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 Group 16SrXII-A Phytoplasma Causing Stolbur Disease in Saponaria officinalis Plants in Serbia.

Saponaria officinalis L. (Caryophyllaceae; also known as bouncingbet or soapwort) is a perennial medicinal plant important for the pharmaceutical industry and used as an expectorant, alterative, laxative, and ointment for some skin diseases and arthritic conditions. S. officinalis plants with typical symptoms (23% in 2011 and 47% in 2012) of phytoplasma infection were observed in Pancevo plantation, Serbia. The symptoms appeared in May with leaves changing color from green to brown with severe reddening and necrosis. Severely diseased plants died. The infected plants had a significant reduction in biomass and quality. To investigate the presence of phytoplasma, total DNA was extracted from 10 symptomatic and four asymptomatic plants by a CTAB method. The nested PCR was carried out using phytoplasma-specific primer set P1/16S-SR followed by R16F2n/R16R2, targeting the 16S rRNA gene sequence of 1.5 and 1.2 kb in length, respectively. The amplicons of expected size were obtained from the symptomatic plants, but not from the asymptomatic plants. To obtain restriction fragment length polymorphism (RFLP) patterns, the R16F2n/R2 amplicons were digested with AluI, TruI1, HpaII, and HhaI endonucleases. The resulting patterns indicated that seven plants were infected by a Stolbur phytoplasma belonging to the 16SrXII-A subgroup, since it had the identical RFLP pattern as the STOL reference strain. The 1.2 kb nested PCR products of representative isolate Sap7 were purified using PCR purification kit (Fermentas, Vilnius, Lithuania) according to the recommended protocol and sequenced using facilities of IMGGI SeqService, Belgrade, Serbia. The obtained sequence was deposited in the NCBI database (GenBank Accession No. JX866951). The phytoplasma 16S rRNA gene sequence from Sap7 had a sequence identity of 97% with GenBank accessions GQ273961.1 ('Euonymus japonicus' phytoplasma), JX311953.1 (Candidatus Phytoplasma solani clone 5043), JQ412100.1 (Iranian alfalfa phytoplasma M21), and JN561702.1 ('Convolvulus arvensis' stolbur phytoplasma clone P1/P7-Conv2/2010-Bg). To our knowledge, this is the first report of a natural infection of S. officinalis by 16SrXII-A subgroup (Stolbur) phytoplasma in Serbia. As cited by Lee et al. (1), the 16SrI-M subgroup phytoplasma in S. officinalis sample was already detected in Lithuania by Valiunas (2). The identification of phytoplasma in the Pancevo plantation caused the intensification of our biological control tests and efforts to reduce the ecological and economic impacts of these phytoplasmas. References: (1) I. M. Lee et al. Int. J. Syst. Evol. Microbiol. 54:1037, 2004. (2) D. Valiunas. PhD thesis, Institute of Botany, Vilnius, Lithuania, 2003.

First Report of Oidium neolycopersici on Greenhouse Tomatoes in Serbia.

In September 2011, tomato (Solanum lycopersicum L. 'Big Beef') plants showing typical symptoms of powdery mildew were collected in a greenhouse in the vicinity of Padinska Skela (District of City of Belgrade) in Serbia. Numerous circular, white colonies of powdery mildew were observed predominantly on the adaxial surface of the leaves, the petioles, and the stems. The foliage of infected plants turned yellow and necrotic, which was followed by rapid defoliation. Disease incidence was estimated by counting plants with powdery mildew symptoms in a random batch of 100 plants in four replicates and estimated to be extremely high, approaching 90%. Tomato plants ('Novosadski Jabucar') were inoculated with conidia released from diseased tomato leaves positioned above the tomato leaves and maintained at 25°C with a 14-h photoperiod. Healthy tomato plants from the same lot, which were not exposed to the conidia shower, were used as negative control. The first white fungal colonies appeared on the leaves of the inoculated plants within 4 to 7 days after inoculation, while no fungal growth was observed in the control plants. To determine the morphological characteristics of the pathogen, surface mycelium was removed with small strips of clear adhesive tape and examined using light microscopy. Microscopic observation revealed mycelium with lobed appressoria and hyaline, ellipsoid-ovoid or doliform conidia (32.5 to 47.5 × 17.5 to 25 µm) with no distinct firosin bodies and which produced sub-terminal germ tubes. Conidia were produced on the unbranched, erect conidiophores (82.5 to 150 µm) consisting of a cylindrical foot-cell followed by one to three short cells. No chasmothecia were found. On the basis of morphological characteristics, the pathogen was identified as Oidium neolycopersici (4), which was confirmed by internal transcribed spacer (ITS) sequence analysis. Total DNA was extracted directly from the whitish spots of superficial mycelium on the leaves with a DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. PCR amplification and sequencing were performed with primers ITS1F and ITS4 (1). The nucleotide sequence of the representative isolate 809-11 (Accession No. JQ619840) shared 100% identity with 16 O. neolycopersici isolates deposited in GenBank from different parts of the world. Tomato powdery mildew caused by O. neolycopersici is present in many European (4) and other countries around the world (3) and is becoming economically very important as majority of the tomato cultivars have shown to be susceptible (2). To our knowledge, this is the first report of O. neolycopersici in Serbia. Because tomato is a very popular and widely grown vegetable in Serbia, the presence of a new and potentially harmful disease could endanger greenhouse as well as open field tomato production. References: (1) J. H. Cunnington et al. Australas. Plant Pathol. 32:421, 2003. (2) T. Jankovics et al. Phytopathology 98:529, 2008. (3) H. Jones et al. Mol. Plant Pathol. 2:303, 2001. (4) L. Kiss et al. Mycol. Res. 105:684, 2001.