First Report of Neofusicoccum parvum Associated with Grapevine Trunk Diseases in Croatia.

In September 2010, during survey of diseased grapevines (Vitis vinifera L.) in vineyards at localities Zmajevac (BZ), Orahovica (SO), Cilipi (KC), and Novalja (PN), symptoms characteristic of grapevine trunk diseases (GTD) (3) were observed, showing on cross-sectioned cordons and trunks as brown, wedge-shaped perennial cankers and/or dark streaking of the wood. In Croatia, these symptoms were traditionally associated with Eutypa Tul. & C.Tul. and with fungi from Diaporthaceae (2). From affected grapevines (cvs. Grasevina, Pinot bijeli, Malvazija dubrovacka, and Gegic), samples of symptomatic cordons and trunks were collected (n ≥ 35). To isolate the causal agents from the samples, woodchips of symptomatic tissue, surface-sterilized in 2% sodium hypochlorite for 2 min, were placed on potato dextrose agar amended with streptomycin sulphate (50 µg/ml) and incubated for 7 days at 25°C in darkness. A percentage of samples (72, 15, 27, and 54% from BZ, SO, KC, and PN, respectively) yielded fungal colonies with abundant aerial mycelium, initially white, but turning olivaceous grey after 5 days. From these colonies, monohyphal isolates were obtained and pycnidial formation stimulated by cultivation on 2% water agar with stems of plant species Foeniculum vulgare Mill. at 25°C under diffuse light for 3 weeks. Pycnidia contained conidia that were hyaline, unicellular, ellipsoid with round apices and truncated bases, and thin walled with smooth surface. Dimensions of conidia (n ≥ 50) were (12.8) 15.3 ± 1.4 (17.6) × (5.4) 6.3 ± 0.8 (7.6) µm, with length/width ratio (2.0) 2.5 ± 0.5 (3.2). Based on morphological data, species Neofusicoccum parvum (Pennycook & Samuels) Crous, Slippers & A.J.L. Phillips was suspected (1). For molecular identification, isolates BZ330, SO334, KC342, and PN121 were used for PCR to amplify internal transcribed spacer region and partial translation elongation factor 1-alpha gene, using primers ITS5/ITS4 and EF1-728F/EF1-986R, respectively. Obtained sequences were shown to be identical between the four isolates (GenBank: KF296318, KF296319) and when compared with sequences for reference N. parvum isolate CMW9080 (AY236942, AY236887) they showed >99% homology, confirming the isolates as species N. parvum. Pathogenicity tests were done by inoculation of detached green shoots (GS) and lignified canes (LC) (n = 5) of grapevine cv. Skrlet by either mycelial plugs of the same four isolates, or sterile agar plugs for the controls. Inoculated GS were kept in flasks with sterile water in a glasshouse for 10 days, and LC in humid dark chambers for 30 days, at 25°C. Resulting vascular necrosis measured 62 to 81 mm (GS) and 215 to 246 mm (LC), but was absent on controls. Koch's postulates were satisfied by successful reisolation of N. parvum only from plants inoculated with mycelial plugs. N. parvum has been recognized as a serious grapevine pathogen, causing similar symptoms worldwide (3). To our knowledge, this is the first report of N. parvum associated with GTD in Croatia, and due to its relatively high incidence at surveyed localities, it could present considerable threat, particularly for neighboring vine growing regions. Diplodia seriata De Not., a weak pathogen (3), was also identified from a percentage of samples in this survey. References: (1) P. W. Crous et al. Stud. Mycol. 55:235, 2006. (2) J. Kaliterna et al. Arh. Hig. Rada Toksikol. 63:471, 2012. (3) J. R. Urbez-Torres. Phytopathol. Mediterr. 50(Suppl.):S5, 2011.

First Report of Diplodia seriata as Causal Agent of Olive Dieback in Croatia.

In August 2010, a dieback of young olive (Olea europea L.) trees (cvs. Pendolino and Leccino) occurred in two orchards in Istria, Croatia. According to the producers, low temperatures during the winter severely damaged the plants and led to their decline. Distinctive symptoms, assumed fungal infection, were observed in internal tissue of stems and branches. Elongated brown necrosis, sometimes with black streaks, was visible under the bark, therefore Verticillium wilt was suspected. Of 1,086 trees in two orchards (4 ha), 165 (15%) showed symptoms. To isolate the causal agent, surface-sterilized wood chips of symptomatic tissue were placed on potato dextrose agar (PDA). Fungal colonies resembling Botryosphaeriaceae spp. grew from all wood fragments placed on PDA, and from these colonies, monohyphal isolates were obtained. For morphological identification, pycnidial formation was stimulated by growing the isolates on 2% water agar that included stems of plant species Foeniculum vulgare Mill. at room temperature under diffuse light. Pycnidia contained conidia that initially showed as hyaline, becoming light to dark brown as they matured, ovoid with truncated or rounded base and obtuse apex, aseptate, with wall moderately thick, externally smooth, roughened on the inner surface, and 22.8 to 23.5 × 9.6 to 10.5 µm. On the basis of these morphological characters, fungal species Diplodia seriata (teleomorph "Botryosphaeria" obtusa) was suspected (3). For molecular identification, four isolates (MN3, MN4, MN5, and MN6) were used for PCR to amplify the internal transcribed spacer (ITS) region and partial translation elongation factor 1-alpha (EF1-α) gene, using primers ITS4/ITS5 and EF1-728F/EF1-986R, respectively. Sequencing was performed with those amplified genes, then sequences were deposited in GenBank. Comparison of these sequences with GenBank sequences for referent D. seriata isolate CBS 112555 (AY259094 and AY573220) (3) showed 100% homology. On the basis of molecular data, the isolates were confirmed to be species D. seriata De Not. Pathogenicity tests were performed by inoculation of 2-year-old olive plants, six plants per tested cultivar (Pendolino and Leccino). For every cultivar, four plants were wounded and mycelium plugs from D. seriata cultures on PDA were placed on the wounds and sealed with Parafilm. Two control plants per tested cultivar were inoculated with sterile PDA plugs. After 2 months, six of eight inoculated plants wilted completely, and under the bark, brown necrosis was observed. D. seriata was constantly reisolated from the inoculated plants and fulfilled Koch's postulates and confirmed pathogenicity of D. seriata on olive as causal agent of olive dieback. Control plants showed no symptoms of the disease. This fungus has been recognized as the cause of fruit rot of olive (1) and branch canker or dieback in Spain (2). To our knowledge, this is the first report of D. seriata as a pathogen of olive in Croatia. Also, this is one of the first reports of D. seriata as the cause of olive dieback in the world, while Moral et al. (1,2) mostly reported it as the cause of olive fruit rot. Since the same symptoms of olive dieback were observed at other localities in Croatia, the disease could represent a serious threat, particularly for young olive orchards. References: (1) J. Moral et al. Plant Dis. 92:311, 2008. (2) J. Moral et al. Phytopathology 100:1340, 2010. (3) A. J. L. Phillips et al. Fungal Divers. 25:141, 2007.

First Report of the Golden Potato Cyst Nematode, Globodera rostochiensis, in Bosnia and Herzegovina.

The golden potato cyst nematode, Globodera rostochiensis (Wollenweber, 1923) Behrens, 1975, is the most problematic pest of potato (Solanum tuberosum L. subsp. tuberosum) worldwide and it is recognized as an A1 plant health quarantine species. In Bosnia and Herzegovina, there have been no surveys for the presence of potato cyst nematodes (PCN) in ware potato lands, although they have been documented in the nearby countries of Serbia and Croatia (3). To determine the presence and distribution of PCN and to prevent further spread or increase of PCN populations, a survey in ware potato land was undertaken in 2008, 2009, and 2010. Soil samples consisting of 100 cores of 4 to 5 ml of soil were taken in a grid pattern throughout the plots. Samples of approximately 500 g were processed in the laboratory. The samples were air dried and cysts were extracted using a Spears's flotation method (4) from a 100-ml subsample. Species identification was based on morphometrical characters (2) and real-time PCR where the ribosomal internal transcribed spacer (ITS5, PITSr3) region was amplified and detected by the Sequence Dection Software v1.3 (Applied Biosystems, Carlsbad, CA) (1). In 2008, soil sampling was conducted in five locations and PCN were not detected in any of 10 soil samples taken. In June 2009, 17 soil samples were collected from seven locations. Two viable cysts of G. rostochiensis were detected in one soil sample from Tihaljina (Zaside; E 6447659.560, N 4797392.101, altitude 130 m). A total of 110 soil samples were collected from 90 locations in May and June 2010. Five viable cysts of G. rostochiensis were detected in one soil sample taken from Capljina (Oplicici - Turajlovina; E 6485169, N 4775964, altitude 222 m). On the basis of the survey results, G. rostochiensis is not widely spread but the cysts are detectable, indicating that the infestation is relatively recent and that there is a threat of further spread of this quarantine species. Consequently, producers need to grow resistant potato cultivars and implement additional control measures to keep the nematode populations below damaging levels. References: (1) J. Bacic et al. Russ. J. Nematol. 16:1, 61, 2008. (2) C. C. Fleming and T. O. Powers. Potato Cyst Nematodes: Biology, Distribution and Control. R. J. Marks and B. B. Brodie, eds.CAB International, Wallingford, 1998. (3) D. Grubisic et al. J. Pest Sci. 80:21, 2007. (4) J. F. Spears. Agr. Handb. No. 353. U.S. Dep. Agric., 1968.