effective against all isolates. On Rp1-D, three isolates were virulent and an intermediate interaction was observed with three other isolates. This represents a considerable level of phenotypic diversity in the P. sorghi population. AFLP analysis of the genetic variability in P. sorghi is currently being conducted.
Analysis of soil samples from central Mexico for potential suppressiveness to Phytophthora infestans. H. MAYTON (1), C. Jones (2), M. Cadena-Hinojosa (3), J. Thies (2), and W. E. Fry (1). (1) Dept. Plant Pathology, Cornell University, Ithaca, NY 14853; (2) Dept. Crop and Soil Science, Cornell University, Ithaca, NY 14853; (3) Campo Experimental Valle de Mexico, CIR-CENTRO INIFAP, Chapingo, Mexico, 56230.
Soil samples from the central highlands of Mexico were evaluated for suppressive activity to Phytophthora infestans sporangia in soil. A New York soil was used for comparative analysis. Even though both mating types are known to cohabit these regions of Mexico, no oospores were detected in the soils. Through infectivity bioassays it was determined that infectivity was higher in sterile versus non-sterile soils. Significant differences in the duration and degree of infectivity of P. infestans were observed among the various soils. Soils collected from the Toluca valley appeared to be less hospitable to the pathogen than the mountain soils sampled, where wild Solanum species are commonly infected with P. infestans. Bacterial diversity in the different soil types was investigated using terminal restriction fragment length polymorphism analysis (T-RFLPs). Each soil community was different from that of the others.
Occurrence of fungicide resistance in Podosphaera xanthii on Long Island, NY, in 2004 and impact on cucurbit powdery mildew control. M. T. MCGRATH. Dept. Plant Pathology, Cornell Univ., Riverhead, NY 11901.
Resistance (R) to quinone-outside-inhibiting (QoI) fungicides was first detected in Podosphaera xanthii, cucurbit powdery mildew (PM) fungus, in the US in 2002. In 2004, R to QoI, DMI (demethylation inhibiting), and MBC (methyl benzimidazole carbamate) fungicides in P. xanthii was examined in commercial fields and research plots. A fungicide sensitivity bioassay was done in 3 spring summer squash crops and 4 pumpkin crops on 29 July, when PM was starting to develop in pumpkin. The frequency of QoI R was 15-84%, MBC R was 31-91%, and moderate DMI R was 15-84%. A QoI was applied only in 1 of the squash crops beforehand. Isolates were collected 3 times from research plots planted to pumpkin and subjected to a leaf disk fungicide assay. Frequency of QoI R was 14% on 9 Aug when symptoms were first seen in the experiment and the fungicide treatments began, 45% on 23 Aug where a DMI plus sulfur (S) was applied in alternation with quinoxyfen plus S, and 71% where the DMI plus S was alternated with a QoI plus S. Frequency of QoI R on 23 Sep was 90% and 100%, respectively. Control of PM on lower leaf surfaces on 13 Sep was 69% and 93%, respectively.
Volatile metabolic profiling to detect and discriminate diseases of mango fruit. M. MOALEMIYAN, A. Vikram, and A. Kushalappa. Dept Plant Sci., McGill Univ. Ste. Anne de Bellevue, QC, Canada H9X 3V9.
Volatile metabolites from headspace gas of mango fruits, cv. Tommy Atkins, wounded and inoculated with Colletotrichum gleosporioides (Cg) or Lasiodiplodia theobromae (Lt) or non-inoculated controls (Ck) were profiled using a GC/mass spect. to develop a technology to discriminate diseases caused by the above pathogens. Over 200 peaks were detected. Among these, 34 compounds were consistent among replicates, including seven that were specific to one or more diseases/inoculations. 1-pentanol and boronic acid ethyl were unique to Lt-inoculated mangoes while thujol was unique to Cg-inoculated mangoes. 1-butanol, ethyl propanoate, and styrene were detected only in pathogen-inoculated mangoes. Methyl octanoate was common to mangoes inoculated with Cg, non-wounded Ck, and wounded Ck. The significant mass ions and relatively consistent compounds were subjected to discriminant analysis to develop models to predict diseases/inoculations. The disease discriminatory compounds and discriminant analysis models could be used in the early detection and discrimination of postharvest mango diseases, cv. Tommy Atkins.
Tombusviruses isolated from water draining forest stands in New Zealand. S. S. MUKHERJEE (1), J. D. Castello (1), Tony Lough (2), and Douglas Hopcroft (3). (1) Faculty of Environmental & Forest Biology, SUNY College of Environmental Science & Forestry, Syracuse, NY 13210; (2) Agrigenesis, Auckland, New Zealand; (3) HortResearch, Palmerston North, New Zealand.
Plant viruses were isolated from surface waters of the North Island of New Zealand using Zeta Plus 50S membrane filtration. Morphology (30 nm icosahedra), A(260/280) values of 1.64, & buoyant densities in CsCl of 1.35 g/cc suggested that they were tombusviruses. Maximum parsimony trees using an 820 bp fragment within the p33 gene, and the entire 1100 bp coat protein gene suggested two distinct isolates. The first is an apparently new species of the genus Tombusvirus with maximum coat protein gene sequence similarity of 73% to artichoke mottled crinkle virus. We propose the name Turitea Creek Tombusvirus. The second virus is an isolate of TBSV with 88% coat protein sequence similarity to TBSV, cherry strain. To our knowledge, this is the first report of tombusviruses isolated from New Zealand.
Anti-sporulant activity of trifloxystrobin on nectarine scab twig lesions. E. MURDAY, N. Lalancette, and K. A. Foster. Rutgers University, Agricultural Research and Extension Center, Bridgeton, NJ.
The influence of bloom applications of trifloxystrobin fungicide (Flint 50WG) on sporulation of overwintering twig lesions caused by Fusicladosporium carpophilum was studied in a ‘Redgold’ orchard during 2005. Treatments consisted of two consecutive applications at pink and bloom, bloom and petal fall, and petal fall and shuck-split to 3 × 3 tree plots arranged in a RCBD with three replicates. Five sporulation assessments were subsequently conducted from May through August by removing ten infected twigs from each plot and incubating them for 24 h at 25°C and RH > 95%. Conidial production was estimated using a hemacytometer. All treatments significantly reduced sporulation relative to the non-treated control and conidial production (#conidia/twig) was reduced by 64 to 70% over the entire season. Concomitant reductions in fruit disease incidence and severity were also observed. These results indicate that the use of anti-sporulant fungicides at bloom is an effective strategy for reducing inoculum production on twigs prior to and during the subsequent fruit growth and development period.
Geographic distribution and diversity of Phytophthora spp. associated with bleeding cankers of European beech, Fagus sylvatica. A. H. NELSON, J. E. Weiland, and G. W. Hudler. Dept. Plant Path., Cornell University, Ithaca, NY.
European beech (Fagus sylvatica) are common in managed landscapes throughout the northeastern United States. The death of mature specimens has been reported for at least 50 years, although a cause has not been determined. Often, the first indication of decline is the presence of bleeding cankers on the lower trunk. Surveys have revealed cankers are widespread, affecting about 25% of mature European beech in the Northeast. These symptoms, along with ELISA results and literature reports, suggested the involvement of a species of Phytophthora. Attempts to isolate from the cankers consistently yielded two members of the genus Phytophthora; P. citricola and P. cactorum. Identification of isolates was conducted using morphological characteristics as well as phylogenetic analyses comparing the beta tubulin and cytochrome c oxidase genes and the ITS regions. Both species were found associated with diseased trees throughout the Northeast, often infecting trees at the same site, though they were never recovered from the same tree. Current work is focused on completing Koch’s postulates with both species.
Characterization of HIPM (HrpN-Interacting Protein from Malus) in apple. C.-S. Oh and S. V. Beer. Dept. Plant Pathology, Cornell University, 334 Plant Science Building, Ithaca, NY 14853, USA.
HrpN (harpin) of Erwinia amylovora, the first bacterial cell-free elicitor of the hypersensitive reaction (HR), is critical to the development of fire blight of apple and pear. Moreover, HrpN promotes growth and induces systemic acquired resistance (SAR) after many plants are sprayed with the protein. To elucidate the mechanisms underlying the effects of HrpN, we sought an HrpN-interacting protein(s) in apple using a yeast two-hybrid assay. We found a single positive clone, designated HIPM (HrpN-Interacting Protein from Malus), which encodes a 6.5-kDa protein. Deletion analysis showed that the N-terminal 198 of 403 amino acids of the HrpN protein are required for interaction with HIPM. HIPM orthologs were found in Arabidopsis thaliana (AtHIPM) and rice (OsHIPM). Domain analysis showed that HIPM has a functional signal peptide. GFP-labeled HIPM associated, in clusters, with plasma membranes. The HIPM gene is expressed constitutively; it is expressed more strongly in apple flowers than in leaves and stems. Silencing of the HIPM gene in apple is underway as an approach to determine its function.
A multiplex PCR assay for detection of Clavibacter michiganensis subsp. michiganensis, Pseudomonas syringae pv. tomato and Xanthomonas axonopodis pv. vesicatoria. Z. ÖZDEMIR. Dept. Plant Protection, Adnan Menderes University, Aydın, Turkey.
A multiplex PCR assay has been developed for detection of Clavibacter michiganensis subsp. michiganensis (Cmm), Pseudomonas syringae pv. tomato (Pst) and Xanthomonas axonopodis pv. vesicatoria (Xav) in a single PCR tube. Previously reported primers of CMM-5-CMM-6 for Cmm, primer 1-primer 2 for Pseudomonas syringae pathovars and Pst, and RST2-RST3 designed from parts of hrp gene of Xanthomonas campestris pv. vesicatoria for Xav were employed. For multiplex PCR, annealing temperatures were tested by gradient PCR and primer concentrations were investigated. Temperatures of 59 ± 1°C were found optimal for annealing. Primer concentrations of 0.36 µM for Cmm, 0.30 µM for Xav, and 0.12 µM for Pst in 25 µl reaction volume produced approximately equal PCR amplification yield. Determination of detection thresholds from pure cultures for multiplex PCR assay is in progress. Optimization of multiplex PCR conditions from pure cultures could be a preliminary step for assays of these pathogens from tomato seeds.
Defining the biological functions of the Potato leafroll virus readthrough protein. KARI PETER and Stewart Gray. Cornell Univ. and USDA-ARS, Ithaca, NY 14853.
Potato leafroll virus (PLRV) particles contain 180 coat protein monomers, with a percentage containing a readthrough (RT) extension located on the particle’s surface. In other luteoviruses, the conserved RT N-terminal domain has been linked to aphid transmission, viral accumulation and movement. The effect of small deletions in the PLRV RT N-terminal domain was the focus of this study. Fourteen site-directed mutants were generated from a cloned PLRV cDNA and delivered to plants by agroinoculation. All mutant viruses accumulated locally in infiltrated N. benthamiana tissue. All mutants expressed RT in the plant; however, only 3 mutants incorporated RT into the virion when purified. These mutants, as well as 4 non-incorporating RT mutants, were examined further for systemic infection in 3 host species. All mutants, regardless of RT incorporation, moved systemically in each host. None were aphid transmissible despite RT incorporation and when bypassing the midgut. The RT protein’s biological properties are sensitive to small changes in the N-terminal domain, which isn’t required for systemic movement, yet incorporation doesn’t ensure aphid transmission.
Natural epidemic of fireblight in a newly planted orchard and effect of pruning on disease development. V. Philion (1), J. Charest (2), and V. TOUSSAINT (3). (1) IRDA, St-Hyacinthe, Qc; (2) MAPAQ, Marievielle, Québec; (3) CRDH, St-Jean, Qc.
In 2005 we experienced a fireblight epidemic (Erwinia amylovora) in a newly planted orchard at IRDA St-Bruno research station. Because the trees were stored in a cold chamber prior to planting, bloom was delayed 2 wks and coincided with a period of high temperature, which triggered infection. First symptoms were visible on June 30th and disease progressed until August. The epidemic was monitored during the summer and 6 eradication strategies were compared. In July, we could clearly observe a rapid disease progress in the untreated plots, which underlines the need for pruning trees as soon as possible. Initial disease severity was proportional to bloom intensity and was highest on Paulared, intermediate on Empire and Cortland, and low on McIntosh. The rate of disease increase was similar for Paulared, Empire and Cortland but lower for McIntosh. Since bloom in the year of planting is expected to occur when temperatures are favorable to E. amylovora, our future recommendations to growers will stress the need for treatment or complete removal of flowers on susceptible cultivar/rootstock combinations.
Variability of isolates of M. gramnicola obtained from diverse geographic regions. R. R. POKHAREL (1), G. S. Abawi (1), J. M. Duxbury (2), J. A. Brito (3), and C. D. Smart (1). (1) Dept of Plant Pathology, Cornell University, Geneva, NY; (2) Dept. of Crop and Soil Science, Cornell University, Ithaca, NY; (3) Florida Dept. of Agriculture and Consumer Service, FL.
Isolates of Meloidogyne graminicola (Mg) collected in Nepal, India, Bangladesh and Florida were compared according to morphometric measurements of second-stage juveniles (J2), host range, virulence to rice and ITS sequencing. The mean of body length, stylet length, A, B and C values for j2 of all isolates were generally within the range described for Mg. Two Nepalese and two Indian isolates were the most virulent to rice cvs. Labelle and LA 110, respectively, whereas the Florida isolate was the least virulent on both cultivars. Results of the host range study indicated that several rice varieties were poor hosts to the Florida isolate, but were excellent hosts to the other isolates. The Florida isolate exhibited a similar perennial pattern and belonged to the same clade in the parsimony analysis of the ITS region, although there was minor sequence difference between these isolates. These results suggest the possible existence of different races within this nematode species.
Quaternary ammonium sanitizer eliminates airborne spores of Penicillium expansum. A. L. Rugh and D. A. ROSENBERGER. Cornell University’s Hudson Valley Lab, Highland, NY 12525.
Penicillium expansum causes blue mold decay, the major postharvest disease of apples. Spore densities in packinghouse air gradually increase during winter months when packinghouses are rarely vented. The airborne spores can infect fruit as they are packed. Studies were conducted to measure the settling rate of spores in undisturbed air in a plastic-covered hoop greenhouse measuring 7.5 × 10.9 m with a height of 3.1 m. Spore trapping with a Burkard volumetric spore trap showed that most spores settled to the floor within four hours of release regardless of whether the spore load was generated by manually dislodging spores from a Petri plate culture in front of a fan, by releasing spores into air with an atomizer, or by using fans to resuspend spores that previously settled to the floor. Spraying uncoated concrete floors after spores had settled with 200 ppm quaternary ammonium (Deccosan 315, Cerexagri, Inc., King of Prussia, PA) at a rate of 115 ml of solution per square m of floor eliminated spores in the test chamber and could prove useful for reducing airborne inoculum in apple packinghouses. Spraying floors prior to spore settling was not effective.
A selective medium for recovering Penicillium from soil. A. L. RUGH and D. A. Rosenberger. Cornell University’s Hudson Valley Lab, Highland, NY 12525.
Penicillium expansum causes a postharvest decay of apples and is common in orchard soils, but populations in eastern soils have not been quantified. We developed DG18-P agar, a modified DG18 agar, for recovering Penicillium species from soils. DG18-P contains 155 g glycerol, 10 g glucose, 5 g peptone, 1 g monobasic potassium phosphate, 0.5 g magnesium sulfate heptahydrate, 2 micrograms dichloran dissolved in 1 ml ethanol, 15 g agar, 0.1 g chloramphenicol, 1.0 ml tergitol NP-10 nonionic in 1000 ml distilled water. Dichloran is added after autoclaving. This medium suppresses fast growing fungi such as Trichoderma and Mucor species and eliminates most bacteria, yeasts, and actinomycetes. When plates of DG18-P and acidified potato dextrose agar were similarly inoculated with known numbers of P. expansum spores, colony counts were similar on both media. DG18-P does not eliminate other species of soil-inhabiting Penicillium, so quantification of P. expansum from soil dilution plates must be based on sub-sampling and colony morphology on Czapek yeast extract agar. DG18-P is being used to study Penicillium densities in soil, on wooden bins, and on apple fruit in the field.
Oxalic acid secretion and extracellular oxalate regulation by brown rot wood decay fungi. J. S. SCHILLING and J. Jellison. Dept. of Biological Sciences, University of Maine, Orono, ME 04469.
Brown rot fungi depolymerize wood non-enzymatically by combining iron (II) and hydrogen peroxide to produce hydroxyl radicals. We are studying the role of fungal oxalic acid secretion in lowering pH and mobilizing iron because it is unknown if these functions are mechanistic or incidental. We tested for oxalate optimization by subjecting wood pre-treated with 0-100 mM oxalate to several brown rot species. In agar microcosms, brown rot fungi equalized wood pH and oxalate regardless of time-zero level. In soil-block microcosms, however, oxalate catabolism dynamics were more variable between microcosms, suggesting soil effects. Because these same fungi over-produce oxalate when grown on metal-amended agar, we tested effects of soil-relevant forms of Fe, Al, and Cu on these brown rot dynamics. While agar oxalate levels were affected by metal treatments, wood oxalate was equal among treatments despite significant metal translocation into the wood. Our work suggests that brown rot fungi regulate extracellular oxalate during wood decay, and this has implications both on in-service wood preservation and on forest biogeochemistry.
Impact of Sirococcus clavigignenti-juglandacearum on health of butternut. T. Schmalz and D. R. BERGDAHL. University of Vermont, Burlington, VT 05405.
Butternut canker, caused by Sirococcus clavigignenti-juglandacearum (SCJ), results in extensive decline and mortality of butternut (Juglans cinerea) throughout its range in North America. On permanent plots in northern Vermont, SCJ infection and butternut mortality rates were 92% and 12%, respectively (1993-1996). Reduced crown vigor and secondary pathogens, especially Armillaria sp., were associated with SCJ infection. In 2001-02, infections increased to 96% and mortality to 41%. Logistic regression models found significant associations among mortality and canker presence, root rots, crown class, and stocking level. When examined in 2002, trees that exhibited main stem or root cankering in 1996 were more likely to be dead and have Armillaria root rot and increased epicormic branching compared to uninfected trees, especially in understocked stands. Suppressed trees were most likely to die, and dominant trees had highest rates of Armillaria root rot, other root rots, and heart/trunk rots. Butternut canker plays a significant role in increased mortality, occurrence of secondary pathogens, and reduced overall health of trees.
Population levels of Aspergillus niger in muck soil in relation to the inoculum load required for infection of onion seedlings. A. M. SEYB and J. W. Lorbeer. Dept. Plant Pathology, Cornell University, Ithaca, NY 14853.
Black mold of onion, caused by A. niger, can be a serious pre-emergence and post-harvest problem. Several infection pathways have been suggested to explain disease development, including infection of the root system via A. niger in the soil. This study investigated the relationship between levels of inoculum in muck soil and the incidence of seedling infection. The spatial distribution of A. niger was determined in two New York onion fields in Orange County. The distribution of inoculum was aggregated but was not related to the direction of cultivation/the rows. The levels of inoculum ranged between 0 to 3.7 × 10(^4) spores per gram (oven dry weight) in the first field and 0 to 3.9 × 10(^4) spores per gram (oven dry weight) in the second field. A laboratory-based trial was conducted in which onion seedlings grown in muck soil and inoculated with different levels of A. niger inoculum were incubated at 15, 20, 25 and 30°C. Higher levels of inoculum were required for seedling infection at the lower temperatures (15 and 20°C) than at 25 and 30°C. Temperature significantly affects the inoculum potential of A. niger and therefore infection.
Meta-analysis of yield losses due to common rust in sweet corn. D. A. SHAH and H. R. Dillard. Dept. of Plant Path., NYSAES, Geneva, NY 14456.
Meta-analysis is the quantitative synthesis of the results of several independent studies, with the objective of deriving more information from the pooled results than that provided by each individual study alone. Nineteen of 69 published studies from 1977 to 2004 that reported on common rust (Puccinia sorghi) in sweet corn were identified as meeting criteria for inclusion in a meta-analysis. Percent yield loss (by weight) was calculated from presented data, and restricted (intercept = 0) linear regression was used to relate % yield loss to % rust severity. Individual regression slope estimates and their associated variances were the basis of a random effects meta-analysis, in which “study” was specified as the random effect. The overall slope estimate was 0.5045, with a 95% confidence interval of (0.2311, 0.7780). Interpretation of these results suggests that every 10% increase in rust severity results in a 2.3 to 7.8% reduction in yield. The between-study variability was further examined by meta-regression using state, study year, variety, minimum severity, maximum severity and severity range as individual covariates. Variety explained 77% of the between-study variability in slope.
Phenotypic and genetic variation of Plectosporium tabacinum isolates from southern New England. S. L. SLINSKI and R. L. Wick. Dept. of Microbiology, University of Massachusetts, Amherst.
Plectosporium blight caused by Plectosporium tabacinum is an emerging disease of cucurbits that has become relatively common in MA and CT since 1999. The objectives of this research were to gather information on genetic, phenotypic and physiological variability, and fungicide resistance. Ten to thirty single spore isolates have been collected from each of 17 sites in MA and CT in 2004. Optimum and cardinal temperature for growth on potato dextrose agar were 25, 13 and 33°C respectively. Optimum pH for mycelial growth was between 6.5 and 8.0. Of several culture media examined growth was best on carrot and V8 agar. P. tabacinum was sensitive to nystatin and cyclohexamide; moderately sensitive to PCNB and chloroamphenicol; and resistant to chlorotetracycline HCL, streptomycin sulfate and neomycin sulfate. Spores began to germinate at four hours at 25°C in both the light and in the dark. The growth rate of P. tabacinum isolates on fungicide amended media varied, but no apparent fungicide resistance was observed. All isolates tested were vegetatively compatible.
Recent changes to a model of Venturia inaequalis ascospore maturation. A. Stensvand (1), H. Eikemo (1), D. M. GADOURY (2), and R. C. Seem (2). (1) Norwegian Crop Research Institute, 1432 Ås, Norway; and (2) Cornell Univ., NYSAES, Geneva, NY 14456.
A model to estimate ascospore maturity of Venturia inaequalis (Phytopath. 72:1073-1080) was recently revised (Plant Dis. 89:198-202) to reflect the impact of rain-free periods after bud break, but delayed spore maturity may also be associated with dry weather before bud break. Our objective was to quantify the impact of late-winter dryness upon final stages of pseudothecial maturation. Scabbed leaves from 12 countries exposed to a range of moisture prior to bud break were placed in a common controlled environment. Ascospores were harvested weekly until the supply was depleted. The number of pre-bud break rain events affected the initial date of ascospore maturity, but not the subsequent rate. For sites where rain >2 mm fell on ca 15 days during the month prior to bud break, no adjustment of the model was necessary. To improve performance at drier sites, 10 degree days were added to the model biofix for each additional day with <2 mm rain. At wetter sites 10 degree days were subtracted for each additional wet day. These preliminary changes were evaluated with additional field data.
Histopathology of hybrid poplar stems inoculated with Septoria musiva. J. E. WEILAND (1) and G. R. Stanosz (2). (1) Dept. Plant Path., Cornell Univ., Ithaca, NY; (2) Dept. Plant Path., Univ. Wisconsin, Madison, WI.
Septoria musiva causes cankers that limit production of susceptible hybrid poplars in eastern North America. Despite variation in gross responses of poplar clones to S. musiva, knowledge of differences at the tissue level is limited. Branches of field-grown clones DN34 (resistant) and NC11505 (susceptible) were inoculated and samples were collected for histology after 7 weeks. Data from nonwounded and wounded controls, and wound-inoculated stems were analyzed for effects of clone and treatment. Wound-inoculated stems of both clones were greater in diameter than control stems. The length of first-formed necrophylactic periderm (NP), measured in transverse section, did not differ by treatment in DN34. In contrast, NPs were shorter in wound- inoculated stems than in wounded control stems of NC11505. In wound-inoculated stems, DN34 usually developed a single NP that formed deeper within the stem than in wounded controls. However, NC11505 developed successive NPs with the first-formed NP closer to the wound surface than in wounded control stems. In general, NPs of DN34 were thicker than those formed by NC11505.
Two years of research on biological control of fire blight in New York. N. A. WERNER and H. S. Aldwinckle. Cornell University, Geneva, NY 14456.
The blossom blight phase of fire blight, caused by Erwinia amylovora, was managed by applying the bacterial antagonists Pseudomonas fluorescens A506, Pantoea agglomerans C9-1 or P. agglomerans E325 to open blooms in New York in 2004 and 2005. Treatments were made at 20-30 percent and 70-80 percent bloom in both years. Applications of A506 and C9-1 were also made 24 hr after inoculation in 2004, and A506 was applied with the surfactant Breakthru at 1-5 percent bloom in 2005. Temperatures at 20-30 percent bloom in 2005 averaged 15°C lower than in 2004 and a frost occurred 24 hr prior to the 70-80 percent application. In 2004, 87 percent of blossom clusters were blighted on non-treated, inoculated trees, compared to only 33 percent infection in 2005. This probably reflects the difference in max and min temperatures on the day of inoculation, 30°C and 18°C in 2004 versus 13°C and 5°C in 2005. C9-1 provided 34 percent control in 2004 and less than one percent control in 2005. E325 and A506 provided 17 percent and 8 percent control in 2004, respectively and no control in 2005. The standard control material, streptomycin, provided 44 percent and 80 percent control in 2004 and 2005, respectively.