EFFICACY OF METAMITRON IN APPLE THINNING IN SERBIA.

The thinning of fruits is a required pomotechnical measure in intensive fruit production which ensures the production of good quality fruits and high yields. Metamitron, known as inhibitor of photosynthesis, has been successfully used in the thinning of apple fruits. This study had the aim to determine the efficacy of metamitron on the thinning of apple fruits in the agroecological conditions of Serbia and to evaluate the possibility of its practical application. Two varieties of apples that are widely grown in Serbia, dared and Golden Delicious, have been chosen for this research. The experiments were carried out during 2011 and 2012 according to the EPPO PP 1/158 (3) method. Metamitron has shown a good efficacy in the thinning of apple fruits. The effect of metamitron on the thinning of apple fruits depends on multiple factors, pri- marily the application dose, time of application, apple variety, but also on the number of fruits developed. The best efficacy on the Idared variety was in plots where metamitron was applied at a dose of 1.1 kg ha⁻¹, once (in the growth stage when the fruits were 8 mm in diameter) or twice (in the growth stages when the fruits were 8 mm and 12 mm in diameter), when the number of developed fruits per tree is smaller, or 1.65 kg ha⁻¹ applied once when the fruits are 12 mm in size when a larger number of fruits per tree is developed. On the Golden Delicious variety, the best efficacy was in treatments when metamitron was applied twice (in the growth stages when the fruits were 8 mm and 12 mm in diameter) in quantities of 1.1 kg ha⁻¹, when less fruits per tree were formed or 1.65 kg ha⁻¹, applied once or twice when a larger number of fruits per tree were formed.

First Report of Colletotrichum clavatum Causing Quince Anthracnose in Serbia.

Quince (Cydonia oblonga Mill.) tree is traditionally grown in Serbia. The fruits are used for compote, marmalade, and brandy production. In December 2012, quince fruits cv. Leskovacka with symptoms of postharvest anthracnose were collected in a storage facility in the area of Sabac, western Serbia. The symptoms were observed on fruits approximately 2 months after harvest. The incidence of the disease was about 3%, but the symptoms were severe. Affected fruits showed sunken, dark brown to black lesions with orange conidial masses produced in black acervuli. Small pieces (3 to 5 mm) of necrotic tissue were surface sterilized for 1 min in 1% NaOCl, washed twice with sterile distilled water, and placed on potato dextrose agar (PDA). Macroscopic and microscopic morphology characteristics of three isolates were observed after growth on PDA for 7 days at 25°C under a 12-h photoperiod. Fungal colonies developed white to gray dense aerial mycelium with orange conidial masses in the center of the colony. Conidia were hyaline, aseptate, clavate with rounded distal apices, 15.2 (12.8 to 16.8) × 4.5 (4.0 to 5.2) µm (mean L/W ratio = 3.3, n = 100). Morphological characteristics are consistent with the description of Colletotrichum clavatum (2). Fungal isolates were also characterized by sequencing of the internal transcribed spacer (ITS) rDNA region using ITS1/IT4 primers and ß-tubuline 2 gene using T1/T2 primers. The nucleotide sequences were deposited in GenBank (ITS Accession Nos. KF908866, KF908867, and KF908868; ß-tubuline 2 gene KF908869, KF908870, and KF908871). BLAST analyses of ITS and ß-tubuline 2 gene sequences showed that isolates from quince were 100% identical to other C. clavatum in GenBank (ITS JN121126, JN121130, JN121132, and JN121180; ß-tubuline 2 gene JN121213 to 17, JN121219, JN121228, JN121261 to 62, and JN121266 to 69), thus confirming the morphological identification. To fulfill Koch's postulates, asymptomatic fruits of quince cv. Leskovacka (five fruits per isolate) were surface sterilized with 70% ethanol, wounded with a sterile needle, and inoculated with 50 µl of a spore suspension (1 × 106 conidia/ml). Five control fruits were inoculated with 50 µl of sterile distilled water. The experiment was repeated twice. After 10 days of incubation in plastic containers, under high humidity (>90% RH) at 25°C, typical anthracnose symptoms developed on inoculated fruits, while control fruits remained symptomless. The isolates recovered from symptomatic fruits showed the same morphological features as original isolates. C. clavatum previously indicated as group B (3), or genetic group A4 within the C. acutatum sensu lato complex (4), is responsible for olive anthracnose in some Mediterranean countries (1,2), and has been reported as causal agent of anthracnose on a wide range of other hosts including woody and herbaceous plants, ornamentals, and fruit trees worldwide (4). To our knowledge, this is the first report of C. clavatum in Serbia, and the first report of quince anthracnose caused by this pathogen in Europe. Anthracnose caused by C. clavatum can endanger the production and storage of quince in the future, and may require investigation of new disease management practices to control this fungus. References: (1) S. O. Cacciola et al. J. Plant Pathol. 94:29, 2012. (2) R. Faedda et al. Phytopathol. Mediterr. 50:283, 2011. (3) R. Lardner et al. Mycol. Res. 103:275, 1999. (4) S. Sreenivasaprasad and P. Talhinhas. Mol. Plant Pathol. 6:361, 2005.

First Report of Broccoli Soft Rot Caused by Pectobacterium carotovorum subsp. carotovorum in Serbia.

In September 2012, soft rot symptoms on broccoli (Brassica oleracea L. var. italica Plenck) were observed in several commercial fields in the western part of Serbia. Following the first harvest, water-soaked areas developed on broccoli stem tissue and progressed into soft rot decay of entire plants. The incidence of disease was approximately 30%. In Serbia, broccoli is grown on smaller fields compared to other vegetables, but its production and consumption increased significantly in recent years. From the diseased tissue, shiny, grayish white, round colonies were isolated on nutrient agar. Six non-fluorescent, gram-negative, facultative anaerobic, oxidase-negative, and catalase-positive bacterial strains were chosen for further identification. All strains caused soft rot on potato and carrot slices and did not induce hypersensitive reaction on tobacco leaves. They grew at 37°C and in yeast salts broth medium containing 5% NaCl (2), did not produce acid from α-methyl glucoside, but utilized lactose and trehalose, and did not produce indole or lecitinase. Investigated strains formed light red, 1.5-mm-diameter colonies on Logan's medium (2), and did not produce blue pigmented indigoidine on glucose yeast calcium carbonate agar (2) nor "fried egg" colonies on potato dextrose agar. Based on biochemical and physiological characteristics (1) and ITS-PCR and ITS-RFLP analysis (4), the strains were identified as Pectobacterium carotovorum subsp. carotovorum. The 16S rRNA gene sequence from two strains (GenBank KC527051 and KC527052) showed 100% identity with sequences of P. carotovorum subsp. carotovorum previously deposited in GenBank (3). Pathogenicity of the strains was confirmed by inoculation of broccoli head tissue fragments. Three florets per strain were inoculated by pricking the petals with a syringe and hypodermic needle and depositing a droplet of bacterial suspension (approx. 1 × 108 CFU/ml) at the point of inoculation. Sterile distilled water was used as a negative control. Inoculated florets were placed in a sealed plastic container and incubated in high humidity conditions at 28°C. Tissue discoloration and soft rot developed around the inoculation point within 48 to 72 h. No symptoms developed on control florets. Identity of bacterial strains reisolated from inoculated plant tissues was confirmed by ITS-PCR using G1/L1 primers followed by digestion of PCR products with Rsa I restriction enzyme (4). In Serbia, P. carotovorum subsp. carotovorum has been isolated from potato, some vegetable crops, and ornamentals, but not from broccoli until now. References: (1) S. H. De Boer and A. Kelman. Page 56 in: Laboratory Guide for Identification of Plant Pathogenic Bacteria, 3rd ed. N. W. Schaad et al., eds. The American Phytopathological Society, St. Paul, MN, 2001. (2) P. C. Fahy and A. C. Hayward. Page 337 in: Plant Bacterial Diseases: A Diagnostic Guide. P. C. Fahy and G. J. Persley eds. Academic Press, New York, 1983. (3) S. Nabhan et al. J. Appl. Microbiol. 113: 904, 2012. (4) I. K. Toth et. al. Appl. Environ. Microbiol. 67:4070, 2001.

Tissue Necrosis of Sour Cherry Fruits, An Unusual Disease Occurrence Caused by a Phoma sp. in Serbia.

Following frequent rains during May and June of 1999, a high incidence of an unknown disease on sour cherry (Prunus cerasus) fruits was observed in western Serbia (Sabac). Initially, small, water-soaked, dark green spots appeared on the fruit tissue similar to infections caused by Pseudomonas syringae or a Colletotrichum sp. (1,2). Later, spots enlarged, coalesced, and became necrotic and covered most of the fruit surface. The diseased tissue was slightly depressed and fruit shriveled and dried. Approximately 60 to 80% of the total yield was affected. Numerous hyaline, unicellular, and ellipsoidal conidia were observed with a microscope. Orange spore masses often occurred on the necrotic tissue following rain. Symptoms of the disease were not expressed during the dry spring of 2000, but were observed in rainy 2001. The most susceptible cultivar was Haiman's Rubin. Disease symptoms were absent on the domestic cultivar Oblacinska. Placing small necrotic fragments on potato dextrose agar (PDA) resulted in grayish fungal colonies that produced black, globose pycnidia with hyaline, unicellular, and ellipsoidal conidia under daylight conditions that were similar to those formed on necrotic sour cherry fruit in the orchard. At 25°C, colonies covered the entire 9-cm diameter surface of PDA after 5 to 6 days. No teleomorph was observed. Bacterial isolations on nutrient agar were unsuccessful. Unripe sour cherry fruits were punctured with a sterile needle and small colony fragments of the fungus were placed over the wound. Symptoms typical of those on fruit found in orchards were observed after 7 days of incubation at 25°C. No symptoms developed on control fruits punctured with a sterile needle. The fungus was reisolated from the inoculated fruits and shown to be identical to the original strains. On the basis of pathogenicity and morphological characteristics of the strains investigated, the fungus was determined to be a Phoma sp. (3), also known as sour cherry necrosis in other fruit-tree-growing regions. To our knowledge, this is the first report of the fungus on sour cherry fruit in Serbia. References: (1) J. Balaz and M. Arsenijevic. Proc.Int. Conf. Plant. Path. Bact. 7:515, 1990. (2) M. Ivanovic and D. Ivanovic. Zast. Bilja 201:211, 1992. (3) B. C. Sutton. Pages 379-391 in: Coelomycetes. CMI, Kew, Surrey, England, 1980.

Native Vaccinium spp. and Gaylussacia spp. infested by Rhagoletis mendax (Diptera: Tephritidae) in the Great Lakes Region: a potential source of inoculum for infestation of cultivated blueberries.

In this study, we addressed the question of whether or not native stands of blueberry (Vaccinium spp.) and/or huckleberry (Gaylussacia spp.) support populations of blueberry maggot, Rhagoletis mendax Curran, in the Great Lakes region. Infestation of commercial blueberries by the blueberry maggot, R. mendax, is a serious problem in many areas where blueberries are grown. In the past 10-20 yr, commercial bighbush blueberry, Vaccinium corymbosum L., production has expanded into places such as southern Ontario and southern Quebec where blueberry maggot had not previously been reported. In the mid-1990s, isolated infestations of commercial highbush blueberry were reported in southern Ontario. Because R. mendax was not considered endemic to that area, it was widely assumed that the pests had come into the fields via movement from exotic localities. Here we present an alternative hypothesis, that the blueberry maggots infesting newly established highbush plantations are derived from native blueberries growing in the vicinity. To test this hypothesis, in 1997-1999, we sampled potential native hosts for R. mendax (Vaccinium spp. and Gaylussacia spp.) from 31 localities in the Great Lakes region, primarily in Michigan and Ontario. R. mendax was reared from fruits of native hosts collected at four sites in Michigan and one site each in Ontario, Indiana, and Ohio. V. corymbosum was the predominant host infested, with infestation of this host observed at five of the seven sites. However, two huckleberry species [Gaylussacia baccata (Wangenheim) K. Koch, and Gaylussacia dumosa (Andersson) Torrey & Gray] had the highest rates of infestation that we observed (25.4 and 17.6%, respectively). These data represent the first published reports of R. mendax infesting native host plants in the Great Lakes region, and support the hypothesis that infestations observed in commercial fields may have originated from infested native host plants.