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Molecular Characterisation and Toxicity Analysis of Indigenous Bacillus thuringiensis Berliner Isolates against Cucurbit Fruit Fly Maggots, Zeugodacus cucurbitae (Coquilett) (Diptera: Tephritidae)

T. Sharmitha1, V. Balasubramani2, T. Elaiyabharathi3, M. Raveendran4, L. Pugalendhi5, E. Kokiladevi6
1Department of Agricultural Entomology, Tamil Nadu Agricultural University, Coimbatore-641 003, Tamil Nadu, India.
2Controller of Examinations, Tamil Nadu Agricultural University, Coimbatore-641 003, Tamil Nadu, India.
3Department of Medicinal and Aromatic Crops, Tamil Nadu Agricultural University, Coimbatore-641 003, Tamil Nadu, India.
4Director of Research, Tamil Nadu Agricultural University, Coimbatore-641 003, Tamil Nadu, India.
5Department of Vegetable Science, Tamil Nadu Agricultural University, Coimbatore-641 003, Tamil Nadu, India.
6Department of Plant Biotechnology, Tamil Nadu Agricultural University, Coimbatore-641 003, Tamil Nadu, India.

Background: Indiscriminate use of insecticides against fruit flies (Tephritidae: Diptera) has led to the development of residue and resistance and there is a need to try alternate eco-friendly management practices. Bacillus thuringiensis (Bt) is the most widely used bacterium forming proteins active against insects of different orders. Thus, the present study was framed to characterize and evaluate 50 indigenous Bt isolates against cucurbit fruit fly maggots, Zeugodacus cucurbitae

Methods: Colony morphology was observed visually, while crystal morphology was observed through a microscope. The cry gene content of the Bt isolates was screened by Polymerase Chain Reaction and the proteins harbored were assessed by SDS- PAGE analysis. Full diet contamination method was used to evaluate the toxicity of Bt isolates against cucurbit fruit fly maggots. 

Result: Uniform full white, off-white and creamy white colony colours were found except one isolate each, exhibiting full white at centre surrounded by off white and off white at centre surrounded by full white. Spherical shape crystal (45.10%) was predominant followed by cuboidal (29.41%), bipyramidal (17.65%), rectangular (3.92%) and minute crystal attached to spore (3.92%). Insecticidal proteins varied from ~15 to >200kDa in size with one to four and more distinct bands per isolate. PCR screening revealed cry4Aa in 2 isolates, cry4Ba and cry11Aa in one isolate each, cyt1 in 1 isolate. The standard strain Bti 4Q2 (100% mortality of maggots) and the indigenous Bt isolates, T166 (92.59%), T184 (91.11%) and T60 (90.48%) were found to be significantly toxic.

Cucurbits occupy 66 percent of the vegetable land in India as commercial and kitchen garden crops, producing only 11 per cent of the total vegetable production (Nasiruddin et al., 2004). The genus Bactrocera with more than 40 species is the major constraint, among the 200 species of Tephritidae listed in India (Kapoor, 2002). Among which, the cucurbit fruit fly/melon fruit fly, Zeugodacus (Bactrocera) cucurbitae (Coquillett) causes 30 to 100 per cent loss (Dhillon et al., 2005).
With the increasing awareness among farmers for organic cucurbit production, cucurbit fruit flies have been managed with several eco-friendly practices (Ansari et al., 2012; Nelson, 2019), but they are uneconomical with 25 per cent of the production cost spent solely to manage cucurbit fruit fly (Nasiruddin et al., 2004). Bacillus thuringiensis Berliner (Bt) of the family Bacillaceae forms resistant spores of proteins against insects of different orders (Schnepf et al., 1998). This remains a possibility and also imposes the need for the search of native Bt isolates with activity against the cucurbit fruit fly, Z. cucurbitae.
Laboratory maintenance of insect culture
Laboratory culture of Z. cucurbitae was established initially from TNAU orchard (11°0'31"N latitude: 76°55'39"E longitude) from the fruit fly infested cucurbits showing symptoms such as distorted shape and oozing of resinous fluid (Lal et al., 2014). The freshly emerged adult flies from the infested cucurbits were identified as the cucurbit fruit fly, Z. cucurbitae based on its taxonomic characters (Prabhakar et al., 2012), sexed (Mir et al., 2014) and released into cages (42 cm length × 42 cm width × 42 cm height). Cucumber slices (ca. 1mm thickness) were cut and placed in piles of 3-4 slices in petri dishes and kept inside cages with adults for oviposition. The cucumber slices in piles were replaced every 24 hours for fresh oviposition (Liu et al., 2020). Adults were fed with 10 percent sugar solution suspended with vitamin E through a piece of soaked clean cotton wick placed inside a glass vial (2.3 cm diameter × 5.3 cm height). The cucumber slices in petri dishes with eggs were placed in plastic boxes for the development of maggots. Cucumber slices were replaced or added when necessary and the maggots were fed ad-lib. throughout the study. As the maggots reached the third instar and were about to pupate, the plastic boxes were transformed in to trays containing sand of 2 cm height to facilitate pupation. Pupae were sieved from the sand after 2 days and placed inside cages in petri dishes for adult emergence. The insect culture was maintained under controlled conditions of temperature, humidity and photoperiod (25±2°C, 70±10% RH and 12 L: 12D) at the insect bioassay laboratory (Department of Plant Biotechnology, TNAU, Coimbatore, India) and was reared for ten successive generations before initiating bioassay experiments.
Bt isolates and growth conditions
Indigenous Bt isolates (n=50) and negative Bt check (4Q7) were obtained from the Bt laboratory, Department of Plant Biotechnology, Centre for Plant Molecular Biology and Biotechnology, Tamil Nadu Agricultural University, Coimbatore, India. The standard reference strain of Bacillus thuringiensis subsp. israelensis used in the study as positive check was obtained from Bacillus Genetic Stock Centre (BGSC, Columbus, Ohio) (Supplementary Table 1). The bacterial cultures were revived, sub-cultured and stored for further use (Ramalakshmi and Udayasuriyan, 2010).

Supplementary Table 1: Details of indigenous Bt isolates and reference strains used in the study.

Characterisation of bacterial colony and crystal morphology
Colony shape, surface, colour, margin and elevation of individual Bt isolates were examined. Crystals were documented for their morphology using Leica (DM 1000LED, DFC295, Germany) microscopy (Sharif and Alaeddinoglu, 1988; Ramalakshmi and Udayasuriyan, 2010).
Spore crystal mixture isolation from Bt isolates
Spore crystal mixture was isolated based on the method described by Ramalakshmi and Udayasuriyan (2010).
Characterization of protein
The SC mixtures were analysed for the presence of d-endotoxins by SDS-PAGE (Sodium dodecyl sulphate poly acrylamide gel electrophoresis) (Laemmli, 1970). Pre stained three colour protein marker (Puregene, Genetix Biotech Asia Pvt Ltd.,) which spans wide range of molecular weights from 10 to 315 kDa was used to document the molecular weight of the proteins.
Insecticidal gene profiling of Bt isolates
In Bt cultures, genomic DNA was extracted using the technique outlined by Kalman et al., (1993). Polymerase chain reaction (PCR) was performed (20 µl) with gene-specific primers for the genes cry4Aa, cry4Ba, cry10Aa, cry11Aa and cyt1. The amplification conditions were programmed according to the specifications mentioned in Supplementary Table 3. The amplified products were observed for the expected size of amplicon of each gene (Supplementary Table 2) (Ben-Dov et al., 1997).

Supplementary Table 2: Details of primers used in the study for screening indigenous Bt isolates.


Supplementary Table 3: PCR conditions for screening Bt isolates for specific genes.

Toxicity of Bt isolates against cucurbit fruit fly maggots
ELISA reader (Biotek-Powerwave XS) was used to estimate the concentration of protein in SC mixtures by Broadford’s reagent method (He, 2011). Artificial diet was used to evaluate the toxicity of SC mixtures against cucurbit fruit fly, Z. cucurbitae maggots. The proportion of various ingredients used in preparation of artificial diet (AD) is 2.52g pumpkin, 8.84 g common bean powder, 2.27g yeast extract powder, 3.53 g sugar (sucrose), 0.63 g agar, 0.21 g vitamin E capsule, 200 µl Zincovit and 100 ml of sterile distilled water (composition for 100ml of AD). Agar and other homogenized ingredients (pumpkin, common bean powder, yeast extract and sucrose) were boiled in separate containers, mixed together, allowed to cool, then vitamins were added, blended again and distributed into containers.
Bioassay was conducted using different concentrations of protein (0.03 to 2.3 µg of protein/μl of SC mixture isolated) with the standard reference strain Bti 4Q2. Based on the result, the estimated protein concentration of indigenous Bt isolates was equalised to a concentration of 0.7 µg/μl and the toxicity was evaluated by whole diet contamination (Aboussaid et al., 2010) by mixing 150 μl of quantified SC mixture to 500 μl of AD solidified (the maggots were fed ad lib. throughout the experiment) in plastic cups (2.3 cm height × 3 cm diameter) with a glass rod under sterile conditions.
To perform in vitro bioassay, the Z. cucurbitae eggs (less than 24 h old) of the same cohort were collected from the piles of cucumber slices removed from the cages after 12 hours of exposure to oviposition. Eggs were removed using fine brush and placed over fresh cucumber slices. Maggots newly emerged from the eggs were harvested by placing the cucumber slices in sterile distilled water (Kaur et al., 2010). The maggots were released into plastic cups with AD contaminated with SC mixture using a fine camel hair brush without any physical damage. Three replications were maintained (10 maggots/replication). Observation was made for 7 days. Standard strain, Bti 4Q2 was the positive check, acrystalliferous strain 4Q7 was the negative check and water was used as control (Ilias et al., 2013).
Statistical analysis
The experimental design was completely randomized (CRD). The cumulative mortality was expressed in percentage, computed using formula (Abbott, 1925) when the mortality in control exceeded 5 percent but was less than 20 per cent. The data was analysed by one way ANOVA using AGRES (version 7.01). Least significant difference (LSD) was used to determine the statistical significance. The research was carried out from 2020 to 2022.
Colony and crystal morphology of Bt isolates
Colony colour was observed as full white (15.38%), creamy white (11.54%) and off white (69.23%). One isolate each was found to express full white colour at the centre encircled by off white and creamy white colour at the centre encircled by off white (Table 1). Navya et al., (2021) have reported the predominant occurrence of off-white colonies (53 isolates) followed by full white (7 isolates) favoring the present findings. Occurrence of creamy white colonies is accorded by Gothandaraman et al., (2022). The colony surface was observed to be glossy (9.62%), fried egg (40.38%) and smooth (50%). Circular (37 isolates) and irregular shaped (15) colonies were observed. Elevation was flat (55.77 %) and raised (44.23%). Colony margins appeared entire (22 isolates) and undulate (30) (Table 1). Colony type was observed as fried egg/mucoid/smooth with raised and flat elevation with entire/undulate margins and appeared in circular/irregular shapes (Navya et al., 2021; Gothandaraman et al., 2022) in line with the present findings. Crystal morphology revealed the dominant occurrence of spherical crystals (45.10%) followed by cuboidal (29.41%) and bipyramidal (17.65%) (Table 2). This is supported by the occurrence of spherical crystals in 99 per cent of the isolates against mosquitoes (Mahalakshmi et al., 2012).

Table 1: Colony morphology of Indigenous Bt isolates.


Table 2: Crystal morphology of Indigenous Bt isolates.

Protein profiles of Bt isolates
Insecticidal proteins with varying molecular weights ranging from ~15 to >200 kDa were found. The banding pattern was observed to have one band (11.76%), two to three (17.65%) and four or more bands (52.94%) (Table 3). Diversified protein patterns having molecular weights between ~20 and more than 200 kDa and variable number of protein bands above one have been reported earlier (Navya et al., 2021; Gothandaraman et al., 2022) in line with the current study.

Table 3: Insecticidal protein distribution in Indigenous Bt isolates from SDS-PAGE analysis.

Distribution of dipteran-specific insecticidal genes in Bt isolates
PCR screening was done for five dipteran-specific genes (cry4Aa, cry4Ba, cry10Aa, cry11Aa and cyt1). Two isolates viz., T166 and T184 were positive for cry4Aa. The isolate T60 was positive for cry4Ba and the isolate T166 was positive for cry11Aa gene. Among the 50 isolates screened, the gene cyt1 was positive in 1 isolate (T166). The remaining 47 number of indigenous Bt isolates did not show the presence of these five genes screened. No isolate showed PCR positive result for the gene cry10Aa. One isolate, T166 was positive for three genes viz., cry4Aa, cry11Aa and cyt1 in combination (Table 4). Jain et al., (2017) reported diverse cry gene profiles with the occurrence of cry4 (84.14%) and cry11 (39.28%) genes. Genes viz., cry 4A, cyt 1, cry 10, cry 11 and cry 4B were found in B. thuringiensis strain B474 (Geetha et al., 2007).

Table 4: Insecticidal gene distribution in indigenous Bt isolates from PCR screening.

Toxicity of Bt isolates against Z. cucurbitae maggots
Observations on insecticidal activity of Bti 4Q2 at different concentrations revealed, 100 percent mortality of maggots at an initial concentration of 0.7µg/µl at seventh day after treatment (DAT) (Supplementary Fig 1). Three indigenous Bt isolates viz., T60, T166 and T184 produced 90.48, 92.59 and 91.11 percent mortality respectively (Table 5) (Supplementary Fig 2). Bt strain from biotype kurstaki (96%) and the indigenous  Bt JSc 1 (93%) harbouring cry1A type genes exhibited higher toxicity on 3rd instar Z. cucurbitae maggots (Shishir et al., 2015). Bt 13.4 toxin exhibited mortality (100 %) against Mediterranean fruit fly, Ceratitis capitata (Wiedemann) neonate maggots 7 days after exposure, in accordance with the present result (Aboussaid et al., 2010).

Supplementary Fig 1: Toxicity of Bacillus thuringiensis subsp. israelensis (HD 500) at different concentrations of insecticidal protein(s) against cucurbit fruit fly maggots.


Table 5: Toxicity of indigenous Bt isolates against maggots of cucurbit fruit fly, Z. cucurbitae.


Supplementary Fig 2: In vitro insect bioassay with spore-crystal mixtures of Bt isolates against neonate cucurbit fruit fly, Zeugodacus cucurbitae maggots showing difference in mortality and feeding of artificial diet on 7 days after treatment.

Among the 50 indigenous Bt isolates evaluated, three were highly toxic with more than 90 per cent mortality of cucurbit fruit fly maggots. Insecticidal activity was found to persist in few isolates despite the absence of five genes screened in the present study, but with the expression of distinct protein bands coded by other genes.
The authors declare that they have no conflict of interest.

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