Thirty-six strains of Xanthomonas campestris pv. campestris (Xcc) isolated from cabbage, kale and broccoli were identified according to their pathogenicity, phenotypic and genotypic characterization. Pathogenicity was confirmed by the injection method with a hypodermic syringe into the mesophilic tissue of cabbage leaves. All strains were Gramnegative, aerobic, catalase-positive, oxidase-negative, grew at 35°C, produced levan, H2S and indole, did not reduce nitrate, hydrolyzed Tween 80, starch, gelatin and esculin and did not show tolerance to 0.1 and 0.02% TTC. The strains produced acid from d-arabinose, arginine, dulcitol, galactose, d-glucose, maltose, mannose, sorbitol, sucrose and xylose. The genetic characterization was based on the sequence analyses of 16S rDNA and ERIC and BOX PCR. Strains of different pathovars were also used to compare PCR resulting patterns. BOX-PCR of the strains from kale and broccoli, obtained using (GTG)5 primer, yielded patterns with a high similarity level to pathovar reference strain Xcc. The strains from cabbage yielded BOX and ERIC product patterns, distinguishing them from the other tested strains and reference strains. 16S rDNA of the representative strains was closely related to Xcc strain ATCC 33913. ERIC PCR and BOX using (GTG)5 primer generated different Xcc patterns and were effective in distinguishing strains from different plant hosts. [Projekat Ministarstva nauke Republike Srbije, br. III43010 i br. III46007
In September 2010, leaves of oilseed rape (Brassica napus L.) with v-shaped, necrotic lesions on the leaf margins surrounded by yellow halos were collected. Symptoms were observed on the domestic cultivar Slavica (IFVC, Novi Sad) located in the Bačka region, Vojvodina, Serbia, from a 3-ha field. Average disease incidence on 3-month-old plants was 45% (15 to 75%). Diseased leaves were rinsed in sterilized distilled water (SDW) and dried at room temperature for isolations. Leaf sections taken from the margin of necrotic leaf tissue were macerated in SDW and the extract was streaked onto yeast extract-dextrose-calcium carbonate (YDC) agar. Plates were incubated at 28°C for 3 days. Colonies were yellow, translucent, circular, and raised. Ten representative strains tested further were all gram-negative, catalase-positive, and oxidase-negative. The partial 16S rDNA sequence of a representative strain, TUr1, was amplified using primers fD1 and rD1 (2), and determined using the IMGGI SeqService facility in Belgrade. The 1,510-bp 16S rDNA sequence of TUr1 was compared to that of known strains in the NCBI GenBank database, and showed greatest similarity with that of Xanthomonas campestris pv. campestris (Xcc) strains ATCC 33913 and B100 (99% homology). Pathogenicity of 10 strains grown for 48 h on YDC at 28°C was completed using each of three methods: spraying a bacterial suspension (108 cfu/ml) onto the leaf surfaces of oilseed rape plants, stabbing the major veins of each of the first two true leaves with the tip of a sterile toothpick that had been dipped into a colony of the appropriate strain, and immersing cotyledons of the plants into a bacterial suspension (108 cfu/ml). All three tests were performed on 4-week-old oilseed rape plants of the cultivar Slavica. SDW was used for the negative control treatment for each method of inoculation. Reference strain Xcc NCPPB 1144 was used as a positive control treatment. Tests plants (two for each method of inoculation and each bacterial strain or control treatment) were maintained in a greenhouse at 25 ± 1°C and 80% relative humidity by keeping the plants in plastic bags. Two control plants for each of the negative and positive control treatments for each inoculation method were also enclosed in separate plastic bags. The bacterial strains and reference strain caused yellow lesions on inoculated plants that turned necrotic starting about 7 days after inoculation (DAI). The spots coalesced within 21 DAI to form necrotic areas. Plants inoculated with SDW remained symptomless. Reisolations were done onto YDC as described above. Reisolated strains showed the same colony morphology as described above. The bacterial strains grew at 35°C; produced levan from sucrose, hydrogen sulfide, and indole; did not reduce nitrate; hydrolyzed Tween 80; starch, gelatin, and aesculin; did not show tolerance to 0.10 and 0.02% triphenyl-tetrazolium chloride; and produced acid from d-arabinose, arginine, dulcitol, galactose, d-glucose, maltose, mannose, sorbitol, sucrose, and xylose (1). All strains tested by Plate Trapped Antigen-ELISAs (ADGEN Phytodiagnostics, Neogen Europe Ltd., Scotland) reacted with Xcc-specific polyclonal antibodies. Based on these tests, the strains were identified as Xcc. To our knowledge, this is the first report of this pathogen causing black rot of oilseed rape in Serbia. References: (1) T. B. Adhikariand and R. Basnyat. Eur. J. Plant Pathol. 105:303, 1999. (2) W. G. Weisburg et al. J. Bacteriol. 173:697, 1991.
Plants of alfalfa (Medicago sativa) exhibiting general stunting, proliferation and phyllody associated with leaf yellowing and reddening were observed in three localities of Central Serbia. Phytoplasma strains belonging to 16SrIII-B and 16SrXII-A groups were detected and identified by RFLP and sequence analysis of 16S rDNA. Stolbur phytoplasma tuf gene RFLP analysis showed the presence of the TufAY-b-type phytoplasma subgroup in 80% of symptomatic samples. This is the first report of 16SrIII-B and 16SrXII-A phytoplasma groups affecting alfalfa in Serbia.
Talaromyces minioluteus is one of the important species of genus Talaromyces, which has cosmopolitan distribution and is encountered on a wide range of different habitats. This species has not been considered as an important plant pathogen, even though it has been isolated from various plant hosts. Fruits and vegetables with Penicillium-like mold symptoms were collected from 2015 to 2017 from markets in Serbia. Isolates originating from quince, tomato, and orange fruits, onion bulbs, and potato tubers were identified and characterized on a morphological, physiological, and molecular level. Morphological and physiological examination included observing micromorphology, testing growth on six different media and at five different temperatures, and production of three enzymes. Molecular identification and characterization were performed using four molecular markers: internal transcribed spacer, β-tubulin, calmodulin, and DNA-dependent RNA polymerase II second largest subunit. The results of morphological and molecular analyses were in agreement, and they proved that the obtained isolates are T. minioluteus. In the pathogenicity assay, T. minioluteus was confirmed as a pathogen of all species tested with the exception of potato tubers. This is the first report of T. minioluteus as a postharvest plant pathogen on quince, tomato, and orange fruit and onion bulbs. Also, this is the first record of T. minioluteus in Serbia.
The essential oil of Lavandula stoechas was examined by GC and GC-MS. Discs (5 mmi.d.) of the tested fungi (Alternaria alternata, Fusarium oxysporum and Botritys cinerea) were inoculated separately onto each assay plate and incubated at 25 o C for 7 days. The oil yield of dried parts (v/dw) obtained by hydrodistillation was 2.9%. Thirty-two compounds representing 98.3% of the essential oil were determined. Linalool (49.9%), linalyl acetate (14.4%), lavandulyl acetate (5.7%), α-terpineol (5.6%), terpinene-4-ol (5.1%), lavandulol (3.7%), (E)-β-ocimene (2.6%) and (Z)-β-ocimene (2.4%) were identified as the main constituents of the oil. In addition, both doses of the lavender oil showed varying levels of inhibitory effects on the mycelial growth of tested fungi used in the experiment. The results demonstrated the strongest effect on B.cinerea, followed by A.alternata and F.oxysporum. The inhibitory effect is probably dependent on the concentration of essential oils.
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