Synergistic effect of naturally occurring Granulosis virus isolates (

Background: To manage the cabbage butterfly, Pieris brassicae (L.) (Lepidoptera: Pieridae), it is not wise to use insecticides on leafy vegetables which are eaten mostly fresh. During the past decades, the efforts to manage the pest, through chemical insecticides have raised serious health. Investigations were carried out to isolate naturally occurring GVs (PbGV) as a potent biopesticide against P. brassicae and to explore their efficacy with the application of phagostimulants. Results: Among the four naturally occurring isolates obtained from Northwestern Himalayas, Sudhmahadev isolate was found to be the most promising based on virulence and speed of kill against all the instars tested in the laboratory, showing the natural incidence of PbGV infection in field conditions. In concentration and time–response bioassay, all the isolates of P. brassicae Granulosis virus were found high virulent against second instar larvae of cabbage butterfly. Therefore, for enhanced efficacy of PBGV, its combined application with phagostimulants (Lepidium sativum + Teepol + jaggery) or sticker (Teepol + jaggery), applied in field trials, resulted into greater mortality of larval instars than the single one. Overall, the results indicated that the introduction of a more isolates PBGV strain into populations of P. brassicae could be of vital importance for eco-friendly suppression of this pest globally with the combination of phagostimulants. The application virus alone with the pre-standardized concentration of 1 × 10 OBs/ha did not reduce the larval population density to the desirable extent in the greenhouse chamber and therefore was not included in field experiments. Overall, the most promising treatments in reducing the larval population of the pest were PbGV + Teepol + B. thuringiensis (93.49 and 91.39%) and PbGV + Teepol + L. sativum (88.79 and 86.97%) over control in both greenhouse and field trials, respectively. Conclusions: In this study, the native isolates of PbGV from different target locations to test their efficacy against different instars of P. brassicae were explored. Using native PBGV isolates with phagostimulant combinations played an important role for regulating the pest effectively. These phagostimulants not only protected the OBs from degradation in the presence of sunlight but also increased the speed of killing. The biocontrol potential of PbGV in both laboratory and field conditions indicated that baculoviruses are sustainable alternative to chemical insecticides. © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. Open Access Egyptian Journal of Biological Pest Control *Correspondence: bhatbari@rediffmail.com 2 Division of Entomology, Department of Horticulture, SKUAST-Kashmir, Srinagar, J&K 190025, India Full list of author information is available at the end of the article Page 2 of 9 Kour et al. Egyptian Journal of Biological Pest Control (2022) 32:5 Background The cabbage butterfly, Pieris brassicae (L.) (Lepidoptera: Pieridae) is a cosmopolitan species with Palearctic distribution and causes extensive damage at all the growing stages of Cole crops due to their voracious feeding habit (Ohno et al. 2020). During the past decades, the efforts to manage this insect pest were dominated by chemical insecticides. However, increasing failures of chemical pesticides and the health hazard problems posed by their indiscriminate use in the field reaffirmed the need to focus attention on alternative and eco-friendly pest management tactics. Entomopathogenic viruses (EPVs) appear to be an ideal candidate as biological control agent against insect pests (Lacey 2012). Moreover, baculoviruses are naturally involved in the regulation of insect populations and individual isolates often have a very narrow host range and, therefore, no side effects or harmful effects on mammals, birds, other animals, aquatic systems and beneficial insects. Also, these microbes may establish themselves in the pest population and exert long-term protection. Therefore, their use is being encouraged by international concerns for a reduction in pesticides within the environment, also as by the event of resistance to chemical insecticides by target pests. However, their usage is hampered due to the inactivation of OBs in the presence of sunlight and the slow speed of action to kill the insect. Nearly 50% of the viral activity of HaNPV was reduced when exposed for 6  h. However, by 36  h all the isolates had lost their activity by about 70% (Mehrvar et  al. 2007). Similarly, when a highly purified preparation of P. brassicae virus was exposed to direct sunlight, total inactivation of the virus occurred between 12 and 19 h (David et al. 1968). These viruses have greater significance for bioprospecting as most of these baculoviruses are registered and used mainly in many countries. Despite the successes of the past, there is a continuous need to discover and develop new strains/races of baculovirus due to vast differences in their pathogenicity and virulence (Gupta et al. 2013; Cory and Myers 2003). In this regard, the different native geographical isolates must be tested against insect populations from the locality of the program to meet the future needs of food and fiber production and concurrent reduction in the use of chemical pesticides. Since native isolates are always preferred for sustainability, adaptability, and efficacy in a given agro-ecosystem owing to an adaptive advantage to retain high infectivity toward the local population via host–pathogen co-evolution process (Barrera et al. 2011). Therefore, the present study aimed to isolate and identify the P. brassicae Granulosis virus from different geographical regions of Jammu and Kashmir, India, and to assess their potential under field conditions.

Page 2 of 9 Kour et al. Egyptian Journal of Biological Pest Control (2022) 32:5 Background The cabbage butterfly, Pieris brassicae (L.) (Lepidoptera: Pieridae) is a cosmopolitan species with Palearctic distribution and causes extensive damage at all the growing stages of Cole crops due to their voracious feeding habit (Ohno et al. 2020).
During the past decades, the efforts to manage this insect pest were dominated by chemical insecticides. However, increasing failures of chemical pesticides and the health hazard problems posed by their indiscriminate use in the field reaffirmed the need to focus attention on alternative and eco-friendly pest management tactics. Entomopathogenic viruses (EPVs) appear to be an ideal candidate as biological control agent against insect pests (Lacey 2012). Moreover, baculoviruses are naturally involved in the regulation of insect populations and individual isolates often have a very narrow host range and, therefore, no side effects or harmful effects on mammals, birds, other animals, aquatic systems and beneficial insects. Also, these microbes may establish themselves in the pest population and exert long-term protection. Therefore, their use is being encouraged by international concerns for a reduction in pesticides within the environment, also as by the event of resistance to chemical insecticides by target pests. However, their usage is hampered due to the inactivation of OBs in the presence of sunlight and the slow speed of action to kill the insect. Nearly 50% of the viral activity of HaNPV was reduced when exposed for 6 h. However, by 36 h all the isolates had lost their activity by about 70% (Mehrvar et al. 2007). Similarly, when a highly purified preparation of P. brassicae virus was exposed to direct sunlight, total inactivation of the virus occurred between 12 and 19 h (David et al. 1968).
These viruses have greater significance for bioprospecting as most of these baculoviruses are registered and used mainly in many countries. Despite the successes of the past, there is a continuous need to discover and develop new strains/races of baculovirus due to vast differences in their pathogenicity and virulence (Gupta et al. 2013;Cory and Myers 2003). In this regard, the different native geographical isolates must be tested against insect populations from the locality of the program to meet the future needs of food and fiber production and concurrent reduction in the use of chemical pesticides. Since native isolates are always preferred for sustainability, adaptability, and efficacy in a given agro-ecosystem owing to an adaptive advantage to retain high infectivity toward the local population via host-pathogen co-evolution process (Barrera et al. 2011). Therefore, the present study aimed to isolate and identify the P. brassicae Granulosis virus from different geographical regions of Jammu and Kashmir, India, and to assess their potential under field conditions.

Establishment of laboratory culture of P. brassicae
The nucleus culture of P. brassicae was maintained in the Biocontrol laboratory, Division of Entomology at SKUAST-J, FOA-Chatha, India, by collecting the adults from the field. The adults were kept in Nylon mesh cages (10 × 9 × 7 ft) provided with potted cabbage, cauliflower, Knol khol, and some flowering plants of Nasturtium sp. Leaves with eggs were kept in sterilized glass jars (50 × 30 cm diameter) covered with muslin cloth for further rearing. Uneaten food and fecal matter were removed daily to maintain the hygiene conditions. The colony was maintained at a temperature of 26 ± 20 °C and 70 ± 10% RH and L/D (16:8) photoperiod.

Virus-isolation, purification, and standardization
Intensive exploratory surveys were conducted from different zones of J & K, viz. Chatha, Poonch, Sudhmahadev, and Kashmir, India, during 2014-2020 for the presence of native GV isolates of Pieris brassicae. During the survey, live and dead specimens were collected individually in sterile glass vials and were kept at − 20 °C till further use. It is pertinent to mention here that the Himachal Pradesh (H.P) isolate of PbGV was procured from Dr. Pankaj Sood, Principal Scientist, KVK Mandi, Himachal Pradesh, India. The extraction, purification, and standardization of OBs were done as per the methods given by Moore (2002) and Hussain et al. (2020). Preliminary baculovirus identification was done by a light microscopy by spreading smears from infected larvae thinly across a microscope slide, followed by Giemsa staining and then examined through a phase-contrast microscope at 1000 × magnification under oil immersion (Gupta et al. 2013). Enumeration of OBs in the viral suspension was done with the help of the Petroff-Hausser counting chamber (Lacey 2012). The virus was also identified through electron microscopy as per the methods of Sood and Prabhakar (2012) and Hussain et al. (2019).

Concentration-response and time-response bioassays: selection of a potent isolate
For selection, the virus isolates were bio-assayed on 12-h pre-starved, 30 freshly molted first-, second-, and thirdinstar host larvae via leaf disc method. Six different Keywords: Pieris brassicae, Baculoviruses, Biopesticides, Field evaluation Page 3 of 9 Kour et al. Egyptian Journal of Biological Pest Control (2022) 32:5 virus concentrations, viz. 8 × 10 2 , 3.6 × 10 3 , 7.2 × 10 4 , 1.44 × 10 5 , 2.88 × 10 6 and 5.76 × 10 7 OBs/larvae, were used in the study. Serial dilutions with distilled water were performed to achieve the desired concentrations. For time-response, the second, third, and fourth larval instars were inoculated with a concentration of 1.44 × 10 5 OBs/larva. Ten μl aliquots of each viral concentration from each viral isolate were spread evenly onto each leaf disc with the help of a brush. Control larvae were fed a leaf disc inoculated with water. Larvae ate the whole leaf disc, were transferred to fresh uncontaminated leaves and reared at 28 ± 2 °C, 60% RH, and 16L/8D photoperiod. Bioassay of each viral concentration and a corresponding experimental control group was replicated 3 times in case of concentration-response and 7 times in case of the time-response. Larvae were observed daily for virus-induced mortality until pupation.

Virus propagation
For in vivo production of PbGV, laboratory-reared third instar healthy larvae were inoculated by 7.2 × 10 4 OBs/ larva (optimum inoculum concentration). To avoid contamination, only moribund larvae were harvested into plastic bags and held at − 20 °C till further use. Frozen larvae were thawed for 24 h at 4 °C and then blended with water at high speed for 10 s to release the OB's. The crude concentrate was filtered through a three-layered muslin cloth to remove debris and stored at − 20 °C for further studies.

Field trails Greenhouse pot experiment
A laboratory pot experiment was conducted to evaluate the efficacy of PbGV against P. brassicae on cabbage by preparing different aqueous formulations of PbGV with different substances. The plants were grown in pots under lab conditions encaged in nylon mesh for aeration. The virus @ 1 × 10 12 OBs/ml was sprayed on the plants uniformly with a hand sprayer. All the additives were added to GV before spraying. Third instar larvae (50) released on plants immediately after drying of spray fluid on plants. The larval mortality was observed after 1, 2, and 3 weeks of application. Each experiment was replicated 5 times with 5 plants (6 weeks old) per replicate, and mean larval mortality at different intervals was recorded.

Field efficacy
To check whether the performance of an isolate in the laboratory is entirely reflective of its performance, 2 trials (I and II) were conducted in the field to generate the efficacy data of the most virulent strain.

Trial 2
Another trial was conducted at Farmer's field at Marh area of Jammu, Jammu, and Kashmir on Knol khol, which is another major host of P. brassicae. Five treatments (as applied in Trail 1) were assigned to 4 replicates in randomized block design. A crude preparation of the virus from infected larval cadavers was applied at dusk with the motorized knapsack sprayer, while the control plots were sprayed with water in both trails. Five plants of cabbage were randomly selected from each plot and observations were recorded after first, second, and third weeks of spray.

Data analysis
Data were subjected to one-way ANOVA using statistical software SPSS on the personal computer. The data on mortality were analyzed through one-way ANOVA and further subjected to post hoc tests for comparison of means. The mortality among the isolates was compared through the nonparametric Kruskal-Wallis H test. Larval mortality was analyzed using Probit analysis to estimate the median lethal concentration (LC 50 ) values that cause 50% mortality in test population. Median survival time (ST 50 ) was calculated using a log-rank test under Kaplan-Meier analyses.
Page 4 of 9 Kour et al. Egyptian Journal of Biological Pest Control (2022) 32:5 Results A microscopic examination through Giemsa staining of the tissue smears and hemolymph of the infected larvae collected from all the locations revealed the presence of OBs. The amplification of total DNA with gran gene-specific primers resulted in successful amplification of all the isolates of PbGV with a readable sequence of 500 bp. As such, the Himachal Pradesh, Poonch, Chatha, and Sudhmahadev isolates were named as PbGV-IND HP, PbGV-IND PH, PbGV-IND CH, and PbGV-IND SD, respectively, by adopting the most commonly used methods (Erlandson 2009). The pathogenicity of all the isolates was confirmed through Koch's postulates, and symptoms typical to GV were observed. The natural incidence of GV in P. brassicae larvae varied from 0.95 to 6.88%. The highest incidence and lowest incidence of 6.88 and 0.95% were reported on cabbage from Sudhmahadev and Chatha, respectively. However, no natural incidence of GV was observed in the Kashmir division of Jammu and Kashmir.
In concentration-response bioassay, all the strains of P. brassicae Granulosis virus were found to be highly virulent against second ( Table 2).
The results obtained from the experimental trials of SKUAST-J revealed nonsignificant differences in the pretreatment population of P. brassicae in both trials. However, all the treatments were proved significantly superior  Page 6 of 9 Kour et al. Egyptian Journal of Biological Pest Control (2022) 32:5 over control after first (F = 12.28, df = 4, 15, p = 0.00), second (F = 16.88, df = 4, 15, p = 0.00) and third larval instars of P. brassicae (F = 19.28, df = 4, 15, p = 0.00) week of spray in trial I at Chatha. Following 7 days post-treatment application, Spinosad 20 ES (4.6 larvae/plant) and PbGV + Bacillus thuringiensis (10.67 larvae/plant) were found to be the most effective treatment in reducing the larval population. However, the efficiency of Spinosad decreased with time and a drastic increase in the population dynamics of P. brassicae was observed after the second and third weeks of spray. In contrast, 60.2-93.9% and 49.32-88.79% reduction in the larval population density in the PbGV + Teepol + Bacillus thuringiensis and PbGV + Teepol + Lepidium, respectively, were observed over control after first to third weeks of spray (Fig. 1A).
Similarly, in trail II at Marh significant differences between all the treatments and control blocks were observed after first (F = 15.81, df = 4, 15, p = 0.00), second (F = 21.69, df = 4, 15, p = 0.00) and third (F = 47.16, df = 4, 15, p = 0.00) week of spray. A similar trend of decreased in the Spinosad efficacy was observed after 7 days post-spray. Similarly, PbGV + Teepol + Bacillus thuringiensis and PbGV + Teepol + Lepidium were found superior from all other treatments. The magnitude of reduction increased from 36.60 to 86.97% and 59.57 to 91.39%, respectively, over control after the first to third week of spray, respectively (Fig. 1B). Page 7 of 9 Kour et al. Egyptian Journal of Biological Pest Control (2022) 32:5

Discussion
Interest in baculoviruses as potential biopesticides has certainly increased the screening of insect populations for new and potentially more efficacious isolates or strains. It is a well-known fact that the geographical isolate with different biological activities influences the effectiveness of the virus as a biopesticide. In this regard, 4 different GV isolates of P. brassicae were screened against the P. brassicae population of Jammu and Kashmir during the present study, which exhibited a differential degree of pathogenicity. Although nonsignificant differences were observed in median lethal concentrations (LC 50 ), Sudhmahadev isolate exhibited a 38.74-48.28% reduction in the LC 50 , as compared to other isolates. The ST 50 estimates of 4 different isolates of PbGV against second, third, and fourth larval instars of P. brassicae varied (from 2.96 to 5.33), (5.90 to 7.43) and 7.73 to 11.83 days, respectively. Therefore, in all the tested isolates, the Sudhmahadev isolate of PbGV was selected for further study because of its high infectivity and low ST 50 values. Marked differences within the virulence of baculoviruses isolated from different sites are reported by several researchers (Gupta et al. 2013). Mehrvar et al. (2007) reported variation in the concentration and time-response mortality among different isolates of Helicoverpa armigera (Hb.) nucleopolyhedrovirus. Such differences are not unusual among virus isolates collected from the same species and from different geographical locations (Cory et al. 2005). Furthermore, native isolates tend to be more pathogenic to local populations in comparison with exotic ones (Barrera et al. 2011) owing to an adaptive advantage to retain high infectivity toward the local population via the process of host-pathogen coevolution (Barrera et al. 2011). In other instances, baculovirus isolates with varying genotypes particularly the presence or absence of key genes (Crook 1981) can display differences in viral virulence (Eberle et al. 2009). In contrast, Ali et al. (2018) reported nonsignificant differences between 2 strains of S. litura (SpltNPVPak-BNG and SpltNPV-G1 strain) in terms of concentration-mortality response (LC 50 ). However, time-to-death (LT 50 ) was significantly shorter for SpltNPV-Pak-BNG than for SpltNPV-G1, thus suggesting that it may be genetically heterogeneous with other wild types from other geographic regions. Further studies on their polypeptide profiling and gene sequencing may reveal deeper insights on this attribute. In addition, it was also evident from the results that the median survival time (ST 50 ) increased with the age of larvae in all the isolates tested, i.e., susceptibility of the P. brassicae larvae is inversely proportional to the age of the insect. A rapid decrease in larval susceptibility was reported by Payne (1982) against the biggest larval instars in the case of both NPVs and GVs. These age-dependent differences in susceptibility were also reported by many researchers (Sood and Prabhakar 2012). This may be due to the less developed defense system of insects at their young stage than at the oldest stage and midgut-based barriers to infection in older larvae such as lesser permeability of the midgut peritrophic membrane, The speed of kill of baculoviruses is of great concern because the chemical insecticides produce quicker results. Another constrain in the usage of baculovirus is the inactivation of OBs in the presence of Sunlight. When a highly purified preparation of the known virus of P. brassicae was exposed to direct sunlight, only 7-33% of the initial virus deposits remained 1 day after application (Tatchell and Payne 1984). Many fields and laboratory studies have demonstrated that microbial insecticides are inactivated by solar UV radiation (Mehrvar et al. 2007). These results emphasize the importance of finding some means of screening or protecting the virus from UV radiation. Therefore, small-scale trials were conducted in the laboratory and field to evaluate the efficacy of PbGV in combination with certain botanical and microbial insecticides against P. brassicae. Several materials have been tested for the ability to protect deposits of viruses from ultraviolet light thereby extending the activity of the virus after application. In the present study, PbGV + Bacillus thuringiensis and PbGV + Lepidium + Jaggery were found to be the most effective treatments in reducing the larval population of P. brassicae. Enhanced efficacy of PbGV in combination with B. thuringiensis might be due to the latent infections, i.e., due to stress imparted by B. thuringiensis on P. brassicae larvae resulting in inactivated infection of PbGV. These results are in line with Aruga (1963) who reported stress as the main cause of activation of ingested viruses which results in the outbreak of the disease in the host insect populations. The role of stressors in activating latent viral infection has also been reported earlier (Williams et al. 2017). For instance, S. exempt larvae infected by bacteria were markedly more susceptible to SpexNPV than bacteriafree larvae (Graham et al. 2012;Mahmoud et al. 2012). Enhanced effectiveness of NPV/GV with the addition of B. thuringiensis was reported by Sood et al., (2011). The synergetic effect of Lepidium sativum might be due to hydro colloidal-encapsulation of the baculovirus, where OBs were embedded within micro granules which protect the OBs from degradation in the presence of sunlight (Alfazairy et al. 2014). To improve the efficacy of baculovirus certain experiments were carried out by several researchers (Devi 2011).
Overall, the results indicated that the introduction of a more virulent GV strain into populations of P.