A comprehensive study on characterization of elite Neem chemotypes through mycofloral, tissue-cultural, ecomorphological and molecular analyses using azadirachtin-A as a biomarker.

Azadirachtin-A (Aza-A), a tetranortriterpenoid, found in minuscule amounts in the Neem seed-kernels, has proved to be a potent biopesticide. Given the vast biodiversity of Azadirachta indica (Neem) in India, this study is an overview of four main aspects that corroborate with each other in identifying elite Neem chemotypes based on their Aza-A content. These biomarkers included mycofloral, tissue-cultural, ecomorphometrical and molecular analyses on accessions from five ecogeographically different regions in Andhra Pradesh, India, which high-lighted the characteristics of trees that yielded the highest Aza-A. In essence, extremely-arid-alkaline regions with maximum soil pH (8.05) yielded trees with the highest amount of this biopesticide. Likewise, both VAM and soil fungal diversity and frequency exhibited maximal values in their rhizosphere, whereas it exhibited the least values for percentage moisture and also for several micronutrients measured (P2O5, Zn, Fe and Cu). In vitro studies on seeds with high versus low Aza-A content gave sturdier seedlings in the former; with profusely coiled roots and fibirillar foliage in tissue-culture; in addition to these seeds being more viable. Furthermore, their cotyledons alone exhibited significant amount of Aza-A, as measured by HPLC. Besides this significant difference, the impact of growth factors culminated not only in the variations of several secondary metabolites, but also differences in DNA patterns from various parts of a single in vitro plant. Ecomorphometric analyses clearly indicated that at least eight parameters (seed diameter, soil pH, percentage moisture, K2O, P2O5, Zn, lower lobe serrations and upper-lobe-distance of leaves) were significantly related to the quantitative variations in Aza-A. Finally, PCR analyses exhibited a habitat-based molecular concordance of ISSR and FISSR profiles with Aza-A content among the Neem chemotypes. Their relatedness was based on dendrograms constructed by UPGMA algorithms using similarity-index-values.