Enhanced biocontrol activity of Trichoderma virens transformants constitutively coexpressing β‐1,3‐ and β‐1,6‐glucanase genes

Abstract

Evidence for the role of chitinases, proteases and beta-1,3- and beta-1,6-glucanases in mycoparasitism by Trichoderma species has been well documented. Moreover, constitutive over-expression of genes encoding individual cell-wall-degrading enzymes (CWDEs) has been shown to improve the potential of biological agents. In this study, we generated transformants of T. virens in which beta-1,3- and beta-1,6-glucanase genes, TvBgn2 and TvBgn3, respectively, were constitutively coexpressed in the same genetic T. virens Gv29.8 wild-type background. The double over-expression transformants (dOEs) grow and sporulate slower than the wild-type (WT). However, the reduction in growth did not seem to affect their mycoparasitic and biocontrol capabilities, as dOEs displayed much higher levels of total beta-1,3- and beta-1,6-glucanase activity than the WT. This higher enzymatic activity of dOEs positively correlated with observed in vitro inhibition of Pythium ultimum and Rhizoctonia solani mycelia, and with enhanced bioprotection of cotton seedlings against P. ultimum, R. solani and Rhizopus oryzae. Besides effective biocontrol of all pathogens at an original inoculum level, the performance of dOEs was highly enhanced (up to 312% of WT performance) when pathogen pressure was greater (i.e. concentration of inoculum was higher or pathogens applied in combination). These results demonstrate that the strategy of introducing multiple lytic enzyme-encoding genes through transformation of a given biocontrol strain can be successfully used to achieve better biocontrol.

Saprotrophic competitiveness and biocontrol fitness of a genetically modified strain of the plant-growth-promoting fungus Trichoderma hamatum GD12

“…In addition to their biocontrol properties, certain strains have been shown to enhance crop productivity by stimulating plant growth (Contreras-Cornejo et al, 2009;Harman et al, 2004;Ortíz-Castro et al, 2009;Vinale et al, 2008Vinale et al, , 2009. While genetic modification of Trichoderma strains by constitutive overexpression of chitinase, b-glucanase and proteinase genes has allowed the development of strains with improved biocontrol capabilities (Flores et al, 1997;Baek et al, 1999;Limó n et al, 1999;Viterbo et al, 2001;Djonović et al, 2007), less attention has been paid to enhancing the plantgrowth-promoting (P-G-P) activities of these fungi via genetic modification, and the impact that any such modification might have on their saprotrophic competence and fitness as biocontrol agents.…”

“…In addition to their biocontrol properties, certain strains have been shown to enhance crop productivity by stimulating plant growth (Contreras-Cornejo et al, 2009;Harman et al, 2004;Ortíz-Castro et al, 2009;Vinale et al, 2008Vinale et al, , 2009. While genetic modification of Trichoderma strains by constitutive overexpression of chitinase, b-glucanase and proteinase genes has allowed the development of strains with improved biocontrol capabilities (Flores et al, 1997;Baek et al, 1999;Limó n et al, 1999;Viterbo et al, 2001;Djonović et al, 2007), less attention has been paid to enhancing the plantgrowth-promoting (P-G-P) activities of these fungi via genetic modification, and the impact that any such modification might have on their saprotrophic competence and fitness as biocontrol agents.In a previous study, we showed that a naturally occurring strain of Trichoderma hamatum was able to promote plant growth in low pH, nutrient-poor peat soils (Thornton, 2008). These soils contain a significant pool of nitrogen sequestered in chitin of insect and fungal origin (Kerley & Read, 1998), and fungal N-acetyl-b-D-glucosaminidase has been shown to be a key chitinolytic enzyme releasing sequestered nitrogen for assimilation by plants in peat ecosystems (Leake & Read, 1990;Kerley & Read, 1995, 1998Read & Perez-Moreno, 2003;Lindahl & Taylor, 2004).…”

Indução de resistência sistêmica à antracnose em feijoeiro-comum pela raça delta avirulenta de Colletotrichum lindemuthianum

“…O aumento da atividade das enzimas avaliadas neste trabalho contribui para a resistência do feijoeiro ao C. lindemuthianum, pela ação hidrolítica sobre betaglucanos e quitina presentes na parede celular de muitos fungos (Chet, 1993;Djonovic et al, 2007). O envolvimento dessas enzimas na defesa ativa das plantas contra patógenos está apoiado em trabalhos que demonstraram, in vitro, que elas inibem o crescimento de microrganismos (Schlumbaum et al, 1986;Mauch et al, 1988;Xiuyun & Tzi, 2005).…”

“…Embora vários produtos de biocontrole estejam disponíveis, ainda há demanda por novos (Montesinos, 2003;Djonovic et al, 2007), e o entendimento de seus mecanismos de proteção tem grande importância para subsidiar o seu uso.…”

“…Mecanismos baseados, principalmente, em competição por espaço e por nutrientes, micoparasitismo, antibiose e elicitação de defesas da planta contribuem, simultânea ou subsequentemente, para o biocontrole de microrganismos (Janisiewicz & Korsten, 2002;Djonovic et al, 2007 Em plantas infectadas por microrganismos potencialmente patogênicos, há primeiro a acumulação de proteínas induzidas patogenicamente, ou proteínas-PR (Hartleb et al, 1997). Após a penetração do patógeno, há formação de um ou mais tipos de estruturas, que apresentam maior ou menor sucesso na defesa da planta, e podem apresentar poderosa atividade antifúngica (Hartleb et al, 1997;Karban & Kuc, 1999;Xiuyun & Tzi, 2005).…”