A New Approach to Analyzing Endophytic Actinobacterial Population in the Roots of Banana Plants (Musa sp., AAA)

Yanyan Zhai, Wenfeng Wang, Hongming Tan, Lixiang Cao

Yanyan Zhai, School of Life Sciences, Sun Yat-sen University, Guangzhou, 510275, China
Wenfeng Wang, School of Environmental Science and Engineering, Sun Yat-sen University, Guangzhou, 510275, China
Hongming Tan, School of Life Sciences, Sun Yat-sen University, Guangzhou, 510275, China
Lixiang Cao, School of Life Sciences, Sun Yat-sen University, Guangzhou, 510275, China

Correspondence to: Lixiang Cao, School of Life Sciences, Sun Yat-sen University, Guangzhou, 510275, China.
Email: lssclx@163.com
Telephone: +86-20-84110238
Fax: +86-20-84036215
Received: April 14, 2016
Revised: June 19, 2016
Accepted: June 21, 2016
Published online: September 14, 2016


AIM: To elucidate the endophytic actinobacterial populations of healthy banana plant roots.

METHODS: A surface sterilization protocol was adopted to remove rhizoplane bacterial DNAs and total DNAs of surface-sterilized roots were purified and analyzed by Illumina-based sequencing.

RESULTS and CONCLUSION: A total of 2152102 bacterial sequences remained with an average length of 448bp, 31952 different actinobacterial OTUs at the 97% similarity level were obtained, and the dominant families were Nocardioidaceae, PseudoNocardiaceae and Nocardiaceae, which accounted for 56.37%, 14.36% and 9.77%, respectively. At genus level, Pseudonocardia (11.83%), Rhodococcus (9.74%), Nocardioides (3.06%) were the most abundant taxa. The results indicated that surface sterilization approach with formaldehyde (36%) and Illumina-based sequencing is suitable for analysis of endophytic actinobacterial populations in banana roots.

Key words: Banana roots; Endophytic actinobacterial; Surface-sterilization; Illumina-based analysis

© 2016 The Authors. Published by ACT Publishing Group Ltd.

Zhai Y, Wang W, Tan H, Cao L. A New Approach to Analyzing Endophytic Actinobacterial Population in the Roots of Banana Plants (Musa sp., AAA). Journal of Biochemistry and Molecular Biology Research 2016; 2(3): 180-184 Available from: URL: http: //www.ghrnet.org/index.php/jbmbr/article/view/1675


Bananas and plantains grown in the tropic and subtropic regions are among the important crops worldwide[1]. Nevertheless, fusarium wilt of banana (Panama disease) is one of the most devastating diseases of banana. It is a major problem throughout most of the banana production regions of the world. Since the Fusarium clamydospores persist in the soil for decades, no cultural or agronomic practices that are useful for the growth susceptible cultivars on infested soils have been identified[1]. No effective chemical method exists to control fusarium wilt of banana; however, biological control using endophytes provides promising perspective for sustainable control of fusarium wilt[2].

Endophytes are thought to be a sub-population of the rhizosphere microbiome, but they also have characteristics distinct from rhizosphere bacteria[3]. The analysis of core Arabidopsis thaliana root microbiome suggested that although various different soil types altered the bacterial endophyte microbiome, actinobacteria were consistently enriched in the endosphere compared with the rhizosphere[4]. Endophytic non-filamentous actinobacteria had been isolated from growing shoot tips of banana[5,6]. Although the healthy-promoting roles of filamentous actinobacteria were widely accepted[7], the diversity of actinobacteria among the banana root microbiota is still poorly understand.

The actinobacteria were bacteria with high G+C content, and they grow slowly than other bacteria, the cultivation methods and E. coli 16S rRNA genes might low estimate the actinobacterial taxa[8]. Deciphering the plant actinobacteriome is critical to identify actinobacteria that can be exploited for improving plant growth and health[9]. More comprehensive information on banana actinobacteriome would be obtained by high-throughput sequencing approaches.

The use of high-throughput sequencing technologies has been widely adopted as they allow identification of thousands to millions of sequences in a sample, revealing the abundances of even rare microbial species[3]. Illumina has fewer errors than 454 sequencing and it could provide a higher phylogenetic resolution than 454 based approaches[10]. The advantage of Illumina to provide 30 times more reads would enable us to perform in depth sequencing of samples in one run, making it an excellent tool for endophytic actinobacteria diversity.

To develop new approach to analyzing endophytic actinobacterial populations in banana roots, a new surface sterilization procedure and new actinobacteria specific primers were designed in the study. The Illumina-based analysis illustrated that the new approach was suitable for analyzing endophytic actinobacterial populations in healthy banana roots.

Material and Methods

Sample Collection

Four to five month-old field grown suckers of healthy banana plants (Musa sp., AAA, Giant Cavendish cv. Baxi,) were collected from a banana plantation in the suburbs of Guangzhou, China. The roots of healthy banana plants in fields without wilting symptoms were used for total DNA extraction. The plants (n = 8) were dug out carefully to ensure that maximal amount of root materials was collected. The root samples were placed in plastic bags and processed after surface sterilization within 4 h of collection.

Surface Sterilization

The root samples were washed with tap water to remove soil particles and sterilized by immersion in 36% formaldehyde solution for 7 min. Then, the sterilized roots were rinsed three times with demineralized sterile water (vortex for 2 min per rinse) to remove the surface sterilization agents.

DNA Extraction

The total DNA was extracted using E.Z.N.A. HP Plant DNA Kit (Omega) and according to the manufacture’s instruction. Total DNA concentration and purity were monitored on 1% agarose gels.

Amplicon Generation and Illumina MiSeq sequencing

The actinobacteria specific primers 243F (5′ - GGATGAGCCCG CGGCCTA - 3′) and A3R (5′ - CCAGCCCCACCTTCGAC - 3′) were used to amplify actinobacteria 16S rRNA gene, and this could produce a 1.21 kb sequence fragment. The PCR reaction mixture (25 µL) contained 1 × PCR buffer (Takara), 200 µM dNTP, 0.2 µM of each primer, 3 mM MgCl2, 2.5 U Taq DNA polymerase (Takara). Thermal cycling conditions were as follows: an initial denaturation at 94ºC for 5 min, followed by 35 cycles at 94ºC for 30 s, 56ºC for 40 s, and 72ºC for 90 s, with a final extension at 72ºC for 10 min. The PCR products were further sequenced with the primers S-D-Bact-0341-b-S-17 (5’- CCTACGGGNGGCWGCAG - 3’) and S-D-Bact-0785-a-A-21 (5’ - GACTACHVGGGTATCTAATCC - 3’) targeting the V3-V4 hyper variable regions of bacterial 16S rRNA genes[11]. Both forward and reverse primers were tagged with adapter, pad and linker sequencing. Each barcode sequence was added to the reverse primer for pooling multiple samples into one run of sequencing. All PCR reactions were performed in a total volume of 30μL containing 15 μL Phusion® High-Fidelity PCR Master Mix (New England Biolabs) and 0.5 units of AccuPrimer TM Taq DNA Polymerase (Life Technologies, USA); 0.2 μM of forward and reverse primers, and 10 ng template DNA. Thermal cycling conditions were as follows: an initial denaturation at 98 ℃ for 1 min, followed by 30 cycles at 98℃ for 10 s, 50℃ for 30 s, and 72℃ C for 60 s, with a final extension at 72℃ for 5 min.

Following amplification, 5 μL of PCR product was used to successful amplification using 1% agarose gel electrophoresis. The triplicate PCR reaction were combined and the pooled mixtures was purified with GeneJET Gel Extraction Kit (Thermo Scientific) and analyzed on an Agilent 2100 Bioanalyzer using High Sensitivity DNA Chips (Agilent Technologies, germany) for size distribution. The sequencing libraries were generated using NEB Next® Ultra™ DNA Library Prep Kit for Illumina (NEB, USA) following manufacturer’s recommendations and index codes were added. The library quality was assessed on the Qubit@ 2.0 Fluorometer (Thermo Scientific) and Agilent Bioanalyzer 2100 system (Agilent Technologies, Germany). Finally, the library was sequenced on an Illumina MiSeq platform at Magigen biotechnology Co. Ltd, Guangzhou, China.

Combination and data preprocessing

Forward and reverse sequences were merged by overlapping paired-end reads using FLASH (V1.2.7, http: //ccb.jhu.edu/software/FLASH/)[12]. All sequences reads with the same tag were assigned to the same sample according to the unique barcodes (raw tags). The raw tags were further strictly filtered by previous methods[13] and the quality of clean tags were detected by Qiime (V1.7.0, http: //qiime.org/index.html)[11], and the low quality tags were removed. The tags with chimera were detected and removed using UCHIME Algorithm, (http: //www.drive5.com/usearch/manual/uchime_algo.html)[14,15]. The effective sequences were then clustered into operational taxonomic units (OTU) at 97% sequence similarity using the UPARSE-OTU and UPARSE-OTUref algorithms of UPARSE software package (Uparse v7.0.1001, http: //drive5.com/uparse/)[16]. Finally, the RDP classifier was used to assign representative sequence to the microbial taxa[17]. Sequence data have been deposited in the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) under the accession number SRP061867.

Statistical analysis

Cluster analysis was preceded by principal component analysis (PCA) using the QIIME software package. QIIME calculates both weighted and unweighted unifrac distance, which are phylogenetic measures of beta diversity[18], the phylogenetic relations among different microbial taxa were further displayed by KRONA[19].


Validation of surface sterilization

To validate the surface sterilization, the sterilized roots were stirred in 10 mL sterile water. The copy numbers of whole bacterial 16S rRNA gene and bacterial 16S rRNA gene segments were further quantified by MPN (most probable number)-PCR and real-time PCR, respectively[20,21]. Results from MPN-PCR illustrated that sterilization by formaldehyde (36%) for 7 min removed 99.99% of rhizoplane bacterial whole 16S rRNA genes. Results from real-time PCR indicated that the rhizoplane 16S rRNA gene copies of roots sterilized with formaldehyde (36%) for 7 min were similar to those in negative controls (without DNA template).

Actinobacterial species richness and diversity

After qualify filtering the raw reads, 2152102 bacterial sequences remained with an average length of 450 bp, 31952 different bacterial OTUs at the 97% similarity level were obtained (Table 1).

The alpha diversity indices calculated from bacterial OTUs of indicated that the banana roots contained more diverse actinobacteria (Table 2).

Bacterial representative sequences of each OTU were classified into the domain bacteria, more than 99.99% of the total data set was belonged to Actinobacteria (Figure 1).

The Actinobacteria was also the most dominant actinobacterial class observed, at 99.08%, and others belong to Thermoleophilia (0.82%) (Figure 2).

The dominant orders were Actinomycetales (99.07%) and Solirubrobacterales (0.82%) (Figure 3).

The Nocardioidaceae was the most domain family observed, at 56.37%, PseudoNocardiaceae was the second most abundant family, at14.36%, others belonged to Nocardiaceae, (9.77%), Microbacteriaceae (3.77%), Dietziaceae (2.67%), Dermabacteraceae (1.35%), Micrococcaceae (1.18%), Micromonosporaceae (1.01%), Mycobacteriaceae (0.91%), Actinosynnemataceae (0.85%), Corynebacteriaceae (0.70%), Kineosporiaceae (0.30%), Cellulomonadaceae (0.10%) and Promicromonosporaceae (0.10%) (Figure 4).

The actinobacterial genera Pseudonocardia, were the most detected bacterial genera in the banana roots, at 11.83%. Other frequently detected generas belonged to Rhodococcus (9.74%), Nocardioides (3.06%), Pimelobacter (2.78%), Dietzia (2.04%), Brachybacterium (1.27%), Microbacterium (1.03%), Mycobacterium (0.91%), Corynebacterium (0.7%), Citricoccus (0.54%), Micrococcus (0.28%), Quadrisphaera (0.27%), Agrococcus (0.27%), Arthrobacter (0.22%) and Leucobacter (0.15%) (Figure 5).

About 64.48 % OTUs belonged to the unidentified actinobacterial taxa, they might be belonged to novel actinobacterial species.


Results from MPN-PCR and real-time PCR illustrated that surface sterilization protocol could remove all the rhizoplane bacterial 16S rRNA genes and the protocol with formaldehyde (36%) for 7 min could be used to remove rhizoplane bacterial whole 16S rRNA genes.

The actinobacteria are rich source of biologically active natural products and are widely distributed in different terrestrial and marine habitats[8], their growth rates were lower than other endophyic bacteria and fungi[7]. The previous culture-dependent and culture-independent methods based on E. coli 16S rRNA gene might underestimate the diversity of endophytic actinobacteria. Pseudonocardia were the most detected bacterial genera in the banana roots, others belonged to Rhodococcus (9.74%), Nocardioides (3.06%). The actinobacterial taxa were not consistent with previous results. Streptomyces was the most frequently isolated genus from surface-sterilized banana roots[7], nevertheless, it can not be isolated or detected in previous studies[1,6,22]. In the study, total 11 reads belonged to Streptomyces or Streptomycetaceae were detected by Illumina-based analysis. Arabidopsis thaliana hosts a genotype-specific core microbiome dominated by Actinobacteria, however, the removal efficiency of rhizosplane bacterial DNA was not demonstrated in these studies[4]. The relative abundance of sequences identified as Streptomyces spp. was possibly biased by the extraction method as these actinobacteria have robust spores[23]. Our results indicated that the proportion of Streptomyces sequences was low among the actinobacterial sequences in banana root. The dominance of actinobacteria in Arabidopsis thaliana roots did not derived from extraction methods, but from the rhizoplane Streptomyces, Other actinobacterial analysis based on 16S rRNA gene library showed that Streptomyces could be detected from wheat or rice roots[24,25]. However, hypochlorites were used to surface sterilize plant roots and the removal efficiency of rhizosplane bacterial DNA was not further demonstrated[26,27]. Probably, some rhizoplane S

treptomyces spp. were misconsidered as endophytic streptomycetes in roots.

In the study, 99.99% of OTUs belonged to actinobacteria and high resolution actinobacteriome were obtained. The strategies of surface sterilization with formaldehyde (36%) and Illumina-based sequencing is suitable for analysis of endophytic actinobacterial populations in further studies.


The authors have not conflicts of interest to declare.


1Rossmann B, Müller H, Smalla K, Mpiira S, Tumuhairwe JB, Staver C, Berg G. Banana-associated microbial communities in Uganda are highly diverse but dominated by Enterobacteriaceae. Applied and Environmental Microbiology 2012; 78: 4933-4941.

2Wang YG, Xia QY, Gu WL, Sun JB, Zhang H, Lu XH, Lu J, Peng M, Zhang X. Isolation of a strong promoter fragment from endophytic Enterobacter cloacae and verification of its promoter activity when its host strain colonizes banana plants. Applied Microbiology and Biotechnology 2012; 93: 1585-1599.

3Turner TR, James EK, Poole PS. The plant microbiome. Genome Biology 2013; 14: 209.

4Lundberg DS, Lebeis SL, Paredes SH, Yourstone S, Gehring J, Malfatti S, Tremblay J, Engelbrektson A, Kunin V, del Rio TG, Edgar RC, Eickhorst T, Ley RE, Hugenholtz P, Tringe SG, Dangl JL. Defining the core Arabidopsis thaliana root microbiome. Nature 2012; 488: 86-90.

5Thomas P, Swarna GK, Patil P,Rawal RD.Ubiquitous presence of normally non-culturable endophytic bacteria in field shoot-tips of banana and their gradual activation to quiescent cultivable form in tissue cultures. Plant Cell, Tissure and Organ Culture 2008a; 93: 39-54.

6Thomas P, Soly TA. Endophytic bacteria associated with growing shoot tips of banana (Musa sp.) cv. Grand Naine and the affinity of endophytes to the host. Microbial Ecology 2009; 58: 952-964.

7Cao LX, Qiu ZQ, You JL, Tan HM, Zhou SN. Isolation and characterization of endophytic streptomycete antagonists of fusarium wilt pathogen from surface-sterilized banana roots. FEMS Microbiology Letters 2005; 247: 147-152.

8Zhang Y, Tan HM, Deng QL,Cao LX. Actinobacterial flora in feces of healthy cottontail rabbits (Sylvilagus auduboni). Probiotics and Antimicrobial Proteins 2015; 7: 9-13.

9Mendes R, Garbeva P&Raaijmakers JM. The rhizosphere microbiome: significance of plant beneficial, plant pathogenic, and human pathogenic microorganisms. FEMS Microbiology Reviews 2013; 37: 634-663.

10Shi Y, Yang H, Zhang T, Sun J,Lou K. Illumina-based analysis of endophytic bacterial diversity and space-time dynamics in sugar beet on the north slope of Tianshan mountain. Applied Microbiology and Biotechnology 2014; 98: 6375-6385.

11Caporaso JG, Lauber CL, Walters WA, Berg-Lyons D, Huntley J, Fierer N, Owens SM, Betley J, Fraser L, Bauer M, Gormley N, Gilbert JA, Smith G, Knight R. Ultra-high-throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms. The ISME journal 2012; 6: 1621-1624.

12Magoč T&Salzberg SL. FLASH: fast length adjustment of short reads to improve genome assemblies. Bioinformatics 2011; 27: 2957-2963.

13Bokulich NA, Subramanian S, Faith JJ, Gevers D, Gordon JI, Knight R, Mills DA, Caporaso JG.Quality-filtering vastly improves diversity estimates from illumine amplicon sequencing. Nature Methods 2013; 10: 57-59.

14Edgar RC, Haas BJ, Clemente JC, Quince C, Knight R. UCHIME improves sensitivity and speed of chimera detection. Bioinformatics 2011; 27: 2194-2200.

15Haas B J, Gevers D, Ear AM., Feldgarden M, Ward DV, Giannoukos G, Ciulla D, Tabbaa D, Highlander SK, Sodergren E, Methé B, DeSantis TZ, Petrosino JF, Knight R, Birren BW. Chimeric 16S rRNA sequence formation and detection in Sanger and 454-pyrosequenced PCR amplicons. Genome Research 2011; 21: 494-504.

16Edgar RC.UPARSE: highly accurate OTU sequences from microbial amplicon reads. Nature methods 2013; 10: 996-998.

17Wang Q, Garrity GM, Tiedje JM, Cole JR. Naïve Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Applied and Environmental Microbiology 2007; 73: 5261-5267.

18Kõljalg U, Nilsson RH, Abarenkov K, Tedersoo L, Taylor AFS, Bahram M et al. Towards a unified paradigm for sequence-based identification of fungi. Molecular ecology 2013; 22: 5271-5277.

19Ondov BD, Bergman NH, Phillippy AM. Interactive metagenomic visualization in a Web browser. BMC bioinformatics 2011; 12: 385.

20Nadkarni MA, Martin FE, Jacques NA, Hunter N. Determination of bacterial load by real-time PCR using a broad-range (universal) probe and primers set. Microbiology 2002; 148: 257-266.

21Luan XY, Chen JX, Liu Y, Li Y, Jia JT, Liu R, Zhang XH. Rapid Quantitative Detection of Vibrio parahaemolyticus in Seafood by MPN-PCR. Current Microbiology 2008; 3: 218-221.

22Thomas P, Swarna GK, Roy PK, Patil P. Identification of culturable and originally non-culturable endophytic bacteria isolated from shoot tip cultures of banana cv. Grand Naine. Plant Cell, Tissure and Organ Culture 2008b; 93: 55-63.

23Hirsch PR, Mauchline TH. Who’s who in the plant root microbiome? Nature Biotechnology 2012; 30: 961-962.

24Conn VM, Franco CMM. Analysis of the endophytic actinobacterial population in the roots of wheat (Triticum aestivum L.) by terminal restriction fragment length polymorphism and sequencing of 16S rRNA clones. Applied and Environmental Microbiology 2004; 70: 1787-1794.

25Tian XL, Cao LX, Tan HM, Han WQ, Chen M, Liu YH,Zhou SN. Diversity of cultivated and uncultivated actinobacterial endophytes in the stems and roots of rice. Microbial Ecology 2007; 53: 700-707.

26Sessitsch A, Reiter B, Pfeifer U, Wilhelm E. Cultivation-independent population analysis of bacterial endophytes in three potato varieties based on eubacterial and Actinomycetes-specific PCR of 16S rRNA genes. FEMS Microbiology Ecology 2002; 39: 23-32.

27Sessitsch A, Hardoim P, Döring J, Weilharter A, Krause A, Woyke T, Mitter B, Hauberg-Lotte L, Friedrich F, Rahalkar M, Hurek T, Sarkar A, Bodrossy L, van Overbeek L, Brar D, van Elsas JD,Reinhold-Hurek B. Functional characteristics of an endophyte community colonizing rice roots as revealed by metagenomic analysis. Molecular Plant-Microbe Interactions 2012; 25: 28-36.

Peer reviewer: MKyoung Sik Park, Assistant Professor, Department of Biomedical Science, Cheongju University, 298 Daesung-ro, Chungwon-gu, Cheongju-si, Chungbuk 360-764, KOREA.


  • There are currently no refbacks.