Two ralstonia-solanacearum-resistant endophytic bacillus velezensis strains and application thereof
A technology for tobacco bacterial wilt disease and Bacillus, which is applied in the field of two endophytic Bacillus velesi that antagonizes tobacco bacterial wilt disease, can solve the problems of tobacco bacterial wilt disease without very effective measures and the like, and achieve good colonization ability , the effect of good development prospects and application potential
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Embodiment 1
[0026] Screening and identification of antagonistic endophytic bacteria
[0027] 1. Isolation of tobacco endophytes
[0028] Healthy tobacco plants were selected from Yunyan 87 bacterial wilt disease-infected tobacco fields, and endophytic bacteria in roots and stems were isolated. Weigh 0.5g of roots and stems for surface disinfection: 75% ethanol for 10s-30s, rinse twice with sterile water, 0.1% mercuric chloride for 1-3min, rinse with sterile water three times (Li Yanchang et al. 2011), then add 5ml Grind well with sterile water, dilute 10 times and spread on LB plates, culture at 37°C for 48 hours, pick bacteria with different colony shapes to isolate and purify, and store the purified strains in glycerol tubes at -20°C.
[0029] 2. Screening of antagonistic endophytic bacteria
[0030] Inoculate Ralstia solanacearum in LB, culture overnight at 37°C, 180 rpm, dilute 10 times, and spray evenly on the LB plate. Pick a single colony of endophytic bacteria cultured at 37°C ...
Embodiment 2
[0038] Colonization Test of Endophytic Antagonistic Bacteria in Tobacco Plants
[0039] 1. Screening of antagonistic bacteria resistant to rifampicin
[0040] Add 2 μg / ml rifampicin to the LB solid medium, take 100 μl of the bacterial solution and spread it, and incubate at 37°C for 2-4 days. After the colony grows, select a single colony and transfer it to a 5 μg / ml rifampicin-resistant plate. Resistant colonies were screened after cultivation. Gradually increase the concentration of rifampicin to 10, 20, 40, 60 μg / ml respectively, and finally obtain the anti-60 μg / ml rifampicin strain. The resistant strains were continuously passaged 3-5 times on LB plates supplemented with 60 μg / ml rifampicin to stabilize the resistance of the resistant strains. The antagonistic ability of rifampicin-resistant strains against R. solanacearum was detected, and the resistant strains with similar antibacterial activity to the original strain were selected for colonization research.
[0041]...
Embodiment 3
[0044] In 2016, a field experiment was carried out in Liangfeng Village, Jiaoyuan Town, Xuan'en County. Strains R-3 and R-9 were inoculated at 1% in 2.5L fermentation medium, fermented at 37°C and 180rpm for 48h, diluted 10 times, and 200ml was taken and poured into the rhizosphere soil of tobacco plants in the field. 100 tobacco plants in each plot, and the condition investigation was carried out after two months. The tobacco plants were irrigated with 4% kasugamycin and clear water respectively as drug control and blank control. Three plots were set up for each treatment.
[0045] After 2 months of watering treatment, the incidence of bacterial wilt in each treatment was investigated, and the incidence rate, disease index, and control effect were calculated. The results are shown in Table 1. The control effects of R-3 and R-9 were 55.72% and 50.71%, respectively, significantly higher than that of kasugamycin.
[0046] Table 1 Field control effect of endophytic antagonisti...
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