Bio-insecticidal Activity of Bacillus thuringiensis Isolates on the Tomato Borer, Tuta absoluta (Meyrick) and their Molecular Identification

Twelve bacterial isolates were isolated from dead larvae of Tuta absoluta (4th instar) infested tomato cultivated fields at Fayoum Governorate, Egypt. All isolates were preliminarily identified as members of the genus Bacillus, based on their Morphological and biochemical characteristics. According to the results of the pathogenicity of Bacillus isolates against different larval instars of T absoluta, the 12 isolates revealed varying efficiencies and the isolates B-1, B-2, B-3 and B-4 showed high mortality rates of 93.3, 90, 86.7 and 80% on day7, respectively on the 4th instar larvae. Also, protecto (Bacillus thuringiensis subsp. kurstaki) recorded high mortality rate, when the 4th instar larvae were treated by 2 gm /2 liter of water (96.7% on 5th day post treatment). Isolate B-12 recorded the lowest percentage of mortality (13.3%) of the 4th instar larvae. In addition, there was a significant decrease in egg hatching percentage reached 44.12 %, compared with the control. Meanwhile, adult emergence was decreased after treatment when only 38 and 30 adults emerged from the cages contained tomato plants infested by eggs with B-1 and protecto as compared with the control (253 adults). Further genetic identification of 12 isolates was performed using randomly amplified poylmorphic DNA (RAPD) markers to determine their genetic diversity pattern. Different random primers were used for RAPD amplification, which generated a total of 52 fragments; of these 42 were polymorphic and 10 monomorphic. The primers OPA02, OPA04, and OPA07 produced 100% polymorphic fragments, whereas primers OPA1, OPA3, OPA05, OPA06, OPA08 and OPA09 produced 1, 3, 1, 2, 1 and 2 monomorphic fragments, respectively. When the RAPD banding pattern data was subjected to dendrogram construction, the 4 isolates fell into two separate clusters, cluster I and cluster II, which includes 1 and 3 B. thuringiensis isolates, respectively. The RAPD technique was shown to be effective in differentiating closely related isolates and applied to confirm the identification of Bacillus isolates by API system to reveal the phylogenetic relationships among the isolates.