The Potential Activities of Two Bacillus thuringiensis Strains Against the Neonate Larvae of Pectinophera gossypiella

The current study was conducted to investigate the biological activities of the two strains of Bacillus thuringiensis (Bacillus thuringiensis var. Kurstaki 1(Bt K1) and Bacillus thuringiensis var. Kurstaki 2 (Bt K2)) against the newly hatched (neonate) larvae of the pink bollworm, Pectinophera gossypiella. The two strains exhibited their toxicity against the treated larvae. Also, the lethal effect was extended in the resulted stages, pupae and adults. Based on LC50 for total mortality, Bt K1 was more potent than Bt K2 where LC50 was 2.21x1010 and 3.11x1010, respectively. However, the two strains were revealed a reduction of pupation and adult emergence %.  Irrespective of the strain, Bt significantly decreased larval duration and significantly increased pupal duration. No effect was recorded on morphogenesis. In the present study,it was broadly that Bacillus thuringiensis showed its ability in the control of Pectinophora gossypiella.


INTRODUCTION
The pink bollworm, Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae) is considered the main insect pests infested the cotton plants causing decreasing qualities and quantities of the cotton yield (Jaleel et al., 2014;Parmar and Patel 2016;Moustafa et al., 2019). This pest is difficult to control with insecticides (Lykouressis et al., 2005).
Bacillus thuringiensis (Bt) is the most commonly used biopesticide worldwide (Osman et al., 2015). Bt can induce mortality, effects on growth and reproduction (Barker 1998;Erb et al., 2001;Huang et al., 2018). Although many bacteria cause diseases to insects (Contwell 1974), only a few are used commercially as control agents. Some bacteria have been isolated from soil, insect habitats (Ohba et al., 1979;McSpadden Gardner 2004), insect larvae (Abou El-Ela 1996), or stored products (Kares 1991). Despite most species of Bacillus are harmless saprophytes, two species viz., B. thuringiensis and B. cereus are considered important in the field of controlling some plant insects (Gray et al., 2006).
The use of entomopathogenic Bt as an insect biological control agent has received worldwide attention (Legwaila et al., 2015;Opisa et al. ,2018). The aim of the present study is to evaluate the susceptibility of P. gossypiella to entomopathogenic bacteria.
The current study was conducted to investigate the biological activities of the two strains of Bacillus thuringiensis (Bacillus thuringiensis var. Kurstaki 1(Bt K1) and Bacillus thuringiensis var. Kurstaki 2 (Bt K2)) against the newly hatched (neonate) larvae of the pink bollworm, Pectinophera gossypiella.
The two strains exhibited their toxicity against the treated larvae. Also, the lethal effect was extended in the resulted stages, pupae and adults. Based on LC50 for total mortality, Bt K1 was more potent than Bt K2 where LC50 was 2.21x10 10 and 3.11x10 10 , respectively. However, the two strains were revealed a reduction of pupation and adult emergence %. Irrespective of the strain, Bt significantly decreased larval duration and significantly increased pupal duration. No effect was recorded on morphogenesis.
In the present study, it was broadly that Bacillus thuringiensis showed its ability in the control of Pectinophora gossypiella.

Rearing of Insect:
A culture of the pink bollworm, P. gossypiella (Sanders) (Lepidoptera: Gelechiidae ) was reared under constant laboratory conditions of ( 27±1 o C and 65 % R.H ) in the rearing room at the Bio Insecticides Production Unit, Plant Protection Research Institute, DoKki, Giza, Egypt. The neonate larvae were reared on an artificial diet described by (Rashad et al. 1993). The pupae were kept in clean glass villas without diet which was plugged with cotton until moths emerge. Entomopathogenic Bacteria: Bacillus thuringensis, kurstaki (K1 and K2) obtained from producing bioinsecticides, plant protection research institute Agriculture research center, Egypt.

Insect Treatment:
Early 4 th larval instar was immersed in five concentrations of bacteria for 30-60 Seconds and then transferred to sterile filter paper to dry. Four replicates (each replicate contained ten 4th larval instar). A control experiment was done, but larvae were immersed in distilled water. 4th larval instar was transferred by sterile forceps to glass tubes (2×7 cm) containing an untreated artificial diet. Tubes were plugged with cotton wool and incubated at 27±1 o C and 65 % R.H. The mortality was recorded daily until pupation and adult emergence.

Studied Criteria:
Mortalities, Pupation rate and adult emergence rate were expressed as %. The duration was recorded as mean days±SD.

Corrected Mortality:
The total mortality percentages were corrected against those of the control by Abbott's formula (Abbott, 1925) as follows: The corrected percentages of mortalities were plotted versus the corresponding concentrations on the logarithmic probability paper to obtain the corresponding Logconcentration probit lines. The lethal concentration of 50 % (LC50) of treated insects was determined from the established regression lines (Finney 1971).

Statistical Analysis of Data:
All obtained data were statically analyzed by Student's t-distribution by using (SPSS) computer program to test the significance of the difference between means ± SD.

c) LC50:
Depending on the data of LC50 for the total mortality of P. gossypiella after-treatment of the newly hatched larvae, Bt K1 was more potent than Bt K2 where LC50 was 2.21x10 10 and 3.11x10 10 , respectively (Table 3).

Development Effects After Newly Hatched (neonate) Larvae of P. gossypiella:
Table (4) reveals the effect of Bt K1and Bt K2 on the larval and pupal development after-treatment of the newly hatched larvae (Neonate) of P. gossypeilla. Bt, irrespective of the strain, significantly decreased larval duration and significantly increased pupal duration.
For the larval duration, the highest reduction was recorded at the highest concentration of Bt K1 by 9.90±0.78 at 10 12 (Bacteria/ml) vs. 14.23±0.33 of control larvae. Also, the same highest concentration of Bt K1 induced the highest increased of pupal duration as 10.25±0.29 compared to 7.24±0.31 of control pupae. No effect was recorded on morphogenesis.

DISCUSSION
Bt is the most commonly used bio-pesticide worldwide (Osman et al., 2015). B. thuringiensis is very well-known as a bio-control agent especially its crystal protein against many insects (Schnepf 1998). Despite most species of Bacillus are harmless saprophytes, two species viz., B. thuringiensis and B. cereus are considered medically and environmentally important especially in the field of controlling some plant insects (Gray et al., 2006).
The use of Bt became a vital component in integrated pest management. Bt proved to be the best alternative to pesticides (Gonzalez et al., 2011).

b-Bt and Disturbance of Development and Metamorphosis:
However, the current study recorded the effect of Bt K1 and Bt K2 on reduction of pupation and adult emergence %. Also, Bt irrespective of the strain significantly decreased larval duration and significantly increased pupal duration. These data were in harmony with other studies as Bt. significantly prolonged the larval duration of P. gossypiella and insignificant increase the pupal duration (Abbas et al., 2017). The tested biocide Btk (Dipel 2×) caused different influences on all biological aspects of pink bollworm which decreasing larval duration, pupation percentage and adult emergence (Hegab and zaki 2012). Furthermore, their latent effect caused the lowest pupation % resulted from treated P. gossypiella larvae by Staphyloccus sciuri and Micrococcus luteus (Abou-zeid et al., 2015). The percentages of pupation and adult emergence of P. gossypiella were negatively correlated with the increase of spore-crystal concentration and positively with the increase in the concentration of the supernatant of B. cereus (Mahfouz and Abou El-Ela 2011). The biocide Protecto from Bacillus thuringiensis Subsp Kurstari alone significantly increased the larval and pupal period of S. littoralis. It significantly decreased pupation and adult emergence %. (Abdel-Rahim 2011). The effects of Bt on larval, pupal and adult durations and adult emergence of H. armigera were significantly different (Fite et al., 2019).
Disturbance in development, metamorphosis and inducing mortalities of P. gosyypiella after treatment by Bt may result from its mode of action. After ingestion of Btk, the active toxin is known to bind to and destroy the midgut epithelium, resulting in rapid gut paralysis, which causes the larva to stop feeding within hours in the most sensitive species (Talekar 1992). Btk-affected larvae die from starvation, which may take several days. Since Btk does not kill rapidly, users may incorrectly assume that it is ineffective if treatments are assessed a day or two after application (Legwaila et al., 2015). However, Imam (2018) proved the effect of bacterial isolate Bacillus thuringiensis on the midgut of the 4 th larval instar of the pink bollworm, treated with LC50 CFU/ml. The study showed several histological changes; some epithelial cells were disintegrated, vacuolated and their cell boundaries were destructed and separated from the basement membrane.
On the other hand, larval mortality, according to (Yoshinori and Kaya 1993), is probably due to either the septicemia in which the bacterial spores invade the hemocoel, multiply, produce toxin and subsequent kill the insect; or due to the toxemia in which the bacteria produce toxin and confined to the gut lumen. Mortality in infected larvae may also be due to the deficiency in the excretory system due to Malpighian tubules infection (Lotfy 1988).

CONCLUSIONS
Bacteria are one of the microbial insect pathogens and are considered a non-chemical alternative for the suppression of insect pests. The current study broadly showed that Bt K1and Bt K2 have a toxic potential against P. gossypiella. However, the bacteria-induced developmental disturbance to the immature stages. Further study is needed to show some light about the mode of action of bacteria.