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Synergistic effect of entomopathogens against Spodoptera litura (Fabricius) under laboratory and greenhouse conditions



Entomopathogens such as nematodes, bacteria and fungi are well recognized for their biocontrol potential. This study was carried out to examine the insecticidal properties of the Heterorhabditis bacteriophora Poinar, Beauveria bassiana Balsamo-Crivelli, Bacillus thuringiensis Berliner, individually and in combination against 3rd instar larvae of Spodoptera litura Fabricius (Noctuidae: Lepidoptera) under controlled laboratory and greenhouse conditions at Eternal University, Baru Sahib, Sirmaur, Himachal Pradesh.


The results demonstrated that the combined applications of the tested entomopathogens resulted in 100% insect mortality under the laboratory conditions. Among the individual concentrations, applications of 200 IJs/ml were noticed highly virulent with (98%) mortality, followed by B. thuringiensis (96%) and then by B. bassiana (92%). However, single treatments were also evaluated that further showed a highest mortality in the target pest by H. bacteriophora, followed by B. thuringiensis. Among the combined treatments by H. bacteriophora plus B. thuringiensis (200 IJs + 1 × 1012 CFU/cm2) more effective caused (100%) mortality were noticed in the laboratory and (28%) under the greenhouse conditions than H. bacteriophora plus B. bassiana (200 IJs + 1 × 1010 conidia/cm2) that caused (100%) mortality and (34%) damage under both, laboratory and greenhouse conditions.


Laboratory bioassay and greenhouse evaluation tests demonstrated that the combined sprayed treatments showed reliable and fast synergism. This study could be recommended to the farmers to control the pest.


Spodoptera litura (Fabricius,1755) (Lepidoptera: Noctuidae), known as tobacco caterpillar, beet armyworm, lesser armyworm, small mottled willow, cutworm and pigweed caterpillar, is the most serious insect pest in the countries like Japan, China, India, Pakistan (Ghaffar et al. 2002) and South Asia (Qin et al. 2004). It is a very destructive and polyphagous insect pest that causes damage to various crops such as potato, cotton, capsicum, tomato, soybean, okra, clover and onion (Saleem et al. 2016). The larvae feed on leaves of the cultivated plants that lead to complete defoliation in the early stage causing severe crop damage in India (Firake and Behere 2020). Commercially important vegetable Capsicum (Capsicum annuum Linnaeus) (Solanales: Solanaceae) grown worldwide is highly infested by S. litura (Baikar and Naik 2016). Constant use of pesticides leads to environmental contamination and pesticide residues in all foodstuffs all over the world (WHO 2017). This leads to develop safer, novel, biodegradable biopesticides as insecticidal alternatives (Chaudhary et al. 2017).

Entomopathogenic nematodes (EPNs) are considered as non-chemical alternatives of pesticides. They belong to family Heterorhabditidae and Steinernematidae and mostly genera Heterorhabditis and Steinernema (Razia and Sivaramakrishnan 2014). Infective juvenile of Heterorhabditis bacteriophora Poinar, 1976 (Rhabditida: Heterorhabditidae), kills their host insect within 24–72 h (Vashisth et al. 2013). Juvenile reaches the hemocoel of the host insect, releases its bacteria that multiply in hemolymph and liberates many factors virulent against host insect including antimicrobial compounds, hydrolytic enzymes, complexes of toxins and hemolysins (Ribeiro and Vaz 2019).

Entomopathogenic fungus (EPF) such as Beauveria bassiana Balsamo-Crivelli, 1912 (Hypocreales: Cordycipitaceae), is a pathogenic against many insects pest widely (Butt et al. 2001) and is alternated to synthetic insecticides (Maina et al. 2018) that infect the insect by entering through their cuticle. B. bassiana is non-obligatory saprophyte in nature that can also survive as endophytes of plants (Bing and Lewis 1992). It produces a number of mono-nucleated single celled submerged conidia, aerial conidia and blastospores (Holder et al. 2007). The conidia of the fungus come in close contact with the host insect cuticle for further germination and proliferation (Ortiz-Urquiza and Keyhani 2013). It is used for the management of bacterial or viral diseases caused by grasshoppers and locusts (Quesada-Moraga 2002).

Entomopathogenic bacteria (EPB) Bacillus thuringiensis (Bt) Berliner, 1915 (Bacillales: Bacillaceae) along with several microbial species proven as successful biocontrol agent. It is the spore forming bacteria which is omnipresent and forms crystal proteins. These crystal proteins are δ-endotoxin and are toxic for insects. It is found in grain storage facilities, insect-rearing facilities, sericulture farms and grain dust from flour mills (insect-rich environments). During spore formation, it also produces proteins which are released into the environment after crystallizing it degrades the cell wall. It expedites insect death, when these crystals are ingested by insect. The Cry proteins have diversifying nature and mainly target insects belonging to order Diptera (flies and mosquitoes), Lepidoptera (butterflies and moths), Coleoptera (beetles and weevils) and Hymenoptera (wasps and bees) (Bravo et al. 2007). EPNs, EPF and EPB are used as biopesticides against a variety of insects (Thakur et al. 2021). Although there are several synthetic chemical insecticides available in the market, S. litura develop resistance against various pesticides so there is an urgent need to develop various management strategies that are eco-friendly and user safe. In this investigation, a mixture of the EPNs (H. bacteriophora), EPB (B. thuringiensis), EPF (B. bassiana), each singly and in combination, was applied as control measures against S. litura (3rd instar) under controlled laboratory and greenhouse conditions.


Rearing of Spodoptera litura

This work was performed in the Department of Zoology and Entomology, Eternal University, Sirmour (30.7537° N, 77.2965° E, 1900 m altitude), state of Himachal Pradesh, India. Adults and larvae of S. litura (target insect) were collected from University Agricultural fields and farmer’s fields near Eternal University. Castor leaves were provided to feed the larvae. The culture was maintained at 28 ± 1 °C temperature and 70% relative humidity (Patil et al. 2014). Emerged adults were transferred to chimneys for oviposition that contained 15% sucrose solution for feeding of moths (Santharam 1985). Female laid eggs in cluster after 3–4 days.

Entomopathogenic nematode EPNs

Heterorhabditis bacteriophora were cultured on Corcyra cephalonica Stainton, 1866 (Lepidoptera: Pyralidae) larvae (Chaudhuri and Senapati 2017). These baits were further used for mass multiplication of EPNs (Orozco et al. 2014). Emerged nematodes were accumulated from the white traps and stored in disinfected distilled water at 14 ± 1 °C for 5–15 days before use (Shapiro-Ilan et al. 2002). The suspension containing nematodes was also poured over the sterilized synthetic sponge pieces and stored at 10 °C for future use (Ramakuwela et al. 2015).

Entomopathogenic Fungi and Bacteria

Commercially available formulations of B. bassiana Daman (1.0% W.P. Strain: IPL/BB/MI/01, International Panaacea Limited, New Delhi, India) and B. thuringiensis var. kurstaki Mahastra (0.5% W.P., Strain: DOR BT-1, International Panaacea Limited, New Delhi, India) were purchased from the local market for the bioassay experiment.

Bio-efficacy studies under the laboratory

Bio-efficacy of different treatments of EPNs (H. bacteriophora), EPF (B. bassiana) and EPB (B. thuringiensis) were applied against S. litura larvae (3rd instar), alone and in combination (Table 1) under laboratory conditions. Bioassay test was executed using 5 different treatments at different concentrations along with absolute control under a polystyrene tray having 48 wells per tray. The experiment was replicated 5 times, and the insect larval mortality was assessed every 24 h. of applications.

Table 1 List of tested entomopathogens applied against 3rd instar larvae of Spodoptera litura under laboratory conditions

Bioassay studies under greenhouse conditions

Single applications of H. bacteriophora, B. bassiana and B. thuringiensis

The vulnerability of H. bacteriophora, B. bassiana and B. thuringiensis against 3rd instar larvae of the target pest was assessed in capsicum plants under greenhouse conditions at 30 ± 2 °C temperature and 60% relative humidity. The capsicum plants were grown under polyhouse conditions with a randomized block design. The area was divided into small plots (3 × 3 m) containing 16 capsicum plants per plot. As the plants attained vegetative stage, the larvae (3rd instar) were transferred 12 h. prior to the treatment applications. The IJs, conidia and spores were mixed in water individually. Surfactant Tween 20 (0.3%) was added and applied with hand sprayer. Data over leaf damage and mortality were noted after 24 h up to 5 consecutive days. Four different concentrations of each biocontrol agent: H. bacteriophora (50 IJs, 100 IJs, 150 IJs and 200 IJs/cm2), B. bassiana (1 × 104, 1 × 106, 1 × 108 and 1 × 1010 conidia/cm2) and B. thuringiensis (1 × 103, 1 × 106, 1 × 109 and 1 × 1012 CFU/cm2), were applied along with control (absolute) (Table 2) in each plot. The experiment was replicated thrice per treatment. The study was conducted for 2 consecutive years, and the data over the mortality were pooled for the statistical analysis.

Table 2 Tested biocontrol pathogens and the concentrations applied against 3rd instar larvae of Spodoptera litura under greenhouse conditions

Combined applications of H. bacteriophora, B. bassiana and B. thuringiensis

Experiment was accomplished to evaluate the efficiency of combined concentrations of entomopathogens against S. litura larvae (3rd instar) under the greenhouse conditions (Table 3). During this experiment also, insect larvae were relocated 12 h. before treatment application. The combined concentrations of IJs + fungal conidia (150 IJs + 1 × 1010 conidia/cm2 and 200 IJs + 1 × 1010 conidia/cm2) and IJs + bacterial spores (150 IJs + 1 × 1012 CFU/cm2 and 200 IJs + 1 × 1012 CFU/cm2) were suspended in water containing Tween 20 (0.3%) and applied over the capsicum leaves. Result variables such as leaf damage and larval mortality were noted 24 h post treatment applications for 5 days after 3 consecutive sprays. The data calculated over the leaf damage percentage and larval mortality were recorded.

Table 3 Combined concentrations of entomopathogens’ applied against 3rd instar larvae of Spodoptera litura under greenhouse conditions

Statistical analysis

Statistical analysis of variance ANOVA was enumerated. ANOVA was evaluated using OPSTAT software developed by HAU, Haryana. Statistical significance was calculated at level P < 0.05. Statistical differences among the treatments were considered.


Bio-efficacy of entomopathogens under laboratory

Pathogenic potential of different tested treatments of entomopathogens was assessed against 3rd instar larvae of S. litura, alone and in combination under controlled conditions. The data obtained over the mortality are represented in Fig. 1, Table 4. Mortality percentage increased in all the treatments as exposure time increased. In H. bacteriophora, the highest larval mortality (98%) was recorded at the highest treatment of 200 IJs/ml after 96 h. of nematode exposure. However, (48%) mortality was observed after 96 h., in the lowest concentration (50 IJs/ml) of nematode (Fig. 1A). Significant differences were recorded in the mortality percentage by EPNs (F = 16.91, df = 4, P value < 0.001). In treatments with B. bassiana, maximum mortality (92%) in treatment 1 × 1010 conidia/ml and minimum mortality (45%) (Table 5) were detected in treatment 1 × 104 conidia/ml after 96 h. of inoculation (Fig. 1B). Statistically significant differences were observed in insect mortality rates (F = 20.43, df = 4, P < 0.001). The larvae treated with different concentrations of B. thuringiensis recorded (96%) insect mortality at the highest concentration 1 × 1012 CFU/ml, followed by the low concentrations (Table 6). Minimum mortality rate (54%) was noticed in the lowest concentration 1 × 103 CFU/ml after 96 h. of incubation (Fig. 1C). Considerable differences in the percent mortality were recorded in all treatments of B. thuringiensis, (F = 32.35, df = 4, P value < 0.001).

Fig. 1
figure 1

Application of entomopathogens as a biocontrol agent against 3rd instar larvae of Spodoptera litura under laboratory conditions. Insect mortality percentage by using A Heterorhabditis bacteriophora, B Beauveria bassiana, C Bacillus thuringiensis, D H. bacteriophora + B. bassiana, E H. bacteriophora + B. thuringiensis

Table 4 Pathogenic potential of Heterorhabditis bacteriophora against 3rd instar larvae of Spodoptera litura under laboratory conditions
Table 5 Pathogenic potential of Beauveria bassiana against 3rd instar larvae of Spodoptera litura under laboratory conditions
Table 6 Pathogenic potential of Bacillus thuringiensis against 3rd instar larvae of Spodoptera litura under laboratory conditions

Combined concentrations of treatments showed differential impact over the mortality. Treatments of EPNs and EPF, 200 IJs + 1 × 1010 conidia/ml gave a maximum of (100%) mortality rate, followed by (96%) in the treatment of 150 IJs + 1 × 1010 conidia/ml (Table 7). Minimum mortality rate (74%) was perceived at the lowest concentration 50 IJs + 1 × 1010 conidia/ml (Fig. 1D). Significantly increased mortality percent were recorded after treated with combined concentrations of H. bacteriophora + B. bassiana (F = 160.10, df = 4, P value < 0.001). In another experiment of combined concentrations of EPNs and EPB, 150IJs + 1 × 1012 CFU/ml and 200 IJs + 1 × 1012 CFU/ml, both high concentrations caused (100%) larval mortality after 96 h. of exposure (Table 8). Even minimum mortality rate (78%) was recorded at the concentration 50 IJs + 1 × 1012 CFU/ml (Fig. 1E). The combined mixture of H. bacteriophora + B. thuringiensis showed synergistic impact on S. litura larvae (3rd instar) and significant differentiation in the larval mortality was recorded (F = 238.12, df = 4, P value < 0.001) under the laboratory bioassay study.

Table 7 Potential of Heterorhabditis bacteriophora + Beauveria bassiana against 3rd instar larvae of Spodoptera litura under laboratory conditions
Table 8 Potential of Heterorhabditis bacteriophora + Bacillus thuringiensis against 3rd instar larvae of Spodoptera litura under laboratory conditions

Bio-efficacy of entomopathogens single and combined mixture under greenhouse conditions

Pathogenic potential of entomopathogens were also evaluated under greenhouse conditions. Capsicum plants sprayed with H. bacteriophora IJs showed significantly reduced damage in contrast to control. Minimum damage percentage was detected at plot treated with 200 IJs/cm2 concentration. Even though the damage percentages were quite higher (35%) but significantly lower than the control (F = 10.39, df = 4, P value < 0.001) (Fig. 2A). In treatment with B. bassiana also, (43%) damage was recorded at the highest concentration (1 × 1010 conidia/cm2) after 3rd application of the biocontrol agent. However, it showed protective effect on capsicum plant against non-treated plants (F = 11.79, df = 4, P value < 0.001) (Fig. 2B). Another experiment with B. thuringiensis, (41%) damage was recorded after 3rd spray of 1 × 1012 CFU/cm2. Significantly reduced damage percentage was recorded at the highest treatment in contradiction of control (F = 10.39, df = 4, P value < 0.001) (Fig. 2C).

Fig. 2
figure 2

Application of entomopathogens on capsicum plants against Spodoptera litura (3rd instar) As: A Heterorhabditis bacteriophora, B Beauveria bassiana, C Bacillus thuringiensis, D H. bacteriophora + B. bassiana and H. bacteriophora + B. thuringiensis

Combined mixture of concentrations containing H. bacteriophora plus B. bassiana and H. bacteriophora plus B. thuringiensis reduced the damage percentage and synergistic impact of these entomopathogens, in contradiction of non-treated plants. Significant variations were recorded. Least damage (28%) was detected in concentration 200 IJs + 1 × 1012 CFU/cm2, while (34%) damage was encountered in 200 IJs + 1 × 1010 conidia/cm2. The highest (96%) damage was detected in the control (Fig. 2D).

Additionally, the data recorded over the mortality in S. litura larvae (3rd instar) via H. bacteriophora also demonstrated that application of 200 IJ/cm2 caused significant mortality in insect larvae after 3rd spray. The percent mortality ranged between (35 and 93%) at 5 days after 3rd application of infective juveniles (F = 28.71, df = 4, P < 0.001) (Fig. 3A). Applications with B. bassiana also showed variation in the percent mortality in contradiction to control. In treatment B. bassiana, insect mortality percentage ranged between (30 and 88%) (F = 26.01, df = 4, P value < 0.001) (Fig. 3B). Regarding the applications of different concentration of B. thuringiensis, treatment of 1 × 1012 CFU/ml resulted in a significant mortality rate in insect larvae. Mortality rates increased from (38 to 90%) after 3rd spray; in contrast, no larval deaths were observed in the control (F = 25.33, df = 4, P value < 0.001) (Fig. 3C).

Fig. 3
figure 3

Application of entomopathogens on larval mortality in capsicum plants against Spodoptera litura (3rd instar) As: A Heterorhabditis bacteriophora, B Beauveria bassiana, C Bacillus thuringiensis, D H. bacteriophora + B. bassiana and H. bacteriophora + B. thuringiensis

Capsicum plants treated with combined concentrations of H. bacteriophora plus B. bassiana showed improved insect mortality. Furthermore, H. bacteriophora plus B. thuringiensis also demonstrated increased insect mortality rates. The least mortality (41%) was detected at 150 IJs + 1 × 1010 conidia/cm2, and the maximum one (98%) was evidenced at 200 IJs + 1 × 1012 CFU/cm2. Statistically significant results were achieved in the combined concentrations of all entomopathogens (F = 108.39, df = 4, P < 0.001) (Fig. 3D). All larvae were alive in control. The combined mixture of entomopathogens was found synergistic and had potential of reducing the insect population.


In the present study, the pathogenicity potential of H. bacteriophora, B. bassiana and B. thuringiensis was evaluated individually and in combined treatment against S. litura (3rd instar) under controlled laboratory and greenhouse conditions. Foliar spray of H. bacteriophora was quite effective in managing 3rd instar larvae under both controlled conditions. Hussaini (2005) noticed better efficacy in H. bacteriophora than H. indica against S. litura. He reported 40 and 20% mortality with H. bacteriophora and H. indica after 72 h. of exposure. Divya et al. (2010) conducted in laboratory bioassay study on H. indica against S. litura (3rd instar). Yadav et al (2017) also reported that EPNs (S. carpocapsae) were responsible for causing 100% larval mortality in S. litura at a concentration of 400 IJs after 96 h. of exposure. Khan et al. (2020) evaluated the biocontrol potential of EPNs against four lepidopteran insect pests including S. litura. They reported EPNs as significant biocontrol agents which is highly virulent against insect population. Sun et al. (2021) recorded 100% mortality within 48 h in S. litura larvae when exposed to ten different isolates of Steinernema and Heterorhabditis.

B. bassiana showed significant mortality against S. litura under the laboratory conditions. The treatments were also effective in managing the insect in the greenhouse. Similar observations were recorded by Indriyanti et al. (2017) at Salatiga. They reported that B. bassiana is a powerful and eco-friendly biopesticides in managing the S. litura population. Moorthi et al. (2011) found that foliar sprays of B. bassiana were highly virulent against S. litura. Erawati et al. (2018) reported that application of B. bassiana against S. litura resulted in 50–60% less leaf damage than control and recommended it as effective biocontrol agent for the management of leaf-eating larvae. Ullah et al. (2019) also described the biocontrol prospective of EPF against S. litura. El-Husseini (2019) also reported B. bassiana as an effective biocontrol agent against Spodoptera exigua under the laboratory as well as open field conditions. Fergani and Refaei (2021) conducted a bioassay experiment by using B. bassiana against different developmental stages of Spodoptera littoralis in the laboratory conditions. They recommended B. bassiana as an effective biological control agent for Spodoptera management.

Another treatment of B. thuringiensis also evidenced high insect mortality in controlled at greenhouse conditions. Similar study was carried by Sajid et al. (2020) who reported 100% mortality in S. litura (2nd instars) by the applications of B. thuringiensis. Vimala Devi et al. (2021) reported that water dispersible granules of B. thuringiensis mixed with Tween20 and starch were highly effective against S. litura larvae. Singh et al. (2021) reported that cry proteins of B. thuringiensis were highly virulent against lepidopteran insect pest including S. litura. The combined treatment of the mixture containing H. bacteriophora plus B. bassiana and H. bacteriophora + B. thuringiensis demonstrated the effectiveness of these treatments in insect management with reduced damage percentage. The combined mixtures of treatments were recorded highly synergistic. Karthikeyan et al. (2016) demonstrated that the combination of B. thuringiensis with X. bovienii resulted in 100% larval mortality after 60 h. Synergistic and additive connections were detected in the combined mixture of treatments. H. downesi and S. carpocapsae were the EPNs used to manage destructive insect pests. Beside this, commercial strains of M. brunneum and B. bassiana were also employed. Sáenz-Aponte et al. (2020) also demonstrated that the combined applications of H. bacteriophora along with fungi including B. bassiana and M. anisopliae were highly effective in controlling the diamond back moth in the greenhouse and in field conditions.


This study was carried to investigate the pathogenicity prospective of entomopathogens against polyphagous insect species, S. litura under laboratory and greenhouse conditions. The outcome of this study concluded that the combined mixture of native EPNs, EPF and EPB showed high virulence toward S. litura in bioassay experiment and under greenhouse. The highly reliable and fast synergism observed in combined concentration containing H. bacteriophora plus B. bassiana and H. bacteriophora plus B. thuringiensis under controlled conditions and in greenhouse conditions. The study suggested that the synergistic blend of these 3 entomopathogens could be best biological control means to overcome local pest problems.

Availability of data and materials

All data generated and analyzed for the current study are presented in this manuscript, and the corresponding authors have no objection to the availability of data and materials.



Entomopathogenic bacteria


Entomopathogenic fungus


Entomopathogenic nematodes

B. thuringiensis :

Bacillus thuringiensis

B. bassiana :

Beauveria bassiana

H. bacteriophora :

Heterorhabditis bacteriophora

S. litura :

Spodoptera litura

S. carpocapsae :

Steinernema carpocapsae

S. feltiae :

Steinernema feltiae


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This research work carried out at Eternal University Baru Sahib, Himachal Pradesh. Authors acknowledge the financial assistance provided by Department of Science and Technology (DST), Govt. of India (SP/YO/506/2018-G). The authors are thankful to Dr. H.S. Dhaliwal, former Vice Chancellor, Eternal University Baru Sahib, for providing his guidance. The authors acknowledge the Vice chancellor Dr. Davinder Singh and Prof. A.S. Ahluwalia for providing the necessary laboratory facilities. The authors are also thankful to the local farmer for cooperation during the field collection.


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PT, SS and SK conducted the experiment and wrote the manuscript. SS, ANY and AE-LH collaborated with useful suggestions in biocontrol. NT gave concept. All authors have read and approved the final manuscript.

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Correspondence to Abd El-Latif Hesham.

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Thakur, N., Tomar, P., Sharma, S. et al. Synergistic effect of entomopathogens against Spodoptera litura (Fabricius) under laboratory and greenhouse conditions. Egypt J Biol Pest Control 32, 39 (2022).

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