Characterization and gene cloning of a novel serine protease with nematicidal activity fromTrichoderma pseudokoningii SMF2

Abstract

Trichoderma pseudokoningii SMF2 is a biocontrol fungus with inhibitory ability against phytopathogenic fungi. Here, a crude extract of strain SMF2 in a solid ferment exhibited strong nematicidal activity against Meloidogyne incognita, and a novel serine protease SprT with nematicidal activity was purified from the crude extract. Protease SprT has a molecular mass of 31 kDa, a pH optimum of 8.5, and a temperature optimum of 60-65 degrees C. It had good thermostability, and was stable in an alkaline environment. SprT could degrade bovine serum albumin, lysozyme, and gelatin, and its activity was enhanced by many metal ions. The cuticles of nematodes treated by protease SprT obviously crimpled. Purified protease SprT could kill juveniles of M. incognita and inhibit egg hatch, suggesting that it is involved in the nematicidal process of T. pseudokoningii SMF2. The full-length cDNA gene-encoding protease SprT was cloned by rapid amplification of cDNA ends. Sequence analysis showed that SprT is a monodomain subtilase containing 284 amino acid residues. It had higher identities and a closer relation to the nematicidal serine proteases (59-69%) from nematode parasitic fungi than to the serine proteases (<50%) from Trichoderma. Protease SprT represents the first well-characterized subtilase with nematicidal activity from Trichoderma.

Identification of a novel fungus, Trichoderma asperellum GDFS1009, and comprehensive evaluation of its biocontrol efficacy

“…Each disc was inverted and placed on the center of a 9-cm Petri dish with 1.5% skim milk and 2.5% agar. After incubation at 28°C for 36h, proteolytic circle was observed [26–27]. …”

Biology and biotechnology of Trichoderma

“…apply lytic enzymes (Kubicek et al 2001; Viterbo et al 2002), proteolytic enzymes (Kredics et al 2005; Suarez et al 2007; Chen et al 2009), ABC transporter membrane pumps (Ruocco et al 2009), diffusible or volatile metabolites (Calistru et al 1997; Eziashi et al 2006), and other secondary metabolites (Reino et al 2008) as active measures against their hosts or they succeed by their impairing growth conditions of pathogens (Benitez et al 2004). Interestingly, the success of these actions is not independent of the surrounding temperature (Mukherjee and Raghu 1997), which can be crucial for the use as a biocontrol agent in different climates.…”

Comparative Genomics Provide Insights into Evolution of Trichoderma Nutrition Style

“…It was reclassified as T . pseudokoningii based on molecular data and morphological data (Chen et al 2009). The secondary metabolites, peptaibols, and the extracellular enzymes secreted by SMF2 are thought to be important factors that contribute to the inhibitory ability against pathogens.…”