Background

The tomato fruit borer, Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae), is one of the most destructive insect pest which attacks a great variety of food, fiber, oil seed, fodder, and horticultural crops of more than 300 plant species (Arora et al. 2005). This pest is polyphagous and highly mobile in nature and continues to spread to new environments around the world (Koch et al. 2003). The polyphagy, high mobility, high fecundity, and facultative diapause help H. armigera to attain the status of a major pest (Fite et al. 2020). In India, H. armigera feeds on 181 cultivated and uncultivated species belonging to 45 families (Wubneh 2016). Infestations that reach the Economic Threshold Level (ETL) are normally managed by synthetic insecticides; however, H. armigera has developed resistance to numerous insecticides (Musser et al. 2015).

Biocontrol agents are being mass-produced and applied for ecofriendly management of this pest owing to a reduction in pesticides in the environment as well as the development of resistance to chemical insecticides (Martin and Oliveira 2005). The microbial control agents viz. entomopathogenic viruses (NPV and GV), entomopathogenic bacteria (Bacillus thuringiensis Berliner), entomopathogenic fungi (Beauveria bassiana (Bals.-Criv.) Vuill. and Metarhizium anisopliae (Metchnikoff) Sorokin), and entomopathogenic nematodes (Steinernemma and Heterorhabiditis) are widely used for eco-friendly management of H. armigera (Eroglu 2022). The entomopathogenic viruses (EPV) are naturally involved in the regulation of insect pest populations, including many agricultural, horticultural, and forest pests, and individual virus isolates have a very narrow host range, making them harmless to non-target organisms, humans, and the environment (Groner 1986). The H. armigera Nucleopolyhedrovirus (HearNPV) is extremely effective against H. armigera larvae and a component of integrated pest management program in many crops (Kalia et al. 2001). The widespread use of HearNPV in India and abroad is well documented and the demand for commercial formulation of these viruses is increasing. The HearNPV isolates have been reported from temperate (Sofi 2020), sub-tropical (Gupta et al. 2010) and tropical (Jeyarani et al. 2010) regions of India, however, their comparative evaluation under laboratory and field conditions against the H. armigera larval population has not been conducted till now and the same was investigated in this study.

Methods

Helicoverpa armigera

Laboratory culture of H. armigera was established from field collected larvae from experimental farm of FoA, Wadura. The 1st instar larvae were reared on Rumex (Rumex obtusifolius) in large Petri plates that were lined with a tissue paper at the bottom. Larvae were regularly monitored for their growth. The leaves were supplemented regularly to ensure sufficient food and proper care was taken for sanitation. The culture was maintained at 28 ± 2 °C, 60% R.H., and 16L: 8D photoperiod. From 2nd instar onwards, the larvae were reared on the semi-synthetic diet (Chitneni 2008) and transferred into individual glass vials or multi-well plates to avoid cannibalism. The pupae for culture maintenance were transferred into plastic jars (7 cm × 5 cm × 12 cm) for adults’ emergence. After 7 to 8 days, adults were separated based on sex and released into oviposition cages (45 cm length: 30 cm diameter) in 1:2 ratio of males to females. Adults were provided by 10% honey solution as food using cotton swabs that was kept hanging inside the jar. Oviposition cages were lined internally with muslin cloth for egg laying. The larvae on hatching were maintained as described earlier.

Helicoverpa armigera NPV isolates

The temperate, subtropical and tropical HearNPV isolates available with the Division of Entomology, Faculty of Agriculture, SKUAST-Kashmir were used in the present investigation. The temperate HearNPV was previously isolated from H. armigera larval population in Kashmir (Sofi 2020). The subtropical and tropical HearNPV isolates were provided by Division of Entomology, Faculty of Agriculture, SKUAST-Jammu and Indian Council of Agricultural Research—National Bureau of Agricultural Insect Resources (ICAR-NBAIR), Bangalore, respectively. The HearNPV isolates were mass multiplied on laboratory reared late 3rd instar healthy H. armigera larvae that were inoculated with the crude virus of temperate, subtropical and tropical isolates, individually. All precautions were taken to prevent any cross-contamination. Semi- synthetic diet was prepared and cut into pieces of 1 cm2 and 0.5 cm thick. Virus inoculation was done by the surface contamination method as given by Evans and Shapiro (1997). The larvae were allowed to feed on the virus contaminated diet and subsequently on the healthy diet. The NPV infected moribund larvae were collected and the isolation and purification of HearNPV was conducted as per the methods described by Gani et al. (2021).

Concentration response and time response bioassays

The temperate, subtropical and tropical HearNPV isolates were bio-assayed on laboratory reared freshly molted second-instar host larvae that were starved for 12 h and then inoculated with six concentrations of HearNPV viz., 1.0 × 102, 1.0 × 103, 1.0 × 104, 1.0 × 105, 1.0 × 106 and 1.0 × 107 OBs/larva, using semi-synthetic diet as described earlier. For time-response the larvae were inoculated with the median lethal concentration (LC50) in order to produce similar mortality rates and facilitate comparisons among the isolates. Serial dilutions with distilled water were performed to achieve the desired concentrations. Ten microliter (µl) of each viral concentration from each viral isolate was spread on each diet plug using a blunt end of glass rod. Control larvae were fed a diet plug sprayed with distilled water and healthy larval extract. The diet plugs were air-dried and then placed individually in the wells of the multi-well plates. One larva was then placed in each well. Larvae having eaten the entire diet plug within 24 h, were transferred to fresh uncontaminated diet and reared at 28 ± 2 °C, 60% RH, and 16L: 8D photoperiod. Larvae that did not consume the entire plug were discarded. Bioassay of each viral concentration and a corresponding experimental control group was replicated three times in case of concentration–response and 7 times in case of time-response with 20 larvae per replicate. The virus induced mortality was recorded daily starting from third day to the tenth day after inoculation. Mortality caused by virus was diagnosed from typical virus disease characteristics and confirmed by microscopic examination through Giemsa staining.

Field trial

The tomato plants (variety: S2) were grown in the experimental farm of Division of Entomology, Faculty of Agriculture, Wadura, Jammu & Kashmir. The pest incidence was monitored visually and the treatments were started when the pest incidence was noticed. Only a single application of the virus was made to the plants at vegetative period. The crude preparation of the HearNPV from infected larval cadavers was applied at dusk with the motorized knapsack sprayer using the treatments viz. 2 × 104, 2 × 105, 2 × 106, 2 × 107, 2 × 108 and 2 × 109 OBs/plot. The control plots were sprayed with distilled water and healthy larval extract. The treatments were replicated three times in a way that each plant represents a single independent replicate in Randomized Complete-Block Design (RCBD). The plot size was 1 × 2 m and the spacing between the plants was 45 × 30 cm. Observations on the number of live larvae was recorded at one day before spray and then three, seven and ten days after spray. The per cent larval mortality on 3rd, 7th and 10th day and cumulative mortality (%) in treated and control plants were calculated.

Statistical analysis

All analyses were performed utilizing SPSS software. The data on mortality was analysed through one-way ANOVA and further subjected to post hoc tests for comparison of means. Probit analysis was performed for the calculation of median lethal concentration (LC50 ± 95% C.L.). Median Survival times (ST50) and corresponding confidence interval was calculated using log rank test under Kaplan–Meier analysis. The data on mortality of larvae (%) in the treatment and control plants in the field was analysed by one-way ANOVA and further subjected to post hoc tests.

Results

Bioassays

The Kashmir, Jammu and Bangalore isolates of HearNPV were named as HearNPV-IND-K, HearNPV-IND-J and HearNPV-IND-B, respectively. The virulence of three HearNPV isolates against 2nd instar H. armigera larvae were determined under laboratory conditions. The mortality in the larvae was confirmed by signs and symptoms of the NPV disease and also by microscopic examination.

The mortality of 2nd instar H. armigera larvae with the three different HearNPV isolates at concentrations of 1 × 102, 1 × 103, 1 × 104, 1 × 105, 1 × 106 and 1 × 107 OBs/larva ranged from 13.45–33.40, 25.00–46.70, 33.20–51.60, 43.00–58.50, 51.60–63.35, 56.60–73.25%, respectively, with significant differences between the concentrations in each isolate (HearNPV-IND-K: F = 108.308, df = 7, 16; p < 0.05; HearNPV-IND-J, F = 37.434, df = 7, 16; p < 0.05; HearNPV-IND-B: F = 28.590, df = 7, 16; p < 0.05). It was found that the mortality of NPV infected larvae increased with increase in the concentration with a significant positive correlation (r values; HearNPV-IND-K = 0.591; HearNPV-IND-J = 0.572; HearNPV-IND-B = 0.558) between concentration and mortality. The highest mortality was observed with the HearNPV-IND-K isolate (73.25%), followed by HearNPV-IND-J isolate (61.70%) and HearNPV-IND-B (56.60%) isolate at a concentration of 1 × 107 OBs/larva. A mortality rate of 0–1.66% was observed in the larvae in control group.

Probit analysis of the mortality data of H. armigera larvae with the HearNPV-IND-K, HearNPV-IND-J and HearNPV-IND-B isolates showed median lethal concentration (LC50) values of 4.62 × 103, 5.99 × 104 and 7.24 × 104 OBs/larva, respectively, at 10th day post inoculation with significant differences among them as evidenced by non-overlapping confidence limits (Table 1). The ascending order of LC50 values were HearNPV-IND-K < HearNPV-IND-J < HearNPV-IND-B isolate. The HearNPV-IND-K isolate had the lowest LC50 value of 4.62 × 103 OBs/larva revealing that it is highly virulent to H. armigera larvae than HearNPV-IND-J and HearNPV-IND-B isolates. The contribution of different HearNPV isolates towards the mortality of H. armigera larvae varied up to the magnitude of 95–96%.

Table 1 Concentration response bioassays of three different HearNPV isolates against Helicoverpa armigera larvae over a period of 10 days
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The time response bioassays were conducted using the LC50 value of the three HearNPV isolates. The first virus induced mortality was observed on day 3 and continued up to day 10 post-inoculation. After 10th day, no further virus induced mortality was observed. The daily mortality observed on day 1 to day 10 varied from 0.00 to 12.83% with significant differences (HearNPV-IND-K, F = 9.407; df = 9, 12; p = 0.00; HearNPV-IND-J, F = 5.724; df = 9, 12; p = 0.01; HearNPV-IND-B, F = 1.62; df = 9, 12; p = 0.369) between the days in each isolate. The cumulative mortality (%) caused by HearNPV-IND-K, HearNPV-IND-J and HearNPV-IND-B isolates over a period of 10 days was 50.66, 49.12 and 49.02%, respectively (Fig. 1).

Fig. 1
figure 1

Comparative daily mortality (%) of three different HearNPV isolates against 2nd instar Helicoverpa armigera larvae in time response bioassays. The first virus induced mortality was observed on day 3 with the HearNPV-IND-K isolate and day 5 with the other two isolates. The virus induced mortality peaked on 7th day with the HearNPV-IND-K and HearNPV-IND-J isolates and 10th day with the HearNPV-IND-B isolate. After 10th day, no further virus induced mortality was observed with all isolates

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The statistical analysis related to the time-response bioassays revealed that the ST50 values followed the same pattern as that of LC50 in comparing the virus isolates. The ST50 values of HearNPV-IND-K, HearNPV-IND-J and HearNPV-IND-B isolates against 2nd instar H. armigera larvae were 8.32, 8.94 and 9.10 days, respectively, at their respective LC50, with significant differences among them as evidenced by the non-overlapping confidence limits. The HearNPV-IND-K isolate had the highest speed of kill, followed by HearNPV-IND-J and HearNPV-IND-B isolates (Table 2).

Table 2 Time response bioassays of three different HearNPV isolates against Helicoverpa armigera larvae at their respective median lethal concentrations
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Field trial

To determine whether the performance of HearNPV isolates under laboratory conditions was entirely reflective of their performance under field conditions, the experiments were conducted on tomato crop at Faculty of Agriculture, Wadura. The cumulative mortality (%) of H. armigera larvae with HearNPV-IND-K, HearNPV-IND-J and HearNPV-IND-B isolates at concentrations of 2 × 104, 2 × 105, 2 × 106, 2 × 107, 2 × 108 and 2 × 109 OBs/plot ranged from 22.00–77.82, 20.64–55.11, 11.68–44.42%, respectively, with significant differences {HearNPV-K (F = 1.034, df = 23, 16; p < 0.01) HearNPV-J (F = 2.638, df = 23,16; p < 0.01) HearNPV-B (F = 2.211, df = 23, 16; p < 0.01)} among the concentrations in each isolate. The highest mortality of 77.82% was observed with the HearNPV-IND-K isolate at a concentration of 2 × 109 OBs/plot. The larval mortality observed in the control plots varied from 3 to 5.43% (Fig. 2). It’s concluded that the local HearNPV isolate was more virulent and had the potential for development of effective microbial control agent against H. armigera in Kashmir, India.

Fig. 2
figure 2

Comparative mortality (%) of three different HearNPV isolates against Helicoverpa armigera larvae on tomato plants under field conditions (DW = Distilled water; HLE = Healthy larval extract). The highest mortality of 77.82% was observed with the HearNPV-IND-K isolate at a concentration of 2 × 109 OBs/plot

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Discussion

Baculoviruses play a major role in the suppression of a variety of insect pests in agro-ecosystems. They act as natural regulators of pest population dynamics of economically important insect pests primarily in the order Lepidoptera such as H. armigera, Spodoptera litura (Fabricius), Autographa californica (Speyer) and Cydia pomonella (Linnaeus) (Williams et al. 2017). They have been developed as a promising biocontrol agent of insect pests in agriculture due to their specificity, high virulence, persistence in the environment and eco-friendly nature (Malinga and Laing 2022). The HearNPV has been found widely in nature and the local isolates was mass produced and applied to different crops for management of H. armigera in India. The commercial formulations of HearNPV viz. Helicovex® (Corteva Agriscience, USA; Andermatt Biocontrol, Brazil), Heligen® (Agbitech LLC., USA), Gemstar® (Certis USA) and Diplomata (Koppert, Brazil) are being used for eco-friendly management of H. armigera in different parts of the world.

The HearNPV isolates used in this study were found virulent to the 2nd instar H. armigera larvae. The HearNPV-IND-K isolate was found highly effective, followed by HearNPV-IND-J and HearNPV-IND-B isolates against H. armigera larvae. The HearNPV was reported to cause 90–100% mortality against neonate and 2nd instar H. armigera larvae (Ginting et al. 2018). The HearNPV was more effective against early instar larvae of H. zea and caused 99% mortality in 1st–3rd larval instars in 4–6 days and only 35% mortality in 4th and 5th larval instars (Black et al. 2022). The variation in biological activity of NPV isolates reported in this study and also by other authors is attributed to the genotype of the virus and the strain of the host (Kitchin and Bouwer 2018). Further, insects have evolved methods to inhibit or block virus replication and the early instars are more susceptible than late instars due to the increased presence of anti-microbial peptides, gut proteases, midgut-based mechanism and developmental resistance (Sauer et al. 2021). The LC50 values of the HearNPV-IND-K, HearNPV-IND-J and HearNPV-IND-B isolates against the 2nd instar H. armigera larvae were 4.62 × 103, 5.99 × 104 and 7.26 × 104 OBs/larva, respectively. Our findings corroborate with many other studies on differences in the LC50 values of NPVs against the host larvae (Ali et al. 2018). It was found that the HearNPV-IND-K isolate had the highest speed of kill, followed by HearNPV-IND-J and HearNPV-IND-B isolates. Obtained results revealed that the virulence of the HearNPV varied with respect to the isolate and the HearNPV-IND-K was found highly effective against the 2nd instar H. armigera larvae. It is a well-established fact that the baculoviruses isolated from the same species at different locations frequently vary in their biological activity i.e., pathogenicity and virulence (Erlandson 2009). These differences in biological activity are attributed to a number of factors. The significant differences in virus infectivity can be associated with virus structural changes or genetic composition, particularly the presence or absence of key genes and their functions (Fleischmann 1996). The variation in the pathogenicity of baculoviruses isolated from different hosts from different parts of the world has also been confirmed through functional genomics studies (Eberle et al. 2009). Therefore, the HearNPV-IND-K isolate may be genetically heterogeneous from the other two isolates. Another reason for the highest efficacy of HearNPV-IND-K isolate against the H. armigera was that this isolate was isolated from local host population and hence is well established and adapted in agro-ecological conditions of Kashmir.

The cumulative mortality (%) of H. armigera larvae with HearNPV-IND-K, HearNPV-IND-J and HearNPV-IND-B isolates at different concentrations ranged from 22.00–77.82, 20.64–55.11, 11.68–44.42%, respectively, with significant differences between the concentrations in each isolate under field conditions. The highest mortality of 77.82% was observed with the HearNPV-IND-K isolate at a concentration of 2 × 109 OBs/plot. The field application of HearNPV at 1 × 1012 OBs ha−1 resulted in effective management of H. armigera in India and Thailand on a variety of crops (Arrizubieta et al. 2016). Previous attempts to use HearNPV for management of H. armigera have usually met with success and it was found that the local isolates were more infective against host populations presumably as a result of continuous host–pathogen coevolution (Escribano et al. 1999).

Conclusions

The results indicated that the local isolate of HearNPV was likely better suited for the development as a biological insecticide for eco-friendly management of H. armigera in Kashmir. Future studies should be focussed towards the development of HearNPV-IND-K based formulations against H. armigera and their commercialization for conventional and organic farming in Kashmir.