Pairwise Sequence Comparisons Research Articles

Yeast species diversity in apple juice for cider production evidenced by culture-based method.

Identification of yeasts isolated from apple juices of two cider houses (one located in a plain area and one in an alpine area) was carried out by culture-based method. Wallerstein Laboratory Nutrient Agar was used as medium for isolation and preliminary yeasts identification. A total of 20 species of yeasts belonging to ten different genera were identified using both BLAST algorithm for pairwise sequence comparison and phylogenetic approaches. A wide variety of non-Saccharomyces species was found. Interestingly, Candida railenensis, Candida cylindracea, Hanseniaspora meyeri, Hanseniaspora pseudoguilliermondii, and Metschnikowia sinensis were recovered for the first time in the yeast community of an apple environment. Phylogenetic analysis revealed a better resolution in identifying Metschnikowia and Moesziomyces isolates than comparative analysis using the GenBank or YeastIP gene databases. This study provides important data on yeast microbiota of apple juice and evidenced differences between two geographical cider production areas in terms of species composition.

Revisiting the Parvilucifera infectans / P. sinerae (Alveolata, Perkinsozoa) species complex, two parasitoids of dinoflagellates

Members of the family Parviluciferaceae (Alveolata, Perkinsozoa) are the well-known dinoflagellate parasitoids along with Amoebophrya ceratii species complex and parasitic chytrid Dinomyces arenysensis and contain six species across three genera (i.e., Parvilucifera infectans, P. sinerae, P. rostrata, and P. corolla, Dinovorax pyriformis, and Snorkelia prorocentri) so far. Among Parvilucifera species, the two species, P. infectans and P. sinerae, are very similar or almost identical each other morphologically and genetically, thereby make it difficult to distinguish between the two. The only main difference between the two species known so far is the number of sporangium wall (i.e., 2 layers in P. infectans vs. 3 layers in P. sinerae). During sampling in Masan bay, Korea during the spring season of 2015, the dinoflagellate Akashiwo sanguinea cells infected by the parasite Parvilucifera were observed and this host-parasite system was established in culture. Using this culture, its morphological and ultrastructural features with special emphasis on the variation in the number of sporangium wall over developmental times, were investigated. In addition, the sequences of rDNA regions and β-tubulin genes were determined. The result clearly demonstrated that the trophocyte at 36 h was covered with 4 layers, and then outer layer of the sporocyte gradually degraded over time, resulting in wall structure consisting of two layers, with even processes being detached from 7-day-old sporangium with smooth surface, indicating that the difference in the number of layers seems not to be an appropriate ultrastructural character for distinguishing P. infectans and P. sinerae. While pairwise comparison of the large subunit rDNA sequences showed 100% identity among P. infectans / P. sinerae species complex, genetic differences were found in the small subunit (SSU) rDNA sequences but the differences were relatively small (11–13 nucleotides) compared with those (190–272 nucleotides) found among the rest of Parvilucifera species (P. rostrata and P. corolla). Those small differences in SSU rDNA sequences of P. infectans / P. sinerae species complex may reflect the variations within inter-strains of the same species from different geographical areas. Taken together, all morphological, ultrastructural, and molecular data from the present study suggest that they are the same species. Key words: inter-strain variation; parasitism; parasitoid; sporangium; synonymy

Phenylobacterium terrae sp. nov., isolated from a soil sample of Khyber-Pakhtun-Khwa, Pakistan.

A Gram-stain negative, aerobic, motile, non-spore-forming and rod-shaped bacterial strain, designated YIM 730227T, was isolated from a soil sample, collected from Karak district, Khyber-Pakhtun-Khwa, Pakistan. The bacterium was characterized using a polyphasic taxonomic approach. Pairwise comparison of the 16S rRNA gene sequences showed that strain YIM 730227T is closely related to Phenylobacterium lituiforme FaiI3T (97.5% sequence similarity), Phenylobacterium muchangponense A8T (97.4%), Phenylobacterium panacis DCY109T (97.1%), Phenylobacterium immobile ET (97.1%) and Phenylobacterium composti 4T-6T (97.0%), while also sharing 98.0% sequence similarity with Phenylobacterium hankyongense HKS-05T after NCBI blast, showing it represents a member of the family Caulobacteraceae. The major respiratory quinone was Q-10 and the major fatty acids were C16:0, summed feature 8 (comprising C18:1ω7c and/or C18:1ω6c), C18:1ω7c 11-methyl and C17:0. The polar lipids were phosphatidylglycerol, unidentified glycolipids, phospholipid and unidentified lipid. The G + C content of the genomic DNA was 68.2mol%. The DNA-DNA relatedness values of strain YIM 730227T with P. hankyongense HKS-05T, P. lituiforme FaiI3T, P. muchangponense A8T, P. panacis DCY109T, P. immobile ET and P. composti 4T-6T were 31.3 ± 0.6, 26.1 ± 0.2, 24.3 ± 0.1, 21.8 ± 0.9, 19.8 ± 0.6 and 18.2 ± 1.1%, respectively, values lower than 70%. Besides the morphological and chemotaxonomic characteristics, phylogenetic analyses of 16S rRNA gene sequences and the biochemical characteristics indicated that the strain YIM 730227T represents a novel member of the genus Phenylobacterium, for which the name Phenylobacterium terrae sp. nov. (type strain YIM 730227T = KCTC62324T = CGMCC 1.16326T) is proposed.

Identification and characterization of Candidatus Phytoplasma trifolii (16SrVI-D) inducing shoot proliferation disease of potato in India

Potato (Solanum tuberosum) is a widely cultivated and commercially important vegetable crop widely grown in India. Profuse shoot proliferation symptoms were observed in potato plants of two varieties, Kufri Khyati and Kufri Surya at the Research plots of All India Co-ordinated Research Project on Potato, Orissa University of Agriculture and Technology, Bhubaneswar, Odisha, India during February, 2017. The symptomatic plants were tested for phytoplasma etiology. Nested and semi-nested PCR assays primed by primer pairs P1/P7 and 3Far/3Rev for 16S rRNA gene and secAF1/R3 for secA gene amplified DNA fragments of 1.3 kb (3Far/3Rev) and 840 bp (secAF1/R3), respectively from all the symptomatic potato samples but not from any asymptomatic plants. The 16S rDNA sequences of potato tuber (Acc. no. KY815100, Kufri Khyati; MG566064, Kufri Surya) and shoot phytoplasma strains (Acc. no. KY815099, Kufri Khyati; MG566063, Kufri Surya) shared 100% nucleotide sequence identity with 16S rDNA sequence of identified phytoplasma strains belonging to 16SrVI-D subgroup associated with Lens culinaris witches’ broom (Acc. no. KY439869), brinjal little leaf (Acc. no. KX284698) and Brassica oleracea var. capitata witches’ broom (Acc. no. KX671553) in pair wise sequence comparison. Sequence comparison of 840 bp of secA gene of potato phytoplasma strains from tuber of variety, Kufri Khyati (Acc. no. KY815101) and variety Kufri Surya (Acc. no. MG566065) showed 99% sequence identity to secA gene sequences of brinjal little leaf phytoplasma strains (Acc. no. KX610808, KX894792, KY064175), which belonged to 16SrVI-D subgroup. Phylogenetic analysis of 16S rRNA and secA gene sequences, and virtual RFLP analysis of 16S rDNA sequences of potato shoot proliferation phytoplasma strain confirmed their clustering and grouping with strains of clover proliferation subgroup D. Our results confirmed potato as a new host of 16SrVI-D subgroup of phytoplasmas in the world. The 16SrVI-D strain is wide spread in India and cause significant yield losses and hence poses serious socio-economic threat to commercially grown potato crops.

Georgenia deserti sp. nov., a halotolerant actinobacterium isolated from a desert sample.

Strain SYSU D8004T was isolated from a sample collected from an arid area in Saudi Arabia. The isolate was Gram-stain-positive, non-motile, aerobic and non-spore-forming. It could grow at 4-45 °C, at pH 6.0-10.0 and in the presence of up to 17 % (w/v) NaCl. Pairwise comparison of the 16S rRNA gene sequences indicated that strain SYSU D8004T shared highest sequence similarity with Georgenia halophila YIM 93316T (96.5 %). Menaquinone MK-8(H4) was detected as the respiratory quinone. The polar lipid profile of strain SYSU D8004T consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, two phosphatidylinositol mannosides, two unidentified phospholipids and an unidentified glycolipid. Strain SYSU D8004T contained anteiso-C15 : 0, iso-C15 : 0 and C14 : 0 as the predominant fatty acids (>10 %). Galactose, glucose and rhamnose were detected as whole-cell sugars. Based on analyses of the phenotypic, genotypic and phylogenetic characteristics, it was determined that strain SYSU D8004T could be differentiated from other closely related members of the genus Georgenia. Strain SYSU D8004T is therefore considered to represent a novel species of the genus Georgenia, for which the name Georgenia deserti sp. nov. is proposed. The type strain is SYSU D8004T (=CGMCC 1.15793T=KCTC 39987T).

Molecular characterization of phytoplasmaassociated with four important ornamental plant species in India and identification of natural potential spread sources.

Phytoplasma suspected symptoms of phyllody, witches' broom, leaf yellowing, stunting and little leaf were observed in Chrysanthemum morifolium, Bougainvillea glabra, Jasminum sambac and Callistephus chinensis during survey of flower nurseries and experimental ornamental fields at Delhi, Maharashtra, Tamil Nadu and Karnataka from 2014 to 2016. Pleomorphic bodies typical to phytoplasma structures were observed in the phloem sieve elements of ultrathin sections of all the four symptomatic ornamental plants (stem tissue) in transmission electron microscope. Amplification of 1.8 and 1.2kb phytoplasma DNA products was observed in all the four test plants in PCR assays using universal primer pairs P1/P7 followed by nested primer pair R16F2n/R16R2, respectively. Pairwise sequence comparison, phylogeny and virtual RFLP analysis of 16S rDNA sequences confirmed the association of two phytoplasma subgroups (16SrI-B and 16SrII-D) in four ornamental plant species. 'Ca. P. aurantifolia' subgroup D (16SrII-D) was found associated with chrysanthemum phyllody and leaf yellowing at Delhi and Tamil Nadu, bougainvillea little leaf and yellowing at Delhi and Chinese aster phyllody at Bengaluru, Karnataka. However, jasmine little leaf and yellowing at Bengaluru, Karnataka and chrysanthemum stunting at Pune were found to be associated with 'Ca. P. asteris' subgroup B-related strains (16SrI-B). The identification of 16SrII-D subgroup phytoplasma infecting bougainvillea and 16SrI-B subgroup infecting jasmine are the new reports to the world. Besides weed species, Cannabis sativa showing witches' broom in jasmine fields at Bengaluru and Parthenium hysterophorus showing witches' broom symptoms in chrysanthemum fields at Delhi were identified to be caused by phytoplasma strains classified under subgroups 16SrI-B and 16SrII-D, respectively, by PCR assays and 16Sr DNA sequence comparison analysis. Among the three major leafhopper species identified, only Hishimonas phycitis was identified positive for 16SrI-B and 16SrII-D subgroups of phytoplasmas from chrysanthemum fields at Delhi and jasmine fields at Bengaluru, respectively. The identity of similar phytoplasma strains infecting ornamental species in leafhopper and the weed species in the present study suggested that H. phycitis and weeds may act as potential natural sources for secondary spread of the identified phytoplasma strains.

Chevrier's Field Mouse (Apodemus chevrieri) and Père David's Vole (Eothenomys melanogaster) in China Carry Orthohepeviruses that form Two Putative Novel Genotypes Within the Species Orthohepevirus C.

Hepatitis E virus (HEV) is the prototype of the family Hepeviridae and the causative agent of common acute viral hepatitis. Genetically diverse HEV-related viruses have been detected in a variety of mammals and some of them may have zoonotic potential. In this study, we tested 278 specimens collected from seven wild small mammal species in Yunnan province, China, for the presence and prevalence of orthohepevirus by broad-spectrum reverse transcription (RT)-PCR. HEV-related sequences were detected in two rodent species, including Chevrier's field mouse (Apodemus chevrieri, family Muridae) and Père David's vole (Eothenomys melanogaster, family Cricetidae), with the infection rates of 29.20% (59/202) and 7.27% (4/55), respectively. Further four representative full-length genomes were generated: two each from Chevrier's field mouse (named RdHEVAc14 and RdHEVAc86) and Père David's vole (RdHEVEm40 and RdHEVEm67). Phylogenetic analyses and pairwise distance comparisons of whole genome sequences and amino acid sequences of the gene coding regions showed that orthohepeviruses identified in Chinese Chevrier's field mouse and Père David's vole belonged to the species Orthohepevirus C but were highly divergent from the two assigned genotypes: HEV-C1 derived from rat and shrew, and HEV-C2 derived from ferret and possibly mink. Quantitative real-time RT-PCR demonstrated that these newly discovered orthohepeviruses had hepatic tropism. In summary, our work discovered two putative novel genotypes orthohepeviruses preliminarily named HEV-C3 and HEV-C4 within the species Orthohepevirus C, which expands our understanding of orthohepevirus infection in the order Rodentia and gives new insights into the origin, evolution, and host range of orthohepevirus.

Saccharopolyspora deserti sp. nov., a novel halotolerant actinobacterium isolated from a desert.

Strain SYSU D8010T was isolated from a desert sand sample collected in Saudi Arabia. The taxonomic position of the isolate was investigated by the polyphasic taxonomic approach. The isolate was found to be Gram-positive and aerobic. The strain was able to grow at 14-40 °C, pH 5.0-9.0 and in the presence of up to 22 % (w/v) NaCl. Strain SYSU D8010T contained meso-diaminopimelic acid as cell-wall diamino acid, and arabinose, fucose, galactose, glucose and rhamnose as the whole-cell sugars. The primary polar lipids were diphosphatidylglycerol, phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol and phosphatidylinositolmannosides. Menaquinone MK-9(H4) was detected as the respiratory quinone; and anteiso-C17 : 0, iso-C16 : 0, iso-C15 : 0 and iso-C17 : 0 as the predominant fatty acids. Pairwise comparison of the 16S rRNA gene sequences indicated that strain SYSU D8010T had a sequence similarity of 97.8 % to Saccharopolyspora halophila YIM 90500T. The genomic DNA G+C content of strain SYSU D8010T was determined to be 69.9 mol%. Based on the analyses of the phenotypic, genotypic and phylogenetic characteristics, strain SYSU D8010T was determined to represent a novel species of the genus Saccharopolyspora, for which the name Saccharopolyspora deserti sp. nov. is proposed. The type strain of the species is SYSU D8010T (=KCTC 39989T=CPCC 204620T).

Improving pairwise comparison of protein sequences with domain co-occurrence.

Comparing and aligning protein sequences is an essential task in bioinformatics. More specifically, local alignment tools like BLAST are widely used for identifying conserved protein sub-sequences, which likely correspond to protein domains or functional motifs. However, to limit the number of false positives, these tools are used with stringent sequence-similarity thresholds and hence can miss several hits, especially for species that are phylogenetically distant from reference organisms. A solution to this problem is then to integrate additional contextual information to the procedure. Here, we propose to use domain co-occurrence to increase the sensitivity of pairwise sequence comparisons. Domain co-occurrence is a strong feature of proteins, since most protein domains tend to appear with a limited number of other domains on the same protein. We propose a method to take this information into account in a typical BLAST analysis and to construct new domain families on the basis of these results. We used Plasmodium falciparum as a case study to evaluate our method. The experimental findings showed an increase of 14% of the number of significant BLAST hits and an increase of 25% of the proteome area that can be covered with a domain. Our method identified 2240 new domains for which, in most cases, no model of the Pfam database could be linked. Moreover, our study of the quality of the new domains in terms of alignment and physicochemical properties show that they are close to that of standard Pfam domains. Source code of the proposed approach and supplementary data are available at: https://gite.lirmm.fr/menichelli/pairwise-comparison-with-cooccurrence

Diversity of phytoplasmas infecting different crops and weeds in Tripuraregion of India

Phytoplasma suspected symptoms of little leaf, chlorosis, yellowing, phyllody, witches’ broom, flat stem, flower virescence and stunting were observed in sixteen plant species during surveys in fields, gardens, fruit orchards and nurseries of six districts of Tripura, India in 2017–2018. Out of 15 symptomatic plant samples belonging to different species, 12 were positive through nested PCR assays for phytoplasma presence. Pair wise sequence comparison, phylogeny and virtual RFLP analysis of phytoplasma partial 16S rDNA sequences of these 12 samples indicated the presence of different phytoplasma groups and subgroups (16SrI-B, 16SrII-D, 16SrVI-D, 16SrXIV-A 16SrI, 16SrIX and 16SrXI). ‘Candidatus Phytoplasma asteris’-related phytoplasmas (16SrI-B) were identified in sesame phyllody affected plants at South Tripura and in crape jasmine, Celosia argentea and Zinnia elegans from Unakoti, Dhalai, North Tripura and West Tripura district. In West Tripura ‘Ca. P. australasiae'related phytoplasmas (16SrII-D) were identified with Jatropha hastata and bitter gourd; ‘Ca. P. trifolii’-related phytoplasmas (16SrVI-D) were identified in chili and brinjal and ‘Ca. P. cynodontis’-related phytoplasmas (16SrXIV-A) were identified in palm grass samples from Sipahijala district. ‘Ca. P. phoenicium’-related phytoplasmas (16SrIX) were identified in cowpea; ‘Ca. P. oryzae’-related phytoplasmas were identified in arecanut from North Tripura and ‘Ca. P. asteris’-related phytoplasmas were detected in Toona ciliata from Jampui Hills. This study suggests the presence of a wide genetic diversity of phytoplasmas infecting different crops and weeds in the Tripura state of India.

Sesame phyllody disease-a potential emerging threat to sesamecultivation in the north east region of India

Sesame phyllody disease associated with the presence of phytoplasmas is becoming a serious problem to many sesame growing countries of the world including India. However no systematic study on the disease has been done to assess the disease incidence, characterization of the associated phytoplasma strains and effect on the yield in north eastern states of India. Therefore an extensive survey was conducted from 2015 to 2017 in seven north eastern states of India covering 52 districts to record the incidence of the disease and to perform phytoplasma associated molecular characterization. The infected plants showed symptoms of virescence, phyllody, floral proliferation, flattened stem, splitting of capsules and witches’ broom. The disease incidence varied from 2% to 29% in different locations. The highest disease incidence (29%) was observed in the Sonitpur district of Assam in the variety ST-1683. Sesame phyllody infection was found to reduce the number of pod and seed yield per plant from 72.82%-100% and 57.20%-100%, respectively. A total of 96 symptomatic sesame samples were collected and analysed on extracted DNA with polymerase chain reaction using phytoplasma specific primers, amplifying 16S rRNA and secA genes. PCR and sequencing analysis confirmed the presence of ‘Candidatus Phytoplasma asteris’-related strains in all the symptomatic samples. Pairwise sequence comparison and phylogenetic analyses of 21 strains on 16S rRNA gene and 9 strains on secA gene sequences confirmed the phytoplasma classification in the aster yellows groups. Among the 7 leafhopper species collected only Hishimonus phycitis tested positive for sesame phyllody phytoplasma presence. Seven species of weeds and wavy leaf basket grass (Oplismenus undulatifolius) and flax (Linum usitatissimum) in and around the infected sesame fields were also positive for phytoplasma presence indicating them as potential reservoir hosts of the phytoplasma associated with sesame phyllody disease in the north east region of India.

Fish red blood cells express immune genes and responses

The erythrocytes or red blood cells (RBCs) and leucocytes or white blood cells (WBCs) are the most abundant cell types in the circulatory system of vertebrates. The RBCs are responsible for gas exchange while WBCs are solely involved with immunity. In this study, we present evidence that fish RBCs despite their respiratory role they also express immune-related genes and responses in the teleost fish, Nile tilapia (Oreochromis niloticus). Fluorescent staining identified the presence of numerous mitochondria and lysosomes in tilapia RBCs. RNA sequencing of tilapia RBCs and WBCs led to the assembly and annotation of 20.876 transcripts of which 1017 and 3628 transcripts were specific to RBCs and WBCs, respectively. Pairwise sequence comparisons identified 3251 transcripts that were significantly differential expressed of which 707 (21.75%) were up-regulated and 2544 (78.25%) down-regulated in RBCs when compared to WBCs. 809 transcripts were assigned to 16 immune system pathways, 21 and 163 transcripts were specifically expressed in tilapia RBCs and WBCs respectively, and 23 and 233 transcripts were highly expressed in RBCs and WBCs respectively. Expression of 7 immune genes (interferon regulatory factors (irf) 1–5 and 9 and tlr3) in both WBCs and RBCs was validated by RT-PCR analysis. Quantitative real-time PCR revealed that all genes were up-regulated in poly(I:C)-challenged WBCs and irf (1, 3, 4, and 9) and tlr3 were also significantly up-regulated in poly(I:C)-challenged RBCs. Therefore, fish RBCs expressed immunity genes and responses and they may play a complementary role in vertebrate immunity.

First Report of Johnsongrass mosaic virus Infecting Urochloa mosambicensis in Ethiopia

Urochloa mosambicensis (Hack.) Dandy, commonly known as sabi grass, is a perennial grass with a creeping and stoloniferous habit. Predominantly used as a cereal in southern Africa, sabi grass is also commonly used for a variety of purposes including forage for livestock production, protection against soil erosion, and in the rehabilitation of mine sites. Its fast-growing nature combined with drought resistance, salinity tolerance, ability to grow on different soil types, fertility levels and environments, tolerance to heavy grazing, and good seed production capacity all help to demonstrate that it has great potential as a pasture grass for the semiarid tropical regions. The Forage Genebank of the International Livestock Research Institute in Ethiopia maintains a total of seven accessions of Urochloa germplasm at their field site in Zwai, on the border of Oromia and the Southern Nations, Nationalities, and Peoples regions. During routine disease surveillance of the field, a typical mosaic symptom was observed on some of the Urochloa accessions. Consequently, both symptomatic and asymptomatic leaf samples from all accessions were collected and tested for virus infection by dot-blot assay using in-house antisera developed to detect two strains (Is-14982 and Is-16840) of Elephant grass mosaic virus (Mih and Hanson 2004) and a Brachiaria potyvirus antiserum received from the International Center for Tropical Agriculture, Colombia. Two accessions of U. mosambicensis (7233, 16665) that showed significant mosaic symptoms produced a positive reaction with the Brachiaria potyvirus antiserum and a weak reaction with the antiserum to Is-14892; one accession (16665) that showed minor symptoms produced a weak reaction only with the antiserum to Is-16840 and not with the Brachiaria potyvirus antiserum; and the other four asymptomatic accessions did not give any reaction with any of these antisera. The association of virus only with two accessions (7233, 16665) of U. mosambicensis was further confirmed by reverse-transcription (RT) PCR using a pair of universal primers, NIb2F/NIb3R designed for potyvirus (Zheng et al. 2010). The initial BLAST analysis of the sequences generated by direct sequencing of both the PCR products indicated the presence of Johnsongrass mosaic virus (JGMV) in these grass accessions. For more accurate determination of the associated virus, a pair of JGMV-specific primers; GBV125F (5′-GGAACAACCGTCTATCTGACG-3′) and GBV106R (5′-GTGGTGAGAAGCTCACTCTTA-3′) were designed to conserved domains flanking the viral coat protein (CP) gene, using the sequences available in the NCBI database. RT-PCR analysis produced a specific ∼1.5 kb amplification product indicative of JGMV in both accessions, and the PCR product from accession 7233 was then fully sequenced. A pairwise comparison of the CP sequences of the present isolate, ZG-181 (GenBank accession no. KY659307) with other JGMV isolates from the NCBI database showed the sequence shared 87.6 and 87.9% amino acid sequence identity with Brazilian isolates from Panicum maximum (KT289893) and Pennisetum purpureum (KT833782), respectively. A phylogenetic analysis of the available JGMV CP sequences separated the present isolate together with the Brazilian isolates of JGMV in a cluster that was distinct from the cluster formed by isolates from Australia and the United States. This study confirms the presence of JGMV in an important grass species in Ethiopia; JGMV could also pose a threat for other forage grasses and crops, and therefore this finding needs to be studied further to understand the potential impact. To the best of our knowledge, this is the first report of JGMV infection in U. mosambicensis in Ethiopia.

Tepidimonas sediminis sp. nov. and Tepidimonas alkaliphilus sp. nov., two novel moderately thermophilic species isolated from a hot spring.

The taxonomic positions of two moderately thermophilic bacteria, strains YIM 72259T and YIM 72238T, isolated from a hot spring sediment sample were determined using polyphasic taxonomic approaches. Pairwise comparison of the 16S rRNA gene sequences showed that strains YIM 72259T and YIM 72238T are closely related to Tepidimonas ignava SPS-1037T (97.0 and 97.5%, respectively), Tepidimonas taiwanensis I1-1T (96.5 and 97.3%), while sharing 98.0% sequence similarity with each other. The DNA-DNA relatedness values of strains YIM 72238T with T. ignava SPS-1037T and T. taiwanensis I1-1T were determined to be 25.2±1.2 and 24.6±3.1%, respectively, while the two strains (YIM 72259T and YIM 72238T) had a relatedness value of 38.3±4.4% between themselves. The two strains were aerobic, Gram-stain negative, motile by means of a polar flagellum and formed non-pigmented colonies. The DNA G+C contents of strains YIM 72259T and YIM 72238T were determined to be 71.6 and 68.9mol%, respectively. The major fatty acids in both strains were identified as (>10%) C16:0, C17:0 and summed feature 8, while the strain YIM 72238T contained a moderately high amount of summed feature 3 as well. The polar lipid profiles for strains YIM 72259T and YIM 72238T consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, an unidentified aminolipid and several unidentified phospholipids. Besides the morphological and chemotaxonomic characteristics, phylogenetic analyses of the 16S rRNA gene sequences and the biochemical characteristics indicated that the two strains represented two novel members of the genus Tepidimonas, for which the name Tepidimonas sediminis sp. nov. [type strain YIM 72259T (=NBRC 112410T=CGMCC 1.15971T) and Tepidimonas alkaliphilus sp. nov. [type strain YIM 72238T (=KCTC 52717T=CCTCC AB2017168T)] are proposed.

Molecular Characterization of QX-Like and Variant Infectious Bronchitis Virus Strains in Malaysia Based on Partial Genomic Sequences Comprising the S-3a/3b-E-M-Intergenic Region-5a/5b-N Gene Order.

Infectious bronchitis virus (IBV) is one of the major poultry pathogens of global importance. However, the prevalence of IBV strains in Malaysia is poorly characterized. The partial genomic sequences (6.8 kb) comprising the S-3a/3b-E-M-intergenic region-5a/5b-N gene order of 11 Malaysian IBVs isolated in 2014 and 2015 were sequenced using next-generation sequencing technology. Phylogenetic and pairwise sequence comparison analysis showed that the isolated IBVs are divided into two groups. Group 1 (IBS124/2015, IBS125/2015, IBS126/2015, IBS130/2015, IBS131/2015, IBS138/2015, and IBS142/2015) shared 90%-95% nucleotide and deduced amino acid similarities to the QX-like strain. Among these isolates, IBS142/2015 is the first IBV detected in Sarawak state located in East Malaysia (Borneo Island). Meanwhile, IBV isolates in Group 2 (IBS037A/2015, IBS037B/2015, IBS051/2015, and IBS180/2015) were 91.62% and 89.09% identical to Malaysian variant strain MH5365/95 (EU086600) at nucleotide and amino acid levels, respectively. In addition, all studied IBVs were distinctly separate from Massachusetts (70%-72% amino acid similarity) and European strains including 793/B, Italy-02, and D274 (68%-73% amino acid similarity). Viruses in Group 1 have the insertion of three amino acids at positions 23, 121, and 122 of the S1 protein and recombinant events detected at nucleotide position 4354-5864, with major parental sequence derived from QX-like (CK-CH-IBYZ-2011) and a minor parental sequence derived from Massachusetts vaccine strain (H120). This study demonstrated coexistence of the IBV Malaysian variant strain along with the QX-like strain in Malaysia.

Isolation and Characteristics of the Arkansas-Type Infectious Bronchitis Virus in China.

Two infectious bronchitis virus (IBV) strains, designated as γCoV/ck/China/I0712/11 (I0712/11) and γCoV/ck/China/I0108/17 (I0108/17), were isolated from diseased chicken flocks in different provinces in China and genotyped as Arkansas (Ark)-type viruses with three other Chinese Ark field strains, the Jilin vaccine strain, and the American Ark- and Ark DPI-like viruses. Complete genomic sequence analysis and pairwise comparison of nucleotide sequences encoding the S1 subunit of the spike protein and other structural and accessory proteins revealed that Chinese Ark field isolates were genetically closely related to the Jilin vaccine and American ArkDPI11 strains, although extensive nucleotide changes were found across the genomes of Chinese Ark field isolates. This suggests that Chinese Ark-type isolates are derived from the Jilin vaccine, and have diverged and evolved independently by point mutations since introduction into China. It is also possible that the Chinese Ark viruses have arisen as a result of different introductions of American ArkDPI11-like strains from the United States; this hypothesis requires further investigation. Pathogenicity testing showed that Chinese Ark viruses had comparable virulence to that of the Massachusetts-type M41 strain, although they had lower affinity for the kidneys of chickens than the M41 strain had. Although Ark-type viruses are not widespread in China, surveillance and updating the currently applied vaccination strategy for sound protection against IBV disease are important because this type of virus has caused heavy economic losses in the United States.

Preliminary Classification of Novel Hemorrhagic Fever-Causing Viruses Using Sequence-Based PAirwise Sequence Comparison (PASC) Analysis.

During the last decade, genome sequence-based classification of viruses has become increasingly prominent. Viruses can be even classified based on coding-complete genome sequence data alone. Nevertheless, classification remains arduous as experts are required to establish phylogenetic trees to depict the evolutionary relationships of such sequences for preliminary taxonomic placement. Pairwise sequence comparison (PASC) of genomes is one of several novel methods for establishing relationships among viruses. This method, provided by the US National Center for Biotechnology Information as an open-access tool, circumvents phylogenetics, and yet PASC results are often in agreement with those of phylogenetic analyses. Computationally inexpensive, PASC can be easily performed by non-taxonomists. Here we describe how to use the PASC tool for the preliminary classification of novel viral hemorrhagic fever-causing viruses.

Complete genome sequence of datura leaf curl virus, a novel begomovirus infecting Datura innoxia in Sudan, related to begomoviruses causing tomato yellow leaf curl disease.

Several species in the genus Datura (family Solanaceae) are hosts for begomoviruses (family Geminiviridae), both in the New World and the Old World. Here, we report the genome characterization of two isolates of a novel Old World monopartite begomovirus infecting Datura innoxia in Sudan, which we propose to be named "datura leaf curl virus" (DaLCV). Pairwise sequence comparisons and phylogenetic analysis showed that DaLCV isolates are related to begomoviruses causing tomato yellow leaf curl disease, a global menace for tomato crops. Thus, the novel begomovirus could pose an additional threat to tomato cultivation in Africa.

Comparative Analysis of Sequence Clustering Methods for Deduplication of Biological Databases

The massive volumes of data in biological sequence databases provide a remarkable resource for large-scale biological studies. However, the underlying data quality of these resources is a critical concern. A particular challenge is duplication, in which multiple records have similar sequences, creating a high level of redundancy that impacts database storage, curation, and search. Biological database deduplication has two direct applications: for database curation, where detected duplicates are removed to improve curation efficiency, and for database search, where detected duplicate sequences may be flagged but remain available to support analysis. Clustering methods have been widely applied to biological sequences for database deduplication. Since an exhaustive all-by-all pairwise comparison of sequences cannot scale for a high volume of data, heuristic approaches have been recruited, such as the use of simple similarity thresholds. In this article, we present a comparison between CD-HIT and UCLUST, the two best-known clustering tools for sequence database deduplication. Our contributions include a detailed assessment of the redundancy remaining after deduplication, application of standard clustering evaluation metrics to quantify the cohesion and separation of the clusters generated by each method, and a biological case study that assesses intracluster function annotation consistency to demonstrate the impact of these factors on a practical application of the sequence clustering methods. Our results show that the trade-off between efficiency and accuracy becomes acute when low threshold values are used and when cluster sizes are large. This evaluation leads to practical recommendations for users for more effective uses of the sequence clustering tools for deduplication.

Use of secA Gene for Characterization of Phytoplasmas Associated with Sugarcane Grassy Shoot Disease in India

Surveys of commercial sugarcane varieties were conducted to the phytoplasma disease incidence in eight major sugarcane growing states of India (Uttarakhand, Uttar Pradesh, Maharashtra, Bihar, Assam, Chhattisgarh, Haryana and Tamil Nadu) during 2014–2015. Leaves from 24 symptomatic sugarcane plants of eight varieties showing grassy shoot and chlorosis symptoms, and of 8 non-symptomatic plants were collected and analyzed for phytoplasma presence using 16S rRNA and secA gene-specific primers. Amplification of 1.8- and 1.2-kb products using nested primers (P1/P7 and R16F2n/R16R2) of 16S rRNA gene and 880- and 480-bp products using secA gene-specific primer pairs (SecAfor1/SecArev3 and SecAfor2/SecArev3) was obtained for all the 24 symptomatic sugarcane samples. Pairwise sequence comparison, phylogenetic and in silico RFLP analysis of partial 16S rRNA and secA gene sequences of eight strains of sugarcane grassy shoot phytoplasma representative of the eight states confirmed the association of ‘Candidatus phytoplasma oryzae’-related strains (16SrXI-B) with symptomatic sugarcane varieties. The study confirmed that secA gene-specific primers could be employed for molecular characterization of phytoplasmas associated with sugarcane grassy shoot phytoplasmas belonging to 16SrXI group.