Non-pathognic ralstonia solanacearum strain of transferred bacteriophage trp574 gene, and preparation method for and application of non-pathognic ralstonia solanacearum strain
A non-pathogenic, phage technology, applied in the field of R. solanacearum control, can solve the problems of pollution, chemical control of R. solanacearum, and the control effect needs to be improved.
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Embodiment 1
[0035] The preparation of the solanacearum engineering bacterium of embodiment 1 transfer phage trp574 gene
[0036] 1. Construction of recombinant plasmids
[0037] The inventor discovered a transcription regulator orf30 by sequencing the genome of the lytic phage P574 in the early stage, and further designed the amplification primers for the target gene orf30 according to the P574 genome information, using the phage DNA as a template, and using the phage DNA as a template for PCR amplification. A target fragment of about 688bp was obtained, and the target fragment was sequenced, and it was found that the orf30 sequence was 555bp, its nucleotide sequence was shown in SEQ ID NO: 1, and its encoded amino acid sequence was shown in SEQ ID NO: 2; BALST comparison search found that orf30 only has 82% homology with Ralstonia phage GP4, protein search results show that it has a transcriptional regulator (Transcriptional regulator) conserved sequence, and the highest amino acid seque...
Embodiment 2
[0060] Embodiment 2 Tobacco bacterial wilt control pot experiment
[0061] 1. Method
[0062] Ralstonia solanacearum culture: Activate Tb1546 stored at -80°C with TTC medium, culture at 30°C for 48 hours to obtain toxic colonies of Ralstonia solanacearum, pick a single colony and culture it in LB liquid medium for 24 hours, and set aside. Similarly, the activated orf30-Tb1546 strain was cultured with LB liquid for 24-48 hours and set aside. Potted plant inoculation: The time was June 26, 2019, in the bacterial research room of South China Agricultural University. 15kg of soil is filled in every pot, and 2 tobacco seedlings with 6 leaf ages are planted, and each handles 48 tobacco plants, totally 24 pots. Inoculate engineering bacteria orf30-Tb1546 strain first, inoculate 200mL of engineering bacteria solution in each pot, the concentration is 2×10 6 CFU / mL. The next day, the concentration of 5 × 10 6 CFU / mL of R. solanacearum Tb1546 was sprayed on the root circumference ...
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