Biocontrol activities of rhizobacteria associated with apple, apricot and kiwi rhizosphere against bacterial canker caused by Clavibacter michiganensis

  • Swati GautamEmail author
  • Rashmi Sharma
  • Anjali Chauhan
  • C. K. Shirkot
  • Rajesh Kaushal
Research Article


Bacterial canker, a destructive disease caused by Clavibacter michiganensis subsp. michiganensis causes significant economic losses to tomato production worldwide. Biological control has been proposed as an alternative to current chemical protectants, however, a little headway has so far been made in developing biocontrol methods against this destructive bacterial pathogen. To narrow this knowledge gap, we investigated the antagonistic capacity of different rhizobacterial isolates from three horticultural crops viz., apple, apricot and strawberry against C. michiganensis under in vitro conditions. The potential antagonistic strains showing in vitro inhibition against C. michiganensis were further screened for multifarious plant growth promoting and biocontrol activities (P-solubilization, IAA production, siderophore production, lytic enzyme activity, and ability to fix atmospheric nitrogen). Increase in concentration of cell free supernatant from 0.25 to 1.00% (v/v) revealed significant increase in antagonistic activity against C. michiganensis with most prominent increase being observed in isolate S1 with 9.90 mm zone of inhibition at 0.25% concentration, which increased to 15.40 mm with increase in concentration to 1.0%. Five most efficient antagonists identified under in vitro conditions were tested for biocontrol potential against C. michiganensis under net house conditions. Isolate S1 showed maximum reduction in disease incidence (70.00%) with minimum disease severity recorded (28.55%), besides significant increase in various plant growth parameters. The isolate was identified as Bacillus amyloliquefaciens by 16S rDNA sequencing. Therefore, strain S1 holds considerable biocontrol as well as growth promoting potential and could be used as a potential biocontrol agent against bacterial canker of tomato.


Bacterial canker Bacillus amyloliquefaciens Biocontrol Clavibacter michiganensis Tomato 



  1. Agrawal K, Sharma DK, Jain VK (2012) Seed-borne bacterial diseases of tomato (Lycopersicon esculentum mill.) and their control measures: a review. Int J Food Agric Vet Sci 2:173–182Google Scholar
  2. Ahemed M, Khan MS (2011) Functional aspects of plant growth promoting rhizobacteria: recent advancements. Insight Microbiol 1:39–54CrossRefGoogle Scholar
  3. Ahmed Sheikh HM (2010) Antimicrobial activity of certain bacteria and fungi isolated from soil mixed with human saliva against pathogenic microbes causing dermatological diseases. Saudi J Biol Sci 17:331–339CrossRefGoogle Scholar
  4. Ali B, Sabri AN, Hasnain S (2010) Rhizobacterial potential to alter auxin content and growth of Vigna radiata (L.). World J Microbiol Biotechnol 26:1379–1384CrossRefGoogle Scholar
  5. Altschul SF, Thomas LM, Alejandro AS, Jinghui Z (1997) Gapped BLAST and PSIBLAST: a new generation of protein database search programs. Nucleic Acid Res 25:3389–3402CrossRefGoogle Scholar
  6. Alyie N, Fininsa C, Hikias Y (2008) Evaluation of rhizosphere bacterial antagonist for their potential to bioprotect potato (Solanum tuberosum) against bacterial wilt (R. solanacearum). Biol Control 47:282–288CrossRefGoogle Scholar
  7. Amaresan N, Jayakumar V, Krishna Kumar, Thajuddin N (2012) Endophytic bacteria from tomato and chilli, their diversity and antagonistic potential against Ralstonia solanacearum. Arch Phytopathol Plant Prot 45:344–355CrossRefGoogle Scholar
  8. Andreote FD, Azevedo JL, Araújo WL (2009) Assessing the diversity of bacterial communities associated with plants. Braz J Microbiol 40:417–432CrossRefGoogle Scholar
  9. Arguelles-Arias A, Ongena M, Halimi B, Lara Y, Brans A, Joris B (2009) Bacillus amyloliquefaciens GA1 as a source of potent antibiotics and other secondary metabolites for biocontrol of plant pathogens. Microb Cell Fact. CrossRefPubMedPubMedCentralGoogle Scholar
  10. Aslantas R, Cakmakci R, Sahin F (2007) Effect of plant growth promoting rhizobacteria on young apple tree growth and fruit yield under orchard conditions. Sci Hortic 111:371–377CrossRefGoogle Scholar
  11. Baldani VLD, Döbereiner J (1980) Host-plant specificity in the interaction of cereals with Azospirillum spp. Soil Biol Biochem 12:433–439CrossRefGoogle Scholar
  12. Bloemberg GV, Lugtenberg BJJ (2001) Molecular basis of plant growth promotion and biocontrol by rhizobacteria. Curr Opin Plant Biol 4:343–350CrossRefGoogle Scholar
  13. Boudyach EH, Fatmi M, Okhayat O, Benizri E, Aoumar AAB (2001) Selection of antagonistic bacteria of Clavibacter michiganensis subsp. michiganensis and evaluation of their efficiency against bacterial canker of tomato. Biocontrol Sci Technol 11:141–149CrossRefGoogle Scholar
  14. Calis O, Bayan Y, Celik D (2012) Characterization of resistant tomato mutants to bacterial canker disease. Afr J Biotechnol 11:8070–8075Google Scholar
  15. Chakraborty U, Chakraborty BN, Chakraborty AP, Sunar K, Dey PL (2013) Plant growth promoting rhizobacteria mediated improvement of health status of tea plants. Indian J Biotechnol 12:20–31Google Scholar
  16. Chen L, Chopra T, Kaye K (2009) Pathogens resistant to antibacterial agents. Infect Dis Clin N Am 23:817–845CrossRefGoogle Scholar
  17. Conrath U, Thulke O, Katz V, Schwindling S, Kohler A (2001) Priming as a mechanism in induced systemic resistance of plants. Eur J Plant Pathol 107:113–119CrossRefGoogle Scholar
  18. Dahiya N, Tewari R, Hoondal GS (2006) Biotechnological aspects of chitinolytic enzymes. Appl Microbiol Biotechnol 7:773–782CrossRefGoogle Scholar
  19. Deshwal VK, Kumar P (2013) Production of plant growth promoting substances by Pseudomonads. J Acad Ind Res 2:221–225Google Scholar
  20. Dhingra OD, Mizubuti ESG, Santana FM (2003) Chaetomium globosum for reducing primary inoculum of Diaporthe phaseolorum f. sp. meridionalis in soil-surface soybean stable in field condition. Biol Control 26:302–310CrossRefGoogle Scholar
  21. Ding C, Shen Q, Zhang R, Chan W (2013) Evaluation of rhizosphere bacteria and derived bioorganic fertilizers as potential biocontrol agents against bacterial wilt (Ralstonia solanacearum) of potato. Plant Soil 366:453–466CrossRefGoogle Scholar
  22. Fleming HP, Etchells JL, Costilus RH (1975) Microbial inhibition by an isolate of Pediococcus from cucumber brines. Appl Microbiol 30:1040–1042PubMedPubMedCentralGoogle Scholar
  23. Gautam S, Chauhan A, Sharma R, Sehgal R, Shirkot CK (2019) Potential of Bacillus amyloliquefaciens for biocontrol of bacterial canker of tomato incited by Clavibacter michiganensis ssp. michiganensis. Microb Pathog 130:196–203CrossRefGoogle Scholar
  24. Ghose TK (1987) Measurement of cellulase activities. Pure Appl Chem 59:257–268CrossRefGoogle Scholar
  25. Girish N, Umesha S (2005) Effect of plant growth promoting rhizobacteria on bacterial canker of tomato. Arch Phytopathol Plant Prot 38:235–243CrossRefGoogle Scholar
  26. Gordon SA, Paleg LG (1957) Quantitative measurement of indole acetic acid. Physiol Plant 10:37–48CrossRefGoogle Scholar
  27. Haggag MW (2010) Role of entophytic microorganisms in biocontrol of plant diseases. Life Sci J 7:57–62Google Scholar
  28. Hameeda B, Reddy YHK, Rupela OP, Kumar GN, Reddy G (2006) Effect of carbon substrates on rock phosphate solubilization by bacteria from composts and macrofauna. Curr Microbiol 53:298–302CrossRefGoogle Scholar
  29. Idris ES, 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 Microbe Interact 20:619–626CrossRefGoogle Scholar
  30. Joseph S, Jisha MS (2009) Buffering reduces phosphate solubilizing ability of selected strains of bacteria. World J Agric Sci 5:135–137Google Scholar
  31. Kumar A, Kumar A, Devi S, Patil S, Payal C, Negi S (2012) Isolation, screening and characterization of bacteria from rhizospheric soils for different plant growth promotion (PGP) activities: an in vitro study. Recent Res Sci Technol 4:1–5Google Scholar
  32. Kumar A, Maurya BR, Raghuwanshi R (2015) Characterization of bacteria strains and their impact on plant growth promotion and yield of wheat and microbial populations of soil. Afr J Agric Res 10:1367–1375CrossRefGoogle Scholar
  33. Kurabachew H, Wydra K (2013) Characterization of plant growth promoting rhizobacteria and their potential as bioprotectant against tomato bacterial wilt caused by Ralstonia solanacearum. Biol Control 67:75–83CrossRefGoogle Scholar
  34. Lanteigne C, Gadkar VJ, Wallon T, Novinscak A, Filion M (2012) Production of DAPG and HCN by Pseudomonas sp. LBUM300 contributes to the biological control of bacterial canker of tomato. Phytopathology 102:967–973CrossRefGoogle Scholar
  35. Lemessa F, Zeller W (2007) Screening rhizobacteria for biological control of Ralstonia solanacearum in Ethiopia. Biol Control 42:336–344CrossRefGoogle Scholar
  36. Maksimov IV, Abizgil’dina RR, Pusenkova LI (2011) Plant growth promoting rhizobacteria as alternative to chemical crop protectors from pathogens (review). Appl Biochem Microbiol 47:333–345CrossRefGoogle Scholar
  37. Marcic SM, Gatermann KH, Frohwitter J, Eichenlaub R, Todorovic B, Rekanovic E, Potocnik I (2012) Characterization of Clavibacter michiganensis subsp. michiganensis strains from recent outbreaks of bacterial wilt and canker in Serbia. Eur J Plant Pathol 134:697–711CrossRefGoogle Scholar
  38. Mehta P, Chauhan A, Mahajan R, Mahajan PK, Shirkot CK (2010) Strain of Bacillus circulans isolated from apple rhizosphere showing plant growth promoting potential. Curr Sci 98:538–542Google Scholar
  39. Mehta P, Walia A, Kulshreshtha S, Chauhan A, Shirkot CK (2013) Efficiency of plant growth promoting P-solubilizing Bacillus circulans CB7 for enhancement of tomato growth under net house conditions. J Basic Microbiol 53:1–12CrossRefGoogle Scholar
  40. Mitchell JK, Carter WE (2000) Modeling antimicrobial activity Clorox™ using an agar-diffusion test: a new twist on an old experiment. Bioscene 26:9–13Google Scholar
  41. Miyazawa J, Kawabata T, Ogasawara N (1998) Induction of an acidic isozyme of peroxidise and acquired resistance to wilt disease in response to treatment of tomato roots with 2-furoic acid, 4-hydroxy benzoic hydrazide or salicylic hydrazide. Physiol Mol Plant Pathol 52:115–126CrossRefGoogle Scholar
  42. Neeraja C, Anil K, Purushotham P, Suma K, Sarma P, Moerschbacher BM, Podile AR (2010) Biotechnological approaches to develop bacterial chitinases as a bioshield against fungal diseases of plants. Crit Rev Biotechnol 30:231–241CrossRefGoogle Scholar
  43. Nguyen NV, Kim YJ, Oh KT, Jung WJ, Park RD (2008) Antifungal activity of chitinases from Trichoderma aureoviride DY-59 and Rhizopus microspores VS-9. Curr Microbiol 56:28–32CrossRefGoogle Scholar
  44. Patel DK, Archana G, Kumar GN (2008) Variation in the nature of organic acid secretion and mineral phosphate solubilization by Citrobacter sp. DHRSS in the presence of different sugars. Curr Microbiol 56:168–174CrossRefGoogle Scholar
  45. Pikovskaya RI (1948) Mobilization of phosphorus in soil in connection with the vital activity of some microbial species. Mikrobiologiya 7:362–370Google Scholar
  46. Raaijmakers JM, Paulitz TC, Steinberg C, Alabouvette C, Moënne-Loccoz Y (2009) The rhizosphere: a playground and battlefield for soilborne pathogens and beneficial microorganisms. Plant Soil 321:341–361CrossRefGoogle Scholar
  47. Raaijmakers JM, De Bruijn I, Nybroe O, Ongena M (2010) Natural functions of lipopeptides from Bacillus and Pseudomonas: more than surfactants and antibiotics. FEMS Microbiol Rev 34:1037–1062CrossRefGoogle Scholar
  48. Ramos Solano B, Garcia JAL, Villaraco AG, Algar E, Cristobal JG, Manero FJG (2010) Siderophore and chitinase producing isolates from the rhizosphere of Nicotiana glauca Graham enhance growth and induce systemic resistance in Solanum lycopersicum L. Plant Soil 334:189–197CrossRefGoogle Scholar
  49. Rangajaran S, Saleena LM, Vasudevan P, Nair S (2003) Biological suppression of rice diseases by Pseudomonas spp. under saline soil conditions. Plant Soil 251:73–82CrossRefGoogle Scholar
  50. Robert WK, Selintrennikoff CP (1988) Plant and bacterial chitinase differ in antifungal activity. J Gen Microbiol 134:169–176Google Scholar
  51. Romero AM, Correa OS, Moccia S, Rivas JG (2003) Effect of Azospirillum-mediated plant growth promotion on the development of bacterial diseases on fresh-market and cherry tomato. J Appl Microbiol 95:832–838CrossRefGoogle Scholar
  52. Ryu RJ, Patten CL (2008) Aromatic amino acid-dependent expression of indole-3-pyruvate decarboxylase is regulated by TyrR in Enterobacter cloacae UW5. J Bacteriol 190:7200–7208CrossRefGoogle Scholar
  53. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, New YorkGoogle Scholar
  54. Sarala IS, Shetty S (2005) Clavibacter michiganensis subsp. michiganensis in tomato fields of Karnataka, India. Adv Plant Sci 18:581–589Google Scholar
  55. Schwyn B, Neilands JB (1987) Universal chemical assay for the detection and determination of siderophores. Anal Biochem 160:47–56CrossRefGoogle Scholar
  56. Sharma R, Chauhan A, Shirkot CK (2015) Characterization of plant growth promoting Bacillus strains and their potential as crop protectants against Phytophthora capsici in tomato. Biol Agric Hortic. CrossRefGoogle Scholar
  57. Shenge KC, Mabagala RB, Mortensen CN (2010) Current status of bacterial speck and spot diseases of tomato in three tomato-growing regions of Tanzania. J Agric Ext Rural Dev 2:84–88Google Scholar
  58. Singh G, Bharat NK (2017) Studies on bacterial canker (Clavibacter michiganensis subsp. michiganensis) of tomato (Solanum lycopersicum). Int J Curr Microbiol Appl Sci 6:317–323Google Scholar
  59. Singh G, Bharat NK, Sharma M (2015) Occurrence of bacterial canker of tomato in Himachal Pradesh, India: identification and molecular characterization of the pathogen. Bioscan 10:1753–1757Google Scholar
  60. Singh G, Bharat NK, Bhardwaj RK, Singh SP (2017) Evaluation of germplasm and biocontrol agents against bacterial canker (Clavibacter michiganensis subsp. michiganensis) of tomato in Himachal Pradesh. Int J Pure Appl Biosci 5:740–748CrossRefGoogle Scholar
  61. Soylu S, Baysal O, Soylu M (2003) Induction of disease resistance by the plant activator, Acibenzolar-S-methyl (ASM), against bacterial canker (Clavibacter michiganensis ssp. michiganensis) in tomato seedlings. Plant Sci 165:1069–1075CrossRefGoogle Scholar
  62. Suresh P, Pallavi P, Srinivas V, Kumar SP, Chandra Jeevan, Ram Reddy S (2010) Plant growth promoting activities of fluorescent Pseudomonads associated with some crop plants. Afr J Microbiol Res 4:1491–1494Google Scholar
  63. Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680CrossRefGoogle Scholar
  64. Umesha S (2006) Occurrence of bacterial canker in tomato fields of Karnataka and effect of biological seed treatment on disease incidence. Crop Prot 25:375–381CrossRefGoogle Scholar
  65. Van Loon LC (1997) Induced resistance in plants and the role of pathogenesis-related proteins. Eur J Plant Pathol 103:753–765CrossRefGoogle Scholar
  66. Villacieros M, Power B, Sanchez-Contreras M, Loret J, Oruzebal RI, Martin M, Franandez-Pinas F, Bouile I, Whelan C, Dowling DN, Rivilla R (2003) Colonization behaviour of Pseudomonas fluorescens and Sinorhizobium meloti in the alfalfa (Medicago sativa) rhizosphere. Plant Soil 251:47–54CrossRefGoogle Scholar
  67. Vitullo D, Di Pietro A, Romano A, Lanzotti V, Lima G (2012) Role of new bacterial surfactins in the antifungal interaction between Bacillus amyloliquefaciens and Fusarium oxysporum. Plant Pathol 61:689–699CrossRefGoogle Scholar
  68. Wahyudi AT, Astuti RP, Widyawati A, Meryandini A, Nawangsih A (2011) Characterization of Bacillus sp. strains isolated from rhizosphere of soybean plants for their use as potential plant growth for promoting rhizobacteria. J Microbiol Antimicrob 3:34–40Google Scholar
  69. Walia A, Mehta P, Chauhan A, Shirkot CK (2013) Effect of Bacillus subtilis strain CKT1 as inoculum on growth of tomato seedlings under net house conditions. Proc Natl Acad Sci India Sect B Biol Sci 84:145–155CrossRefGoogle Scholar
  70. Yin XM, Jin ZQ, Xu BY, Ma WH, Fu YG, Wang JB (2011) Characterization of early events in banana root infected with the GFP-tagged Fusarium oxysporum f. sp. cubense. Acta Hortic 897:371–376CrossRefGoogle Scholar
  71. Yoshida S, Hiradate S, Tsukamato T, Hatakeda K, Shirata A (2000) Antimicrobial activity of culture filtrate of Bacillus amyloliquefaciens RC-2 isolated from Mulberry leaves. Biol Control 92:181–187Google Scholar
  72. Yuan J, Ruan Y, Wang B, Zhang J, Waseem R, Huang Q, Shen Q (2013) Plant growth-promoting rhizobacteria strain Bacillus amyloliquefaciens NJN-6-enriched bio-organic fertilizer suppressed Fusarium wilt and promoted the growth of banana plants. J Agric Food Chem 61:3774–3780CrossRefGoogle Scholar

Copyright information

© Indian Phytopathological Society 2019

Authors and Affiliations

  1. 1.Department of Basic SciencesDr. YSP UHFSolanIndia
  2. 2.Department of MicrobiologyDAV UniversityJalandharIndia
  3. 3.Department of Soil Science and Water ManagementDr. YSP UHFSolanIndia

Personalised recommendations