Comunicación presentada en el 5th International Conference on Mycorrhiza, 23-27, July 2006, Granad

Aguirreolea Morales, Jone

Garmendia, Idoia

Goicoechea Preboste, Nieves

Sánchez Díaz, Manuel

Repositorio Institucional de la Universidad de Alicante

DEFENCE-RELATED ENZYMES IN PEPPER ROOTS DURING INTERACTIONS WITH ARBUSCULAR MYCORRHIZAL FUNGI AND/OR  Verticillium dahliaeGarmendia López, I.(*), Aguirreolea Morales, J., Sánchez-Díaz, M. and Goicoechea Preboste, N. Departamento de Biología Vegetal. Facultades de Ciencias y Farmacia. University of Navarra. c/Irunlarrea s/n. 31008-Pamplona(*)Present address: Dpto. Ciencias de la Tierra y del Medio Ambiente. Facultad de Ciencias. University of Alicante. Apto de correos, 99. 03080-AlicanteMATERIAL AND METHODS• Experimental designSEEDSINTRODUCTION The active defence responses of plants include the activation of genes that are coding for enzymes of the phenylpropanoid pathway or for pathogenesis-related (PR) proteins, some of them with chitinase, chitosanase and β-1,3-glucanase activities (Pozo et al., 2002) that degrade wall of many pathogenic fungi (Van Loon and Van Strien, 1999).Previous studies have described that the arbuscular mycorrhizal fungus (AMF) Glomusdeserticola (Trappe, Bloss and Menge) can reduce the deleterious effect of Verticillium dahliae Kleb. on Capsicum annuum L. cv. Piquillogrowth and yield (Garmendia et al., 2004). The establishment of the mycorrhizal association involves the production of plant defence related proteins that could contribute to the bioprotection (Lambais, 2000). Our first objective was to study if V. dahliaeinduced defence mechanisms in roots before infected-pepper developed visible symptoms of disease. The second aim was to determine if AMF induced defence-related enzymatic activities in pepper roots before or after pathogen’s attack.- Disease assessment- AMF colonization- Plant growth parameters- Root enzymatic activities:Phenylalanine ammonia-lyase (PAL) (EC 4.1.1.5)Chitinase (EC 3.2.1.14)β-1,3-glucanase (EC 3.2.1.39)Chitosanase (EC 3.2.1.99)Peroxidase (EC 1.11.1.7)Superoxide dismutase (SOD) (EC 1.15.1.1)DETERMINATIONSTreatments:NM-V: non-mycorrhizal healthy plantsNM+V: non-mycorrhizal plants inoculated with V. dahliaeM-V: mycorrhizal healthy plantsM+V: mycorrhizal plants inoculated with V. dahliaeGrowth conditions:25/15ºC day/night14h photoperiodSoil-sand-vermiculite (1:2.5:2.5 v/v/v) Nutrition: LANS solution 1 monthNon-mycorrhizal(NM)G. deserticola(M)2 monthsTRANSPLANTINGINOCULATION WITH AMFINOCULATION WITH          V. dahliae (+V) Firstharvest7 days after inoculation with V. dahliae14 days after inoculation with V. dahliaeNM-V NM+VM-V M+VTreatments Shoot DM(g plant-1)Root DM(g plant-1)NM 0.87 ± 0.26 a 0.47 ± 0.14 aM 0.90 ± 0.29 a 0.43 ± 0.13 aMycorrhizal colonization(%)-25.32 ± 6.76 Table 1. Mycorrhizal colonization (%), shoot dry matter (DM) (g plant-1) and root DM (g plant-1) in non-mycorrhizal (NM) and mycorrhizal (M) plants just before inoculating Verticillium dahliae. Means ± SD (n = 3 plants) were compared with Student’s t-test within each column. Values followed by a common letter are not significantly different (p ≤ 0.05).Photograph 1. Isolation of V. dahliae from cross–stem sections of inoculated non-mycorrhizal (NM) and mycorrhizal (M) pepper plants on Messiaen culture medium 14 days after the pathogen inoculation. Verticillium-infected plants did not show any visible disease symptom.NM +VM +VFigure 1. Phenylalanine ammonia-lyase (PAL) activities (nmol trans-cinnamic acid g-1 DM h-1) in roots from non-mycorrhizal (NM) (a) and mycorrhizal (M) (b) plants inoculated (+V) or not (-V) with Verticillium dahliae. Means ± SD (n = 3 plants) just before inoculating Verticillium were compared with Student’s t-test and data on days 7 and 14 after pathogen inoculation were analysed by Tukey-b-test. Histograms followed by a common letter are not significantly different (p ≤ 0.05). PAL activities were assayed according to Dunn et al. (1998) with slight modifications.PAL (nmoltrans-cinnamicacidg-1DMh-1)aaba abc01002003004005006007000 7 14Days after pathogen inoculationNM-VNM+Vabbaaba0 7 14M-VM+V(a) (b)Figure 2. Acidic (a) and basic (b) chitinase activities after separation of proteins under native conditions using the Davis and the Reisfeld systems, respectively, in 15% (w/v) polyacrylamide gels containing 0.01% (w/v) glycol chitin as substrate. Roots extracts in McIlvaine buffer (7 µg of protein per sample) from non-mycorrhizal (NM) and mycorrhizal (M) plants inoculated (+V) or not (-V) with Verticillium dahliae were analysed just before inoculating the pathogen as well as 1 and 2 weeks later. Bars indicate constitutive isoforms and arrows mark additional isoforms. Days after pathogen inoculationNM M NM M NM M NM M NM M-V -V+V +V0 7 14(a)(b)Figure 3. Acidic (a) and basic (b) β-1,3-glucanase activities after separation of proteins under native conditions using the Davis and the Reisfeld systems, respectively, in 15% (w/v) polyacrylamide gels containing β-glucans as substrate. Roots extracts in McIlvaine buffer (7 µg of protein per sample) from non-mycorrhizal (NM) and mycorrhizal (M) plants inoculated (+V) or not (-V) with Verticillium dahliaewere analysed just before inoculating the pathogen as well as 1 and 2 weeks later. Bars indicate constitutive isoforms and arrows mark additional isoforms. Days after pathogen inoculation (b)(a)MNM M NM M NM M NM M NM-V -V+V +V0 7 14Figure 4. Peroxidase activity after separation of proteins under native conditions using the Davis system in 15% (w/v) polyacrylamide gels. Root protein extracts in McIlvaine buffer (7 µg of protein per sample) from non-mycorrhizal (NM) and mycorrhizal (M) plants inoculated (+V) or not (-V) with Verticillium dahliae were analysed just before inoculating the pathogen as well as 1 and 2 weeks later. After protein separation, gels were incubated in 0.1 M Tris-HCl (pH 7.6) with 4-chloro-1-naphthol and H2O2. Bars indicate constitutive isoforms and arrows mark additional isoforms. Days after pathogen inoculationMNM M NM M NM M NM M NM-V -V+V +V0 7 14? Mycorrhizal-specific induction of new isoforms of acidic chitinases and SOD together with enhanced PAL and peroxidaseactivities two weeks after pathogen inoculation could be involved in the biocontrolof Verticillium-induced wilt in pepper (C. annuum L. cv. Piquillo) by Glomus deserticola. ? Verticillium dahliae neither stimulated the phenylpropanoid pathway nor elicited hydrolytic activities in non-mycorrhizal pepper roots one and two weeks after its inoculation.? No signal corresponding to chitosanaseactivity was detected in any treatment.ACKNOWLEDGEMENTSThe authors are grateful to the Estación Experimental del Zaidín (CSIC, Granada) and, especially, to Dr. C. Azcón-Aguilar, Dr. R. Calvente. Fundación Universitaria de Navarra (FUNA) and Gobierno Vasco (AE-2000) supported this work.Figure 5. Superoxide dismutase (SOD) isoenzymes after separation of proteins under native conditions using the Davis system in 15% (w/v) polyacrylamide gels. Root protein extracts in McIlvaine buffer (7 µg of protein per sample) from non-mycorrhizal (NM) and mycorrhizal (M) plants inoculated (+V) or not (-V) with Verticillium dahliae were analysed just before inoculating the pathogen as well as 1 and 2 weeks later. Bars indicate constitutive isoforms and arrows mark additional isoforms. Days after pathogen inoculationMNM M NM M NM M NM M-V -V+V0 7 14+VNMCONCLUSIONSREFERENCESDavis BJ (1964) Disc electrophoresis. II. Method and           application to human serum proteins. Ann. NY Acad. Sci. 121: 404-427.Dunn DC, Duncan LW and Romeo JT (1998) Changes in arginine, PAL activity, and nematode behaviour in salinity-stressed citrus. Phytochemistry 49: 413-417.Garmendia I, Goicoechea N and Aguirreolea J (2004) Plant phenology influences the effect of mycorrhizal fungi on the development of Verticillium-induced wilt in pepper. Eur. J. Plant Pathol. 110: 227-238.Lambais MR (2000) Regulation of plant defense-related genes in arbuscular mycorrhizae. In: GK Podila and DD Douds (eds), Current Advances in mycorrhizae Research. APS Press, Minnesota, USA, pp.45-59.Pozo MJ, Slezack-Deschaumes S, Dumas-Gaudot E, Gianinazzi S and Azcón-Aguilar C (2002) Plant defenseresponses induced by arbuscular mycorrhizal fungi. In: S Gianinazzi, H Schüepp, JM Barea and K Haselwandter (eds), Mycorrhizal Technology in Agriculture. From Genes to Bioproducts. Birkhäuser Verlag, Berlin, pp. 525-534.Reisfeld RA, Lewis VJ and Williams DE (1962) Disk electrophoresis of basic proteins and peptides on polyacrylamidegels. Nature 195: 281-283.Van Loon LC and Van Strien EA (1999) The families of pathogenesis-related proteins, their activities, and comparative analysis of PR-1 type protein. Physiol. Mol. Plant Pathol. 55: 85-97.2

Defence-related enzymes in pepper roots during interactions with arbuscular mycorrhizal fungi and/or Verticillium dahliae

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