Norman W. Schaad

Detection of Clavibacter michiganensis subsp. sepedonicus in potato tubers by BIO-PCR and an automated real-time fluorescence detection system.

Ring rot of potato, caused by Clavibacter michiganensis subsp. sepedonicus, is one of the most regulated diseases of potatoes world wide. The organism is often difficult to detect in symptomless tubers because of low populations and slow competitive growth on available media. Polymerase chain reaction (PCR) primers and a fluorescent probe for use in the Perkin Elmer 7700 automated real time PCR detection system (TaqMan) were designed from a C. michiganensis subsp. sepedonicus-specific genomic DNA fragment for development of a BIO-PCR assay for C. michiganensis subsp. sepedonicus in potato tubers. Results of screening the primers with strains of C. michiganensis subsp. sepedonicus and other bacteria showed the primers to be specific. A total of 30 naturally infected ring rot suspect tubers were sampled by the core extract, shaker incubation procedure and assayed by (i) plating aliquots onto agar media, (ii) classical PCR, and (iii) BIO-PCR. In all, 4 tubers were positive by agar plating and pathogenicity tests, 8 by classical TaqMan PCR, and 26 by TaqMan BIO-PCR. We conclude that BIO-PCR combined with the TaqMan automated closed detection system is a rapid, reliable method of assaying large numbers of potato tuber extracts for C. michiganensis subsp. sepedonicus. Furthermore, for a large central laboratory running large numbers of PCR assays, the high-throughput TaqMan system can reduce costs per sample over the more labor-intensive classical PCR.

Pseudomonas syringae and related pathogens

Since the early 1990s, bacterial blight has emerged as an important disease of cantaloupe in France, particularly in the south-west but has caused epidemics in all the production regions of this country. The pathogen is a complex composed of strains identified as Pseudomonas syringae pv. aptata (70% of the strains) and the remaining resembling P. viridijlava (2%), P. s. pv. lachrymans (1%) or P. syringae in general but with no correspondence to a known pathovar. To investigate the epidemiology of this disease and to develop disease control strategies, we have attempted to identify sources of inoculum in the field, factors contributing to the explosive development of the disease, resistance of the bacterium to copper pesticides, and sources of disease resistance in Cucumis melo. Based on our observations and on the description of possible outbreaks of this disease elsewhere in the world, we report here a hypothetical scenario of the critical factors triggering disease development and of the potential efficiency of different control strategies.

Multilocus Sequence Typing Reveals Two Evolutionary Lineages of Acidovorax avenae subsp. citrulli

Acidovorax avenae subsp. citrulli, causal agent of bacterial fruit blotch, has caused considerable damage to the watermelon and melon industry in China and the United States. Understanding the emergence and spread of this pathogen is important for controlling the disease. To build a fingerprinting database for reliable identification and tracking of strains of A. avenae subsp. citrulli, a multilocus sequence typing (MLST) scheme was developed using seven conserved loci. The study included 8 original strains from the 1978 description of A. avenae subsp. citrulli, 51 from China, and 34 from worldwide collections. Two major clonal complexes (CCs), CC1 and CC2, were identified within A. avenae subsp. citrulli; 48 strains typed as CC1 and 45 as CC2. All eight original 1978 strains isolated from watermelon and melon grouped in CC1. CC2 strains were predominant in the worldwide collection and all but five were isolated from watermelon. In China, a major seed producer for melon and watermelon, the predominant strains were CC1 and were found nearly equally on melon and watermelon.

Pseudomonas syringae Pathovars and Related Pathogens – Identification, Epidemiology and Genomics

Preface. Sponsors and Donors. Section 1. Identification and Detection. 1. Current technologies for Pseudomonas spp. and Ralstonia solanacearum detection and molecular typing: M.M. Lopez et al. 2. Siderophore uses in Pseudomonas syringae identification: A. Bultreys, I. Gheysen. 3. Chlorophyll fluorescene imaging for detection of Bean response to Pseudomonas syringae in asymptomatic leaf areas: L. Rodrigues-Moreno et al. 4. Sensitive detection of Ralstonia solanacearum (race 3) using serological methods and Biolog automated system: A.E. Tawfik et al. Section 2. Epidemiology and Disease Management. 5. Epidemiological basis for an efficient control of Pseudomonas savastanoi pv. savastanoi on olive trees: J.M. Quesada et al. 6. Pseudomonas syringae pv. syringae on kiwifruit plants: its role and its control: A. Rossetti and G.M. Balestra. 7. Head rot of cauliflower caused by Pseudomonas fluerescens in southern Italy: P. Lo Cantore and N.S. Iacobellis. 8. Internalization and survival of Pseudomonas corrugata from flowers to fruits and seeds of tomato plants: G. Cirvilleri et al. 9. Copper and streptomycin resistance in pseudomonas strains isolated from pipfruit and stone fruit orchards in New Zealand: J.L. Vanneste et al. 10. Basal defence in Arabidopsis agains Pseudomonas syringae pv. phaseolicola: Beyond FLS2?: A. Forsyth et al. 11. Agrobacterium suppresses P. syringae-elicited salicylate production in Nicotiana tabacum leaves: A. Rico and G.M. Preston. 12. Characterization of an inhibitory strain of Pseudomonas syringae pv. syringae withpotential as a biocontrol agent for bacterial blight on soybean: S.D. Braun and B. Volksch. 13. Characterization of the inhibitory strain Pantoea sp. 48b/90 with potential as a biocontrol agent for bacterial plant pathogens: B. Volksch and U. Sammer. 14. Pseudomonas syringae: Prospects for its use as a weed biocontrol agent: B.M. Thompson et al. 15. Analysis of Pseudomonas syringae populations and identification of strains as potential biocontrol agents against postharvest rot of different fruits: G. Cirvilleri et al. Section 3. Pathogenesis and Determinants of Pathogenicity. 16. The distribution of multiple exopolysaccharides in Pseudomonas syringae biofilms: H. Laue et al. 17. Impact of temperature on the regulation of coronatine biosyntesis in Pseudomonas syringae: Y. Braun et al. 18. Role of flagelling glycosylation in bacterial virulence: Y. Ichinose et al. 19. Genetic relatedness among the different genetic lineages of Pseudomonas syringae pv. phaseolicola: M.E. Fuhrer et al. 20. WLIP and Analogues of Tolaasin I, Lipodepsipeptides from Pseudomonas reactans and Pseudomonas tolaasii: A Comparison of their Activity on Natural and Model Membranes: R. Paletti et al. 21. Competitive index in mixed infection: a sensitive and accurate method to quantify growth of pseudomonas syringae in different plants: A.P. Macho et al. Section 4. Genomics and Molecular Characterization. 22. Genomic analysis of Pseudomonas syringae pathovars: Identification of virulence genes and associated regulatory elements using pattern-based searches and genome comparison: W. Lindeberg et al. 23. Gene ontology (GO) for microbe-host interactions and its

An Assessment Model for Rating High-Threat Crop Pathogens

ABSTRACT Natural, accidental, and deliberate introductions of nonindigenous crop pathogens have become increasingly recognized as threats to the U.S. economy. Given the large number of pathogens that could be introduced, development of rapid detection methods and control strategies for every potential agent would be extremely difficult and costly. Thus, to ensure the most effective direction of resources a list of high-threat pathogens is needed. We address development of a pathogen threat assessment model based on the analytic hierarchy process (AHP) that can be applied world-wide, using the United States as an illustrative example. Previously, the AHP has been shown to work well for strategic planning and risk assessment. Using the collective knowledge of subject matter expert panels incorporated into commercial decision-making software, 17 biological and economic criteria were determined and given weights for assessing the threat of accidental or deliberately introduced pathogens. The rating model can be applied by experts on particular crops to develop threat lists, especially those of high priority, based on the current knowledge of individual diseases.

Draft Genome Sequence of Xanthomonas arboricola Strain 3004, a Causal Agent of Bacterial Disease on Barley

ABSTRACT We report here the annotated genome sequence of Xanthomonas arboricola strain 3004, isolated from barley leaves with symptoms of streak and capable of infecting other plant species. We sequenced the genome of X. arboricola strain 3004 to improve the understanding of molecular mechanisms of the pathogenesis and evolution of the genus Xanthomonas.

First Report of Citrus Canker Caused by Xanthomonas citri in Somalia

Xanthomonas citri (synonym = Xanthomonas axonopodis pv. citri) (3) has been reported in several countries in Africa (1) but not Somalia. During 2006 and 2007, hyperplasia-type lesions, often surrounded by a water-soaked margin and yellow halo, typical of citrus canker caused by X. citri were found on 8- to 10-year-old lime (Citrus limetta) and grapefruit (Citrus × paradisi Macfed.) trees in northern and southern Somalia, respectively. Ten leaf samples diagnosed presumptively as citrus canker by Xac ImmunoStrip test kits (Agdia, Elkhart, IN) were mailed to the USDA Foreign Disease-Weed Science Research Unit at Ft. Detrick, MD. To confirm the identification of X. citri, isolations were made from several lesions from each sample onto yeast-dextrose-CaCO3 (YDC) agar (2). Yellow, xanthomonad-like mucoid, convex colonies were purified and stored on YDC slants. Phenotypic tests were done as described (2), and real-time PCR assays were done using primers XCit8F and XCit5R with probe XCitP2 (N. W. Schaad, unpublished). For pathogenicity tests, cultures were grown overnight in liquid nutrient broth-yeast (4) medium adjusted to contain 1 × 105 CFU/ml and inoculated into leaves of lime seedlings with the blunt end of a 2-ml syringe. After 21 to 30 days in a lighted dew chamber (Model I-60DLM; Percival Scientific, Inc. Perry, IA) at 30/23°C day/night, symptoms were recorded. Cultures of sample S-1 (northern Somalia) from lime were phenotypically atypical of X. citri, PCR negative, and nonpathogenic. However, cultures of samples 3 to 7 (southern Somalia) from grapefruit were typical of X. citri and PCR positive; cultures 3 and 4 were tested for pathogenicity and produced erumpent lesions on lime. Isolations onto YDC agar resulted in typical mucoid, convex, yellow, PCR-positive colonies. To our knowledge, this is the first report of X. citri on citrus plants in Somalia. Strains S3 and S4 have been deposited in ICPB at Ft. Detrick, MD as ICPB 11650 and 11651, respectively. References: (1) J. F. Bradbury. Guide to Plant Pathogenic Bacteria. CAB International, Egham, UK, 1986. (2) N. W. Schaad et al. Xanthomonas. Page 175 in: Laboratory Guide for Identification of Plant Pathogenic Bacteria. 3rd ed. N. W. Schaad et al. eds. American Phytopathological Society, St. Paul. MN. 2001. (3) N. W. Schaad et al. Syst. Appl. Microbiol. 29:690, 2006. (4) A. K. Vidaver. Appl. Microbiol. 15:1523, 1967.

Emerging Plant Pathogenic Bacteria and Global Warming

Several bacteria, previously classified as non-fluorescent, oxidase positive pseudomonads, Ralstonia, Acidovorax, and Burkholderia have emerged as serious problems worldwide. Perhaps the most destructive is R. solanacearum (RS), a soilborne pathogen with a very wide host range. RS race 3, biovar 2 infects potato and geranium during cooler weather making it an additional threat. Acidovorax avenae subsp. avenae has emerged as a disease of upland rice in Southern Europe during periods of high temperatures and B. gladioli has emerged as a serious pathogen of orchids in Thailand. B. andropogonis has been identified for the first time on jojoba in eastern Australia; the plant grows under very high temperatures and the disease occurs in nursery stock grown under overhead watering. Burkholderia glumae is emerging as a problem on rice in the southern United States and the pathogen has become much more prevalent in southern South Korea during periods of high temperatures. Why the increase in these bacteria? One possible explanation is global warming. A common trait among them is an optimum growth tempera- ture of 32-36°C; most grow well up to 41°C, whereas most other plant patho- genicbacteria grow best at lower temperatures. An increase in extreme weather conditions, including extended heat waves, long periods of rain, and storms such as hurricanes appear to favor these high-temperature bacteria. There has been a gradual increase in the mean global temperature over the past century. Recent summer temperatures in the southern USA. have been 1-2°C higher than that which is optimum for many crops. Evidence is growing that the increase in the occurrence of extreme weather, including heat waves, continual rains, and hurricanes is caused by rising CO 2 levels from expanding world economies. Several heat waves have occurred in Europe and the US in recent years. The year 1995 was the warmest since global records began in 1856, and the 2003 heat wave killed hundreds in Europe. An increase in violent and extreme summer storms has occurred the past several years. Ice core studies, photographic recordings of retreating glaciers, and recorded temperatures provide solid evidence that global warming is occurring at

Use of a Seed Scarifier for Detection and Enumeration of Galls of Anguina and Rathayibacter Species in Orchard Grass Seed

Seed galls, caused by Anguina spp., are normally detected visually in cereals such as wheat and barley. However, in grasses such as orchard grass, the presence of galls induced by Anguina or Rathayibacter spp. are difficult to detect visually due to their infrequent occurrence and masking by lemmas and paleas. To develop improved seed assays for the presence of the nematode and bacterial galls, a small scarifier was used to remove lemmas and paleas without causing major damage to seeds or galls. Following scarification, the galls were visually identified and manually counted under a dissecting microscope. Using the scarifier, several orchard grass seed lots were screened for Anguina and Rathayibacter spp. The percentage of samples of orchard grass seed harvested in the Willamette Valley of Oregon during 1996, 1997, and 2000 containing galls of Anguina sp. were 37, 46, and 48, respectively. The percentage of samples containing bacterial galls with Rathayibacter sp. was 27, 31, and 40, respectively. Total galls with Anguina sp. per 25 g of orchard grass sample ranged from 1 to 24. The mean of Anguina sp. galls per sample in 1996, 1997, and 2000 were 4, 5, and 5, respectively. Total galls with bacteria per 25 g of sample ranged from 27 to 40; mean number of galls per sample in 1996, 1997, and 2000 were, 6, 5, and 11, respectively. This is the first report confirming the presence of Rathayibacter sp. galls in orchard grass in Oregon.

ANTIGENIC GLYCOPROTEINS IN THE TELIOSPORE WALL OF TILLETIA INDICA

We describe the partial characterization of immunoreactive glycoproteins in the teliospore wall of Tilletia indica, the causal agent of Karnal bunt of wheat (Triticum aestivum ). Proteins from phenol-ex- tracted and precipitated spore wall preparations were used as inject antigens to immunize New Zealand rabbits. Immunoblots of crude preparations of spore wall proteins probed with anti-T indica spore wall IgG showed that the antibodies recognized a 37-43 kDa band present in the spore wall fraction of T. indica. A weakly immunoreactive band of the same mobility was detected in the spore wall of T barclay- ana, a closely related fungal pathogen and causal agent of kernel smut of rice. The 37-43 kDa poly- peptide could not be detected in the teliospore walls of the related fungal wheat pathogens T controversa and T tritici. Differential centrifugation of pulverized