Colonization of cacao seedlings by Trichoderma stromaticum, a mycoparasite of the witches’ broom pathogen, and its influence on plant growth and resistance J.T. De Souza a,b, * , B.A. Bailey b , A.W.V. Pomella c,1 , E.F. Erbe d , C.A. Murphy d , H. Bae b , P.K. Hebbar e a Wye Research and Education Center, University of Maryland, Queenstown, MD 21658, USA b USDA-ARS, Sustainable Perennial Crops Laboratory, BARC-West, Beltsville, MD 20705, USA c Almirante Cacau Agrı ´cola Come ´rcio e Exportac ßa ˜o Ltda, Caixa Postal 55, 45630-000 Itajuı ´pe, BA, Brazil d USDA-ARS, Henry A. Wallace Beltsville Agricultural Research Center-East, Electron Microscopy Unit, Beltsville, MD 20705, USA e Masterfoods, Hackettstown, NJ 07840, USA Received 20 July 2007; accepted 15 January 2008 Available online 26 January 2008 Abstract Trichoderma stromaticum, a mycoparasite of the cacao witches‘ broom pathogen Moniliophthora perniciosa, is being used in Bahia, Brazil to control the disease under field conditions. The endophytic potential of this biocontrol agent was studied in both shoot and root tissues of sterile and nonsterile cacao and bean seedlings. Agar plate and light and electron microscopy studies showed that T. stromat- icum is able to colonize extensively both cacao and bean plants grown under sterile conditions. However, colonization was lower when plants were grown under nonsterile conditions, especially in the shoot tissues. Recovery of T. stromaticum from field-grown trees showed that isolates belonging to genetic group II are more persistent (>120 days) as endophytes than isolates from group I. Endophytic colo- nization of cacao plants by T. stromaticum did not result in plant growth promotion nor induced resistance against M. perniciosa on seedlings that had been treated 30 days prior to application of the pathogen. These results were confirmed by Northern blot studies, where the fungus was unable to alter the expression of selected genes involved in plant defense such as ChiB, a putative class VII chi- tinase, Glu-1, a putative endo-1,4-b-glucanase, Caf-1, a putative caffeine synthase, and Per-1 apoplastic quiacol peroxidase, genes involved in the regulation of plant growth TcORFX-1(fw2.2-like) and TcLhca-1(photosystem I 24 kDa protein) involved in energy pro- duction. This study indicates that induced resistance and growth promotion are not responsible for the activity of T. stromaticum in the biocontrol of the witches’ broom pathogen. Ó 2008 Elsevier Inc. All rights reserved. Keywords: Theobroma cacao; Trichoderma stromaticum; Witches’ broom disease; Biological control; Endophytes; Colonization 1. Introduction Trichoderma stromaticum is a biological control agent of Moniliophthora (ex Crinipellis) perniciosa (Aime and Phillips-Mora, 2005), the causal agent of witches’ broom disease of cacao (Theobroma cacao). This biocontrol agent is being used on an experimental basis in Brazil since 1999 as part of the integrated management of cacao (Samuels et al., 2000; Sanogo et al., 2002; De Souza et al., 2006). The witches’ broom disease has been responsible for the rapid decline in cacao production in Bahia, Brazil, by almost 75% since its introduction (Pereira et al., 1990). The pathogen is able to infect all meristematic tissues of the plant, including vegetative flushes, flower cushions, 1049-9644/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.biocontrol.2008.01.010 * Corresponding author. Present address: Universidade Federal do Reco ˆ ncavo da Bahia (UFRB), CCAAB, Campus Universita ´rio de Cruz das Almas, 44380-000 Cruz das Almas, BA, Brazil. E-mail address: jorgetdes@yahoo.com.br (J.T. De Souza). 1 Present address: Sementes Farroupilha, Caixa Postal 90, 38702-054 Patos de Minas, MG, Brazil. www.elsevier.com/locate/ybcon Available online at www.sciencedirect.com Biological Control 46 (2008) 36–45