Distribution Of Endophytic Bacteria In Plants By FISH And The Effect Of The Number Of Bacteria On Diseased Plants

Endophytic bacteria XG32, DLJ1, DP24, and SZ2 strains which saved in laboratory were selected as subjects to investigate the cloning and distribution in the aseptic plants through aseptic system and fluorescence in situ hybridization. The distribution of endophytic bacteria preventing of fungal diseases of plants also have been researched.The strains have been chosen according to the antagonism of endophytic bacteria and several fungal diseases, and ACC deaminase activity and the endogenous of endophytic bacteria. The strains included XG32, DLJ1 and DP24 which on Phytophthora capsici have significant inhibitory effect. The SZ2 strain could antagonistize against Sclerotinia strongly. The four strains catch with ACC deaminase activity, and can be found in the colonization of cucumber aseptic.Fluorescence in situ hybridization was applied to study the the cloning and distribution of the strain XG32 in the cucumber aseptic and strain DP24 in the capsicum aseptic in vivo. Firstly, several specific probes were designed theoretically at the base on the mid-specific sequence of the 16S rRNA of the strain XG32 and the strain DP24 with the GenBank database, BLAST program, DNAclub software and Oligo software. Then the most specific probes were selected to test assessed by the analysis software. Secondly, number of strains which similar or adjacent to the two strains were used to certify the specificity of the probe by repeated hybridization in the basis of the suitable conditions of hybridization. The result indicated that appropriate conditions for hybridization of the XG32 probe were 35% formamide and 1.2mol/L NaCl in hybridization buffer,42℃hybridization temperature,0.02mol/L NaCl in washing buffer. The appropriate conditions for hybridization of the DP24 probe were 20% formamide and 0.9mol/L NaCl in hybridization buffer,44℃hybridization temperature,0.02mol/L NaCl in washing buffer. The hybridization signal was clear between the two probes and the two strains. To sum up, the probe XG32 and DP24 were the specific probes of the strain XG32 and the strain DP24.The two probes with the Cy5 labeled for red fluorescence, and test with the plant tissue inoculated with bacteria by fluorescence in situ hybridization. The results showed that the fluorescence in situ hybridization results with the plate method basically consistent with the distribution of bacteria in both the number and distribution of parts of line, only a slight fewer of the quantity of bacteria.The strain SZ2 can be found in the colonization of cucumber and were distributed in roots, stems and leaves.Aseptic in vitro plant tissue tests showed that the bacterial suspension inhibit the Sclerotinia infection. The cucumber aseptic which inoculated to the strain SZ2 could inhibit the infection of the Sclerotinia, and the positively correlated with the amount of the bacteria. The SZ2 strain was identified as Bacillus cereus by physiological and biochemical tests and molecular identification of 16S rDNA.