Skip to main content
SpringerLink
Log in

Flagellin of Bacillus amyloliquefaciens works as a resistance inducer against groundnut bud necrosis virus in chilli (Capsicum annuum L.)

  • Original Article
  • Published:
Archives of Virology Aims and scope Submit manuscript

Abstract

Groundnut bud necrosis virus (GBNV), a member of the genus Tospovirus, has an extensive host range and is associated with necrosis disease of chilli (Capsicum annuum L.), which is a major threat to commercial production. Plant growth promoting rhizobacteria (PGPR) have been investigated for their antiviral activity in several crops and for their potential use in viral disease management. However, the microbial mechanisms associated with PGPR in triggered immunity against plant viruses have rarely been studied. To understand the innate immune responses activated by Bacillus spp. against GBNV, we studied microbe-associated molecular pattern (MAMP) triggered immunity (MTI) in chilli using transient expression of the flagellin gene of Bacillus amyloliquefaciens CRN9 from Agrobacterium clones, which also induced the expression of EAS1 gene transcripts coding for epi-aristolochene synthase, which is responsible for the accumulation of capsidiol phytoalexin. In addition, the transcript levels of WRKY33 transcription factor and salicylic acid (SA)-responsive defense genes such as NPR1, PAL, PO and SAR8.2 were increased. Jasmonate (JA)-responsive genes, viz., PDF, and LOX genes, were also upregulated in chilli plants challenged with GBNV. Further analysis revealed significant induction of these genes in chilli plants treated with B. amyloliquefaciens CRN9 and benzothiadiazole (BTH). The transcript levels of defense response genes and pathogenesis-related proteins were significantly higher in plants treated with Bacillus and BTH and remained significantly higher at 72 h post-inoculation and compared to the inoculated control. The plants treated with flagellin using the agrodrench method and exogenous treatment with B. amyloliquefaciens and BTH showed resistance to GBNV upon mechanical inoculation and a reduced virus titre which was confirmed by qPCR assays. Thus, transient expression of flagellin, a MAMP molecule from B. amyloliquefaciens CRN9, is able to trigger innate immunity and restrain virus growth in chilli via induced systemic resistance (ISR) activated by both the SA and JA/ET signalling pathways.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  1. Biswas K, Hallan V, Zaidi AA, Pandey PK (2013) Molecular evidence of Cucumber mosaic virus subgroup II infecting Capsicum annuum L. in the Western region of India. Curr Discov 2(2):97–105

    Google Scholar 

  2. Kunkalikar SR, Sudarsana P, Rajagopalan P, Zehr UB, Ravi KS (2010) Biological and molecular characterization of Capsicum chlorosis virus infecting chilli and tomato in India. Arch Virol 155(7):1047–1057

    Article  CAS  PubMed  Google Scholar 

  3. Sushmitha C, Bhat SK (2014) Pepper vein banding virus-an over view. Int J Sci Res 3(6):30–31

    Google Scholar 

  4. Gopal K, Muniyappa V, Jagadeeshwar R (2011) Weed and crop plants as reservoirs of peanut bud necrosis tospovirus and its occurrence in South India. Arch Phytopathol Plant Prot 44(12):1213–1224

    Article  Google Scholar 

  5. Sahu AK, Chitra N, Mishra R, Verma R, Gaur RK (2016) Molecular evidence of Chilli vein mottle virus and Chilli leaf curl virus simultaneously from naturally infected chilli plant (Capsicum annuum L.). Indian J Biotechnol 15:266–268

    Google Scholar 

  6. Sharma A, Kulshrestha S (2016) Molecular characterization of tospoviruses associated with ringspot disease in bell pepper from different districts of Himachal Pradesh. Virus Dis 27(2):188–192

    Article  Google Scholar 

  7. Pavithra BS, Krishnareddy M, Rangaswamy KT (2016) Detection and partial characterization of Groundnut bud necrosis virus in chilli. Int J Sci Nat 7(4):843–847

    Google Scholar 

  8. Kunkalikar SR, Poojari S, Arun BM, Rajagopalan PA, Chen TC, Yeh SD, Naidu RA, Zehr UB, Ravi KS (2011) Importance and genetic diversity of vegetable-infecting tospoviruses in India. Virology 101(3):367–376

    Google Scholar 

  9. Haokip BD, Alice D, Nagendran K, Renukadevi P, Karthikeyan G (2016) Detection of Capsicum chlorosis virus (CaCV), an emerging virus infecting chilli in Tamil Nadu, India. Vegetos 29:1–4

    Article  Google Scholar 

  10. Mishra S, Jagadeesh KS, Krishnaraj PU, Prem S (2014) Biocontrol of Tomato leaf curl virus (ToLCV) in tomato with chitosan supplemented formulations of Pseudomonas sp. under field conditions. Aust J Crop Sci 8:347–355

    CAS  Google Scholar 

  11. Vinodkumar S, Nakkeeran S, Renukadevi P, Mohankumar S (2018) Diversity and antiviral potential of rhizospheric and endophytic Bacillus species and phyto-antiviral principles against tobacco streak virus in cotton. Agric Ecosyst Environ 267:42–51

    Article  CAS  Google Scholar 

  12. Huckelhoven R (2007) Cell wall-associated mechanisms of disease resistance and susceptibility. Annu Rev Phytopathol 45:101–127

    Article  PubMed  CAS  Google Scholar 

  13. Beris D, Theologidis I, Skandalis N, Vassilakos N (2018) Bacillus amyloliquefaciens strain MBI600 induces salicylic acid dependent resistance in tomato plants against Tomato spotted wilt virus and Potato virus Y. Sci Rep 8:10320. https://doi.org/10.1038/s41598-018-28677-3

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Subramanian KS, Narayanasamy P (1973) Mechanical transmission of Whitefly borne yellow mosaic virus of Lablab niger Medikus. Curr Sci 47:92–93

    Google Scholar 

  15. Widana Gamage SMK, McGrath DJ, Persley DM, Dietzgen RG (2016) Transcriptome analysis of Capsicum chlorosis virus-induced hypersensitive resistance response in bell Capsicum. PLoS ONE 11(7):e0159085. https://doi.org/10.1371/journal.pone.0159085

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Hobbs HA, Reddy DVR, Rajeshwari R, Reddy AS (1987) Use of direct antigen coating and protein A coating ELISA procedures for detection of three peanut viruses. Plant Dis 71:747–749

    Article  Google Scholar 

  17. Bacon WC, Hinton DM (2002) Endophytic and biological control potential of Bacillus mojavensis and related species. Biol Control 23:274–284

    Article  CAS  Google Scholar 

  18. Weisburg WG, Barns SM, Pelletier DA, Lane DJ (1991) 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol 173(2):697–703

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Vinodkumar S, Nakkeeran S, Renukadevi P, Malathi VG (2017) Biocontrol potentials of antimicrobial peptide producing Bacillus species: multifaceted antagonists for the management of stem rot of carnation caused by Sclerotinia sclerotiorum. Front Microbiol 8:446. https://doi.org/10.3389/fmicb.2017.00446

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Jefferson RA (1987) Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol Biol Rep 5:387–405

    Article  CAS  Google Scholar 

  21. Ditta G, Stanfield S, Corbin D, Helinski D (1980) Broad host range DNA cloning system for gram-negative bacteria: construction of a gene bank of Rhizobium meliloti. Proc Natl Acad Sci USA 77:7347–7351

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Ryu CM, Anand A, Kang L, Mysore KS (2004) Agrodrench: a novel and effective agroinoculation method for virus-induced gene silencing in roots and diverse Solanaceous species. Plant J 40:322–331

    Article  CAS  PubMed  Google Scholar 

  23. Mishra R, Nanda S, Rout E, Chand SK, Mohanty JN, Joshi RK (2017) Differential expression of defense-related genes in chilli pepper infected with anthracnose pathogen Colletotrichum truncatum. Physiol Mol Plant Pathol 97:1–10

    Article  CAS  Google Scholar 

  24. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2-∆∆Ct Method. Methods 25:402–408

    Article  CAS  PubMed  Google Scholar 

  25. Sain S, Chadha M (2012) Evaluation of improved lines of tomato for yield performance and disease resistance under open field conditions. Indian J Hortic 69(2):185–194

    Google Scholar 

  26. Berges R, Rott M, Seemuller E (2000) Range of phytoplasma concentration in various plant hosts as determined by competitive polymerase chain reaction. Phytopathology 90:1145–1152

    Article  CAS  PubMed  Google Scholar 

  27. Jain RK, Pandey AN, Krishnareddy M, Mandal B (2005) Immunodiagnosis of groundnut and watermelon bud necrosis viruses using polyclonal antiserum to recombinant nucleocapsid protein of Groundnut bud necrosis virus. J Virol Methods 130:162–164

    Article  CAS  PubMed  Google Scholar 

  28. Lian L, Xie L, Zheng L, Lin Q (2011) Induction of systemic resistance in tobacco against Tobacco mosaic virus by Bacillus spp. Biocontrol Sci Technol 21:281–292

    Article  Google Scholar 

  29. Hongwei L, Xueling D, Chao W, Hongjiao KE, Zhou W, Yunpeng W, Hongxia L, Jianhua G (2016) Control of tomato yellow leaf curl virus disease by Enterobacter asburiae BQ9 as a result of priming plant resistance in tomatoes. Turk J Biol 40:150–159

    Article  CAS  Google Scholar 

  30. Gomez-Gomez L, Boller T (2000) FLS2: an LRR receptor-like kinase involved in the perception of the bacterial elicitor flagellin in Arabidopsis. Mol Cell 5:1003–1011

    Article  CAS  PubMed  Google Scholar 

  31. Boller T, Felix G (2009) A renaissance of elicitors: perception of microbe-associated molecular patterns and danger signals by pattern-recognition receptors. Annu Rev Plant Biol 60:379–406

    Article  CAS  PubMed  Google Scholar 

  32. Ciarroni S, Clarke CR, Liu H, Levi NE, Mazzaglia A, Balestr GM, Vinatzer BA (2018) A recombinant flagellin fragment, which includes the epitopes flg22 and flgII-28, provides a useful tool to study flagellin-triggered immunity. J Gen Plant Pathol 84:169–175

    Article  CAS  Google Scholar 

  33. Ma Y, Zhao Y, Berkowit GA (2017) Intracellular Ca2+ is important for flagellin-triggered defense in Arabidopsis and involves inositol polyphosphate signaling. J Exp Bot. https://doi.org/10.1093/jxb/erx176

    Article  PubMed  PubMed Central  Google Scholar 

  34. Rosli HG, Zheng Y, Pombo MA, Zhong S, Bombarely A, Fei Z, Collmer A, Martin GB (2013) Transcriptomics-based screen for genes induced by flagellin and repressed by pathogen effectors identifies a cell wall-associated kinase involved in plant immunity. Genome Biol 14:139

    Article  CAS  Google Scholar 

  35. Paramo GZ, Moctezuma MPC, Pineda EG, Yin S, Chappell J, Gloria EL (2000) Isolation of an elicitor-stimulated 5-epi-aristolochene synthase gene (gPEAS1) from chili pepper (Capsicum annuum). Physiol Plant 110:410–418

    Article  Google Scholar 

  36. Jingyuan Z, Xuexiao Z, Zhenchuan M, Bingyan X (2011) A novel pepper (Capsicum annuum L.) WRKY Gene, CaWRKY30, is involved in pathogen stress responses. J Plant Biol 54:329–337

    Article  CAS  Google Scholar 

  37. Huh SU, Lee GJ, Jung HH, Kim Y, Kim YJ, Paek KH (2015) Capsicum annuum transcription factor WRKYa positively regulates defense response upon TMV infection and is a substrate of CaMK1 and CaMK2. Sci Rep 5:7981. https://doi.org/10.1038/srep07981

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Ward ER, Uknes SJ, Williams SC, Dincher SS, Wiederhold DL, Alexander DC, Ahl-Goy P, Metraux J, Ryals JA (1991) Coordinate gene activity in response to agents that induce systemic acquired resistance. Plant Cell 3:1085–1094

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Lee SC, Hwang BK (2003) Identification of the pepper SAR8.2 gene as a molecular marker for pathogen infection, abiotic elicitors and environmental stresses in Capsicum annuum. Planta 216:387–396

    Article  CAS  PubMed  Google Scholar 

  40. Lee SC, Hwang BK (2006) CASAR82A, a pathogen-induced pepper SAR 8.2, exhibits an antifungal activity and its overexpression enhances disease resistance and stress tolerance. Plant Mol Biol 61:95–109

    Article  CAS  PubMed  Google Scholar 

  41. Lee J, Rudd JJ, Macioszek VK, Scheel D (2004) Dynamic changes in the localization of MAPK cascade components controlling pathogenesis-related (PR) gene expression during innate immunity in parsley. J Biol Chem 279:22440–22448

    Article  CAS  PubMed  Google Scholar 

  42. Hwang IS, Hwang BK (2010) The pepper 9-lipoxygenase gene CaLOXl functions in defense and cell death responses to microbial pathogens. Plant Physiol 152:948–967

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Seo HH, Park S, Park S, Oh BJ, Back K, Han O, Kim JI, Kim YS (2014) Overexpression of a defensin enhances resistance to a fruit-specific anthracnose fungus in pepper. PLoS ONE 9(5):e97936. https://doi.org/10.1371/journal.pone.0097936

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Kachroo P, Yoshioka K, Shah J, Dooner HK, Klessig DF (2000) Resistance to Turnip vrinkle virus in Arabidopsis is regulated by two host genes and is salicylic acid dependent but NPR1, ethylene, and jasmonate independent. Plant Cell 12:677–690

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Ahn IP, Park K, Kim HH (2002) Rhizobacteria-induced resistance perturbs viral disease progress and triggers defense-related gene expression. Mol Cells 13(2):302–308

    CAS  PubMed  Google Scholar 

  46. Murphy JF, Zehnder GW, Schuster DJ, Sikora EJ, Polston JE, Kloepper JW (2000) Plant growth-promoting rhizobacterial mediated protection in tomato against Tomato mottle virus. Plant Dis 84:779–784

    Article  PubMed  Google Scholar 

  47. Vasanthi VJ, Kandan A, Ramanathan A, Raguchander T, Balasubramanian P, Samiyappan R (2000) Induced systemic resistance to Tomato leaf curl virus and increased yield in tomato by plant growth promoting rhizobacteria under field conditions. Arch Phytopathol Plant Prot 43(15):1463–1472

    Article  Google Scholar 

  48. Lee GH, Ryu CM (2016) Spraying of leaf colonizing Bacillus amyloliquefaciens protects pepper from Cucumber mosaic virus. Plant Dis 100(10):2099–2105

    Article  CAS  PubMed  Google Scholar 

  49. Vanthana M, Nakkeeran S, Malathi VG, Renukadevi P, Vinodkumar S (2019) Induction of in planta resistance by flagellin (Flg) and elongation factor-TU (EF-Tu) of Bacillus amyloliquefaciens (VB7) against groundnut bud necrosis virus in tomato. Microb Pathog 137:103757

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The authors are grateful for the financial support provided under “Dr. A.P.J. Abdul Kalam Fellowship” sponsored by School of Post Graduate Studies, Tamil Nadu Agricultural University, Coimbatore-641 003 for accomplishing this work. The authors also acknowledge DST-FIST, Department of Science and Technology, Government of India, New Delhi, for the infrastructure facilities for conducting the experiments.

Author information

Authors and Affiliations

  1. Department of Plant Pathology, Tamil Nadu Agricultural University, Coimbatore, 641 003, India

    S. Rajamanickam & S. Nakkeeran

Authors
  1. S. Rajamanickam
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. S. Nakkeeran
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to S. Rajamanickam.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical standards

This article does not contain any studies with human participants or animals performed by any of the authors.

Additional information

Handling Editor: Massimo Turina.

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 56 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Rajamanickam, S., Nakkeeran, S. Flagellin of Bacillus amyloliquefaciens works as a resistance inducer against groundnut bud necrosis virus in chilli (Capsicum annuum L.). Arch Virol 165, 1585–1597 (2020). https://doi.org/10.1007/s00705-020-04645-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00705-020-04645-z

Search

Navigation