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

Active Publication Date: 2018-03-09
湖北省烟草公司恩施州公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, there are still no very effective measures for the prevention and treatment of tobacco bacterial wilt. The comprehensive control policy of combining agricultural control, biological

Method used

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  • Two ralstonia-solanacearum-resistant endophytic bacillus velezensis strains and application thereof
  • Two ralstonia-solanacearum-resistant endophytic bacillus velezensis strains and application thereof
  • Two ralstonia-solanacearum-resistant endophytic bacillus velezensis strains and application thereof

Examples

Experimental program
Comparison scheme
Effect test

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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Abstract

The invention relates to two ralstonia-solanacearum-resistant endophytic bacillus velezensis strains and application thereof. By separation, screening and purification in roots of healthy tobacco plants in a ralstonia solanacearum occurrence area of the Enshi Prefecture, two biocontrol bacillus endophyticus strains R-3 and R-9 are obtained, have evident resistance effects on ralstonia solanacearumand are both capable of generating high-temperature-resistant ralstonia-solanacearum-resistant substances. By gyrB gene sequence identification and phylogenetic tree construction, results show that the R-3 and R-9 are both bacillus velezensis; by potted plant root irrigation experimental verification, the bacilli R-3 and R-9 have great colonization performance in tobacco roots and can enter stemsthrough root transmission; by field experimental verification, bacillus velezensis R-3 and R-9 are effective in ralstonia solanacearum prevention and treatment, remarkably superior to chemical agentkasugamycin and free of pollution. The biocontrol endophyte research field is further enriched by researching on the bacilli R-3 and R-9, and a guiding significance to ralstonia solanacearum prevention and treatment is achieved.

Description

technical field [0001] The invention relates to the technical field of microbial engineering, in particular to two strains of endophytic Bacillus veles that antagonize tobacco bacterial wilt and the application thereof. Background technique [0002] Tobacco bacterial wilt, also known as tobacco blast, is a soil-borne bacterial disease caused by Ralstonia solanacearum, and is also a typical vascular disease. The most notable symptom is wilting. It can cause the death of the whole tobacco plant and is one of the devastating diseases of tobacco, which seriously affects the yield of tobacco leaves (Feng Ji et al. 2016). Tobacco bacterial wilt is particularly serious in tropical and subtropical regions, and it generally occurs in the Yangtze River Basin and the tobacco-growing areas south of my country. Due to climate and other reasons, in recent years, the damage range of tobacco bacterial wilt has gradually spread from south to north, and the disease has continued to intensify...

Claims

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Application Information

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IPC IPC(8): C12N1/20A01N63/00A01P1/00C12R1/07
CPCA01N63/00C12N1/205C12R2001/07
Inventor 王瑞赵秀云舒照鹤姜乾坤施河丽黄勇彭阁陈红华
Owner 湖北省烟草公司恩施州公司
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