1Department of Biology, and 2Department of Chemistry, University of Turku, Finland; 3Department of Organic Chemistry, Abo Akademi, Akatemiankatu 1, 20500 Turku, Finland

Background and objectives
Clavibacter xyli subsp. cynodontis is a fastidious Gram-positive bacterium found as a xylem-limited parasite of bermuda grass. When artificially inoculated, C. xyli subsp. cynodontis is able to colonize the xylem of many other plants including maize and sugarcane. We are studying the molecular biology of this bacterium and the interaction between it and its host plants. As specific plant molecules are known to induce gene expression of many plant-associated bacterial species [1], we decided to test the effect of host plant xylem sap on C. xyli subsp. cynodontis.

Materials and methods
Instead of bermuda grass, we used maize as the host for C. xyli subsp. cynodontis and as the source of xylem fluid because of the larger plant size. We collected xylem fluid of 5-6-week-old maize by root pressure aspiration, filter-sterilized it and stored at -20C. We fractionated the components of xylem sap of uninfected plants first by size, using centrifugable ultrafiltration devices. The >10 kDa molecules were washed with deionized water and phosphate buffer, and stored at 4C. The <10 kDa fraction was subsequently fractionated based on hydrophilicity using low-pressure reverse-phase chromatography, and the hydrophilic compounds then fractionated on an HPLC gel filtration column (Ultrahydrogel DP, Waters). All the fractions of maize xylem sap were filter-sterilized and tested on C. xyli subsp. cynodontis to track the growth-enhancing activity. Samples of the fractions were added to liquid culture medium [2], 1/10 of the volume, and the growth of bacteria was observed by measuring the OD650 of each culture.

Results and conclusions
We discovered that the growth of C. xyli subsp. cynodontis in vitro is enhanced by host plant xylem fluid. The logarithmic phase growth rate increases, and, when taking bacterial inoculum from a culture more than a week old, the bacteria do not grow in liquid medium unless it is conditioned with the plant material. Moreover, even fresh C. xyli subsp. cynodontis cells do not grow in liquid medium without the plant factors if the medium has been stored over 2 months at RT. Additional nutrients like sugars or group B vitamins did not cause the same kind of growth induction. Instead, a root extract of bermuda grass prepared by soaking washed roots in water had a similar effect on C. xyli subsp. cynodontis as maize xylem sap.

The xylem sap of maize infected by C. xyli subsp. cynodontis had the same amount of enhancing activity as uninfected sap. This implies that this bacterium does not produce or make the host produce compounds that would further enhance bacterial growth.

We found that maize xylem fluid contains two distinct factors that enhance the growth of C. xyli subsp. cynodontis. One of them is a labile low molecular-weight organic compound, whose NMR, Raman and mass spectra point to an amino-substituted carbohydrate having a polymer-like repeating structure. The other enhancer is 10-90 kDa in size, and its activity is eliminated by protease treatment and reduced by heating to 65C, suggesting that it is a heat-sensitive protein. We think that these molecules may act as signals to C. xyli subsp. cynodontis, enabling the bacterium to sense when it reaches the plant xylem. The compound characterized as a carbohydrate may also fill some special nutritional demands of this fastidious bacterium.

1. Peters NK, Verma DPS, 1990. Molecular Plant-Microbe Interactions 3, 4-8.
2. Haapalainen ML, Kobets N, Piruzian E, Metzler MC, 1997. FEMS Microbiology Letters (in press).