Isolation, identification, characterization, and screening of rhizospheric bacteria for herbicidal activity

  • Charles Oluwaseun Adetunji
  • Julius Kola Oloke
  • Gandham Prasad
  • Oluwasesan Micheal Bello
  • Osarenkhoe Omorefosa Osemwegie
  • Mishra Pradeep
  • Ravinder Sing Jolly


The consistent application of agrochemical herbicides has been reported to impact negatively on human health, environment, and food safety, and facilitated the emergence of weed resistances. Rhizosphere bacteria (RB) of different crops were screened for antagonism against Amaranthus hybridus L. (pigweed) and Echinochloa crus-galli (L.) Beauv. (barnyard grass) using necrosis assay technique. A total of eight rhizosphere bacterial isolates (B1–B8) produced different degrees of leaf necrosis on target weeds with isolate B2 manifesting the most significant necrotic activity. The rhizospheric bacterium (B2) with the highest necrotic activity was identified using 16S rRNA sequencing technique and further investigated. Molecular, morphological, and biochemical characterizations confirmed B2 isolate to be Pseudomonas aeruginosa. On isolation with ethyl acetate, separation, defatting, purification, and flash chromatography, seven different fractions (fraction 1–fraction 7) were obtained out of which fraction 4 showed the highest necrotic activity in necrosis assay experiment. Preparative HPLC of fraction 4 resulted in a pure compound that completely inhibited seed germination and seedling development of pigweed and barnyard grass but remained non-antagonistic to other tested soil fungi used in this study. The result obtained from this present study consequently confirmed the antagonistic behavior of rhizosphere-inhabiting P. aeruginosa to the target weeds and qualified the suitability of bacterium as good alternative source of bioherbicide. Potential herbicidal formulation from P. aeruginosa will help reduce crop loss due to weed challenges while offering a partial solution to the use of agrochemicals and food security.


16S rRNA gene DNA sequencing Deleterious rhizosphere bacteria Phylogenetic tree PHPLC and bioherbicide 



The authors are grateful to the Council of Scientific and Industrial Research (CSIR), New Delhi, India, and The World Academy of Science (TWAS), Italy, for providing the necessary facilities and opportunity to carry out this research. Special thanks to Mr. Rajul Tomar and the whole staff of Microbial Type Culture Collection and Gene Bank (MTCC), Institute of Microbial Technology, Sector 39A, Chandigarh, India for their contribution to the molecular aspect of this work. Also, I like to appreciate Dr. Adejumo Isaac and Miss Onikanni Olayinka for their input in the statistical analysis.


  1. Abouziena HFH, Omar AAM, Sharma SD, Singh M (2009) Efficacy comparison of some new natural product herbicides for weed control at two growth stages. Weed Technol 23(3):431–437CrossRefGoogle Scholar
  2. Adeosun JO, Dauda CK, Gezui MA, Odunze AC, Amapu IY, Kudp T (2009) On-farm weed management in upland rice in three villages of Katsina State of Nigeria. African Crop Science Society. Afr Crop Sci Conf Proc 9:625–629Google Scholar
  3. Adetunji CO, Oloke JK (2013) Efficacy of freshly prepared pesta granular formulations from the multi-combination of wild and mutant strain of Lasiodiplodia pseudotheobromae and Pseudomonas aeruginosa. Albanian J Agric Sci 12(4):555–563Google Scholar
  4. Adigun JA, Lagoke STO (2003) Assessment of critical period of weed interference in transplanted rainfed and irrigated tomatoes in the Nigerian Northern Guinea Savanna. Niger J Plant Prot 21:89–100Google Scholar
  5. Adigun J, Osipitan AO, Lagoke ST, Adeyemi RO, Afolami SO (2014) Growth and yield performance of cowpea (Vigna unguiculata (L.) Walp) as influenced by row-spacing and period of weed interference in South-West Nigeria. J Agric Sci 6(4):188–198Google Scholar
  6. Al-Hinai AH, Al-Sadi AM, Al-Bahry MAS, Al-said FA, Al-Harthi SA, Deadman ML (2010) Isolation and characterization of Pseudomonas aeruginosa with antagonistic activity against Pythium aphanidermatum. J Plant Pathol 92(3):653–660Google Scholar
  7. Allen VB, Randall GP (2014) Management of vegetation by alternative practices in fields and roadsides. Int J Agron 2014:1–12. doi: 10.1155/2014/207828 Google Scholar
  8. Altschul SF (1997) Gapped BLAST & PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25(17):3389–3402CrossRefPubMedPubMedCentralGoogle Scholar
  9. Asiwe J, Kutu RF (2007) Effect of plant spacing on yield weeds insect infestation and leaf bright of Bambara groundnut. Proc Afr Crop Sci Soc 4:1947–1950Google Scholar
  10. Baghestani MA, Zand E, Soufizadeh S, Bagherani N, Deihimfard R (2007) Weed control and wheat (Triticum aestivum L.) yield under application of 2, 4-D plus carfentrazone-ethyl and florasulam plus flumetsulam: evaluation of the efficacy. Crop Prot 26(12):1759–1764CrossRefGoogle Scholar
  11. Bolton H, Elliott LF (1989) Toxin production by a rhizobacterial Pseudomonas sp. that inhibits wheat root growth. Plant Soil 114(2):269–278CrossRefGoogle Scholar
  12. Brainard DC, Curran WS, Bellinder RR (2013) Temperature and relative humidity affect weed response to vinegar and clove oil. Weed Technol 27(1):156–164CrossRefGoogle Scholar
  13. Caldwell CJ, Hynes RK, Boyetchko SM, Korber DR (2012) Colonization and bioherbicidal activity on green foxtail by Pseudomonas fluorescens BRG100 in a pesta formulation. Can J Microbiol 58(1):1–9CrossRefPubMedGoogle Scholar
  14. Cappuccino JG, Sherman N (1992) Microbiology, a laboratory manual. The Benjamin Cummings Publishing Company Inc., California, p 462Google Scholar
  15. Cattelan AJ, Hartel PG, Fuhrmann JJ (1999) Screening for plant growth-promoting rhizobacteria to promote early soybean growth. Soil Sci Soc Am J 63(6):1670–1680CrossRefGoogle Scholar
  16. Charudattan R (1991) The mycoherbicide approach with plant pathogens. In: TeBeest DO (ed) Microbial control of weeds. Chapman & Hall, New York, pp 24–57. doi: 10.1007/978-1-4615-9680-6_2
  17. Chikoye D, Schulz S, Ekeleme F (2004) Evaluation of integrated weed management practices for maize in the northern guinea savanna of Nigeria. Crop Prot 23(10):895–900CrossRefGoogle Scholar
  18. Crump NS, Ash GJ, Fagan RJ (1999) The development of an Australian bioherbicide. 12 Australian Weed Conference, pp 235237Google Scholar
  19. Dueñas M, González-Manzano S, Surco-Laos F, González-Paramas A, Santos-Buelga C (2012) Characterization of sulfated quercetin and epicatechin metabolites. J Agric Food Chem 60(14):3592–3598CrossRefPubMedGoogle Scholar
  20. Ekeleme E, Kamara AY, Oikeh SO, Chikoye D, Omoigui LO (2007) Effect of weed competition on upland rice production in north-eastern Nigeria. Afr Crop Sci Conf Proc 8:61–65Google Scholar
  21. Evidente A, Motta A (2001) Phytotoxins from fungi, pathogenic for agrarian forestal and weedy plants. In: Tringali C (ed) Bioactive compounds from natural source. Taylor& Francis, London, pp 473–525Google Scholar
  22. FAOSTAT (2013) Food and agricultural organization of the United States. Date retrieved, 27th March, 2017.
  23. Fawole MO, Oso BA (2004) Biochemical test. Laboratory manual of microbiology. Spectrum Books Limited, Ibadan, pp 14–17Google Scholar
  24. Flores-Vargas RD, O’Hara GW (2006) Isolation and characterization of rhizosphere bacteria with potential for biological control of weeds in vineyards. J Appl Microbiol 100(5):946–954CrossRefPubMedGoogle Scholar
  25. Gurusiddaiah S, Gealy DR, Kennedy AC, Ogg AG (1994) Isolation and characterization of metabolites from Pseudomonas fluorescens-D7 for control of downy brome (Bromus tectorum). Weed Sci 42:492–501Google Scholar
  26. Hamid AA, Aiyelaagbe OO, Balogun GA (2011) Herbicides and its applications. Adv Nat Appl Sci 5(2):201–213Google Scholar
  27. Harding DP, Raizada MN (2015) Controlling weeds with fungi, bacteria and viruses: a review. Front Plant Sci 6:1–14CrossRefGoogle Scholar
  28. Heap I (2005) The international survey of herbicide resistant weeds. Web page:
  29. Ivany JA (2010) Acetic acid for weed control in potato (Solanum tuberosum L.) Can J Plant Sci 90(4):537–542CrossRefGoogle Scholar
  30. Javaid A (2010) Herbicidal potential of allelopathic plants and fungi against Parthenium hysterophorus—a review. Allelopath J 25(2):331–344Google Scholar
  31. Javaid A, Adrees H (2009) Parthenium management by cultural filtrates of phytopathogenic fungi. Nat Prod Res 23(16):1541–1551CrossRefPubMedGoogle Scholar
  32. Javaid A, Ali S (2011) Herbicidal activity of culture filtrates of Trichoderma spp. against two problematic weeds of wheat. Nat Prod Res 25(7):730–740CrossRefPubMedGoogle Scholar
  33. Javaid A, Shafique S, Shafique S (2010) Herbicidal effects of extracts and residue incorporation of Daturametelagainst partheniumweed. Nat Prod Res 24(15):1426–1437Google Scholar
  34. Javaid A, Shafique S, Shafique S (2011) Management of Parthenium hysterophorus (Asteraceae) by Withania somnifera (Solanaceae). Nat Prod Res 25(4):407–416CrossRefPubMedGoogle Scholar
  35. Johnson WC III, Boudreau MA, Davis JW (2013) Combinations of corn gluten meal, clove oil, and sweep cultivation are ineffective for weed control in organic peanut production. Weed Technol 27(2):417–421CrossRefGoogle Scholar
  36. Kaewchai S, Soytong K, Hyde KD (2009) Mycofungicides and fungal biofertilizers. Fungal Divers 38:25–50.
  37. Kennedy AC, Stubbs TL (2007) Management effects on the incidence of jointed goat grass inhibitory rhizobacteria. Biol Control 40(2):213–221Google Scholar
  38. Kennedy AC, Young FL, Elliott LF, Douglas CL (1991) Rhizobacteria suppressive to the weed downy brome. Soil Sci Soc Am J 55(3):722–727Google Scholar
  39. Kloepper JW, Schroth MN, Miller TD (1980)  Effects of Rhizosphere Colonization by Plant Growth-Promoting Rhizobacteria on Potato Plant Development and Yield. Phytopathology 70(11):1078-1082Google Scholar
  40. Kordali S, Cakir A, Sutay S (2007) Inhibitory effects of monoterpenes on seed germination and seedling growth. Zeitschrift fur Naturforschung (C) 62(3):207–214Google Scholar
  41. Kordali S, Cakir A, Ozer H, Cakmakci R, Kesdek M, Mete E (2008) Antifungal, phytotoxic and insecticidal properties of essential oil isolated from Turkish Origanum acutidens and its three components, carvacrol, thymol and p-cymene. Bioresour Technol 99(18):8788–8795CrossRefPubMedGoogle Scholar
  42. Kremer RJ (2005) The role of bioherbicides in weed management. Biopestic Int 1(3,4):127–141Google Scholar
  43. Kremer RJ, Kennedy AC (1996) Rhizobacteria as biocontrol agents of weeds. Weed Technol 10:601–609Google Scholar
  44. Kremer RJ, Begonia MFT, Stanley L, Lanham ET (1990) Characterization of rhizobacteria associated with weed seedlings. Appl Environ Microbiol 56(4):1649–1655PubMedPubMedCentralGoogle Scholar
  45. Lugtenberg BJJ, Dekkers LC (1999) What make Pseudomonas bacteria rhizosphere competent? Environ Microbiol 1(1):9–13CrossRefPubMedGoogle Scholar
  46. Makhan SB, Simerjit K, Tarundeep K, Tarlok S, Megh S, Amit JJ (2013) Control of broadleaf weeds with post-emergence herbicides in four barley (Hordeum spp.) cultivars. Crop Prot 43:216–222.
  47. Mark R, Behrens NM, Sarbani C, Razvan D, Wen Z, Bradley J, LaVallee PL, Herman TE, Clemente DP (2007) Weeks dicamba resistance: enlarging and preserving biotechnology-based weed management strategies. Science 316(5828):1185–1188. doi: 10.1126/science.1141596 CrossRefGoogle Scholar
  48. McPhail KL, Armstrong DJ, Azevedo MD, Banowetz GM, Mills DI (2010) 4-Formylaminooxyvinylglycine, an herbicidal germination arrest factor (GAF) from Pseudomonas rhizosphere bacteria. J Nat Prod 73(11):1853–1857CrossRefPubMedPubMedCentralGoogle Scholar
  49. Mejri D, Gamalero E, Souissi T (2013) Formulation development of the deleterious rhizobacterium Pseudomonas trivialisX33d for biocontrol of brome (Bromus diandrus) in durum wheat. J Appl Microbiol 114(1):219–228. doi: 10.1111/jam.12036
  50. Muhammad AS, Muhammad M, Muhammad AUL, Abid N (2012) Efficacy of various herbicides against weeds in wheat (Triticum aestivum L.) Afr J Biotechnol 11(4):791–799. doi: 10.5897/AJB11.3274 CrossRefGoogle Scholar
  51. Nurse RE, Hamill AS, Swanton CJ, Tardif FJ, Sikkema PH (2007) Weed control and yield response to foramsulfuron in corn. Weed Technol 21(2):453–458CrossRefGoogle Scholar
  52. Poston DH, Wilson HP, Hines TE (2000) Imidazolinone resistance in several Amaranthus hybridus populations. Weed Sci 48(4):508–513CrossRefGoogle Scholar
  53. Sacchi CT, Whitney AM, Mayer LW, Morey R, Steigerwalt A, Boras A, Weyant RS, Popovic T (2002) Sequencing of 16S rRNA gene: a rapid tool for identification of Bacillus anthracis. Emerg Infect Dis 8(10):1117–1123CrossRefPubMedPubMedCentralGoogle Scholar
  54. Schippers B, Bakker AW, Bakker PAHM (1987) Interactions of deleterious and beneficial rhizosphere microorganisms and the effect of cropping practices. Annu Rev Phytopathol 25:339–358CrossRefGoogle Scholar
  55. Sessitsch A, Gyamfi S, Tscherko D, Gerzabek MH, Kandeler E (2004) Activity of microorganisms in the rhizosphere of herbicide treated and untreated transgenic glufosinate-tolerant and wild type oilseeds rape grown in containment. Plant Soil 266(1):105–116. doi: 10.1007/s11104-005-7077-4
  56. Takim FO, Amodu AA (2013) Quantitative estimate of weeds of sugarcane (Saccharum officinarum L.) crop in Ilorin, southern Guinea savannah of Nigeria. Ethiop J Environ Stud Manag 6(6):611–619Google Scholar
  57. Takim FO, Ahmadu MS, Omotosho SB (2015) Efficacy of Ametryn herbicides on weeds of sugarcane in southern guinea savanna zone of Nigeria. Niger J Agric, Food Environ 11(3):147–151Google Scholar
  58. Usman A, Elemo KA, Lagoke STO, Adigun JA (2002) Nitrogen and weed management in maize intercropped with upland rice. J Sustain Agric 21(1):5–16. doi: 10.1300/J064v21n01_03 CrossRefGoogle Scholar
  59. Van Driesche RG, Carruthers RI, Center T, Hoddle MS, Hough-Goldstein J, Morin L, Smith DL (2010) Classical biological control for the protection of natural ecosystem. Biol Control 154(S1):2–33Google Scholar
  60. Xiaoya C, Mengmeng G (2016) Bioherbicides in organic agriculture. Horticulturae 2(3):1–10Google Scholar
  61. Yang J, Hong-zhe CAO, Wang WV (2014) Isolation, identification, and herbicidal activity of metabolites produced by Pseudomonas aeruginosa CB-4. J Integr Agric 13(8):1719–1726CrossRefGoogle Scholar
  62. Zermane N, Souissi T, Kroschel JR, Sikora R (2007) Biocontrol of broomrape (Orobanchecrenata Forsk. and Orobanchefoetida Poir.) by Pseudomonas fluorescens isolate Bf7-9from the faba bean rhizosphere. Biocontrol Sci Tech 17(5):483–497Google Scholar
  63. Zhang LH, Zhang JL, Liu YC, Cao ZY, Han JM, Yang J, Dong JG (2013) Isolation and structural speculation of herbicide-active compounds from the metabolites of Pythium aphanidermatum. J Integr Agric 12:1026–1032CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2017

Authors and Affiliations

  • Charles Oluwaseun Adetunji
    • 1
  • Julius Kola Oloke
    • 2
  • Gandham Prasad
    • 3
  • Oluwasesan Micheal Bello
    • 4
  • Osarenkhoe Omorefosa Osemwegie
    • 1
  • Mishra Pradeep
    • 5
  • Ravinder Sing Jolly
    • 5
  1. 1.Department of Biological Sciences, Applied Microbiology, Biotechnology and Nanotechnology LaboratoryLandmark UniversityOmu AranNigeria
  2. 2.Department of Pure and Applied BiologyLadoke Akintola University of TechnologyOgbomosoNigeria
  3. 3.Microbial Type Culture Collection and Gene Bank, CSIR-Institute of Microbial TechnologyChandigarhIndia
  4. 4.Department of Applied ChemistryFederal University Dutsin-MaDutsin-MaNigeria
  5. 5.Department of Bioorganic LaboratoryInstitute of Microbial TechnologyChandigarhIndia

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