Sanja Radonjić

Diversity and localization of bacterial symbionts in three whitefly species (Hemiptera: Aleyrodidae) from the east coast of the Adriatic Sea

Several whitefly species (Hemiptera: Aleyrodidae) are cosmopolitan phloem-feeders that cause serious damage in numerous agricultural crops. All whitefly species harbor a primary bacterial symbiont and a diverse array of secondary symbionts which may influence several aspects of the insects biology. We surveyed infections by secondary symbionts in Bemisia tabaci (Gennadius), Trialeurodes vaporariorum (Westwood) and Siphoninus phillyreae (Haliday) from areas in the east cost of the Adriatic Sea. Both the Middle East-Asia Minor 1 (MEAM1) and Mediterranean (MED) B. tabaci genetic groups were detected in Montenegro, whereas only the MED was confirmed in Croatia. Trialeurodes vaporariorum and S. phillyreae were found in all areas surveyed. MEAM1 and MED exhibited similarity to previously reported infections, while populations of T. vaporariorum from Montenegro harbored Rickettsia, Wolbachia and Cardinium in addition to previously reported Hamiltonella and Arsenopnohus. Siphoninus phillyreae harbored Hamiltonella, Wolbachia, Cardinium and Arsenophonus, with the latter appearing in two alleles. Multiple infections of all symbionts were common in the three insect species tested, with some reaching near fixation. Florescent in situ hybridization showed new localization patterns for Hamiltonella in S. phillyreae, and the morphology of the bacteriosome differed from that observed in other whitefly species. Our results show new infections with bacterial symbionts in the whitefly species studied. Infections with the same symbionts in reproductively isolated whitefly species confirm complex relationships between whiteflies and bacterial symbionts, and suggest possible horizontal transfer of some of these bacteria.

Wolbachia infection in natural populations of Dictyophara europaea, an alternative vector of grapevine Flavescence dorée phytoplasma: effects and interactions

The European lantern fly, Dictyophara europaea, is an alternative vector of the Flavescence doree phytoplasma (FDp) disease of grapevine in European vineyards, enabling infection initiation from wild reservoir compartment (Clematis vitalba). Heretofore recorded rate of D. europaea FDp-infection has been very low (3%), making it less epidemiologically significant than would be expected based on reservoir plant infection rate (30%). In this study we present findings on a heavily FDp-infected D. europaea population (>60%), on the natural Wolbachia infection of populations with low FDp-infection rates (DeWo+) and on Wolbachia absence in highly FDp-infected population (DeWo−). We examine several possible causes underlying the differences in vector infection rates: (a) population genetic characteristics of D. europaea and correlation with Wolbachia strain wEur natural infections, (b) Wolbachia effects on fitness components of DeWo+ laboratory colony and (c) rate of reservoir plant FDp-infection and differences in FDp genotypes harboured by low and highly infected vector populations. The vector genetic diversity level was found to be lower in DeWo+ than in uninfected individuals and to exhibit a different evolution of fixed haplotypes. All DeWo+ populations were infected with the same strain of wEur. The FDp was found to be genetically diversified (five genotypes) but had no relation to infection rates. We did not find evidence of fitness upgrades with regard to Wolbachia infection status. Although more experimentation is needed, it seems that Wolbachia confers protection against FDp or is in competition with FDp according to the observed correlations: low FDp-infected vector populations are infected with Wolbachia and vice versa.

First report of alder yellows phytoplasma infecting common and grey alder (Alnus glutinosa and A. incana) in Montenegro.

Alder yellows phytoplasmas (AldYp) of the 16SrV-group associated with common alder (Alnus glutinosa) and grey alder (A. incana) are closely related to the grapevine yellows (GY)-associated quarantine phytoplasma Flavescence dorée (FDp). AldYp have been reported in several countries where epidemic appearance of FDp has been confirmed (France, Italy, and Serbia) (1,2). To date, the presence of 16SrV-group of phytoplasmas has not been reported in Montenegro; however, the main vector of FD phytoplasma, Scaphoideus titanus, has been identified in Montenegrin vineyards since 2008. During a survey in September 2011, in the northern part of Montenegro, 12 symptomatic alder trees showing symptoms of leaf discoloration, ranging from yellow to light green, were sampled. Six samples, each comprising several symptomatic leaves, were collected from A. glutinosa at streamside in woodlands near the town Kolašin and other six samples from A. incana close to the river Lim near the town of Bijelo Polje. Leaves of six young A. glutinosa seedlings were used as controls. Total DNA was extracted from fresh leaf midribs and petioles using the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany). Nested PCR assay was conducted on 16S rRNA gene using phytoplasma generic primers P1/P7 and F2n/R2 followed by RFLP with MseI endonuclease (Fermentas, Vilnius, Lithuania) (3). Confirmation of identification and characterization of phytoplasma positive samples was performed by amplifying the non-ribosomal metionine aminopeptidase (map) gene using FD9f5/MAPr1 and FD9f6/MAPr2 primer set (1), specific for the members of the 16SrV group phytoplasmas. Amplification products were sequenced and deposited in GenBank (KC188998 through 9001). Comparison of the map gene sequences was performed by phylogenetic analysis along with 20 reference sequences of the 16SrV-group members (1), using the neighbor-joining method in MEGA5 software (4). 16S rRNA gene amplification revealed the presence of phytoplasmas in 11 out of 12 symptomatic samples, while Mse I restriction analysis and comparison with reference strains (AldYp and FDp from Serbia) enabled affiliation of detected phytoplasmas to the 16SrV-group. None of the controls were positive for any phytoplasma. Phylogenetic analysis of the Montenegrin AldYp map gene sequences revealed presence of four different strains clustering within the previously defined clusters of the 16SrV-group members (1). Three different strains associated with symptomatic A. glutinosa were identified and they clustered either within the FD1, FD2, or PGY-C cluster, while a single detected strain from A. incana proved to be identical with PGY-A isolate of AldY phytoplasma infecting grapevine in Germany (AM384892). To our knowledge, this is the first report of the association of 16SrV-group phytoplasmas with common and grey alder in Montenegro, as well as the first report of FD-related phytoplasmas in Montenegro. Since alder trees are considered as a possible natural reservoir of the FD phytoplasmas (1), the finding of alders naturally infected with strains related to the FDp (FD1 and FD2 clusters) indicate a possible threat of economic importance to the grape production in Montenegro, which should be addressed in further research. References: (1) G. Arnaud et al. Appl. Environ. Microbiol. 73:4001, 2007. (2) T. Cvrkovic et al. Plant Pathol. 57:773, 2008. (3) I-M. Lee et al. Int. J. Syst. Evol. Bacteriol. 48:1153, 1998. (4) K. Tamura et al. Mol. Biol. Evol. 28:2731, 2011.

Distribution and molecular identification of Tuta absoluta (Meyrick, 1917) (Lepidoptera, Gelechiidae) populations in Bosnia and Herzegovina and Montenegro

Abstract Since 2010, the South American tomato moth - Tuta absoluta (MEYRICK, 1917) (Lepidoptera, Gelechiidae) - has been recorded in Bosnia and Herzegovina (B&H) and Montenegro. In 2012, pheromone traps were placed among tomato crops growing in greenhouses and open fields in 14 localities in B&H and Montenegro; the pest was caught at all the sites. Adults were caught in both greenhouses and open fields in Trebinje (B&H) on 4th June 2012 and in Ulcinj (Montenegro) in mid-May. Species identification was confirmed by means of morphological characters of adults and male genitalia. In addition, molecular identification of specimens from B&H and Montenegro was performed using mitochondrial (mtDNA) markers.

Dispersion of the Mediterranean fruit fly Ceratitis capitata Wiedem. (Diptera: Tephritidae) in Mandarin orchards on Montenegrin seacoast.

The Mediterranean fruit fly Ceratitis capitata Widem. has been an established pest on the Montenegrin seacoast for more than ten years, although with variable abundance in different years and localities. From an economic aspect, its most important host in Montenegro is the mandarin unshiu (Citrus unshiu Marc.), particularly its cultivar Owari. Dispersion of C. capitata in citrus orchards (prevailingly mandarin) was monitored on Baosici, Lastva Grbaljska and Bar localities during 2003 and 2004. The results of this study showed that, during both years, peripheral-row trees (primarily the first row) in citrus orchards were more exposed to attacks by C. capitata than middle and last rows. In 2003, the average number of larvae in mandarin fruits in first rows varied from 11.4±0.59 to 40.1±0.67, from 7.04±0.47 to 28.8±0.48 and from 2.9±0.07 to 17.3±0.54 on the localities of Baosici, Lastva Grbaljska and Bar, respectively. On the same localities, it ranged from 7.4±0.34 to 16.9±0.4, from 0.0 to 18.7±0.32 and from 0.0 to 9.93±0.56 in middle rows, and from 3.0±0.28 to 16.8±0.77, from 0.0 to 20.9±0.38 and from 0.0 to 13.1±0.39 in last rows. Data collected at Baosici, Lastva Grbaljska and Bar in 2003 also suggest that the average number of larvae per mandarin fruit in first rows was 1.78-2.08 times higher than in middle rows, and 1.25-1.77 times higher than in last rows. In 2004, the average number of larvae in mandarin fruits in first rows varied from 7.3±0.27 to 8.3±0.45, from 7.2±0.23 to 17.6±0.59 and from 3.8±0.1 to 8.8±0.25 on the localities of Baosici, Lastva Grbaljska and Bar, respectively. On these localities, it ranged from 1.7 ±0.17 to 3.3±0.19, from 1.1±0.12 to 3.5±0.8 and from 0.0 to 0.8±0.14 in middle rows, and from 1.7±0.17 to 3.6±0.32, from 0.0 to 4.0±0.26 and from 0.0 to 0.2±0.06 in last rows. Data collected in 2004 also showed that the average number of larvae in mandarin fruits in first rows on the same localities was 3.12-15.75 times higher than in middle rows, and 2.94 -6.3 times higher than in last rows.

Intensity of attack caused by Mediterranean fruit fly Ceratitis capitata Wiedem. (Diptera, Tephritidae) on mandarin along the Montenegrin seacoast.

Along the Montenegrin seacoast, all cultivated citrus species (mandarin, orange, lemon, grapefruit), fig, loquat and ziziphus were detected as host plants of the Ceratitis capitata Wiedem. Among those found, in economic sense, the most important host plant is mandarin Unsiu. Intensity of the attack were monitored on mandarin in 2003 and 2004 in localities Baosici, Lastva Grbaljska and Bar, and was determined as higher in 2003. The first symptoms of attack were detected, depending on locality, from middle of September until beginning of October, one to two months earlier then in 2004. The maximum number of larvae per fruit were detected in October and November (average from 13.44±0.16 to 22.82±0.13). Time of the first symptoms appearence on mandarin in September and October 2003 and in November 2004, indicate on crucial importance of alternative host plants (figs and loquat) for reproduction and increasing of C. capitata population, because later during a season its intensity of attack on mandarin, depend largely on their presence.