PowerPoint Presentation

[Audio] Welcome, my name is Dr. M. K. Prasanna Kumar, Professor at Department of Plant Pathology, University of Agricultural Sciences, GKVK, Bangalore, INDIA. Today, I'm happy to present our work titled " Decoding microbiome and metabolome of Panchagavya- an indigenous fermented bio-formulation" contributed by our team H. B. Mahesh, P. Buela Parivallal, Pramesh D, Puneeth M. E, Aditya Sarangi, Manasa K. G, Sushil K. Middha and Sahana N. Banakar..

[Audio] The word ' Panchagavya' is derived from Sanskrit, meaning " five ingredients from a cow," which includes dung, milk, urine, curd, and clarified butter ( ghee). It has been reported that the 'Panchagavya' application enhances plant growth, nutrient uptake, reduces the biotic and abiotic stresses in crop plants. Preparation of Panchagavya formulation involves several fermentation processes with a consortium of beneficial bacteria. In such bio-formulations, cow dung is an important constituent that acts as a source of bacterial inoculum. The Panchagavya formulations can be applied to crop plants in many methods, of which foliar spray is widely used. As the bio-formulation can be suspended or dissolved in water, and therefore can be used along with irrigation. In organic farming, it is used as a foliar spray with a concentration of 3%. The Panchagavya is also used as a biofertilizer at 1: 100 (Panchagavya: soil; v/wt), promoting plant growth..

[Audio] 7 kg of fresh cow dung, 7 liters of cow's urine and 10 liters of water were added and left in a tank ( iron and aluminium containers should not be used) and the mixture was stirred thoroughly everyday. On 16th day, again one kg of fresh cow dung was added. Then 2 liters of milk, 2 liters of sour curd and one liter of ghee that were obtained from cow, were added to the above mixture. The stirring process was continued for 7 days more. On the 18th day, Panchagavya solution was ready for use. Cow dung acts as a medium for the growth of beneficial microbes. Cow's urine provides nitrogen which is essential for crop growth. Milk provides protein, fat, carbohydrates, amino acids and calcium. Curd provides lactobacillus which acts as a catalyst in the digestion of organic wastes. Ghee provides vitamin A and B, calcium and fat..

[Audio] With the recent advancement in metagenomic sequencing, constructing the whole microbiome is easier and more comprehensive than the 16S rRNA sequencing of only culturable bacteria. However, metagenomics reveals only the microbial populations; therefore, metabolomics is required to study the in situ microbial metabolic activity and their capabilities. Therefore, with the aid of metabolomics, we can identify the plant growth-promoting compounds, including hormones, which can be further screened with high-throughput metabolite identification technologies such as Liquid Chromatography Mass Spectrometry ( LCMS). In this study, we report an integrated approach to connect the microbial population assemblages of the Panchagavya by metagenomics and identifying its potential plant-growth-promoting molecules through LCMS attached to the ' meltin' library. Furthermore, by combining different omics technologies, we have revealed the overall function of these microbes in Panchagavya..

[Audio] The metagenome sequencing approach was used in this study to understand the diversity and function of microbial niches, which could not be addressed using conventional methods. We have employed a Metagenomic sequencing approach for profiling all the associated microbiota of Panchagavya. The analysis revealed that Panchagavya harbours a wide range of bacterial genera. Further, functional classification of identified bacterial genera revealed the abundance of bacteria involved in PGPR, ISR, biocontrol of plant pathogens, nutrient recycling in soil, and production of plant hormones. Among the PGPRs, L. mucosae was abundantly found in the bio-formulation along with several other genera. This study thus provides the microbiological evidence for the enhanced plant growth after applying Panchagavya to the crop plants as reported previously [ 5,32]. Our study also identified several bacterial genera associated with the induction of ISR in crop plants. In addition, several bacterial genera which can degrade complex carbohydrates such as cellulose, hemicellulose, lignin were identified, indicating the usefulness of Panchagavya in recycling/maintaining organic carbon in the soil..

Taxonomic identity and their abundance of different bacteria identified in the Panchagavya bio-formulation..

Taxonomic identity and their abundance of different bacteria identified in the Panchagavya bio-formulation. A) Circos containing three halves a. The first halve representing, Micro- organisims classified into 10 groups depending on their potential role b. The second halve representing, genes involved in the amino acid biosynthesis (AAB), amino acid degradation (AAD), amine and polyamine biosynthesis (APAB), amino-sugar metabolism (ASM), Bacterial outer membrane biogenesis (BOMB), cell wall biogenesis (CWB), co-factor biogenesis (CFB), Glycan degradation (GD), Glycan Metabolism (GM) and Metabolic intermediate biosynthesis (MIB) pathways are represented. c. The third halve representing, Genes present in the organism classified for potential role other than pathways. B) Circular diagram indicates the potential role of identified bacteria along with their read depth..

Venn-diagram depicting the functional annotation of predicted protein in to major functions. A) Clustered Ortholog Group (COGs) analysis classified the predicted proteins in to three major functional groups. B) Sub-system annotation of the predicted proteins..

Microbial community composition of Panchagavya formulation assessment using CCMetagen revealed 18% of the reads are of eukaryotic origin..

[Audio] The Extraction of metabolites for LC-MS/ MS analysis was done using Multi-solvent and aqueous followed by LC-MS/MS analysis using QTRAP 6500 mass spectrometer Agilent 1290 Infinity II LC system and finally the analysis of metabolome data using XCMS online tool Metlin' metabolite database..

[Audio] The XCMS was used to decipher the metabolites of the Panchagavya formulation. A total of 5737 features were identified in XCMS metabolite analysis ( 2035 in aqueous extract method and 1783 in multi-solvent extract method), of which 2970 were identified in the Metlin database..

[Audio] The essential plant hormones, like gibberellic acid and its eight other derivatives such as Gibberellin A70, Gibberellin A45, Gibberellin A61, Gibberellin A51, Gibberellin A4, Gibberellin A20, Gibberellin A29-catabolite, and Gibberellin A14 aldehyde were recorded. In addition, several compounds such as Kaurenol, Strigol, cis- Zeatin 9-glucoside., etc., belonging to different chemical groups which are associated with plant-growth-promoting and biotic stress tolerance were also recorded in the Panchagavya formulation..

[Audio] Panchagavya helps in the production of synthetic pesticide-free food. It also maintains and restores crop production levels when the field changes from inorganic to organic farming practices within a year. It enhances the shelf-life, taste of fruits, grains, and vegetables and yields better and safe quality food products. Our study has identified several bacterial genera in the Panchagavya which have not been characterized so far for any role in the soil and plant system, which necessitates a systematic study to characterize such potential bacteria..

[Audio] For the first time, updated molecular techniques were used to validate and elucidate the effect of Panchagavya. Metagenomics was used to decipher the bacterial microbiome structure, which showed promising results for their existence and abundance in Panchagavya ( number of reads of particular taxa; the gene annotations and functional annotations were predicted). On the other hand, the metabolome predicted the byproducts of numerous plant growth promoters as well as other compounds released and fermented by these bacteria. According to these findings, Panchagavya is a promising manure that contains helpful microorganisms and plant development promoters. As a result, Panchagavya is a bio-formulation that is both ecologically benign and effective in improving soil and crop health..