Introduction

The attack by insect pests on plants has been a major challenge among the various biotic stresses leading to severe losses of crops. Thus, protection of crops from insect pests is highly important for sustained food production and is mainly achieved by using chemical insecticides. The increasing environmental concerns related to use of these chemicals have necessitated the search for novel bio-pesticides as pest management tools. The bio-pesticide formulations based on fungi, bacteria, viruses and some plant extracts have been demonstrated to be effective against insect pests and are safe for non-target organisms and environment (Mazid et al. 2011). The bacterial bio-pesticides currently form the major part of the growing international microbial pesticide market. The use of rhizospheric bacterial isolates, especially those belonging to genus Bacillus have received major attention as biocontrol agents because of their ability to produce variety of biomolecules such as kanosamines, lipopeptides and proteins with diverse modes of action (Nicholson 2002; Ongena and Jacques 2008).

Bacillus thuringiensis (Berliner) has been successfully used to control lepidopteran, dipteran and coleopteran pests and has dominated the microbial control of insect pests (Ferro et al. 1997; Lacey et al. 2001; Bravo et al. 2011). B. thuringiensis (Bt) produces crystal proteins which are highly toxic to insects (Aranda et al. 1996). The other entomopathogenic bacteria developed commercially as insecticidal agents include Gram positive bacteria like Bacillus sphaericus (Garrity and Lilburn), Bacillus subtilis (Cohn), Bacillus firmus (Bredemann and Werner), Paenibacillus popilliae (Pettersson) and Gram negative bacteria like Pseudomonas sp. (Migula), Chromobacterium sp. (Schroter) and Serratia entomophila (Grimont) (Martin and Blackburn 2008; Jurat-Fuentes and Jackson 2012; Chattopadhyay and Sen 2013). The insecticidal activity of most of these entomopathogenic bacteria is mainly associated with toxins (Nielsen-Leroux et al. 2002). The different toxins like rhamnolipids, orfamide, viscosin, pyoverdine, etc., produced by Pseudomonas protegens, P. fluorescence, P. chlororaphis, P. entomophila and P. syringae have been reported earlier for their insecticidal activity (Kupferschmied et al. 2013). However, there are reports indicating development of resistance in insects to bacterial toxins attributed to their regular use over the years (Singh et al. 2004; Su and Mulla 2004). Thus, there is need to explore novel bacterial strains for insecticidal potential.

Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae) is a polyphagous pest feeding on more than 150 species of host plants (Ahmed et al. 2013). It is becoming a serious pest of various economically important crops, viz., groundnut, cotton, cabbage, cauliflower, chilli, castor, tobacco and pulses (Tuan et al. 2014). The use of chemical insecticides is the primary method to control insect pests; however, this pest has developed resistance to most of the commercially available insecticides (Tong et al. 2013). The present study explores the insecticidal potential of isolate Bacillus vallismortis R2 (Roberts) (Bacillales: Bacillaceae) against S. litura. The intact cells and the extracellular biomolecules produced by the isolate were analyzed for their insecticidal potential against S. litura. Different strains of B. vallismortis have been earlier reported as biocontrol agents for plant pathogenic fungi (Zhao et al. 2010; Kaur et al. 2015); however, this is a first effort to explore its insecticidal potential against a polyphagous pest, S. litura.

Materials and methods

Microorganisms and growth conditions

The bacterial strain R2 was isolated from soil samples collected from rhizosphere of wheat and maintained on yeast malt extract (YME) agar medium. It was identified as B. vallismortis and the nucleotide sequence of gene coding for 16S rRNA has been submitted to GenBank, with Accession No. KC283030 (Kaur et al. 2015). The cells of R2 were preserved in 20% glycerol and kept at − 80 °C for long-term preservation.

Insect rearing

Egg masses and larvae of S. litura collected from the cauliflower fields around Amritsar (Punjab), India, were used to establish a laboratory culture. Rearing was carried out in jars (15 cm × 10 cm) at 25 ± 2 °C and 65 ± 5% relative humidity (RH). The larvae were fed on leaves of castor (Ricinus communis) and the diet was changed daily till pupation. Pupae were transferred to pupation jars (15 cm × 10 cm) containing 2–3 cm layer of sterilized moist sand. The adults just after emergence were shifted to oviposition jars similar to pupation jars, and provided with a cotton swab soaked with honey solution (1 part honey to 4 parts water) as food. The oviposition jars were lined with filter paper to facilitate egg laying. The newly hatched larvae were immediately transferred to fresh castor leaves and from this stock culture, larvae were used for further experiments.

Preparation of cell suspension

Bacterial cells were inoculated to 200 ml yeast malt extract (YME) broth in 500 ml Erlenmeyer flask. The flask was incubated at 30 °C, 180 rotations per minute (rpm) for 48 h. The activated cells were harvested by centrifugation at 10,000 rpm for 10 min and the pellet was washed thrice with phosphate buffer saline (PBS) (pH 7.0). The pellet was re-suspended in same buffer and suspension was diluted to OD600 of 0.5, 1.0, 1.5 and 2.0. The colony forming unit (CFU) in cell suspension with respective OD600 was determined by standard plate count method and was equivalent to C1 = 7.0 × 107, C2 = 2.6 × 108, C3 = 6.8 × 108, C4 = 1.8 × 109 (CFU)/ml, respectively. These concentrations were further used for experiment as described in following section:

Effect of cells of R2 on S. litura

The insecticidal potential of R2 cells was evaluated by feeding the 2nd instar larvae (6 days old) of S. litura on castor leaves treated with different concentrations of R2 cells, i.e., C1, C2, C3, and C4 (as mentioned above). The larvae fed on leaves treated with PBS buffer served as control. The experiment was replicated six times with five larvae/replication (n = 30). The larvae were kept individually in plastic containers (4 cm × 6 cm) and incubated at 25 ± 2 °C temperature and 65 ± 5% RH. The larvae were provided with fresh treated/untreated leaves at a regular interval of 2 days till pupation. The survival of larvae was recorded daily. Observations were also made on development period, pupation and adult emergence.

Preparation of acid-precipitated biomolecules (APB)

The preparation of APB was done as per protocol described earlier (Kaur et al. 2015). The methanol dissolved stock solution (1% w/v) of APB was filter-sterilized (Acrodisc 13 mm with 0.2 µm Supor Membrane, Pal Life Sciences, USA). The appropriate volumes of this stock solution were added to diet to achieve initial concentrations of C1 = 50, C2 = 100, C3 = 150, C4 = 200, C5 = 250 µg/ml.

Effect of APB of R2 on S. litura

To study the effect of APB of R2 on S. litura larvae, the artificial diet was supplemented with different concentrations of APB as described above. The artificial diet was prepared with slight modifications (Gupta et al. 2005). The ingredients of diet were wheat bran, kidney bean flour, yeast, methyl para-benzoic acid, ascorbic acid, sorbic acid, etc. The diet with methanol but without APB served as control. The 2nd instar (6 days old) larvae were reared on amended and unamended diets at 25 ± 2 °C temperature and 65 ± 5% RH. The experiment was replicated six times with five larvae/replication (n = 30). The larvae were kept individually in plastic containers (4 cm × 6 cm) to avoid cannibalism and the diet was changed on alternate days. The larvae were checked daily for their survival. The larvae that survived were further observed for various biological parameters, viz., development period, pupation and adult emergence. The sub-lethal effects of APB on each developmental stage such as larval, pupal and adult deformities were also recorded.

Effect of APB of R2 on nutritional physiology of S. litura

The effect of APB on food utilization of 2nd instar larvae of S. litura was studied as per procedure described in literature (Farrar et al. 1989; Waldbauer 1968). The artificial diet was prepared and amended with five different concentrations of APB of R2 (C1–C5) as described above. The diet without APB and with methanol served as control. The larvae starved for 3–4 h were weighed individually and placed in plastic containers (4 cm × 6 cm) containing known amount of control or treated diets. The temperature and humidity conditions were maintained at 25 ± 2 °C and 65 ± 5%, respectively. The experiment was carried out using 15 larvae for each treatment and the observations were made after 72 h on larval weight, residual diet and faecal matter. The overall change in each variable was compared with the last recorded value. At the end of each experiment, the dry weight of the larvae was determined by incubating at 60 ± 2 °C for 72 h. Similarly, the dry weight of diet and faecal matter was recorded to determine the loss of water under experimental conditions. The various nutritional parameters were calculated using the following formulae:

$${\text{RGR}} = \frac{{{\text{Change}}\;{\text{in}}\;{\text{larval}}\;{\text{dry}}\;{\text{weight}}/{\text{day}}}}{{{\text{Starting}}\;{\text{larval}}\;{\text{dry}}\;{\text{weight}}}},$$
$${\text{RCR}} = \frac{{{\text{Change}}\;{\text{in}}\;{\text{diet}}\;{\text{dry}}\;{\text{weight/day}}}}{{{\text{Starting}}\;{\text{larval}}\;{\text{dry}}\;{\text{weight}}}},$$
$${\text{ECI}} = \frac{{{\text{Dry}}\;{\text{weight}}\;{\text{gain}}\;{\text{of}}\;{\text{larva}}}}{{{\text{Dry}}\;{\text{weight}}\;{\text{of}}\;{\text{food}}\;{\text{ingested}}}} \times 100,$$
$${\text{ECD}} = \frac{{{\text{Dry}}\;{\text{weight}}\;{\text{gain}}\;{\text{of}}\;{\text{larva}}}}{{{\text{Dry}}\;{\text{weight}}\;{\text{of}}\;{\text{food}}\;{\text{ingested}} - {\text{Dry}}\;{\text{weight}}\;{\text{of}}\;{\text{frass}}}} \times 100,$$
$${\text{AD}} = \frac{{{\text{Dry}}\;{\text{weight}}\;{\text{of}}\;{\text{food}}\;{\text{ingested}} - {\text{Dry}}\;{\text{weight}}\;{\text{of}}\;{\text{frass}}}}{{{\text{Dry}}\;{\text{weight}}\;{\text{of}}\;{\text{food}}\;{\text{ingested}}}} \times 100,$$

where RGR is the relative growth rate, RCR is the relative consumption rate, ECI is the efficiency of conversion of ingested food, ECD is the efficiency of conversion of digested food, and AD is the approximate digestibility.

Statistical analysis

The experiment was conducted as a completely randomized block design. All the values were represented as their mean ± standard error. The data for bioassay studies and nutritional indices were subjected to one way analysis of variance (ANOVA). The differences between treatments were determined by Tukey’s test at p ≤ 0.05. SPSS software for Windows version 16.0 (SPSS Inc, Chicago) and Microsoft Office Excel 2007 (Microsoft Corp., USA) were used to perform the statistical analysis.

Results

Effect of cells of R2 on S. litura

The feeding of larvae of S. litura with castor leaves impregnated with different concentrations of cells of isolate R2, adversely affected their survival and development. The rate of larval mortality increased in a dose-dependent manner (Fig. 1) with higher concentrations (C3 and C4) resulting in 11- to 13-fold increase over control (F = 14.1, p ≥ 0.05). The larvae feeding on leaves treated with cells of R2 became sluggish, stopped feeding and turned black, soft and flaccid after death. The larvae fed on leaves impregnated with 1.8 × 109 CFU ml−1 took 1.7 days more to pupate (F = 6.4, p ≤ 0.05), whereas no significant delay was observed in larvae fed with lower concentrations of R2 cells. Similarly, the pupal development was significantly delayed in larvae reared on castor leaves treated with 6.8 × 108 and 1.8 × 109 CFU/ml of R2 (F = 45.9, p ≤ 0.05). The total development period of S. litura tended to prolong with significant effect at higher concentrations (F = 61.81, p ≤ 0.05). As compared to control, it took 4.2 and 6.0 days more for larvae to develop into adults when reared on leaves treated with 6.8 × 108 and 1.8 × 109 CFU/ml of R2, respectively. The adverse effects of cells of R2 were also observed on adult emergence as all the treatments differed significantly from control with only 25.0–58.3% adults emerging from larvae reared on treated leaves as compared to 100.0% emergence of adults in control (F = 12.5, p ≤ 0.05) (Table 1).

Fig. 1
figure 1

Larvicidal activity of different concentrations of cell suspension of R2 against S. litura (C1 = 7.0 × 107, C2 = 2.6 × 108, C3 = 6.8 × 108 and C4 = 1.8 × 109 CFU/ml). The error bars represent standard error and the means followed by different letters (a, b, c) within a column are significantly different; Tukey’s test, p ≤ 0.05

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Table 1 Effect of different concentrations of cell suspension of R2 on development and adult emergence of S. litura
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Effect of APB of R2 on S. litura

The insecticidal potential of the APB was evaluated by feeding the larvae artificial diet impregnated with different concentrations of APB. A progressive increase in larval mortality was observed with increasing concentration of APB in artificial diet. The 200 and 250 µg ml−1 supplements of APB to larval diet caused 73.3 and 76.6% larval mortality, respectively as compared to 3.3% observed in control (Fig. 2). The symptoms like cessation of feeding and dead larvae, under the influence of APB were similar to those observed for larvae infected with cells of R2. The APB was also found to have growth inhibitory influence on S. litura as the larval and pupal development period extended significantly in all the treatments as compared to control (Table 2). The overall development period from larva to adult emergence extended significantly under the influence of APB. The larvae fed on diet supplemented with 250 µg ml−1 APB took 32.8 days to develop into adults in comparison to 21.9 days for control larvae (F = 144.2, p ≤ 0.05) with 44.4% adult emergence (F = 2.7, p ≤ 0.05). The percentage of morphologically deformed adults having crumpled and underdeveloped wings was significantly higher at all the concentrations of APB (F = 3.0, p ≤ 0.05) (Fig. 3).

Fig. 2
figure 2

Larvicidal activity of different concentrations of APB of R2 against S. litura (C1 = 50, C2 = 100, C3 = 150, C4 = 200 and C5 = 250 µg/ml of APB). The error bars represent standard error and the means followed by different letters (a, b, c) within a column are significantly different; Tukey’s test, p ≤ 0.05

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Table 2 Effect of different concentrations of APB of R2 on development and adult emergence of S. litura
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Fig. 3
figure 3

Deformed adults (%) of S. litura due to different concentrations of APB of R2. The error bars represent standard error and the means followed by different letters (a, b, c) within a column are significantly different; Tukey’s test, p ≤ 0.05

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Effect of APB on nutritional physiology of S. litura

The addition of APB to diet negatively influenced the nutritional physiology of S. litura larvae. The consumption of APB-supplemented diet lead to a significant decrease of RGR by 17.1–65.6% over control (F = 7.2, p ≤ 0.05). A significant reduction in RCR was recorded with maximum effects at the highest concentration (250 µg ml−1) that resulted in 41.7% decrease over control (F = 6.5, p ≤ 0.05). The inhibitory effect of bacterial biomolecules was also detected on ECI and ECD. All the treatments differed significantly from control except for the lowest concentration (ECI: F = 3.8, p ≤ 0.05; ECD: F = 3.1, p ≤ 0.05). The ECI and ECD values of larvae feeding on diet amended with 100–250 µg/ml of APB were 1.5–1.7 and 1.2–1.5 times lower than control respectively (Table 3). Similarly, AD decreased significantly when 100–250 µg/ml of APB was added to the diet (F = 9.4, p ≤ 0.05). The reduced values of RGR, RCR, ECI, ECD and AD indicate deterrent as well as toxic effect of the APB on S. litura.

Table 3 Influence of different concentrations of APB of isolate R2 on growth, feeding and food utilization of S. litura larvae after 3 days of feeding
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Discussion

The insecticidal potential of bacteria, particularly B. thuringiensis (Bt), B. subtilis and Bacillus cereus (Frankland) has earlier been reported against various insect pests belonging to the orders Lepidoptera, Coleoptera and Diptera (Polanczyk et al. 2000; Assie et al. 2002; Selvakumar et al. 2007; Strongman et al. 1997; Chatterjee et al. 2010). The results of the present study demonstrated that both intact cells and APB of B. vallismortis strain R2 adversely affected the survival and development of S. litura. These adverse effects may be due to production of CLPs, viz., surfactin, iturin A and fengycins by B. vallismortis R2 (Kaur et al. 2017). The insecticidal potential of CLPs like surfactin and iturin A from different strains of B. subtilis against various insects, viz., Drosophila melanogaster (Meigen), Ephestia kuehniella (Zeller) and Culex quinquefasciatus (Say) has been documented previously (Assie et al. 2002; Geetha and Manonmani 2010; Ghribi et al. 2012). However, there are no reports on insecticidal potential of CLPs from B. vallismortis against S. litura. In comparison to the intact cells, the APB of R2 was found to have more larvicidal effect.

The consumption of intact cells as well as APB of R2 caused cessation of feeding in larvae followed by paralysis and death. The dead larvae were soft, flaccid with intact integument having blackish colour, which are typical symptoms of bacterial infection (Huang et al. 2009; Vega and Kaya 2012). The results of present study also indicated the growth inhibitory effects of cells and APB of R2. Besides significant influence on development, the isolate R2 also reduced the adult emergence. The extended development period of Spodoptera spp. under the influence of secondary metabolites from Bacillus spp. has earlier been documented by Chandrasekaran et al. (2012). Similarly, exposure of larvae of S. littoralis to B. thuringiensis and B. subtilis delayed the development of insect (Mohamed et al. 2005; Abd El-Salam et al. 2011). The toxicity of APB of R2 was also manifested in the form of morphologically deformed S. litura adults with crumpled and underdeveloped wings. Deformities in Musca domestica vicina (Macq) have also been demonstrated due to B. thuringiensis israelensis (Abozinadah et al. 2011). Previously, the morphologically deformed adults of S. littoralis showed reduced longevity and failed to reproduce, thus significantly affecting the population build-up (Martinez and van Emden 2001). Thus, the cells and APB of R2 can be further used for developing bio-formulation for controlling insect pest at field scale level.

The inhibitory effects of APB were also detected on nutritional physiology of S. litura. The decrease in relative consumption and growth rate of larvae feeding on APB amended diet indicates the antifeedant effect of metabolites of B. vallismortis R2. The toxicity of R2 biomolecules is also evident from significant drop in ECI and ECD values as more food is being utilized for energy required for detoxification and less is being converted to biomass that resulted in reduced RGR. Thus, reduction in growth of larvae may not be entirely due to antifeedant activity of APB but partly due to toxic effect of biomolecules produced by B. vallismortis. A significant reduction in RGR and RCR of Spodoptera spp. due to secondary metabolites from B. subtilis had been documented earlier (Chandrasekaran et al. 2012). The toxins produced by different strains of B. sphaericus have been reported to target the midgut epithelium of insects which is the major site for synthesis and secretion of digestive enzymes (Carpusca et al. 2006; Park et al. 2010; Senthil-Nathan 2013). Different metabolites from genus Bacillus have been demonstrated to cause structural disorganization and atrophy of midgut of insect leading to their death in 2–5 days following the toxin consumption (Soberon et al. 2009; Ruiu et al. 2012). Thus, it is possible that decreased nutritional/food utilization efficiency of S. litura after consumption of APB of R2 may be due to adverse effects on midgut epithelium and disruption of enzyme secretion and nutritional absorption.

The present study reveals the insecticidal potential of both cells and APB of R2. The insecticidal activity of R2 may be attributed to production of extracellular CLPs (exotoxins) which are reported to have antifungal potential (Kaur et al. 2015, 2017). The biodegradable and less toxic nature of these biomolecules than chemical pesticides are particularly suitable for developing environmental-friendly formulations. Thus, the isolate B. vallismortis R2, efficient in arresting the growth and development of S. litura, can be a suitable candidate for integrated pest management strategies and helpful in reducing the dependence on synthetic pesticides. Further studies on effect of individual purified biomolecule against S. litura as well as their mode of action remain to be elucidated.