A new Helicoverpa armigera Nucleopolyhedrovirus isolate from Heliothis peltigera (Denis & Schiffermuller) (Lepidoptera: Noctuidae) in Turkey

This study reports a new Helicoverpa armigera nucleopolyhedrovirus (NPV) isolated from Heliothis peltigera (Denis & Schiffermuller), collected in the vicinity of Adana, Turkey. Infection was confirmed by tissue polymerase chain reaction and sequence analysis. Results showed that dead H. peltigera larvae contain Helicoverpa armigera nucleopolyhedrovirus. Thus, the isolate was named as HearNPV-TR. Microscopy studies indicated that occlusion bodies were 0.73 to 1.66 μm in diameter. The nucleocapsids are approximately 184 × 41 nm in size. The genome of HearNPV-TR was digested with KpnI and XhoI enzymes and calculated as 130.5 kb. Phylogenetic analysis showed that HearNPV-TR has close relation with the H. armigera SNPV-1073 China isolate. The Kimura analysis confirmed that the isolate is a variant of H. armigera NPV. Bioassays were performed using six different concentrations (1 × 103 to 1 × 108 occlusion bodies (OBs)/mL) on 2nd instar larvae of H. peltigera, H. armigera, Heliothis viriplaca, Heliothis nubigera. LC50 values were calculated to be 9.5 × 103, 1.9 × 104, 8.6 × 104 and 9.2 × 104 OBs/mL within 14 days, respectively. Results showed that it is a promising biocontrol agent against Heliothinae species.


Introduction
The bordered straw, Heliothis peltigera (Denis & Schiffermuller) is one of the most serious pests infesting various economic crops such as safflower, cotton, soybean, corn, bean, and herbs (Manjunath et al., 1976;Iqbal and Mohyuddin, 1990;Saeidi and Adam, 2011). H. peltigera is distributed in the paleosubtropical regions (Hacker, 1989); however, as a result of climatic changes, such as global warming, it is currently becoming a major problem in the European countries (Ragionieri et al., 2017). Cultural methods, natural enemies, and some chemical agents are used in the control of H. peltigera (Simoglou et al., 2017). The application of cultural control methods and natural enemies may be insufficient in epidemics of pest. Additionally, chemical pesticides damage nontarget organisms and environment, and it causes increase of insect resistance against chemicals (Ffrench-Constant et al., 2004).
Baculoviruses are important biocontrol agents of insect pests in agriculture due to their narrow host range, persistence in the environment, being eco-friendly, and having high virulence (Fuxa, 2004;Wang et al., 2005). They are divided into two groups as nucleopolyhedrosis viruses (NPV) and granulosis viruses (GV) according to their polyhedrin and granule morphology (Ackermann and Smirnoff, 1983). Molecular classification of baculoviruses is based on the use of conserved gene regions of polyhedrine (polh), late expression factor 8 (lef8), and late expression factor 9 (lef9) (Jehle et al., 2006).
Baculoviruses are used broadly as bio-insecticides to control Heliothinae species in various vegetables and cotton (Ignoffo, 1973;Cunningham, 1998;Yang, 2012;Mironidis et al., 2013). At present, there are many registered products belonging to Helicoverpa armigera and Helicoverpa zea nucleopolyhedroviruses in the world (Erlandson et al., 2007;Yang, 2012). They are both used against their own hosts and other Heliothinae species (Beas-Catena et al., 2014). However, application of exotic virus isolates may cause adverse effects on native isolates (Munoz et al., 1997). Moreover, native isolates can be more pathogenic as per exotic isolates against local populations (Barrera et al., 2011;Cabodevilla et al., 2011). For these reasons, it is important to identify native isolates that indicate high virulence in each geographical region and use them against native pests (Figueiredo et al., 2009).
In this study, a novel H. armigera NPV isolate was recorded the first time in H. peltigera host collected from safflower fields in Adana, Turkey. So far, no record of microbial control agent has been mentioned from this pest. Morphological and molecular features of this isolate were determined and insecticidal activity tests, using various concentrations, were performed against all larval stages of four Heliothinae species (H. peltigera, armigera, Heliothis viriplaca, Heliothis nubigera) distributed in Turkey. Results showed that HearNPV-TR isolate is a promising biocontrol agent that could be used to control Heliothinae species.

Virus detection and propagation
In July 2014, infestations of safflower crops by H. peltigera were observed in the vicinity of Adana, Turkey. Symptoms of larval feeding damage were observed on capitulum of safflower plants. A total of 357 larvae were collected from the field and brought to the laboratory. During laboratory rearing, five larvae died in a short time with typical baculovirus infection signs. Dead larvae were checked for the presence of baculovirus occlusion bodies (OBs) under light microscope (Nikon eclipse E600).
Tissue PCR was performed to confirm the presence of baculovirus infections molecularly. Dead larvae were homogenized in sterile water and filtered through cheesecloth to remove the larval debris. The filtered OBs were purified as described by Munoz et al. (1997). OBs were dissolved using 0.1 M NaOH (pH 12.5) and subsequently, neutralized with 100 mM Tris-HCl (pH 8). The Phire Animal Tissue Direct PCR Kit (Thermo Fisher Scientific, Lithuania) was used to amplify the conserved regions of the baculovirus lef8, lef9, and polh genes. These genes were identified in all completely sequenced baculovirus genomes. Thus, they were detected in baculovirus phylogeny analysis (Jehle et al., 2006). The degenerate primer sets of these genes are shown in Table  1. Tissue-PCR reaction mixture was prepared following the manufacturer's standard protocol for animal tissues. Amplified PCR products were sequenced by Macrogen Company (the Netherlands). Sequences were first blasted for alignment of the baculoviruses in NCBI (The National Center for Biotechnology Information) web site, subsequently registered to the GenBank.
The virus was propagated freshly in the laboratory in healthy H. peltigera larvae. Insects were infected individually with diet (Poitout and Bues, 1974) discs contaminated with 10 µL virus suspension in a concentration of 10 5 OBs/mL and maintained at 25 °C to develop infection. The OBs were purified from infected larvae as described by Ishii et al. (2003) and stored at -20 °C.

Electron microscopy studies
OB morphology was studied under scanning electron microscope and transmission electron microscope. Twenty-five microliters of the OB suspension was spread on a stub and dried at 37 °C for 16 h. The stub was covered with gold dust (Quorum Technology SC7620-CF) and examined to measure the sizes of OBs (Agilent Technologies).
For transmission electron microscopic examination, previously isolated OBs were washed twice with PBS and precipitated by high-speed centrifugation. The precipitated OB pellet was fixed in a fixative containing 2% glutaraldehyde, 2% paraformaldehyde in a 0.05 M pH 7.2 cacodylate buffer + 0.001 M CaCl 2 for 2 h, postfixed in 1% OsO 4 in the same fixative for 1 h, and subsequently embedded in resin. Ultrathin sections were obtained from OB blocks embedded in resin using an Ultracut S Ultramicrotome (Reichert, Munich, Germany). The sections, stained in 1% uranyl acetate and 1% lead citrate, were examined with a transmission (JEOL JEM 1220) electron microscope.

DNA extraction and restriction endonuclease profile
Viral DNA was extracted according to the method described by Reed et al. (2003) and dialyzed for 24 h at 4 °C against a 0.1 × TE buffer (10mM Tris/HCl, 1mM EDTA, pH 7.5) following phenol:chloroform:isoamyl alcohol extraction. The buffer was changed every 12 h. The extracted DNA was quantified using a spectrophotometer (Thermo Scientific, NanoDrop 2000).
To determine the restriction profile of viral genome, 2 µg of DNA was digested with KpnI and XhoI enzymes for 4 h at 37 °C. The digested fragments were separated in 0.6% agarose gel at 15 V for 16 h. The agarose gel was then stained with ethidium bromide for 15 min and analyzed under UV light.

Phylogeny and Kimura-2 parameter analysis
In order to show the position of the HearNPV-TR among the other Heliothinae NPVs in GenBank, Kimura-2 parameter analysis was performed. For this reason, polh, lef8, and lef9 gene sequences of all isolates were concatenated to obtain a single phylogenetic tree of these genes. The nucleotide sequences were assembled and edited using BioEdit version 7.0.5.3 (Hall, 1999). The MEGA program (version 6.0) and the maximum composite likelihood method with 1000 bootstrap replicates were used for phylogenetic analysis. H. armigera granulosis virus isolate was used as an outgroup. Distance matrices between HearNPV-TR and other Heliothinae species were determined for partial lef8, lef9, and polh sequences using the pairwise distance calculation of MEGA version 6.0 applying the Kimura-2 parameter model. H. armigera granulosis virus, Trichoplusia ni nucleopolyhedrovirus, and Pseudoplusia includens nucleopolyhedrovirus were also included in the Kimura-2 parameter analysis.

Biological tests
H. peltigera and H. armigera larvae were collected from safflower and sunflower fields, respectively, in Çukurova Region of Turkey. Heliothis viriplaca and Heliothis nubigera larvae, which are legume pests, were collected from the chickpea and lentil fields in the Southeastern Anatolia Region of Turkey, respectively. A generation of each larvae were reared to use for insecticidal tests and to determine the host range of HearNPV-TR in a climate cabinet at 26 °C and 65% humidity. Six different virus concentrations (1 × 10 3 , 1 × 10 4 , 1 × 10 5 , 1 × 10 6 , 1 × 10 7 , 1 × 10 8 OBs/mL -1 ) were used for biological tests. Second instar of H. peltigera, H. armigera, H. viriplaca, and H. nubigera larvae were infected with virus contaminated semisynthetic diets. For the control group, sterile water was applied on diet. Thirty larvae were used for each test and the experiments were repeated 3 times. Mortality was assessed daily for 14 days. The mortality rates were determined by Abbott's formula (Abbott, 1925), and the LC 50 values were determined using probit regression analysis in SPSS (24.0 version).

Virus detection and microscopy
Light microscopy examinations of dead H. peltigera larvae, collected from safflower fields in Adana, Turkey, showed typical baculovirus OBs as bright crystals. To verify baculovirus infection, partial sequences of lef8, lef9, and polh genes were amplified by tissue PCR. Amplified products of the expected sizes (lef8: 800 bp, lef9: 350 bp, and polh: 420 bp) were sequenced. Blast analysis of sequence results showed that deaths of the H. peltigera larvae were due to H. armigera nucleopolyhedrovirus infection. Thus, we have designated the isolate as HearNPV-TR. The sequences were registered to the GenBank and the accession numbers for lef8 (MG870624), lef9 (MG870625), and polh (MH161372) genes were obtained.
Scanning electron microscopy studies showed that OBs were irregularly shaped and ranged in size from 0.73 to 1.66 μm (Figure 1a). Transmission electron microscopy examinations of HearNPV-TR OBs demonstrated several virions with a single nucleocapsid packaged within a single viral envelope and confirmed that HearNPV-TR was a single nucleocapsid-type virus (SNPV). The dimensions of rod-shaped nucleocapsids were approximately 184 × 41 nm (Figure 1b).

Restriction endonuclease profile
The DNA of the HearNPV-TR, digested with KpnI and XhoI restriction enzymes, yielded six and seven fragments, respectively. These fragments were designated from A to G on Figure 2. Lambda (λ) DNA, digested with NarI and HindIII enzymes, was used as a molecular size marker. The sizes of the fragments relative to λ DNA/NarI and λ DNA/ HindIII fragments are given in Table 2. Genome size of HearNPV-TR was calculated to be nearly 130.5 kbp.

Phylogeny and Kimura-2 parameter analysis
Phylogenetic analysis was performed to indicate the relationship of HearNPV-TR isolated from H. peltigera with the other Heliothinae NPVs (Table 3). The phylogenetic tree showed close relation of HearNPV-TR with H. armigera SNPV-1073 China isolate (Figure 3).
Kimura-2 parameter analysis of nucleotide distances indicated that the viral isolate in this study is a H. armigera NPV isolate. If the value of the nucleotide locus distances is less than 0.015, two baculoviruses are considered to be the same species and more than 0.050 are considered to be different species (Jehle et al., 2006). Distances between HearNPV-TR and other HearNPV isolates in GenBank were less than 0.015. However, distances between H. armigera GV and the other NPVs were greater than 0.050 (Table 4).

Discussion
In this study, we detected a nucleopolyhedrovirus infection in H. peltigera larvae. As baculovirus isolates were named according to the host from which they were isolated, the name of this viral isolate was originally thought to be H.   (Eroglu et al., 2018). However, the previous one was determined as a multinucleopolyhedrovirus. Therefore, the current virus isolate is totally different than the previous isolate. Electron microscopy studies are important for the morphological identification of baculovirus isolates. Scanning electron microscope results indicated HearNPV-TR OBs have sizes between 0.73 and 1.66 μm. When these OB sizes were compared to the OB sizes of other Heliothinae NPV isolates in the literature, it was obvious that HearNPV-TR has OB sizes smaller than those of Indian isolates (Somasekar et al., 1993) and China isolate (Tang et al., 2012). Indian isolates have OB sizes changing between 1.6 and 2.4 mm. China isolate has an OB size of approximately 2 mm. However, OB sizes of HearNPV-TR is larger than the OB size of Argentinian isolate (Ferrelli et al., 2016) which is between 0.6 and 1.2 mm. On the other hand, previous Turkish isolate, H. armigera NPV-O1, has OB sizes between 0.85 and 1.25 μm (Eroglu et al., 2018).
TEM results revealed that HearNPV-TR has nucleocapsid sizes of 184 nm in length and 41 nm in width.    Nucleocapsid sizes were also compared with the other Heliothinae NPV isolates in the literature. This comparison showed that HearNPV-TR has smaller nucleocapsid size than those of India isolate (Sridhar et al., 2011), China isolate (Tang et al., 2012), and previous Turkish isolate (Eroglu et al., 2018) which have nucleocapsid lengths and widths of 277 × 41 nm, 230 × 50 nm, and 279 × 56 nm, respectively. These results showed that the nucleocapsid size of HearNPV-TR isolate is significantly smaller than those of the other HearNPV isolates. This result suggested that nucleocapsid sizes varied between isolates obtained from different geographical regions. HearNPV-TR was further characterized by restriction endonuclease digestion of the viral DNA. The size of the genome digested with KpnI and XhoI enzymes calculated to be approximately 130.5 kbp. This size is in accordance with the size of the viral genomes reported for Helicoverpa isolates in GenBank. Digestion with the same restriction enzymes was also reported for HearNPV China isolate (Chen et al., 2000). China isolate has similar restriction profile with HearNPV-TR for KpnI digestion, but digestion with XhoI enzyme produced 7 fragments for HearNPV -TR, 9 fragments for HearNPV China isolate. However, the genome size of HearNPV China isolate (130.1 kb) is almost at the same size with HearNPV-TR genome (130.5 kb).
To see the position of HearNPV-TR between the other Heliothinae species in the GenBank, a phylogenetic tree was constructed using concatenated sequences of partial lef8, lef9, and polh genes. In the tree, HearNPV-TR was located near H. armigera SNPV-1073 China isolate (Rowley et al., 2011). Studies performed in the literature showed that Helicoverpa NPVs obtained from different hosts of Helicoverpa species are actually reported to be variants of each other (Jehle et al., 2006;Rowley et al., 2011;Wennmann et al., 2018). Thus, we concluded that HearNPV-TR was a H. armigera NPV variant. This result was also confirmed by the result of Kimura-2 parameter analysis performed by concatenated sequences of conserved partial genes (lef8, lef9, and polh).
The LC 50 values of these Heliothinae species were obtained as 9.5 × 10 3 , 1.9 × 10 4 , 8.6 × 10 4 , and 9.2 × 10 4 OBs/ mL, respectively. Kumar et al. (2011) also mentioned a similar result for their Indian HearNPV isolate which had produced a LC 50 value of 2.3 × 10 4 OBs/mL against the 2nd instar H. armigera larva. Furthermore we have found that HearNPV-TR is highly effective against all Heliothinae species found in Turkey (H. peltigera, H. viriplaca, and H. nubigera). According to these results, it is possible to conclude that HearNPV-TR isolate is an encouraging biological control agent against Heliothinae species.