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. Prabhu and Mahalingam (2017) have prepared an additional formulation of some substances (starch, tinopal, tween 80) and nucleopolyhedrovirus (1 × 109 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 × 103 and 1 × 108 OBs/ml−1 against 2nd instar H. armigera larvae. The mortality rate was calculated to be (98%) for the maximum concentration (1 × 108 OBs/ml−1) and the LC50 value was determined to be 1.5 × 103 OBs/ml−1 after 14 d. In this study, 8 different concentrations (1 × 103 –1 × 1010 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.
In the pot assays, Elamathi et al. (2012) applied 109 OBs/ml−1 concentrations of various H. armigera NPV formulations on marigold seedlings against H. armigera. After 96 h. the mortality rate was (86%) for 4 g formulation. Gupta et al. (2010) performed with 3 × 1012 OBs/ml−1 concentration of 3 different H. armigera NPV India isolates (Samba, Udheywalla, Chenani) containing tinopal (0.1%) and jaggery (1%) on tomato seedlings. The mortality rates of 3rd instar H. armigera larvae were determined as 9% (Samba), 86% (Udheywalla), and 81% (Chenani) after 9 days. In this study oil-based formulation and commercial products were applied with 1 × 108 OBs/ml−1 concentration on lettuce seedlings against 3rd instar H. armigera. The mortality rates were calculated as (92%) (Oil formulation) and (90%) (Commercial product) for 10 d. As a result, it was observed that the oil-containing formulation had almost the same virulence as a commercially available NPV product in the market.