Short communication Spore production in solid-state fermentation of rice by Clonostachys rosea, a biopesticide for gray mold of strawberries Graciele Viccini a , Michael Mannich a , Deise Maria Fontana Capalbo b , Rosa Valdebenito-Sanhueza c , David Alexander Mitchell a, * a Departamento de Bioquı ´mica e Biologia Molecular, Universidade Federal do Parana ´, Caixa Postal 19046 Centro Polite ´cnico, Curitiba 81531-990, Parana ´, Brazil b EMBRAPA CNPMA Jaguariu ´na, SP, Brazil c EMBRAPA CNPUV Bento Gonc ¸alves, RS, Brazil Received 8 December 2005; received in revised form 30 June 2006; accepted 12 July 2006 Abstract Gray mold caused by Botrytis cinerea is an important disease of strawberry. Clonostachys rosea is a mycoparasite of B. cinerea that reduces fruit losses when used as a biocontrol agent. Since spore production by C. rosea has not been optimized, we investigated factors affecting sporulation under aseptic conditions on white rice grains. The greatest spore production in glass flasks, 3.4  10 9 spores/g-dry-matter (gDM), occurred with an initial moisture content of 46% (w/w wet basis), inoculated with 1  10 6 spores/gDM and hand shaken every 15 days. However, a lower inoculum density (9  10 3 spores/gDM) and no shaking also gave acceptable sporulation. In plastic bags 1.1  10 8 spores/gDM were produced in 15 days, suggesting that larger scale production may be feasible: with this spore content, 24 m 2 of incubator space would produce sufficient spores for the continued treatment of 1 ha of strawberry plants. # 2006 Elsevier Ltd. All rights reserved. Keywords: Clonostachys rosea; Botrytis cinerea; Biological control; Grey mould; Strawberry 1. Introduction Strawberry (Fragaria  ananassa Duch.) is highly appre- ciated in many countries, including Brazil, where it is frequently produced in greenhouses. The conditions in green- houses favor the growth of the ‘‘gray mold’’, Botrytis cinerea Pers.:Fr., which can cause pre- and post-harvest losses of 50% or more [1]. Control with fungicides is difficult because the plants have fruits at all stages of development and it is not possible to ensure a pre-harvest interval free of fungicide. In any case, chemical control has become difficult due to the development of resistant strains as well as increased concern of consumers towards pesticide use [2–4]. The non-pathogenic, saprophytic fungus Gliocladium roseum Bainer, now reclassified as Clonostachys rosea (Link:Fr.) Schroers, Samuels, Seifert & W. Gams [5], is a mycoparasite that has emerged as an effective antagonist of B. cinerea and promises to improve disease management in several crops [6–9]. In the specific case of strawberries, C. rosea is efficacious in reducing fruit losses due to B. cinerea [1,6,10,11]. An efficient, standardized process for mass production of C. rosea spores is needed, but studies have focused on the efficacy of spores in disease management rather than production of the spores themselves. Spores are generally produced by solid-state fermentation (SSF) on wheat grains [6,8,9,11]. SSF has two main advantages over submerged liquid fermentation. Firstly, fungal spores produced by SSF are typically more robust and have longer shelf-lives than those produced by liquid fermentation [12,13]. Secondly, SSF processes can be under- taken by relatively unskilled workers and it may therefore be possible to transfer the production technology to farmer cooperatives that work under the guidance of government institutions that provide support to farmers, like EMBRAPA in Brazil. Solid substrates other than wheat have been tested, including rice, a mixture of sphagnum peat and wheat bran, potato dextrose agar, corn meal, poultry layer mash and oat bran [7,13–15]. The aim of the current work was to optimize the conditions for spore production by C. rosea in SSF of white rice. www.elsevier.com/locate/procbio Process Biochemistry 42 (2007) 275–278 * Corresponding author. Tel.: +55 41 361 1658; fax: +55 41 266 2042. E-mail address: davidmitchell@ufpr.br (D.A. Mitchell). 1359-5113/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2006.07.006