ABSTRACT
In 2008 and 2009, necrotic bark lesions at the root collar and lower stem associated with root rot, reduced growth, and wilting were observed on container-grown common box (Buxus sempervirens L.), lavender (Lavandula angustifolia Mill. 'Hidcote'), and Port-Orford-cedar (Chamaecyparis lawsoniana (A. Murray) Parl. 'Columnaris') in three ornamental nurseries in western Hungary. Number of affected plants ranged from approximately 100 (Port-Orford-cedar) to 250 (lavender). Isolations from necrotic root collars of each host plant species yielded four Phytophthora isolates developing uniform colonies on carrot agar with a maximum growth temperature of 35 to 36°C. The isolates were homothallic with smooth-walled oogonia (32.2 ± 2.3 to 35.9 ± 3.5 µm), aplerotic oospores (27.5 ± 1.8 to 32.1 ± 3.1 µm) and both amphigynous and paragynous antheridia, and produced chlamydospores (25.8 ± 3.9 to 29.1 ± 5.2 µm) and papillate sporangia (35.2 ± 2.5 to 43.5 ± 5.6 µm long and 27.6 ± 2.2 to 32.0 ± 3.8 µm wide), mostly obpyriform to nearly spherical or rarely distorted with two or three apices. In spring water, sporangia were both caducous with short pedicel and non-caducous. Multiplex ITS-PCR assay of DNA from all isolates, using primers specific for P. nicotianae (NICF1 and NICR2.1) and P. cactorum (CACTF1 and CACTR1) (1), amplified DNA fragments of the expected size for each Phytophthora species. In addition, isoenzyme analysis revealed a characteristic banding pattern of one heterodimer and two homodimer bands at both loci of the dimeric enzyme malate dehydrogenase. These bands comigrated with those of P. × pelgrandis (Gerlach et al.) (CBS 123385) and isolate PD 93/1339 (courtesy of W. A. Man in 't Veld), two natural hybrid strains of P. nicotianae and P. cactorum (2,3), proving that our four isolates can be referred to as this interspecific hybrid. Pathogenicity was tested on 1- or 3-year-old plants of the original host species and cultivars (for common box, cv. Faulkner was used). Cultures were grown for 4 to 6 weeks at 20°C on autoclaved millet grains moistened with V8 broth. Infested and uninfested grains were mixed with autoclaved soil in a ratio of 6% (w/v), and the mixes were used as potting media for transplanting five treated and five control plants per isolate, respectively. Plants were kept in a growth chamber (20°C, 70% RH, 12-h photoperiod). Pots were flooded for 24 h on the 1st and 21st day after transplanting. All plants in infested potting mix showed symptoms of wilt associated with basal stem and root necrosis, similar to those observed on the plants from the field, within 2 and 3 months on lavender and both common box or Port-Orford-cedar, respectively. Additionally, a reduction of root weight ranging from 35 to 68% compared to the control was recorded. Growth reduction was significant at P ≤ 0.019 according to Mann Whitney test. Control plants remained healthy. The same Phytophthora hybrid was reisolated solely from inoculated plants. In Europe, hybrid isolates of P. nicotianae × P. cactorum have been reported on several ornamental plants, including lavender, in the Netherlands and Germany (2,3). However, to our knowledge, this is the first report of this hybrid in Hungary and as a pathogen of common box and Port-Orford-cedar in the world. References: (1) P. J. M. Bonants et al. Phytopathology 90:867, 2000. (2) W. A. Man in 't Veld et al. Phytopathology 88:922, 1998. (3) H. I. Nirenberg et al. Mycologia 101:220, 2009.
ABSTRACT
From 2007 to 2009, necrotic bark lesions at the root collar and lower stem associated with root rot were observed on container-grown cork-bark fir (Abies lasiocarpa var. arizonica, Port-Orford-cedar (Chamaecyparis lawsoniana (A. Murray) Parl. 'Barabits Gold'), lavender (Lavandula angustifolia Mill. 'Hidcote'), flowering currant (Ribes sanguineum Pursh 'King Edward VII'), and lilac (Syringa vulgaris L. 'Belle de Nancy') in six ornamental nurseries in western Hungary. Symptoms also included reduced growth, wilting, and desiccation of branches. Mortality of affected plants ranged from a low level in flowering currant to a high frequency in cork-bark fir (1,000 plants; 50%) and lavender (2,500 plants; 50%). Isolations from necrotic tissues onto PARPB medium (1) yielded 13 isolates of a Phytophthora sp. None of the isolates produced sexual structures, sporangia, or chlamydospores but developed slightly stellate or patternless colonies with loose, aerial mycelium on nonselective carrot agar (CA) at 25°C. One isolate from each host species was further characterized and tested for pathogenicity. Growth on CA was fastest at 25°C (7.9 to 8.6 mm per day) and no growth occurred below 5°C or above 34°C. In nonsterile stream water, persistent, mono- and bipapillate, mostly ovoid, rarely distorted sporangia, measuring 40.5 to 49.4 × 29.8 to 37.3 µm were produced. In pairings on CA with A1 and A2 strains of P. cambivora and P. nicotianae, used as testers, none of our isolates produced gametangia. On the basis of these characteristics, the pathogen from ornamentals appeared to be P. citrophthora (Smith & Smith) Leon. (1). Species identity of all 13 isolates was determined in single-round PCR assays with the P. citrophthora-specific primer-pair Pc2B/Pc7 (2) and/or by sequencing the rDNA internal transcribed spacer (ITS) regions amplified with the universal ITS1/ITS4 primers. The primers Pc2B and Pc7 generated a single DNA fragment of the expected size (approximately 210 bp), and the rDNA ITS sequences (NCBI Accession Nos. GU723282, GU723284, GU723285, and GU723287) showed 99 to 100% homology with many GenBank sequences (e.g., HQ697232) of P. citrophthora as the closest match. Pathogenicity to the original host plant cultivars was tested on 2- or 3-year-old healthy plants potted in sterile soil and inoculated at the root collar (2 replicates per isolate). A 4-mm-diameter bark plug was removed and a mycelial disc of the same size from an actively growing CA culture was placed into the hole. Control plants received sterile CA plugs. Inoculation points were sealed with sterile moist cotton and Parafilm, covered with sterile soil, and then the plants were kept in a greenhouse at 24 ± 4°C. Symptoms identical to those observed on the naturally diseased hosts developed on inoculated plants within 3 months. Control plants remained healthy. P. citrophthora could be reisolated only from the infected plants. The pathogen is polyphagous, widely distributed (1), and has been associated with woody ornamentals in nurseries (3). However, to our knowledge, this is the first record of P. citrophthora in Hungary. The pathogen has to be considered as a threat to ornamental production within Hungary. References: (1) D. C. Erwin and O. K. Ribeiro. Phytophthora Diseases Worldwide. The American Phytopathological Society, St. Paul, MN, 1996. (2) A. Ippolito et al. Eur. J. Plant Pathol. 108:855, 2002. (3) B. W. Schwingle et al. Plant Dis. 91:97, 2007.
ABSTRACT
A high performance liquid chromatographic (HPLC) technique is described for the determination of residue levels of the anthelmintic drug phenothiazine in sheep tissues. Phenothiazine was administered to sheep which were slaughtered after withholding periods of 24, 48, and 72 h. Residues of phenothiazine were then extracted from tissue samples by homogenization in methanol. The HPLC analysis of the extracts involved separation on a 10 micrometer silica column using a mobile phase of 0.3% n-propanol in cyclohexane. The lower limit of detection by ultraviolet absorption at 254 nm was 0.05 ppm.