Serbian Isolates Research Articles

Identification and Characterization of Eutypa lata on Grapevine in Serbia.

In the period from late May 2004 to late May 2010, grapevine (Vitis vinifera L.) between 11 and 22 years old was observed for the incidence of symptoms of shortened shoots and zigzag internodes, with tiny, chlorotic leaves, torn and bended edges, with necrosis along the edges of leaves and dead internervous tissue. These symptoms are conspicuous especially when the vine is in the F phases of development. Later, in the course of vegetation, the dying of infected shoots and branches or covering of symptoms by a new foliage mass is perceived. Foliar symptoms are initially confined to one arm of infected vines; however, as the disease progresses, symptoms may spread throughout the entire vine. If a cross-section is made of an infected trunk, the canker appears as a wedge-shaped area of discolored wood spreading to the center of the trunk. In the period from 2004 to 2010, disease incidence was high, approaching 20%, and decline-affected cordons, vine branches, or whole plants was very high, resulting in losses of more than 35%. The loss created by a decline in grapevine quality is not included in this estimate. In this study, the causal agent was diagnosed as Eutypa lata (anamorph Libertella blepharis), on the basis of morphology of conidia of a Libertella anamorph on a 4- to 6-week-old culture on PDA (1) and by molecular identification. Molecular identification was performed by PCR and RFLP analysis and supplemented by sequence analysis. Total DNA was isolated from cultured mycelia of fungi using CTAB extraction protocol. PCR reaction was performed by universal ITS1/ITS4; the primer pair and RFLP patterns were determined after restriction with AluI (3). For specific identification of E. lata, the primer pair Lata 1/Lata 2.2 (2) were used and the 385-bp fragment was detected from analyzed isolates. Five selected isolates were purified and a fragment encompassing ITS1, ITS2, and 5.8S rDNA gene was sequenced. Sequences were deposited in the NCBI database under Accession Nos. JQ041699, JQ041700, JQ041701, JQ041702, and JQ041703. Sequence comparisons revealed high nucleotide identity among isolates (99.6 to 100%). When aligned with other E. lata isolates retrieved from the NCBI database, Serbian isolates show the highest nucleotide identity with the isolates from North America (AY462541, AY462540, AY662393, AY662392) and Australia (EU835166, EU835163, EU835162, EU835161, EU835160, EU835159, EU835156). A pathogenicity test was performed in February 2006 in a greenhouse at room temperature (approximately 22°C) and included inoculation rooted cuttings of grapevine (cultivars Cabernet Sauvignon, Prokupac, and Drenak) plants by mycelium. Agar plugs containing mycelium were inserted into 5 mm diameter holes drilled in the main stem of the rootlings and sealed by wrapping with Parafilm. Uninoculated control vines treated with a sterile agar plug were included in the experiment. Foliar symptoms and discoloring of wood beneath and above the inoculation site, inoculated plants, was observed. Reisolation and reinoculation were performed 27 months later, and 54 months later the pathogenicity test was confirmed (4). To our knowledge, this is the first report of death of infected cordons of grapevine by E. lata in Serbia.

First Report of Tomato spotted wilt virus Infecting Onion and Garlic in Serbia

In June 2011, extensive bleaching and numerous small whitish spots on leaves were observed in an onion (Allium cepa) seed crop as well as chlorotic spots and streaks in the neighboring garlic (A. sativum) bulb crop in the Aleksandrovo locality (Central Banat District, Serbia). Affected plants occurred throughout the field and disease incidence was estimated at 60% in the onion and 40% in the garlic crop. A high population of Thrips tabaci that was found in both crops, and local necrotic spots on Petunia × hybrida mechanically inoculated with infected onion or garlic sap by a chilled 0.01 M phosphate buffer, pH 7.0, containing 0.1% sodium sulfite (1), suggested the presence of a Tospovirus. For these reasons, sampled symptomatic onion and garlic plants were tested for the presence of Tomato spotted wilt virus (TSWV) and Iris yellow spot virus (IYSV) using commercial double-antibody sandwich-ELISA diagnostic kits (Bioreba AG, Reinach, Switzerland). Commercial positive and negative controls and extracts from healthy onion and garlic tissue were included in each ELISA. Of the 18 onion and 10 garlic plants tested, 16 and 7 samples, respectively, were positive for TSWV, and all were negative for IYSV. The identity of TSWV was further confirmed by conventional reverse transcription (RT)-PCR analysis. Total RNAs were extracted with an RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) and RT-PCR was performed with the One-Step RT-PCR Kit (Qiagen) using TSWV-specific forward (5'-GGTTAAGCTCACTAAGAAARCA-3') and reverse primers (5'-TTTAACYCCRAACATTTCATAGA-3'), designed to amplify a 738-bp fragment of the nucleocapsid protein (N) gene. Total RNAs obtained from plants infected with a Serbian isolate of TSWV (GenBank Accession No. GQ373173) and healthy onion garlic plants were used as positive and negative controls, respectively. An amplicon of the expected size was produced from the 16 onion and 7 garlic ELISA-positive plants, but not from healthy controls. The amplified products derived from the two selected isolates, 114-11 from onion and 115-11 from garlic, were sequenced directly after purification with the QIAquick PCR Purification kit (Qiagen); the sequences obtained were allocated GenBank Accession Nos. JQ619234 and JQ619235, respectively. Sequence analysis of the partial N gene, conducted with MEGA5 software (4), revealed 99.9% nucleotide identity (100% amino acid identity) between the two Serbian Allium isolates. Serbian onion and garlic isolates showed the highest nucleotide identities of 100% and 99.9% with Serbian summer squash isolate (JF303081) and tobacco isolate from Montenegro (GU369729), respectively. Well-established in many European countries, TSWV has been reported as an important constraint to the production of tomato, pepper, tobacco, and ornamentals (2), but the information on TSWV naturally infecting Allium spp. is limited. The presence of TSWV on onion and garlic in Serbia revealed that its known host range has expanded in Europe. To our knowledge, other than Marchoux's unpublished data (3), there are no other reports of garlic as a natural host of TSWV. The TSWV presence on Allium spp. represents a serious threat for these crops in Serbia, considering that it is prevalent in other crops in the area and its vectors are widespread.

Carboxy‐terminal sequence variation of LMP1 gene in Epstein–Barr‐virus‐associated mononucleosis and tumors from Serbian patients

Seven strains of Epstein-Barr virus (EBV) are defined based on C-terminal sequence variations of the latent membrane protein 1 (LMP1). Some strains, especially those with a 30-bp deletion, are thought to be related to tumorigenic activity and geographical localization. The aims of the study were to determine the prevalence of different LMP1 strains and to investigate sequence variation in the C-terminal region of LMP1 in Serbian isolates. This study included 53 EBV-DNA-positive plasma and tissue block samples from patients with mononucleosis syndrome, renal transplantation, and tumors, mostly nasopharyngeal carcinoma. The sequence of the 506-bp fragment of LMP1 C terminus was used for phylogenetic analyses and identification of LMP1 strains, deletions, and mutations. The majority of isolates were non-deleted (66%), and the rest had 30-bp, rare 69-bp, or yet unknown 27-bp deletions, which were not related to malignant or non-malignant isolate origin. However, the majority of 69-bp deletion isolates were derived from patients with nasopharyngeal carcinoma. Less than five 33-bp repeats were found in the majority of non-deleted isolates (68.6%), whereas most 69-bp deletion isolates (75%) had five or six repeats. Serbian isolates were assigned to four LMP1 strains: B95-8 (32.1%), China 1 (24.5%), North Carolina (NC; 18.9%), and Mediterranean (Med; 24.5%). In NC isolates, three new mutations unique for this strain were identified. EBV EBNA2 genotypes 1 and 2 were both found, with dominance of genotype 1 (90.7%). This study demonstrated noticeable geographical-associated characteristics in the LMP1 C terminus of investigated isolates.

First Report of Zucchini yellow mosaic virus in Watermelon in Serbia.

During a survey of cucurbit viruses in the Gornji Tavankut locality (North Backa District), Serbia in June 2011, field-grown (a surface of 1.8 ha) watermelon plants (Citrullus lanatus [Thunb.] Matsum and Nakai) with mild mosaic symptoms were observed. Large numbers of Aphis gossypii were colonizing the crop. A total of 26 samples, six from plants exhibiting mosaic and 20 from asymptomatic plants, were analyzed by double-antibody sandwich-ELISA using polyclonal antisera virus (Bioreba AG, Reinach, Switzerland) against three cucurbit-infecting viruses known to infect Cucurbita pepo in Serbia: Zucchini yellow mosaic virus (ZYMV), Cucumber mosaic virus, and Watermelon mosaic virus (3). Commercial positive and negative controls were included in ELISA analysis. Only six symptomatic samples tested positive for ZYMV, but no other tested viruses were found. The virus was mechanically transmitted from a representative ELISA-positive watermelon sample (550-11) to five plants of C. pepo 'Ezra F1' and severe mosaic was noticed 10 days after inoculation. For further confirmation of ZYMV infection, total RNA from a naturally infected watermelon plant and symptomatic C. pepo 'Ezra F1' plants were extracted with the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany). Reverse transcription (RT)-PCR was performed with the One-Step RT-PCR Kit (Qiagen) using primer pair ZY-2 and ZY-3 (2). Total RNA obtained from a Serbian isolate of ZYMV from pumpkin (GenBank Accession No. HM072432) and healthy watermelon plants were used as positive and negative controls, respectively. The expected sizes of the RT-PCR products (1,186 bp) were amplified from naturally and mechanically infected symptomatic samples, but not from healthy tissues. The amplified product that derived from isolate 550-11 was purified (QIAquick PCR Purification Kit, Qiagen), sequenced in both directions, deposited in GenBank (Accession No. JN561294), and subjected to sequence analysis using MEGA4 software. Sequence comparisons revealed a high nucleotide identity of 99.9 to 99.8% and 100 to 99.6% amino acid identity for the CP gene with Serbian ZYMV isolates from C. pepo (Accession Nos. JF308188, HM072431, and HM072432). The nucleotide and deduced amino acid sequences of the entire CP gene (837 nt) of the Serbian ZYMV isolate from watermelon shared 99.9 to 93.7% and 100 to 96.8% identity, respectively, with innumerous isolates of ZYMV deposited in the GenBank (e.g., Accession Nos. AJ420012-17 and FJ705262). To our knowledge, this is the first report of ZYMV spreading its host range to watermelon in Serbia. ZYMV infection has been responsible for severe epidemics on cucurbits throughout the world (1). The presence of ZYMV on watermelon could therefore represent a serious threat for this valuable crop in Serbia, especially considering that it is prevalent in other cucurbit crops in the country and the vectors are widespread.

Complete genome sequences and phylogenetic analysis of Hepatitis B virus isolates from Serbia

Although the genotype and subgenotype distribution of HBV isolates from Serbia has previously been reported, data about whole genome sequences from this area are scarce. This study included plasma samples from 5 chronically infected patients. Full genome amplification of the HBV isolates was performed by nested-PCR using 7 primer pairs, and the whole genome nucleotide sequences were obtained by direct sequencing. Two complete genome sequences belonged to D2 subgenotype (ayw3 HBsAg subtype), one to D1 (ayw2) and two to A2 (adw2). All 5 Serbian isolates clustered with sequences from the expected geographic regions and had nucleotide and coded protein length in accordance to their assigned genotypes, except for one HBeAg-negative isolate displaying G1896A mutation leading to a premature stop codon in the Pre-C region. The first complete genome sequences of HBV D1, D2 and A2 subgenotypes from Serbian patients showed characteristics similar to the nucleotide sequences of HBV isolates from other European and Middle East countries.

Mycelial compatibility of Hungarian Macrophomina phaseolina isolates

The charcoal root disease caused by Macrophomina phaseolina (Tassi) Goidanich leads to considerable damage in hot, dry seasons in many parts of the world, including Hungary. The present study investigated the mycelial compatibility of 53 Macrophomina phaseolina isolates, collected from sunflower, maize and soybean in different regions of Hungary, in order to characterize the diversity of the pathogen. Compatible isolates were identified by growing the isolates in direct contact with each other on potato-dextrose agar medium under laboratory conditions. Most isolates were compatible. Only 24 pairs of all the possible paired combinations showed incompatible relationships. Even geographically distant isolates were found to be compatible. Some Serbian isolates were compatible with all the Hungarian isolates and one Spanish isolate tested in this work. The latter exhibited incompatibility only with a single Hungarian isolate. These results suggest that the same or very similar genotypes may spread over long distances, probably through the transportation of seeds or crops contaminated with microsclerotia. This is the first report on the compatibility of Macrophomina phaseolina isolates in Hungary.

Genetic diversity and effect of temperature and pH on the growth of Macrophomina phaseolina isolates from sunflower fields in Hungary

The effects of temperature and pH on the growth of 45 Hungarian Macrophomina phaseolina isolates from different locations and hosts were compared on the basis of their genetic diversity. One Spanish and two Serbian isolates were also included in the experiment. The most favourable temperature regimes for the development of the isolates ranged between 25 and 35 °C. The optimal pH for the pathogen varied between 4.0 and 6.0, but growth was observed on potato dextrose agar even at pH values of 3.0, 7.0 and 8.0. RAPD analysis with 13 different primer pairs generated 148 unambiguous bands. RFLP analysis involving 8 different restriction endonucleases was performed on a 1550 bp fragment of the rDNA region containing internal transcribed spacers (ITS1, ITS2), the 5.8S rDNA and part of the 25S rDNA. The greatest genetic distance values were obtained for three isolates, two from Hungary and one from Spain, which had similar values, but were quite distinct from all the others. A strong positive correlation was observed between the genetic distances and the growth parameters measured at various temperatures, and between the geographical data and the growth data sets at different pH values, but the correlation was less strong in the latter case. While Hungarian M. phaseolina populations are thought to reproduce clonally, the present results indicate the coexistence of different haplotypes in this area, and besides the geographical dominance of a given haplotype it was found that a closer genetic relationship might exist between spatially distinct haplotypes.

Molecular characterization and phylogenetic analysis of full-genome HBV subgenotype D3 sequences from Serbia

Hepatitis B virus (HBV) is classified into 8 genotypes with distinct geographical distribution. Genotype D (HBV/D) has the widest distribution area and is comprised of 7 subgenotypes. Subgenotypes D1, D2 and D3 appear worldwide, while D4–D7 have a more restricted distribution. Within the Mediterranean area, HBV/D and subgenotype D3 are the most prevalent. The purpose of this study was to characterize the full genome of Serbian HBV/D3 isolates by comparison and phylogenetic analysis with HBV/D3 sequences (66 samples) found in GeneBank/DDBJ databases from different parts of the world. Isolates were obtained from three patients diagnosed with chronic hepatitis B (HBsAg+). All three isolates have two very rare nucleotide substitutions, A929T and T150A, which indicate the same ancestor. Phylogenetic analysis of HBV/D3 genome sequences throughout the world follows an ethno-geographical origin of isolates with rare exceptions, which could be explained by human travelling and migration. The geographically close but ethnically different Serbian and Italian isolates clustered in the same subnode, and on a common branch with strains from Northern Canada. To test the apparently close HBV phylogenetic relationship between completely separated patients from Serbia and Northern Canada we analyzed in depth a 440bp region of the HBsAg from Canadian (n=73) and Serbian (n=70) isolates. The constructed parsimony tree revealed that strains from Serbia and Northern Canada fell along the same branch which indicates independent evolution within regions of each country. Considering that HBsAg sequence has limited variability for phylogenetic analyses, our hypothesis needs further confirmation with more HBV complete genome sequences.

First report of Trichinella britovi in Serbia

AbstractIn Europe, Serbia ranks among countries with a high prevalence of Trichinella infection in pigs, which continues to be a serious human health problem. While in some Balkan countries, more than one Trichinella species/genotype has been described in both the sylvatic and domestic cycles, these data are lacking for Serbia. To date, only a few Serbian isolates of Trichinella have been genetically specified, and all were classified as T. spiralis. Although transmission of Trichinella from domestic pigs to wildlife could be assumed, neither the infection status nor the species of Trichinella circulating among wildlife in Serbia has been investigated. This study shows the presence of two Trichinella species, T. spiralis and T. britovi, in wild animals originating from five districts in Serbia, where Trichinella infections in domestic pigs and humans have been recorded. Trichinella spiralis was detected in jackals (n = 3), red foxes (n = 2) and a wild cat (n = 1). We also established that wolves (n = 4) and red foxes (n = 2) serve as sylvatic reservoirs for T. britovi. This is the first report on the presence of T. britovi in Serbia.

Phytophthora ramorum Occurrence in Ornamentals in Serbia.

In a survey to determine the presence of Phytophthora ramorum in Serbia, ornamentals from garden centers, nurseries, and private and public gardens, as well as imported plant material, were inspected. In total, 577 plant, soil, and potting media samples were tested using various detection methods: lateral flow diagnostic test, enzyme-linked immunosorbent assay, conventional polymerase chain reaction, and isolation, followed by identification based on growth characteristics in culture and morphological features. P. ramorum was not detected in any of the 162 soil or potting media tested by the baiting method. P. ramorum was detected in 12 Rhododendron samples from one private garden in Zemun (City of Belgrade District) exhibiting symptoms of leaf necrosis and blight and petiole necrosis, and in three samples of Pieris spp. from one garden center exhibiting symptoms of leaf necrosis. Eight Phytophthora isolates were obtained from the positive Rhododendron plants and three isolates from Pieris plants, and all were identified as P. ramorum on the basis of their uniform morphological and growth characteristics. P. ramorum conformation was also made by sequencing of the internal transcribed spacer regions for a single isolate taken from one infected rhododendron and one pieris plant. Serbian isolates were determined as A1 mating type, due to formation of a few typical sexual structures when crossed with the A2 mating type of P. cinnamomi and P. cryptogea. Pathogenicity test on nonwounded detached leaves of 19 popular ornamentals, as well as the most frequently imported ones, revealed that 10 host species were susceptible, including Robinia pseudoacacia, which is widely distributed in Serbia. During this study, Cotoneaster horizontalis and C. dammeri were determined to be new experimental hosts of P. ramorum. This article provides evidence of P. ramorum introduction into Serbia. Although P. ramorum has not been detected in Serbian production nurseries, its presence outdoors might cause severe damages on susceptible common urban plants in public green and natural ecosystems.

Bordetella pertussis vaccine strains and circulating isolates in Serbia

In Serbia, whole cell pertussis vaccine was introduced in 1957. Current composition of the vaccine has been used since 1985 and contains four autochthonous strains of Bordetella pertussis isolated from 1957 to 1984. To monitor changes in bacterial population, 70 isolates collected from 1953 to 2000 were studied together with the vaccine strains. The methods included serotyping of fimbriae (Fim), genotyping of pertactin ( prn) and pertussis toxin S1 subunit ( ptxA), and pulsed-field gel electrophoresis analysis. Shift from ptxA2 to ptxA1 has been observed in isolates since the late of 1960s. All isolates from 1980 to 1984 harbored ptxA1. Re-appearance of the ptxA2 allele followed an addition of the two strains harboring ptxA1 in the vaccine in 1985. The allele prn1 was predominant among the Serbian isolates, though prn3 and prn11 have been detected since 1981 and 1984. The allele prn2 was found only in two strains isolated in 2000. Serotype Fim2.3 disappeared before 1980 and serotype Fim2 became predominant since then. The Serbian vaccine strains showed differences in ptxA and prn. The results of this present study indicate that the B. pertussis population in Serbia is different from other vaccinated populations and that this difference may be related to the vaccine used.

Incidence and Distribution of Iris yellow spot virus on Onion in Serbia.

In a survey to determine the presence and distribution of Iris yellow spot virus (IYSV) in greenhouse ornamentals and onion field crops in 14 districts of Serbia as well as on imported ornamental plants, 1,574 samples were collected and analyzed by double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). IYSV was not detected in nearly 1,200 plant samples collected from 39 genera of ornamentals grown in greenhouses in Serbia or imported from other countries during 2005 to 2007. The virus was detected in samples from an onion seed crop in the Sirig locality (South Bačka District) that showed symptoms resembling those caused by IYSV and in samples without IYSV-like symptoms from an onion bulb crop in the Obrenovac locality (City of Belgrade District). Mechanical transmission of IYSV isolates was difficult, and only the isolate 605-SRB could infect four plant species, but not in all replications. No virus transmission could be demonstrated in 5,000 tested seeds originating from IYSV-infected onion crops. For further confirmation of IYSV, the nucleotide sequence of its nucleocapsid (NC) gene was obtained by reverse transcription-polymerase chain reaction (RT-PCR) in symptomatic onion samples as well as in symptomless leaves of Nicotiana benthamiana. Four previously developed primers were tested to determine their suitability for routine detection of Serbian IYSV isolates. Phylogenetic analysis showed clustering of isolates 605-SRB and 622-SRB from the onion seed crop and isolate 283-SRB from the onion bulb crop into two distant clades. The analysis indicated that Serbian isolates of IYSV do not share a recent common ancestor and that they represent two distinct lineages of IYSV in Serbia. Considering that onion is one of the most important and traditionally grown vegetable crops in Serbia, IYSV represents a potentially devastating pathogen in this country.