| Plant parasitic nematode is one of the most important infectious pathogens in agriculture production, which cause severe loses in crop production. Chemical nematocides have traditionally been widely used to control nematode diseases in the field, but they have undesirable health and environmental risks due to their toxicity and persistence. An alternative method for protection of plants against nematodes is the use of microorganisms as bicontrol agents. BC2000 is one of these bacteria isolated from rhizosphere soil.BC2000 has been proved to have functions on plant growth-promoting and antagonism against plant parasitic nematodes. Its potential value of being used as a biocontrol agent has been confirmed by series of in door or field trial. To understand the infection mechanism of this potential biocontrol agent and its ecological characteristics in the field, a specific marker is required to distinguish target bacteria from indigenous microbe, gfp is one of these kinds of promising biomarker. The aim of this research was use gfp as a fluorescent biomarker, to monitoring the colonization pattern of gfp-tagged BC2001 in the rhizosphere and plant tissue, and investigation the biological characteristics, antagonism against root knot nematode and plant growth promoting ability.BC2000 was identified using 16S rRNA gene cloning and sequencing technology, the sequence was analyzed by blasting the database in the GenBank, result showed that the 16S rDNA sequence of BC2000 shared high similarity with that of Alcaligenes faecalis. After antibiotic tolerance test, BC2000 was chromosomally tagged with gfp and luxAB by electroporation through a pUTmini-Tn5 transposon vector containing the gfp/luxAB dual markers. One transformant, designated as BC2001, exhibited strong fluorescence under fluorescence stereomicroscopy, and was chosen for further study.Chromosomal insertion of the marker gene in BC2001 was confirmed by PCR amplification and Southern blotting. Expression of luxAB gene in BC2001 was demonstrated by examining the luciferase activity of BC2001 during growth in LB medium.The growth rate of BC2001 and BC2000 was evaluated, and the gfp-taggedstrain BC2001 showed slightly slower growth rate than BC2000. 30 °C and pH7.0 were optimal for the growth of BC2000 and BC2001, and gfp expression in BC2001, although BC2001 was more sensitive to growth temperature compare to the wild type strain. Fluorescence stability of gfp gene in BC2001 kept 100% in selective and nonselective LB plates, and no green fluorescence phenotype lost after 120 h continuous transfer. Cell number of BC2001 decreased during the incubation time in soil at both of temperature 28°C and 35 °C, but the trend towards better cell survival at 28 °C than at 35°C.The anti-nematode function of BC2001 was tested by treating M. incognita with BC2001 suspension and cells alone. A strong inhibition on the egg hatching and a significant death effect on the second stage larva were observed. Pot experiments with tomato showed that GFP-tagged BC2001 promoted the plant growth, enhanced the yields and reduced the root-knot nematode galling on the roots.Two inoculation treatments with BC2001, the germinating seeds (radical just emerged) dressing and the seedlings inoculation, exhibited a significant impact on growth and galling of tomato. Inoculation of tomato seedlings with BC2001 alone promoted vegetative growth at late growth stage of the plant, while the germinating seeds not only promoted vegetative and reproductive growth but also had better suppression of root-knot nematode, and increasing the yield markedly.To monitoring the g/^-tagged BC2001 in soil, tomato rhizosphere and plant tissue, different microscopes were used to visualize the gfp fluorescence. A single cell could be detected in soil suspension with the epifluorescence microscope, and g/￡>-tagged cells were found to attach on the tomato root surface under the fluorescence steromicroscope. The fluorescence intensity of GFP from BC2001 cells was much higher than from gfp plasmid host strain E.coli CC118. 5d after the seeds inoculated with BC2001, assemblages of bacteria were observed on the surface of germinated seed hair and inside the root tissue around the 1- to 1.5-cm region of the root tip under the scanning confocal laser microscope (SCLM). SCLM pictures indicated that BC2001 not only adhered to rootlets of tomato seeds but also penetrated into root tissue, moving forward to vascular system of stem along with the growth of the plant. The result of plate counting to monitoring numbers of culturable cells in rhizosphere indicated that BC2001 kept dominant status in rhizosphere soil after introduced cells by the seedlings inoculation, whereas the seed inoculation was better for BC001 cells to colonize in the plant tissue such as root or basal stem. The 700bp gfp fragment was amplified by PCR...
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