Partial Nucleotide Sequences Research Articles

Distribution of a devastating fungal pathogen in mangrove forests of southern Iran

An epidemic of canker and dieback was found on Avicennia marina (Forssk.) Vierth and Rhizophora mucronata Lam. in the mangrove forests located in three different regions (the Qeshm Island, Sirik Port and Khamir Port) of Hormozgan province, on the southern coast of Iran, Persian Gulf. For A. marina, the highest mean of disease incidence was estimated in the Sirik Port (60.6%), followed by the Qeshm Island (50.4%) and Khamir Port (45.2%). Based on a dieback ordinal rating scale from 0 to 5, disease severity had the highest mean in the Sirik Port (3.1), followed by the Qeshm Island (2.7) and Khamir Port (2.5). The species R. mucronata was more affected by the disease than A. marina with the mean disease incidence of 62.5% and the mean disease severity of 3.2. A fungus was consistently recovered from the symptomatic tissues which was identified as Neoscytalidium dimidiatum based on morphological and molecular studies. Phylogenetic analysis of partial nucleotide sequence of the internal transcribed spacer (ITS) region (ITS1–5.8S-ITS2) validated the identity of the putative pathogenic fungus. Koch's postulates were fulfilled using healthy detached branches of both mangrove species inoculated with mycelium plugs of N. dimidiatum. This study is the first report worldwide concerning the occurrence of canker and dieback on A. marina and R. mucronata caused by N. dimidiatum.

Molecular characterization of a unique variant of avian infectious bronchitis virus in Tunisia

Avian infectious bronchitis virus (IBV) is responsible of significant economic losses for poultry industry around the world, through evolution of its pathogenicity, inadequacy of vaccines, and virus evasion. Such evasion is related to the unstable nature of its RNA, in particular the S glycoprotein encoding gene, which raises great challenges with regard to the control of the disease, along with the lack of proof reading mechanisms of the RNA polymerase. The emergence of new variants might be a reason for the endemic outbreaks that are being reported in Tunisia, in addition to poor vaccination techniques and ineffective prophylactic programs.In the present study, partial nucleotide sequences of the S1 glycoprotein gene and the 3′-untranslated region (UTR) of 2 Tunisian isolates, TN1011/16 and TN1012/16, identified in 2016, were determined. Specific mutations were found in S1 gene as well as in 3′UTR region. Phylogenetic analysis of the S1 nucleotide sequences showed that both isolates are closely related to the Algerian strains, and formed a common cluster within the genotype I. In addition, these isolates were non-recombinant ones, confirming that they are unique variants. Based on their S1 gene sequences, TN1011/16 and TN1012/16 strains were distant from the H120 vaccine strain, commercially used in Tunisia along with the variant vaccine 793B type (4/91). A comparison between nucleotide sequences of their 3′UTR region and S1 gene showed a difference in IBV classification. The obtained results have confirmed that the IBVsequence continues to drift and brings valuable information in relation with its evolution, vaccine development and better control of the disease.

Phylogenetic study of living fossil Psilotum nudum L. from Himalayan range of Pakistan using DNA barcodes

The genus Psilotum belongs to Psilotaceae family, with about 133 to 189 species having outstanding social, economic and medically important relict plants. However, being living fossil plant, many hurdles come in way while trying to discern its phylogenetics reconstruction. The partial conserve nucleotide sequences of rbcLa, trnA, trnV, matK, ITS, ycF3 and rpoB genes were used to select the best suitable barcodes in Psilotum nudum L. The amplified fragments were subjected to Sanger sequencing and then Maximum likelihood, Maximum Parsimony and Neighbor Joining trees were generated using bioinformatics software. i.e., MUSCLE, BioEdit and Mega-7. Current population showed the best match between P. complanatum (KY099851.1), population reported from New Zealand and P. nudum (F384430.1) with identity value 100 and 99% having E-value 0.0 and 0.0 by rbcLa and trnA barcode region, respectively. The findings showed that P. nudum L. sequences are ever first reported from the Himalaya regions of Pakistan and could be utilized for further comparison among different populations of this relict plant species across the globe.

Phylogenetic variability of Human Metapneumovirus in patients with acute respiratory infections in Cameroon, 2011–2014

BackgroundIdentified in 2001, Human Metapneumovirus (HMPV) is a Pneumovirus associated with acute lower and upper respiratory infections in all age groups and especially in newborns, elderly and immunocompromised subjects. Data are still limited in sub-Saharan African countries genetic characterization of this respiratory virus. This study reports the genetic variability of HMPV strains in Cameroonian children for 3 consecutive epidemic seasons (September 2011–October 2014). MethodsA prospective surveillance was conducted to identify inpatient and outpatient children less than 15 years with respiratory symptoms ≤5 days. The nasopharyngeal samples were tested for HMPV using a multiplex polymerase chain reaction. Viral distribution and demographic data were analyzed statistically. Positive samples for HMPV were amplified by semi-nested polymerize chain reaction and then partially sequenced at the G gene. Phylogenetic analyzes were performed on the partial nucleotide and protein sequences of the G gene. ResultsFrom September 2011 to October 2014, 822 children under 15 years were enrolled in the study. HMPV was identified in each of 3.9% (32/822) of children. HMPV were detected throughout the year. HMPV-A (73.3%; 11/15) was predominant compared to HMPV-B (26.7; 4/15). Cameroonian HMPV strains are grouped among the members of genotype A2b (for HMPV-A), B1 and B2 (for HMPV-B). ConclusionThis study suggests that about 4% of ARI recorded in children in Cameroon are caused by HMPV. The present study is also the first report on the genetic variability of the G gene of HMPV strains in the region. Although this work partially fills gaps for some information, additional studies are required to clarify the molecular epidemiology and evolutionary pattern of HMPV in sub-Saharan Africa in general and more particularly in Cameroon.

A new sinistral species of the land-snail genus Satsuma (Pulmonata: Camaenidae) from China

ABSTRACTThe species of Satsuma are mostly endemic to East Asia except for one species distributed in Batan Island of the Philippines. More than 99% of the known species of this genus are endemic to the island environment. Only three species are currently known to occur on the mainland. Here we describe a new sinistral Satsuma species from China, Satsuma guandi n. sp. We studied the shell morphology and genital anatomy of the new species and reconstructed the molecular phylogeny of the genus based on partial nucleotide sequences of the mitochondrial gene cytochrome c oxidase subunit I and nuclear markers from the ribosomal RNA cistrons (the internal transcribed spacer and the external transcribed spacer regions). The new species differs from other sinistral Satsuma species by having a strongly angulated shell and an open dark brownish-red umbilicus. The new species also differed from all other sinistral congeners in details of its reproductive anatomy. The molecular analysis supports the validity of the new species within the genus Satsuma.

Molecular characterization and phylogenetic analysis of human pathogenic Leptospira species circulating in a tertiary care hospital of Western Uttar Pradesh in India

ABSTRACTLeptospirosis is identified as an important reemerging zoonotic disease distributed worldwide, caused by Leptospira. This study was carried out to explore the genetic characterization and its phylogenetic analysis of circulating Leptospira species, among the Aligarh region of western Uttar Pradesh in India, utilizing secY gene-based nucleotide sequence. A total of 190 human samples were included in the study. Positive samples were identified by ELISA, MAT and PCR. MAT was carried out utilizing local circulating Leptospira serovars. Four positive samples including two MAT positive samples were subjected to DNA sequencing for further confirmation and phylogenetic tree was constructed. Out of the total of 190 samples, 24 patients were found positive by ELISA and 29 by PCR. Two samples were found reactive in MAT with L. interrogans serovars like hebdomadis and copenhageni. Phylogenetic analysis of four isolates based on partial secY gene nucleotide sequences revealed that species obtained from the Aligarh region clustered with the several published pathogenic Leptospira interrogans, while some of our isolates nucleotide sequences also clustered with the published sequence of intermediate and saprophytic Leptospira serovars like Leptospira inadai and Leptospira meyeri. This pilot study will help us to decipher the present scenario of circulating serovars of leptospira as well as to identify the nucleotide changes in secY gene, in this region.

Botryosphaeriaceae species associated with the pickly pear cactus, Nopalea cochenillifera

The production of cactus prickly pear (Nopalea cochenillifera) as a foraging plant in the Brazilian Northeastern region is limited by diseases for which knowledge of etiology and diversity of pathogens is scarce. In this work Botryosphaeriaceae fungi isolated from cladode spots of cactus prickly pear were characterized based on morphology and phylogeny using on partial nucleotide sequences of ITS-rDNA, EF1-α e β-tub. Four species were identified, of which three caused a brown spot, Lasiodiplodia theobromae, L. jatrophicola and Neofusicoccum batangarum and one, Neoscytalidium hyalinum, caused a squamous spot firstly reported to occur in N. cochenillifera. We report for the first time worldwide L. jatrophicola and N. hyalinum species causing spots on cactus prickly pear.

Record of Some Digeneans, Infecting Freshwater Fishes, Snails, and Amphibians in India, Using 28S Ribosomal DNA Marker

Eleven species of digenetic trematodes are recorded herein in an attempt to chart the trematode biodiversity in Kakraiya Lake of District Barabanki, U.P., infecting freshwater fishes, snails, and frogs. The species are Opisthorchis attuai and Isoparorchis hypselobagri from Wallago attu; Diplostomulum minutum from Heteropneustes fossilis; Buchephalopsis karvai from Xenentodon cancila; Cephalogonimus mukerjius, and Kaurma sp. from Pila globosa; Pseudodiplodiscoides murtyi from Bellamya bengalensis; Mesocoelium sociale from Duttaphrynus melanostictus; Diplodiscus amphichrus, Halipegus mehransis, and Halipegus barabankiensis n.sp. from Euphlyctis cyanophlyctis. Among the eight species considered as valid so far O. attuai, I. hypselobagri, M. sociale, B. karvai, P. murtyi, D. amphichrus, H. mehransis and C. mukherjius have been described well only at the adult stage, whereas the morphology of D. minutum, and Kaurma sp. at the metacercarial stage has been described, which further awaits to trace their life cycle. The present results show that H. barabankiensis represents distinct entities from a molecular and morphological point of views, which leads to the description of a new species. All these trematode species were subjected to morphological, morphometric, and molecular analysis. Partial nucleotide sequences of the 28S ribosomal gene were obtained from these trematode species and deposited in the GenBank. The phylogenetic reconstruction based on the 28S rRNA gene placed the studied species within the respective families, and their validity is discussed.

Sequence Variations and Molecular Phylogeny of some Red Sea Parrotfishes (Scaridae) Using Mitochondrial Gene Sequences

Targeted DNA fragments were isolated from four scarids species; Scarus collana Ruppell 1835; Scarus frenatus Lacepede 1802; Scarus (Chlorurus) sordidus Forsskal 1775 and Scarus niger Forsskal 1775 using mitochondrial 16S rRNA gene-specific primers. Analysis of obtained partial 16S rRNA gene nucleotide sequences showed a high level of nucleotide identity in the studied regions. This reflects a close genetic relationship and shared ancestry among studied parrotfishes. Nucleotide compositions of partial 16S rRNA gene nucleotide sequence biased towards adenine and similar preference towards thymine, cytocine, and guanine. Also, base constitutions revealed preference towards higher DNA conservations. Phylogenetic analysis displayed patterns of assembly for studied species, and other included related taxa, which reflect their similar genetic makeup and their tendency to have similar niches. The phylogenetic trees revealed two evolutionary lineages splitting Scaridae and Wrasses which assumed that Scaridae should maintain the family status. Obtained data could be beneficial for parootfishes classification, conservation, and their needed environments. Therefore, the acquisition of nucleotide sequences from other parrotfishes using the developed mt16S rRNA gene-specific primers utilized here would contribute in the future to the phylogenetic

First Report of Shoot Blight on Fragrant Pear Caused by Pantoea ananatis in China

Fragrant pears are important economic plants in Xinjiang region, a major agricultural production area in China. In March 2017, a strange shoot blight disease on fragrant pear was initially observed in Kolar, Xinjiang. The incidence of the disease was up to 50% in certain orchards, and the total incidence area was approximately 26,000 ha of commercial fields. The disease developed from blossoms to shoots and larger branches. More disease was observed on old and wounded trees. Symptoms included wilted shoot and leaves. When infections spread to larger tree limbs, the bark of branches became darkened and water-soaked. After bark shaving, dark xylem tissue was visible. Shoot samples were collected from diseased plants (Pyrus sinkiangensis Yu) with characteristic symptoms. The shoot samples were surface sterilized with 75% ethanol and 1% sodium hypochlorite, rinsed three times in sterilized water, cut into small pieces, and then soaked in 10 ml of sterile phosphate buffered saline (PBS) for 30 min. The suspension was streaked onto a nutrient agar plate and incubated at 30°C. After 48 h, dominating light yellow, round flat colonies with smooth surfaces were picked and restreaked for purification. The bacteria were identified by polymerase chain reaction amplified 16S rRNA gene and further by multilocus sequence analysis of partial nucleotide sequences of the genes rpoB and infB using rpoB CM7-F/CM31b-R (1,000 bp) and infB 01-F/02-R (1,030 bp) primer pairs, respectively (Brady et al. 2008). BLAST analysis showed that the 16S rRNA (NCBI accession no. MK128507), rpoB (MK135821), and infB (MK135822) gene sequences shared 100, 100, and 99% similarity to that of Pantoea ananatis strains BSP9 (KF360069.1), 15320 (MH015163.1), and BRT98 (MH015108.1), respectively. In April 2018, 3-year-old fragrant pear trees were inoculated with the P. ananatis in a separated garden. The branches of trees were injured with cuts of 2 to 3 cm in the bark. Autoclaved cotton balls were soaked with bacterial suspensions of 10⁸ CFU/ml and attached to the wounds with Parafilm. The bacterial suspensions were inoculated on three trees, and an additional two trees were treated with PBS instead as a negative control. Three shoots were inoculated per tree. The trees were then grown under natural conditions. After 12 weeks, symptoms resembling bacterial shoot blight symptoms had developed on all inoculated shoots. Control trees did not develop symptoms. Reisolations were performed from symptomatic tissues, and isolates were identified as P. ananatis using the techniques for molecular identification as mentioned above. P. ananatis was initially discovered on pineapple causing fruitlet rot in the Philippines in 1928 (Serrano 1928), and it is regarded as an emerging pathogen based on the increasing number of reports of diseases occurring on previously unrecorded hosts in different parts of the world. Coutinho et al. (2002) reported similar symptoms including shoot wilt and die-back after the infection of Eucalyptus trees with P. ananatis. In 2017, isolation and identification of brown rot bacteria P. ananatis was reported on imported succulent plants in China (Li et al. 2017). To our knowledge, this is the first report of P. ananatis causing shoot blight on fragrant pear in China. This study expanded the host range and geographic location of P. ananatis as a plant pathogen and suggests that commercial fragrant pear producers should take early control actions to prevent spread of and reduce losses caused by the disease.

First Report of Phytophthora palmivora Causing Black Pod on a Herrania sp. in Brazil

During inspections in a germplasm collection in the municipality of Barro Preto, Bahia State, Brazil, it was observed that about 30% of the pods of a Herrania sp. (Malvaceae) genotype showed black pod rot in 2017. Symptoms initially appeared as brown, circular lesions that quickly enlarged at high humidity, and a white mycelium covered the diseased pod surface. The putative pathogen was isolated from six symptomatic pods on 2% water agar containing ampicillin, rifampicin, nystatin, and pentachloronitrobenzene. A hyphal-tip culture from a morphologically representative isolate was obtained on carrot agar (CA) and deposited in the Colecao de Fungos Fitopatogenicos of the Universidade de Brasilia (accession no. CCUB1089). The colony on CA was white and slightly cottony with sparse aerial mycelium. Sporangia (n = 50) were ellipsoid, deciduous, 32.5 to 55.5 µm long, and 25.5 to 39 μm wide, and papillae were 6.0 to 7.0 µm thick. Globose, terminal chlamydospores measuring 16.5 to 39.5 µm in diameter were formed abundantly on CA. The specimen was identified as mating type A1 by pairing with A1 and A2 tester strains of Phytophthora capsici and P. nicotianae. Partial nucleotide sequences of the internal transcribed spacer (ITS), β-tubulin (βt), elongation factor (EF), heat shock protein 90 (HSP90), and cytochrome oxidase subunit 2 (coxII) genomic regions were obtained and deposited in GenBank (accession nos. MH807778 to MH807782). In the Phytophthora database (Park et al. 2008), the ITS, coxII, βt, EF, and HSP90 sequences matched P. palmivora (isolate PD_00022) with 100.0, 100.0, 99.5, 99.8, and 98.9% identities (accession nos. HQ261635, GU222099, EU080339, EU080340, and EU080342, respectively). Additionally, our isolate clustered with other P. palmivora isolates in a Bayesian phylogenetic tree based on concatenated sequences (ITS, coxII, βt, EF, and HSP90) (study S23112 deposited in TreeBASE). Thus, based on morphological and molecular data, the pathogen was identified as P. palmivora. To confirm pathogenicity, mycelial plugs (5 mm) from a 7-day-old culture (grown on CA at 25°C) were placed onto three nonwounded pods of Herrania sp., Theobroma cacao (clone CCN51), T. grandiflorum, T. bicolor, T. speciosum, and T. subincanum. On control pods, CA discs were used. Pods were kept for 5 days in a humid chamber at 25°C. Inoculated pods showed symptoms similar to those observed in the field, and P. palmivora was successfully reisolated from all symptomatic pods. Control pods were symptomless. P. palmivora has been previously reported on Herrania sp. only in West Africa (Erwin and Ribeiro 1996). In Brazil, this pathogen is infecting Annona squamosa, Carica papaya, Citrus sp., Cocos nucifera, Ficus carica, Hevea brasiliensis, Solanum lycopersicum, Piper nigrum, T. cacao, and T. grandiflorum (dos Santos et al. 2017; Farr and Rossman 2018; Zentmyer et al. 1973). The pathogenicity of P. palmivora on T. bicolor, T. speciosum, and T. subincanum (and the absence of reports on these hosts) reinforces that this pathogen can infect species closely related to cacao. This is the first report of P. palmivora causing black pod on a Herrania sp. in Brazil.

Molecular investigation of two novel bacteriophages of a facultative methylotroph, Raoultella ornithinolytica: first report of Raoultella phages.

Bacteria of the genus Raoultella are known to inhabit aquatic environments and can be found in medical samples. The pathogenicity of Raoultella ornithinolytica isolates in human has become increasingly important, and several cases of infections have been reported recently. However, there are no reports of isolation of bacteriophages infecting this bacterium. In this study, two novel phages (ISF3 and ISF6) of a methylotrophic Raoultella strain were isolated from sewage. To characterize the isolated phages, morphological features, protein profiles, restriction digestion patterns, and partial genome sequences were studied. Despite morphological differences, electron microscopy revealed that both phages had an icosahedral capsid connected to a contractile tail, suggesting that ISF3 and ISF6 both belong to the family Myoviridae. Partial nucleotide sequences of the ISF3 genome showed 99% to 100% identity to DNA of Klebsiella pneumonia phages KP15, KP27 and BMBT1; however, the restriction digestion profiles of ISF3 genome digested by EcoRI and EcoRV differed from those of Klebsiella phages KP15 and KP27. A partial sequence alignment showed that ISF6 can be classified as a member of a new viral genus due to its significant differences from other previously identified phages. To the best of our knowledge, this is the first report of the isolation and characterization of the specific Raoultella phages that have potential to be used as new pharmaceuticals against R. ornithinolytica.

Temporal dynamics of Human mastadenovirus species in cases of respiratory illness in southern Brazil.

Human mastadenovirus (HAdV) genus is related to several diseases, among them upper and lower respiratory tract illness. HAdV species B, C, D, and E are mainly associated with respiratory infections. The goal of this work was to identify the HAdV species associated with respiratory infections in hospitalized patients from southern Brazil. Samples were collected from 1996 to 2004 and 2011 to 2017. During this period, 28,524 samples were collected, and 9983 were positive for respiratory viruses, being 435 for HAdV. From these 435 samples, 57 were selected for characterization of HAdV species. For screening the presence of HAdV, a partial sequence of the DNA polymerase gene (DNApol gene) was amplified by nested PCR. Partial nucleotide sequencing was performed in positive samples, and HAdV (DNApol gene) was detected in 53 samples: species B (28; 49.1%), C (16; 28.0%), D (2; 3.5%), E (5; 8.7%), and untyped (2; 3.5%). Specie D was found only in 2017 and specie E in 2011 and 2012. The age of the patients ranged from < 1 to 81years old, and 62.3% were male. No relationship between gender or age and identified HAdV species were observed. In addition, in the period of 2013-2017, 18 samples from patients who died were analyzed: 11 were related to species B, 4 to C, and 2 to D and 1 remained untyped. Circulation of HAdV species D and E varied over the years, but species B and C were present throughout the evaluated period. In addition, respiratory infections by HAdV affect elderly and children mainly.

Two new taxa of the Auriscalpium vulgare species complex with substrate preferences

Basidiomes of the cone-inhabiting Auriscalpium species, usually regarded as A. vulgare Gray, are widely distributed across the Northern Hemisphere and easily recognized because of their unique macro-morphology. However, phylogenetic diversity patterns among different geographic populations and different substrates are completely unknown. In this study, samples of A. vulgare s.l. in different areas of China were studied phylogenetically, morphologically, and ecologically. For comparison, European collections of A. vulgare were included. Our phylogenetic analyses, inferred from partial nucleotide sequences including the internal transcribed spacers (ITS) 1 and 2 with the 5.8S nrDNA, the large subunit of nuclear ribosomal RNA (nrLSU), and the region between the conserved domain 6 and 7 of the gene for the second largest subunit of RNA polymerase II (RPB2), indicated that three phylogenetic species can be recognized, which correspond well with morphological and ecological evidence. Therefore, three species, having distinct substrate preferences, are documented here, namely the well-known A. vulgare, and the newly described species A. orientale and A. microsporum. Auriscalpium vulgare is widely distributed throughout the northern temperate Eurasia and North America, while the new species have been known mainly from subtropical East Asia and northeastern India to date.

Chabaudstrongylus ninhae (Trichostrongylidae: Cooperiinae) and Oesophagostomum muntiacum (Chabertiidae: Oesophagostominae) in feral alien Reeves's muntjacs on Izu-Oshima Island, Tokyo, Japan.

The naturalization of alien Reeves's muntjacs (Muntiacus reevesi) on Izu-Oshima Island, Tokyo, Japan, has proceeded intensively over the last five decades. To clarify whether the gastrointestinal helminths of these animals were brought from their original endemic area or were newly acquired in Japan, 32 Reeves's muntjacs trapped on the island were parasitologically examined. In addition to Gongylonema pulchrum in the oesophagus (34.4% prevalence), Chabaudstrongylus ninhae (Dróżdż, 1967) (Trichostrongylidae: Cooperiinae) and Oesophagostomum muntiacum Jian, 1989 (Chabertiidae: Oesophagostominae) were prevalent in the small (28.1%) and large (46.9%) intestines, respectively. For the first time, these trichostrongylid or chabertiid worms were genetically characterized based on partial nucleotide sequences of the nuclear ribosomal RNA gene (rDNA) and mitochondrial DNA cytochrome c oxidase subunit 1 gene (cox-1), and the phylogenetic relationships with other members of their family were explored. Since these two intestinal nematode species are inherent in muntjacs, this study demonstrates a new distribution of exotic helminth species in Japan in accordance with the naturalization of alien mammalian hosts. The molecular genetic data collected here could assist the taxonomic assessment of morphological variants in different Muntiacus spp. and/or of different geographical origins. Furthermore, our data may help to define the phylogenetic relationships among such isolates.

Sea stars of the genus Henricia Gray, 1840 (Echinodermata, Asteroidea) from Vostok Bay, Sea of Japan.

We report seven species of the genus Henricia Gray, 1840 that were found in Vostok Bay, and two species from adjacent area, known from museum collection or seen in underwater footage. while existing literature reported no confirmed species from this area. Most of these species: H. djakonovi, H. alexeyi, H. densispina, H. hayashii, H. granulifera, H. pacifica, H. asiatica, and H. oculata robusta were reported from the Sea of Japan previously. H. nipponica, known from Japan, is reported from Russian seas for the first time. All studied taxa are re-described here using a range of morphological characters and partial 16S rRNA nucleotide sequences, life colorations of several species are reported for the first time, and an identification key is provided. Lectotype designations are fixed for studied series of species described by AM Djakonov.

Biochemical and genetic polymorphism of Bromopsis inermis populations under chronic radiation exposure.

For the subsequent assessment of the genetic mechanisms responsible for the resistance of plants to chronic irradiation, the analysis of RAPD-cDNA with the subsequent isolation, cloning, and sequencing of expressed polymorphic sequences is a promising technique. A study was conducted on Bromopsis inermis populations that have been growing for a long time in the EURT area. Using RAPD primers, we studied the genetic spectra of plants. In analysing the UPGMA algorithm, we identified two well-distinguishable clusters with a high level of bootstrap support (> 85%): background samples hit the first, and impact samples hit the second. Our data indicate a decrease in diversity in the most polluted population, as well as the appearance of new alleles in chronically irradiated samples of the B. inermis. Smooth brome seedlings were characterised by the content of anthocyanins, comparable with other types of cereals. In the gradient of chronic irradiation, the relative content of anthocyanins was not significantly changed. For the first time, the partial nucleotide sequences of the key genes of anthocyanin biosynthesis (Chi and F3h) in the brome were determined, these sequences were found to be 191 and 356bp in length, respectively, and were cloned and sequenced. Three copies of the Chi gene were identified in the B. inermis genome. One copy (BiChi-1) clustered with the sequences of the Aegilops tauschii gene (D genome), and the other two copies (BiChi-2 and BiChi-3) formed a separate cluster in the Pooideae subfamily adjacent to Hordeum vulgare. In the copy of BiChi-1, a complete deletion of intron 1 was detected. For the F3h gene, one copy of the B. inermis gene was obtained, which forms a separate branch in the subfamily Pooideae.

Identification of six grouper species under the genus Epinephelus (Bloch, 1793) from Indian waters using PCR-RFLP of cytochrome c oxidase I (COI) gene fragment

Groupers are biologically diverse, commercially important and highly regarded of all fish species associated with reef fisheries globally. Given the huge demand and high value in live fish trade, restaurants and supermarkets around the world, the accurate identification of the closely related grouper species is crucial to prevent any adulteration. Partial nucleotide sequence data of the mitochondrial cytochrome c oxidase I (COI) gene, which is globally accepted as fish DNA barcoding gene, was generated for six commercially important grouper species belonging to the genus, Epinephelus viz. E. fuscoguttatus, E. coioides, E. logispinis, E. malabaricus, E. bleekeri and E. tauvina collected from Indian waters. Based on the analysis of 704 bp COI gene sequences, a PCR-RFLP method was developed to identify all the six species accurately. The PCR products of COI gene fragment was digested with selected restriction enzymes viz. HinfI, AseI, HindIII and BsaJI. Restriction endonuclease HinfI was enabled to produce species-specific fragments for E. coioides, E. malabaricus, E. logispinis and E. bleekeri. The result shows that AseI restriction enzyme could be used to differentiate E. fuscoguttatus from other five species. The expected and actual molecular weight of the digested products was similar indicating the authenticity of the methods followed. This is the first report on differentiating closely related grouper species using above mentioned restriction endonucleases and COI gene fragment. PCR-RFLP profiles generated could be useful for detecting seafood fraud by identifying the grouper species under the genus Epinephelus using samples from fresh, frozen, cooked and even processed products.

Persistent development of adomavirus and aquareovirus in a novel cell line from marbled eel with petechial skin haemorrhage.

In Taiwan, a petechial haemorrhage disease associated with mortality has affected marbled eels (Anguilla marmorata). The eels were revealed to be infected with adomavirus (MEAdoV, previously recognized as a polyoma-like virus). In this study, cell line DMEPF-5 was established from the pectoral fin of a diseased eel. DMEPF-5 was passaged >70 times and thoroughly proliferated in L-15 medium containing 2%-15% foetal bovine serum at 20-30°C. Transcripts of neural cell adhesion molecule 1 and nestin genes, and nucleic acids of MEAdoV and a novel reovirus (MERV) in the cells were demonstrated by reverse transcription-polymerase chain reaction analysis. Phylogenetic analysis revealed that the AdoV LO8 proteins mostly relate to adenovirus adenain, whereas MERV is close to American grass carp reovirus in Aquareovirus G, based on a partial VP2 nucleotide sequence. DMEPF-5 cells are susceptible to additional viral infection. Taken together, the marbled eels with the haemorrhagic disease have coinfection with MEAdoV and MERV, and the pathogenic role of MEAdoV and MERV warrants research. DMEPF-5 has gene expression associated with mesenchymal stem and progenitor cells and is the first cell line persistently infected with adomavirus and aquareovirus. DMEPF-5 can facilitate studies of such viruses and haemorrhagic disease.

Circulation of Chikungunya virus East-Central-South Africa genotype during an outbreak in 2016-17 in Piaui State, Northeast Brazil.

ABSTRACTChikungunya virus (CHIKV) is an arbovirus that emerged in the Americas in 2013. Infection with CHIKV is symptomatic in most of the cases and patients can develop chronic arthralgia that lasts from months to years in over 40% of the cases. The East-Central-South Africa (ECSA) genotype was introduced in Brazil in 2014, in Bahia State. Here we report the circulation of the CHIKV ECSA genotype in Piaui State, Northeast Brazil, during the years 2016-2017. The phylogenetic analysis revealed a single introduction of this lineage probably in 2015 and its maintenance at least until 2017. This analysis has also demonstrated the proximity of this genotype with isolates from neighboring States, and its partial nucleotide sequence of the viral E1 gene revealed a synapomorphy synonyms. This finding highlights the spread of the ECSA genotype in Brazil and supports its circulation in the Brazilian Northeast.