Islolation and Characterization of Plant Based Pesticides

Isolation and Characterization of Plant Based Pesticides With the pace of constant population growth, the demand for sufficient and safer food is continuously increasing around the globe. On the other hand, global loss to crops due to pests, diseases and weeds is significantly high, warranting excessive use of pesticides, threatning environmnet and food safety. The most frequently used pesticides are synthetic posing several associated pre and post application problems such as residual toxicity that results in compromising the safety of food and causing insect resistance. An alternative approach may be to utilize plant’s secondary metabolites that plants actually synthesize in their defense against pests and pathogens. The major aim of current research study was, therefore, to identify, isolate, and characterize at biochemical and molecular level the potent insecticidal compounds from plant sources. To achieve this aim, seven plants namely Cinnamomum camphora, Eucalyptus sideroxylon, Isodon rugosus, Boenninghausenia albiflora, Calotropis procera, Daphne mucronata, and Tagetes minuta were selected. The crude and purified extracts of each of these plants were used to screen for their toxic effects against six economically important agricultural pests, each representing a separate insect order; Acyrthosiphon pisum (Hemiptera), Drosophila melanogaster (Diptera), Tribolium castaneum (Coleoptera), Spodoptera exigua (Lepidoptera), Schizaphis graminum (Hemiptera) and Bactrocera zonata (Diptera). Aphids were the most susceptible insects with 100% mortality observed after 24 h for all the plant extracts tested. Further bioassays with lower concentrations of the plant extracts against aphids revealed that the extracts from Isodon rugosus (Lamiaceae) (LC50 36.2 ppm and LC90 102.1 ppm) and Daphne mucronata (Thymelaeaceae) (LC50 126.2 ppm and LC90 197.5 ppm) found out to be the most toxic to aphids, A. pisum. These most toxic and active plant extracts were further fractionated in different solvent fractions on polarity basis and their insecticidal activity was further evaluated. While all fractions showed considerable mortality in aphids, the most active was the butanol fraction from Isodon rugosus with an LC50 of 18 ppm and LC90 of 48.2 ppm. Further bioactivity guided fractionation of the butanol fraction results in isolation of bioactive principle compound that was identified through various spectroscopic techniques as rosmarinic acid with LC50 0.2 ppm and LC90 5.4 ppm. There was no significant difference between LCs of purified rosmarinic acid and of commercial rosmarinic acid. Further, two key genes, hydroxyphenylpyruvate reductase and rosmarinic acid synthase, known to involve in biosynthesis of rosmarinic acid were targeted to clone from Isodon rugosus. Only one of these genes, hydroxyphenylpyruvate reductase was successfully cloned in Isodon rugosus which consequently will open the way to explore all other genes responsible for biosynthesis of rosmarinic acid. The molecular knowledge regarding biosynthetic pathway will help in biotechnological production of rosmarinic acid and to produce aphid resistant plants through genetic engineering approaches. Considering the high mortality rate in aphids to a significantly low concentration of the rosmarinic acid from Isodon rugosus, could be exploited and further developed as a potential eco-friendly plant-based insecticide against sucking insect pests.