Strains In GenBank Research Articles

Complete genome analyses of the first porcine rotavirus group H identified from a South African pig does not provide evidence for recent interspecies transmission events

Rotaviruses (RVs) are classified into eight species/groups (RVA–RVH) according to the migration patterns of their 11 genome segments, as well as by serological and molecular properties of Viral Protein 6 (VP6). In 1997 a new unclassified RV was reported infecting adults in Bangladesh and China. This virus was initially named novel adult diarrhoea rotavirus (ADRV-N), but later renamed as RVH. Since then, RVH has been detected in humans only very sporadically. However, RVH is increasingly being detected in pig populations in the USA, Brazil and Japan, but not yet in Africa. Unfortunately, whole genome sequence data of porcine RVH strains in GenBank is currently restricted to a single strain (SKA-1) from Japan.Porcine diarrhoeic samples were collected in South Africa and analysed for rotavirus using an RVA ELISA and electropherotyping by PAGE. One sample displayed a 4:2:1:1:1:1:1 migration pattern, typical for RVH. In order to further investigate this strain, sequence-independent amplification followed by random sequencing using the 454/Roche GS FLX Sequencer was performed, resulting in the second complete porcine RVH strain (MRC-DPRU1575) available in databases.Phylogenetically, all segments of MRC-DPRU1575 clustered closely with the SKA-1 strain and in some segments with known porcine RVH strains from Brazil and the USA. In contrast, the porcine RVH strains were only distantly related to human RVH strains from Asia and a partial RVH-like strain recently detected in bats from Cameroon.Overall, strain MRC-DPRU1575 is the first complete genome of a porcine RVH from Africa and allows for the development of improved RVH screening methods. Our analyses indicate that RVH strains cluster according to their host species, not suggesting any evidence of recent interspecies transmission events. However, more RVH genomes from a wider host range are needed to better understand their evolutionary pathways and zoonotic potential.

Prokaryotic High-Level Expression System in Producing Adhesin Recombinant Protein E of Nontypeable Haemophilus influenzae.

Background:Adhesion protein E (PE) of Haemophilus influenzae is a 16 - 18 kDa protein with 160 amino acids which causes adhesion to epithelial cells and acts as a major factor in pathogenesis.Objectives:In this study, we performed cloning, expression and purification of PE as a candidate antigen for vaccine design upon further study.Materials and Methods:At first, the pe gene of NTHi ATCC 49766 strain (483 bp) was amplified by PCR. Then, to sequence the resulted amplicon, it was cloned into TA vector (pTZ57R/T). In the next step, the sequenced gene was sub-cloned in pBAD/gIII A vector and transformed into competent Escherichia coli TOP10. For overexpression, the recombinant bacteria were grown in broth medium containing arabinose and the recombinant protein was purified using metal affinity chromatography (Ni-nitrilotriacetic acid) (Ni-NTA agarose). Finally, the protein was detected using sodium dodecyl sulfate polyacrylamide gel electrophores (SDS-PAG) and confirmed by western blotting.Results:The cloned gene was confirmed by PCR, restriction digestion and sequencing. The sequenced gene was searched for homology in GenBank and 99% similarity was found to the already deposited genes in GenBank. Then we obtained PE using Ni-NTA agarose with up to 7 mg/mL concentration.Conclusions:The pe gene was successfully cloned and confirmed by sequencing. Finally, PE was obtained with high concentration. Due to high homology and similarity among the pe gene from NTHi ATCC 49766 and other NTHi strains in GenBank, we believe that the protein is a universal antigen to be used as a vaccine design candidate and further studies to evaluate its immunogenicity is underway.

Development of a genome copy specific RT-qPCR assay for divergent strains of type 2 porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome virus (PRRSV) became a significant pathogen of swine upon its emergence in the late 1980s and since then has exemplified a rapidly evolving, constantly re-emerging pathogen. In addition to the challenges faced in development of vaccines and diagnostics, research on the basic molecular pathogenesis of PRRSV is also restrained by the ability to accurately and comparatively quantitate levels of replication in different tissues and between strains. This is further complicated by the presence of non-genomic RNA within infected tissues which are generally detected with equivalent efficiency by RT-qPCR based techniques, thereby introducing inherent error in these measurements that may differ significantly by tissue and strain. To address this, an RT-qPCR based technique was developed which targets the viral RNA-dependent RNA polymerase gene (nsp9) which is unique to genomic RNA, being absent from all subgenomic and heteroclite RNAs. This assay targets a region of considerable sequence conservation, and based on sequence only, should be quantitative for approximately 40% of all Type 2 PRRSV strains in GenBank for which nsp9 sequence is available. The assay was demonstrated to be linear over nine orders of magnitude (1010–102 copies) and can be readily adapted for multiplex detection of additional divergent PRRSV strains. This assay will add significantly to the ability to assess and compare PRRSV replication in a variety of tissues and between divergent strains, including highly pathogenic strains of considerable concern to the global pork industry.

Molecular Identification 0f 16s Ribosomal RNA Gene of Helicobacter pylori Isolated from Gastric Biopsies in Sudan

H. pylori are a ubiquitous microorganism infecting up to half of the world’s population. A total of 81 gastric biopsies taken from patients complaining of gastric disorders in Khartoum state, Sudan screened for H.pylori. Eighteen samples (22.2%) yielded positive culture results. The majority of them were males. Also results indicated higher prevalence of H. pylori in patients with gastritis. Further identification performed using PCR targeted a region of 16S ribosomal RNA gene of H. pylori and gene amplified on 12 samples. Six of isolated sequences subjected to BLAST analysis that showed high similarity to GenBank strains of H. pylori.Multiple sequence alignments were performed between isolated 16S rRNA gene sequences and most related H.pylori strains deposited on GenBank. One isolate differed on one base-pair substitution (G-A) from other isolates and selected reference H.pylori strains. Phylogenetic analysis based on 16S rRNA gene sequences reflects that H.pylori could be originated from Africa.

Cytospora species associated with walnut canker disease in China, with description of a new species C. gigalocus

Cytospora species associated with canker disease are presently difficult to identify because of lack of ex-types cultures with molecular data, few distinguishable characters, and only Internal transcribed spacer (ITS) sequence data is available for most Cytospora strains in GenBank. We report on Cytospora species from the walnut tree in China. Collections were subjected to morphological and phylogenetic study. The relatedness of species associated with walnut canker were established using combined ITS, nrLSU, β-tubulin, and actin gene sequence data. Cytospora atrocirrhata, Cytospora chrysosperma, Cytospora sacculus, and a new species, Cytospora gigalocus, were identified causing canker disease of walnut. Cytospora gigalocus is formally described and compared with most similar species. Cytospora chrysosperma and C. sacculus have previously been recorded from walnut, whereas C. atrocirrhata is reported as associated with walnut canker for the first time. This is the first study that has established the Cytospora species causing walnut canker in China using a multi-phasic approach. All species are recorded as being associated with walnut canker disease in China for the first time.

Porcine coronavirus HKU15 detected in 9 US states, 2014.

Porcine coronavirus HKU15 detected in 9 US states, 2014.

Molecular characterization and phylogenetic analysis of the causative agent of hemoplasma infection in small Indian Mongoose (Herpestes Javanicus).

Hemoplasmas are the trivial name for a group of erythrocyte-parasitizing bacteria of the genus Mycoplasma. This study is the first report of hemoplasma infection in Small Indian Mongoose (Herpestes Javanicus) based on molecular analysis of 16S rDNA. Whole blood samples were collected by sterile methods, from 14 live captured mongooses, in the south of Iran. Candidatus Mycoplasma turicensis (CMt)-like hemoplasma was detected in blood samples from one animal tested. BLAST search and phylogenetic analysis of partial 16S rDNA sequence (933bp) of the hemoplasma from Small Indian mongoose (KJ530704) revealed only 96-97% identity to the previously described CMt followed by 95% and 91% similarity with Mycoplasma coccoides and Mycoplasma haemomuris, respectively. Accordingly, the Iranian mongoose CMt isolate showed a high intra-specific genetic variation compared to all previously reported CMt strains in GenBank. Further molecular studies using multiple phylogenetic markers are required to characterize the exact species of Mongoose-derived hemoplasma.

Bioinformatics Analysis on Homology of CagM Protein in <i>Helicobacter pylori </i>Cag Pathogenicity Island

[Objective]This paper aims to make a bioinformatics analysis on the homology ofcagMnucleotide sequence and protein sequence inHelicobacter pyloricag pathogenicity island, calculate the protein conservatism and evolutionary process, and research the functions of the protein. [Methods]Use Clustal W software to make multiple sequence analysis, edit the results with GeneDOC software, and detect the variable sites and homology.[Results] The homology betweencagMgene and the nucleotide sequence of other HP bacterial strains in GenBank is 96%~98%, the homology betweencagMgene andcagMgene is 98%~99%, so the nucleotide sequence and protein sequence have high conservatism.[Conclusion]CagM gene is very conservative and it plays an important role in the process ofH. Pylorievolution; CagM gene is very conservative and it has important functions in helicobacter pylori; as an important means of bioinformatics, homology not only can calculate the protein conservatism and evolutionary process, but also can find the similar functions of the protein.

16S rRNA Gene-based Identification and Antibiotic Resistance Profiling ofAcinetobacter baumanniifrom River Water Isolate, Dehradun, India

Acinetobacter baumannii represents the most clinically relevant opportunistic pathogen. This study describes to 16S rDNA sequence-based identification of Acinetobacter spp. from river water. The strain showed 99% identity with that of several A. baumannii reference strains in GenBank. The phylogenetic analysis indicated that A. baumannii W29 strain was related to strains within the A. baumannii, and yet have a distinct line of descent, representing a novel species. The study revealed multi drug resistance in the species to cephalosporins, carbapenems and other classes of antibiotics (Pencillins,p-Lactam/3-Lactamase inhibitor combinations, aminoglycosides). The occurrence of A. baumannii in river water sources has serious epidemiological implications and a potential human health risk.

Molecular and morphological criteria for revision of the genus Microcoleus (Oscillatoriales, Cyanobacteria).

Ninety-two strains of Microcoleus vaginatus (=nomenclatural-type species of the genus Microcoleus Desmazières ex Gomont) and Phormidium autumnale Trevisan ex Gomont from a wide diversity of regions and biotopes were examined using a combination of morphological and molecular methods. Phylogenies based on the 16S rDNA and 16S-23S ITS (partial) demonstrated that the 92 strains, together with a number of strains in GenBank, were members of a highly supported monophyletic clade of strains (Bayesian posterior probability = 1.0) distant from the species-cluster containing the generitype of Phormidium. Similarity of the 16S rRNA gene exceeded 95.5% among all members of the Microcoleus clade, but was less than 95% between any Microcoleus strains and species outside of the clade (e.g., Phormidium sensu stricto). These findings, which are in agreement with earlier studies on these taxa, necessitate the revision of Microcoleus to include P.autumnale. Furthermore, the cluster of Phormidium species in the P.autumnale group (known as Group VII) must be moved into Microcoleus as well, and these nomenclatural transfers are included in this study. The main diacritical characters defining Microcoleus are related to the cytomorphology of trichomes, including: narrowed trichome ends, calyptra, cells shorter than wide up to more or less isodiametric, and facultative presence of sheaths. The majority of species are 4-10μm in diameter. The possession of multiple trichomes in a common sheath is present facultatively in many but not all species.

Immunodetection of Helicobacter pylori-specific proteins in oral and gastric Candida yeasts.

Human gastric epithelium and immunocytes have been recognized as the sole specialized eukaryotic cells that host Helicobacter pylori (H. pylori). The aim of this study was to provide further evidence for our previous proposal regarding the occurrence of H.pylori inside the yeast vacuole, verifying the viability of the intravacuolar H.pylori by western blotting. Light microscopy and polymerase chain reaction (PCR) were used for primary detection of nonculturable H.pylori in 11 Candida yeasts (six oral and five gastric). Boiling was used for extraction of proteins from yeasts and the control H.pylori. Western blot analysis was recruited to assess the occurrence of H.pylori-specific proteins in protein pool of yeasts, using IgY-Hp raised in hens and IgG1-Hp raised in mice. The fast-moving bacterium-like bodies (BLBs) were identified as H.pylori by amplification of H.pylori 16S rRNA, ureAB, vacA s1, and ahpC genes from the whole DNA of yeasts. Analysis of the sequenced products of 16S rRNA gene amplified from the yeast and H.pylori isolates of patient #2 showed 100 % homology with the corresponding sequences of the reference H. pylori strains in GenBank. According to published data, it was plausible to assign the H.pylori-specific proteins, detected by western blot analysis, as thiol peroxidase (21 kDa), peroxiredoxin (AhpC) (26 kDa), urease-A subunit (UreA) (32 kDa), vacuolating cytotoxin A (VacA) small subunit (36 kDa), and VacA large subunit (56 kDa). Results of this study show that inside yeast, H.pylori expresses proteins and is viable. These proteins appear to serve as powerful tools to help H.pylori to establish in the vacuole of yeast where it can reach nutrients and multiply. The intimate relationship between H.pylori and Candida yeast which began long time ago, could have led to the establishment of H.pylori inside the yeast vacuole before invading human cells.

Nested PCR Detection and Phylogenetic Analysis of <i>Babesia bovis</i> and <i>Babesia bigemina</i> in Cattle from Peri-Urban Localities in Gauteng Province, South Africa

ABSTRACTBabesia bovis and Babesia bigemina are tick-borne hemoparasites causing babesiosis in cattle worldwide. This study was aimed at providing information about the occurrence and geographical distribution of B. bovis and B. bigemina species in cattle from Gauteng province, South Africa. A total of 268 blood samples collected from apparently healthy animals in 14 different peri-urban localities were tested using previously established nested PCR assays for the detection of B. bovis and B. bigemina species-specific genes encoding rhoptry-associated protein 1 (RAP-1) and SpeI-AvaI restriction fragment, respectively. Nested PCR assays revealed that the overall prevalence was 35.5% (95% confidence interval [CI]=± 5.73) and 76.1% (95% CI=± 5.11) for B. bovis and B. bigemina, respectively. PCR results were corroborated by sequencing amplicons of randomly selected samples. The neighbor-joining trees were constructed to study the phylogenetic relationship between B. bovis and B. bigemina sequences of randomly selected isolates. Analysis of phylogram inferred with B. bovis RAP-1 sequences indicated a close relationship between our isolates and GenBank strains. On the other hand, a tree constructed with B. bigemina gp45 sequences revealed a high degree of polymorphism among the B. bigemina isolates investigated in this study. Taken together, the results presented in this work indicate the high incidence of Babesia parasites in cattle from previously uncharacterised peri-urban areas of the Gauteng province. These findings suggest that effective preventative and control measures are essential to curtail the spread of Babesia infections among cattle populations in Gauteng.

Cloning and enhancing production of a detergent- and organic-solvent-resistant nattokinase from Bacillus subtilis VTCC-DVN-12-01 by using an eight-protease-gene-deficient Bacillus subtilis WB800

BackgroundNattokinases/Subtilisins (EC 3.4.21.62) belong to the second large family of serine proteases, which gain significant attention and play important role in many biotechnology processes. Thus, a number of nattokinases/subtilisins from various Bacillus species, especially from B. subtilis strains, extensively have been investigated to understand their biochemical and physical properties as well as to improve the production for industrial application. The purpose of this study was to clone a nattokinase gene from Bacillus subtilis strain VTCC-DVN-12-01, enhance its production in B. subtilis WB800, which is deficient in eight extracellular proteases and characterize its physicochemical properties for potential application in organic synthesis and detergent production.ResultsA gene coding for the nattokinase (Nk) from B. subtilis strain VTCC-DVN-12-01 consisted of an ORF of 1146 nucleotides, encoding a pre-pro-protein enzyme (30-aa pre-signal peptide, 76-aa pro-peptide and 275-aa mature protein with a predicted molecular mass of 27.7 kDa and pI 6.6). The nattokinase showed 98-99% identity with other nattokinases/subtilisins from B. subtilis strains in GenBank. Nk was expressed in B. subtilis WB800 under the control of acoA promoter at a high level of 600 mg protein per liter culture medium which is highest yield of proteins expressed in any extracellular-protease-deficient B. subtilis system till date. Nk was purified to homogeneity with 3.25 fold purification, a specific activity of 12.7 U/mg, and a recovery of 54.17%. The purified Nk was identified by MALDI-TOF mass spectrometry through three peptides, which showed 100% identity to corresponding peptides of the B. subtilis nattokinase (CAC41625). An optimal activity for Nk was observed at 65°C and pH 9. The nattokinase was stable at temperature up to 50°C and in pH range of 5–11 and retained more than 85% of its initial activity after incubation for 1 h. Mg2+ activated Nk up to 162% of its activity. The addition of Triton X-100, Tween 20, and Tween 80 showed an activation of Nk up to 141% of its initial activity but SDS strongly inhibited. The enzyme was highly resistant to organic solvents.ConclusionsOur findings demonstrated that an eight-protease-gene-deficient Bacillus subtilis WB800 could overproduce the nattokinase from B. subtilis VTCC-DVN-12-01. Due to high resistance to detergents and organic solvents of this nattokinase, it could be potentially applied in organic synthesis and detergent production.

Mortality and pathology of hybrid catfish, Clarias macrocephalus (Günther) × Clarias gariepinus (Burchell), associated with Edwardsiella ictaluri infection in southern Thailand

Enteric septicaemia of catfish (ESC) caused by Edwardsiella ictaluri is becoming an increasing problem in aquaculture and has been reported worldwide in a variety of fish species. This study reports ESC in hybrid catfish, Clarias macrocephalus (Günther)×Clarias gariepinus (Burchell), cultured in southern Thailand. The bacteria were identified as E.ictaluri by conventional and rapid identification systems, as well as by genetic and phylogenetic characterization. Analysis of 16S rRNA indicated 100% homology to the 16S rRNA sequence of several E.ictaluri strains in GenBank. Plasmid profiles demonstrated 4.0- and 5.6-kb plasmids, compared with the 4.8- and 5.6-kb plasmids in the US isolates, and representative genes of three of the four known pathogenicity islands of US isolates were present. Serologically, lipopolysaccharide (LPS) purified from the Thai isolates was not recognized by a monoclonal antibody against the LPS of US isolates. Fish experimentally infected with E.ictaluri showed 23-100% mortality within 14days with a 168-h LD50 of 6.92×10(7) CFUmL(-1) by immersion and a 96-h LD50 of 1.58×10(6) CFUfish(-1) by intraperitoneal injection. Examination of tissue sections obtained from both naturally and experimentally infected fish indicated that infection of hybrid catfish with E.ictaluri produced lesions in several organs including liver, kidney, spleen, heart and brain. Histopathology findings included cellular necrosis, focal haemorrhage, infiltration of lymphocytes and multifocal granulomatous inflammation in the infected organs.

Genetic characterization of VP1 gene of seven Sacbrood virus isolated from three provinces in northern China during the years 2008–2012

The genetic diversity of seven northern China isolated Sacbrood virus strains (SBV) has been analyzed, and hypervariable regions of the VP1 gene of 7 SBV were sequenced and characterized, in order to obtain epidemiological and immunological information, and to suggest typing criteria for SBV. Sequence analysis of hypervariable regions of the VP1 gene in the genome of these isolates revealed a sequence homology of 91.0–99.3% among all seven local SBV isolates from Apis cerana from China, with a similarity of 93.3–100.0% in deduced amino acid sequences. These local isolates shared 87.4–92.8% sequence homology with six SBV reference strains in GenBank (including two SBV reference strains from Apis cerana from China), which represents a 91.8–97.6% similarity in deduced amino acid sequences. Genetic analysis also showed that five SBV strains from Apis cerana from China had a 13-amino-acid deletion at amino acid positions 287–299, and two SBV strains infecting the Korean honeybee had a 17-amino-acid deletion at amino acid positions 284–300 in comparison with other SBV. Phylogenetic analysis revealed two major groups (AC genotype SBV infecting Apis cerana and AM genotype SBV infecting Apis mellifera). The AC genotype could be further divided into subgroups. Based on the results of phylogenetic analysis, a similarity scan of SBV nucleotide sequences was carried out by using Simplot software and results in similar results. Our results suggest possible typing criteria for SBV based on the phylogenetic tree and sequence homology, and also that the virus has host specificity and regional variations.

First Report of Candidatus Phytoplasma solani on Blackberry (Rubus fruticosus) in Bulgaria.

While performing a routine field survey on 2-year-old canes of Rubus fruticosus (cv. Evergreen Thornless) in the region of Plovdiv (central southern Bulgaria), severe stunting of single or grouped plants (3 to 4 in a row) was found in late August of 2009. It was noteworthy that the leaves of these plants were curved upwards and stayed green until the end of the season. The bushy aspect of the diseased plants led to the assumption of a phytoplasma origin; therefore, specific PCR and sequence based identification methods were applied on leaves, petioles, and stems from three infected Rubus plants grown in different rows of the field (midsummer, nine samples in total) and the same number of asymptomatic samples. Partial amplification of the 16S ribosomal RNA gene with generic phytoplasma primer pairs P1/P7 and fu5/ru3 (3), followed by a nested PCR specific for all members of the Phytoplasma stolbur subgroup by means of the stol11 primers (1), and an RFLP analysis of the tuf gene (elongation factor Tu) fragment produced with PCR primers tufAY/r tufAY (3), were used for the identification and characterization of the pathogen. All target amplicons were also sequenced by Macrogen (Seoul, South Korea) following gel purification (Nucleospoin Plant II, Macher-Nagel). Identical sequences were obtained from each of the P1/P7-derived amplicons (100% homology between samples) and a consensus 1,142 bp sequence was delineated and submitted to NCBI GenBank with accession no. JF293091. It had the highest similarity (99 to 100%) to sequences of 'Bois noir' phytoplasma (e.g. HQ589193; Candidatus Phytoplasma solani, position 29 to 1,171). The fu5/ru3 amplicons produced sequences that showed 99.5% homology to the Ca. Phytoplasma solani strains of a southern Russian and Romanian phytoplasma survey on different hosts (potato, tomato, Convolvulus) (GenBank Accession No. HM449999 to HM4450002). The stolbur specific primers also produced an amplicon in all samples and again the consensus sequence was identified (100% homology between the samples) and deposited in GenBank (JN561701). RFLP analysis of the tuf gene with the enzymes HindIII, HinfI, HpaII, and TaqI (Fermentas) produced the same profile types for the different samples and clearly allocated the phytoplasma in the tuf type-b (VKII), according to (2). This type is commonly reported as associated with bindweed (Convolvulus arvensis). Additionally, the sequenced tufAY fragment also confirmed a 100% correspondence with the submitted Tu elongation factor fragments of Ca. Phytoplasma solani strains in GenBank. No phytoplasma was detected in symptomless blackberry plants that were sampled from the same plot. In the molecular identification tests, a stolbur phytoplasma control (potato isolate), a Rubus stunt (EY subgroup, 16SrV) and an apple proliferation phytoplasma (AP subgroup, 16SrX) were used as controls. Based on the symptoms and the laboratory results, we concluded that the Rubus plants were infected by Ca. Phytoplasma solani, a species belonging to the stolbur subgroup (16SrXII-A). To our knowledge, this is the first report of Ca. Phytoplasma solani on Rubus fruticosus in Bulgaria. The disease is not likely to be an isolated case in the future because of the pathogen's spread on other hosts and the expected increase in blackberry fields.

First Report of Sclerotinia Stem Blight Caused by Sclerotinia trifoliorum on Trifolium ambiguum in Poland.

Kura clover (Trifolium ambiguum M. Bieb.), a rhizomatous, persistent legume native to the Caucasus region, has received recent attention in North America and New Zealand as a pasture and silage crop. It is reported to be resistant to most pathogens affecting other clovers, including Sclerotinia trifoliorum Eriks. (3,4), one of the most destructive pathogens of clovers in northern Europe. Kura clover (cv. KTA202) was established in May 2009 near Mochełek, Poland (53° 13' N, 17° 51' E) on a Luvisol soil. By May 2011, 70% of plants grown in an experimental field (350 m2) had died, and 20% of the remaining plants were yellow and wilted. At crowns and the lower stem regions, wet, brown lesions with delicate white mycelium were observed. Lesion development was followed by death of the entire plant in a few days. By early June, only a few asymptomatic plants per square meter remained in the field. Tissue fragments of 20 symptomatic plants were surface-sterilized with 1% NaOCl for 1 min and plated on potato dextrose agar (PDA). A fungus with morphological characteristics of S. trifoliorum was consistently isolated. DNA isolation from sclerotia was performed with the DNeasy Plant Mini Kit (Qiagen, USA). Amplification and sequencing of the ITS region of rDNA was performed with primers ITS1/ITS4. NCBI Blast analysis of the 542-bp segment showed a 99% homology with most of S. trifoliorum and S. sclerotiorum strains in GenBank (e.g., AY547267.1 and EU082466.1). Sequence of isolate St0211TA was deposited in GenBank (Accession No. JQ743329). To determine growth rate of hyphae, morphology, and dimensions of sclerotia, colonies were grown in three replications on PDA at 20 ± 1°C in the dark. S. trifoliorum (CBS 122377) and S. sclerotiorum from our local collection were used as controls. Mean growth rate of S. trifoliorum isolates (20.5 mm/day) was slower compared to S. sclerotiorum (32.3 mm/day). Sclerotia began to form on delicate and smooth mycelium of S. trifoliorum on the entire surface of the plate in 7 to 8 days. Sclerotial size on day 28 was 2.0 to 9.0 × 2.0 to 7.0 mm (average 4.2 × 3.6 mm). Ultimately, the identification of S. trifoliorum was confirmed on the basis of ascospore morphology. Apothecia grew from sclerotia in wet sand at 12°C after 12 weeks. Asci contained dimorphic ascospores: four larger 13.0 to 16.0 × 6.0 to 9.0 mm (average 14.1 × 7.4 μm) and four smaller 10.0 to 12.0 × 5.4 to 6.0 mm (average 10.6 × 6.0 μm), typical for this species (1). Isolate St0511TA, which most intensively produced apothecia, was deposited in CBS (No. 133234). Koch's postulates were fulfilled by pathogenicity tests carried out on 2-week-old T. ambiguum seedlings grown in pots (6 × 30 plants), sprayed with a mycelial fragment suspension, and incubated at 15°C (2). Brown, wet spots with delicate white mycelium were observed on cotyledons after 3 days. After 5 days, approximately 10% of cotyledons were killed and mycelium appeared on stems and leaves, and after 10 days, 73% of seedlings were dead. S. trifoliorum was reisolated from all symptomatic tissues. To our knowledge, this is the first report of S. trifoliorum stem blight on T. ambiguum in the field.

Severe Outbreak of Bacterial Blight Caused by Xanthomonas arboricola pv. corylina on Hazelnut cv. Tonda di Giffoni in Central Italy.

Hazelnut (Corylus avellana L.) is one of the most economically important tree crops in Italy. Xanthomonas arboricola pv. corylina (Xac) causes bacterial blight of hazelnut (4). During early summer 2010, a survey of three orchards (5 ha total) containing 4-year-old hazelnut trees (cv. Tonda di Giffoni) in Viterbo Province, Latium region, Italy, showed an 80 to 100% incidence of bacterial blight. Initially, water-soaked, necrotic spots were visible on leaves, fruit involucres, and shells, followed by lateral shoot dieback and development of cankers as longitudinal bark cracks on twigs, branches, and main trunks. Brown necrosis of the cambium was observed when bark tissue was removed. By late summer, necrosis had extended down main branches to the trunk, causing complete girdling of branches. Symptomatic tissues were collected from leaves, branches, and trunks, sections were surface-sterilized in 1% NaOCl for 1 min followed by two rinses in sterile distilled water (SDW, each for 1 min), and each section was then crushed in SDW. A loopful of the suspension was streaked onto yeast extract-dextrose-calcium carbonate agar medium (YDCA). Thirty six (12 from each type of tissue) yellow-mucoid, shiny, round bacterial colonies, each approximately 2 mm in diameter, were subcultured on YDCA. All strains were gram-negative and aerobic; negative for indole, lecithinase, urease, tyrosinase, and nitrate reduction; and positive for catalase, growth in 2% NaCl in nutrient broth, and growth at 35°C. All strains produced dark green pigment on succinate-quinate (SQ) medium. Inoculum of each of 15 isolates was prepared in nutrient broth, and washed cells from late log-phase cultures used to prepare a bacterial suspension of each isolate for inoculation of 2-year-old potted hazelnut plants cv. Tonda di Giffoni. A suspension of 106 CFU/ml for each isolate was sprayed onto leaves (10 ml/plant), and drops of inoculum were placed on wounds made on twigs with a sterile scalpel (0.10 μl/wound). For each isolate, three plants were inoculated per inoculation method. Inoculations with two reference strains of Xac (Xaco 1 from central Italy (3) and NCPPB 2896 from England) and SDW were performed on the same number of plants for positive and negative control treatments, respectively. Inoculated plants were maintained at 26 ± 1°C in a greenhouse. After 21 days, all inoculated plants had developed symptoms on leaves, while cankers developed on twigs after 40 days. Positive control plants developed the same symptoms, while negative control plants remained asymptomatic. Bacteria recovered from lesions on plants inoculated with the test strains or positive control strains had the same morphological and physiological characteristics as the original strains. No bacteria were recovered from negative control plants. Total DNA was extracted from bacterial suspensions and 16S rDNA amplified using universal primers (2). Sequences (GenBank Accession Nos. JQ861273, JQ861274, and JQ861275 for strains Xaco VT3 to VT5) had 99 to 100% identity with 16S rDNA sequences of Xac strains in GenBank. In Italy, Xac was reported by Petri in 1932 in Latium, and later in other regions on several hazelnut cultivars (1). However, to our knowledge, this is the first report of the disease causing severe damage in Italy.

Molecular characterization and phylogenetic analysis of porcine epidemic diarrhea virus (PEDV) field strains in south China

A total of 127 porcine samples were collected from 48 farms in six provinces in south China. The positive rate of porcine epidemic diarrhea virus (PEDV) was 43.0 % (55/127), and the co-infection rate of PEDV and transmissible gastroenteritis virus (TGEV) was 12.0 % (15/127). The partial S gene and complete M gene were amplified from PEDV-positive strains by RT-PCR, cloned, sequenced and compared with each other, as well as with the reference strains in GenBank. Sequence homology results of the partial S gene and complete M gene showed that all south China field PEDV strains had nucleotide (deduced amino acid) sequence identities of 86.7-98.7 % (83.2-99.3 %) and 96.1-100 % (95.0-100%), respectively, with the foreign reference strains reported in GenBank. Phylogenetic analysis of the partial S gene showed that all the south China PEDV strains and two Thailand strains (08UB01 and 08RB07) belong to the same group and differ genetically from European strains and early domestic strains. Phylogenetic analysis of the complete M gene showed that all south China PEDV strains have a close relationship with most of the strains in Korea and Thailand, but differ genetically from the vaccine strain (CV777).

Identification and characterization of a Bacillus subtilis strain TS06 as bio-control agent of strawberry replant disease (Fusarium and Verticilium wilts)

Replant disease is a major limitation for strawberry production in greenhouse. Bio-control may be a good way to cope with the replant diseases. Here, we report identification and characterization of a bacterial strain TS06 that may be used as a bio-control agent against the replant diseases in strawberry. TS06 was identified as a new strain of Bacillus subtilis based on the homology of its 16S rRNA and gyrA sequences to the reference strains in Genbank. It has a 7 bp difference from the most homologous strain B. subtilis NRRL B-23978. TS06 formed a monophyletic group with species of the B. subtilis complex group in the 16S rDNA sequence analysis, and shared similarity values of 99.9% with the B. subtilis type strains in the sequence analysis of the gyrA genes. TS06 was demonstrated to be a potent inhibitor to Fusarium oxysporum and Verticillium dahliae . There were 89.6 and 84.5% growth inhibition of TS06 on F. oxysporum and V.dahliae respectively in the antagonism test of TS06 strain in vitro . The mycelia growth inhibition rate of TS06 for F. oxysporum and V.dahliae reached 65.96 and 68.45% respectively, in the 40% TS06 culture filtrate treatments. Strain TS06 reduced the average percentage of strawberry diseases ( Fusarium and Verticillium Wilts) by 88.94 and 79.94% respectively in greenhouse. In summary, B. subtilis strain TS06 has a broad antifungal spectrum and synchronously bio-control the two severe replant diseases (Fusarium and Verticilium Wilts), and TS06 can be developed as a biocontrol agent to be applied in strawberry production. Key words : Bacillus subtilis , strain TS06, biological control, strawberry Fusarium wilt , strawberry Verticillium wilt.