MOLECULAR TOOLS TO DELINEATE THE INTERACTIONS BETWEEN BACTERIA AND FUNGI

DC NASEBY and JM LYNCH

University of Surrey, Guildford, UK

A number of methods have been applied in the literature to evaluate the interactions between soil bacteria and fungi. This is of the greatest importance with the application biocontrol agents of soil borne plant pathogens. The use of marker genes, such as lac, gus, lux, and gfp, have expanded knowledge of the ecology of biocontrol agents and to a lesser extent plant pathogens. Such markers have been used in a number of microcosm and field studies of biocontrol agents within the EU IMPACT programme, results of which will be presented. A number of authors have manipulated genes involved in the biocontrol of plant pathogens to investigate the nature of or improve the biocontrol activity. For example, Carrol et al. [2] used a Tn5 insertion to suppress the production of the antibiotic diacetyl phloroglucinol by P. fluorescens F 113; this reduced the biocontrol properties of this strain and a number of ecological and impact studies have been performed using it [3]. Antibiotic overproducers have also been constructed and used in similar studies [4].

Some authors have successfully used a molecular approach to demonstrate the interactions between fungi and bacteria. For instance, Bianciotto et al. [1] showed that that the cytoplasm of the arbuscular-mycorrhizal fungus, Gigaspora margarita harbors a bacterial endosymbiont. They used a sequence of rRNA from the bacteria to identify it as a rRNA group II pseudomonad (genus Burkholderia). PCR assays with specifically designed oligonucleotides were used to check that the sequence came from the bacteria.

Fungal genetic targets for identification, classification and biodiversity or impact studies have been poorly described. Molecular methods have immense potential in the quantification of microbial diversity in environmental samples; 16S rRNA has shown particular promise with bacteria, but as yet the fungi lack a universal probe. The use of 18S rDNA of fungi may fill this hole, data from collaborative work with HV Tichy,(TUEV et GmbH, Germany), on the impact of a biocontrol strain of Pseudomonas fluorescens on the indigenous fungal community using 18S rDNA will be presented.

References
1. Bianciotto V, Bandi C, Minerdi D, Sironi M, Tichy HV, Bonfante P, (1996) An obligately endosymbiotic mycorrhizal fungus itself harbors obligately intracellular bacteria. Applied and Environmental Microbiology 62, 3005-3010.
2. Carroll H, Moenneloccoz Y, Dowling DN, Ogara F, (1995). Mutational disruption of the biosynthesis genes-coding for the antifungal metabolite 2,4-diacetylphloroglucinol does not influence the ecological fitness of Pseudomonas fluorescens fl 13 in the rhizosphere of sugar-beets. Applied and Environmental Microbiology 61, 3002-3007.
3. Naseby DC and Lynch JM, (1998) Soil enzymes and microbial population structure to determine the impact of wild type and genetically modified Pseudomonas fluorescens in the rhizosphere of pea. Molecular Ecology (in press).
4. Natsch A, Keel C, Hebecker N, Laasik E, Defago G, (1997). Influence of biocontrol strain Pseudomonas fluorescens CHAO and its antibiotic overproducing derivative on the diversity of resident root colonizing pseudomonads. FEMs Microbiology Ecology 23, 341-352.
5. Niemann S, Keel C, Puhler A, Selbitschka W, (1997). Biocontrol strain Pseudomonas flourescens CHAO and its genetically modified derivative with enhanced biocontrol capability exert comparable effects on the structure of a Sinorhizobium meliloti population in gnotobiotic systems. Biology and Fertility of Soils 25, 240-244.