Diversity of indigenous Bacillus thuringiensis isolates toxic to the diamondback moth, Plutella xylostella (L.) (Plutellidae: Lepidoptera)

Abstract

Background

Toxins from the Bacillus thuringiensis (Bt) bacterium are employed as an alternative to synthetic pesticides in pest management. The greatest threat to the long-term viability of Bt toxins is resistance evolution in the target pests. Genetic diversity and toxicity of Bt isolates were studied in this work in order to find Bt isolates with novel cry genes.

Results

In terms of colony morphology, among a total of 60 isolates, 51 isolates had off-white colour colonies with typical fried egg appearance, irregular shape, flat and undulate margin. Different crystal shapes, viz. spherical (88.13%), bipyramidal (49.15%), cuboidal (42.37%), rectangular, and crystals attached to spores (3.38%) were observed among Bt isolates. SDS-PAGE analysis of spore crystal mixture showed the presence of proteins with various molecular weights ranging from 124 to 26 kDa. PCR screening with cry1, cry2, cry9 and vip3A1 primers showed isolates with varied insecticidal gene combinations. Bt isolates containing cry1 genes were found to be abundant (30), followed by cry2 (9) and vip3A1 (9). Cry9 was absent in all the 60 isolates tested. Insecticidal activity of spore crystal mixtures ranged from 0 to 100% mortality. Furthermore, 12 isolates were found to be highly toxic against the larvae of diamondback moth, Plutella xylostella (L.) (Plutellidae: Lepidoptera) with 100% mortality, at 25 µg/ml in leaf disc bioassay.

Conclusions

The present work established the diversity of Bt isolates and confirmed the importance of continuous exploration of new Bt isolates for novel genes. Further, research needs to be carried out to unveil the hidden potential of these toxic isolates.

Background

The most widely used biopesticide, Bacillus thuringiensis (Berliner) (Bacillaceae: Bacillales) (Bt), is a gram-positive, spore-forming entomopathogenic bacterium usually found in soil, leaf surfaces, grain dusts, dead insects and aquatic environments (Gupta et al. 2021). Bt can be distinguished from other Bacillus spp. due to its capacity to produce insecticidal proteins during sporulation phase as parasporal inclusions, which predominantly comprises of Cry and Cyt toxins (Schnepf et al. 1998). Numerous Bt strains that showed toxic activity towards wide range of insects including Lepidoptera, Coleoptera, Diptera, Homoptera, Mallophaga, Hymenoptera and Nematodes (Sandeep et al. 2020) have been isolated and eventually used to develop biopesticides. At present, 400 Bt-based formulations made up of insecticidal proteins and spores have been registered in the market. These toxins are employed in pest management in the form of foliar sprays or by delivering them to the target insect through the expression of toxin encoding genes in the transgenic plants (George and Crickmore 2012). Biopesticides are applied at 10–50 g/acre or 10²⁰ molecules/acre, while chemical pesticides such as organophosphates and pyrethroids are applied at 8 × 10²⁴ and 3 × 10²² molecules/acre, respectively. By this, the molecular potency of Bt toxins is 80,000 and 300 times higher than organophosphates and pyrethroids (Singh et al. 2021). Based on the amino acid sequence homology, 80 holotypes of crystal proteins (Cry1–Cry80), 4 holotypes of vegetative insecticidal proteins (Vip1–Vip4) and 7 holotypes of cytolytic proteins (Cyt1–Cyt7) of Bt have been identified (Crickmore et al. 2018). Cry proteins are highly selective to their target insect, innocuous to humans, non-target pests, vertebrates and rapidly degrade in the environment. Therefore, Bt is an effective alternative for the control of insect pests in agriculture. However, the primary disadvantage in the long-term use of Bt toxins contribute in decreasing susceptibility of pest population. It has been shown that insects developed resistance to Bt toxins in the laboratory and in field to Bt sprays (Jurat-Fuentes et al. 2021). As a result, isolation of Bt strains with novel toxins has gained significant importance for providing alternatives to these problems. With this background, the present study was carried out to screen novel Bt isolates that could be toxic to the diamondback moth, P. xylostella larvae under controlled conditions.

Methods

Insect culture

A laboratory culture of P. xylostella was established initially from pupae obtained from National Bureau of Agricultural Insect Resources, Bangalore, India. The pupae were transferred to the rearing cages (45 × 45 × 45 cm) for the emergence of adults. The newly emerged adults of P. xylostella were fed on 10% sugar solution, and mustard seedlings were provided as oviposition substrate and the hatched larvae were fed by fresh cauliflower leaves (CFL-1522, Syngenta) maintained under greenhouse conditions without any insecticide application. The larvae were placed in a (30 × 30 × 30 cm) larval rearing cage with a glass top, wooden bottom and mesh on all 4 sides. The insect culture was maintained at the Insect Bioassay laboratory (Department of Plant Biotechnology, TNAU, Coimbatore, India) under controlled conditions of temperature, humidity and photoperiod (25 ± 1 °C, 75 ± 5% RH and 16: 8 h (L: D).

Bt isolates and growth conditions

Sixty native Bt isolates were obtained from Department of Plant Biotechnology, Centre for Plant Molecular Biology and Biotechnology, TNAU, Coimbatore, India. The reference strain Bacillus thuringiensis subsp. Kurstaki HD1 originally obtained from Bacillus Genetic Stock Center (Columbus, Ohio) was used. In order to obtain single colonies, all the isolates were subcultured on T3 agar media plates using the quadrant streak method and incubated at 30 °C for 12–14 h. Single colony was picked using sterile loop and transferred to the test tube containing 5 ml of T3 broth, incubated at 30 °C for 24 h at 200 rpm and stored as sterile 50% glycerol stocks at − 20 °C for further use.

Isolation of spore–crystal mixture for toxicity analysis

The spore crystal mixture was isolated from 58 Bt isolates and a reference strain, HD1 as a positive control and an acrystalliferous strain 78/11 as a negative control. To obtain the spore crystal mixture from each isolate, a single colony of Bt culture from T3 agar plates was inoculated into culture tubes containing 5 ml of T3 broth and kept for overnight incubation in a shaking incubator (Orbitek, Scigenics Biotech Pvt. Ltd, Chennai, India) maintained at 30 °C and 200 rpm. From the overnight grown cultures, 1% inoculum (250 µl) was added to the 250-ml conical flask containing 25 ml of T3 broth in a shaking incubator maintained at 30 °C, 200 rpm for 48–60 h. The growth and lysis of the bacterial cells were checked using the phase contrast microscope after 48 h. When more than 90% cells have lysed, culture was centrifuged at 4 °C for 10 min at 10,000 rpm (Centrifuge 5810R, Eppendorf, Germany) and resulting pellet was suspended in 25 ml of ice-cold Tris–EDTA buffer [Tris 10 mM, EDTA 1 mM, pH 8.0 with 1 mM phenyl methyl sulphonyl fluoride (PMSF)] and washed once with 25 ml of ice-cold 0.5 M NaCl and centrifuged for 10 min, followed by 2 washes with 25 ml Tris–EDTA buffer with 0.5 mM PMSF at the same speed and time (Ramalakshmi and Udayasuriyan 2010). Finally, the pellet was suspended in 500 µl of sterile distilled water containing 10 µl of 1 mM PMSF and stored at − 20 °C as aliquots of 50 µl for later use.

Colony and crystal morphology

Colony morphology pertaining to colour, surface and margin of each single bacterial colony was observed from the culture plate maintained at 30 °C for 24 h. To examine the shape of the parasporal crystalline inclusions using glass slides, culture smears were prepared on a glass slide, heat fixed and stained with Coomassie brilliant blue stain (0.133% Coomassie Brilliant Blue G250 in 50% acetic acid) for about one min. Smears were washed gently in running water, blot-dried and observed through the phase contrast microscope.

Protein analysis

Protein analysis using sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) was done for spore crystal mixture from all the Bt samples. SDS-PAGE was performed by following the standard protocol (Laemmli 1970), using 10% separating gel and 4% stacking gel. The spore crystal mixtures were mixed by loading buffer (4x) containing 0.25 M Tris HCl pH 6.8, 8% SDS, 40% glycerol, 0.5% bromophenol blue in the ratio 4:1, respectively. Then, samples were boiled for 2 min before loading into wells. The molecular mass of the proteins was estimated using pre-stained three-colour protein ladder (Puregene, Genetix Biotech Asia Pvt. Ltd.) covering a wide range molecular weight from 10 to 315 kDa.

Detection of cry genes in Bt isolates

Total genomic DNA was isolated from a single bacterial colony of all isolates according to method described by Sambrook and Russell (2001) and used as a template for the amplification of cry1, cry2, cry9 and vip3A1 genes. The isolated DNA was quantified through NanoDrop (Genova Nano, Jenway), and the integrity was evaluated by agarose gel electrophoresis (1%). Each polymerase chain reaction (PCR) mixture contained 20–50 ng template DNA, 1 µM of each primer (Ben-Dov et al. 1997) and 10 µl of 2 × PCR Master Mix (Smart prime) consisting of dNTPs, Taq polymerase and PCR buffer, and the final volume was made up to 20 µl with sterile distilled water. PCR amplification was performed in a thermal cycler (ProFlex PCR system, Applied Biosystems). The PCR amplicons were separated in 1% agarose gel using 1 kb DNA ladder. The amplified products were visualized under UV transilluminator (Bio-Rad).

Insect toxicity assays with Bt isolates

Insecticidal activity of the spore crystal mixtures of all the Bt isolates was tested against 3-day-old P. xylostella larvae. In order to conduct the bioassay, the protein concentration was estimated using Bradford method (Bradford 1976) and the suspension was diluted to 25 µg/ml of concentration in all the isolates before conducting toxicity tests. In all the cases, spore crystal mixture was smeared on small discs (25 mm diameter) of cauliflower leaves and allowed to dry. To prevent desiccation, all the leaf discs were placed on the wet surface of Whatman filter paper discs in plastic cups (30 mm diameter). For each isolate, 3 replications with 10 larvae per replicate were used. The standard strain, Bt subsp. kurstaki (Btk) HD1 and acrystalliferous strain 78/11 were used as positive and negative controls, respectively. Insect mortality was recorded at 24-h interval for 3 days, and cumulative mortality at 72 h was taken as mortality for comparing isolates.

Statistical analysis

Experiments were carried out in a completely randomized design (CRD). Single-dose bioassays were statistically analysed by one-way analysis of variance (ANOVA) using AGRES statistical software version 7.01, and significant differences between means were determined by Duncan’s multiple range test (p = 0.05).

Results

Characterization based on colony and crystal morphology

Bt isolates were characterized based on colony colour, surface, shape, elevation, margin, cry gene content and protein profile. Among the 60 isolates, 53 isolates were off-white in colour, whereas 7 were full white. Colony surface was found to be fried egg type with flat elevation and undulate margin in 51 isolates, while smooth, raised and entire in 9 isolates. The colony shape of 53 isolates appeared as irregular whereas circular in 7 isolates (Table 1). Phase contrast microscopic (100x) observations (Iscope, Euromex) showed the presence of spores and crystal inclusions. Out of 51 isolates, crystal shape appearance was varied from spherical (88.13%), bipyramidal (49.15%), cuboidal (42.37%), rectangular (3.38%) and minute crystals attached to spores (3.38%) (Table 2).

Table 1 Colony morphology of indigenous Bacillus thuringiensis isolates

Table 2 Crystal morphology of Bacillus thuringiensis isolates

Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE)

SDS-PAGE analysis of parasporal crystal proteins revealed various banding patterns among the Bt isolates. Bt isolates carried proteins of different molecular weights, viz. ⁓ 124 kDa (20 isolates), ⁓ 90 kDa (8 isolates), ⁓ 70 kDa (50 isolates), ⁓ 60 kDa (2 isolates), ⁓ 55 kDa (2 isolates), ⁓ 45 kDa (11 isolates) and ⁓ 26 kDa (15 isolates) (Table 3).

Table 3 Crystal protein profile and cry genes distribution in native Bt isolates

Distribution of lepidopteran specific cry and vip genes in Bt isolates

PCR analysis for four lepidopteran specific genes (cry1, cry2, cry9 and vip3A1) showed the presence of only cry1, cry2 and vip3A1 genes but not cry9 gene. Out of 51 isolates, cry1, cry2 and vip3A1 gene were found to be present in 30, 9 and 9 isolates, respectively. Fourteen isolates showed the presence of more than one gene, in different combinations (Table 3).

Insecticidal activity against P. xylostella larvae

From the toxicity results, it was evident that 12 Bt isolates, viz. RM12, RM17, RM22, RM29, T15, T20, T29, T32, T152, T191, T380 and T405, showed 100% mortality on par with the positive standard check, HD1 (Tables 4 & 5) after 72 h, against 3-day-old larvae of P. xylostella. Two isolates; RM14, T354 exhibited 90 and 93.33% mortality, respectively. Bt isolates RM3, RM27, RM28, VB7 and VB16 showed a very least toxicity (< 10%) against P. xylostella larvae.

Table 4 Mortality of diamondback moth, Plutella xylostella caused by indigenous Bt isolates

Table 5 Grouping of Bt isolates based on mortality of Plutella xylostella larvae at 25 µg/ml

Discussion

Bt collections exhibiting distinct level of toxicity to different insect pests are a good source of novel genes encoding for insecticidal proteins which could be employed in pest management. Bt isolates featured matt to granular surface textures with circular to irregular colonies and entire or undulate edges (Logan and De Vos 2011). Bt formed colonies on agar plates giving fried egg appearance as reported earlier (Zothansanga et al. 2016). The presence of parasporal crystalline inclusions is the common way to identify Bt (Rampersad and Ammons 2005). In the present study, spherical crystals were found in more frequency (88.13%) than bipyramidal crystals (49.15%). These findings are in accordance with reports by Reyaz et al. (2017) from India and Nair et al. (2018) from Qatar. Apart from these shapes, in the present study cuboidal crystals and crystals attached to spore were found in 42.37 and 3.38% isolates, respectively, as reported earlier by Rampersad and Ammons (2005). On the other hand, Ramalakshmi and Udayasuriyan (2010) observed more frequent occurrence of cuboidal (27%) and bipyramidal crystals (21%) among the 316 Bt isolates tested. In terms of crystal morphology, isolates that are considered to be highly toxic produced 2 types of crystals, viz. bipyramidal and cuboidal, which is consistent with previous reports on crystal shape (Boonmee et al. 2019). All these variations in the morphology of crystalline inclusions suggested the presence of diversity in the studied isolates.

Carozzi et al. (1991) introduced PCR-based identification of cry genes to predict the insecticidal activity of Bt isolates, and it has since become a common tool. In the present study, cry1 was more prevalent than cry2 and vip3A1 genes, in accordance with earlier reports (Lone et al. 2017). Among all the Bt isolates, only 16 showed the presence of single gene, while other positive isolates confirmed the presence of both cry and vip genes in combinations. Earlier, Ben-Dov et al. (1997) reported the tendency of occurrence of cry1 and cry2 genes together.

Bt isolates containing inter group crystalline protein encoding genes are ideal candidates for the development of broad-spectrum biopesticides. The findings presented here are similar to the previous reports (Sahin et al. 2018). All the 12 isolates that showed 100% mortality including positive control (HD1) were PCR-positive for cry1. These findings are comparable to those of Salama et al. (2015) who confirmed that, most isolates were PCR-positive for cry1 gene. In the present study, vip3A1-positive isolates have been identified to contain cry1 and cry2 genes also, in agreement with previous studies (Sahin et al. 2018).

SDS-PAGE analysis of all the 59 Bt isolates revealed the existence of diversity in our collection. Among these, 22 isolates produced one protein band only while 25 isolates produced 2 separate protein bands with varying molecular weights. Out of 59 Bt isolates, 50 produced proteins of ⁓ 70 kDa, as reported earlier by Reyaz et al. (2017). The bands observed at 124 kDa were next major proteins in the present study. Thirty Bt isolates produced protein bands in the range of 26 to 60 kDa. This wide range of protein banding pattern in these isolates suggested the variable biological activity and specificity for different insect pests. Ramalakshmi and Udayasuriyan (2010) found 2 protein bands of ~ 135 and ~ 65 kDa along with proteins of 95, 43 and 30 kDa in their Bt isolates. Diverse electrophoretic patterns in Cry proteins with molecular weights ranging from 20 to 160 kDa were reported earlier by Swamy et al. (2013).

Analysis of insecticidal activity of spore/crystal mixtures is an effective way to identify native Bt isolates with entomocidal properties (Sahin et al. 2018). In this study, 12 isolates were found to be highly toxic causing 100% mortality to P. xylostella larvae after 3 days. Kahrizeh et al. (2017) reported that 9 out of 148 isolates were highly toxic to P. xylostella larvae exhibiting 78.3 to 100% mortality. Monnerat et al. (2007) reported that 20 out of 1400 Bt isolates were found to be 100% effective to 1-day-old 2^nd instar P. xylostella larvae after 5 days in selective bioassays. Among a total of 104 Bt isolates, 13isolates caused mortality that ranged between 30 and 100% against P. xylostella neonates (Silva et al. 2012).

Conclusions

The present work showed the diversity among the Bt isolates, in terms of protein content, colony and crystal morphology, cry gene content and toxicity to P. xylostella. The presence of more than one cry or vip gene in the isolates suggested that they had a high frequency of genetic information exchange. The existence of crystals in isolates that were not PCR-positive with the primer tested suggests that these isolates include cry/cyt genes that code for insecticidal proteins unique to other target insects from other lepidopteran families. The potency of toxic isolates in the present study (RM12, RM17, RM22, RM29, T15, T20, T29, T32, T152, T191, T380 and T405) to P. xylostella may be due to variations in amino acid sequences of cry or vip proteins, accumulation of cry toxins or a combination of these factors. Detailed studies on toxicity, spectrum of activity and speed of kill will help to decide the need for whole genome sequencing of the particular Bt isolate, isolation and cloning of novel cry genes. Such novel Cry proteins could be developed in to a biopesticide formulation for direct use in IPM or in development of newer transgenic crops.