RESEARCH LETTER Real-time RT-PCR expression analysis of chitinase and endoglucanase genes in the three-way interaction between the biocontrol strain Clonostachys rosea IK726, Botrytis cinerea and strawberry Mojtaba Mamarabadi, Birgit Jensen, Dan Funck Jensen & Mette L ¨ ubeck Plant Pathology Section, Department of Plant Biology, Faculty of Life Sciences, University of Copenhagen, Copenhagen, Denmark Correspondence: Mette L ¨ ubeck, Department of Life Science, Aalborg University, Lautrupvang 15, DK-2750 Ballerup, Denmark. Tel.: 145 9940 9440; e-mail: mette.lubeck@hotmail.com Present addresses: Mojtaba Mamarabadi, Faculty of Agriculture, Shahrood University of Technology, Shahrood, Iran. Dan Funck Jensen, Department of Forest Mycology and Pathology, Swedish University of Agricultural Sciences, SLU, Uppsala, Sweden. Mette L ¨ ubeck, Department of Life Science, Aalborg University, Lautrupvang 15, DK-2750 Ballerup, Denmark. Received 10 January 2008; accepted 30 April 2008. First published online 13 June 2008. DOI:10.1111/j.1574-6968.2008.01228.x Editor: Bernard Paul Keywords real-time RT-PCR; chitinase; endoglucanase; Clonostachys rosea ; Botrytis cinerea . Abstract Clonostachys rosea is a well-known biocontrol agent against Botrytis cinerea, the causal agent of gray mold in strawberry. The activity of cell wall-degrading enzymes might play a significant role for successful biocontrol by C. rosea. The expression pattern of four chitinases, and two endoglucanase genes from C. rosea strain IK726 was analyzed using real-time RT-PCR in vitro and in strawberry leaves during interaction with B. cinerea. Specific primers were designed for b-tubulin genes from C. rosea and B. cinerea, respectively, and a gene encoding a DNA- binding protein (DBP) from strawberry, allowing in situ activity assessment of each fungus in vitro and during their interaction on strawberry leaves. Growth of B. cinerea was inhibited in all pathogen–antagonist interactions while the activity of IK726 was slightly increased. In all in vitro interactions, four of the six genes were upregulated while no change in expression of two endochitinases was measured. In strawberry leaves, the chitinase genes were upregulated 2–12-fold, except one of the endochitinases, whereas no change in expression of the two endoglucanases was measured. The results suggest that three out of four chitinase genes of IK726 are involved in biocontrol on leaves. This is the first example of monitoring of expression of chitinolytic genes in interactions between biocontrol agents and pathogens in plant material. Introduction Botrytis cinerea Pers. ex. Fr. is one of the most important pathogens in vegetable and fruit production, causing gray mold on various plant parts, including shoots, leaves, flowers and fruits (Jarvis, 1980; Maude, 1980). Control of B. cinerea is normally carried out by the application of fungicides. However, there is public concern related to the use of fungicides and furthermore fungicide resistance has been reported in B. cinerea (Washington et al., 1992). These problems support the need for alternative methods, which must be safe and able to replace fungicide treatments fully or partially (Wilson & Wisniewski, 1989). Biological control is a promising alternative for management of B. cinerea in both pre- and postharvest stages in crop production (Sutton, 1994; Paulitz & Belanger, 2001; Nobre et al., 2005). There are many reports of the efficacy of filamentous fungi, yeasts and bacteria antagonists against the pathogen (Leifert et al., 1993; Elad et al., 1994; K¨ ohl et al., 1998). Among the large group of antagonists, Clonostachys rosea Schroers, FEMS Microbiol Lett 285 (2008) 101–110 c  2008 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved Downloaded from https://academic.oup.com/femsle/article-abstract/285/1/101/594378 by guest on 02 June 2020