Humboldt University Berlin, Faculty for Aghculture and Horticulture, D-10099 Berlin, Germany

Background and objectives
The root-colonizing bacterium Bacillus subtilis FZB249D has been registered in Germany as plant-strengthening agent to improve plant health to biocontrol the pathogenesis of different root-infecting fungi, bacteria and also some abiotic stress factors. Seed or seedplant applications to introduce B. ;subtilis into the rhizosphere resulted in remarkably better plant growth and health, compared with unbacterized plants, on exposure to natural substrate infestations, e.g. Rhizoctonia solani, Fusarium oxysporum, Scierotinia scierotiorum, Pythium spp. and Phytophthora spp., as well as toxic stresses (salt, unspecific toxin), in field and greenhouse experiments with potatoes, maize, sunflowers, tomatoes, cucumbers and some ornamental plants. Studies to understand the mechanisms of these effects, which may be based on metabolic influences from the root colonizing bacteria on plant activities, have been made.

Materials and methods
To identify relevant bacterial exogenic metabolites, standardized culture filtrates (CF) were produced from two active B. ;subtilis isolates. Samples were taken from the three different steps of the bacterial fermentation process - the logarithmic, transition and stationary phase - and each were tested with 0.1% and 1% in nutrient solution tubes, in which 8-day-old tomato seedlings were grown under controlled climatic conditions for 10 ;days. Shoot and root growth (fresh and dry matter) of test seedlings were calculated. Following CF, pretreated seedlings were cleaned and transferred into nutrient solution tubes inoculated with microconidia of the pathogen F. ;oxysporum f. ;sp. radicis-lycopersici; 5, 7 and 10 ;days after incubation the number of diseased, collapsed test seedlings were counted and invasion of the fungus in the xylem vessels was determined. In CF-treated seedlings changes of the phytohormone level (IAA) and enzymatic activities (chitinase, peroxidase, 9-1,3-glucanase) were measured. All CFs tested also had direct antibiotic activity against the pathogen. The transition CF were further fractionated by HPLC in a number of fractions with only main substance peak (Gl-G6). These fractions were tested each in the same way as described for the complex CF. The IAA precursor indole-3-pyruvic acid (1PyA) was found in one CF . IPyA was also tested in the system, compared with classical phytohormones (AA, IBA, kinetin).

Results and conclusions
The 0.1% CF from the logarithmic and transition fermentation phases of both bacterial isolates significantly promoted the root growth of tomato seedlings after 10nbsp;days' cultivation. However, the 1% CF inhibited the growth of plants, because of the higher levels of antibiotic substances present. Precultivations of test seedlings with the 0.1% CF from the transition phase reduced significantly the plant invasion of the postinoculated test pathogen (to about 50%) and the number of killed plants, although the 0.1% CF did not have any antibiotic activity. The pretreated seedlings showed an increased level of IAA and increased activity of alpha-1,3-glucanase. Among the non-antibiotic CF fractions tested, G3, G5 and G6 did stimulate the test plant root growth in similar way. Fraction G3, containing special protein(s) in the range of 10-15 ;kDa, enhanced not only the root growth but also induced resistance and tolerance towards the test pathogen (60% after 7 ;days incubation). As with the G3 fraction, test plant pretreatments with IPyA stimulated plant growth and reduced significantly the pathogenesis of the test fungus. This could not be found by respective applications of classical phytohormones, such as IAA, IBA or kinetin. After the G3 and IPyA application the IAA level and 1,3-glucanase activity also increased in treated seedlings. It would appear that B. ;subtilis, in interaction with plant roots, produces specific exogenic bacterial metabolites (special protein(s), IPyA) that appear to act as elicitors for plant resistance and tolerance combined with growth promotion by changing the phytohormone balance in the plant system.

1. Dolej ST, 1998. Dissertation, Agriculture-Horticulture Faculty, Humboldt University, Berlin.
2. Alemayehu M, 1997. Dissertation, Agriculture-Horticulture Faculty, Humboldt University, Berlin.