Efficacy of native Bacillus isolates against different larval instars of fall armyworm, Spodoptera frugiperda alone and in combination

Fall armyworm (FAW) Spodoptera frugiperda (J.E. Smith) Lepidoptera: Noctuidae is an invasive polyphagous pest that causes severe damage to several agricultural crops. The use of pesticides is limited because of their mode of feeding and resistant development. Hence, the present work aimed to determine the pathogenicity of entomopathogenic bacteria (Bacillus spp.) against FAW in terms of mortality and growth inhibition. In this study, initially 49 native Bacillus isolates, isolated from diverse habitats in India, along with five reference strains, were screened for their efficacy against neonates of S. frugiperda under controlled laboratory conditions, followed by virulence and combinatorial bioassays. Five native Bacillus isolates (VKK1, VKK5, S16C2, S25C1, and SOIL 20) showed mortality in the range of 35.49–65.52% against neonates of S. frugiperda at single concentration (1000 μg g−1 of diet). These five isolates, along with one reference strain Btk-HD1 (Bacillus thuringiensis serovar kurstaki strain HD1), were further tested to find the median lethal concentration (LC50) for neonates of S. frugiperda. Among these, native Bt strain VKK5 showed the lowest LC50 (718.40 µg/g of diet) and HD1 showed the highest LC50 (3352 µg/g of diet). Combinatorial bioassay against neonate and third instar larvae showed that the combination of VKK5 and VKK1 had an additive effect. Moreover, growth inhibition was also recorded. The combination of Bt strains leads to an enhancement of pathogenicity toward FAW larvae at the initial stage of development, and in later stages, it affects their growth and development. Thus, biocontrol of FAW by entomopathogenic bacteria (Bt) can play a vital role in the effective management of FAW.


Background
The Bacillus genus has a wide genetic diversity ranging from seawater to soil, and it is even found in extreme environments (Usta 2013).Bacteria of the genus Bacillus are of greater agricultural importance due to their capacity to produce lipopeptides (LPs), which are active against insects, mites, nematodes, and phytopathogens (Penha et al. 2020).B. thuringiensis (Bt) is a Grampositive, facultative anaerobic, spore-forming bacteria that produces parasporal crystals during its sporulation phase, which produce protein toxins to kill insects of different groups with high host specificity and environmental safety (Sanahuja et al. 2011).Other than Bt, exploration of Bacillus sp.against insect pests was carried out for several pests.
The fall armyworm (FAW), Spodoptera frugiperda (J.E.Smith) (Lepidoptera: Noctuidae), is a migratory polyphagous pest native to the neotropics of the Americas and has created a great threat to food security in invaded countries (Bruce 2020).In India, the FAW was observed for the first time in 2018 (Ganiger et al. 2018).It was majorly associated with maize crops across different regions in the country (Deshmukh et al. 2020).Within a span of 2 years, FAW has established itself throughout the country and had wreaked havoc on major staple and cash crops such as maize, sorghum, sugarcane, rice, cotton, soybean, peanut, millets, and ginger (Shankar and Adachi 2019).Two types of strains were reported in FAW, referred to as rice and corn strains (Pashley 1988).In India, only the rice (R) strain was detected up to December 2018 and was found to feed on maize (Swamy et al. 2018).Later in January 2019, the occurrence of the corn (C) strain on sugarcane was also reported from India (Nayyar et al. 2021).It has affected 26 Indian states out of a total of 29 states (Delanthabettu et al. 2022).Centre for Agriculture and Bioscience International (2022) reported the occurrence of FAW in Africa (48 nations), Asia (27 nations), Europe (6 nations), North America (32 nations), Oceania (6 nations), and South America (13 nations).
Being a highly polyphagous pest, its management strategies mostly rely on the use of pesticides, which has led to the development of resistance to permethrin, cypermethrin, carbaryl, thiodicarb, chlorpyrifos, and dichlorvos (Zhang et al. 2019).Hence, biopesticides are the most prominent method for controlling this pest due to their specificity and efficacy in the management of agricultural pests.In this context, the present study was conducted on the screening of native Bacillus spp.isolated from diverse habitats in India against FAW, followed by combinatorial bioassays in terms of larval mortality and growth inhibition.In this context, the present study was conducted on the screening of native Bacillus spp.isolated from diverse habitat of India against FAW, followed by combinatorial bioassays in terms of larval mortality and growth inhibition.

Insect collection and rearing
Fall armyworm (FAW) larvae were collected from a maize field at the Indian Agricultural Research Institute, New Delhi, and reared on a kabuli gram-based semi-synthetic diet till pupation in the laboratory as per Gopalakrishnan and Kalia (2022).On emergence, five pairs of adults were transferred to a mating jar (20 cm height and 15 cm diameter) covered with muslin cloth containing a 10% fortified honey solution for adult feeding and folded paper strips for egg laying.Egg masses were collected daily and kept in a separate container for hatching.On hatching, neonates were transferred to a semi-synthetic diet in a plastic container and the lid was closed tight with tissue paper to prevent the escape of larvae.After 7 days of feeding, the larvae were separated and placed in individual plastic containers having one small cube of diet to prevent cannibalism of the larvae.Later on, pupae were collected and kept in plastic containers lined with blotting paper until adult emergence.The neonates (< 24 h old) and later instars were used for bioassays.The culture was maintained under controlled laboratory conditions, i.e., at 27 ± 1 °C and 70 ± 5% relative humidity and a 14L:10D photoperiod.

Screening of Bacillus isolates against neonates of fall armyworm
Screening bioassays were carried out by the diet incorporation method by using acetone precipitated spore/spore crystal complexes (weight basis) of Bacillus isolates that were isolated from silo dust, insect cadavers, live insects, plant endophytes (neem), and various agricultural ecosystems of the north eastern region of India (Sibsagar and Jorhat, Assam), and also five reference strains were used for screening bioassays.Acetone precipitated spore crystal complex of forty nine Bacillus isolates, viz., B. thuringiensis (14), B. subtilis (13), B. wiedmanni (3), B. cereus (2), B. flexus (1), B. megaterium (1), B. thermophilus (2), Bacillus spp.( 7), B. paramycoides (2), Aneurinobacillus migulans (1), A. aneurinilyticus (2), Lysinibacillus sphaericus (1), and 5 reference strains, viz., B. thuringiensis serovar kurstaki (HD1, HD73), B. thuringiensis serovar thuringiensis (HD2, HD14), B. thuringiensis serovar aizawai HD137 were taken from the bacterial stock of the National Facility for Insect Rearing and Xenobioticcum-Transgenic Bioassay, Division of Entomology, IARI, New Delhi.These isolates were screened at a single concentration (1000 μg g −1 of diet) by the diet incorporation method using spore/spore crystal complex as per Daravath et al (2015).The toxin-incorporated diet was transferred to small plastic containers (5 × 2 cm).Each container was served as one replicate, with three replications per treatment.Ten neonates were released on the treated diet per replicate.The diet in the control was mixed with the same volume of sterilized distilled water (SDW).All the bioassays were conducted under controlled conditions of 27 ± 1 °C, 70 ± 5% RH, and a 14L:10D photoperiod.Mortality data was recorded every 24 h till 7th day after treatment, and corrected percent mortality was calculated on 7th day using Abbott's formula (1925).

Bioassay with a combination of different Bacillus thuringiensis strains against neonates of fall armyworm
In this study, the Zwittermicin A-positive isolates of native Bt i.e., VKK-LE1, VKK-LE2, and VKK1 (data not shown) were combined with the most potential Bt isolate, VKK5.Combinatorial bioassays were carried out by two methods i.e., by using Bt cell suspensions (1 × 10 8 cells/g of diet in VKK5, VKK1, VKK-LE1 and VKK-LE2; 1 × 10 4 cells/g of each isolate in combination of VKK5 + VKK1, VKK5 + VKK-LE1,VKK5 + VKK-LE2) and another by using acetone precipitated spore crystal complex (weight basis) (1000 μg g −1 of diet in VKK5, VKK1, VKK-LE1 and VKK-LE2; for combination bioassay 500 μg g −1 of diet of each isolate (VKK5 + VKK1, VKK5 + VKK-LE1, VKK5 + VKK-LE2).For cell suspension, each Bt strain was inoculated in Luria Bertani broth (LB) and incubated at 30 °C for 72 h in an incubator shaker (200 rpm).After 72 h, the bacterial colonies in suspension were centrifuged, and pure colonies of bacteria collected as pellets in the centrifuge tube were re-suspended in SDW.The number of viable spores was estimated by counting colony forming units (cfu) on LA plates after overnight incubation at 30 °C.The Bt incorporated in the diet in the form of Bt cell suspensions (1 × 10 8 cells/g of diet) and spore crystal complexes (1000 μg g −1 of diet) was used for bioassay against neonates of FAW.Mortality data was recorded every day till 7th day after treatment.Corrected percent mortality on 7th day was calculated using Abbott's formula (1925).

Mortality and sublethal effects of Bt strains alone and their combinations on third and last-instar larvae of FAW
The combinatorial bioassays were carried out on third and last-instar larvae of FAW to examine mortality as well as growth reduction using most potential combination (VKK5 and VKK1) against neonates of FAW.This bioassay was also performed with both acetone precipitated spore crystal complex (weight basis) and cell suspensions (cfu/ml) as mentioned above.The observations were made on percent mortality and differences in larval weight in third instar and last-instar larvae after 7 and 2 days of treatment, respectively.The survived larvae were observed till adult emergence for sub-lethal effects.

Co-toxicity of a combination of different Bt isolates against fall armyworm larvae
The interaction between Bt strains in relation to larval mortality was differentiated according to the co-toxicity factor (Mansour et al 1966) as follows: where % expected mortality = the sum of % mortality in each Bt isolate alone and % observed mortality = % mortality in combination bioassay.A co-toxicity factor of > 20 is considered synergistic, a co-toxicity factor of < − 20 is considered antagonistic, and co-toxicity factor of an intermediate value between − 20 and + 20 is considered as additive.

Confirmation of the Bt strain in the infected larvae
To verify that the strains employed in the bioassays were the cause of the larval mortality, larvae that had exhibited typical symptoms were collected.Each deceased larva was packed into a 1.5 ml micro-centrifuge tube after surface sterilization with 70% ethyl alcohol and then submerged in sterile water.Larvae were then homogenized with 100 µl SDW and spread on a nutrient agar (NA) plate supplemented with ampicillin at 50 μg/ml (these Bt strains are resistant to ampicillin) and incubated at 30 °C for 72 h.The bacterial colonies grown on NA plates were checked with original Bt colonies for colony morphology; further confirmation of spore crystals inside the bacterial cells of re-isolated Bt colonies was done by using a phase contrast microscope.

Statistical analysis
The data on corrected percent mortality of all bioassays and larval weight were subjected to analysis of variance (ANOVA) at 5% level of significance using statistical analysis system (SAS) version 4.2 (SAS Institute Inc. Cary, USA).The significantly different means (< 0.05) were separated using Duncan's Multiple Range (DMRT) test.The LC 50 values for bioassays were calculated using the maximum likelihood programme (MLP) 3.01 (Ross 2000).

Screening of native Bacillus isolates against neonates of fall armyworm
Perusal of the mortality data in (  S28C2).Out of 49 native Bacillus isolates, 2 showed more than 50% mortality, 13 showed 30-50% mortality, and 5 showed less than 15% mortality.The results revealed that there was a variation in virulence among Bt strains as well as other Bacillus isolates from different habitats toward FAW neonates.

Computation of the median lethal concentration (LC 50 ) of potential Bacillus spp. against neonates of fall armyworm
Perusal of LC 50 data (Table 2) showed that LC 50 values of the spore crystal form of Bacillus spp isolates varied from 718.40 μg/g of diet (BtVKK5) to 3352 μg/g of diet (Btk-HD1) against neonates of FAW.Among the studied Bacillus spp.isolates, BtVKK5 was found to be the most toxic with a minimum LC 50 of 718.40 μg/g of diet, followed by BtVKK1 (995.59 μg/g of diet).Btk-HD1 was found to be at par with other B. subtilis isolates (SOIL20) but significantly different from other native Bacillus isolates.

Combined effect of different Bt isolates on neonates of the fall armyworm
Mortality results of the combinatorial bioassay (Fig. 1) showed that in cell suspensions (1 × 10 8 cells/g of diet), the mortality was doubled in the combination of VKK5 + VKK1 (92.86%) as compared to VKK5 alone (46.43%).The corrected percent mortality in VKK1 (32.14%) was less than half of the percentage mortality obtained in its combination with VKK5.The VKK-LE1 and VKK-LE2 strains were able to cause 25 and 17.85%  mortality, respectively, while their combination with VKK5 increased the mortality by more than two-fold.In the cell suspension bioassay, the mortality recorded in Bt isolates alone and in combination with BtVKK5 was statistically different from each other except for combinations of VKK5 + VKK-LE1 and VKK5 + VKK-LE2.
The spore crystal complex bioassay results (Fig. 1) showed that the percent mortality obtained in combination of VKK5 + VKK1 (93.11%) was the maximum, followed by VKK5 + VKK-LE1 (75.87%), which were statistically different from each other.Comparing the results of VKK1 with the combination of VKK5 and VKK1, the percent mortality was nearly doubled.The results obtained clearly showed that the combination of VKK5 with VKK1, VKK-LE1, and VKK-LE2 increases the virulence of VKK5 against neonates of FAW.Among those, the highest mortality was observed in VKK5 + VKK1 in both cell suspension (92.86%) and spore crystal complex (93.11%) bioassays.

Mortality and growth reduction in third and last larval instars fed on a Bt-incorporated diet
The results of a combinatorial bioassay of VKK5 and VKK1 against third and last larval instars of FAW in both cell suspension (1 × 10 8 cells/g of diet) and spore crystal complex (1000 μg g −1 of diet) revealed that in last instar larvae no mortality was observed.Perusal of data in (Fig. 2) showed that there was a significant increase in mortality with the combination of VKK5 and VKK1 when compared to the treatments alone in third-instar larvae in both cell suspension and spore crystal complex bioassays.
Difference in larval size was observed in both cell suspension and spore crystal complex bioassays (Fig. 4A).The larval weight was significantly reduced in all the treatments, especially in the combination of VKK5 + VKK1 (180.69,158.83 mg) when compared to control (461.35,463.80 mg) (Fig. 3), in both cell suspension and spore crystal form, respectively.The weight of the larvae attained after bioassay was statistically at par in VKK5 (248.60 ± 25.65 mg) and VKK1 (254.71 ± 21.23 mg) in cell suspension (1 × 10 8 cells/g of diet) bioassay.In the last instar larval bioassay, the weight of the combination of VKK5 + VKK1 was very low in both cell suspension (162.80 mg) and spore crystal complex (135.05mg) bioassays when compared to all other treatments and control (420.88 mg).The weight attained in larvae fed on the diet incorporated with VKK5 (206.80,225.75 mg) was significantly different when compared to VKK1 (308.10,272.30mg) in both cell suspension and spore crystal complex bioassays of last instar larvae (Fig. 3).The size of pupae was reduced in all the treatments than the control.Subsequently, small pupae (Fig. 4B) and malformed adults (Fig. 4C) were formed in the larvae that fed on the combination of VKK5 and VKK1.

Co-toxicity factor of a combination of Bt isolates in neonates and third-instar larvae of FAW
The co-toxicity factor was calculated for the combined effect of different Bt isolates (Table 3).The results revealed that, in the neonate bioassay, the combination of VKK5 and VKK1 in cell suspension has an additive effect with a co-toxicity factor of 18.19%.All the isolates in combination with BtVKK5 in both cell suspension and spore crystal complex showed additive effects to different extents.Third-instar larvae also showed an additive effect with a co-toxicity factor of − 12.50 and − 20% in both cell suspension and spore crystal Fig. 2 Corrected percent mortality of combinatorial bioassay in spore crystal and cell suspension form of Bacillus thuringiensis strains against 3 rd instar larvae of Spodoptera frugiperda.*Corrected percent mortality bar's followed by different small alphabets and capital alphabets are significantly different at 5% level in 1 × 10 8 cells/g of diet and 1000 µg/g of diet, respectively Fig. 3 Mean larval weight attained in 3rd and last larval instars of Spodoptera frugiperda after 4th day and 2nd day of combinatorial bioassay of Bacillus thuringiensis strains.*Mean larval weight bar's followed by different small alphabets and capital alphabets are significantly different at 5% level in 1 × 10 8 cells/g of diet and 1000 µg/g of diet, respectively form, respectively (Table 3).The results of this study indicated that there was a strong additive effect in the toxicity of native Bt isolates, which produced mortality in FAW larvae.Apart from mortality, native Bt isolates can produce sublethal effects on the growth and development of FAW larvae, which remain until adult emergence.

Bt confirmation in the infected larvae
The neonate larvae feeding on a diet treated with native Bacillus isolates during the bioassay experiment became lethargic, stopped feeding, and turned black when compared to the control (Fig. 5A, B).But in the case of Bt treatment, in addition to the above symptoms, the gut region of larvae turned a black colour (Fig. 5C, D).The larvae were found dead on the surface of the diet and became flaccid after death.The dead larva was homogenized and plated on NA plates.The bacterial colonies obtained on NA plates (Fig. 5E) were confirmed for spore crystals inside the bacterial cells by using a phase contrast microscope (Fig. 5F).The results proved that the Bt strains were responsible for the mortality of neonates, and the physical changes were induced by B. thuringiensis infection.Koch's postulates were fulfilled by the confirmation of the Bt strain after re-isolation from the infected larval gut.

Discussions
Fall armyworm is a highly polyphagous pest, and its management methods are not well developed in India.Bacillus isolates have a range of functions in ecology, biotechnology, industrial, and clinical microbiology.Soilassociated Bacillus strains are the source of industrial enzymes and may be vital for the cycling of organic matter in soil environments (Avsar et al. 2017).Bt has been isolated from a variety of environments, including soil, dead insects, grains, deciduous and coniferous leaves, and stored product dust (Rajashekhar et al. 2017).In the present study, the susceptibility of FAW against different  Bacillus sp. from diverse habitats was explored.The pathogenicity of 49 native Bacillus isolates, which were isolated from silo dust, insect cadavers, live insects, plant endophytes (neem), and various agricultural ecosystems of the north eastern region of India was tested against neonates of S. frugiperda.Two Bt isolates, viz., VKK5 and VKK1, recorded > 50% mortality, whereas isolates S16C2, S25C1, and SOIL 20 caused > 35% mortality in the diet incorporation bioassay.The results revealed that other than Bt, several Bacillus spp.also showed pathogenicity toward neonates of FAW.Studies observed that Bacillus spp., which were isolated from soil, also had a larvicidal effect against FAW (Handayani et al. 2023).There are several studies exploring the susceptibility of S. frugiperda to Cry toxins and Bt strains (Maheesha et al. 2021).Several other Bt subspecies were also reported to cause mortality in second instar FAW larvae, viz., Bt dendrolimus HD37, Bt aizawai HD68, Bt kurstaki HD73, and Bt darmstadiensis HD146, in in-vivo assays.According to studies performed by Hernandez (1988), subspecies of Bt kurstaki, Bt aizawai, and Bt thuringiensis caused death rates of 80, 100 and 70% against FAW neonates at (3 × 10 7 cells/ml), respectively.The composition of the crystals and their toxic potential may be associated with the variations in these strains' toxicity to S. frugiperda (Polanczyk et al. 2000).
Present study reported that the pathogenicity of native strains was higher than that of reference strains.Similar kinds of studies with Bacillus isolates stated that the commercially available B. thuringiensis subsp.kurstaki product, Dipel, was not promising against larvae of fall armyworm, with 48.86% mortality and an LC 50 of 116.239 (Priyanka et al. 2021).Obtained results are in accordance with the results of Delanthabettu et al (2022), who found that some strains were much more efficient than the reference strain (HD1) in killing the FAW larvae.Bt isolates were showing mortality in the range of 6.9-65.52%.
In present study, the combined effects of different Bt strains are evident.Kausarmalik and Rizwana (2014) stated the combined effect of different Bt strains on the management of the red flour beetle.There are reports indicating that certain strains of B. thuringiensis produce a compound that potentiates another B. thuringiensis activity (Manker et al. 2002).In combination bioassays of third and last larval instars, mortality was observed only in third instar larvae but not in last instar larvae.The differences in vulnerability and death rates across developmental instar larvae may be connected to their morphological features, sizes, behaviors, and immunological defence systems, as previously stated by Elbrense et al (2021).The extent of synergism between spores and toxins of B. thuringiensis depends on the strain of insect, the type of spore, the set of toxins, the presence of other materials such as formulation ingredients, and the concentrations of spores and toxins (Liu et al. 1998).In this study, the larvae fed on Bt incorporated diet showed significant reductions in growth and development.A study by Li et al (2015) showed that B. amyloliquefaciens, deterred feeding by FAW larvae and caused a significant decrease in the weight of larvae.In other investigations, sublethal dosages of Bt induced reductions in consumption and delays in development in S. frugiperda (Lambert et al. 1996), S. littoralis (Regev et al. 1996), andS. exigua (Lopes Lastra et al. 1995).However, these effects were temporary, and their intensity decreased with the growth of the larvae.This makes it evident that, in addition to having a fatal impact on hosts, entomopathogens may change an insect's physiology, which prevents them from consuming food and reproducing (Polanczyk and Alves 2005).

Conclusions
The majority of chemical insecticides now in use were unable to control FAW and they also had some negative environmental effects.Because of its effectiveness and lack of negative effects on natural enemies, the use of Bt against FAW is gaining interest.The biocidal property of Bt is caused by the spore-crystal complex.Besides Bt, other Bacillus spp.exhibit entomocidal activity against FAW, which needs further exploration.The combination of Bt strains increased the efficacy on S. frugiperda in terms of mortality and growth inhibition in neonates, third and last larval instars.The combination of Bt strains led to an enhancement of pathogenicity toward FAW larvae at the initial stage of development, and in later stages, it affects their growth and development.Therefore, biological control by entomopathogenic bacteria (Bt) and its combination is the unavoidable choice to manage the FAW in an ecofriendly way.

Fig. 1
Fig.1Corrected percent mortality of combinatorial bioassay in spore crystal and cell suspension form of Bacillus thuringiensis strains against neonates of Spodoptera frugiperda.*Corrected percent mortality bar's followed by different small alphabets and capital alphabets are significantly different at 5% level in 1 × 10 8 cells/g of diet and 1000 µg/g of diet, respectively

Fig. 4
Fig. 4 Growth and development of larvae which fed with native Bacillus thuringiensis strains incorporated diet in combinatorial bioassay A Differences in larval size; B Differences in pupal size; C Malformation in adults

Fig. 5
Fig. 5 Isolation of Bacillus thuringiensis from dead larva and visualization of spore crystal by phase contrast microscope A, B Control larvae not showing any gut discoloration; C, D Gut discoloration in fall armyworm larva due to infection of various B. thuringiensis strains; E Plates of colonies reisolated from dead larvae; F Spore and crystal of re-isolated B. thuringiensis cells from gut of dead larvae under phase contrast microscope

Table 1
Efficacy of Bacillus isolates from diverse sources against neonates of Spodoptera frugiperda in terms of percent mortality B. cereus (S4C1) and B. thuringiensis (S17C3, S31C5,

Table 1
(continued)Means followed by same letter within column alphabets are not significantly different at 5% level.*-Sequence of these strains yet to submit in NCBI

Table 2
Median lethal concentration (LC 50 ) of potential Bacillus isolates against neonates of Spodoptera frugiperda on 7th day after treatment

Table 3
Combinatorial effect of native Bacillus thuringiensis isolates against neonates and third instar larvae of Spodoptera frugiperda