ABSTRACT
From 2006 to 2008, several similar Phytophthora isolates were obtained from roots of mature Quercus robur and other tree species (Acer platanoides, Fraxinus excelsior, Q. rubra, and Tilia cordata) in forests and parks in several areas in the Czech Republic. The trees were characterized by chlorotic and reduced foliage, crown dieback, and reduced root hairs. Several isolates of Phytophthora were obtained from necrotic roots of these trees and identified as Phytophthora plurivora Jung & Burgess (1). Isolated colonies grown on V8A medium were radiate to slightly chrysanthemum shaped with limited aerial mycelium in the center. Optimum growth was at 25°C, minimum at 5°C and maximum at 32°C. Radial growth of colonies averaged 6.4 mm/day at 20°C. The isolates were homothallic and produced abundant smooth-walled, spherical oogonia (23.3 to 29.1 µm in diameter), oospores were nearly plerotic or plerotic (21.8 to 26.9 µm in diameter), and the oospore wall was 1.2 to 1.4 µm thick. Antheridia were usually paragynous and measured 8.4 to 12 × 6.5 to 8 µm, but amphigynous antheridia were occasionally observed. Noncaducous, semipapillate sporangia formed on simple or sympodial sporangiophores, were obpyriform, ovoid, ellipsoid or irregular in shape, and occasionally distorted with more than one apex. Sporangia dimensions were 33 to 65 × 24 to 33 µm; L/B ratio 1.2 to 1.6 (-2.1). Comparison of DNA sequences of internal transcribed spacer (ITS) regions of isolates (representative strain GenBank Accession No. FJ952382) confirmed the 100% identity of P. plurivora (1). The soil infestation test was conducted using a P. plurivora isolate acquired from roots of Q. robur and 20 3-year-old plants of Q. robur. Sterilized millet seeds colonized by pathogen with the method as described (2) were used as inoculation medium and added into sterilized peat substrate at the rate of 0.5% (vol/vol). The plants were cultivated in 5.8-liter pots in a greenhouse (20°C, 16-h/8-h photoperiod). After 4 months, the roots of all plants were washed, dried, and weighed. The root biomass of 20 infected plants was significantly reduced by approximately 25% on average compared with the control 20 plants (P < 0.05, t-test, Statistica 7.1). The pathogen was consistently reisolated from the roots of infected plants but not from control plants. Stem inoculation tests were conducted with 20 replicates in each group of 2-year-old plants of oak, maple, ash, and lime and isolates acquired from the hosts. On each seedling, a 5-mm-diameter bark plug was removed 5 cm above the collar. The inoculum (5-mm-diameter V8A agar plug with actively growing mycelium) was applied to the exposed substrate. The wounds were sealed with Parafilm. Stem necrosis developed in all cases after 1 to 2 weeks, whereas control plants remained healthy. The pathogen was successfully reisolated from necrotic stem tissues. To our knowledge, this is the first report of P. plurivora causing root rot on oak, maple, ash, and lime in the Czech Republic. On the basis of the host range and distribution of P. plurivora in the Czech Republic, it can be assumed that, as elsewhere in Europe (1), this pathogen is widespread and is a common cause of decline of many tree species. References: (1) T. Jung and T. I. Burgess. Persoonia 22:95, 2009. (2) C. Robin et al. Plant Pathol. 50:708, 2001.
ABSTRACT
During 2007 and the spring of 2008, a disease of poplars (Populus spp.) resembling the Dothichiza canker was found in plantations of fast-growing trees in central Bohemia and in southern Moravia where it was more abundant. The yellowish brown-to-brown, round or elongated cankers occurred on damaged shoots and twigs. Tissues directly under the bark were discolored and turned black. As the cankers enlarged, infected shoots and twigs died after several months. Small, black, gregarious pycnidia were observed under the bark or in lenticels after several weeks. The disease occurred on Populus nigra, P. × euroamericana cvs. Regenerata, Robusta, Brabantica, Spreewald, CZ-425/58, Blanc du Poitou, and Flaschlanden, and other Populus spp. Isolates of a species of Phoma were acquired by culturing damaged tissues on agar plates containing 3% oatmeal agar (OA) and 2% malt agar. Initial identification of the isolates was done by cultural and morphological characteristics (1). Colonies were floccose, aerial mycelium was olivaceous gray to gray, reverse olivaceous gray sometimes with darker tones at the margins or in the colony center, and NaOH reaction was negative. The growth rate was 42 to 56 in diameter after 7 days at 20°C on OA (optimum temperature for growth was 22°C with a minimum of 1°C and a maximum of 28 to 29°C). Pycnidia in culture scattered, were globose or subglobose, obviously with one nonpapillate ostiolum, olivaceous black or black, 120 to 370 µm in diameter, and conidial exudate was whitish. Phialides were globose to ampulliform and 3 to 7 × 3 to 6 µm. Conidia were hyaline, ellipsoidal, often guttulate, 3.1 to 7.8 × 1.9 to 3.1 µm, and L/B ratio 1.4:3.1. Septate conidia occurred only on natural substrate up to 10.6 × 3.9 µm. Morphological and cultural characteristics resembled those of P. exiqua var. populi Gruyter & P. Scheer (1). The internal transcribed spacer (ITS) sequence (GenBank Accession No EU562206) for the representative isolate (CCF No 3759) confirmed 100% identity to P. exigua. Pathogenicity was confirmed with 1-year-old P. nigra plants during a 2-month greenhouse experiment at 15 to 20°C. Fifteen replicate plants were wounded (5-mm diameter), inoculated with 5-mm OA plugs from actively growing colonies (isolate CCF No 3759), and sealed by Parafilming. An additional 15 control plants following wounding were inoculated with a sterile agar plug. After 3 to 4 weeks, yellowish or brownish necrotic lesions ranging from 1 to 1.5 cm long developed on all inoculated plants. The pathogen was successfully reisolated from lesions and the control plants were asymptomatic. P. exigua var. populi is considered an opportunistic poplar and willow pathogen (2) that becomes more important in winter (1). The pathogen potentially invades host tissues damaged by frost, sun scald, or weakened by excessive transpiration during sunny winter days. To our knowledge, this is the first record of the pathogen on poplars in the Czech Republic, which may have an economic impact on short-rotation coppice plantations. References: (1) J. de Gruyter and P. Scheer. J. Phytopathol. 146:411, 1998. (2) H. A. van der Aa et al. Persoonia 17:435, 2000.
ABSTRACT
During the summer and autumn of 2006, a disease of rhododendron plants (Ericaceae) was found in nurseries and public gardens in several areas of the Czech Republic. Leaves of damaged plants showed dark brown-to-black lesions extending along the mid-rib and commonly spreading to petioles and shoots. The infected shoots turned black and died. The cankers on branches, stems, and collars were characterized by reddish, brownish, or blackish discoloration. The disease was identified on Rhododendron catawbiense, R. repens, and other Rhododendron spp. After plating pieces of symptomatic tissue on PARPNH medium (2), several isolates of a homothallic Phytophthora sp. were acquired. Ten representative isolates of the pathogen were cultivated on V8A plates and examined for cultural and morphological characteristics. Colonies had a stellate pattern of growth with sparse aerial mycelium at 20°C; optimum temperature for growth was 25 to 28°C, minimum was 4°C, and maximum was 33°C. Radial growth was 14 mm per day at 20°C on V8A. The isolates produced terminal, spherical, smooth-walled oogonia, which were 19 to 37 µm in diameter. Oospores were plerotic (17 to 32 µm) with walls 2 to 4 µm thick; antheridia were paragynous. Single, terminal, noncaducous, semipapillate sporangia were formed on simple (occasionally sympodial) sporangiophores in nonsterile soil filtrate. The sporangia (28 to 61 × 24 to 35 µm, L:B ratio 1.5) were mostly obpyriform, rarely obovoid, or ovoid-ellipsoid. Morphological and cultural characters resembled those described for Phytophthora citricola Sawada (1). The ITS sequences of the rDNA of the two representative isolates (GenBank Accession Nos. EF194772 and EF194773) showed 100% homology to P. citricola sequences obtained from GenBank, thus the identity was confirmed as P. citricola. Both specimens were deposited in CCF (Culture Collection of Fungi, Charles University, Prague, Czech Republic). To confirm the pathogenicity of isolates, Koch's postulates were tested using 40 3-year-old potted rhododendron (R. catawbiense and R. repens) plants and the two P. citricola strains deposited in CCF. Surfaces of attached healthy leaves were disinfected with 95% ethanol and gently abraded with a sterile scalpel near the mid-rib. Agar plugs from the margin of a 5-day-old colony grown on carrot agar were placed on leaf surfaces and also inserted under flaps of stem tissues made with a sterile scalpel. The leaves and stems were then sealed with Parafilm. Control plants were treated in the same manner with sterile agar plugs. All plants were watered with deionized water, covered with a plastic bag, and maintained in a greenhouse at 21°C for 6 weeks. All inoculated plants exhibited necrotic lesions on leaves and stems around the points of inoculation after 4 days, whereas the control plants remained healthy. The pathogen was consistently reisolated from symptomatic plants. P. citricola is well known as a pathogen of rhododendron (1), but to our knowledge, this is the first report of P. citricola on Rhododendron sp. in the Czech Republic. P. citricola has been found at five different locations and in the most frequently isolated Phytophthora spp. from rhododendron in the Czech Republic. References: (1) D. C. Erwin and O. K. Ribeiro. Phytophthora Diseases Worldwide. The American Phytopathological Society. St. Paul, MN, 1996. (2) T. Jung et al. Eur. J. For. Pathol. 26:253, 1996.
ABSTRACT
Impatiens necrotic spot virus (INSV) was found for the first time in the Czech Republic in 1999 in Columnea sp. and Curcuma sp. plants that were imported. They were grown in a garden among other ornamental crops. By June 2000, INSV was detected in 17 ornamental plant species in several gardening establishments never having received imported plants. Frankliniella occidentalis thrips were present at the sites of INSV infection. Natural INSV infection was found also in Stellaria media, which is an important weed species in our conditions. INSV was identified by enzyme-linked immunosorbent assay using polyclonal antibodies to INSV from Loewe Biochemica, Germany, and from Bioreba, Switzerland. The virus was transmitted in sap to Nicotiana benthamiana and Datura stramonium (1). Virus particles typical of a tospovirus were detected by electron microscopy. INSV is the second tospovirus member occurring in the Czech Republic. Tomato spotted wilt virus (TSWV) has occurred in this country since 1992 (2) and to date has been detected in more than 100 plant species including ornamentals, vegetables, and weeds. TSWV became a serious problem in glasshouse grown crops. From the epidemiological point of view the situation of INSV dissemination seems to be similar to that of TSWV. References: (1) M. Daughtrey et al. Plant Dis. 81:1220, 1997. (2) J. Mertelik and V. Mokra. Acta Virol. 42:348, 1998.
