1Department of Plant Sciences, University of Oxford, Oxford OX1 3RB, UK; 2Plant Breeding International, Cambridge CB2 2LQ, UK

Background and objectives
In studies with a laboratory isolate of the fungal pathogen Stagonospora (Septoria) nodorum (SG), the causal organism of glume blotch of wheat, three bacterial isolates were found to be closely associated with the pycnidiospores of the fungus. When co-inoculated with the fungus, two of the three increased fungal pathogenicity, whilst the third had no effect. The aim of this project was to determine whether, under field conditions, bacteria are normally associated with the pathogen and, if so, the role such bacteria play in pathogenicity.

Results and discussion
Nineteen fresh isolates of S. nodorum and associated bacteria were obtained from infected wheat leaves from field trials at PBI and NIAB, Cambridge, and ADAS, south-west Cornwall. Six of these isolates were from naturally infected material. Identification using the API system showed that although a range of different bacterial isolates were involved, only one type of bacterium was associated with each isolate of S. nodorum. Enterobacter agglomerans was the most common isolate, particularly in natural infections. Pathogenicity tests using detached leaf assays were repeated several times, using various combinations of bacterial isolates with spore suspensions from axenic cultures of fresh isolates of S. nodorum. Differences between leaflets inoculated with droplets of spores plus and minus bacteria were most obvious in tests where the spore concentrations were very low. In assays using concentrations of 1x103 and 1x104 spores/ml, differences became apparent after 5 days. By day 13, pycnidiospores were present in three out of four leaflets inoculated with the fungus, together with one of the bacterial isolates from the SG laboratory culture of S. nodorum, namely X. maltophilia and Erwinia spp. or either of two of the bacterial isolates from natural infectious, namely E. agglomerans and Erwinia rhapontica. In contrast, pycnidiospores were only present in one out of four leaflets inoculated with fungal spore suspensions alone.

In assays for enzymes involved in the first steps of host penetration, lipase activity, determined using Difco lipase reagent with Bacto spirit blue agar, was detected in all the bacterial isolates, but no lipase activity was detected in germinating spores of S. nodorum. In contrast, endogenous cutinase activity was found in spores of all the isolates of S. nodorum , using the artificial substrate p-nitrophenyl butyrate, but production of cutinases by the bacteria was limited to a few isolates, notably those that increased pathogenicity.

To our knowledge this is the first time that bacteria associated with a fungal pathogen have been shown to increase patbogenicity of the fungus. The presence among phylloplane microorganisms of bacteria that are antagonistic to fungi is well known [1]; it should therefore not be surprising to find agonistic bacteria, i.e. those that help the fungus. Indeed, our findings parallel those of Garbaye and co-workers [2] who demonstrated that 'helper bacteria' play a positive role in the establishment of ectomycorrhizal infections of rootlets. The lack of endogenous lipase in spores of S. nodorum and the ready production of lipases by the most effective bacterial isolates could account in part for their synergistic effect.

1. Whipps JM, 1997. Advances in Botanical Research 26, 1-134.
2. Girbaye J, 1994. New Phytologist 128, 197-210.