Plant and Soil 232: 215–226, 2001. © 2001 Kluwer Academic Publishers. Printed in the Netherlands. 215 An integrated approach for the evaluation of biological control of the complex Polymyxa betae/Beet Necrotic Yellow Vein Virus, by means of seed inoculants R. Resca 1 , M. Basaglia 2 , S. Poggiolini 3 , P. Vian 2 , S. Bardin 6 , U. F. Walsh 4 , C. M. Enriquez Barreiros 1 , F. O’Gara 4 , M. P. Nuti 5 , S. Casella 2 & U. Peruch 3, ∗ 1 Eridania B´ eghin-Say, Centro Ricerche, Massalombarda RA, Italy, 2 Dipartimento di Biotecnologie Agrarie, Universit` a di Padova, Italy, 3 Agronomica, Ravenna, Italy, 4 BIOMERIT Research Centre, National University of Ireland, UCC, Cork, Ireland, 5 Dipartimento di Chimica e Biotecnologie Agrarie, Universit` a di Pisa, Italy 6 Lethbridge Research Centre, Agriculture and Agri-Food Canada, Lethbridge, Alberta, Canada Key words: biocontrol, 2,4-diacetylphloroglucinol, genetically modified microorganism, environmental impact, Pseudomonas fluorescens, rhizomania Abstract Rhizomania is an extremely severe sugarbeet disease caused by the complex Polymyxa betae/Beet Necrotic Yellow Vein Virus (BNYVV). A relatively small number of recently introduced sugarbeet cultivars characterized by a high tolerance to rhizomania are available on the market. An integrated approach was therefore developed using Pseudomonas fluorescens biological control agents (BCAs) in order to improve yield performance of cultivars characterized by a medium tolerance to the disease. A genetically modified biological control agent, Pseudo- monas fluorescens F113Rif (pCUGP), was developed for enhanced production of the antimicrobial metabolite 2,4-diacetylphloroglucinol (Phl) and lacking an antibiotic resistance marker gene, making the strain suitable for field release. The ability of synthetic Phl and P. fluorescens F113Rif (pCUGP) to antagonize the fungal vector, P. betae, was assessed in microcosm trials. Results encouraged the preparation of multiple field trials in a soil naturally infested with P. betae/BNYVV, to determine the biocontrol efficacy of P. fluorescens F113Rif (pCUGP) and to assess its impact on sugarbeet yield and quality and on the indigenous microbial population. While the col- onization ability of P. fluorescens F113Rif (pCUGP) was satisfactory at sugarbeet emergence (2.5×10 6 CFU g -1 root), control of rhizomania was not achieved. Inoculation of sugarbeet with Pseudomonas fluorescens F113Rif (pCUGP) did not affect crop yield and quality nor affect the numbers of selected microbial populations. Introduction There is increasing interest in the use of bacterial and fungal biocontrol agents for managing soil-borne phytopathogens, partly as a response to public con- cerns regarding the continued application of envir- onmentally hazardous agro-chemicals and also be- cause of a lack of effective controls (Cook, 1993). Among the ways in which bacteria can control the growth and activity of fungal phytopathogens is via the production of antimicrobial metabolites including ∗ Corresponding author. Eridana, Coordinamento Sviluppo Ag- ricolo, Via de Lavoro 17, 44100 Ferrara, Italy. Tel.: +39 532 590596; Fax: +39 532 590209; E-mail: uperuch@it.ebsworld.com 2,4-diacetylphloroglucinol (Phl), pyoluteorin (Plt) and hydrogen cyanide (Voisard et al., 1994). The identific- ation of the link between antimicrobial metabolite(s) production and the suppression of plant disease has stimulated the construction of genetically modified (GM) strains able to overproduce antimicrobial com- pounds (Dowling and O’Gara, 1994). Maurhofer et al. (1995) demonstrated that the GM Phl and pyoluteorin overproducing strain P. fluorescens CHA0 (pME3090) significantly increased protection of cucumber from Fusarium oxysporum f.sp. cucumerinum and Phomop- sis sclerotioides, compared to the wild-type strain. The antifungal metabolite overproducing strain, how- ever, resulted in reduced growth when inoculated on tobacco and sweet corn.