An environmentally safe and tolerant microbial insecticide utilizing Helicoverpa armigera single nucleopolyhedrovirus (HearNPV-TR)

Baculoviruses are significant biocontrol agents of pests in agriculture; however, ultraviolet light B (UV-B) and high temperature inactivate them in the environment within a short time. In this study, formulations of Helicoverpa armigera nucleopolyhedrovirus Turkey isolate (HearNPV-TR) were prepared. Crude virus, the oil-based formulations of the virus, and a commercial H. armigera NPV product were exposed to UV-B light and various temperatures (28, 35, and 42 °C) at different periods (0, 1, 3, 5 h.). While the UV-B application decreased the virulence of crude virus up to 32% after 5 h. exposure, it decreased the virulence of oil-based formulation by (15%). Similarly, the virulence of the crude virus by 70% at 42 °C, while was reduced by 10% in the formulations at the same temperature. Therefore, 5% oil formulation was enough to keep the virulence of the virus underexposed to UV-B and high temperatures. LC95 values were calculated as 1 × 108 OBs/ml−1 in concentration–response tests, and mortality rates were 90 and 92% in pot assays for commercial product and 5% oil formulation, respectively. The results showed that 5% oil formulation of HearNPV-TR was an important biocontrol preparation to protect virus virulence under suppressive environmental conditions.


Background
Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) larvae are polyphagous pests in agricultural products that cause economic losses to economic crops worldwide (Sivasupramaniam et al. 2007). Chemical insecticides have been used to control H. armigera; however, their uses are no longer favored due to the negative effects on the environment and human health, as well as insects' resistance build-up (Whalon et al. 2018). As an alternative to chemical pesticides for the control of H. armigera, baculoviruses have been preferred due to the narrow host range and being environmentally friendly (Wang et al. 2005). To date, about 1000 baculoviruses have been isolated from pests and more than 60 commercial products have been developed (Beas-Catena et al. 2014).
Although baculoviruses are important bio-pesticide material, they are inactivated shortly due to the high temperature and the UV light in the natural environment (Priyadharshini 2009). It is indicated that ultraviolet B (280-320 nm wavelength) in the sunlight is one of the important factors influencing the infectivity of baculovirus (Ignoffo et al. 1974). Therefore, it has been shown that some protective substances added to the crude virus increased the protection of the virus against suppressive environmental conditions (Shapiro et al. 2012). The clay-based materials contained in the solid formulations, like kaolin, decrease the infectivity of the inclusion body because it increases the pH. In this case, the formulation shortens the persistence of the virus and reduces its virulence. On the other hand, it has been shown that the oilbased formulations are more stable on the foliage against environmental factors, such as rain and wind, and have high shelf life due to low pH levels (Batista et al. 2001).
In this study, oil-based formulations of the H. armigera single nucleopolyhedrovirus (HearNPV-TR) isolated from Turkey were prepared to make the crude virus tolerant to UV-B and temperature. It was determined that the highest tolerance from formulations exposed to UV-B and heat at different times contained 5% oil. Concentration-response tests and pot assay were performed against H. armigera larvae. According to the results obtained, the tolerance of the crude virus to natural environmental conditions was retained by using an oil-based formulation.

Virus source, production, and purification
The HearNPV-TR was isolated in earlier studies (Eroglu et al. 2019) and obtained from the Department of Biology at Karadeniz Technical University, Trabzon, Turkey. Besides, whole-genome analysis of the HearNPV-TR isolate was determined (Eroglu et al. 2020). For virus production, H. armigera larvae were collected from a tomato crop in the vicinity of Gümüşhane, Turkey, between July and August 2018. Larvae were normalized at laboratory conditions for a generation before they were used in the bioassays. They were placed individually into plastic cups (25 ml) containing nearly100 mg of artificial diet (Nagarkatti and Prakash 1974). Approximately 100 3rd instar larvae were infected with the droplet method for virus production (Hughes et al. 1986). The infected larvae were incubated at 26 °C and 60% RH. One week after the infection, dead larvae were collected and transferred to a falcon tube and homogenized in dH 2 O at a ratio of 1: 2. The solution was filtered through cheesecloth and centrifuged at 5000 × g for 30 min. Pellet, suspended with dH 2 O was loaded on 30% sucrose and centrifuged at 5000 × g for 30 min. The pellet was re-suspended with dH 2 O. The concentration of the stock solution was determined to be 1 × 10 10 OBs/ml −1 on the Thoma's hemocytometer.

Oil-based formulation of HearNPV-TR
The oil-based formulations were prepared by mixing the 25 ml of the HearNPV-TR (1 × 10 10 OBs/ml −1 ) solution with 5, 7.5, and 10 ml of sunflower oil, and 1 ml of emulsifier (E323 soy lecithin). After that, 10 ml feeding stimulant (E422 glycerine), 1 ml of thickener (E415 xanthan gum), 0.5 ml of spreader sticker (Silwet L-77), 0.2 ml of optical brightener (calcofluor white M2R), and 1 ml of a physiological stress factor for larvae (E284 boric acid) were added and brought to 100 ml with sterile dH 2 O. The mixture was homogenized in a vortex, and the pH of the formulation was measured. This formulation was transferred into a glass bottle, sheltered from light, and stored at − 20 °C.

Exposure to ultraviolet B and heat assays
The UV-B lamp of 15 watts, 306 nm (Fotodyne Inc., Germany) was used for exposure to UV light in laboratory conditions. The crude HearNPV-TR, commercial HearNPV (Helicoverpa armigera NPV) product, 3 oilbased formulations (5, 7.5, and 10%) whose final concentrations were 1 × 10 9 OBs/ml −1 and control group (only dH 2 O) were exposed to 300 mm above the test dishes UV-B irradiation in 24 well plates for 0, 1, 3, and 5 h. Three replicates were prepared for each specimen. The infectivity of the crude virus, commercial product, and oil formulation after being exposed to UV-B were then applied against 30 3rd instar H. armigera larvae using the leaf disk bioassay method (Akhanaev et al. 2017). For this application, 10 µl of each solution was inoculated onto fresh 4 mm 2 diameter lettuce disks cut with a cork borer. Then, leaf disks were placed on the 24 well plates. Third instar H. armigera larvae were placed on each leaf disk and continued incubation at 26 °C. The crude HearNPV-TR, commercial product, 3 oil-based formulations, and control group were incubated in 24 well plates for 0, 1, 3, and 5 h. at 28, 35, 42 °C at 60% RH. Temperature values were selected according to the summer temperatures of the region where H. armigera is common. The specimens exposed to the temperature were inoculated to the larvae in the same bioassay conditions.

Concentration-response tests
The 5% oil-containing formulation was selected for the concentration-response test as it had a sufficient tolerance against UV-B and high temperature. In order to compare the infectivity of commercial products, 5% oil formulation, and control group, they were used in the leaf disk method. The concentrations of viral solutions were calculated by a Thoma's hemocytometer, and eight different concentrations (10 3 to 10 10 OBs/ml −1 ) were prepared. The control group was treated with dH 2 O only. Thirty larvae of H. armigera were used for each test, and the experiments were performed in three replicates. Infected larvae and control groups were reared on fresh lettuce leaves. Larvae were incubated at 26 °C and 60% RH. All groups were examined for a 2-week period. Mortality rates were calculated by Abbott's formula (Abbott 1925). The lethal concentration (LC 50 and LC 95 ) was determined by SPSS 24.0 software.

Pot assay
Lettuce seedlings in (80 × 29 × 20 cm) plastic oblong pots containing a mixture of sterile soil and peat (3: 2) were grown for 20 days before use. The experiment was carried out in a growth chamber under a 16 h light 8 h dark at 28 °C per day. After 20 days, 20 leaf lettuce plants were used for inoculation. The pots were covered with cheesecloth. The spreader sticker (0.5 ml, Silwet L-77) contained in the formulation was added to the control group to ensure that the droplets were evenly distributed to the leaf surface. All solutions were used according to LC 95 values (1 × 10 8 OBs/ml −1 ), which spread on leaf surfaces using a handheld sprayer bottle. Thirty larvae of 3rd instar H. armigera were placed in each pot.

Exposure to ultraviolet B and heat assays
The crude HearNPV-TR, oil formulations of the virus, and commercial products were exposed to UV-B light at different periods (0, 1, 3and 5 h.). After then, 3rd instar H. armigera larvae were infected with the solutions, and observed for 14 days. The mortality rates of larvae are indicated in Fig. 1a-d. Before UV-B irradiation, the mortality rate of the crude virus was detected as (98%) on larvae while oil formulations and the commercial product showed (100%) mortality against 3rd instar H. armigera larvae. After 1, 3, and 5 h. of exposure to UV-B irradiation, mortality rates of the crude virus on larvae were 52, 39, and 32%, respectively, while mortality values of the oil formulations and commercial products were above (85%) on the larvae even after 5 h. exposure. So, it was determined that the virulence effect of the crude HearNPV-TR decreased when exposed to UV-B light. However, the oil formulations protected the crude HearNPV-TR from the negative effects of UV-B light significantly (Fig. 1a). The crude virus, oil formulations, and commercial products were also exposed to 3 different temperatures (28, 35, and 42 °C) for 0, 1, 3, and 5 h. The temperatures of 28 and 35 °C did not cause an important change in the virulence effect of crude virus and formulations (Fig. 1b,  c). However, the virulence of the crude HearNPV-TR remarkably decreased at 42 °C. After exposing the crude virus to 42 °C for 1, 3, and 5 h, mortality rates on the larvae were calculated as 73, 70, and 68%, respectively. Furthermore, oil formulations and commercial products exposed to 42 °C showed (90%) mortality on the larvae even after 5 h (Fig. 1d). Results of UV-B and heat assays showed that 5% oil formulation was sufficient to protect the virus against environmental conditions. So, 5% oil formulation was selected for concentration-response tests and pot assays.

Pot assay
The LC 95 (1 × 10 8 OBs/ml −1 ) concentration of the products was used for the pot assay. The 5% oil formulation, commercial product, and control group were exposed to natural sunlight on 3rd instar H. armigera larvae. Mortality values of 5% oil formulation and the commercial  Commercial product 100% 1.5 × 10 4 1.1 × 10 8 2.0 2 5% oil formulation 100% 1.9 × 10 4 1.6 × 10 8 2.0 2 Fig. 3 Mortality rates of control group, 5% oil formulation and commercial product in lettuce seedlings against 3rd instar Helicoverpa armigera larvae Page 5 of 7 Eroglu and Demırbag Egyptian Journal of Biological Pest Control (2022) 32:53 product showed 92 and 90% protection, respectively, for 10 days (Fig. 3). The protection levels of the formulations and control group are shown in Fig. 4. In the pots, where the 5% oil formulation and commercial product were applied, the larvae began to die from 4th day and the lettuce seedlings were not damaged. However, in the control group, larvae were fed with lettuce seedlings, and consumption all seedlings were at the end of the 10th day.

Discussion
Up to now, the oil-based formulation has not been produced in the formulations against Heliothis species produced from other baculoviruses are proved highly tolerant to environmental factors (such as UV, heat, wind, and rain) (Shapiro, et al. 2012).
The most significant feature that should be in a formulation is long-term stability (Airenne et al. 2013). One of the factors, the pH value, significantly affects the shelf life of the baculovirus formulations. The pH of the solution prior to the addition of NPV should be 5-7. Because OBs structures dissolve in alkaline substrates, it causes virulence to decrease in a short time (Batista et al. 2001). Generally, cotton seed oil and corn oil were used in the oil-based baculovirus formulations (Shapiro et al. 2012). The sunflower oil was chosen to be used in this study because of the low cost. As well, some natural additives other than oil made the formulation more effective. In previous studies, the use of various feeding stimulants such as glycerol was found to be beneficial in the level of the effect of the virus. They were reported that glycerol facilitated consumption by insects as well as accelerated the death rate because it enhances the taste of the virus (Narendrakumar et al. 2017). Baculoviruses may be suspended with a thickening agent (xanthan gum) in liquid formulations; however, surfactants must also be included to aid in the fluidity (Ríos-Velasco et al. 2012). Besides, surfactant substances such as Silwet L-77 allow the formulation to spread evenly across the entire leaf surface (Camacho et al. 2015). The optical brighteners, especially calcofluor, tinopal, leucophor, and blankophor are both protect baculoviruses from UV and damage to the peritrophic membrane of insects. Thus, these substances are added to the formulations to accelerate baculovirus infection (Ibargutxi et al. 2008). In addition, boric acid used as a source of physiological stress in larvae increased the virulence (Bhutia et al. 2012).
An oil-based formulation prepared from the crude virus which would be relatively more resistant under abiotic conditions such as ultraviolet light, rain, wind, and temperature was purposed. Therefore, some natural ingredients such as glycerol, xanthan gum, Silwet L-77, calcofluor white M2R, and boric acid were added to the baculovirus origin and sunflower oil-based formulation.
The UV-B and temperature tolerance of 3 different oil formulations (5, 7.5, and 10%) against 3rd instar H. armigera larvae were determined. Besides, these data were compared to crude HearNPV-TR and commercial products. Thus, it was determined that the 5% oil-containing formulation provided an adequate tolerance to UV-B and high temperature.
To protect the OBs from environmental conditions, so far, many different products have been formulated.

Fig. 4
Morphological effects of control group, 5% oil formulation and commercial product on 3rd instar Helicoverpa armigera larvae in lettuce seedlings. a Before application for all test groups, b after application, c Foliage consumption of H. armigera larvae on untreated lettuce seedlings, d, e. A cadaver of H. armigera larvae died after application of the d 5% oil formulation, and e commercial product Page 6 of 7 Eroglu and Demırbag Egyptian Journal of Biological Pest Control (2022) 32:53 Prabhu and Mahalingam (2017) have prepared an additional formulation of some substances (starch, tinopal, tween 80) and nucleopolyhedrovirus (1 × 10 9 OBs/ml −1 ) isolated from Diaphania pulverulentalis. The prepared formulation initially (0. min) had a 98% mortality rate but decreased to 66% after 1 h of UV in laboratory conditions. Batista et al. (2001) prepared 2 different formulations as emulsifiable oil (EO) and wettable powder (WP) of Anticarsia gemmatalis NPV. The crude virus, WP, and EO were exposed to UV in the laboratory for 5 min. The mortality rates of crude virus, WP, EO which were not exposed to UV-B were initially 92, 99, and 98%, whereas those exposed to UV decrease to 19, 37, and 37% in 5 min. In this study, the formulation containing 5% sunflower oil showed enough protection even after 5 h. UV-B exposure (84%), while the virulence of crude HearNPV-TR reduced to 31%.
In the temperature assays, the virulence of the crude HearNPV-TR decreased to 68% at the highest temperature (42 °C) and the longest time (5 h.), whereas the virulence of the 5% oil formulation remained almost the same (85%). Thus, it was determined that the sunflower oil-based formulation made the crude virus tolerant to UV-B and high temperature. Eroglu et al. (2019) applied different concentrations of crude HearNPV-TR between 1 × 10 3 and 1 × 10 8 OBs/ ml −1 against 2nd instar H. armigera larvae. The mortality rate was calculated to be (98%) for the maximum concentration (1 × 10 8 OBs/ml −1 ) and the LC 50 value was determined to be 1.5 × 10 3 OBs/ml −1 after 14 d. In this study, 8 different concentrations (1 × 10 3 -1 × 10 10 OBs/ml −1 ) of 5% oil-based formulation and commercial products were used against 3rd instar H. armigera larvae. For the highest concentration, the mortality values were (100%) (formulation) and (100%) (Commercial product). According to the mortality rates in this study, high virulence was obtained at lower concentration than the concentrations used in the literature against H. armigera larvae.

Conclusions
Among entomopathogenics, viruses, baculoviruses have been used for the control of pest insects. Earlier studies have shown that the crude virus is required to be formulated so that it would not be inactivated due to sunlight UV-B radiation and high temperature in its nature applications. In this study, a sunflower oil-based formulation of HearNPV-TR with UV-B and high-temperature tolerance against H. armigera was produced. The results showed that this formulation was a promising viral control agent also it is ecofriendly and lower cost than chemical products.