Natural extracts from pepper, wild rue and clove can activate defenses against pathogens in tomato plants

Abstract

Tomato is an important species grown in many countries, either in fields or greenhouses. Despite decades of improvement, it is still susceptible to diseases, thus requiring the use of chemical pesticides, especially in greenhouses. Nevertheless it is imperative to reduce the use of environmental-unfriendly phytochemicals and favor less toxic tools to fight pathogens. Plants possess elaborate mechanisms against diseases that can lead to resistance. In the present work, we investigate the induction of plant defenses by means of extracts from plants widespread and easy to find, also known for their antimicrobial properties. Aqueous extracts of pepper ‘Rocoto’, wild rue and ethanolic extracts of clove powder (whose inhibiting effect was assessed on Oidium sp. spores) were tested on tomato plants for their ability to induce expression of different defense genes (PRs and regulatory proteins) after spraying. As revealed by RT-qPCR, all extracts were able to induce mRNA accumulation of different PR and MAPK regulators for several hours upon treatment, with clove and wild rue being the strongest. This effect could also be reproduced in tomato plants after a second treatment, 15 days after the first. The same extracts were tested in tomato and tobacco plants via leaf infiltration, showing necrotic symptoms associated with the hypersensitive response, thus confirming the priming capacity of the extracts. The involvement of salicylic acid (SA) in these responses was verified by HPLC analysis and in SA-depleted transgenic tobacco (NahG). The results obtained suggest that natural antimicrobial extracts can be used to induce plant defenses and protect valuable crops. At the same time these low-cost extracts do not pose a threat to the environment or the farmer and can help reduce the farming costs, especially in developing countries.

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Abbreviations

BABA:

β-aminobutirric acid

BTH:

benzo (1,2,3) thiadiazole-7-carbothioic acid

GUS:

β-glucuronidase

INA:

2,6-dichloroisonicotinic acid

JA:

jasmonic acid

MAPK:

mitogen-activated protein kinase

NaDC:

sodium deoxycholate

PR:

pathogenesis-related proteins

RT-qPCR:

reverse transcription quantitative PCR

SA:

salicylic acid

WT:

wild type

References

  1. Al-Ani, R. A., Adhab, M. A., & Nawar, H. H. (2012). Antibacterial activity of clove, cinnamon, and datura extracts against Erwinia carotovora subsp. atroseptica causative agent of black stem and soft rot on potato. Journal of Medicinal Plant Research, 6, 1891–1895.

    Google Scholar 

  2. Baccelli, I., & Mauch-Mani, B. (2016). Beta-aminobutyric acid priming of plant defense: the role of ABA and other hormones. Plant Molecular Biology, 91, 703–711. doi:10.1007/s11103-015-0406-y.

    CAS  Article  PubMed  Google Scholar 

  3. Balasubramanian, V., Vashisht, D., Cletus, J., & Sakthivel, N. (2012). Plant β-1,3-glucanases: their biological functions and transgenic expression against phytopathogenic fungi. Biotechnology Letters, 34, 1983–1990. doi:10.1007/s10529-012-1012-6.

    CAS  Article  PubMed  Google Scholar 

  4. Benouaret, R., Goujon, E., Trivella, A., Richard, C., Ledoigt, G., Joubert, J. M., Mery-Bernardon, A., & Goupil, P. (2014). Water extracts from winery by-products as tobacco defense inducers. Ecotox., 23, 1574–1581. doi:10.1007/s10646-014-1298-3.

    CAS  Article  Google Scholar 

  5. Borges, A. A., & Sandalio, L. M. (2015). Induced resistance for plant defense. Frontiers in Plant Science, 6, 109. doi:10.3389/fpls.2015.00109.

    Article  PubMed  PubMed Central  Google Scholar 

  6. Careaga, M., Fernández, E., Dorantes, L., Mota, L., Jaramillo, M. E., & Hernandez-Sanchez, H. (2003). Antibacterial activity of capsicum extract against Salmonella typhimurium and Pseudomonas aeruginosa inoculated in raw beef meat. International Journal of Food Microbiology, 83, 331–335.

    Article  PubMed  Google Scholar 

  7. Chaturvedi R., Venables B., Petros R.A., Nalam V., Li M., Wang X., Takemoto L. J and Shah J. (2012). An abietane diterpenoid is a potent activator of systemic acquired resistance. The Plant Journal 71, 161–172

  8. Conrath, U. (2009). Priming of induced plant defense responses. Advances in Botanical Research, 51, 61–395. doi:10.1016/S0065-2296(09)51009-9.

    Google Scholar 

  9. Conrath, U., Beckers, G. J. M., Langenbach, C. J. G., & Jaskiewicz, M. R. (2015). Priming for enhanced defense. Annual Review of Phytopathology, 53, 97–119.

    CAS  Article  PubMed  Google Scholar 

  10. Dangl, J. L., Horvath, D. M., & Staskawicz, B. J. (2013). Pivoting the plant immune system from dissection to deployment. Science, 341, 746–751. doi:10.1126/science.1236011.

    CAS  Article  PubMed  Google Scholar 

  11. Danhash, N., Wagemakers, C. A., van Kan, J. A., & de Wit, P. J. (1993). Molecular characterization of four chitinase cDNAs obtained from Cladosporium Fulvum-infected tomato. Plant Molecular Biology, 22, 1017–1029.

    CAS  Article  PubMed  Google Scholar 

  12. Degrave, A., Fagard, M., Perino, C., Brisset, M. N., Gaubert, S., Laroche, S., Patrit, O., & Barny, M.-A. (2008). Erwinia amylovora type three–secreted proteins trigger cell death and defense responses in Arabidopsis thaliana. Molecular Plant-Microbe Interactions, 21, 1076–1086.

    CAS  Article  PubMed  Google Scholar 

  13. Denancé, N., Sánchez-Vallet, A., Goffner, D., & Molina, A. (2013). Disease resistance or growth: the role of plant hormones in balancing immune responses and fitness costs. Frontiers in Plant Science, 4, 155. doi:10.3389/fpls.2013.00155.

    Article  PubMed  PubMed Central  Google Scholar 

  14. Dorantes, L., Colmenero, R., Hernandez, H., Mota, L., Jaramillo, M. E., Fernandez, E., & Solano, C. (2000). Inhibition of growth of some foodborne pathogenic bacteria by Capsicum annum extracts. International Journal of Food Microbiology, 57, 125–128.

    Article  Google Scholar 

  15. Floryszak-Wieczorek, J., Magdalena, A.-J. M., & Abramowski, D. (2015). BABA-primed defense responses to Phytophthora infestans in the next vegetative progeny of potato. Frontiers in Plant Science, 6, 844. doi:10.3389/fpls.2015.00844.

    Article  PubMed  PubMed Central  Google Scholar 

  16. Friedrich, L., Vernooij, B., Gaffney, T., Morse, A., & Ryals, J. (1995). Characterization of tobacco plants expressing a bacterial salicylate hydroxylase gene. Plant Molecular Biology, 29, 959–968.

    CAS  Article  PubMed  Google Scholar 

  17. Frost, C. J., Mescher, M. C., Carlson, J. E., & De Moraes, C. M. (2008). Plant defense priming against herbivores: getting ready for a different battle. Plant Physiology, 146, 818–824. doi:10.1104/pp.107.113027. geNorm .https://genorm.cmgg.be/

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  18. Glazebrook, J. (2005). Contrasting mechanisms of defense against biotrophic and necrotrophic pathogens. Annual Review of Phytopathology, 43, 205–227. doi:10.1146/annurev.phyto.43.040204.135923.

    CAS  Article  PubMed  Google Scholar 

  19. Görlach, J., Volrath, S., Knauf-Beiter, G., Hengy, G., Beckhove, U., Kogel, K.-H., Oostendorp, M., Staub, T., Ward, E., Kessmann, H., & Ryals, J. (1996). Benzothiadiazole, a novel class of inducers of systemic acquired resistance, activates gene expression and disease resistance in wheat. The Plant Cell, 8, 629–643.

    Article  PubMed  PubMed Central  Google Scholar 

  20. Grüner, R., & Pfitzner, U. M. (1994). The upstream region of the gene for the pathogenesis-related protein 1a from tobacco responds to environmental as well as to developmental signals in transgenic plants. European Journal of Biochemistry, 220, 247–255.

    Article  PubMed  Google Scholar 

  21. Herms, S., Seehaus, K., Koehle, H., & Conrath, U. (2002). A strobilurin fungicide enhances the resistance of tobacco against tobacco mosaic virus and pseudomonas syringae pv tabaci. Plant Physiology, 130, 120–127.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  22. INEC (Instituto Nacional de Estadística y Censos) (2012). Encuesta de Producción Agropecuaria Continua: http://www.ecuadorencifras.gob.ec/documentos/web-inec/Estadisticas_agropecuarias/espac/Publicaciones-espac/INDICE_PUBLICACION%20ESPAC%202012.xlsx.

  23. Jaramillo J., Rodríguez V., Guzmán M., Zapata M., & Rengifo T. (2007). Manual técnico: buenas prácticas agrícolas (BPA) en la producción de tomate bajo condiciones protegidas. FAO online document. ftp://ftp.fao.org/docrep/fao/010/a1374s/a1374s02.pdf.

  24. Jeschke, P. (2016). Progress of modern agricultural chemistry and future prospects. Pest Management Science, 72, 433–455. doi:10.1002/ps.4190.

    CAS  Article  PubMed  Google Scholar 

  25. Kessmann, H., Staub, T., Hofmann, C., Maetzke, T., Herzog, J., Ward, E., Uknes, S., & Ryals, J. (1994). Induction of systemic acquired disease resistance in plants by chemicals. Annual Review of Phytopathology, 32, 439–459.

    CAS  Article  PubMed  Google Scholar 

  26. Kong, F., Wang, J., Cheng, L., Liu, S., Wu, J., Peng, Z., & Lu, G. (2012). Genome-wide analysis of the mitogen-activated protein kinase gene family in Solanum lycopersicum. Gene, 499, 108–120.

    CAS  Article  PubMed  Google Scholar 

  27. Meng, X., & Zhang, S. (2013). MAPK cascades in plant disease resistance signaling. Annual Review of Phytopathology, 51, 245–266.

    CAS  Article  PubMed  Google Scholar 

  28. Mitsuhara, I., Iwai, T., Seo, S., Yanagawa, Y., Kawahigasi, H., Hirose, S., Ohkawa, Y., & Ohashi, Y. (2008). Characteristic expression of twelve rice PR1 family genes in response to pathogen infection, wounding, and defense-related signal compounds. Molecular Genetics and Genomics, 279, 415–427.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  29. Moloudizargari, M., Mikaili, P., Aghajanshakeri, S., Asghari, M. H., & Shayegh, J. (2013). Pharmacological and therapeutic effects of Peganum harmala and its main alkaloids. Pharmacognosy Reviews, 7, 199–212. doi:10.4103/0973-7847.120524.

    Article  PubMed  PubMed Central  Google Scholar 

  30. Nakagami, H., Pitzschke, A., & Hirt, H. (2005). Emerging MAP kinase pathways in plant stress signalling. Trends in Plant Science, 10, 339–346.

    CAS  Article  PubMed  Google Scholar 

  31. Noda, J., Brito, N., & González, C. (2010). The Botrytis Cinerea xylanase Xyn11A contributes to virulence with its necrotizing activity, not with its catalytic activity. BMC Plant Biology, 10, 38. doi:10.1186/1471-2229-10-38.

    Article  PubMed  PubMed Central  Google Scholar 

  32. Pandey, A., & Singh, P. (2011). Antibacterial activity of Syzygium aromaticum (clove) with metal ion effect against food borne pathogens. Asian Journal of Plant Sciences Research, 1, 69–80.

    Google Scholar 

  33. Saeidi, S., Amini, B. N., Ahmadi, H., & Hassanshahian, M. (2015). Antibacterial activity of some plant extracts against extended- spectrum beta-lactamase producing Escherichia coli isolates. Jundishapur Journal of Microbiology, 8, e15434. doi:10.5812/jjm.15434.

    Article  PubMed  PubMed Central  Google Scholar 

  34. Sarpeleh, A., Sharifi, K., & Sonbolkar, A. (2009). Evidence of antifungal activity of wild rue (Peganum harmala L.) on phytopathogenic fungi. Journal of Plant Diseases and Protection, 116, 208–213.

    Article  Google Scholar 

  35. Seiber, J. N., Coats, J., Duke, S. O., & Gross, A. D. (2014). Biopesticides: state of the art and future opportunities. Journal of Agricultural and Food Chemistry, 62, 11613–11619. doi:10.1021/jf504252n.

    CAS  Article  PubMed  Google Scholar 

  36. Sels, J., Mathys, J., De Coninck, B. M. A., Cammue, B. P. A., & De Bolle, M. F. C. (2008). Plant pathogenesis-related (PR) proteins: a focus on PR peptides. Plant Physiology and Biochemistry, 46, 941–950.

    CAS  Article  PubMed  Google Scholar 

  37. Shah, J., Chaturvedi, R., Chowdhury, Z., Venables, B., & Petros, R. A. (2014). Signaling by small metabolites in systemic acquired resistance. The Plant Journal, 79, 645–658.

    CAS  Article  PubMed  Google Scholar 

  38. Spoel, S. H., & Dong, X. (2012). How do plants achieve immunity? Defence without specialized immune cells. Nature Reviews Immunology, 12, 89–100. doi:10.1038/nri3141.

    CAS  Article  PubMed  Google Scholar 

  39. Stulemeijer, I. J. E., Stratmann, J. W., & Joosten, M. H. A. I. (2007). Tomato mitogen-activated protein kinases LeMPK1, LeMPK2, and LeMPK3 are activated during the Cf-4/Avr4-induced hypersensitive response and have distinct phosphorylation specificities. Plant Physiology, 144, 1481–1494.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  40. Tornero, P., Gadea, J., Conejero, V., & Vera, P. (1997). Two PR-1 genes from tomato are differentially regulated and reveal a novel mode of expression for a pathogenesis-related gene during the hypersensitive response and development. Molecular Plant-Microbe Interactions, 10, 624–634.

    CAS  Article  PubMed  Google Scholar 

  41. Tuzun, S., & Somanchi, A. (2006). The possible role of PR proteins in multigenic and induced systemic resistance. In S. Tuzun & E. Bent (Eds.), Multigenic and induced systemic resistance in plants vol 6 (pp. 112–142). NY: Springer.

    Google Scholar 

  42. van Kan, J. A., Joosten, M. H., Wagemakers, C. A., van den Berg-Velthuis, G. C., & de Wit, P. J. (1992). Differential accumulation of mRNAs encoding extracellular and intracellular PR proteins in tomato induced by virulent and avirulent races of Cladosporium Fulvum. Plant Molecular Biology, 20, 513–527.

    Article  PubMed  Google Scholar 

  43. Van Loon, L. C., & Van Strien, E. A. (1999). The families of pathogenesis-related protein, their activities, and comparative analysis of PR-1 type proteins. Physiological and Molecular Plant Pathology, 55, 85–97.

  44. Vandesompele J., De Preter K., Pattyn F., Poppe B., Van Roy N., De Paepe A. and Speleman F. (2002). Accurate normalization of Real-Time quantitative RT–PCR data by geometric averaging of multiple internal control genes. Genome Biology 3: research0034.1.

  45. Verhagen, B. W., Van Loon, L. C., & Pieterse, C. M. (2006). Induced disease resistance signaling in plants, global science books. Floriculture, Ornamental and Plant Biotechnology, 3, 334–343.

    Google Scholar 

  46. Zarattini, M., De Bastiani, M., Bernacchia, G., Ferro, S., & De Battisti, A. (2015). The use of ECAS in plant protection: a green and efficient antimicrobial approach that primes selected defense genes. Ecotox., 24, 1996–2008. doi:10.1007/s10646-015-1535-4.

    CAS  Article  Google Scholar 

  47. Zarattini, M., Launay, A., Farjad, M., Wénès, E., Taconnat, L., Boutet, S., Bernacchia, G., & Fagard, M. (2016). The bile acid deoxycholate elicits defenses in Arabidopsis and reduces bacterial infection. Molecular Plant Pathology. doi:10.1111/mpp.12416.

    PubMed  Google Scholar 

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Acknowledgements

G. B. would like to thank the Proyecto Prometeo of Senescyt (Secretaría de Educación Superior, Ciencia, Tecnología e Innovación) of Ecuador for funding this work.

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Correspondence to G. Bernacchia.

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Malo, I., De Bastiani, M., Arevalo, P. et al. Natural extracts from pepper, wild rue and clove can activate defenses against pathogens in tomato plants. Eur J Plant Pathol 149, 89–101 (2017). https://doi.org/10.1007/s10658-017-1168-y

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Keywords

  • Quantitative PCR
  • Resistance
  • Antimicrobial
  • Priming
  • Pathogenesis-related proteins