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The cotton bollworm, Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae), is a major pest are agricultural crops, such as cotton, horticultural plants, a range of vegetable crops and forest trees. Many dead and live larvae and pupa samples of H. armigera, an extremely polyphagous pest, were collected from Adana in Turkey. It is observed that cadaveric integuments were fragile, and their body tissues were also liquefied. Studies with phase-contrast microscopy revealed a very intense baculovirus infection in cadavers. Transmission electron microscopy exhibited that the new baculovirus had one nucleocapsid in an envelope in the occlusion bodies. Therefore, the isolate was named Helicoverpa armigera single nucleopolyhedrovirus-D (HearSNPV-D). The phylogenetic analysis according to the polyhedrin and late expression factor-8 genes appeared that the new isolate was in close relationship with HearSNPV China and Turkey isolate. The new isolate was tested on its own host Helicoverpa armigera and six different host insects including Hyphantria cunea, Malacosoma neustria, Lymantria dispar, Thaumetopea pityocampa and Spodoptera exigua larvae. Bioassay studies showed that HearSNPV-D was virulent to H. armigera and had a narrow host range. The LC50 value was determined as 0.7 × 105 and 1.9 × 107 OBs/ml for H. armigera and S. exigua, respectively. As a result of this study, the new HearSNPV-D isolate can be used for the control of H. armigera. Non-significant mortality was observed in non-host species by HearSNPV-D, which may reveal the highly specific nature of HearSNPV-D. Therefore, HearSNPV-D may be successful as a highly specific, environmentally friendly management tool for H. armigera.

The aim of this study, conducted in 2018 and 2020, was to investigate the bioactivity of a local baculovirus isolate, Hyphantria cunea granulovirus (HycuGV), in seven lepidopteran pests. Based on data collected 10 days after exposure, HycuGV was found to infect Malacosoma neustria (L., 1758) (Lepidoptera: Lasiocampidae), Lymantria dispar (L., 1758) (Lepidoptera: Erebidae), Helicoverpa armigera (Hubner, 1805) (Lepidoptera: Noctuidae) and Spodoptera exigua (Hubner, 1808) (Lepidoptera: Noctuidae) larvae as well as its host Hyphantria cunea (Drury, 1773) (Lepidoptera: Erebidae). However, it did not infect Spodoptera littoralis (Boisduval, 1883) (Lepidoptera: Noctuidae) and Cydia pomonella (L., 1758) (Lepidoptera: Tortricidae). A HycuGV dose rate experiment indicated LC50 of 4.7x10(5) occlusion bodies (OBs)/ml in H. cunea, 5.6x10(6) OBs/ml in L. dispar, 7x10(7) OBs/ml in S. exigua, 1.5x10(9) OBs/ml in M. neustria and 7.7x10(9) OBs/ml in H. armigera. HycuGV was infectious to S. exigua and L. dispar, but only provided effective control in M. neustria and H. armigera at high dose rates. These findings demonstrate that HycuGV can be highly effective for control of S. exigua, L. dispar and H. cunea.

Background Chitin, a long-chain polymer of N-acetylglucosamine, is a major structural component of the insect exoskeleton and the peritrophic membrane (PM). Chitinases are able to effectively break down glycosidic bonds of chitin polymer thus can be used in agriculture to control plant pathogen insects. These enzymes can be synthesized by higher plants, animals, protista, bacteria, and viruses. Results In this study, viral and bacterial chitinases were compared for their potential activity on a laboratory test insect. The genes encoding chitinases of Autographa californica nucleopolyhedrovirus (AcNPV) and Cydia pomonella granulovirus (CpGV) were amplified from genomic DNAs by PCR and cloned into the pET-28a (+) expression vector. The chitinase proteins of these 2 viruses (AcNPV-Chi, CpGV-Chi) and Serratia marcescens chitinase C (ChiC) protein which was previously cloned were overexpressed in Escherichia coli. Expressed proteins were purified and confirmed by western blot analysis as 50, 63, and 68 kDa for AcNPV, CpGV, and S. marcescens chitinases, respectively. Enzyme activities of the chitinases were confirmed. Chitinases were also compared to each other in silico. The insecticidal effects of these proteins were evaluated on Galleria mellonella L. larvae. Bioassays were performed on the 3rd instar larvae for each chitinase protein in triplicate. The results showed that although there were differences in enzymatic activities and domain organizations, all 3 microbial chitinases produced almost the same level of insecticidal activity on the test insect. LC50 and LT50 values were compatible with the mortality results. These results were a preanalysis for comparing the effects of microbial chitinases. Conclusion Potential activity experiments should be carried out on more insects to provide detailed information on the insecticidal effects of bacterial and viral chitinases.

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 degrees C. The recPEL S6 showed a maximal activity at pH 10 and at 60 degrees 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 degrees 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.

The gypsy moth (Lymantria dispar L., Lepidoptera: Lymantriidae) is one of the most serious pest of various forestal, food and industrial crops worldwide. We have characterized a new Lymantria dispar nucleopolyhedrovirus (LdMNPV-T4) variant, which was isolated from dead L. dispar larvae in Turkey. Scanning electron microscope observations showed that the polyhedral occlusion bodies (OBs) of the LdMNPV-T4 were irregularly shaped. Transmission electron microscopy revealed that OBs of LdMNPV-T4 were occupied with several virions in which multiple nucleocapsids packaged by viral envelope. Restriction analysis of the LdMNPV-T4 DNA purified from the viral inclusion bodies yielded BamHI, BglII, EcoRI and HindIII fragments. The mean size estimated for the complete LdMNPV-T4 genome was calculated to be 163.3 kb. Phylogenetic analysis of amplified polh, lef-8 and lef-9 sequences showed its relation to the other NPVs from Lymantria species. Mortality values of the LdMNPV-T4 at four different concentrations ag...

Omurgasız iridesan virüs 6 (IIV6), çeşitli zararlı böcek türlerini düşük oranda enfekte edebilir. Rekombinant DNA teknolojisi ile oluşturulan yeni özelliklere sahip virüsler biyolojik kontrol ajanları olarak etkin bir şekilde kullanılabilir. Daha önce IIV6 157L ORF’si yerine birisi yeşil floresan protein geni (rCIV-Δ157L-gfp) ve diğeri de gfp ile birleştirilmiş bir akrep Androctonus australis (Linnaeus, 1758) böcek toksin geni (rCIV-Δ157L/gfp-AaIT) ihtiva eden iki rekombinant IIV6 inşa edilmişti. Bu çalışmada, yaban tip IIV6 ve iki rekombinant virüs, Helicoverpa armigera (Hübner, 1805) (Lepidoptera: Noctuidae), Spodoptera littoralis (Boisduval, 1883) (Lepidoptera: Noctuidae), Lymantria dispar (Linnaeus, 1758) (Lepidoptera: Erebidae), Euproctis chrysorrhoea (Linnaeus, 1758) (Lepidoptera: Erebidae) ve Tenebrio molitor (Linnaeus, 1758) (Coleoptera: ‎Tenebrionidae) larvalarında enfeksiyon oluşturma yeteneği açısından değerlendirildi. Çalışma 2018 ve 2019 yılları arasında Karadeniz Teknik Üniversitesi Biyoloji Bölümü'nde gerçekleştirildi. Her bir böcek larvasını enjekte etmek için virüslerin beş farklı konsantrasyonu (103, 104, 105, 106 ve107 TCID50/ml) kullanıldı. rCIV-Δ157L/gfp-AaIT ile enfekte olmuş S. littoralis dışında tüm larvalar felç oldu. rCIV-Δ157L/gfp-AaIT ile enfekte olmuş böcek larvalarının LC50'si sırasıyla, S. littoralis, T. molitor, L. dispar, H. armigera ve E. chrysorrhoea üzerinde 0.3 x 107, 0.7 x 105, 0.2 x 105, 0.15 x 105, 0.7 x 104 TCID50/ml olarak belirlenmiştir. En yüksek virüs konsantrasyonlarına göre hesaplanan LT50 değerleri, rCIV-Δ157L/gfp-AaIT için S. littoralis, T. molitor, L. dispar, H. armigera ve E. chrysorrhoea'da sırasıyla 10.5, 6.2, 4.7, 7.5 ve 5 gün olarak bulundu. Bu çalışma, rekombinant IIV6'nın Lepidoptera ve Coleoptera takımlarına ait bazı böceklerde patojeniteyi artırdığını göstermiştir., Invertebrate iridescent virus 6 (IIV6) can infect a broad range of pest insect species. Viruses with new features created by recombinant DNA technology can be used effectively as biological control agents. Previously, recombinants have been constructed: IIVs harboring green fluorescent protein gene (gfp) in place of IIV6 157L ORF (rCIV-Δ157L-gfp) and a scorpion Androctonus australis (Linnaeus, 1758) insect toxin gene (AaIT) fused with gfp (rCIV-Δ157L/gfp-AaIT). In this study, wild type IIV6 and the two recombinants, were evaluated for their ability to cause infections on Helicoverpa armigera (Hübner, 1805) (Lepidoptera: Noctuidae), Spodoptera littoralis (Boisduval, 1883) (Lepidoptera: Noctuidae), Lymantria dispar (Linnaeus, 1758) (Lepidoptera: Erebidae), Euproctis chrysorrhoea (Linnaeus, 1758) (Lepidoptera: Erebidae) and Tenebrio molitor (Linnaeus, 1758) (Coleoptera: ‎Tenebrionidae) larvae. This study was performed at Karadeniz Technical University, Department of Biology during 2018 and 2019. Five different concentrations (103, 104, 105, 106 and 107 TCID50/ml) of viruses were used to inject each insect larvae. All larvae, infected with rCIV-Δ157L/gfp-AaIT, became paralyzed, except S. littoralis. The LC50 of insect larvae infected by rCIV-Δ157L/gfp-AaIT were determined as 0.3 x 107, 0.7 x 105, 0.2 x 105, 0.15 x 105, 0.7 x 104 TCID50/ml on S. littoralis, T. molitor, L. dispar, H. armigera and E. chrysorrhoea, respectively. LT50 values, calculated according to the highest virus concentrations, were found as 10.5, 6.2, 4.7, 7.5 and 5 d on S. littoralis, T. molitor, L. dispar, H. armigera and E. chrysorrhoea, respectively, for rCIV-Δ157L/gfp-AaIT. This study showed that recombinant IIV6 has increased pathogenicity on some insects from Lepidoptera and Coleoptera.

Malacosoma neustria (Linnaeus, 1758) (Lepidoptera: Lasiocampidae) causes economic losses in apple, pear, plum, willow, oak and other economically important trees. In this study, an alphabaculovirus was isolated from the larval population of M. neustria from Samsun in the central Black Sea Region of Turkey between 2015 and 2016. Electron microscope analysis of occlusion bodies (OBs) obtained from dead larvae showed that the nucleocapsids of a new isolate (ManeNPV-T4) are multiply enveloped. The Kimura two-parameter analysis and the phylogenetic tree were performed based on concatenated nucleotide and amino acid sequences of the partial lef-8, lef-9 and polh genes from ManeNPV-T4 isolate compared to those of other 51 baculoviruses. Insecticidal activity tests against third instar M. neustria larvae produced 48 to 100% mortalities. The LC 50 of ManeNPV-T4 was 0.78 x 10 3 OBs/ml in M. neustria . Additionally, the isolate caused mortalities lower than 50% in Spodoptera exigua (Hubner, 1808) (Lepidoptera: Noctuidae) , Lymantria dispar (Linnaeus, 1758) (Lepidoptera: Erebidae) , Hyphantria cunea (Drury, 1773) (Lepidoptera: Arctiidae) and Helicoverpa armigera (Hubner, 1805) (Lepidoptera: Noctuidae) larvae. Consequently, the new nucleopolyhedrovirus is infectious on M. neustria larvae and other lepidopterans.

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.

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.

In this study, Lymantria dispar dispar larvae, collected from three different localities in Turkey, were examined for the presence of inclusion bodies under phase contrast and electron microscopes. Inclusion bodies from infected larvae were subjected to polymerase chain reaction using the conserved primers for polyhedrin (polh), late expression factor 8 (lef-8) and late expression factor 9 (lef-9) genes. Sequence analysis confirmed that larvae collected from the three different localities contained multiple nucleopolyhedrosis viruses (MNPVs). These isolates were designated LdMNPV-T1, LdMNPV-T2 and LdMNPV-T3. Phylogenetic analyses of these isolates were performed using target genes polh, lef-8 and lef-9. Restriction endonuclease analysis of the three geographic isolates with EcoRI and PstI enzymes demonstrated some differences existed among the isolates. According to the EcoRI profile, the mean estimated size for the complete genome of each isolate (LdMNPV-T1, LdMNPV-T2 and LdMNPV-T3) was calculated to be approximately 170, 153 and 170kb, respectively. Insecticidal activities of each isolate were tested on L.d.dispar larvae using four different viral concentrations between 10(3) and 10(6)OBs/ml. Results showed that the mortalities for LdMNPV-T1, -T2 and -T3 ranged between 13-53%, 47-100% and 46-93%, respectively. The LC50 and LC95 values of LdMNPV-T2 were not significantly different from the respective corresponding values of the other two isolates. However, isolate LdMNPV-T2 killed larvae with a LC50 value that was lower than the other two isolates. Our results suggested there are promising LdMNPV isolates in Turkey that can be used for microbial control of L.d.dispar larvae.