Skip to main content
SpringerLink
Log in

An efficient protocol for inoculation of Fusarium graminearum in detached leaves of Arabidopsis

  • Original Article
  • Published:
Tropical Plant Pathology Aims and scope Submit manuscript

Abstract

Fusarium graminearum is the main cause of Fusarium head blight, a fungal disease that reduces yield and affects the quality of wheat and other small grains. Genetic resistance is the ideal method for disease control; however, there are not yet cultivars with sufficient resistance levels to withstand an epidemic. Genetic engineering strategies such as gene silencing, host-induced gene silencing (HIGS), overexpression, and genome editing are promising, but many target genes need to be analyzed to find a suitable one. Arabidopsis thaliana is a model plant that is also susceptible to F. graminearum. Although this interaction was reported nearly two decades ago, consistent infection and symptoms are not always obtained. The availability of an efficient inoculation method is essential for studying plant-pathogen interaction. This work aimed at testing protocols for inoculating F. graminearum in detached leaves of A. thaliana which varied in inoculation site (leaf sides), wound (abrasion or hole injury), and inoculum type (mycelium agar disk or spore). We found that a mycelium agar disk placed on an abrasion injury at the adaxial leaf side was the most efficient (highest lesion size, incidence, and easiness to visualize the symptoms caused by the pathogen) method to inoculate F. graminearum in detached leaves of Arabidopsis.

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

Similar content being viewed by others

Data availability

All data generated or analyzed during this study are included in this published article and its supplementary information files.

References

  • Alisaac E, Rathgeb A, Karlovsky P, Mahlein AK (2020) Fusarium Head Blight: effect of infection timing on spread of Fusarium graminearum and spatial distribution of deoxynivalenol within wheat spikes. Microorganisms 9:79

    Article  PubMed Central  CAS  Google Scholar 

  • Andrade CM, Tinoco MLP, Rieth AF, Maia FCO, Aragão FJL (2016) Host-induced gene silencing in the necrotrophic fungal pathogen Sclerotinia sclerotiorum. Plant Pathology 65:626–632

    Article  CAS  Google Scholar 

  • Bates D, Mächler M, Bolker B, Walker S (2015) Fitting linear mixed-effects models using lme4. Journal of Statistical Software 67:1–48

    Article  Google Scholar 

  • Bian C, Duan Y, Wang J, Xiu Q, Wang J, Hou Y, Song X, Zhou M (2020) Validamycin A induces broad-spectrum resistance involving salicylic acid and jasmonic acid/ethylene signaling pathways. Molecular Plant-Microbe Interactions 33:1424–1437

    Article  CAS  PubMed  Google Scholar 

  • Boyes DC, Zayed AM, Ascenzi R, Mccaskill AJ, Hoffman NE, Davis KR, Görlach J (2001) Growth stage-based phenotypic analysis of Arabidopsis: a model for high throughput functional genomics in plants. The Plant Cell 13:1499–1510

    CAS  PubMed  PubMed Central  Google Scholar 

  • Boenisch MJ, Schäfer W (2011) Fusarium graminearum forms mycotoxin producing infection structures on wheat. BMC Plant Biology 11:110

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Brewer HC, Hammond-Kosack K (2015) Host to a stranger: arabidopsis and fusarium ear blight. Trends in Plant Science 20:651–663

    Article  CAS  PubMed  Google Scholar 

  • Brown NA, Urban M, van de Meene AM, Hammond-Kosack KE (2020) The infection biology of Fusarium graminearum: defining the pathways of spikelet to spikelet colonisation in wheat ears. Fungal Biology 114:555–571

    Article  Google Scholar 

  • Chen J, Ullah C, Reichelt M, Beran F, Yang ZL, Gershenzon J, Hammerbacher A, Vassão DG (2020) The phytopathogenic fungus Sclerotinia sclerotiorum detoxifies plant glucosinolate hydrolysis products via an isothiocyanate hydrolase. Nature Communications 11:3090

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Chen X, Steed A, Harden C, Nicholson P (2006) Characterization of Arabidopsis thaliana-Fusarium graminearum interactions and identification of variation in resistance among ecotypes. Molecular Plant Pathology 7:391–403

    Article  CAS  PubMed  Google Scholar 

  • Cuzick A, Urban M, Hammond-Kosack K (2008) Fusarium graminearum gene deletion mutants map1 and tri5 reveal similarities and differences in the pathogenicity requirements to cause disease on Arabidopsis and wheat floral tissue. New Phytologist 177:990–1000

    Article  PubMed  Google Scholar 

  • Del Ponte EM, Spolti P, Ward TJ, Gomes LB, Nicolli CP, Kuhnem PR, Silva CN, Tessmann DJ (2015) Regional and field-specific factors affect the composition of Fusarium head blight pathogens in subtropical no-till wheat agroecosystem of Brazil. Phytopathology 105:246–254

    Article  PubMed  CAS  Google Scholar 

  • Doukhanina EV, Chen S, van der Zalm E, Godzik A, Reed J, Dickman MB (2006) Identification and functional characterization of the BAG protein family in Arabidopsis thaliana. Journal of Biological Chemistry 281:18793–18801

    Article  CAS  PubMed  Google Scholar 

  • Fernández-Bautista N, Domínguez-Núñez JA, Moreno MMC, Berrocal-Lobo M (2016) Plant tissue trypan blue staining during phytopathogen infection. Bio-protocol 6:e2078

    Article  Google Scholar 

  • Guo X, Stotz HU (2007) Defense against Sclerotinia sclerotiorum in Arabidopsis is dependent on jasmonic acid, salicylic acid, and ethylene signaling. Molecular Plant-Microbe Interactions 20:1384–1395

    Article  CAS  PubMed  Google Scholar 

  • Hao G, Bakker MG, Kim HS (2020) Enhanced resistance to Fusarium graminearum in transgenic Arabidopsis plants expressing a modified plant thionin. Phytopathology 110:1056–1066

    Article  CAS  PubMed  Google Scholar 

  • Höfle L, Biedenkopf D, Werner BT, Shrestha A, Jelonek L, Koch A (2019) Study on the efficiency of dsRNAs with increasing length in RNA-based silencing of the Fusarium CYP51 genes. RNA Biology 17:463–473

    Article  CAS  Google Scholar 

  • Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337:816–821

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kidd BN, Foley R, Singh KB, Anderson JP (2021) Foliar resistance to Rhizoctonia solani in Arabidopsis is compromised by simultaneous loss of ethylene, jasmonate and PEN2 mediated defense pathways. Scientific Reports 11:2546

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Koch A, Khalifa W, Langen G, Vilcinskas A, Kogel KH, Imani J (2012) The antimicrobial peptide thanatin reduces fungal infections in Arabidopsis. Journal of Phytopathology 160:606–610

    Article  CAS  Google Scholar 

  • Koch A, Kumar N, Weber L, Keller H, Imani J, Kogel K-H (2013) Host induced gene silencing of cytochrome P450 lanosterol C14α-demethylase-encoding genes confers strong resistance to Fusarium species. Proceedings of the National Academy of Sciences 110:19324-19329

  • Lima MIPM (2010) Giberela e brusone em cereais de inverno. In: Santos HP, Fontaneli RS, Spera ST (eds) Sistema de produção para cereais de inverno sob plantio direto no Sul do Brasil. Embrapa trigo, Passo Fundo, pp 207–224

    Google Scholar 

  • Machado Wood AK, Panwar V, Grimwade-Mann M, Ashfield T, Hammond-Kosack KE, Kanyuka K (2021) The vesicular trafficking system component MIN7 is required for minimizing Fusarium graminearum infection. Journal of Experimental Botany 72:5010–5023

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Makandar R, Nalam V, Chaturvedi R, Jeannotte R, Sparks AA, Shah J (2010) Involvement of salicylate and jasmonate signaling pathways in Arabidopsis interaction with Fusarium graminearum. Molecular Plant-Microbe Interactions 23:861–870

    Article  CAS  PubMed  Google Scholar 

  • Manes N, Brauer EK, Hepworth S, Subramaniam R (2021) MAMP and DAMP signaling contributes resistance to Fusarium graminearum in Arabidopsis. Journal of Experimental Botany 72:6628–6639

    Article  PubMed  Google Scholar 

  • McMullen M, Bergstrom G, De Wolf E, Dill-Macky R, Hershman D, Shaner G, Van Sanford D (2012) A unified effort to fight an enemy of wheat and barley: Fusarium Head Blight. Plant Disease  96:1712–1728

    Article  PubMed  Google Scholar 

  • McMullen M, Jones R, Gallenberg D (1997) Scab of wheat and barley: a re-emerging disease of devastating impact. Plant Disease  81:1340–1348

    Article  PubMed  Google Scholar 

  • Mentges M, Glasenapp A, Boenisch M, Malz S, Henrissat B, Frandsen RJN, Güldener U, Münsterkötter M, Bormann J, Lebrun MH, Schäfer W, Martinez-Rocha AL (2020) Infection cushions of Fusarium graminearum are fungal arsenals for wheat infection. Molecular Plant Pathology 21:1070–1087

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Miller SS, Chabot DMP, Ouellet T, Harris LJ, Fedak G (2004) Use of a Fusarium graminearum strain transformed with green fluorescent protein to study infection in wheat (Triticum aestivum). Canadian Journal of Plant Pathology 26:453–463

    Article  CAS  Google Scholar 

  • Nalam VJ, Alam S, Keereetaweep J, Venables B, Burdan D, Lee H, Trick HN, Sarowar S, Makandar R, Shah J (2015) Facilitation of Fusarium graminearum infection by 9-lipoxygenases in Arabidopsis and wheat. Molecular Plant-Microbe Interactions 28:1142–1152

    Article  CAS  PubMed  Google Scholar 

  • NASC (n.d.) Available at: http://arabidopsis.info/StockInfo?NASC_id=20. Accessed 24 Feb 2021

  • Nguyen TTX, Dehne HW, Steiner U (2016) Maize leaf trichomes represent an entry point of infection for Fusarium species. Fungal Biology 120:895–903

    Article  CAS  PubMed  Google Scholar 

  • Paul NC, Park SW, Liu H, Choi S, Ma J, MacCready JS, Chilvers MI, Sang H (2021) Plant and fungal genome editing to enhance plant disease resistance using the CRISPR/Cas9 system. Frontiers in Plant Science 12:700925

    Article  PubMed  PubMed Central  Google Scholar 

  • Ruan Y, Zhang W, Knox RE, Berraies S, Campbell HL, Ragupathy R, Boyle K, Polley B, Henriquez MA, Burt A, Kumar S, Cuthbert RD, Fobert PR, Buerstmayr H, Depauw RM (2020) Characterization of the genetic architecture for Fusarium head blight resistance in durum wheat: the complex association of resistance, flowering time, and height genes. Frontiers in Plant Science 11:1–17

    Article  Google Scholar 

  • Goswami RS, Kistler HC (2004) Heading for disaster: Fusarium graminearum on cereal crops. Molecular Plant Pathology 5:515–525

    Article  CAS  PubMed  Google Scholar 

  • Sarowar S, Alam ST, Makandar R, Lee H, Trick HN, Dong Y, Shah J (2019) Targeting the pattern-triggered immunity pathway to enhance resistance to Fusarium graminearum. Molecular Plant Pathology 20:626–664

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Skadsen RW, Hohn TM (2004) Use of Fusarium graminearum transformed with gfp to follow infection patterns in barley and Arabidopsis. Physiological and Molecular Plant Pathology 64:45–53

    Article  CAS  Google Scholar 

  • Stevens RB (1960) Cultural practices in disease control. In: Horsfall JG, Dimond AE (eds) Plant pathology, an advanced treatise. Academic, New York, pp 357–429

    Chapter  Google Scholar 

  • Stotz HU, Jikumaru Y, Shimada Y, Sasaki E, Stingl N, Mueller MJ, Kamiya Y (2011) Jasmonate-dependent and COI1-independent defense responses against Sclerotinia sclerotiorum in Arabidopsis thaliana: auxin is part of COI1-independent defense signaling. Plant & Cell Physiology 52:1941–1956

    CAS  Google Scholar 

  • Urban M, Daniels S, Mott E, Hammond-Kosack K (2002) Arabidopsis is susceptible to the cereal ear blight fungal pathogens Fusarium graminearum and Fusarium culmorum. The Plant Journal 32:961–973

    Article  CAS  PubMed  Google Scholar 

  • Wang F, Li X, Li Y, Han J, Chen Y, Zeng J, Su M, Zhuo J, Ren H, Liu H, Hou L, Fan Y, Yan X, Song S, Zhao J, Jin D, Zhang M, Pei Y (2021) Arabidopsis P4 ATPase-mediated cell detoxification confers resistance to Fusarium graminearum and Verticillium dahliae. Nature Communications 12:6426

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wang Q, Buxa SV, Furch A, Friedt W, Gottwald S (2015) Insights into Triticum aestivum seedling root rot caused by Fusarium graminearum. Molecular Plant-Microbe Interactions 28:1288–1303

    Article  CAS  PubMed  Google Scholar 

  • Wang Y, Bouwmeester K, van de Mortel JE, Shan W, Govers F (2013) A novel Arabidopsis-oomycete pathosystem: differential interactions with Phytophthora capsici reveal a role for camalexin, indole glucosinolates and salicylic acid in defence. Plant Cell & Environment 36:1192–1203

    Article  CAS  Google Scholar 

  • Zhang Y, Bai Y, Wu G, Zou S, Chen Y, Gao C, Tang D (2017) Simultaneous modification of three homoeologs of TaEDR1 by genome editing enhances powdery mildew resistance in wheat. The Plant Journal 91:714–724

    Article  CAS  PubMed  Google Scholar 

Download references

Funding

We thank the Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA) and UK Biotechnology and Biological Sciences Research Council (BBSRC) for funding the work through the bilateral BBSRC–EMBRAPA grants (22.15.07.003.00.00 and BB/N018095/1, respectively) and Coordination for the Improvement of Higher Education Personnel (CAPES) for the doctoral scholarship. We thank Dr. Márcio Alves Ferreira for providing the A. thaliana ecotype Ler.

Author information

Authors and Affiliations

  1. Programa de Pós-Graduação em Agronomia, Universidade de Passo Fundo, Rio Grande do Sul, 99052-900, Passo Fundo, Brazil

    Eduardo André Roesler & Carolina Cardoso Deuner

  2. Programa de Pós-Graduação em Genética e Biologia Molecular, Universidade Federal do Rio Grande do Sul, 91501-970, Porto Alegre, Brazil

    Natalia Balbinott

  3. Faculdade de Agronomia e Medicina Veterinária, Universidade de Passo Fundo, Passo Fundo, 99052-900, Rio Grande do Sul, Brazil

    Renata Gabriela Schroeder

  4. Embrapa Trigo, 99050-970, Passo Fundo, Rio Grande do Sul, Brazil

    Elene Yamazaki-Lau, Maria Imaculada Pontes Moreira Lima, Sandra Patussi Brammer & José Maurício Cunha Fernandes

  5. Embrapa Recursos Genéticos e Biotecnologia, 70770-917, Brasília, Distrito Federal, Brazil

    Francisco José Lima Aragão

Authors
  1. Eduardo André Roesler
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Natalia Balbinott
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Renata Gabriela Schroeder
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Elene Yamazaki-Lau
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Maria Imaculada Pontes Moreira Lima
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Sandra Patussi Brammer
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Francisco José Lima Aragão
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. José Maurício Cunha Fernandes
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Carolina Cardoso Deuner
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Conceptualization: EAR, MIPML, EYL, FJLA; formal analysis: EAR; funding acquisition: EYL, JMCF; investigation: EAR, NB, RGS, EYL; methodology: EAR, EYL, MIPML; project administration: JMCF; resources: EYL; supervision: EYL, CCD, MIPML, SPB; visualization: EAR; writing (original draft): EAR, NB, EYL, CCD.

Corresponding author

Correspondence to Elene Yamazaki-Lau.

Ethics declarations

Conflict of interest

On behalf of all authors, the corresponding author declares no competing interests.

Additional information

Publisher’s note

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

Supplementary Information

Below is the link to the electronic supplementary material.

ESM 1

(CSV 13.5 KB)

ESM 2

(DOCX 27.4 KB

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Roesler, E.A., Balbinott, N., Schroeder, R.G. et al. An efficient protocol for inoculation of Fusarium graminearum in detached leaves of Arabidopsis. Trop. plant pathol. 47, 353–361 (2022). https://doi.org/10.1007/s40858-022-00497-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s40858-022-00497-x

Keywords

Search

Navigation