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.


Background
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).
Page 2 of 10 Thakur et al. Egyptian Journal of Biological Pest Control (2022) 32:39 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).
The study was conducted for 2 consecutive years, and the data over the mortality were pooled for the statistical analysis.

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 × 10 10 conidia/ cm 2 and 200 IJs + 1 × 10 10 conidia/cm 2 ) and IJs + bacterial spores (150 IJs + 1 × 10 12 CFU/cm 2 and 200 IJs + 1 × 10 12 CFU/cm 2 ) 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.

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 × 10 10 conidia/ml and minimum mortality (45%) ( Table 5) were detected in treatment 1 × 10 4 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 × 10 12 CFU/ml, followed by the low concentrations (Table 6). Minimum mortality rate (54%) was noticed in the lowest concentration 1 × 10 3 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).

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/cm 2 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 × 10 10 conidia/cm 2 ) after 3rd application of the biocontrol agent. However, it showed protective effect on capsicum plant against nontreated 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 × 10 12 CFU/cm 2 . 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).
Additionally, the data recorded over the mortality in S. litura larvae (3rd instar) via H. bacteriophora also demonstrated that application of 200 IJ/cm 2 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 × 10 12 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).
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 × 10 10 conidia/cm 2 , and the maximum one (98%) was evidenced at 200 IJs + 1 × 10 12 CFU/cm 2 . 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.       Page 7 of 10 Thakur et al. Egyptian Journal of Biological Pest Control (2022) 32:39 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

Conclusion
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.