pp 1–15 | Cite as

Colletotrichum acutatum M11 can suppress the defence response in strawberry plants

  • Rodrigo H. Tomas-Grau
  • Pia Di Peto
  • Nadia R. Chalfoun
  • Carlos F. Grellet-Bournonville
  • Gustavo G. Martos
  • Mario Debes
  • Marta E. Arias
  • Juan C. Díaz-RicciEmail author
Original Article


Main conclusion

Colletotrichum acutatum M11 produces a diffusible compound that suppresses the biochemical, physiological, molecular and anatomical events associated with the defence response induced by the plant defence elicitor AsES.


The fungal pathogen Colletotrichum acutatum, the causal agent of anthracnose disease, causes important economical losses in strawberry crop worldwide and synthetic agrochemicals are used to control it. In this context, the control of the disease using bioproducts is gaining reputation as an alternative of those toxic and pollutant agrochemicals. However, the success of the strategies using bioproducts can be seriously jeopardized in the presence of biological agents exerting a defence suppression effect. In this report, we show that the response defence induced in plant by the elicitor AsES from the fungus Acremonium strictum can be suppressed by a diffusible compound produced by isolate M11 of C. acutatum. Results revealed that strawberry plants treated with conidia of the isolated M11 or the culture supernatant of the isolate M11 suppress: ROS accumulation (e.g., H2O2, O2· and NO), cell wall reinforcement (e.g., lignin and callose), and the up-regulation of defence-related genes (e.g., FaPR1, FaCHI23, FaPDF1.2, FaCAT, FaCDPK, FaCML39) induced by the elicitor AsES. Additionally, we show that the defence suppressing effect causes a systemic sensitization of plants. Results presented here highlights the necessity to make an integral study of the microbiome present in soils and plant biosphere before applying defence activation bioproducts to control crop diseases.


Anthracnose Disease biocontrol Defence elicitor AsES Defence response Fragaria Fungal pathogen Suppressor 



Days post infection


Disease severity rating


Culture filtrate


Potato dextrose broth


Soft mechanical stimulation



This paper was partially supported with grants of the Universidad Nacional de Tucumán (PIUNT 26/D642), Agencia Nacional de Promoción Científica y Tecnológica (PICT 2017-0653), and CONICET (PUE-2016-0104). Authors are grateful to Strawberry Active Germplasm Bank (BGA) from Universidad Nacional de Tucumán (UNT), Cecilia Lemme for providing strawberry plants, and Rafael Gutierrez for technical assistance. RHTG, PDP, and GGM are CONICET fellowships, and NRCh, CFGB, MD and JCDR are members of CONICET.

Supplementary material

425_2019_3203_MOESM1_ESM.jpg (71 kb)
Online Resource S1 Scheme of plant treatments to estimate M11-CF nature (JPEG 70 kb)
425_2019_3203_MOESM2_ESM.jpg (100 kb)
Online Resource S2 Table with primers used in qPCR (JPEG 100 kb)
425_2019_3203_MOESM3_ESM.jpg (72 kb)
Online Resource S3 Effect of the co-inoculation of the isolate SS71 of A. strictum with the virulent isolate M11 of C. acutatum through time (JPEG 72 kb)
425_2019_3203_MOESM4_ESM.jpg (67 kb)
Online Resource S4 Viability of strawberry mesophyllic cells after treatments with M11-CF, SS71-CF and M11-CF + SS71-CF. Different letters represent statistically different values (Tukey test, P < 0.05) (JPEG 67 kb)
425_2019_3203_MOESM5_ESM.jpg (88 kb)
Online Resource S5 Micrographs of conidial suspensions of the isolate SS71 of A. strictum resuspended in PDB (Mock), M11-CF, SS71-CF, M11-CF + SS71-CF or Switch 0.0008%, at 0, 4 and 24 h post treatment (40x). Micrographies are representative of each treatment (Bars = 100 µm). Inserts showing fungal conidia or mycelia hyphae are magnifications of the corresponding micrographs (JPEG 87 kb)
425_2019_3203_MOESM6_ESM.jpg (90 kb)
Online Resource S6 Effect of M11-CF treated with Proteinase K and M11-CF filtered by 1-kDa cut-off membrane. DSR of strawberry plants cv. Pájaro infected with a conidia suspension (1.5 × 106 conidia mL−1) of the virulent isolate M11 of C. acutatum pretreated with the culture filtrates M11-CF treated with a proteinase K and b M11-CF filtered by 1-kDa cut-off membrane. Plants were inoculated with active M11 conidia 48 h post primary treatment with the culture filtrates as mentioned above. Evaluations were performed at 9, 21 and 30 days post infection. Three independent assays were performed (n = 6). Different letters represent statistically different DSR values (Tukey test, P < 0.05) (JPEG 89 kb)
425_2019_3203_MOESM7_ESM.jpg (87 kb)
Online Resource S7 Effect of M11-CF on DCFH fluorescence. a Relative fluorescence change of DCFH (■), DCFH + M11-CF plus Fe2+ (10 µm) and H2O2 (100 µM) (▲), and DCFH + M11-CF without Fe2+ and H2O2 (♦). Fluorescence was measured during 10 min at λex 485 nm and λem 525 nm. b Relative change of DCFH fluorescence measured after 10 min. Each value is an average (± SD) of 5 independent replicates. Different letters represent statistically different values (Tukey test, P < 0.05) (JPEG 86 kb)
425_2019_3203_MOESM8_ESM.jpg (65 kb)
Online Resource S8 M11-CF antioxidant activity. The analysis was carried out by evaluating the reduction of DPPH absorbance at 517 nm. Different concentrations of ascorbic acid (e.g. 1, 5, 10 and 50 µM) were used as standards and PDB was used as control. Three independent assays were performed. Different letters represent statistically different values (Tukey test, P < 0.05) (JPEG 64 kb)


  1. Amil-Ruiz F, Blanco-Portales R, Muñoz-Blanco J, Caballero JL (2011) The strawberry plant defense mechanism: a molecular review. Plant Cell Physiol 52:1873–1903. Google Scholar
  2. Amil-Ruiz F, Garrido-Gala J, Blanco-Portales R, Folta KM, Muñoz-Blanco J, Caballero JL (2013) Identification and validation of reference genes for transcript normalization in strawberry (Fragaria ananassa) defense responses. PLoS ONE 8(8):e70603Google Scholar
  3. Anterola AM, Lewis NG (2002) Trends in lignin modification: a comprehensive analysis of the effects of genetic manipulations/mutations on lignification and vascular integrity. Phytochemistry 61:221–294Google Scholar
  4. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72(1–2):248–254Google Scholar
  5. Bushnell WR (1981) Suppressors of Defense Reactions: A Model for Roles in Specificity. Phytopathology 71(10):1012Google Scholar
  6. Cannon PF, Damm U, Johnston PR, Weir BS (2012) Colletotrichum—current status and future directions. Stud Mycol 73:181–213. Google Scholar
  7. Cessna SG, Sears VE, Dickman MB, Low PS (2000) Oxalic acid, a pathogenicity factor for Sclerotinia sclerotiorum, suppresses the oxidative burst of the host plant. Plant Cell 12:2191–2199. Google Scholar
  8. Chalfoun NR, Castagnaro AP, Díaz Ricci JC (2011) Induced resistance activated by a culture filtrate derived from an avirulent pathogen as a mechanism of biological control of anthracnose in strawberry. Biol Control 58:319–329. Google Scholar
  9. Chalfoun NR, Grellet-Bournonville CF, Martínez-Zamora MG et al (2013) Purification and characterization of AsES protein: a subtilisin secreted by Acremonium strictum is a novel plant defense elicitor. J Biol Chem 288:14098–14113. Google Scholar
  10. Chalfoun NR, Durman SB, Budeguer F et al (2018a) Development of PSP1, a biostimulant based on the elicitor AsES for disease management in monocot and dicot crops. Front Plant Sci 9:1–22. Google Scholar
  11. Chalfoun NR, Durman SB, González-montaner J et al (2018b) Elicitor-based biostimulant PSP1 protects soybean against late season diseases in field trials. Front Plant Sci 9:763. Google Scholar
  12. Creelman RA, Mullet JE (1997) Biosynthesis and action of jasmonates in plants. Annu Rev Plant Physiol Plant Mol Biol 48:355–381. Google Scholar
  13. Cui J, Bahrami AK, Pringle EG et al (2005) Pseudomonas syringae manipulates systemic plant defenses against pathogens and herbivores. Proc Natl Acad Sci USA 102:1791–1796. Google Scholar
  14. Dean R, Van Kan JAL, Pretorius ZA et al (2012) The top 10 fungal pathogens in molecular plant pathology. Mol Plant Pathol 13:414–430. Google Scholar
  15. Delp BR, Milholland RD (1980) Evaluating strawberry plants for resistance to Colletotrichum fragariae. Plant Dis 64(12):1071Google Scholar
  16. Di Rienzo JA (2011) fgStatistics. Statistical software for the analysis of experiments of functional genomics.
  17. Doke N (1983) Involvement of superoxide anion generation in the hypersensitive response of potato tuber tissues to infection with an incompatible race of Phytophthora infestans and to the hyphal wall components. Physiol Plant Pathol 23(3):345–357Google Scholar
  18. Dubiella U, Seybold H, Durian G et al (2013) Calcium-dependent protein kinase/NADPH oxidase activation circuit is required for rapid defense signal propagation. Proc Natl Acad Sci USA 110(21):8744–8799. Google Scholar
  19. Furio RN, Albornoz PL, Coll Y et al (2018) Effect of natural and synthetic brassinosteroids on strawberry immune response against Colletotrichum acutatum. Eur J Plant Pathol 153:167–181. Google Scholar
  20. Geng X, Cheng J, Gangadharan A, Mackey D (2012) The coronatine toxin of Pseudomonas syringae is a multifunctional suppressor of Arabidopsis defense. Plant Cell 24:4763–4774. Google Scholar
  21. Geng X, Jin L, Shimada M et al (2014) The phytotoxin coronatine is a multifunctional component of the virulence armament of Pseudomonas syringae. Planta 240:1149–1165. Google Scholar
  22. Graham MY, Weidner J, Wheeler K et al (2003) Induced expression of pathogenesis-related protein genes in soybean by wounding and the Phytophthora sojae cell wall glucan elicitor. Physiol Mol Plant Pathol 63:141–149. Google Scholar
  23. Guerrero-Molina MF, Lovaisa NC, Salazar SM et al (2015) Physiological, structural and molecular traits activated in strawberry plants after inoculation with the plant growth-promoting bacterium Azospirillum brasilense REC3. Plant Biol 17:766–773. Google Scholar
  24. Guidarelli M, Carbone F, Mourgues F, Perrotta G, Rosati C, Bertolini P, Baraldi E (2011) Colletotrichum acutatum interactions with unripe and ripe strawberry fruits and differential responses at histological and transcriptional levels. Plant Pathol 60(4):685–697Google Scholar
  25. Hael-Conrad V, Abou-Mansour E, Díaz-Ricci JC et al (2015) The novel elicitor AsES triggers a defense response against Botrytis cinerea in Arabidopsis thaliana. Plant Sci 241:120–127. Google Scholar
  26. Hael-Conrad V, Perato SM, Arias ME et al (2017) The elicitor protein AsES induces a systemic acquired resistance response accompanied by systemic microbursts and micro–hypersensitive responses in Fragaria ananassa. Mol Plant Microbe Interact 31:46–60. Google Scholar
  27. Heller J, Tudzynski P (2011) Reactive oxygen species in phytopathogenic fungi: signaling, development, and disease. Annu Rev Phytopathol 49:369–390. Google Scholar
  28. Houterman PM, Cornelissen BJC, Rep M, Cormack BP (2008) Suppression of plant resistance gene-based immunity by a fungal effector. PLoS Pathog 4(5):e1000061Google Scholar
  29. Hukkanen AT, Kokko HI, Buchala AJ, McDougall GJ, Stewart D, Kärenlampi SO, Karjalainen RO (2007) Benzothiadiazole induces the accumulation of phenolics and improves resistance to powdery mildew in strawberries. J Agric Food Chem 55(5):1862–1870Google Scholar
  30. Kårlund A, Salminen JP, Koskinen P et al (2014) Polyphenols in strawberry (Fragaria × ananassa) leaves induced by plant activators. J Agric Food Chem 62:4592–4600. Google Scholar
  31. La Verde V, Dominici P, Astegno A (2018) Towards understanding plant calcium signaling through calmodulin-like proteins: a biochemical and structural perspective. Int J Mol Sci 19:1–18. Google Scholar
  32. Lay FT, Anderson MA (2005) Defensins—components of the innate immune system in plants. Curr Protein Pept Sci 6:85–101. Google Scholar
  33. Lee S, Ishiga Y, Clermont K, Mysore KS (2013a) Coronatine inhibits stomatal closure and delays hypersensitive response cell death induced by nonhost bacterial pathogens. PeerJ 1:e34. Google Scholar
  34. Lee S, Yang DS, Uppalapati SR et al (2013b) Suppression of plant defense responses by extracellular metabolites from Pseudomonas syringae pv. tabaci in Nicotiana benthamiana. BMC Plant Biol 13:65. Google Scholar
  35. Maffei ME, Mithöfer A, Boland W (2007) Before gene expression: early events in plant–insect interaction. Trends Plant Sci 12:310–316. Google Scholar
  36. Mamaní A, Filippone MP, Grellet C et al (2012) Pathogen-induced accumulation of an ellagitannin elicits plant defense response. Mol Plant Microbe Interact 25:1430–1439. Google Scholar
  37. Martos GG, del Terán M, Díaz Ricci JC (2015) The defence elicitor AsES causes a rapid and transient membrane depolarization, a triphasic oxidative burst and the accumulation of nitric oxide. Plant Physiol Biochem 97:443–450. Google Scholar
  38. Martos GG, Mamani A, Filippone MP et al (2018) Ellagitannin HeT obtained from strawberry leaves is oxidized by bacterial membranes and inhibits the respiratory chain. FEBS Open Biol 8:211–218. Google Scholar
  39. McCann HC, Nahal H, Thakur S, Guttman DS (2012) Identification of innate immunity elicitors using molecular signatures of natural selection. Proc Natl Acad Sci USA 109:4215–4220. Google Scholar
  40. Melotto M, Underwood W, Koczan J et al (2006) Plant stomata function in innate immunity against bacterial invasion. Cell 126:969–980. Google Scholar
  41. Mena AJ, De Garcia EP, González MA (1974) Presencia de la antracnosis de la frutilla en la República Argentina. Rev Agron NOA 11:307–312Google Scholar
  42. Mittler R (2017) ROS are good. Trends Plant Sci 22:11–19. Google Scholar
  43. Mittler R, Vanderauwera S, Suzuki N et al (2011) ROS signaling: the new wave? Trends Plant Sci 16:300–309. Google Scholar
  44. Mónaco ME, Salazar SM, Aprea A et al (2000) First report of Colletotrichum gloeosporioides on strawberry in north- western Argentina. Plant Dis 84:595Google Scholar
  45. Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15(3):473–497Google Scholar
  46. Myhre O, Andersen JM, Aarnes H, Fonnum F (2003) Evaluation of the probes 2′,7′-dichlorofluorescin diacetate, luminol, and lucigenin as indicators of reactive species formation. Biochem Pharmacol 65:1575–1582. Google Scholar
  47. Peralta DR, Adler C, Corbalán NS et al (2016) Enterobactin as part of the oxidative stress response repertoire. PLoS One 11:1–15. Google Scholar
  48. Perato SM, Martínez-zamora MG, Salazar SM, Díaz-Ricci JC (2018) The elicitor AsES stimulates ethylene synthesis, induce ripening and enhance protection against disease naturally produced in avocado fruit. Sci Hortic (Amsterdam) 240:288–292. Google Scholar
  49. Pfaffl MW (2001) A new mathematical model for relative quantification in real time RT-PCR. Nucleic Acids Res 29:45Google Scholar
  50. Ranf S, Eschen-Lippold L, Pecher P et al (2011) Interplay between calcium signalling and early signalling elements during defence responses to microbe- or damage-associated molecular patterns. Plant J 68:100–113. Google Scholar
  51. Ramallo CJ, Ploper LD, Ontivero M et al (2000) First report of Colletotrichum acutatum on Strawberry in Northwestern Argentina. Plant Dis 84(6):706Google Scholar
  52. Ramakers C, Ruijter JM, Lekanne Deprez RH, Moorman AFM (2003) Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data. Neurosci Lett 339(1):62–66Google Scholar
  53. Rigano LA, Payette C, Brouillard G et al (2007) Bacterial cyclic β-(1,2)-glucan acts in systemic suppression of plant immune responses. Plant Cell 19:2077–2089. Google Scholar
  54. Romeis T, Herde M (2014) From local to global: CDPKs in systemic defense signaling upon microbial and herbivore attack. Curr Opin Plant Biol 20:1–10. Google Scholar
  55. Salazar SM, Castagnaro AP, Arias ME et al (2007) Induction of a defense response in strawberry mediated by an avirulent strain of Colletotrichum. Eur J Plant Pathol 117:109–122. Google Scholar
  56. Sanders D, Pelloux J, Brownlee C, Harper JF (2002) Calcium at the crossroads of signaling. Plant Cell 14:S401–S417. Google Scholar
  57. Singleton VL, Orthofer R, Lamuela-Raventos RM (1999) Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin-Ciocalteu reagent. Methods Enzymol 299:152–178Google Scholar
  58. Smith BJ (1990) Morphological, cultural, and pathogenic variation among colletotrichum species isolated from strawberry. Plant Dis 74(1):69Google Scholar
  59. Thordal-Christensen H, Zhang Z, Wei Y, Collinge DB (1997) Subcellular localization of H2O2 in plants. H2O2 accumulation in papillae and hypersensitive response during the barley-powdery mildew interaction. Plant J 11(6):1187–1194Google Scholar
  60. Thomma BPHJ, Cammue BPA, Thevissen K (2002) Plant defensins. Planta 216:193–202. Google Scholar
  61. Tomas-Grau RH, Requena-Serra FJ, Hael-Conrad V et al (2018) Soft mechanical stimulation induces a defense response against Botrytis cinerea in strawberry. Plant Cell Rep 37:239–250. Google Scholar
  62. Torres MA, Jones JDG, Dangl JL (2006) Reactive oxygen species signaling in response to pathogens. Plant Physiol 141:373–378. Google Scholar
  63. Tortora ML, Díaz-Ricci JC, Pedraza RO (2012) Protection of strawberry plants (Fragaria ananassa Duch.) against anthracnose disease induced by Azospirillum brasilense. Plant Soil 356:279–290. Google Scholar
  64. Toum L, Torres PS, Gallego SM et al (2016) Coronatine inhibits stomatal closure through guard cell-specific inhibition of NADPH oxidase-dependent ROS production. Front Plant Sci 7:1–12. Google Scholar
  65. van Loon LC, Rep M, Pieterse CMJ (2006) Significance of inducible defense-related proteins in infected plants. Annu Rev Phytopathol 44:135–162. Google Scholar
  66. Vellicce GR, Díaz Ricci JC, Hernández L, Castagnaro AP (2006) Enhanced resistance to botrytis cinerea mediated by the transgenic expression of the chitinase gene ch5B in strawberry. Transgenic Res 15(1):57–68Google Scholar
  67. Wiesel L, Newton AC, Elliott I et al (2014) Molecular effects of resistance elicitors from biological origin and their potential for crop protection. Front Plant Sci 5:1–13. Google Scholar
  68. Williams B, Kabbage M, Kim HJ et al (2011) Tipping the balance: Sclerotinia sclerotiorum secreted oxalic acid suppresses host defenses by manipulating the host redox environment. PLoS Pathog 7(6):e1002107. Google Scholar
  69. Wood RKS (1984) Establishment of infection. Plant Pathol 33:3–12. Google Scholar
  70. Yun MH (2006) Xanthan induces plant susceptibility by suppressing callose deposition. Plant Physiol 141:178–187. Google Scholar
  71. Zeng W, Brutus A, Kremer JM et al (2011) A genetic screen reveals Arabidopsis stomatal and/or apoplastic defenses against Pseudomonas syringae pv. tomato DC3000. PLoS Pathog 7(10):e1002291. Google Scholar
  72. Zheng XY, Spivey NW, Zeng W, Liu PP, Fu ZQ, Klessig DF, He SY, Dong X (2012) Coronatine promotes pseudomonas syringae virulence in plants by activating a signaling cascade that inhibits salicylic acid accumulation. Cell Host Microbe 11(6):587–596Google Scholar
  73. Zhou J, Wang B, Zhu L (2005) Conditioned culture for protoplasts isolated from chrysanthemum: an efficient approach. Colloids Surf B Biointerfaces 45:113–119. Google Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Rodrigo H. Tomas-Grau
    • 1
  • Pia Di Peto
    • 1
  • Nadia R. Chalfoun
    • 1
  • Carlos F. Grellet-Bournonville
    • 1
  • Gustavo G. Martos
    • 1
  • Mario Debes
    • 2
  • Marta E. Arias
    • 2
  • Juan C. Díaz-Ricci
    • 1
    Email author
  1. 1.Instituto Superior de Investigaciones Biológicas (INSIBIO), CONICET-UNT, and Instituto de Química Biológica “Dr. Bernabé Bloj”, Facultad de Bioquímica, Química y Farmacia, UNTSan Miguel de TucumánArgentina
  2. 2.Cátedra de Anatomía Vegetal, Facultad de Ciencias Naturales e Instituto Miguel LilloUniversidad Nacional de TucumánTucumánArgentina

Personalised recommendations