Two short sequences, situated in the bacterial 23S rDNA gene, were used as primers for the PCR detection of Erwinia amylovora bacteria. All 34 E. amylovora strains tested, coming from different geographical and host plant origins and of different virulence, produced a 565‐bp PCR fragment. The E. amylovora bacteria could be discriminated from all other phytobacteria with which no PCR product was observed. Only Escherichia coli bacteria were cross‐recognized by the production of a weaker PCR band of similar size to E. amylovora. In a fast PCR protocol, where two temperatures were cycled, E. amylovora in pure culture could be detected on gel at concentrations as low as 3 × 102 cfu mL–1. This corresponds to a detection limit of 1.5 bacteria per PCR. However, reliable PCR detection in woody host plant tissue was only obtained with PVP/PVPP‐treated sample extracts. Using E. amylovora‐spiked plant extracts and extracts of fruit tree shoots artificially infected with E. amylovora, the PCR detection sensitivity was determined to be 6.6 × 102 cfu mL–1 of extract. Starting from the plant samples, the PCR detection results were visualized on gels within 5 h.
In the fall of 2000, a new blight disease was observed on Buxus spp. in private gardens in Belgium. Since then, more and similar disease samples from other Belgian sites, nurseries, and several garden centers have been received, indicating that this disease is spreading. Similar observations have been made in the U.K. and France, where the disease is widespread and losses are sometimes dramatic (1). Diseased plants have dark brown-to-black leaf spots and streaky, black stem lesions which lead, in some cases, to complete defoliation. On some infected plants new leaves grew in defoliated areas, hiding the original blight symptoms. Infection was mainly observed on Buxus sempervirens cv. Suffruticosa, but B. sempervirens cv. Latifolia raculata, B. microphylla cv. Compacta, and B. microphylla var. japonica cv. Faulkner were also infected. In the U.K., infections have additionally been reported on varieties of B. sempervirens, B. sinica, and B. microphylla (1). On the basis of observed symptoms and comparison of the symptoms with descriptions by Henricot and Culham (2), we identified that this new form of Buxus blight in Belgium is caused by Cylindrocladium buxicola. Sporulating cultures on potato dextrose agar (PDA) had macroconidiophores with stipe extensions terminating in broadly ellipsoidal vesicles with pointed or papillate apices (6.5 to 11 μm in diameter) and a penicillate arrangement of fertile branches each terminating in two to five phialides. Phialides produced clusters of cylindrical conidia (42 to 68 × 4 to 6 μm) that were rounded at both ends and had a single septum. Pathogenicity of the isolate was demonstrated by inoculation of healthy stems and leaves of four 3-year-old plants of B. sempervirens cv. Suffruticosa. On each plant, agar pieces of 1-week-old cultures grown on PDA were placed on five stems and five leaves that had been wounded with a sterile scalpel, then sealed with Parafilm. As a control, five wounded stems and leaves from another B. sempervirens cv. Suffruticosa plant were inoculated with sterile agar plugs. Inoculated plants were incubated in humid chambers (approximately 95% relative humidity) on the laboratory bench. Two weeks after inoculation, no symptoms were visible on the control plant. The inoculated plants showed symptoms as previously described, and C. buxicola was successfully reisolated from diseased tissue completing Koch's postulates. To our knowledge, this is the first record of C. buxicola on Buxus spp. in Belgium. References: (1) B. Henricot et al. Plant Pathol. 49:805, 2000. (2) B. Henricot and A. Culham. Mycologia 94(6):980, 2002.
American ginseng (Panax quinquefolius) is a recently introduced crop in Bulgaria. In autumn 2001, several 2-year-old plants from Stara Zagora County exhibited symptoms of wilting and dying. Laboratory analysis also revealed some browning of the ginseng root surface and discoloration of the vascular tissues. During later stages of the disease, roots became soft, rubbery, and disintegrated. After storage in a humid chamber for 3 to 5 days, roots were covered with a white, cottony mycelium. Following the transfer onto potato dextrose agar, this fungus formed rounded colonies of white, aerial mycelium. Pathogenicity of the isolate was demonstrated by inoculation of roots that were surface-disinfected with alcohol (70%) for 30 s and rinsed with sterile water. Roots were wounded with a scalpel, and agar pieces from a 1-week-old culture were placed under the cortical tissue. Five inoculated root pieces were kept in a humid chamber at 24 to 25°C, and the pathogen was reisolated subsequently from necrotic lesions that developed from wounds. No symptoms were found in the five wounded but noninoculated control roots. The pathogen was reisolated from the diseased tissue to fulfill Koch's postulates. Microscopic examination showed that the pathogen had an aseptate mycelium (mean diameter of 5.3 μm), did not form hyphal swellings or chlamydospores, and had simple sympodial branching of the sporangiophores. Sporangia had a caducous nature with a pedicel length of 4.7 μm (1.7 to 6.7 μm). Sporangia were ovoid to obpyriform in shape, papillate, and nonproliferating measuring 30.6 (26.6 to 40.0) μm × 24.3 (23.3 to 30.0) μm. The length/width ratio varied between 1.25 and 1.3. The fungus was homothallic and produced paragynous antheridia and spherical oogonia with a diameter of 30.6 μm (26.6 to 33.3 μm) on V8 agar and in petri solution. Oospores were aplerotic and spherical (25 to 30 μm in diameter). Based on symptoms and pathogen characteristics (2), the disease was identified as Phytophthora root rot caused by Phytophthora cactorum. Additionally, the identity of the isolate was verified by sequence determination of the ribosomal internal transcribed spacer I region and alignment to the GenBank-EMBL DNA database (1), which revealed 100% sequence similarity with P. cactorum. To our knowledge, this is the first report of P. cactorum on American ginseng in Bulgaria. References: (1) S. F. Altschul et al. Nucleic Acids Res. 25:3389,1997. (2) D. C. Erwin and O. K. Ribeiro. Morphology and identification of Phytophthora species. Pages 96–125 in: Phytophthora Diseases Worldwide. The American Phytopathological Society, St. Paul, MN, 1996.
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