Temporal dynamics of endophytic bacterial and fungal communities during spike development in Piper longum L.

The female spikes (fruits) of Piper longum are widely used in Ayurvedic, Siddha and Unani medicine systems to treat respiratory and digestive disorders. The spikes are rich in piperine, a pharmacologically active amide alkaloid and a potent bioavailability enhancer, which accumulates to the highest level during the dark-green stage of spike development. Plant-associated microbiota influence the plant's fitness, response, and production of economically important metabolites. Considering the economic importance of piperine and other spike-derived alkaloids, understanding microbial community dynamics during spike development would be key to bioprospecting for economically important metabolites. In the present study, the structural diversity of microbial communities associated with early (SI), mid (SII), and late (SIII) stages of spike development in P. longum has been analysed by Illumina-based amplicon sequencing of 16S rRNA gene and ITS region. Results revealed that spike development significantly drives the diversity and abundance of spike-associated microbiota, especially bacterial communities. Cyanobacteria and Ascomycota constituted the most abundant bacterial and fungal phyla, respectively, across all stages of spike development. Interestingly, Halomonas, Kushneria and Haererehalobacter were found to be exclusively associated with SIII (corresponding to economically important) stage of spike development. Sphingomonas, Mortierella, Cladosporium and Vishniacozyma constituted the core microbiome of the spike. Besides, PICRUSt analysis revealed that amino acid metabolism was the most dominant metabolic function attributed to spike-associated bacterial communities. To the best of our knowledge, this is the first study to investigate the endomicrobiome dynamics during spike development in a medicinal plant species. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-023-01352-2.

Surface sterilization for isolation of endophytes: Ensuring what (not) to grow.

Endophytic microbiota opens a magnificent arena of metabolites that served as a potential source of medicines for treating a variety of ailments and having prospective uses in agriculture, food, cosmetics, and many more. There are umpteen reports of endophytes improving the growth and tolerance of plants. In addition, endophytes from lifesaving drug-producing plants such as Taxus, Nothapodytes, Catharanthus, and so forth have the ability to produce host mimicking compounds. To harness these benefits, it is imperative to isolate the true endophytes, not the surface microflora. The foremost step in endophyte isolation is the removal of epiphytic microbes from plant tissues, called as surface sterilization. The success of surface sterilization decides "what to grow" (the endophytes) and "what not to grow" (the epiphytes). It is very crucial to use an appropriate sterilant solution, concentration, and exposure time to ensure thorough surface disinfection with minimal damage to the endophytic diversity. Commonly used surface sterilants include sodium hypochlorite (2%-10%), ethanol (70%-90%), mercuric chloride (0.1%), formaldehyde (40%), and so forth. In addition, the efficiency could further be improved by pretreatment with surfactants such as Triton X-100, Tween 80, and Tween 20. This review comprehensively deals with the various sterilants and sterilization methods for the isolation of endophytic microbes. In addition, the mechanisms and rationale behind using specific surface sterilants have also been elaborated at length.

Exploiting endophytic microbes as micro-factories for plant secondary metabolite production.

Plant secondary metabolites have significant potential applications in a wide range of pharmaceutical, food, and cosmetic industries by providing new chemistries and compounds. However, direct isolation of such compounds from plants has resulted in over-harvesting and loss of biodiversity, currently threatening several medicinal plant species to extinction. With the breakthrough report of taxol production by an endophytic fungus of Taxus brevifolia, a new era in natural product research was established. Since then, the ability of endophytic microbes to produce metabolites similar to those produced by their host plants has been discovered. The plant "endosphere" represents a rich and unique biological niche inhabited by organisms capable of producing a range of desired compounds. In addition, plants growing in diverse habitats and adverse environmental conditions represent a valuable reservoir for obtaining rare microbes with potential applications. Despite being an attractive and sustainable approach for obtaining economically important metabolites, the industrial exploitation of microbial endophytes for the production and isolation of plant secondary metabolites remains in its infancy. The present review provides an updated overview of the prospects, challenges, and possible solutions for using microbial endophytes as micro-factories for obtaining commercially important plant metabolites.Key points• Some "plant" metabolites are rather synthesized by the associated endophytes.• Challenges: Attenuation, silencing of BGCs, unculturability, complex cross-talk.• Solutions: Simulation of in planta habitat, advanced characterization methods.

Targeted 16S rRNA gene and ITS2 amplicon sequencing of leaf and spike tissues of Piper longum identifies new candidates for bioprospecting of bioactive compounds.

Piper longum (also known as Indian long pepper) is widely used in Ayurvedic, Siddha and Unani medicine systems. The principle bioactive compound of this plant is piperine, which mainly accumulates in the fruits called spikes. The report of piperine production by endophytic microbes isolated from Piper sp., motivated us to investigate the endophytic microbial diversity associated with the spikes vis-à-vis leaves (which contain negligible levels of piperine). This is the first report to use metagenomics approach to unravel the endophytic microbial diversity in P. longum. Our results indicate that 2, 56, 631 bacterial OTUs and 1090 fungal OTUs were picked cumulatively from both the tissues. Although bacterial and fungal endophytes occupy the same niche, remarkable differences exist in their diversity and abundance. For instance, the most abundant bacterial genera in spikes were Nocardioides and Pseudonocardia (Phylum Actinobacteria; reported to produce bioactive compounds); while, in leaves were Larkinella and Hymenobacter (Phylum Bacteriodetes). Likewise, the fungal endophytes, Periconia, Cladosporium and Coniothyrium (which have been earlier reported to produce commercially important metabolites including piperine), were also present in high abundance in spikes, in comparison to leaves. Further, the results of PICRUSt analysis reveal the high metabolic potential of spike-associated bacteria for secondary metabolism, namely biosynthesis of alkaloids (including pyridine/piperidine), terpenes, flavonoids and antibiotics. Therefore, our findings indicate that the endophytes abundant or unique in spikes could be explored for bioprospecting of novel/commercially important metabolites; an approach that has both ecological and economical benefits.