Exploring the Potentials of Native Bacillus Thuringiensis (Berliner) Isolates in the Management of Chickpea Pod Borer, Helicoverpa Armigera (Hübner) (Lepidoptera: Noctuidae) in Arsi and East Shoa Zones of Oromia Regional State, Ethiopia
No Thumbnail Available
Date
2024-10
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
Addis Ababa University
Abstract
Chickpea is an important staple food legume that provides proteins and health beneficial phytochemicals across the globe. It also improves soil fertility by hosting some nitrogen fixing soil bacteria. However, its production and productivity are highly affected by different factors including insect pests. Chickpea pod borer, Helicoverpa armigera is the most economically significant insect pest of chickpea globally. An over-reliance on insecticides leads to chemical residue in food, harmful to humans health, and disastrous to the natural ecosystem, which in turn prompts the biological pest management alternatives. Bacillus thuringiensis (Bt) gains popularity as bioinsecticide to control insect pests and vectors of human diseases through its insecticidal crystal toxic proteins encoded as cry and cyt genes. Thus, the objective of this study was to search for native Bacillus thuringiensis isoates that possess lepidopteran active cry genes chiefly cry1 and cry2. Out of the 121 soil samples collected, 23 Bacillus species were isolated and purified on suitable medium. Screening biotoxicity assay was conducted under laboratory along with a reference Bt .var. thuringiensis. Experiments were replicated three times. Ten 3rd instar larvae were used per replicate. Likewise, the ten Bt isolates were morphologically, and biochemically characterised. Further their cry genes profile was analysed using PCR. Five universal primers cry1, cry2, cry3, cry4, cry7, 8 and one specific primer cry9 and 16S rRNA universal primer were used for the analysis of cry genes. Biotoxicity assay was conducted at five concentrations ranging from 0.25-2 mL. LD50, 90 values were 0.84-1.24 mL and 1.54-1.98 mL, respectively. Furthermore, pathogenicity was evaluated in the greenhouse and further verified under field conditions at Kulumsa Agricultural Research Center and Gonde Basic Seed Farm Center in 2023 main crop growing season. Treatments used were Bt concentrations at LD90 in the greenhouse and double of LD90 under field conditions. Bt concentrations first adjusted to 0.5 McFarland standard (1.5x108 cfu/mL) and mixed with 0.5 mL Triton x-100 (0.1%), 1 mL Tween 80 (2%) and one gram milk powder. Control plots sprayed with distilled water. Consequently, ten native Bt isolates were screened based on their larval mortality efficacy that ranged between 27.78% and 75.83% and LT50 values of 45.71-143.9 h. Statistically no significant (p>0.05) difference was observed among the tested native bacterial isolates and the reference Bt.var. thuringiensis. Some of the Bt isolates share the same morphological and biochemicals characteristics, but there are also some variations. All the Bt isolates possessed endospores, insecticidal crystal proteins, rod shaped cells as well as positive to catalase and motility tests.
Similarly, they all were positive to glucose fermentation and citrate utilisation, but negative to urease and indole tests. Two or more cry genes were detected in all Bt isolates, where cry4 (100%) detected followed by cry2 (90%), cry1 (50%), cry9 (40%) and cry7, 8 (10%). All the Bt isolates in the current study amplified 16S rRNA genes. As of the combinations of cry genes, Bt isolates KDL, AUPOS, GHTSW and GHTSW-1 carried (cry2+cry4), AUGHS-1 (cry1+cry4), ZDS (cry2+cry4+cry9), AUGHS-3 (cry1+cry2+cry4), ZDS-3 and AUASG-2 (cry1+cry2+cry4+cry9) and AUSD-1 (cry1+cry2+cry4+cry7,8+cry9). Bt grew under varied values of temperature that ranged from 15 °C- 40 °C. All the Bt isolates showed growth at all the values of the tested temperatures. After 35 °C, the growth of all the Bt isolates declined indicating that 30 °C to 35 °C is the optimum growth temperature range. There was statistically significant (p<0.05) difference between Bt isolates tested and the control plots under the greenhouse conditions. Larval mortality (%), pod damage (%) and grain yield (t/ha) found to be in the range of 66.67±8.70% to 90.48±16.49%, 8.09±2.49% to 36.59±9.71% and 0.91±0.06 t/ha to 2.31±0.54 t/ha, respectively. Whereas, the control plots resulted in 36.59±9.71% pod damage and 0.91±0.06 (t/ha) grain yield. Under field trials, post 1st, pre 2nd and post 2nd Bt sprayed larval population counted per plant both at Gonde and Kulumsa showed statistically (p<0.05) significant variations between Bt isolates and the control plots that ranged from 0.97±0.17 to 3.50±0.23, 0.87±0.25 to 3.91±0.31 and 0.84±0.29 to 2.82±0.14 at Gonde. At Kulumsa, it was found in between 0.99±0.16 and 2.66±0.12, 1.00±0.21 and 2.69±0.20, 0.73±0.17 and 2.70±0.40, separately. Pod damage (%) and grain yield (t/ha) at Gonde ranged from 10.42±6.63 to 49.35±15.02 and 0.75±0.16 to 2.05±0.71, while, at Kulumsa ranged from 8.63±2.57 to 52.25±9.72 and 1.07±0.19 to 2.42±0.02 (t/ha) that was statistically (p<0.05) different from the control plots with the highest 47.53±9.51% pod damage and the lowest 0.97±0.03 (t/ha) grain yield. We concluded that all the Bt isolates tested harboured either cry1 or cry2 or both cry genes in combinations with other cry genes and efficiently reduced larval population, percentage pod damage with higher grain yield (t/ha). Hence, the Bt isolates were identified as potential candidate for H. armigera management and other related insect pests. Therefore, continuous exploration of Bt specific cry gene families is prompted and testing under multiple field trials for commercialisation and development of Bt bioinsecticides.
Description
Keywords
Chickpea Pod Borer, Cry Genes, Crystal Proteins, Mortality, Native Bt Isolates