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

Balakrishnan, Swathi

Girija, D

Mohan, Mahesh

Panchami, P S

Rajeevan, P K

English

Journal of Horticultural Sciences

103Short CommunicationJ. Hortl. Sci.Vol. 13(1) : 103-107, 201816S rRNA gene taxonomic profiling of endophytic bacteriaassociated with phylaenopsis rootsD. Girija*1, P.K. Rajeevan , Swathi Balakrishnan1, P. S. Panchami1 and Mahesh Mohan1Department of Agricultural Microbiology1,Department of Floriculture and Landscapping2Kerala Agricultural University,Vellanikkara, 680656*Email: devakigirija@gmail.comABSTRACTOrchids are one of the main groups of ornamental plants commercially exploited. In thepresent study, we analyzed the diversity of bacterial community in Phalaenopsis root usingmetagenomic approach. The diversity of bacterial taxonomic category was assessed at differentOperational Taxonomic Unit (OTU) levels using Ribosomal Database Project (RDP) pipelineand MG-RAST. At phylum level, Proteobacteria (61.34%) was the most dominant group followedby unclassified derived from bacteria (24.74%) and Actinobacteria (12.52%).  Genus levelanalysis revealed the abundance of Rubrobacter, Pseudomonas and Acinetobacter. The studyrevealed that of the total species detected 50.83 per cent were unclassified, stressing theimportance of metagenomics to assess the diversity of endophytes associated with orchid roots.Keywords: Endophyte, orchid, diversityINTRODUCTIONOrchidaceae is one of the largest plant families,including almost 10% of all flowering plant species.Among these, the monopodial epiphytic Phalaenopsisor ‘Moth Orchid is one of the most popular orchidsdue to its ease of production and blooming year-round.The orchid roots are associated with various fungi andendotrophic bacteria (Teixeira et al., 2015). Apartfrom mycorrhizal fungi, previous reports revealed theabundance of endophytic bacteria on the roots of thecultivated tropical orchids of genera Calanthe,Acampe and Dendrobium (Tsavkelova et al., 2003).Orchids are characterized by low survival rate in thegreen house due to the germination under asymbioticconditions in vitro. Generally, endophytes play animportant role in promoting plant growth and yield,suppress pathogens, aid in removing heavy metalcontaminants, solubilize phosphate or contribute tonitrogen assimilation for plants (Hallmann et al., 2006).Over the past decade, our understanding of microbialdiversity and function in complex environments hasincreased significantly, primarily because of theintroduction of next generation sequencing (NGS)(Lozupone and Knight, 2007). The culture-independent,high-throughput sequencing-based community analysisallows us to observe the microbiome associated withthe plants. Since the endophytes have a strong impacton orchids growth, it is very important to study theirrelationships with plant for developing new strategiesfor orchid conservation and better exploitation of theirmedicinal principles.Therefore, in the present study,we employed NGS technology to unveil the culturableand unculturable endophytic bacteria in Phalaenopsisroot to elucidate the microbial plant colonisation patternand evaluate its microbial diversity.The Phalaenopsis plants grown in Sphagnummoss under green house conditions were collected fromthe Department of Pomology and Floriculture, Collegeof Horticulture, Vellanikkara. Samples wereimmediately transferred and processed for furtherstudies. The roots were detached with sterile knifeand washed with sterile distilled water plus a few dropsof Tween-20 and left for 10–15 min to drain. Thesewere then cut into 4–5 pieces (2–3 cm in size). Surfacesterilization was performed by immersing separatelyin 90% ethanol (5 min), followed by sodiumhypochlorite (3%) solution (2 min), and 75% ethanol(3 min). The disinfected roots were rinsed three timesin sterile distilled water. Total genomic DNA wasextracted from the surface sterilized root tissues usingQIAGEN DNeasy plant kit following themanufacturer ’s protocol. Extracted DNA was104suspended in QIAGN elution buffer and stored at -200C. PCR amplification was carried out to amplifyV3 conserved region of 16S rRNA gene sequencesusing the 16S rRNA gene primers (forward primer 5¹-CCTACGGGNGGCWGCAG-3¹ and reverse 5¹-GACT ACHVGGGTATCTA-3¹). The amplicons werepurified and sequenced on the Illumina Miseq platformat Scigenom Pvt. Ltd. Cochin. The FASTQ sequenceswere filtered to remove chimeric sequences andsingletons to obtain preprocessed reads, which wasthen clustered to obtain OTUs. The chloroplastsequences that comprised of almost 97.5 per cent ofthe total reads were removed using QIIME analysis.Further taxonomic annotation of the 301 OTUsobtained were done using QIIME and MG-RAST tools.Total DNA was isolated from the roots ofPhalaenopsis plants and the presence of 16S rRNAgene was confirmed by amplification with universalprimers. Total raw sequencing reads (paired end) of1,96,595 with average sequence length of 151 bp eachwas obtained from Illumina MiSeq™ sequencer.The abundance of major bacterial groups in eachtaxonomic category is given in Table 1. Altogether,10 bacterial phyla were detected and among these,Proteobacteria (61.34%) was the most dominant groupfollowed by unclassified derived from bacteria(24.74%) and Actinobacteria (12.52%). Readsbelonging to Acidobacteria, Bacteriodetes, Chloroflexi,Cyanobacteria, Spirochaetes, Tenericutes, Firmicutesand Bacteriodetes were found to be the other phylawith less than 1 per cent (Fig 1). The higher abundanceof Proteobacteria in the roots of orchids suggests thatmembers of this phylum are particularly well adaptedto colonize inner plant tissues and establish as rootendophytes. The phylum Proteobacteria comprisesseveral species that promote plant growth and act asbiological control agents of different diseases (Bulgarelliet al., 2013). Actinobacteria play specific roles, forinstance, protecting the host plants against insects anddiseases especially by the production of bioactivecompounds. Firmicutes were found to be metabolicallythe most versatile group with production of multipleenzyme activities. Cyanobacteria are photosynthetic;some are capable of fixing nitrogen and others improvesoil-aggregation stability (Issa et al., 2007), a keyaspect of soil conservation. Results of the presentinvestigation are in agreement with the earlier reportson Proteobacteria, Actinobacteria, Firmicutes andBacteriodes being the prominent phyla in the roots oftree peony (Yang et al., 2017).A total of 7 bacterial classes were identifiedand among them Gammaproteobacteria was themost dominant group (41.90%) followed byunclassified (derived from bacteria) (31.35%)Actinobacteria (25.11%) and Bacilli (1.37%)(Fig 2). A total of 17 bacterial orders were detected.The most dominant group was unclassified derivedfrom bacter ia  (35.99%) followed byPseudomonadales (30.69%) and Rubrobacterales16S rRNA gene taxonomic profiling of endophytic bacteriaJ. Hortl. Sci.Vol. 13(1) : 103-107, 2018Fig. 1. Abundance of endophytic bacteria at phylumlevel constructed in MG-RAST with illuminasequencing data set Fig. 2. Abundance of endophyticbacteria at genuslevel constructed in MG-RAST with illuminasequencing data set105Girija  et alJ. Hortl. Sci.Vol. 13(1) : 103-107, 2018Table 1. Abundance of major taxonomic category from phyla to species level ofendophyticbacteriain Phalaenopsisroot10616S rRNA gene taxonomic profiling of endophytic bacteriaJ. Hortl. Sci.Vol. 13(1) : 103-107, 2018(27.47%). Orders Actinomy etales, Bacillales andEnterobacteriales were also present more than oneper cent. Analyses at family level revealed a totalof 22 bacterial families were present in the sample.Major bacterial families present in the sample wereunclassified der ived from bacter ia  (36%),followed by Rubrobacteraceae (27.48%),Pseudomonadaceae (21.68%) and Moraxellaceae(9.01%). The Gamma Proteobacteria includedPseudomonas ,  Pantoea,  Acinetobacter ,Stenotrophomonas and Xanthomonas making itphylogenetically the most diverse group in thecurrent study. Altogether 31 bacterial genera werepresent.  Unclassified der ived from bacter ia(35.99%) was the most dominant group in thepresent study followed by Rubrobacter (27.47%),Pseudomonas (21.67%) and Acinetobacter (9%).Genus Enterobacter and Aneurinibacillus werealso present at more than one per cent abundance.Genus Rubrobacter is well known to be a radiationresistant bacterium.   Genus Pseudomonas canutilize more than 200 compounds as carbon source,can fix atmospheric N and solubilize P. Several ofthe genera isolated in the current study, includingPantoea ,  Pseudomonas ,  Bacil lus andAcinetobacter have been isolated from differentplants and shown to possess plant growth promotingactivit ies (Trivedi et al. ,  2011). It has beenpreviously observed that  in many cases,Pseudomonas and members ofEnterobacteriaceae are abundant in both the soilenvironment and the plant interior (Spiers et al.,2000).  The prevalence of Pseudomonas andBacillus endosymbionts was also reported inAustralian terrestrial orchids (Wilkinson et al.,1994). The study emphasizes on the importance ofmetagenomics to assess the diversity and role ofendophytic microbes in plants.This study extends the knowledge on thecomposition and diversity in the orchid microbialpopulations. Moreover, most of the endophytesobserved in the present study are perhaps goodproducers of bioactive compounds, which canpromote the growth of orchids in seedling stage andalso in ex vitro acclimatization.Bulgarelli D., Schlaeppi, K., Spaepen, S., Ver Lorenand van Themaat E. 2013. Structure andfunctions of the bacterial microbiota of plants.Annu. Rev. Plant Biol, 64: 807-838.Hallmann, J., Berg, G. and Schulz, B. 2006.  Isolationprocedures for endophytic microorganisms.in:B.E. Schulz, C.C. Boyle, T. Sieber (Eds.),Microbial Root Endophytes, Vol. 9, Springer,Berlin Heidelberg 2006, p. 299–319.Issa, O.M., Defarge, C., Le Bissonnais, Y., Marin,B., Duval, O., Bruand, A., D’Acqui, L.P.Nordenberg, S. and Annerman, M., 2007.Effects of the inoculation of cyanobacteriaon the microstructure and the structuralstability of a tropical soil. Plant Soil, 290:209–219Lozupone, C.A. and Knight, R. Global patterns inbacterial diversity. 2007. Proc. Natl. Acad.Sci.,104: 11436–11440.Spiers, A.J., Buckling, A. and Raineythen, P.B.2000.The causes of Pseudomonas diversity.Microbiology,146:2345–2350.Teixeira da Silva J.A., Tsavkelova, E.A., Zeng, S.,Par thibhan,  S.  and Rao M.V. 2015.Symbiotic in vitro  seed propagationof Dendrobium: fungal and bacterial partnersand their influence on plant growth anddevelopment. Planta, 24:21–22.Trivedi, P.  Spann, T. and Wang, N. 2011. Isolationand characterization of beneficial bacteriaassociated with citrus roots in Florida MicrobEcol., 62:324-336REFERENCES107Girija  et alJ. Hortl. Sci.Vol. 13(1) : 103-107, 2018(MS Received 10 August 2017, Revised 03 April 2018, Accepted 30 June 2018)Tsavkelova, E.A., Klimova, Y.S., Cherdyntseva,T.A. and Netrusov, A.I.2006. Microbialproducers of plant growth stimulators andtheir  pr actical use: a  r eview, Appl.Biochem.Microbiol., 42:133–143.Yang, R.,  Liu,  P. and  Ye, W. 2017. Illumina-basedanalysis of endophytic bacterial diversity oftree peony (Paeonia Sect. Moutan) rootsand leaves. Braz.J.Microbiol.,48: 695-705.Wilkinson, K., Dixon, K., Sivasithamparam, K. andGhisalberti, E., 1994. Effect of IAA onsymbiotic germinationof an Australian orchidand its production by orchid-associatedbacteria. Plant Soil, 159: 291–295.

16S rRNA gene taxonomic profiling of endophytic bacteria associated with phylaenopsis roots

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