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The bioluminescently marked Pseudomonas fluorescens strain 5RL, has been used previously to follow colonisation of soy bean roots (De Weger et al. [1991] Appl. Environ. Microbiol. 57:36-41). In the present paper the method has been further developed and optimized for wheat roots and it is used to get a quick overview of the colonisation patterns of many different root systems at the same time. Colonisation was followed on wheat plants grown in our gnotobiotic sand system (Simons et al., 1996. Mol Plant Microbe Interact 9: 600–607) and the following results were obtained. (i) A spatio-temporal analysis of the colonisation of wheat roots showed that 4 days after planting the highest bacterial activity was observed at the upper part of the root. After 6 days the high bacterial activity at the upper part was further increased, whereas spot-like activities were observed on the lower root parts, possibly due to micro-colonies. (ii) Bacterial mutations causing lack of motility or auxotrophy for amino acids resulted in impaired colonisation of the lower root parts, indicating that motility and prototrophy for the involved amino acid(s) are important factors for wheat root colonisation by strain 5RL. (iii) Coinoculation of strain 5RL with other wild type Pseudomonas strains on the root influenced the colonisation pattern observed for strain 5RL. Colonisation was not visually affected when the competing strain was a poor root coloniser, but was severely reduced when the competing strain was a good root coloniser. The results show that the spatio-temporal colonisation of wheat root by P. fluorescens strain 5RL and derivatives is similar to that of strain WCS365 on tomato. The advantage of the use of lux-marked strains is that the results are obtained much quicker than when conventional methods are used and that the result is supplied as an image of the colonisation pattern of many different roots.