, 162:337 | Cite as

Characterization of an extracellular endopolygalacturonase from the saprobe Mucor ramosissimus Samutsevitsch and its action as trigger of defensive response in tropical plants

  • Maria Rita Marques
  • Marcos S. Buckeridge
  • Marcia R. Braga
  • Sonia M. C. DietrichEmail author


In recent years, interest in the ability of non-pathogenic microorganisms to induce resistance in plants has grown, particularly with respect to their use as environmentally safe controllers of plant disease. In this study, we investigated the capacity of Mucor ramosissimus Samutsevitsch to release pectinases able to degrade cell walls of Palicourea marcgravii St. Hil., a tropical forest native Rubiaceae on which the spores of this saprobic fungus have been found. The fungus was grown in liquid culture medium containing pectin as the sole carbon source and filtrates were analyzed for pectinase activity. An endopolygalacturonase was partially purified by ion exchange chromatography, gel filtration, and preparative isoelectrofocusing, and characterized. This enzyme was more active upon pectic substrates with a low degree of methyl esterification. The products of hydrolysis of different pectic substrates (including pectin from P. marcgravii) by the action of this endopolygalacturonase elicited to different extents the phytoalexin production in soybean cotyledons. Also, the enzyme itself and the products of its action on the pectic fraction of P. marcgravii elicited the production of defensive compounds in the leaves of the plant. These results suggest that, besides the role in recycling organic matter, saprobes may also play an important role in the induction of defensive mechanisms in wild plants by enhancing their non-specific resistance against pathogens. Furthermore, they set the stage for future studies on the role of saprobic fungi in inducing resistance of host plants to pathogens.


cell wall polysaccharides elicitor oligogalacturonides Palicourea marcgravii St. Hil. pectinases Rubiaceae 


  1. 1.
    Moore, JC, Berlow, EL, Coleman, DC, Ruiter, PC, Dong, Q, Johnson, NC, McCann, KS, Melville, K, Morin, PJ, Nadelhofer, K, Rosemond, AD, Post, DM, Sabo, JL, Scow, KM, Vanni, MJ, Wall, DH 2004Detritus, trophic dynamics, and biodiversityEcol Lett7584600CrossRefGoogle Scholar
  2. 2.
    Guenoune, D, Galili, S, Phillips, DA, Volpin, H, Chetokon, Y, Kapulnik, Y 2001The defense response elicited by pathogen Rhizoctonia solani is suppressed by colonization of the AM-fungus Glomus intradices Plant Sci160925932CrossRefPubMedGoogle Scholar
  3. 3.
    Yedidia, I, Shoresh, M, Kerem, Z, Benhamou, N, Kapulnik, Y, Chet, I 2003Concomitant induction of systemic resistance to Pseudomonas syringae pv. lachrymans in cucumber by Trichoderma asperllum (T-203) and the accumulation of phytoalexinsAppl Environ Microbiol6973437353CrossRefPubMedGoogle Scholar
  4. 4.
    Janowicz, K, Mazurkiewicz-Zapalowicz, K, Kaup, G, Kuzna-Grygiel, W 2004Effect of host plant and saprophytic soil fungi on population size of Globodera rostochiensis (Wool.) BehrensRuss J Nematol1218Google Scholar
  5. 5.
    Yedidia, I, Benhamou, N, Chet, I 1999Induction of defense responses in cucumber plants (Cucumis sativus L.) by the biocontrol agent Trichoderma harzianum Appl Environ Microbiol6510611070PubMedGoogle Scholar
  6. 6.
    Howell, CR 2003Mechanisms employed by Trichoderma species in the biological control of plant diseases: The history and evolution of current conceptsPlant Dis8747CrossRefGoogle Scholar
  7. 7.
    Knooge, W 1996Fungal infection of plantsPlant Cell817111722CrossRefGoogle Scholar
  8. 8.
    Dunville, JC, Fry, SC 2000Uronic acid-containing oligosaccharins: Their biosynthesis, degradation, and signaling roles in non-diseased plant tissuesPlant Physiol Biochem38125140CrossRefGoogle Scholar
  9. 9.
    Poinssot, B, Vandelle, E, Bentéjac, M, Adian, M, Levis, C, Brygoo, Y, Garin, J, Sicili, F, Coutos-Thévenot, P, Pugin, A 2003The endopolygalacturonase 1 from Botritys cinerea activates grapevine defense reactions unrelated to this enzymatic activityAm Phytopathol Soc16553564Google Scholar
  10. 10.
    Braga, MR, Young, MCM, Ponte, JVA, Emerenciano, VP, Dietrich, SMC, Gottlieb, OR 1986Phytoalexin induction in plants of tropical environmentBiochem Syst Ecol14507514CrossRefGoogle Scholar
  11. 11.
    Braga, MR, Dietrich, SMC 1991Atividade eliciadora de fitoalexinas em esporos de Trichoderma pseudokoningii RifaiHoehnea18170187Google Scholar
  12. 12.
    Cordeiro-Neto, F, Dietrich, SMC 1992Phytoalexin induction by fungi from the leaf surface of tropical RubiaceaeCiên Cult44342344Google Scholar
  13. 13.
    Costa, APP, Dietrich, SMC 1996Phytoalexin eliciting activity in phytopathogenic and saprophytic fungi — a comparisonCiên Cult48275277Google Scholar
  14. 14.
    Garcéz, WS, Martins, D, Garcéz, FR, Marques, MR, Pereira, AAP, Oliveira, LA, Rondon, JN, Peruca, AD 2000Effect of spores of saprophytic fungi on phytoalexin accumulation in seeds of frogeye spot and canker-resistant and susceptible soybean (Glycine max L.) cultivarsJ Agric Food Chem4836623665CrossRefPubMedGoogle Scholar
  15. 15.
    Ellis, DH 1997ZygomycetesTopley, TWilson, W eds. Topley and Wilson’s Microbiology and Microbial InfectionsEdward ArnoldLondon247277Google Scholar
  16. 16.
    Hesseltine, CW, Anderson, RF 1957Microbiological production of carotenoids. I. Zygospores and carotene produced by intraspecific and interspecifc clones of Choanephoraceae in liquid mediaMycologia49449452CrossRefGoogle Scholar
  17. 17.
    Edwards, M, Dea, ICM, Bulpin, PV, Reid, JSG 1985Xyloglucan (amyloid) mobilisation in the cotyledons of Tropaeolum majus L. seeds following germinationPlanta163133140CrossRefGoogle Scholar
  18. 18.
    English, PD, Maglothin, A, Keegstra, K, Albersheim, P 1972A cell wall-degrading endopolygalacturonase secreted by Colletotrichum lindemuthianum Plant Physiol49293297CrossRefPubMedGoogle Scholar
  19. 19.
    Somogyi, MA 1945New reagent for the determination of sugarJ Biol Chem1606168Google Scholar
  20. 20.
    Konno, H 1988Endopectate lyase from Erwinia aroideae Meth Enzymol161381385CrossRefPubMedGoogle Scholar
  21. 21.
    Fry, SC 1988The Growing Plant Cell Wall: Chemical and Metabolic AnalysisLongmanNew YorkGoogle Scholar
  22. 22.
    Bradford, MM 1976A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein–dye bindingAnal Biochem72248254CrossRefPubMedGoogle Scholar
  23. 23.
    Laemmli, UK 1970Cleavage of the structural protein during the assembly of the head of bacteriophage T4Nature223680685CrossRefGoogle Scholar
  24. 24.
    Dion, AS, Pomenti, AA 1983Ammoniacal silver staining of proteins: Mechanisms of glutaraldehyde enhancementAnal Biochem129490496CrossRefPubMedGoogle Scholar
  25. 25.
    Karr, AL, Albersheim, P 1970Polysaccharide-degrading enzymes are unable to attack plant cell wall without prior action of a wall-modifying enzymePlant Physiol466980CrossRefPubMedGoogle Scholar
  26. 26.
    Braga, MR, Pessoni, RAB, Dietrich, SMC 1998Cell wall polysaccharide composition of leaves of tropical Rubiaceae differing in phytoalexin responseBraz J Plant Physiol107178Google Scholar
  27. 27.
    Moraes, F, Hahn, MG, Braga, MR 2001Comparative analysis of leaf cell-wall polysaccharides of Dialypetalanthus fuscescens and Bathysa meridionalis: Evidence of biochemical similarities between Dialypetalanthaceae and Rubiaceae-CinchonoideaeRevta Brasil Bot24289294CrossRefGoogle Scholar
  28. 28.
    Filisetti-Cozzi, TMCC, Carpita, NC 1991Measurement of uronic acids without interference from neutral sugarsAnal Biochem197157162CrossRefPubMedGoogle Scholar
  29. 29.
    Spiro, MD, Kates, KA, Koller, AL, O’Neill, MA, Albersheim, P, Darvill, AG 1993Purification and characterization of biologically active 1,4-linked α-d-oligogalacturonides after partial digestion of poligalacturonic acid with endopolygalacturonaseCarbohydr Res247920CrossRefGoogle Scholar
  30. 30.
    Ayers, A, Ebel, J, Finelli, F, Berger, N, Albersheim, P 1976Host–pathogen interactions. IX. Quantitative assays of elicitor activity and characterization of the elicitor present in the extracellular medium of cultures of Phytophthora megasperma var. sojae Plant Physiol57751759CrossRefPubMedGoogle Scholar
  31. 31.
    Hahn, MG, Darvill, AG, Albersheim, P 1981Host–pathogen interactions. XIX. The endogenous elicitor, a fragment of a plant cell wall polysaccharide that elicits phytoalexin accumulation in soybeansPlant Physiol6811611169CrossRefPubMedGoogle Scholar
  32. 32.
    Braga, MR, Young, MCM, Ponte, JVA, Dietrich, SMC, Gottilieb, OR 1986Phytoalexin induction in plants of tropical environmentBiochem Syst Ecol14507514CrossRefGoogle Scholar
  33. 33.
    Machinandiarena, MF, Wolsk, EA, Barrera, V, Daleo, GR, Andreu, AB 2005Characterization and in vitro expression of extracellular degradative enzymes from non-pathogenic binucleate Rhizoctonia AG-GMycopathologia159441448CrossRefPubMedGoogle Scholar
  34. 34.
    Annis, SI, Goodwin, PH 1997Recent advances in the molecular genetics of plant cell wall-degrading enzymes produced by plant pathogenic fungiEur J Plant Pathol103114CrossRefGoogle Scholar
  35. 35.
    Riou, C, Freyssinet, G, Fevre, M 1992Purification and characterization of extracellular pectinolytic enzymes produced by Sclerotinia sclerotiorum Appl Environ Microbiol58578583PubMedGoogle Scholar
  36. 36.
    Borin, MDF, Said, S, Fonseca, MJV 1996Purification and biochemical characterization of an extracellular endopolygalacturonase from Penicillium frequentans J Agric Food Chem4416161620CrossRefGoogle Scholar
  37. 37.
    Mohamed, SA, Christensen, TMIE, Mikkelsen, JD 2003New polygalacturonases from Trichoderma reesei: Characterization and their specificities to partially methylated and acetylated pectinsCarbohydr Res338515524CrossRefPubMedGoogle Scholar
  38. 38.
    Markovic, O, Janecek, S 2001Pectin degrading glycoside hydrolases of family 28: Sequence-structural specificities and evolutionProtein Eng14615631CrossRefPubMedGoogle Scholar
  39. 39.
    Silva, CA, Braga, MR 2004Liberação e atividade de moléculas indutoras de fitoalexinas em rubiáceas tropicais: influência da metilesterificação das pectinasRevta Brasil Bot27379393CrossRefGoogle Scholar
  40. 40.
    McAllister, CB, Garcia-Romera, I, Godeas, A, Ocampo, JA 1994Interactions between Trichoderma konningii, Fusarium solani and Glomus mosseae: Effects on plant growth, arbuscular mycorrhizas and the saprophyte inoculantsSoil Biol Biochem2613631367CrossRefGoogle Scholar
  41. 41.
    Camprubi, A, Calvet, C, Estaun, V 1995Growth enhancement of Citrus reshni after inoculation with Glomus intradices and Trichoderma aureoviride and associated effects on microbial populations and enzyme activity in potting mixesPlant Soil173233238CrossRefGoogle Scholar
  42. 42.
    Asiegbu, FO, Denekamp, M, Daniel, G, Johansson, M 1995Immunocytochemical localization of pathogenesis-related proteins in roots of Norway spruce infected with Heterobasidium annosum Eur J Forest Pathol25169178CrossRefGoogle Scholar
  43. 43.
    Heil, M 2002Ecological costs of induced resistanceCurr Opin Plant Biol5345350CrossRefPubMedGoogle Scholar

Copyright information

© Springer 2006

Authors and Affiliations

  • Maria Rita Marques
    • 1
  • Marcos S. Buckeridge
    • 2
  • Marcia R. Braga
    • 3
  • Sonia M. C. Dietrich
    • 3
    • 4
    Email author
  1. 1.Universidade Federal do Mato Grosso do SulCampo GrandeBrazil
  2. 2.Universidade de São PauloSão PauloBrazil
  3. 3.Instituto de BotânicaSeção de Fisiologia e Bioquímica de PlantasSão PauloBrazil
  4. 4.Instituto de BotânicaSeção de Fisiologia e Bioquímica de PlantasSão PauloBrazil

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