Application of D-amino acids in inhibition for biofilms of banana bacterial Erwinia carotovora and culture medium thereof
A technology of bacterial biofilm and soft rot pathogens, applied in the direction of microorganism-based methods, bacteria, microorganisms, etc., can solve the problems of death and harm to the banana industry, and achieve the effects of growth inhibition, environmental protection of pesticides, and reduction of use concentration
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Embodiment 1
[0035] Embodiment 1: the cultivation of bacteria and the cultivation of bacterial biofilm
[0036]Bacterial strain Dickeya zeae MS1 (Identification of Dickeya zeae as a Causal Agent of Bacterial Soft Rot in Banana in China. Plant Disease, 2014, 98(4): 436-442.) was inoculated with glycerol frozen (-80°C) Streak culture on NA solid medium (beef extract 3g, yeast extract 1g, peptone 5g, glucose 10g, agar 18g, water to 1000mL, pH 7.2-7.4) plate; pick a single colony and culture in liquid NA Base (3g beef extract, 1g yeast extract, 5g peptone, 10g glucose, water to 1000mL, pH 7.2-7.4), 180rpm, 32°C.
[0037] Cultivation of Dickeya zeae MS1 to 10 8 CFU / mL; NA liquid medium to dilute the bacterial solution to 1:1000. Take 2mL of the diluted bacterial solution and add it to a polystyrene 24 microwell plate. Each sample has 3 replicates, and cultured at 32°C. After culturing for 6h, 12h, 24h, 36h, 48h, 60h, 72h, 96h, 144h, and 216h, first extract the planktonic bacteria in a 96-wel...
Embodiment 2
[0040] Embodiment 2: Analysis of the effect of different D-amino acids inhibiting banana bacterial soft rot
[0041] Formulated D-valine, D-methionine, D-tryptophan, D-threonine, D-arginine, D-proline, D-leucine, D-serine, D-histidine , D-aspartic acid, D-glutamic acid, D-glutamine, D-phenylalanine, D-alanine, etc. 100mMol / L storage solution; prepare 10mMol / L of D-tyrosine L storage solution, wherein the D-tyrosine storage solution contains 0.1Mol / L HCl. Prepare series of NA liquid media with D-amino acid concentrations according to different concentration ratios; 500μMol / L (abbreviated as μM) concentration of D-tyrosine NA liquid medium (beef extract 3g, yeast extract 1g, peptone 5g, glucose 10g, water to 1000mL, pH 7.2 ~ 7.4); respectively configure 1 , 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17.5 and 20mMol / L (abbreviated mM) of other D-amino acid NA medium (beef extract 3g, yeast extract 1g, peptone 5g, glucose 10g, water to 1000mL, pH 7.2-7.4).
[0042] Inoculate th...
Embodiment 3
[0047] Embodiment 3: the crystal violet staining analysis of D-amino acid inhibition bacterial biofilm effect
[0048] Inoculate the bacterial strain Dickeya zeae MS1 frozen in glycerol (-80°C) into NA solid medium (beef extract 3g, yeast extract 1g, peptone 5g, glucose 10g, agar 18g, water to 1000mL, pH 7.2-7.4 ) plate, streak culture; pick a single colony and culture in NA liquid medium, 180rpm, 32°C.
[0049] Cultivation of Dickeya zeae MS1 to 10 8 CFU / mL; Prepare 20mM concentration of D-threonine and D-alanine NA liquid medium, 200μM and 420μM concentration of D-tyrosine NA liquid medium, 3mM and 5mM concentration of D-tryptophan NA medium, 5mM and 11mM concentration of D-valine NA medium, 7mM and 15mM concentration of D-methionine NA medium; dilute the bacterial solution to 1:1000 with different concentrations of D-amino acid NA medium, set without D-amino acid bacterial liquid control, and sterile liquid NA medium control. Take 0.2mL of the diluted bacterial solution ...
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