Plant Growth Regulation

, Volume 86, Issue 2, pp 287–296 | Cite as

Effects of co-inoculation of two different plant growth-promoting bacteria on duckweed

  • Yusuke Yamakawa
  • Rahul Jog
  • Masaaki MorikawaEmail author
Original paper


Aseptic Lemna minor was soaked for 4 h in pond water where wild L. minor was naturally flourishing. Seven of the eight surface-colonizing bacterial strains were found capable of promoting the growth of L. minor. This high appearance of plant growth-promoting bacteria (PGPB) suggests that association of environmental bacteria is generally beneficial rather than harmful for host plants. One of the PGPB, Pseudomonas sp. Ps6, enhanced the growth of L. minor by 2–2.5-fold in 10 days. This activity was higher than that previously reported for Acinetobacter calcoaceticus P23, which enhanced growth of L. minor by 1.5–2-fold. Ps6 mostly adhered to and colonized the root rather than the frond, a leaf-like structure of duckweed where P23 preferentially adheres. It was expected that these two strains can share niches, coexist, and enhance the growth of duckweed additively upon co-inoculation. However, contrary to expectation, the growth of L. minor was enhanced by only 2.3-fold by co-inoculation of these two bacteria. P23 lowered the initial adhesion of Ps6 cells by 98.2% on the fronds and by 79.5% on the roots. However, initial adhesion of P23 cells to the roots increased dramatically, by 47.2-fold, following co-inoculation with Ps6. However, the number of P23 cells decreased dramatically to 0.7% on the root and to 3.6% on the frond after 10 days, whereas Ps6 cells increased by 12.5-fold on the frond and kept 69% on the root, thereby eventually restoring the population on the plant surfaces. Because duckweed is the fastest growing vascular plant and it is easy to grow an aseptic and axenic plant, the duckweed/bacteria co-culture system will be a model platform for studying multiple interactions among host plants and the associated bacteria.


Lemna minor Plant growth-promoting bacteria Acinetobacter Pseudomonas Three-way symbiosis 



Plant growth-promoting bacteria



We are thankful to Dr. Kyoko Miwa (Hokkaido University) for her critical reading of our manuscript and a number of helpful suggestions. This study was supported by the Advanced Low Carbon Technology Research and Development Program (ALCA) of the Japan Science and Technology Agency (JST) Grant Number JPMJAL1108, Kobayashi International Scholarship Foundation, and Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Number 16K14844.

Author contributions

YY, RJ, and MM conceived and designed the research. YY performed most of the experiments with the help of RJ. YY and MM interpreted the data and wrote the manuscript. RJ revised the manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


  1. Adesemoye AO, Torbert HA, Kloepper JW (2009) Plant growth-promoting rhizobacteria allows reduced application rates of chemical fertilizer. Microb Ecol 58:921–929CrossRefPubMedGoogle Scholar
  2. Appenroth K-J, Ziegler P, Sree KS (2016) Duckweed as a model organism for investigating plant-microbe interactions in an aquatic environment and its applications. Endocytobiosis Cell Res 27:94–106Google Scholar
  3. Berg G, Rybakova D, Grube M, Köberl M (2016) The plant microbiome explored: implications for experimental botany. J Exp Bot 67:995–1002CrossRefPubMedGoogle Scholar
  4. Bhargava N, Sharma P, Capalash N (2012) N-acyl homoserine lactone mediated interspecies interactions between A. baumannii and P. aeruginosa. Biofouling 28:813–822CrossRefPubMedGoogle Scholar
  5. Chang C, Bowman JL, Meyerowitz EM (2016) Field guide to plant model systems. Cell 167:325–339CrossRefPubMedCentralPubMedGoogle Scholar
  6. Cheng JJ, Stomp A-M (2009) Growing duckweed to recover nutrients from wastewaters and for production of fuel ethanol and animal feed. Clean 37:17–26Google Scholar
  7. Erturk Y, Ercisli S, Haznedar A, Cakmakci R (2010) Effects of plant growth promoting rhizobacteria (PGPR) on rooting and root growth of kiwifruit (Actinidia deliciosa) stem cuttings. Biol Res 43:91–98CrossRefPubMedCentralPubMedGoogle Scholar
  8. Gordon SA, Weber RP (1951) Colorimetric estimation of indoleacetic acid. Plant Physiol 26:192–195CrossRefPubMedCentralPubMedGoogle Scholar
  9. Haas D, Blumer C, Keel C (2000) Biocontrol ability of fluorescent pseudomonads genetically dissected: importance of positive feedback regulation. Curr Opin Biotechnol 11:290–297CrossRefPubMedCentralPubMedGoogle Scholar
  10. Idris SE, Iglesias DJ, Talon M, Borriss R (2007) Tryptophan-dependent production of indole-3-acetic acid (IAA) affects level of plant growth promotion by Bacillus amyloliquefaciens FZB42. Mol Plant-Microb Interact 20:619–626CrossRefGoogle Scholar
  11. Innerebner G, Knief C, Vorholt JA (2011) Protection of Arabidopsis thaliana against leaf-pathogenic Pseudomonas syringae by Sphingomonas in a controlled model system. Appl Environ Microbiol 77:3202–3210CrossRefPubMedCentralPubMedGoogle Scholar
  12. Ishizawa H, Kuroda M, Morikawa M, Ike M (2017a) Evaluation of environmental bacterial communities as a factor affecting the growth of duckweed Lemna minor. Biotechnol Biofuels 10:62CrossRefPubMedCentralPubMedGoogle Scholar
  13. Ishizawa H, Kuroda M, Morikawa M, Ike M (2017b) Differential oxidative and antioxidative response of duckweed Lemna minor toward plant growth promoting/inhibiting bacteria. Plant Physiol Biochem 118:667–673CrossRefPubMedGoogle Scholar
  14. Keel C, Schnider U, Maurhofer M, Voisard C, Laville J, Burger U, Wirthner P, Haas D, Défago G (1992) Suppression of root diseases by Pseudomonas fluorescens CHA0: importance of the bacterial secondary metabolite 2,4-diacetylphloroglucinol. Mol Plant-Microb Interact 5:4–13CrossRefGoogle Scholar
  15. Kessler RW, Weiss A, Kuegler S, Hermes C, Wichard T (2018) Macroalgal–bacterial interactions: role of dimethylsulfoniopropionate in microbial gardening by Ulva (Chlorophyta). Mol Ecol 27:1808–1819CrossRefPubMedGoogle Scholar
  16. Körner S, Vermaatb JE, Veenstrac S (2003) The capacity of duckweed to treat wastewater. J Environ Qual 32:1583–1590CrossRefPubMedGoogle Scholar
  17. Kumar S, Stecher G, Tamura K (2016) MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 33:1870–1874CrossRefGoogle Scholar
  18. Ma L-S, Hachani A, Lin J-S, Filloux A, Lai E-M (2014) Agrobacterium tumefaciens deploys a superfamily of Type VI secretion DNase effectors as weapons for interbacterial competition in planta. Cell Host Microbe 16:94–104CrossRefPubMedCentralPubMedGoogle Scholar
  19. Mikkelsen H, Sivaneson M, Filloux A (2011) Key two-component regulatory systems that control biofilm formation in Pseudomonas aeruginosa. Environ Microbiol 13:1666–1681CrossRefPubMedGoogle Scholar
  20. Morikawa M (2006) Beneficial biofilm formation by industrial bacteria Bacillus subtilis and related species. J Biosci Bioeng 101:1–8CrossRefPubMedCentralPubMedGoogle Scholar
  21. National Agricultural Statistics Service (2011) Agricultural statistics 2011. United States Government Printing Office, Washington, DC. ISBN 978-0-16-090545-2Google Scholar
  22. Okada M, Muranaka T, Ito S, Oyama T (2017) Synchrony of plant cellular circadian clocks with heterogeneous properties under light/dark cycles. Sci Rep 7:317CrossRefPubMedCentralPubMedGoogle Scholar
  23. Patil SV, Jayamohan NS, Kumudini BS (2016) Strategic assessment of multiple plant growth promotion traits for shortlisting of fluorescent Pseudomonas spp. and seed priming against ragi blast disease. Plant Growth Regul 80:47–58CrossRefGoogle Scholar
  24. Preston GM (2004) Plant perceptions of plant growth-promoting Pseudomonas. Philos Trans R Soc Lond B 359:907–918CrossRefGoogle Scholar
  25. Rivas R, Abril A, Trujillo ME, Velázquez E (2004) Sphingomonas phyllosphaerae sp. nov., from the phyllosphere of Acacia caven in Argentina. Int J Syst Evol Microbiol 54:2147–2150CrossRefPubMedCentralPubMedGoogle Scholar
  26. Rudrappa T, Czymmek KJ, Paré PW, Bais HP (2008) Root-secreted malic acid recruits beneficial soil bacteria. Plant Physiol 148:1547–1556CrossRefPubMedCentralPubMedGoogle Scholar
  27. Schwyn B, Neilands JB (1987) Universal chemical assay for the detection and determination of siderophores. Anal Biochem 160:47–56CrossRefPubMedGoogle Scholar
  28. Shimada K, Itoh Y, Washio K, Morikawa M (2012) Efficacy of forming biofilms by naphthalene degrading Pseudomonas stutzeri T102 toward bioremediation technology and its molecular mechanisms. Chemosphere 87:226–233CrossRefPubMedCentralPubMedGoogle Scholar
  29. Sundara Rao WVB, Sinha MK (1963) Phosphate dissolving organisms in the soil and rhizosphere. Ind J Agric Sci 33:272–278Google Scholar
  30. Suzuki W, Sugawara M, Miwa K, Morikawa M (2014) Plant growth-promoting bacterium Acinetobacter calcoaceticus P23 increases the chlorophyll content of the monocot Lemna minor (duckweed) and the dicot Lactuca sativa (lettuce). J Biosci Bioeng 118:41–44CrossRefPubMedGoogle Scholar
  31. Toyama T, Tanaka Y, Morikawa M, Mori K (2017a) Comprehensive evaluation of nitrogen removal rate and biomass, ethanol, and methane production yields by combination of four major duckweeds and three types of wastewater effluent. Bioresource Technol 250:464–473CrossRefGoogle Scholar
  32. Toyama T, Kuroda M, Ogata Y, Hachiya Y, Quach A, Tokura K, Tanaka Y, Mori M, Morikawa M, Ike M (2017b) Enhanced biomass production of duckweeds by inoculating a plant growth-promoting bacterium, Acinetobacter calcoaceticus P23, in sterile medium and non-sterile environmental waters. Water Sci Technol 76:1418–1428CrossRefPubMedGoogle Scholar
  33. Tsavkelova EA, Cherdyntseva TA, Netrusov AI (2005) Auxin production by bacteria associated with orchid roots. Microbiology 74:46–53CrossRefGoogle Scholar
  34. Utami D, Kawahata A, Sugawara M, Jog NR, Miwa K, Morikawa M (2018) Effect of exogenous general plant growth regulators on the growth of the duckweed Lemna minor. Front Chem 6:251CrossRefPubMedCentralPubMedGoogle Scholar
  35. Valentini M, Filloux A (2016) Biofilms and cyclic di-GMP (c-di-GMP) signaling: lessons from Pseudomonas aeruginosa and other bacteria. J Biol Chem 291:12547–12555CrossRefPubMedCentralPubMedGoogle Scholar
  36. Xu J, Zhao H, Stomp A-M, Cheng JJ (2012) The production of duckweed as a source of biofuels. Biofuels 3:589–601CrossRefGoogle Scholar
  37. Yamaga F, Washio K, Morikawa M (2010) Sustainable biodegradation of phenol by Acinetobacter calcoaceticus P23 isolated from the rhizosphere of duckweed Lemna aoukikusa. Environ Sci Technol 44:6470–6474CrossRefPubMedGoogle Scholar
  38. Zhang F, Dashti N, Hynes RK, Smith DL (1997) Plant growth-promoting rhizobacteria and soybean [Glycine max (L.) Merr.] growth and physiology at suboptimal root zone temperatures. Ann Botany 79:243–249CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2018

Authors and Affiliations

  1. 1.Graduate School of Environmental ScienceHokkaido UniversitySapporoJapan

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