Purification and characterization of a phytoalexin elicitor from spores of the saprobe Mucor ramosissimus

Abstract

-(Purification and characterization of a phytoalexin elicitor from spores of the saprobe Mucor ramosissimus). Plants accumulate antimicrobial compounds (phytoalexins) in response to a wide variety of microorganisms. Mucor ramosissimus Samutsevitsch is a saprobe capable of inducing phytoalexin production in soybean cotyledons and in the leaves of tropical Rubiaceae on whose surface it has been found. In the present study, the elicitor from M. ramosissimus was partially purified and the activity compared to that of a glucan elicitor isolated from Phytophthora sojae. Optimal isolation of the elicitor (based on fungal growth, yield of spores and elicitor activity) was achieved by autoclaving spores obtained from nine day-old cultures of the fungus. The elicitor was precipitated with ethanol and purified by chromatography on an anion exchange column, which retained the elicitor, and a Concanavalin A-affinity matrix, to which the elicitor did not bind. The purification resulted in a considerable increase (six-fold) in the specific activity of the elicitor. Neutral sugar composition, analyzed by HPLC, revealed the predominance of mannose, followed by glucose and galactose, whereas colorimetric quantification showed the presence of uronic acids. GC-MS analysis of the elicitor revealed the predominance of glucuronic acid and mannose. These results suggest that fragments of mucoran-type polysaccharides are the phytoalexin elicitors present in the spores of the saprobe M. ramosissimus. Our results also indicate for the first time that soybean cotyledon tissues can recognize fragments of glucuronic-acid heteropolymers as phytoalexin elicitors.Key words -elicitor, Mucor ramosissimus, mucoran, phytoalexin, saprobe fungus, soybean RESUMO -(Purificação e caracterização de um eliciador de fitoalexinas de esporos do fungo sapróbio Mucor ramosissimus).Plantas acumulam compostos antimicrobianos (fitoalexinas) em resposta a uma grande variedade de microorganismos. Mucor ramosissimus Samutsevitsch é um fungo sapróbio capaz de induzir a produção de fitoalexinas em soja e em Rubiaceae nativas, sobre a superfície das quais ele é encontrado. Neste estudo, o eliciador de esporos de M. ramosissimus foi parcialmente purificado e sua atividade comparada ao glucano isolado de Phytophthora sojae. O ótimo de isolamento do eliciador (baseado no crescimento do fungo, no rendimento dos esporos e na atividade eliciadora) foi obtido a partir de esporos autoclavados de culturas do fungo com nove dias de idade. O eliciador foi precipitado com etanol e purificado por cromatografia de troca iônica, a qual reteve o eliciador, e por uma matriz de afinidade em Concanavalina-A, à qual o eliciador não se ligou. A purificação resultou em considerável aumento (seis vezes) de atividade específica do eliciador. Análises de açúcares neutros por HPLC revelaram a predominância de manose, seguida de glucose e galactose, enquanto que a quantificação por colorimetria mostrou a presença de ácidos urônicos. Análises do eliciador por GC-MS indicaram a predominân...

Accumulation of Phytoalexins in Beans, Soybeans and Sorghum by Fungal Filtrates

“…As for C. eragrostidis, 3 times more phaseolin was observed with the application of the filtrate in bean hypocotyls compared with the control treatments (water and ASM) ( Figure 2). Similar results were found by Simões et al (2005), who verified five-and six-fold increases in the phytoalexin accumulation in soybean when applying preparations from β-glucan isolated from Phytophthora sojae and from spores of Mucor ramosissimus, respectively. As in this work, Arruda et al (2012) evaluated the effects of different concentrations (1, 2, 4 and 10%) of aqueous extracts of Agaricus blazei, Lentinula edodes and Pycnoporus sanguineus on the induction of glyceolin and observed a higher accumulation of phytoalexins with higher concentrations of the aqueous extracts.…”

Identification of Three Elicitins and a Galactan-Based Complex Polysaccharide from a Concentrated Culture Filtrate of Phytophthora infestans Efficient against Pectobacterium atrosepticum

“…Wolski et al [41] showed that α-glucan from a non-pathogenic Rhizoctonia induces plant defense responses (β-1, 3-glucanase and chitinase activities, callose and lignine deposition) and protects potato sprouts against Rhizoctonia canker and dry rot. By contrast with glucans, there is little published evidence about eliciting activities of galactan-based polysaccharides [42]. Our results are therefore quite original, and warrant further analysis of the fine structure of the complex active polysaccharides within CCF.…”

Resistência de plântulas de pepineiro a Colletotrichum lagenarium induzida por frações de extrato de Saccharomyces cerevisiae

“…A autoclavagem por 30 min a 121°C de esporos do fungo Mucor ramosissimus favoreceu a extração de eliciadores capazes de induzir a síntese de fitoalexinas em soja e em Rubiaceae nativas (Simões et al, 2005). O extrato bruto aquoso do micélio de Penicillium chrysogenum, denominado "Pen", eliciou respostas de defesa e induziu resistência em plantas de Arabidopsis thaliana, tomate, fumo e arroz (Thuerig et al, 2006).…”