Cationic and anionic detergent buffers in sequence yield high-quality genomic DNA from diverse plant species.

Because of the heterogeneity among seedlings of outbreeding species, the use of seedling tissues as a source of DNA is unsuitable for the genomic characterization of elite germplasms. High-quality DNA, free of RNA, proteins, polysaccharides, secondary metabolites, and shearing, is mandatory for downstream molecular biology applications, especially for next-generation genome sequencing and pangenome analysis aiming to capture the complete genetic diversity within a species. The study aimed to accomplish an efficient protocol for the extraction of high-quality DNA suitable for diverse plant species/tissues. We describe a reliable, and consistent protocol suitable for the extraction of DNA from 42 difficult-to-extract plant species belonging to 33 angiosperm (monocot and dicot) families, including tissues such as seeds, roots, endosperm, and flower/fruit tissues. The protocol was first optimized for the outbreeding recalcitrant trees viz., Prosopis cineraria, Conocarpus erectus, and Phoenix dactylifera, which are rich in proteins, polysaccharides, and secondary metabolites, and the quality of the extracted DNA was confirmed by downstream applications. Nine procedures were attempted to extract high-quality, impurities-free DNA from these three plant species. Extraction of the ethanol-precipitated DNA from cetyltrimethylammonium bromide (CTAB) protocol using sodium dodecyl sulfate (SDS) buffer, i.e., the extraction using a cationic (CTAB) detergent followed by an anionic (SDS) detergent was the key for high yield and high purity (1.75-1.85 against A260/280 and an A260/230 ratio of >2) DNA. A vice versa extraction procedure, i.e., SDS buffer followed by CTAB buffer, and also CTAB buffer followed by CTAB, did not yield good-quality DNA. PCR (using different primers) and restriction endonuclease digestion of the DNA extracted from these three plants validated the protocol. The accomplishment of the genome of P. cineraria using the DNA extracted using the modified protocol confirmed its applicability to genomic studies. The optimized protocol successful in extracting high-quality DNA from diverse plant species/tissues extends its applicability and is useful for accomplishing genome sequences of elite germplasm of recalcitrant plant species with quality reads.

Effectiveness of Augmentative Biological Control of Streptomyces griseorubens UAE2 Depends on 1-Aminocyclopropane-1-Carboxylic Acid Deaminase Activity against Neoscytalidium dimidiatum.

To manage stem canker disease on royal poinciana, actinobacterial isolates were used as biological control agents (BCAs) based on their strong in vitro inhibitory effects against Neoscytalidiumdimidiatum. Streptomyces griseorubens UAE2 and Streptomyces wuyuanensis UAE1 had the ability to produce antifungal compounds and cell-wall-degrading enzymes (CWDEs). Only S. griseorubens, however, restored the activity of 1-aminocyclopropane-1-carboxylate (ACC) deaminase (ACCD). In vivo apple fruit bioassay showed that lesion development was successfully constrained by either isolates on fruits inoculated with N. dimidiatum. In our greenhouse and container nursery experiments, S. griseorubens showed almost complete suppression of disease symptoms. This was evident when the preventive treatment of S. griseorubens significantly (p < 0.05) reduced the numbers of conidia of N. dimidiatum and defoliated leaves of royal poinciana seedlings to lesser levels than when S. wuyuanensis was applied, but comparable to control treatments (no pathogen). The disease management of stem canker was also associated with significant (p < 0.05) decreases in ACC levels in royal poinciana stems when S. griseorubens was applied compared to the non-ACCD-producing S. wuyuanensis. This study is the first to report the superiority of antagonistic actinobacteria to enhance their effectiveness as BCAs not only for producing antifungal metabolites and CWDEs but also for secreting ACCD.