Abstract
Endophytes confer unique ecological advantages to their host plants. In this study, we have characterized the diversity of endophytic consortia associated with the GPU-28 (GPU) and Udurumallige (UM) finger millet varieties, which are resistant and susceptible to the blast disease, respectively. Whole genome metagenome sequencing of GPU and UM helped to identify 1029 species (includes obligate endophytes) of microbiota. Among them, 385 and 357 species were unique to GPU and UM, respectively. Remaining 287 species were common to both the varieties. Actinobacteria and other plant-growth promoting bacteria were abundant in GPU as compared to UM. Functional annotation of genes predicted from genomes of endophytes associated with GPU variety showed that many genes had functional role in stress response, secondary metabolism, aromatic compounds, glutathione, and cysteine synthesis pathways as compared to UM. Based on in vitro and in planta studies, Bacillus cereus and Paenibacillus spp. were found to be effective in suppressing the growth of blast disease pathogen Magnaporthe grisea (strain MG03). In the future, these strains could serve as potential biocontrol agents to reduce the incidence of blast disease in finger millet crop.
Similar content being viewed by others
Availability of sequenced data
The raw fastq files for the metagenome data were uploaded in NCBI SRA database with accession number SRP105033.
References
Álvarez C, Bermúdez MÁ, Romero LC et al (2012) Cysteine homeostasis plays an essential role in plant immunity. New Phytol 193:165–177. https://doi.org/10.1111/j.1469-8137.2011.03889.x
Amruta N, Prasanna Kumar MK, Puneeth ME et al (2018) Exploring the potentiality of novel rhizospheric bacterial strains against the rice blast fungus Magnaporthe oryzae. Plant Pathol J 34:126–138. https://doi.org/10.5423/PPJ.OA.11.2017.0242
Anitha A, Rebeeth M (2009) In vitro antifungal activity of Streptomyces griseus against phytopathogenic fungi of tomato field. Acad J Plant Sci 2:119–123
Aranega-Bou P, de la OLeyva M, Finiti I et al (2014) Priming of plant resistance by natural compounds. Hexanoic acid as a model. Front Plant Sci 5:1–12. https://doi.org/10.3389/fpls.2014.00488
Arndt D, Xia J, Liu Y et al (2012) METAGENassist: a comprehensive web server for comparative metagenomics. Nucl Acids Res 40:88–95. https://doi.org/10.1093/nar/gks497
Arnold AE, Mejia LC, Kyllo D et al (2003) Fungal endophytes limit pathogen damage in a tropical tree. Proc Natl Acad Sci 100:15649–15654. https://doi.org/10.1073/pnas.2533483100
Benfradj N, Tounsi S, Boughalleb-MHamdi N (2016) In-vitro evaluation of Antagonists and Fungicides in Controlling Citrus Gummosis Caused by Phytophthora, Phytopythium and Pythium species in Tunisia. Br Microbiol Res J 16:1–14. https://doi.org/10.9734/BMRJ/2016/27247
Bhatt D, Negi M, Sharma P et al (2011) Responses to drought induced oxidative stress in five finger millet varieties differing in their geographical distribution. Physiol Mol Biol Plants 17:347–353. https://doi.org/10.1007/s12298-011-0084-4
Boisvert S, Raymond F, Godzaridis É et al (2012) Ray Meta: scalable de novo metagenome assembly and profiling. Genome Biol. https://doi.org/10.1186/gb-2012-13-12-r122
Bolwerk A, Lagopodi AL, Lugtenberg BJJ, Bloemberg GV (2005) Visualization of interactions between a pathogenic and a beneficial Fusarium strain during biocontrol of tomato foot and root rot. Mol Plant-Microbe Interact 18:710–721. https://doi.org/10.1094/MPMI-18-0710
Braun G, Hirsch U (1992) Communities of parasitic microfungi. In: Winterhoff W (ed) Fungi in vegetation science. Springer, Dordrecht, pp 225–250
Buchfink B, Xie C, Huson DH (2015) Fast and sensitive protein alignment using DIAMOND. Nat Methods 12:59–60. https://doi.org/10.1038/nmeth.3176
Budnik LT, Baur X (2009) The assessment of environmental and occupational exposure to hazardous substances by biomonitoring. Dtsch Arztebl Int 106:91–97. https://doi.org/10.3238/arztebl.2009.0091
Chaturvedi H, Singh V (2016) Potential of bacterial endophytes as plant growth promoting factors. J Plant Pathol Microbiol. https://doi.org/10.4172/2157-7471.1000376
Chiang KS, Liu HI, Bock CH (2017) A discussion on disease severity index values. Part I: warning on inherent errors and suggestions to maximise accuracy. Ann Appl Biol 171:139–154. https://doi.org/10.1111/aab.12362
Doran JL, Leskiw BK, Aippersbach S, Jensen SE (1990) Isolation and characterization of a beta-lactamase-inhibitory protein from Streptomyces clavuligerus and cloning and analysis of the corresponding gene. J Bacteriol 172:4909–4918
Dordas C (2008) Role of nutrients in controlling plant diseases in sustainable agriculture. A review. Agron Sustain Dev 28:33–46. https://doi.org/10.1051/agro:2007051
Dubreuil-Maurizi C, Poinssot B (2012) Role of glutathione in plant signaling under biotic stress. Plant Signal, Behav
Dutta D, Puzari KC, Gogoi R, Dutta P (2014) Endophytes: exploitation as a tool in plant protection. Braz Arch Biol Technol 57:621–629
Esele JP (2002) Diseases of finger millet: a global overview. In: Murphy LJ, Montorsi F (eds) Sorghum and finger millet diseases. Iowa State Press, Iowa, pp 19–26
Etesami H, Alikhani HA (2016) Suppression of the fungal pathogen Magnaporthe grisea by Stenotrophomonas maltophilia, a seed-borne rice (Oryza sativa L.) endophytic bacterium. Arch Agron Soil Sci 62:1271–1284. https://doi.org/10.1080/03650340.2016.1139087
Fiedler H, Bruntner C, Riedlinger J et al (2008) ORIGINAL ARTICLE Proximicin A, B and C, Novel aminofuran antibiotic and anticancer compounds isolated from marine strains of the Actinomycete Verrucosispora. J Antibiot 61:158–163
Frandberg E, Petersson C, Lundgren LN, Schnurer J (2000) Streptomyces halstedii K122 produces the antifungal compounds bafilomycin B1 and C1. Can J Microbiol 46:753–758. https://doi.org/10.1139/w00-050
Gayathri P, Muralikrishnan V (2013) Isolation and characterization of endophytic actinomycetes from mangrove plant for antimicrobial activity. Int J Curr Microbiol Appl Sci 2:78–89
Gerth K, Bedorf N, Irschik H et al (1994) The soraphens: a family of novel antifungal compounds from Sorangium cellulosum (Myxobacteria). I. Soraphen A1 alpha: fermentation, isolation, biological properties. J Antibiot (Tokyo) 47:23–31. https://doi.org/10.7164/antibiotics.47.23
Gerth K, Schummer D, Höfle G, Irschik H, Reichenbach H (1995a) Ratjadon: a new antifungal compound from Sorangium cellulosum (Myxobacteria) production, physico-chemical and biological properties. J Antibiot (Tokyo) 48:973–976. https://doi.org/10.7164/antibiotics.48.973
Gerth K, Washausen P, Hfletf G et al (1995b) The jerangolids : a family of newantifungal compounds from Sorangium cellulosum (Myxobacteria) production, physico-chemical and biological properties of jerangolid A1. Optim Antibiot Product 49:71–75
Gerth K, Bedorf N, Hflen G, Reichenbach H (1996) Epothilons A and B: antifungal and cytotoxic compounds from Sorangium cellulosum (Myxobacteria) production. Physico-chem Biol Prop 1:560–563
Ghanta S, Chattopadhyay S (2011) Glutathione as a signaling molecule another challenge to pathogens. Plant Signal Behav 6:783–788. https://doi.org/10.4161/psb.6.6.15147
Gomez KA, Gomez KA, Gomez AA (1984) Statistical procedures for agricultural research. Wiley, New York
Gond SK, Bergen MS, Torres MS, White JF (2015) Endophytic Bacillus spp. produce antifungal lipopeptides and induce host defence gene expression in maize. Microbiol Res 172:79–87. https://doi.org/10.1016/j.micres.2014.11.004
Grosch R, Scherwinski K, Lottmann J et al (2006) Fungal antagonists of the plant pathogen Rhizoctonia solani: selection, control efficacy and influence on the indigenous microbial community. Mycol Res 110:1464–1474. https://doi.org/10.1016/j.mycres.2006.09.014
Gullner G, Zechmann B (2017) Glutathione in plant growth, development, and stress tolerance. Springer, Cham. https://doi.org/10.1007/978-3-319-66682-2
Gunatilaka AAL (2006) Natural products from plant-associated microorganisms: distribution, structural diversity, bioactivity, and implications of their occurrence. J Nat Prod 69:509–526. https://doi.org/10.1021/np058128n
Handelsman J (2005) Metagenomics: application of genomics to uncultured microorganisms. Microbiol Mol Biol Rev 69:195. https://doi.org/10.1128/MMBR.69.1.195.2005
Huang W, Liu X, Zhou X, et al (2018) Bacillus cereus HS24 suppresses conidia germination of Magnaporthe oryzae by inhibiting the Ca2+ signaling pathway. http://dx.doi.org/10.1101/310516
Hung PQ, Annapurna K (2004) Isolation and characterization of endophytic bacteria in soybean (Glycine Sp.). Omonrice 101:92–101
Huson DH, Mitra S, Ruscheweyh H et al (2011) Integrative analysis of environmental sequences using MEGAN4. Genome Res 21:1552–1560. https://doi.org/10.1101/gr.120618.111.Freely
Hwang BK, Lee JY, Kim BS, Moon SS (1996) Isolation, structure elucidation, and antifungal activity of a manumycin-type antibiotic from Streptomyces flaveus. J Agric Food Chem 44:3653–3657. https://doi.org/10.1021/jf960084o
Jha Y, Subramanian RB (2013) Root associated bacteria from the local variety of rice GJ-17 antagonized the growth of Magnaporthe grisea. Adv Biores 4(1):70–77
Jogaiah S, Kurjogi M, Govind SR et al (2016) Isolation and evaluation of proteolytic actinomycete isolates as novel inducers of pearl millet downy mildew disease protection. Sci Rep 6:1–7. https://doi.org/10.1038/srep30789
Kanehisa M, Goto S, Sato Y et al (2012) KEGG for integration and interpretation of large-scale molecular data sets. Nucl Acids Res 40:109–114. https://doi.org/10.1093/nar/gkr988
Knuat P, Reichenbach H (2000) On the mechanism of action of the myxobacterial fungicide ambruticin. J Antibiot (Tokyo) 53:1182–1190. https://doi.org/10.7164/antibiotics.53.1182
Ladeuze S, Lentz N, Delbrassinne L et al (2011) Antifungal activity displayed by cereulide, the emetic toxin produced by Bacillus cereus. Appl Environ Microbiol 77:2555–2558. https://doi.org/10.1128/AEM.02519-10
Lee CH, Kim BJ, Choi GJ et al (2002) Streptomyces with antifungal activity against rice blast causing fungus, Magnaporthe grisea. J Microbiol Biotechnol 12:1026–1028
Lee JY, Moon SS, Yun BS et al (2004) Thiobutacin, a novel antifungal and antioomycete antibiotic from Lechevalieria aerocolonigenes. J Nat Prod 67:2076–2078. https://doi.org/10.1021/np049786v
Lim SM, Yoon M, Choi GJ et al (2017) Diffusible and volatile antifungal compounds produced by an antagonistic Bacillus velezensis G341 against various phytopathogenic fungi. Plant Pathol J 33:488–498. https://doi.org/10.5423/PPJ.OA.04.2017.0073
Liu CH, Chen X, Liu TT et al (2007) Study of the antifungal activity of Acinetobacter baumannii LCH001 in vitro and identification of its antifungal components. Appl Microbiol Biotechnol 76:459–466. https://doi.org/10.1007/s00253-007-1010-0
Llorens E, García-Agustín P, Lapeña L et al (2017) Advances in induced resistance by natural compounds: towards new options for woody crop protection. Sci Agric 74:90–100. https://doi.org/10.1590/1678-992x-2016-0012
Luckner M (2013) Secondary metabolism in microorganisms, plants and animals. Springer Science & Business Media, Berlin
Ma GBZ, Xia Z, Wu S (2009) Inhibiting effect of seven marine actinomycete strains against vegetable pathogenic microorganisms. Crops 5:3–9
Madhurama G, Sonam D, Urmil PG, Ravindra NK (2014) Diversity and biopotential of endophytic actinomycetes from three medicinal plants in India. Afric J Microbiol Res 8:184–191. https://doi.org/10.5897/AJMR2012.2452
Mahesh H, Meghana S, Shailaja H et al (2016) Acquisition of the grasshopper retro transposon by rice Magnaporthe isolates indicates a dynamic gene flow between rice and non-rice Magnaporthe population. J Pathol Microbiol 1(2):1011
Mander P, Cho SS, Choi YH et al (2016) Purification and characterization of chitinase showing antifungal and biodegradation properties obtained from Streptomyces anulatus CS242. Arch Pharm Res 39:878–886. https://doi.org/10.1007/s12272-016-0747-3
Martin M (2011) Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet J 17:10
Moller Bahnweg G, Sandermann H, Geiger HHEM (1992) A simple and efficient protocol for isolation of high molecular weight DNA from filamentous fungi, fruit bodies, and infected plant tissues. Nucl Acids Res 20:6115–6116
Mousa WK, Raizada MN (2013) The diversity of anti-microbial secondary metabolites produced by fungal endophytes: an interdisciplinary perspective. Front Microbiol 4:1–18. https://doi.org/10.3389/fmicb.2013.00065
Nagaraja A, Gowda J, Krishnappa M, Gowda KTK (2008) GPU 28: a finger millet variety with durable blast resistance. J Mycopathol Res 46:109–111
Netam RS, Tiwari RK, Bahadur ANDS (2014) In vitro and in vivo efficacy of fungicides against Pyricularia grisea causing finger millet blast disease. Int J Plant Prot 7:137–142
Ningthoujam DS, Kshetri P, Sanasam S, Nimaichand S (2009) Screening, identification of best producers and optimization of extracellular proteases from moderately halophilic alkalithermotolerant indigenous actinomycetes. World Appl Sci J 7:907–916
Noguchi H, Taniguchi T, Itoh T (2008) Meta gene annotator: detecting species-specific patterns of ribosomal binding site for precise gene prediction in anonymous prokaryotic and phage genomes. DNA Res 15:387–396. https://doi.org/10.1093/dnares/dsn027
Overbeek R, Olson R, Pusch GD et al (2014) The SEED and the Rapid Annotation of microbial genomes using Subsystems Technology (RAST). Nucl Acids 42:206–214. https://doi.org/10.1093/nar/gkt1226
Pandey SS, Singh S, Pandey H et al (2018) Endophytes of Withania somnifera modulate in planta content and the site of withanolide biosynthesis. Sci Rep 20:1–19. https://doi.org/10.1038/s41598-018-23716-5
Parks DH, Beiko RG (2010) Identifying biologically relevant differences between metagenomic communities. Bioinformatics 26:715–721. https://doi.org/10.1093/bioinformatics/btq041
Paul AK, Banerjee AK (1983) A new antifungal antibiotic produced by Streptomyces galbus. Folia Microbiol (Praha) 28:386–396. https://doi.org/10.1007/BF02879488
Rabeeth M, Anitha A, Srikanth G (2011) Purification of an antifungal endochitinase from a potential biocontrol agent Streptomyces griseus. Pak J Biol Sci 14:788–797. https://doi.org/10.3923/pjbs.2011.788.797
Ravin NV, Mardanov AV, Skryabin KG (2015) Metagenomics as a tool for the investigation of uncultured microorganisms. Russ J Genet 51:431–439. https://doi.org/10.1134/S1022795415050063
Rodriguez RJ, Henson J, Van Volkenburgh E et al (2008) Stress tolerance in plants via habitat-adapted symbiosis. ISME J 2:404–416. https://doi.org/10.1038/ismej.2007.106
Romero LC, Aroca MÁ, Laureano-Marín AM et al (2014) Cysteine and cysteine-related signaling pathways in Arabidopsis thaliana. Mol Plant 7:264–276. https://doi.org/10.1093/mp/sst168
Sastri BN (1950) The Wealth of India. A dictionary of indian raw materials and industrial products. Raw materials. Wealth India A Dict Indian Raw Mater Ind Prod III:160–166
Schloss PD, Handelsman J (2005) Metagenomics for studying unculturable microorganisms: cutting the Gordian knot. Genome Biol 6:6–9. https://doi.org/10.1186/gb-2005-6-8-229
Schulz B, Guske S, Dammann U, Boyle C (1998) Endophyte-host interactions. II. Defining symbiosis of the endophyte-host interaction. Symbiosis Phila Pa (USA) 25:213–227
Selim HMM, Gomaa NM, Essa AMM (2016) Antagonistic effect of endophytic bacteria against some phytopathogens. Egypt J Bot 56:613–626
Sengupta S, Ganguli S, Singh PK (2017) Metagenome analysis of the root endophytic microbial community of Indian rice (O. sativa L.). Genom Data 12:41–43. https://doi.org/10.1016/j.gdata.2017.02.010
Sessitsch A, Hardoim P, Döring J et al (2012) Functional characteristics of an endophyte community colonizing rice roots as revealed by metagenomic analysis. Mol Plant-Microbe Interact 25:28–36. https://doi.org/10.1094/MPMI-08-11-0204
Shankar M, Kurtböke DI, Gillespie-Sasse LMJ et al (1994) Possible roles of competition for thiamine, production of inhibitory compounds, and hyphal interactions in suppression of the take-all fungus by a sterile red fungus. Can J Microbiol 40:478–483. https://doi.org/10.1139/m94-077
Shirke MD, Mahesh HB, Gowda M (2016) Genome-wide comparison of Magnaporthe species reveals a host-specific pattern of secretory proteins and transposable elements. PLoS One 11:1–19. https://doi.org/10.1371/journal.pone.0162458
Singh N, Varma A (2015) Antagonistic Activity of Siderophore Producing Rhizobacteria Isolated from the Semi-Arid Regions of Southern India. Int J Curr Microbiol App Sci 4(9):501–510
Smith GE (1957) Inhibition of Fusarium oxysporum f. lycopersici by a species of Micromonospora isolated from tomato. Phytopathology 47:429–432
Sziderics AH, Rasche F, Trognitz F et al (2007) Bacterial endophytes contribute to abiotic stress adaptation in pepper plants (Capsicum annuum L.). Can J Microbiol 53:1195–1202. https://doi.org/10.1139/W07-082
Tokpah DP, Li H, Newmah JT et al (2017) Biological control of potential antagonistic bacteria isolates to restrict Magnaporthe grisea infection on rice. Afr J Microbiol Res 11:1108–1119. https://doi.org/10.5897/AJMR2017.8562
Upson R, Read DJ, Newsham KK (2009) Nitrogen form influences the response of Deschampsia antarctica to dark septate root endophytes. Mycorrhiza 20:1–11. https://doi.org/10.1007/s00572-009-0260-3
Van Minh N, Woo EE, Kim JY et al (2015) Antifungal substances from Streptomyces sp. A3265 antagonistic to plant pathogenic fungi. Mycobiology 43:333–338. https://doi.org/10.5941/MYCO.2015.43.3.333
Verma SC, Ladha JK, Tripathi AK (2001) Evaluation of plant growth promoting and colonization ability of endophytic diazotrophs from deep water rice. J Biotechnol 91:127–141. https://doi.org/10.1016/S0168-1656(01)00333-9
Vetriventhan M, Upadhyaya HD, Dwivedi SL et al (2015) Finger and foxtail millets. Elsevier Inc., Amsterdam
Vincent JM (1947) Distortion of fungal hyphae in the presence of certain inhibitors. Nature 159:850
Vurukonda SSKP, Giovanardi D, Stefani E (2018) Plant growth promoting and biocontrol activity of Streptomyces spp. As endophytes. Int J Mol Sci. https://doi.org/10.3390/ijms19040952
Waller F, Achatz B, Baltruschat H et al (2005) The endophytic fungus Piriformospora indica reprograms barley to salt-stress tolerance, disease resistance, and higher yield. Proc Natl Acad Sci 102:13386–13391. https://doi.org/10.1073/pnas.0504423102
White TJ, Bruns T, Lee S, Taylor JL (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. Academic Press, San Diego
Xu SJ, Hong SJ, Choi W, Kim BS (2014) Antifungal activity of Paenibacillus kribbensis strain T-9 isolated from soils against several plant pathogenic fungi. Plant Pathol J 30:102–108. https://doi.org/10.5423/PPJ.OA.05.2013.0052
Yeop LJ, Sherman DH, Hwang BK (2008) In vitro antimicrobial and in vivo antioomycete activities of the novel antibiotic thiobutacin Pest Management Science: formerly. Pestic Sci 64(2):172–177
Yoshii A, Moriyama H, Fukuhara T (2012) The novel kasugamycin 2′-N-acetyltransferase gene aac(2′)-IIa, carried by the IncP island, confers kasugamycin resistance to rice-pathogenic bacteria. Appl Env Microbiol 78:5555–5564. https://doi.org/10.1128/AEM.01155-12
Author information
Authors and Affiliations
Contributions
HBM and MKP conceived and conceptualized the study. HBM and RUD collected the plant samples and isolated genomic DNA. RUD and KT performed the in vitro experiments. RCP analyzed the metagenome data. HBM guided metagenome data analysis. MEP assisted in maintaining cultures of endophytes and sequencing. HBM, MKP and BK wrote the manuscript. HBM, KSN and GVB edited the manuscript. KSN, KB and BP contributed in preparing the figures. All authors read and approved the final manuscript for publication.
Corresponding authors
Ethics declarations
Conflict of interest
Author Rajadurai, R. C. was employed by company AgriGenome Laboratory. All other authors declare no competing interests.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Prasannakumar, M.K., Mahesh, H.B., Desai, R.U. et al. Metagenome sequencing of fingermillet-associated microbial consortia provides insights into structural and functional diversity of endophytes. 3 Biotech 10, 15 (2020). https://doi.org/10.1007/s13205-019-2013-0
Received:
Accepted:
Published:
DOI: https://doi.org/10.1007/s13205-019-2013-0