103 Short Communication J. Hortl. Sci. Vol. 13(1) : 103-107, 2018 16S rRNA gene taxonomic profiling of endophytic bacteria associated with phylaenopsis roots D. Girija*1, P.K. Rajeevan , Swathi Balakrishnan1, P. S. Panchami1 and Mahesh Mohan1 Department of Agricultural Microbiology1,Department of Floriculture and Landscapping2 Kerala Agricultural University,Vellanikkara, 680656 *Email: devakigirija@gmail.com ABSTRACT Orchids are one of the main groups of ornamental plants commercially exploited. In the present study, we analyzed the diversity of bacterial community in Phalaenopsis root using metagenomic approach. The diversity of bacterial taxonomic category was assessed at different Operational Taxonomic Unit (OTU) levels using Ribosomal Database Project (RDP) pipeline and MG-RAST. At phylum level, Proteobacteria (61.34%) was the most dominant group followed by unclassified derived from bacteria (24.74%) and Actinobacteria (12.52%). Genus level analysis revealed the abundance of Rubrobacter, Pseudomonas and Acinetobacter. The study revealed that of the total species detected 50.83 per cent were unclassified, stressing the importance of metagenomics to assess the diversity of endophytes associated with orchid roots. Keywords: Endophyte, orchid, diversity INTRODUCTION Orchidaceae is one of the largest plant families, including almost 10% of all flowering plant species. Among these, the monopodial epiphytic Phalaenopsis or ‘Moth Orchid is one of the most popular orchids due to its ease of production and blooming year-round. The orchid roots are associated with various fungi and endotrophic bacteria (Teixeira et al., 2015). Apart from mycorrhizal fungi, previous reports revealed the abundance of endophytic bacteria on the roots of the cultivated tropical orchids of genera Calanthe, Acampe and Dendrobium (Tsavkelova et al., 2003). Orchids are characterized by low survival rate in the green house due to the germination under asymbiotic conditions in vitro. Generally, endophytes play an important role in promoting plant growth and yield, suppress pathogens, aid in removing heavy metal contaminants, solubilize phosphate or contribute to nitrogen assimilation for plants (Hallmann et al., 2006). Over the past decade, our understanding of microbial diversity and function in complex environments has increased significantly, primarily because of the introduction of next generation sequencing (NGS) (Lozupone and Knight, 2007). The culture-independent, high-throughput sequencing-based community analysis allows us to observe the microbiome associated with the plants. Since the endophytes have a strong impact on orchids growth, it is very important to study their relationships with plant for developing new strategies for orchid conservation and better exploitation of their medicinal principles.Therefore, in the present study, we employed NGS technology to unveil the culturable and unculturable endophytic bacteria in Phalaenopsis root to elucidate the microbial plant colonisation pattern and evaluate its microbial diversity. The Phalaenopsis plants grown in Sphagnum moss under green house conditions were collected from the Department of Pomology and Floriculture, College of Hor ticultur e, Vella nikka r a . Sa mples wer e immediately transferred and processed for further studies. The roots were detached with sterile knife and washed with sterile distilled water plus a few drops of Tween-20 and left for 10–15 min to drain. These were then cut into 4–5 pieces (2–3 cm in size). Surface sterilization was performed by immersing separately in 90% etha nol (5 min), followed by sodium hypochlorite (3%) solution (2 min), and 75% ethanol (3 min). The disinfected roots were rinsed three times in sterile distilled water. Total genomic DNA was extracted from the surface sterilized root tissues using QIAGEN DNea sy pla nt kit following the manufa ctur er ’s pr otocol. Extr a cted DNA wa s 104 suspended in QIAGN elution buffer and stored at - 200C. PCR amplification was carried out to amplify V3 conserved region of 16S rRNA gene sequences using the 16S rRNA gene primers (forward primer 5¹- CCTACGGGNGGCWGCAG-3¹ and reverse 5¹- GACT ACHVGGGTATCTA-3¹). The amplicons were purified and sequenced on the Illumina Miseq platform at Scigenom Pvt. Ltd. Cochin. The FASTQ sequences were filtered to remove chimeric sequences and singletons to obtain preprocessed reads, which was then clustered to obtain OTUs. The chloroplast sequences that comprised of almost 97.5 per cent of the total reads were removed using QIIME analysis. Further taxonomic annotation of the 301 OTUs obtained were done using QIIME and MG-RAST tools. Total DNA was isolated from the roots of Phalaenopsis plants and the presence of 16S rRNA gene was confirmed by amplification with universal primers. Total raw sequencing reads (paired end) of 1,96,595 with average sequence length of 151 bp each was obtained from Illumina MiSeq™ sequencer. The abundance of major bacterial groups in each taxonomic category is given in Table 1. Altogether, 10 bacterial phyla were detected and among these, Proteobacteria (61.34%) was the most dominant group followed by unclassified derived from bacteria (24. 74%) and Actinobacter ia (12. 52%). Rea ds belonging to Acidobacteria, Bacteriodetes, Chloroflexi, Cyanobacteria, Spirochaetes, Tenericutes, Firmicutes and Bacteriodetes were found to be the other phyla with less than 1 per cent (Fig 1). The higher abundance of Proteobacteria in the roots of orchids suggests that members of this phylum are particularly well adapted to colonize inner plant tissues and establish as root endophytes. The phylum Proteobacteria comprises several species that promote plant growth and act as biological control agents of different diseases (Bulgarelli et al., 2013). Actinobacteria play specific roles, for instance, protecting the host plants against insects and diseases especially by the production of bioactive compounds. Firmicutes were found to be metabolically the most versatile group with production of multiple enzyme activities. Cyanobacteria are photosynthetic; some are capable of fixing nitrogen and others improve soil-aggregation stability (Issa et al., 2007), a key aspect of soil conservation. Results of the present investigation are in agreement with the earlier reports on Proteobacteria, Actinobacteria, Firmicutes and Bacteriodes being the prominent phyla in the roots of tree peony (Yang et al., 2017). A total of 7 bacterial classes were identified and among them Gammaproteobacteria was the mos t domina nt gr ou p ( 4 1 . 9 0 %) f ollowed b y unclassified (derived fr om ba cter ia ) (31. 35%) Actinoba cter ia (25. 11%) a nd Ba cilli (1. 37%) (Fig 2). A total of 17 bacterial orders were detected. The most dominant group was unclassified derived f r om b a c t er ia ( 3 5 . 9 9 % ) f o llowed b y Pseudomonadales (30.69%) and Rubrobacterales 16S rRNA gene taxonomic profiling of endophytic bacteria J. Hortl. Sci. Vol. 13(1) : 103-107, 2018 Fig. 1. Abundance of endophytic bacteria at phylum level constructed in MG-RAST with illumina sequencing data set Fig. 2. Abundance of endophyticbacteria at genus level constructed in MG-RAST with illumina sequencing data set 105 Girija et al J. Hortl. Sci. Vol. 13(1) : 103-107, 2018 Table 1. Abundance of major taxonomic category from phyla to species level of endophyticbacteriain Phalaenopsisroot 106 16S rRNA gene taxonomic profiling of endophytic bacteria J. Hortl. Sci. Vol. 13(1) : 103-107, 2018 (27.47%). Orders Actinomy etales, Bacillales and Enterobacteriales were also present more than one per cent. Analyses at family level revealed a total of 22 bacterial families were present in the sample. Major bacterial families present in the sample were u nc la s s if ie d der ived f r om b a c t e r ia ( 3 6 % ) , f ollowed b y R u b r ob a c t er a c ea e ( 2 7 . 4 8 % ) , Pseudomonadaceae (21.68%) and Moraxellaceae (9. 01%). The Ga mma Proteoba cter ia included Ps e u d o m o n a s , Pa n t o e a , A c i n e t o b a c t e r , Stenotrophomonas and Xanthomonas making it phylogenetica lly the most diverse group in the current study. Altogether 31 bacterial genera were p r es ent . Unc la s s if ied der ived f r om b a c t er ia (35.99%) was the most domina nt group in the present study followed by Rubrobacter (27.47%), Pseudomonas (21.67%) and Acinetobacter (9%). Genus Enterobacter and Aneurinibacillus were also present at more than one per cent abundance. Genus Rubrobacter is well known to be a radiation resistant bacterium. Genus Pseudomonas can utilize more than 200 compounds as carbon source, can fix atmospheric N and solubilize P. Several of the genera isolated in the current study, including Pa n t o e a , Ps e u d o m o n a s , B a c i l l u s a nd Acinetobacter have been isolated from different plants and shown to possess plant growth promoting a c tivit ies ( Tr ivedi et a l. , 2 011) . I t ha s been p r eviou s ly ob s er ved t ha t in ma ny c a s es , Ps e u d o m o n a s a nd memb er s of Enterobacteriaceae are abundant in both the soil environment and the plant interior (Spiers et al., 2 00 0) . T he pr eva lenc e of Pse ud om o na s a nd Bacil lus endos ymbionts wa s a ls o r epor t ed in Australian terrestrial orchids (Wilkinson et al., 1994). The study emphasizes on the importance of metagenomics to assess the diversity and role of endophytic microbes in plants. This study extends the knowledge on the composition and diversity in the orchid microbial populations. Moreover, most of the endophytes observed in the present study are perhaps good producer s of bioa ctive compounds, which ca n promote the growth of orchids in seedling stage and also in ex vitro acclimatization. Bulgarelli D., Schlaeppi, K., Spaepen, S., Ver Loren and van Themaat E. 2013. Structure and functions of the bacterial microbiota of plants. Annu. Rev. Plant Biol, 64: 807-838. 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