Whole-genome sequence of a novel lytic bacteriophage infecting Clavibacter michiganensis subsp. michiganensis from Turkey.

Clavibacter michiganensis subsp. michiganensis (Cmm) is an important plant-pathogenic bacterium that causes canker and wilt diseases. Biological control of the disease with bacteriophages is an alternative to conventional methods. In this study, Phage33 infecting Cmm was characterized based on morphological and genomic properties. Morphological characteristics such as shape and size were investigated using electron microscopy. The whole genome was sequenced using the Illumina Novaseq 6000 platform and the sequence was assembled and annotated. VICTOR and VIRIDIC were used for determining the phylogeny and comparing viral genomes, respectively. Electron microscopy showed that Phage33 has an icosahedral head with a diameter of ~55 nm and a long, thin, non-contractile tail ~169 nm in length. The genome of Phage33 is 56 324 bp in size, has a GC content of 62.49 % and encodes 67 open reading frames. Thirty-seven ORFs showed high homology to functionally annotated bacteriophage proteins in the NCBI database. The remaining 30 ORFs were identified as hypothetical with unknown functions. The genome contains no antimicrobial resistance, no lysogenicity and no virulence signatures, suggesting that it is a suitable candidate for biocontrol agents. The results of a blastn search showed similarity to the previously reported Xylella phage Sano, with an average nucleotide sequence identity of 92.37 % and query coverage of 91 %. This result was verified using VICTOR and VIRIDIC analysis, and suggests that Phage33 is a new member of the genus Sanovirus under the class Caudoviricetes.

Complete genome sequence analysis and genome organization of Dasychira pudibunda nucleopolyhedrovirus (DapuNPV-T1) from Turkey.

The economically important pale tussock moth Dasychira pudibunda L. (Lepidoptera: Lymantriidae), known as a beech pest in Europe, primarily inflicts damage on beech leaves. In the present study, we aim to reveal the genome characteristics of Dasychira pudibunda nucleopolyhedrovirus (DapuNPV-T1), which was detected for the first time in Turkey and compare it with the reference genome and other baculovirus genomes. The DapuNPV-T1 genome was determined to be a circular, double-stranded DNA molecule with 136,920 bp and a nucleotide distribution of 54.4% G + C. Bioinformatics analysis showed that the DapuNPV-T1 genome contains 163 open reading frames with more than 150 nucleotides. Fifty-four ORFs of unknown function, 6 homologous regions (hrs), 1 AC-rich region, and 3 bro genes (bro-a, bro-b, and bro-c) were determined in the genome sequence. Comparative analysis with other baculovirus strains revealed distinctions in the DapuNPV-T1 genome based on ORF. The gene parity plot and phylogenetic analysis confirmed that DapuNPV-T1 belongs to the alphabaculovirus group Ib. In addition, the DapuNPV-T1 isolate was found to be close to the nucleopolyhedrovirus Poland isolate in Dasychira pudibunda and Orgyia pseudotsugata multiple nucleopolyhedrovirus. With this study, the first genome analysis of DapuNPV from Turkey became the second in the world to enter the literature. Comprehensive information on a wide range of isolates will provide a more detailed overview of baculoviruses and help overcome their shortcomings as biocontrol agents.

Identification of the potential matrix protein of invertebrate iridescent virus 6 (IIV6).

Invertebrate iridescent virus 6 (IIV6) is a nucleocytoplasmic virus with a ∼212 kb linear dsDNA genome that encodes 215 putative open reading frames (ORFs). Proteomic analysis has revealed that the IIV6 virion consists of 54 virally encoded proteins. Interactions among the structural proteins were investigated using the yeast two-hybrid system, revealing that the protein of 415R ORF interacts reciprocally with the potential envelope protein 118L and the major capsid protein 274L. This result suggests that 415R might be a matrix protein that plays a role as a bridge between the capsid and the envelope proteins. To elucidate the function of 415R protein, we determined the localization of 415R in IIV6 structure and analyzed the properties of 415R-silenced IIV6. Specific antibodies produced against 415R protein were used to determine the location of the 415R protein in the virion structure. Both western blot hybridization and immunogold electron microscopy analyses showed that the 415R protein was found in virions treated with Triton X-100, which degrades the viral envelope. The 415R gene was silenced by the RNA interference (RNAi) technique. We used gene-specific dsRNA's to target 415R and showed that this treatment resulted in a significant drop in virus titer. Silencing 415R with dsRNA also reduced the transcription levels of other viral genes. These results provide important data on the role and location of IIV6 415R protein in the virion structure. Additionally, these results may also shed light on the identification of the homologs of 415R among the vertebrate iridoviruses.

Isolation and characterization of a high-efficacy Beauveria bassiana strain from the European tent caterpillar, Malacosoma neustria Linnaeus (Lepidoptera: Lasiocampidae).

The European tent caterpillar, Malacosoma neustria Linnaeus (Lepidoptera: Lasiocampidae), is a worldwide known pest that causes significant losses by feeding on many woody and shrub-like plants during the larval period. In this study, a Beauveria bassiana strain isolated from M. neustria larvae and characterized according to its morphological and molecular properties. Its insecticidal activity was tested on M. neustria larvae. Sequence results of partial ITS (ITS1-5.8S-ITS2) gene region identified the isolate (named as Mn1) as a B. bassiana strain. Phylogenetic analysis showed that Mn1 strain displays close similarity to B. bassiana ARSEF 1848 and ARSEF 751 isolates. According to the concentration-responce tests performed using 1 × 103 - 1 × 109 conidia per mL concentrations, LC50 value of the new strain was calculated as 1 × 105 conidia per mL within 7 days against the larvae of M. neustria in the laboratory conditions. B. bassiana Mn1 strain, characterized in this study, is recorded as the first entomopathogenic fungus isolated from M. neustria, and the results showed that new strain of B. bassiana has a serious potential to be utilized as a good biocontrol agent against M. neustria.

Genome Sequencing and Organization of Three Geographically Different Isolates of Nucleopolyhedrovirus from the Gypsy Moth Reveal Significant Genomic Differences.

Background: The gypsy moth (Lymantria dispar L., Lepidoptera: Erebidae) is a worldwide pest of trees and forests. Lymantria dispar nucleopolyhedrovirus (LdMNPV) belongs to the Baculoviridae family and is an insect virus specific to gypsy moth larvae. In this study, we describe the complete genome sequences of three geographically diverse isolates, H2 (China), J2 (Japan), and T3 (Turkey), of Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV). Methods: The genomes of isolates H2, J2, and T3 were subjected to shotgun pyrosequencing using Roche 454 FLX and assembled using Roche GS De Novo Assembler. Comparative analysis of all isolates was performed using bioinformatics methods. Results: The genomes of LdMNPV-H2, J2, and T3 were 164,746, 162,249, and 162,614 bp in size, had GC content of 57.25%, 57.30%, and 57.46%, and contained 162, 165, and 164 putative open reading frames (ORFs ≥ 150 nt), respectively. Comparison between the reference genome LdMNPV-5/6 (AF081810) and the genomes of LdMNPV-H2, J2, and T3 revealed differences in gene content. Compared with LdMNPV-5/6, ORF5, 6, 8, 10, 31, and 67 were absent in LdMNPV-H2, ORF5, 13, and 66 were absent in LdMNPV-J2, and ORF10, 13, 31, and 67 were absent in LdMNPV-T3. In addition, the gene encoding the mucin-like protein (ORF4) was split into two parts in isolates H2 and T3 and designated ORF4a and ORF4b. Phylogenetic analysis grouped isolates H2 and J2 in a different cluster than isolate T3, which is more closely related to the Turkish and Polish isolates. In addition, H2 was found to be closely related to a South Korean LdMNPV isolate. Conclusion: This study provided a more detailed overview of the relationships between different geographic LdMNPV isolates. The results showed remarkable differences between groups at the genome level.

Genome sequence analysis and organization of the Hyphantria cunea granulovirus (HycuGV-Hc1) from Turkey.

The fall webworm (Hyphantria cunea) impacts a wide variety of crops and cultivated broadleaf plant species. The pest is native to North America, was introduced to Europe and has since spread further as far as central Asia. Despite several attempts to control its distribution, the pest continues to spread causing damage all over the world. A naturally occurring baculovirus, Hyphantria cunea granulovirus (HycuGV-Hc1), isolated from the larvae of H. cunea in Turkey appears to have a potential as microbial control agent against this pest. In this report we describe the complete genome sequence and organization of the granulovirus isolate (HycuGV-Hc1) that infects the larval stages and compare it to other baculovirus genomes. The HycuGV-Hc1 genome is a circular double-stranded DNA of 114,825 bp in size with a nucleotide distribution of 39.3% G + C. Bioinformatics analysis predicted 132 putative open reading frames of (ORFs) ≥ 150 nucleotides. There are 24 ORFs with unknown function. Seven homologous repeated regions (hrs) and two bro genes (bro-1 and bro-2) were identified in the genome. Comparison to other baculovirus genomes, HycuGV-Hc1 revealed some differences in gene content and organization. Gene parity plots and phylogenetics confirmed that HycuGV-Hc1 is a Betabaculovirus and is closely related to Plutella xylostella granulovirus. This study expands our knowledge on the genetic variation of HycuGV isolates and provides further novel knowledge on the nature of granuloviruses.

Biochemical and Molecular Characterizations of a Novel pH- and Temperature-Stable Pectate Lyase from Bacillus amyloliquefaciens S6 for Industrial Application.

In this paper, we report cloning of a pectate lyase gene from Bacillus amyloliquefaciens S6 (pelS6), and biochemical characterization of the recombinant pectate lyase. PelS6 was found to be identical with B. subtilis 168 pel enzyme with 100% amino acid sequence homology. Although these two are genetically very close, they are distinctly different in physiology. pelS6 gene encodes a 421-aa protein with a molecular mass of 65,75 kDa. Enzyme activity increased from 12.8 ± 0.3 to 49.6 ± 0.4 units/mg after cloning. The relative enzyme activity of the recPel S6 ranged from 80% to 100% at pH between 4 and 14. It was quite stable at different temperature values ranging from 15 to 90 °C. The recPEL S6 showed a maximal activity at pH 10 and at 60 °C. 0.5 mM of CaCl2 is the most effective metal ion on the recPEL S6 as demonstrated by its increased relative activity with 473%. recPEL S6 remained stable at - 20 °C for 18 months. In addition recPEL S6 increased juice clarity. This study introduces a novel bacterial pectate lyase enzyme with its characteristic capability of being highly thermostable, thermotolerant, and active over a wide range of pH, meaning that it can work at both acidic and alkaline environments, which are the most preferred properties in the industry.

Complete genome sequence analysis of the Malacosoma neustria nucleopolyhedrovirus from Turkey.

The lackey moth, Malacosoma neustria (Linnaeus, 1758), a worldwide pest, causes extensive economic losses particularly on hazelnut, plum, oak, poplar, and willow trees. A baculovirus, Malacosoma neustria nucleopolyhedrovirus (ManeNPV-T2), has been isolated from the larvae collected in Turkey and appears to have a potential as a microbial control agent. In this study, we describe the complete genome sequence of ManeNPV-T2 and compare it to other sequenced baculovirus genomes. The ManeNPV-T2 genome is a circular double-stranded DNA molecule of 130,202 bp, has 38.2% G + C, and is predicted to contain 131 putative open reading frames (ORFs) each with a coding capacity of more then 50 amino acids. There are 27 ORFs with unknown function of which 6 are unique to ManeNPV-T2. Eleven homologous regions (hrs) and two bro genes (bro-a and bro-b) were identified in the genome. There are two homologues of chaB and nicotinamide riboside kinase-1 genes, separated from themselves with a few nucleotides. Additionally, ac145, thought to be per os infectivity factor (pif) gene, is also found as two homologues. All 38 core genes are found in the ManeNPV-T2 genome. The phylogenetic tree of ManeNPV-T2 in relation to 50 other baculoviruses whose genomes have been completely sequenced showed ManeNPV-T2 to be closely related to the group II NPVs. This study expands our knowledge on baculoviruses, describes the characterization ManeNPV, and ultimately contributes to the registration of this virus as a microbial pesticide.