Revista Argentina de Microbiología ISSN: 0325-7541 [email protected] Asociación Argentina de Microbiología Argentina Ramos, Araceli M.; Gally, Marcela; Szapiro, Gala; Itzcovich, Tatiana; Carabajal, Maira; Levin, Laura In vitro growth and cell wall degrading enzyme production by Argentinean isolates of Macrophomina phaseolina, the causative agent of charcoal rot in corn Revista Argentina de Microbiología, vol. 48, núm. 4, octubre-diciembre, 2016, pp. 267-273 Asociación Argentina de Microbiología Buenos Aires, Argentina Available in: http://www.redalyc.org/articulo.oa?id=213049175002 How to cite Complete issue Scientific Information System More information about this article Network of Scientific Journals from Latin America, the Caribbean, Spain and Portugal Journal's homepage in redalyc.org Non-profit academic project, developed under the open access initiative Rev Argent Microbiol. 2016;48(4):267---273 R E V I S T A A R G E N T I N A D E MICROBIOLOGÍA www.elsevier.es/ram ORIGINAL ARTICLE In vitro growth and cell wall degrading enzyme production by Argentinean isolates of Macrophomina phaseolina , the causative agent of charcoal rot in corn a,∗ b a a Araceli M. Ramos , Marcela Gally , Gala Szapiro , Tatiana Itzcovich , a a Maira Carabajal , Laura Levin a Departamento de Biodiversidad y Biología Experimental, Facultad de Ciencias Exactas y Naturales, INMIBO-CONICET, Universidad de Buenos Aires, Ciudad Autónoma de Buenos Aires, Argentina b Cátedra de Fitopatología, Facultad de Agronomía, Universidad de Buenos Aires, Ciudad Autónoma de Buenos Aires, Argentina Received 20 May 2015; accepted 20 June 2016 Available online 5 November 2016 KEYWORDS Abstract Macrophomina phaseolina is a polyphagous phytopathogen, causing stalk rot on Macrophomina many commercially important species. Damages caused by this pathogen in soybean and maize phaseolina; crops in Argentina during drought and hot weather have increased due its ability to survive Charcoal rot; as sclerotia in soil and crop debris under non-till practices. In this work, we explored the Corn; in vitro production of plant cell wall-degrading enzymes [pectinases (polygalacturonase and Cell wall-degrading polymethylgalacturonase); cellulases (endoglucanase); hemicellulases (endoxylanase) and the enzymes ligninolytic enzyme laccase] by several Argentinean isolates of M. phaseolina, and assessed the pathogenicity of these isolates as a preliminary step to establish the role of these enzymes in M. phaseolina---maize interaction. The isolates were grown in liquid synthetic medium supple- mented with glucose, pectin, carboxymethylcellulose or xylan as carbon sources and/or enzyme inducers and glutamic acid as nitrogen source. Pectinases were the first cell wall-degrading enzymes detected and the activities obtained (polygalacturonase activity was between 0.4 and 1.3 U/ml and polymethylgalacturonase between 0.15 and 1.3 U/ml) were higher than those of cellulases and xylanases, which appeared later and in a lesser magnitude. This sequence would promote initial tissue maceration fol- lowed by cell wall degradation. Laccase was detected in all the isolates evaluated (activity was between 36 U/l and 63 U/l). The aggressiveness of the isolates was tested in maize, sunflower and watermelon seeds, being high on all the plants assayed. This study reports for the first time the potential of different isolates of M. phaseolina to produce plant cell wall-degrading enzymes in submerged fermentation. © 2016 Asociacion´ Argentina de Microbiolog´ıa. Published by Elsevier Espana,˜ S.L.U. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by- nc-nd/4.0/). ∗ Corresponding author. E-mail address: [email protected] (A.M. Ramos). http://dx.doi.org/10.1016/j.ram.2016.06.002 0325-7541/© 2016 Asociacion´ Argentina de Microbiolog´ıa. Published by Elsevier Espana,˜ S.L.U. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 268 A.M. Ramos et al. in vitro PALABRAS CLAVE Crecimiento y producción de enzimas degradadoras de pared celular vegetal Macrophomina de aislamientos argentinos de Macrophomina phaseolina, agente causal de la phaseolina; podredumbre carbonosa en maíz Podredumbre carbonosa; Resumen Macrophomina phaseolina es un fitopatógeno polífago, causante de podredumbre Maíz; carbonosa. Los danos˜ que genera en cultivos de soja y maíz bajo siembra directa en Argentina, Enzimas en períodos secos y calurosos, se incrementaron por su habilidad para sobrevivir como escle- rocios en suelos y restos de cosecha. El propósito del trabajo fue estudiar la producción degradadoras de in vitro de enzimas degradadoras de pared celular vegetal (pectinasas [poligalacturonasa y pared celular vegetal polimetilgalacturonasa]; celulasas [endoglucanasa]; hemicelulasas [endoxilanasa] y la enzima ligninolítica lacasa) de varios aislamientos argentinos de M. phaseolina y evaluar la patogeni- cidad de esos aislamientos, como paso preliminar para establecer el papel de estas enzimas en la interacción M. phaseolina-maíz. Se estudió la cinética de crecimiento del hongo y la de la producción de dichas enzimas en medios de cultivo líquidos sintéticos con ácido glutámico como fuente de nitrógeno y con pectina, carboximetilcelulosa (CMC) o xilano como fuentes de car- bono. Las pectinasas fueron las primeras enzimas detectadas y los máximos títulos registrados (1,4 UE/ml [poligalacturonasa] y 1,2 UE/ml [polimetilgalacturonasa], respectivamente) super- aron a los de celulasas y xilanasas, que aparecieron más tardíamente y en menor magnitud. Esta secuencia promovería la maceración inicial del tejido, seguida luego por la degradación de la pared celular vegetal. Se detectó actividad lacasa en todos los aislamientos (36 a 63 U/l). La agresividad de todos los aislamientos resultó alta en los 3 hospedantes evaluados: semillas de maíz, de girasol y de melón. En este trabajo se investiga por primera vez el potencial de distintos aislamientos de M. phaseolina para producir enzimas degradadoras de pared celular vegetal en cultivo líquido. © 2016 Asociacion´ Argentina de Microbiolog´ıa. Publicado por Elsevier Espana,˜ S.L.U. Este es un art´ıculo Open Access bajo la licencia CC BY-NC-ND (http://creativecommons.org/licenses/by- nc-nd/4.0/). Introduction Many plant pathogenic organisms are capable of degrading cellulose by producing a cellulose complex which involves Fungal plant pathogens secrete a wide range of enzymes the synergistic action of three main enzymatic complexes, such as pectinases, xylanases, cellulases and ligninases that endoglucanase, exoglucanase that releases either glucose or 38 ␤ are able to depolymerize each cell wall component . These cellobiose, and -1,4-glucosidase that hydrolyzes cellobiose 25,27 fungi are of interest for the search of new enzyme activ- and cellodextrins to glucose . ␤ ities due to their potential application in bioconversion Xylan is the major constituent of hemicellulose. -1,4- 17 processes . Hydrolytic enzymes play an important role in Xylans are heteropolysaccharides with a homopolymeric ␤ d plant pathogenicity by facilitating tissue colonization of the backbone chain of 1,4-linked - -xylopyranose units. 3,13 host . O-acetyl, ␣-l-arabinofuranosyl, ␣-1,2-linked glucuronic, or Pectin, a heteropolysaccharide defined as galactosy- 4-O-methylglucuronic acids are the most frequent sub- luronic acid-rich polymers, is composed by ␣-1,4 linked stituents on the backbone. Xylan hydrolysis mainly requires ␤ ␤ galacturonate chains with high percentage of methyl the action of endo- -1,4-xylanase and -xylosidase. How- esterification. Plant pathogenic organisms are capable ever, the presence of other accessory enzymes is needed to 15 of degrading pectin by the combined action of several hydrolyze substituted xylans . enzymes which produce the breakdown of polygalactur- The complex aromatic polymer lignin, a component of onic acid through two enzymatic processes: lyases split the plant secondary cell walls, provides a barrier to fungal ␣ -1,4 glycosidic bond between galacturonic acid residues entry and to the diffusion of fungal toxins and enzymes 8 by transelimination, while polygalacturonases catalyze a into plant cells . The lignin-degrading enzyme, laccase, 33 hydrolytic cleavage . The action of pectinolytic enzymes, is a copper-containing enzyme produced by some fungi and in particular of endopolygalacturonase on cell walls, that catalyzes the oxidation of phenolic substrates. Several 18,20 appears to be a prerequisite for cell wall degradation by phytopathogenic fungi are known to produce lacasse . other enzymes. Therefore, pectinases are the first enzymes Laccase protects phytopathogenic fungi from the plant secreted by most fungal pathogens when attacking plant cell defense system activated in response to a microbial infec- 35 walls, followed by hemicellulases and cellulases . A positive tion, by degrading toxic substances such as phytoalexins, 24 correlation has been established between the production tannins and other phenolic compounds . of pectinolytic enzymes, virulence and disease symptoms in The charcoal rot fungus Macrophomina phaseolina 16,31 several pathosystems . (Tassi) Goid, an Ascomycete which belongs to the family Cellulose is an unbranched glucose polymer composed Botryosphaeriaceae, is a soil-borne pathogen distributed of ␤-1,4-glucose units linked by a ␤-1,4-d-glycosidic bond. worldwide. The pathogen has a wide host range, infecting Cell wall degrading enzyme production by M. phaseolina 269