First Report of the Pale Potato Cyst Nematode Globodera pallida from Bosnia and Herzegovina.

Potato cyst nematodes (PCN), the golden cyst nematode Globodera rostochiensis (Woll.) Bahrens, and the pale potato cyst nematode G. pallida (Stone) stand out by their remarkable and efficient parasitic adaptations causing high economic losses in potato production worldwide. They are A2 quarantine pests in EPPO countries as well as in Bosnia and Herzegovina. Up to now, only G. rostochiensis was reported from Bosnia and Herzegovina in 2011 (1). A systematic survey on the presence of PCN on entire territory of Bosnia and Herzegovina started in 2011. During the PCN survey, each year 120 soil samples of 1.5 liters were collected in the Republic of Srpska, one of the two entities of Bosnia and Herzegovina. The samples were collected by soil auger 10 cm in length with a diameter of 1.5 cm consisting of 100 cores. Entire samples were processed by Seinhorst elutriator (4). In autumn of 2012, PCN viable cysts were found in two soil samples originating from one field. From one sample, 12 cysts were extracted, and 2 from another sample. The morphology of cysts and second stage juveniles and molecular analysis established the identity of this species as pale cyst nematode G. pallida (3). In addition, the sequencing of the ribosomal DNA region revealed unequivocal similarity to G. pallida (GenBank Accession No. HF968449), while PCR-RFLP analysis (2) showed European type of G. pallida. The infested field is located in Rogatica, 70 km east of Sarajevo, which is one of the main seed and potato production areas in Bosnia and Herzegovina. The field history revealed that farm-propagated, farm-saved seed potatoes could spread the nematodes to other fields as well. Therefore, 26 additional samples were taken from the fields that belong to the infested field owner and the surrounding fields, but no cysts were found in additional samples. To determine infestation focus and its size, the infested field (1.1 ha) was divided into 46 plots (25 × 10 m) and resampled by taking samples of 60 cores per plot. The detailed sampling of the field revealed a high infestation of 1 cyst per gram of soil in the infestation focus. The high infestation level and the propagation of farm-own seed potatoes suggest that the introduction of G. pallida might have occurred several years ago, probably with imported seed potatoes. The infested field was subjected to the phytosanitary measure of banning potato production for a period of 6 years with the possibility of its prolongation, if cysts with live content are found afterward. It is expected that the nematode is present in other fields due to the farmer's practices of propagating farm-saved seed potatoes and deficient field machinery hygiene. Therefore, the whole area will be intensively monitored for the presence of PCN in the future. An adequate pest management plan will be prepared after PCN pathotype identification. To our knowledge, only one field was found infested with G. pallida during the 3-year PCN survey in Bosnia and Herzegovina. Strict phytosanitary measures for preventing further PCN introductions and spreading should be intensified. References: (1) I. Ostojic et al. Plant Dis. 95:883, 2011. (2) S. Sirca et al. Phytopathol. Mediterr. 49:361, 2010. (3) A. M. Skantar et al. J. Nematol. 39:133, 2007. (4) J. van Bezooijen. Methods and Techniques for Nematology, Wageningen University, 2006.

First Report of the Pale Potato Cyst Nematode Globodera pallida from Slovenia.

The potato cyst nematodes (PCN) Globodera rostochiensis (Woll.) Behrens and G. pallida (Stone) Behrens are considered the most important nematode threat to potato production worldwide, and they are subject to strict quarantine regulations in many countries. The first report of the PCN in Slovenia dates back to 1971, when a single cyst of G. rostochiensis was detected (3). In the last decade, G. rostochiensis was detected several times, mainly in the central and northern parts of the country (2). Interceptions of G. pallida in imported consignments of ware potato were made several times, but had not been detected in soil in Slovenia. Therefore, the country was declared as a protected zone for G. pallida in the European Union by the national authorities in 2003. During the official PCN systematic survey in autumn 2011, the pale potato cyst nematode, G. pallida, was found in a soil sample. Two viable cysts were extracted and the nematode species was identified. The posterior part of the cysts containing eggs and juveniles were used for morphometrical analysis, while the anterior parts were used for DNA extraction and molecular analyses. The ribosomal internal transcribed spacer (ITS) region was amplified using ITS5 and PITSp4 primers and detected in real-time PCR using ABI7500 (Applied Biosystems, Life Technologies, Carlsbad, CA,) (1). Also, the ribosomal DNA region that extends from the 3' end of the 18S ribosomal subunit and includes all of ITS1, 5.8S, and ITS2, to the 5' end of the 28S ribosomal subunit, was used to generate a DNA sequence. The sequence obtained was compared with those from several Globodera species, revealing unequivocal similarity to G. pallida. The infested soil sample originated from a ware potato field near Ivancna Gorica, central Slovenia. Subsequently, an additional 69 samples were taken from the surroundings and viable G. pallida cysts were found in another five samples taken from two neighboring fields (one of grassland and the other of clover). Three fields, totaling 1.9 ha, were declared as G. pallida-infested. The eradication of the pest will take place by enforcing strict phytosanitary measures. Ware potatoes originating from areas where the pests occur is considered to be the most probable pathway for the introduction of G. pallida in Slovenia. A ware potato processing facility is situated in very close proximity to the infested fields. The waste waters from potato tuber washing were discharging onto the grassland, never used for potato or other field crop production in which the G. pallida infestation was found. The facility processes imported ware potato from several European and non-European countries. This case demonstrates that ware potato may pose a serious risk for the introduction of such pests, and should be therefore subjected to more intensive phytosanitary inspection. References: (1) J. Bacic et al. Russ. J. Nematol. 16:63, 2008. (2) S. Sirca et al. Phytopathol. Mediterr. 49:361, 2010. (3) G. Urek et al. Nematology 5:391, 2003.

Xiphinema rivesi from Slovania Transmit Tobacco ringspot virus and Tomato ringspot virus to Cucumber Bait Plants.

The dagger nematode, Xiphinema rivesi Dalmasso, a member of the X. americanum group, was detected in 2002 for the first time in Slovenia and for the fourth time in Europe (4). X. rivesi is a vector of at least four North American nepoviruses including Cherry rasp leaf virus (CRLV), Tobacco ringspot virus (TRSV), Tomato ringspot virus (ToRSV), and Peach rosette mosaic virus (PRMV) (1,2). All of these viruses are included on the EPPO and EU lists of quarantine organisms, but none of the Xiphinema species found in Europe have been reported to transmit these nepoviruses. Three virus isolates, including TRSV (from Lobelia spp.; virus collection of the Plant Protection Service, Wageningen, The Netherlands), ToRSV (grapevine isolate PV-0381; DSMZ, Braunschweig, Germany), and Arabis mosaic virus (ArMV) (from Vinca spp.; virus collection of the Plant Protection Service), were used in transmission tests with a population of X. rivesi found in Slovenia. X. rivesi is not known to transmit ArMV and this virus was included as a check. The nematodes were extracted from peach orchard soil collected near the village of Dornberk, and transmission tests fulfilled the set of criteria proposed by Trudgill et al. (3). Cucumis sativus cv. Eva, grown in a growth chamber at 25°C, was used as acquisition hosts and transmission bait plants. The acquisition hosts were mechanically inoculated and showing systemic symptoms before the introduction of nematodes. Noninoculated acquisition plants were included as controls. After a 10-day acquisition feeding period, the nematodes were transferred to healthy bait plants and allowed a 14-day inoculation feeding period. X. rivesi transmitted TRSV and ToRSV but not ArMV. TRSV and ToRSV bait plants developed systemic symptoms 4 to 6 weeks after the nematodes were transferred. Transmission of TRSV and ToRSV was confirmed by testing leaf and root sap of bait plants in a double antibody sandwich (DAS)-ELISA. High virus concentrations were detected in the roots and leaves of TRSV and ToRSV symptomatic plants. DAS-ELISA on bait plants from nematodes that had been allowed to feed on ArMV-infected or the virus-free control acquisition plants gave negative results. No symptoms appeared on bait plants used for ArMV transmission or the control bait plants. To our knowledge, this is the first report of transmission of TRSV and ToRSV with a Xiphinema population from Europe. References: (1) D. J. F. Brown et al. Phytopathology 84:646, 1994. (2) L.W. Stobbs et al. Plant Dis. 80:105, 1996. (3) D. L. Trudgill et al. Rev. Nematol. 6:133, 1983. (4) G. Urek et al. Plant Dis. 87:100, 2002.

The Incidence of Root-Knot Nematodes Meloidogyne arenaria, M. incognita, and M. javanica on Vegetables and Weeds in Montenegro.

Root-knot nematodes (Meloidogyne spp.) are common pathogens that parasitize vegetables and other crops and cause significant yield reductions worldwide. In the early spring of 2006, severe plant stunting, chlorosis, and extensive root galling were observed on cucumber plants grown in a greenhouse on Zeta plain, Zetska ravnica, Montenegro. In the summer and autumn of 2006, infected roots of different crops were collected from greenhouses and vegetable production fields of Zeta plain, which represents the largest area of Montenegro's vegetable production. Several vegetable crops were found to be infected with root-knot nematodes, including tomato (Lycopersicon esculentum Mill.), squash (Cucurbita pepo L.), cucumber (Cucumis sativus L.), pepper (Capsicum annuum L.), and lettuce (Lactuca sativa L.). Symptoms on these crops included root-galling, leaf chlorosis, and stunting. Heavily infected tomato plants growing in two greenhouses also displayed early flower and fruit drop. Nematode species were determined based on characterization of (i) female perineal patterns, (ii) male and second-stage juvenile morphology (2,3), and (iii) esterase and malate dehydrogenase phenotypes (PhastSystem; Amersham Biosciences, Piscataway, NJ) from young egg-laying females (1). The most prevalent species was M. incognita, which was isolated from the roots of tomatoes, peppers, cucumbers, and lettuce from nine locations. Meloidogyne arenaria was detected at three locations from the roots of tomatoes grown in a greenhouse and the weeds Convolvulus arvensis L. and Solanum nigrum L., which were growing in open fields in separate locations. M. javanica was found on tomato and squash in the same field where M. arenaria was also found on S. nigrum. M. javanica was isolated from tomato and squash. In this study, we found high incidence of Meloidogyne spp. in intensive vegetable production areas of Montenegro. The implementation of an effective integrated pest management program is essential for future use of infested areas. To our knowledge, this is the first report of M. arenaria, M. incognita, and M. javanica from Montenegro. References: (1) P. R. Esbenshade and A. C. Triantaphyllou. J. Nematol. 17:6, 1985. (2) S. B. Jepson. Identification of Root-Knot Nematodes. CAB International, Wallingford, UK, 1987. (3) G. Karssen. The Plant-Parasitic Nematode Genus Meloidogyne Göldi, 1892 (Tylenchida) in Europe. Koninklijke Brill NV, Leiden, the Netherlands, 2002.