Plant Inoculation with the Fungal Leaf Pathogen Colletotrichum higginsianum

  • Kei Hiruma
  • Yusuke SaijoEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1398)


After Colletotrichum storage methods in Chapter  23, we describe here experimental methods for the inoculation of Colletotrichum higginsianum (C.h.) on Arabidopsis leaves. We put a particular focus on the methods for lesion measurements after the drop-inoculation of the leaves and on C.h. biomass measurements in the leaves by RT-qPCR analysis. As an option, we also briefly describe methods for counting C.h. entry ratio.

Key words

Colletotrichum higginsianum Arabidopsis Lesion measurements Biomass measurements RT-qPCR 


  1. 1.
    O'Connell R, Herbert C, Sreenivasaprasad S, Khatib M, Esquerre-Tugaye MT, Dumas B (2004) A novel Arabidopsis-Colletotrichum pathosystem for the molecular dissection of plant-fungal interactions. Mol Plant Microbe Interact 17(3):272–282CrossRefPubMedGoogle Scholar
  2. 2.
    Narusaka Y, Narusaka M, Park P, Kubo Y, Hirayama T, Seki M, Shiraishi T, Ishida J, Nakashima M, Enju A, Sakurai T, Satou M, Kobayashi M, Shinozaki K (2004) RCH1, a locus in Arabidopsis that confers resistance to the hemibiotrophic fungal pathogen Colletotrichum higginsianum. Mol Plant Microbe Interact 17(7):749–762CrossRefPubMedGoogle Scholar
  3. 3.
    O'Connell RJ, Thon MR, Hacquard S, Amyotte SG, Kleemann J, Torres MF, Damm U, Buiate EA, Epstein L, Alkan N, Altmuller J, Alvarado-Balderrama L, Bauser CA, Becker C, Birren BW, Chen Z, Choi J, Crouch JA, Duvick JP, Farman MA, Gan P, Heiman D, Henrissat B, Howard RJ, Kabbage M, Koch C, Kracher B, Kubo Y, Law AD, Lebrun MH, Lee YH, Miyara I, Moore N, Neumann U, Nordstrom K, Panaccione DG, Panstruga R, Place M, Proctor RH, Prusky D, Rech G, Reinhardt R, Rollins JA, Rounsley S, Schardl CL, Schwartz DC, Shenoy N, Shirasu K, Sikhakolli UR, Stuber K, Sukno SA, Sweigard JA, Takano Y, Takahara H, Trail F, van der Does HC, Voll LM, Will I, Young S, Zeng Q, Zhang J, Zhou S, Dickman MB, Schulze-Lefert P, Loren V, van Themaat E, Ma LJ, Vaillancourt LJ (2012) Lifestyle transitions in plant pathogenic Colletotrichum fungi deciphered by genome and transcriptome analyses. Nat Genet 44(9):1060–1065CrossRefPubMedGoogle Scholar
  4. 4.
    Ross A, Yamada K, Hiruma K, Yamashita-Yamada M, Lu X, Takano Y, Tsuda K, Saijo Y (2014) The Arabidopsis PEPR pathway couples local and systemic plant immunity. EMBO J 33(1):62–75PubMedCentralCrossRefPubMedGoogle Scholar
  5. 5.
    Narusaka M, Shirasu K, Noutoshi Y, Kubo Y, Shiraishi T, Iwabuchi M, Narusaka Y (2009) RRS1 and RPS4 provide a dual Resistance-gene system against fungal and bacterial pathogens. Plant J 60(2):218–226CrossRefPubMedGoogle Scholar
  6. 6.
    Hiruma K, Fukunaga S, Bednarek P, Pislewska-Bednarek M, Watanabe S, Narusaka Y, Shirasu K, Takano Y (2013) Glutathione and tryptophan metabolism are required for Arabidopsis immunity during the hypersensitive response to hemibiotrophs. Proc Natl Acad Sci U S A 110(23):9589–9594PubMedCentralCrossRefPubMedGoogle Scholar
  7. 7.
    Huser A, Takahara H, Schmalenbach W, O’Connell R (2009) Discovery of pathogenicity genes in the crucifer anthracnose fungus Colletotrichum higginsianum, using random insertional mutagenesis. Mol Plant Microbe Interact 22(2):143–156CrossRefPubMedGoogle Scholar
  8. 8.
    Hiruma K, Nishiuchi T, Kato T, Bednarek P, Okuno T, Schulze-Lefert P, Takano Y (2011) Arabidopsis ENHANCED DISEASE RESISTANCE 1 is required for pathogen-induced expression of plant defensins in nonhost resistance, and acts through interference of MYC2-mediated repressor function. Plant J 67(6):980–992CrossRefPubMedGoogle Scholar
  9. 9.
    Schneider CA, Rasband WS, Eliceiri KW (2012) NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9(7):671–675CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Graduate School of Biological SciencesNara Institute of Science and TechnologyIkomaJapan
  2. 2.JST, Precursory Research for Embryonic Science and TechnologyChiyoda-kuJapan

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