Disruption of the <em>Eng18B</em> ENGase Gene in the Fungal Biocontrol Agent <em>Trichoderma atroviride</em> Affects Growth, Conidiation and Antagonistic Ability

<div><p>The recently identified phylogenetic subgroup B5 of fungal glycoside hydrolase family 18 genes encodes enzymes with mannosyl glycoprotein endo-<em>N</em>-acetyl-β-D-glucosaminidase (ENGase)-type activity. Intracellular ENGase activity is associated with the endoplasmic reticulum associated protein degradation pathway (ERAD) of misfolded glycoproteins, although the biological relevance in filamentous fungi is not known. <em>Trichoderma atroviride</em> is a mycoparasitic fungus that is used for biological control of plant pathogenic fungi. The present work is a functional study of the <em>T. atroviride</em> B5-group gene <em>Eng18B</em>, with emphasis on its role in fungal growth and antagonism. A homology model of <em>T. atroviride</em> Eng18B structure predicts a typical glycoside hydrolase family 18 (αβ)₈ barrel architecture. Gene expression analysis shows that <em>Eng18B</em> is induced in dual cultures with the fungal plant pathogens <em>Botrytis cinerea</em> and <em>Rhizoctonia solani</em>, although a basal expression is observed in all growth conditions tested. <em>Eng18B</em> disruption strains had significantly reduced growth rates but higher conidiation rates compared to the wild-type strain. However, growth rates on abiotic stress media were significantly higher in <em>Eng18B</em> disruption strains compared to the wild-type strain. No difference in spore germination, germ-tube morphology or in hyphal branching was detected. Disruption strains produced less biomass in liquid cultures than the wild-type strain when grown with chitin as the sole carbon source. In addition, we determined that Eng18B is required for the antagonistic ability of <em>T. atroviride</em> against the grey mould fungus <em>B. cinerea</em> in dual cultures and that this reduction in antagonistic ability is partly connected to a secreted factor. The phenotypes were recovered by re-introduction of an intact <em>Eng18B</em> gene fragment in mutant strains. A putative role of Eng18B ENGase activity in the endoplasmic reticulum associated protein degradation pathway of endogenous glycoproteins in <em>T. atroviride</em> is discussed in relation to the observed phenotypes.</p>