First Report of Brown Rot Caused by Monilinia fructicola Affecting Peach Orchards in Slovenia.

Monilinia fructicola, the causal agent of brown rot, is a destructive fungal pathogen that affects mainly stone fruits (Prunoideae). It causes fruit rot, blossom wilt, twig blight, and canker formation and is common in North and South America, Australia, and New Zealand. M. fructicola is listed as a quarantine pathogen in the European Union and was absent from this region until 2001 when it was detected in France. In August 2009, mature peaches (Prunus persica cv. Royal Glory) with brown rot were found in a 5-year-old orchard in Goriska, western Slovenia. Symptoms included fruit lesions and mummified fruits. Lesions were brown, round, rapidly extending, and covered with abundant gray-to-buff conidial tufts. The pathogen was isolated in pure culture and identified based on morphological and molecular characters. Colonies on potato dextrose agar (PDA) incubated at 25°C in darkness had an average daily growth rate of 7.7 mm. They were initially colorless and later they were light gray with black stromatal plates and dense, hazel sporogenous mycelium. Colony margins were even. Sporulation was abundant and usually developed in distinct concentric zones. Limoniform conidia, produced in branched chains, measured 10.1 to 17.7 µm (mean = 12.1 µm) × 6.2 to 8.6 µm (mean = 7.3 µm) on PDA. Germinating conidia produced single germ tubes whose mean length ranged from 251 to 415 µm. Microconidia were abundant, globose, and 3 µm in diameter. Morphological characters resembled those described for M. fructicola (1). Morphological identification was confirmed by amplifying genomic DNA of isolates with M. fructicola species-specific primers (2-4). Sequence of the internal transcribed spacer (ITS) region (spanning ITS1 and ITS 2 plus 5.8 rDNA) of a representative isolate was generated using primers ITS1 and ITS4 and deposited in GenBank (Accession No. GU967379). BLAST analysis of the 516-bp PCR product revealed 100% identity with several sequences deposited for M. fructicola in NCBI GenBank. Pathogenicity was tested by inoculating five mature surface-sterilized peaches with 10 µl of a conidial suspension (104 conidia ml-1) obtained from one representative isolate. Sterile distilled water was used as a control. Peaches were wounded prior to inoculation. After 5 days of incubation at room temperature and 100% relative humidity, typical brown rot symptoms developed around the inoculation point, while controls showed no symptoms. M. fructicola was reisolated from lesion margins. Peach and nectarine orchards in a 5-km radius from the outbreak site were surveyed in September 2009 and M. fructicola was confirmed on mummified fruits from seven orchards. The pathogen was not detected in orchards from other regions of the country, where only the two endemic species M. laxa and M. fructigena were present. To our knowledge, this is the first report of M. fructicola associated with brown rot of stone fruits in Slovenia. References: (1) L. R. Batra. Page 106 in: World Species of Monilinia (Fungi): Their Ecology, Biosystematics and Control. J. Cramer, Berlin, 1991. (2) M.-J. Côté et al. Plant Dis. 88:1219, 2004. (3) K. J. D. Hughes et al. EPPO Bull. 30:507, 2000. (4) R. Ioos and P. Frey. Eur. J. Plant Pathol. 106:373, 2000.

First Report of Tomato chlorotic dwarf viroid in Petunia spp. in Slovenia.

In early April 2010, 30 samples of Petunia spp. were taken by phytosanitary inspectors from 22 production sites in Slovenia in the frame of surveying host plants for the presence of Potato spindle tuber viroid (PSTVd). Samples were taken in accordance with the plan of the survey for the year 2010 and were tested for the presence of PSTVd by real-time RT-PCR according to the EPPO protocol (1). At the time of sampling, there were no disease symptoms on the plants. Samples consisted of fully developed leaves collected from as many as five plants. Total RNA was isolated from 50 ± 5 mg of leaf tissue with an RNeasy Plant Mini Kit (Qiagen, Chatsworth, CA). One sample of cv. Surfinia Purple from a production site from the coastal region and another of cv. Surfinia Hot Pink 05 from a production site near Ljubljana, both multiplied through cuttings, were positive by real-time RT-PCR, confirming the presence of PSTVd or Tomato chlorotic dwarf viroid (TCDVd). To identify the viroid, RT-PCR with primer pairs of Shamloul et al. (3) and Di Serio (2) were performed with isolated total RNA of each positive sample. RT-PCR products were obtained only with primer pairs of Shamloul et al. (3). To obtain the full sequence, additional RT-PCR was done for each sample with semi-universal pospiviroid primers Vid-RE/FW (4). RT-PCR products obtained with primer pair of Shamloul et al. (3) and primer pair Vid RE/FW were sequenced (Macrogen, Seoul, Korea). Sequence analysis confirmed the identity of a viroid as TCDVd. Both isolates consisted of 360 nucleotides and were 100% identical to an isolate from tomato deposited in NCBI GenBank under Accession No. AF162131. They showed 98% identity with sequences from petunias (GQ396664, EF582392, EF582393, and DQ859013). The infected Petunia spp. stocks were destroyed. Although the infection of Petunia spp. with TCDVd is symptomless, the infected plants could be a source of infection for tomato and potato. TCDVd infection can cause severe damage on potato and tomato, similar to that caused by infection with PSTVd, to which it is closely related. To our knowledge this is the first finding of TCDVd in Petunia spp. in Slovenia. References: (1) Anonymous. EPPO Bull. 34:257, 2004. (2) F. Di Serio. J. Plant Pathol. 89:297, 2007. (3) A. M. Shamloul et al. Can. J. Plant Pathol. 19:89, 1997. (4) J. Th. J. Verhoeven et al. Eur. J. Plant Pathol. 110:823, 2004.

Identification of Lettuce big-vein associated virus and Mirafiori lettuce big-vein virus Associated with Lettuce Big-Vein Disease in Slovenia.

Lettuce big-vein disease (LBVD) is an important disease of lettuce (Lactuca sativa L.) worldwide. Two viruses are reported to be associated with the disease, Lettuce big-vein associated virus (LBVaV) and Mirafiori lettuce big-vein virus (MLBVV). The majority of publications shows that MLBVV is the causal agent of LBVD, but two plants with big-vein symptoms and infected only with LBVaV were found by Roggero et al. (2). Lettuce plants with big-vein symptoms were observed in a greenhouse in Ljubljana and in a private garden near Sezana. Chlorotic vein banding with deformations of leaves were observed and the plants were generally smaller than those without symptoms. The seedlings at the first location were grown in sterilized soil and showed no such symptoms. Six symptomatic plants and two seedlings without symptoms from the first location and three symptomatic plants from the second location were analyzed for the presence of LBVaV and MLBVV. Total RNA was isolated from 50 mg of lettuce leaf tissue with a RNeasy plant mini kit (Qiagen, Chatsworth, CA). Multiplex reverse transcription-PCR with primers VP248 (5'-CGCCAGGATCTTTGATCCATCTG-3') and VP249 (5'-TTGCGACATGTTCCTCCTCATCG-3') for LBVaV, VP286 (5'-TATCAGCTCACATACTCCCTATCG-3') and VP287 (5'-CAACTAGCTCAGAATACATGCAG-3') for MLBVV, and VP383 and VP389 for internal control (1), was performed. Mixed infections with MLBVV and LBVaV were detected in four plants and only MLBVV was detected in two plants from the first location. None of the viruses was detected in the two seedlings without symptoms. Mixed infections with MLBVV and LBVaV were detected in all analyzed plants from the second location. According to these results, only MLBVV could be correlated with observation of disease symptoms. To our knowledge, this is the first reported association of MLBVV and LBVaV with big-vein disease in Slovenia. MLBVV was the only virus found in all samples with big-vein symptoms. References: (1) J. A. Navarro et al. Phytopathology 94:470. 2004. (2) P. Roggero et al. Eur. J. Plant Pathol. 109:261, 2003.