Identification of antagonistic bacteria

Sodium dodecyl sulfate-proteinase K lysis method was used for DNA extraction, cetyltrimethylammonium bromide was used for precipitation of cell fragment and polysaccharide, and isopropanol precipitation was used for extraction.

Bacterial 16S rDNA was amplified according to the method of Coenye et al.,16 primers were as follows: 27 forward: 5′-AGA GTT TGA TCC TGG CTC AG-3′ and 1541 reverse: 5′-AAG GAG GTG ATC CAC CC-3′. Polymerase chain reaction (PCR) amplification condition was as follows: pre-denaturation was performed at 94 °C for 5 min; denaturation at 94 °C for 40 s; annealing at 55 °C for 50 s; extension at 72 °C for 1 min 15 s; the total number of cycles was 35; termination was done at 72 °C for 10 min. PCR: 0.8 μL Taq (5 U/μL); 10 μL 10× PCR buffer (Mg²⁺ Plus); 8 μL deoxynucleotide triphosphate (dNTP) mixture (2.5 mM each); 2.5 ng template DNA; 2 μL primer F1 (10 μmoL/L); 2 μL primer R1 (10 μmoL/L); ddH₂O was supplemented up to 100 μL. DNA fragments were recovered and purified, and sequence measurement was performed by Shanghai Invitrogen Biotechnology Co. Ltd. in Beijing.

ropB gene was amplified according to the method of Brady et al.,17 forward primer of CM7-f: 5′-AACCAgTTCCgCgTTggCCTg-3′ and reverse primer of CM7-r: 5′-CCTgAACAACACgCTCggA-3′. PCR amplification reaction condition: pre-denaturation was performed at 95 °C for 5 min; denaturation at 95 °C for 35 s; annealing at 55 °C for 1 min 15 s; extension at 72 °C for 1 min 15 s; the total number of cycles was 30; termination was done at 72 °C for 7 min. ropB gene sequence reaction volume was 100 μL: 0.8 μL Taq (5 U/μL); 10 μL 10× PCR buffer (Mg²⁺ Plus); 8 μL dNTP mixture (2.5 mM each); 2.5 ng DNA template; 2 μL F1 primer (10 μmol/L); 2 μL R1 primer (10 μmol/L); ddH₂O was supplemented up to 100 μL. DNA fragments were recovered and purified, and then sequence measurement was performed by Shanghai Invitrogen Biotechnology Co. Ltd.

After sequencing DNA genes, homological BLAST was compared in the National Center for Biotechnology Information database. MEGA4.0 neighbor-joining method was used for the construction of dendrogram, and 1000 times of similarity were calculated. Dendrogram node showed that the value of bootstrap was >50%.18

Physiological and biochemical measurements were performed for two bacterial strains according to the 9th edition of Bergey's Manual of Determinative Bacteriology.19