Potential of standard strains of Bacillus thuringiensis against the tomato pinworm, Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae)

The tomato pinworm Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) is one of the key pests of tomato worldwide, causing an estimated crop loss of 80 to 100%. This pest has developed resistance to several pesticides due to overuse, resulting in control failures in the field. The use of biological insecticides as Bacillus thuringiensis that expressed insecticidal proteins can be an alternative tool by insecticides to suppress the pest population. Laboratory study investigated the efficacy of standard Bacillus thuringiensis (Bt) strains (4D1, 4D4, 4G1, 4K5 and 4XX4) against T. absoluta. Bioassay was conducted using tomato leaf discs treated with spore crystal lysates prepared from the standard strains, and mortality data was subjected to concentration-mortality probit analysis. The LC50 values for Bt 4D1, Bt 4D4 and Bt 4G1 were 6.10, 6.62 and 8.18 μg/ml for the 2nd instar; 9.90, 10.20 and 11.12 μg/ml for the 3rd instar; and 19.82, 23.16 and 24.54 μg/ml for the 4th instar, respectively, while the Bt 4K5 and Bt 4XX4 were not toxic to T. absoluta. This study suggests that Bt strain 4D1 is effective against different larval instars of the pest and can be used in its management.


Background
Tomato pinworm Tuta absoluta (Meyrick, 1917) (Gelechiidae: Lepidoptera) is a tomato pest in South America and recently introduced to India (Shashank et al., 2015). This pest was first reported in 1914 in Peru, and now it is a common pest found in South America (Dilip and Srinivasan, 2019). Since 2006, T. absoluta had invaded Europe, Africa and Asian countries where it has caused significant economic losses of 80-100% both under greenhouse and field conditions (Urbaneja et al., 2013). T. absoluta is one of the most devastating tomato pests because it feeds on foliage, stems, fruits and flowers. Larvae infest all stages of plant growth causing wounds which facilitate the invasion of secondary pathogens (Hatice et al., 2017). The pest species has high reproductive potential with 12 generations in a year and female can lay up to 260 eggs (Ayalew, 2015).
During the last few decades, tomato productivity has been increased worldwide. Heavy reliance on chemical pesticides provide ephemeral benefits, often with adverse side effects and not viable (Hernandez et al., 2011) and, in some instances, actually worsen farmer's overall pest problems, and this pest became resistance to pesticides (Sandeep et al., 2020a). Thus, the major challenge is to increase and sustain crop productivity with less use of pesticides.
Variety of management tactics are used to reduce the pest infestations. The first option is to reduce the pest population through cultural practices, i.e. deep ploughing and trap crops, in order to safeguard the main crop. But chemical management is the most viable method for pest control. Farmers apply huge quantity of insecticides to manage insect pests; consequently, these insects have developed resistance to insecticides (Manivannan et al., 2019). The failure to control this pest may have a strong economic impact, and its recent history of introductions has increased the need for studies to develop strategies for its biological control, by the use of Bacillus thuringiensis (Bt) that express insecticidal proteins .
B. thuringiensis (Berliner), a species of gram-positive sporulating soil bacteria that forms insecticidal crystal (CRY) proteins during sporulation phase of its growth cycle, is the major source for the control of insect pest. The crystals contain one or more endotoxins known as cry proteins, which vary at different Bt strains. Cry and Cyt genes are named by cloning and sequencing from many cry proteins. Each of the Bt strains can carry one or more crystal toxic genes, and therefore, strains of the organism may synthesize one or more crystal proteins, and about 323 holotype crystal proteins are documented as toxic to insects of different orders viz. Lepidoptera, Coleoptera and Diptera (Crickmore, 2017). These crystal proteins are sequestered in bacteria as crystalline inclusions, mediates specific pathogenicity against insects (Schnepf et al., 1998).
B. thuringiensis strains are very effective against all larval stages of T. absoluta (Joel et al., 2011;Molla et al., 2011 andAzra et al., 2015). Cry proteins are highly specific and very effective against the tomato pinworm (Sandeep et al., 2020b and Dakshina and Gary, 2003) and narrow specific to lepidopterans (Hernandez et al., 2011 andMuhammad et al., 2019). Bt has been characterized as being highly specific against several insect orders including Lepidoptera, Diptera and Coleoptera (Xin Zhang et al., 2018). It has been found to be a very effective, environmentally safe insect-specific biopesticide (Palma et al., 2014). With this background, the present study was undertaken to evaluate the potential of 5 standard Bt strains (4D1, 4D4, 4G1, 4K5 and 4XX4) against T. absoluta under laboratory conditions.

Materials and methods
Five standard B. thuringiensis viz. 4D1 (BGSC HD1), 4D4 (BGSC HD73), 4G1 (BGSC HD8), 4K5 (BGSC LM79) and 4XX4 (BGSC YBT-1518) were obtained from Bt collection deposits at Department of Plant Biotechnology, Tamil Nadu Agricultural University, Coimbatore. These strains were originally obtained from Bacillus Genetic Stock Centre, Ohio University, and Columbus, Ohio, USA. All Bt strains were subcultured with four side streaking method on Luria Bertani Agar Media plates and incubated at 30°C for 24 h. Then a single colony was taken from each culture and inoculated in 15 ml test tube containing 5 ml LB broth individually. The test tubes were incubated at 30°C for 24 h with 200 rpm in a shaker. The cultures were stored in sterile 50% glycerol at − 20°C.

Isolation of spore crystal toxins and cry protein solubilization of Bt strains
The spore-crystal mixture of strains were prepared by acetone-lactose co-precipitation method as described by Dulmage et al. (1970). The resulting spore crystal powder was stored at 4°C for further use. Bt culture from glycerol stock was plated in LB agar and incubated for overnight at 30°C. From this culture, a loop was inoculated in to 1.5 ml Eppendorf tube containing sterile water (1 ml) and incubated at 70°C for 1 h to kill other bacteria present in the culture. After 1 h, sterile water with Bt was poured into a test tube containing 5 ml of Plain LB Broth and incubated for 12 h at 30°C. From this overnight culture, 1.25 ml was used for inoculating 125 ml LB Broth in a 250-ml conical flask and incubated at 30°C in an incubated shaker with 200 rpm for 72 h. After 72 h, 6 g of sodium chloride was added to each flask and incubated for 3 h at the same conditions to release the cell contents into the broth. The sporulated broth culture was transferred to refrigerated centrifuge at 4°C and spore crystal mixture was isolated.
The LB broth containing spore-crystal mixture was centrifuged at10,000 rpm for 10 min at 4°C. The pellet was washed once with 20 ml of ice-cold Tris-EDTA buffer [Tris 10 mM, EDTA 1 mM, pH 8.0 with 1 mM phenyl methyl sulphonyl fluoride (PMSF)], once with 20 ml of ice-cold 0.5 M NaCl, followed by 2 more washes with 20 ml of Tris-EDTA buffer with 0.5 mM PMSF by centrifuging at the same speed and time (Ramalakshmi and Udayasuriyan, 2010). The final pellet was solubilized in a solubilizing buffer [50 mM Na 2 CO 3 , pH 10.5 mM (DTT) dithiothreitol] at 30°C for 4 h by shaking and then centrifuged at 10,000 rpm, for 15 min at 4°C. The supernatant containing solubilized protoxin was removed and stored at − 20°C for further use.
This contains pure Cry proteins and their concentrations were estimated as described by (Lowry et al., 1951).
In vitro bio-assay of Bt strains against Tuta absoluta Laboratory experiments were conducted at Horticulture College & Research Institute, Periyakulam, Tamil Nadu Agricultural University. T. absoluta larvae collected, from leaves, stalks and fruits, were packed in plastic bags and brought to the laboratory. Larvae were immediately transferred into a larval rearing cage (45 × 45 × 45 cm) with mesh on all the 4 sides, glass top and wooden bottom. Adult cages (30 × 30 × 30 cm) were used for oviposition only, where leaves of tomato were provided daily as substrate. Adults of T. absoluta were fed by 10% sugar solution, while larvae were fed by tomato leaves, cultivated under greenhouse conditions without any insecticide application. The populations were reared in the laboratory at 25 ± 0.5°C, with a relative humidity of 75 ± 5% and a 12:12 L:D photoperiod.
Potential activity of standard Bt strains was tested on T. absoluta by leaf-dip bioassay method (Dakshina and Gary, 2003). Leaves from 2-month-old pot-cultured tomato plant grown in a greenhouse were used for assay. The healthy tomato (PKM 1) leaves (leaf discs of 1.5 cm diameter) were first washed by distilled water containing 0.02%. Triton X-100 thoroughly, air-dried and dipped in Bt toxin suspension of different strains, whose protein content was previously quantified by Lowry et al. (1951) method. Each leaf disc was dipped for 10 s, allowed to air-dry for a period of 1 h and transferred to clean Petri dishes (6 × 1.5 cm) over a moist filter paper to maintain turgidity of leaves. Single-dose 5-day bioassays with a concentration of 2.5 μg/ml were performed by 10 T. absoluta larvae (2nd, 3rd and 4th instars separately). Ten larvae were released per plate on the leaf discs overlaid on filter paper, using a fine camel hair brush. The concentrations of Bt strains were prepared separately for 4D1, 4D4 (2.5-15 μg/ml) and 4G1, 4K5 and 4XX4 (2.5-25 μg/ml). Forty larvae per treatment were used and each treatment which replicated with 4 subsets. A treatment without Bt protein (treated with 0.02% Tween 20) served as control.

Data analysis
Larval mortality was assessed on 3rd, 4th and 5th days of exposure. Larvae were withdrawn carefully from galleries of tomato leaves and disturbed with a fine camel hair brush; they were considered dead if unable to move the length of their body. Bioassay was conducted under completely randomized design in laboratory conditions. Corrected mortality percentages were worked out by using Abbott's formula (Abbott, 1925) and subjected to probit analysis (Finney, 1971) from EPA Probit Analysis Program (version 1.5).

Results and discussion
The results of probit regression analysis of concentration-response mortality data for the bioassays of Bt strains against T. absoluta were recorded. The slope values of different larval instars varied significantly, indicating variability in the susceptibility to Bt strains among the larval stages. T. absoluta showed variable responses to Bt strains as reflected in the LC 50 values for 2nd, 3rd and 4th larval instars. Bt strains showed toxicity to the 3 larval instars of pinworm. Based on the concentration mortality response to Bt strains (4D1, 4D4 and 4G1), LC 50 values were 6.10, 6.62 and 8.18 μg/ml for the 2nd instar (Table 1); 9.90, 10.20 and 11.12 μg/ml for the 3rd instar (Table 2); and 19.82, 23.16 and 24.54 μg/ml for the 4th instar (Table 3), respectively. The susceptibilities of different larval instars of tomato pinworm to Bt strains were presented in Figs. 1, 2 and 3. At LC 50 concentration, 50% mortality was observed on the 3rd day of treatment for 4D1 and 4D4 and 5th day for 4G1 in all the instars tested. Bioassay with 4K5 and 4XX4 strains of Bt did not show toxicity against the T. absoluta, as there was no difference between the treatment and control. In both control and treatments, larvae fed the same area of leaf tissue (mesophyll) over 5 days. Based on the present study, it is evident that all the 3 larval instars of the pest were susceptible to the Bt strains. The results indicated that susceptibility of larvae decreased with larval developmental stage. Variations in susceptibility of tomato pinworm depend on the age of the insect and susceptibility decreased with increase in the age of the insect. The use of Bt became a vital component in the integrated pest management (IPM), and it has been accepted throughout the world. Already, Bt proved to be the best alternative to the pesticides (Roh et al., 2007 andGonzalez et al., 2011). Different types of agricultural pests were subtle to Bt toxins, and they are essential to notice novel Bt strains to control T. absoluta. The results of the present study exposed a high mortality of the 3 larval instars of T. absoluta that were fed on Bt treated leaves, having value in developing IPM to control tomato pinworm.

Conclusion
The present study confirms that Bt proteins are host specific. Specificity makes Bt proteins safer to non-target organisms including predators and parasitoids, which provide pesticide-free tomato yield with fruit quality and safety.