Compound flora for preventing and treating bacterial wilt

Abstract

The invention discloses a compound flora for preventing and treating bacterial wilt, the compound flora is a combination of pseudomonas sp. Y364, pseudomonas sp. Y832 and bacillus altitudinis 878, the pseudomonas sp. Y364 is Pseudomonas sp., the preservation number is CGMCC (China General Microbiological Culture Collection Center) No.30249, and the preservation number is CGMCC No.30249; the pseudomonas sp. Y832 is Pseudomonas sp., and the preservation number of the pseudomonas sp. Y832 is CGMCC (China General Microbiological Culture Collection Center) No.30247; the bacillus altitudinis 878 is bacillus altitudinis, and the preservation number of the bacillus altitudinis 878 is 30246; the compound flora comprehensively considers the function size and compatibility of each strain, not only has a better control effect, but also can avoid the combination of incompatible strains, and can ensure the stability and synergistic effect of the compound flora, thereby achieving the optimal effect in practical application, and greatly improving the colonization ability of the fungicide in rhizosphere.

Description

Technical Field

[0001] The invention relates to a composite bacterial community for preventing and treating bacterial wilt, and belongs to the technical field of bacterial wilt prevention and treatment. Background Art

[0002] Bacterial wilt can break out in more than 200 plant species from 50 plant families, including many cash crops such as tobacco, tomatoes and potatoes. The disease can easily cause large-scale crop yield reduction or even crop failure. It is a devastating soil-borne disease that has seriously threatened the sustainable development of Solanaceae crops.

[0003] In recent years, the research on plant diseases includes chemical control, physical control, and biological control, among which biological control is a hot topic. Biological control of diseases has the advantages of being economical, pollution-free, and having long-lasting effects, and it is in line with the idea of ​​sustainable agricultural development, and has attracted great attention from researchers at home and abroad. At present, the screening of microbial agents for biological control of diseases has a good prospect for utilization, but the screened agents are greatly affected by human operation factors, and the agents are not easy to colonize in the field, etc. These problems have seriously restricted the large-scale promotion and application of biological control. Summary of the invention

[0004] Based on the above, the present invention provides a composite bacterial community for preventing and controlling bacterial wilt disease, which can enhance the rhizosphere colonization ability of bacterial agents, so as to overcome the shortcomings of the prior art.

[0005] The technical scheme of the invention is: a composite bacterial community for preventing and treating bacterial wilt, which is a combination of Pseudomonas Y364, Pseudomonas Y832 and Bacillus altitudinis Y878; wherein Pseudomonas Y364 is Pseudomonas nicosulfuronedens, with a preservation number of CGMCC No. 30249; Pseudomonas Y832 is Pseudomonas delhiensis, with a preservation number of CGMCC No. 30247; and Bacillus altitudinis Y878 is Bacillus altitudinis, with a preservation number of 30246.

[0006] Preferably, the total cell number ratio of Pseudomonas Y364, Pseudomonas Y832 and Bacillus subtilis Y878 in each milliliter of the composite bacterial population is 1:1:1.

[0007] Beneficial effects of the present invention: The composite bacterial community of the present invention comprehensively considers the functional size and compatibility of each strain, not only has a good prevention and control effect, but also can avoid the combination of incompatible strains, can ensure the stability and synergistic effect of the composite bacterial community, thereby achieving the best effect in practical applications, and greatly improving the colonization ability of the bacterial agent in the rhizosphere. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1Greenhouse efficacy of different treatments against tobacco bacterial wilt;

[0009] Figure 2 Effects of different treatments on tobacco plant bacterial wilt

[0010] Figure 3 30% turbid wells in a 96-well cell culture plate;

[0011] Figure 4 Compatibility test between strains;

[0012] Figure 5 Compatibility results between strains;

[0013] Figure 6 Phylogenetic tree of strains based on 16S rDNA sequences constructed by the neighbor-joining method. A, B, and C are the phylogenetic trees of Y364, Y832, and Y878 based on 16S rDNA, respectively. DETAILED DESCRIPTION

[0014] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below in conjunction with the accompanying drawings. In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present invention, so the present invention is not limited by the specific implementation disclosed below.

[0015] Pseudomonas Y364 is Pseudomonas nicosulfuronedens, with a preservation number of CGMCCNo.30249; Pseudomonas Y832 is Pseudomonas delhiensis, with a preservation number of CGMCCNo.30247; Bacillus altitudinis Y878 is Bacillus altitudinis, with a preservation number of 30246. The above three strains were all deposited in the General Microbiology Center of China Culture Collection Administration on April 2, 2024.

[0016] Experiment: The fermentation liquid of the composite bacterial community (a combination of Pseudomonas Y364, Pseudomonas Y832 and Bacillus subtilis Y878) is an equal volume mixture of each single bacteria. Nine tobacco plants were treated for three times and randomly arranged in the greenhouse. Before transplanting, the roots were soaked in the suspension of antagonistic bacteria for 30 minutes and transplanted into the soil with bacterial wilt. After 9 days, 10 mL of the suspension of antagonistic bacteria was inoculated in the rhizosphere and repeated once every 3 days. The suspension of antagonistic bacteria was replaced with an equal amount of clean water as a control. The effect of bacterial wilt prevention and control was statistically analyzed.

[0017]

[0018] Control effect = (control disease index - treatment disease index) / control disease index × 100%

[0019] Results: The incidence of each treatment is shown in Figure 1 Among them, the composite flora (Y364+Y832+Y878) had the lowest incidence rate ( Figure 2 A), followed by Y364, and CK treatment was the highest. The disease incidence of Y364 and Y878 treatments was lower than that of Y832 treatment, and there was a significant difference. The three were 25.93%, 13.58%, and 23.46% lower than that of CK treatment, and 33.33%, 45.68%, and 35.80% higher than that of the mixed bacterial flora treatment, and there was a significant difference. The disease index of the mixed bacterial flora was the lowest ( Figure 2 B), followed by Y878, and CK was the highest. There was no significant difference in disease index between Y832 and Y878 treatments, both of which were significantly reduced by 15.64% and 18.66% compared with Y364 treatment. The disease index of Y364, Y878 and Y832 treatments were 2.24 times, 2.46 times and 3.65 times that of the composite flora, respectively.

[0020] Since the antagonistic bacteria were screened from the rhizosphere and had a strong chemotactic effect on root secretions, they could use root secretions to enhance their colonization in the rhizosphere, so the control efficiency of the composite bacterial community reached 79.75%. Fu Pinshan et al. (2021) used the main flue-cured tobacco variety "Yunyan 87" as the test object, and through field plot experiments, they clarified the effects of different application methods of composite fluorescent Pseudomonas on the occurrence of tobacco bacterial wilt and the growth and development of tobacco. The final protective effects of applying 5g of bacterial fertilizer in the pot during the transplanting period, 100mL of microbial solution for root irrigation, and 5g of bacterial fertilizer in the pot during the transplanting period and 100mL of microbial solution for root irrigation were 31.70%, 27.35% and 22.39%, respectively. There was no statistically significant difference between them, and the growth of tobacco plants in the microbial agent treatment area was better than that in the control area (Fu Pinshan, Yang Lei, Zhang Shuai, Ran Yuao, Dai Yuhao, Ding Wei. Effects of different application methods of Pseudomonas fluorescens on the occurrence of tobacco bacterial wilt in the field. Plant Doctor, 2021, 34(6):28-32.). Shi Heli et al. (2021) studied 1.5kg / hm 2The 120-day biological control effect of microbial agents on tobacco bacterial wilt can reach 47.15%, which is much lower than the chemotactic microbial flora screened by high-throughput method in this study (Shi Heli, Xiang Bikun, Zuo Mei, Peng Wuxing, Yin Zhongchun, Wang Rui, Tan Jun. Effects of fulvic acid and microbial agents on tobacco bacterial wilt and rhizosphere soil bacterial community. Tobacco Science and Technology, 2021, 54(9): 1-10.). For other crops, Zhou Dengbo et al. (2013) compounded the 4-L-16 strain isolated from rapeseed cake fermentation liquid and the T3-G-59 strain isolated from the healthy banana garden of Meitai, and fermented them in 6 kinds of cake fertilizer substrates. The fermented liquid of bean cake, peanut cake and rapeseed cake has the best control effect on banana wilt, with the control effect reaching 68.22%, 54.21% and 57.94% respectively (Zhou Dengbo. Effect of compound antagonistic bacteria fermented liquid on banana wilt and soil microbial flora. Doctoral dissertation of Hainan University, 2013.). It can be seen that the control effect of the composite flora of the present application reaches 79.75%, while the control effect of the conventional flora can only reach 68.22% at most. It can be seen that the composite flora of the present invention can ensure the stability and synergy of the composite flora, so as to achieve the best effect in practical application and greatly improve the colonization ability of the bacterial agent in the rhizosphere.

[0021] The screening process of the present invention is described in detail below:

[0022] S1 isolates and cultivates antagonistic strains of bacterial wilt by high-throughput separation;

[0023] S11 Collection of sample plants and pretreatment: 30 days after transplanting, the Yanyan 97 tobacco plant samples were completely removed from the soil, and the aboveground parts and impurities of the tobacco plants were removed with a scalpel, leaving only the roots of the tobacco plants. The roots were immersed in sterile water and gently shaken to remove soil particles, etc. The fresh roots were placed in a centrifuge tube filled with PBS solution, shaken at 180 rpm / min for 15 minutes, and the PBS liquid attached to the roots was absorbed with sterile absorbent paper, and this process was repeated three times.

[0024] S12 culture: Weigh 0.03 g of root system into a 2 mL centrifuge tube, add 200 μL of 10 mM MgCl2 solution, grind evenly with a pestle, transfer to a 50 mL sterile centrifuge tube, add 25 mL of 10 mM MgCl2 solution, mix evenly and let stand for 15 min, draw a specific volume of root solution (determined by the dilution factor) and add it to 250 mL of 10% TSB medium, mix evenly and transfer to a 96-well cell culture plate, seal with parafilm and culture in a shaker at 30°C, 100 rpm / min for 48 h.

[0025] S13 screening: From the turbid culture wells of ODC, use an inoculation loop to streak on 50% TSA medium to isolate and purify the strain. Repeat three times for each well. Select the strains with different appearance (including color, shape, edge, etc.) of the colonies on the culture medium and purify them for later use.

[0026] The high-throughput screening method for bacterial strains is based on the mathematical principle of Poisson distribution, that is, under ODC conditions, a single bacterium is extracted from the root system and separated into a culture well of a cell culture plate. After a period of culture, the number of wells that show turbidity reaction accounts for about 30% of the total number of wells. When about 30% of the wells become turbid, it indicates microbial growth, and this dilution concentration is called the "optimal dilution concentration (ODC)". The mathematical binary search method is used to find the ODC: the first setting is 5×10 3 Dilution multiple, 5×10 4 Dilution multiple, 5×10 5 Dilution factor: 5×10 3 and 5×10 4 The turbid pores are close to 100% and 67%, respectively, while 5×10 5 The second setting is 2×10 5 Dilution multiple, 3×10 5 Dilution multiple, 4×10 5 Dilution factor: 2×10 5 About 25% of the pores are turbid, 3×10 5 Turbid pores account for 19%, 4×10 5 11%. The third setting is 7.5×10 4 Multiple, 1.25×10 5 Multiple, 1.75×10 5 The experiment finally determined that the dilution factor close to ODC was 1.25×10 5 The dilution factor is about 31%, and the turbid pores account for about 31%. Figure 3 shown.

[0027] S2 sorts all antagonistic strains according to their antagonistic abilities, and obtains A = {a1, a2, a3…am} to define the solution space;

[0028] 372 strains of rhizospheric bacteria with different morphologies were obtained from the root system, and a total of 109 antagonistic strains with stable antagonistic effects on bacterial wilt pathogens were screened out, accounting for 29.3%, of which 25 strains had an inhibition zone diameter greater than or equal to 20 mm (Table 1 below). The 25 antagonistic bacteria had different inhibitory effects on bacterial wilt, with an inhibition zone diameter ranging from 20.0 to 27.0 mm, with an average of 22.0 mm, while strains with an inhibition zone diameter greater than 22.0 mm accounted for only 32%, and strains with an inhibition zone diameter greater than 25.0 mm accounted for only 20%.

[0029] S3 selects the first three strains from the sorting results as the initial solution S0 = {a1, a2, a3}, and the superposition of all ability assignments is recorded as N0;

[0030] S4 uses chemotaxis as a screening parameter, removes strains without chemotaxis from S0, and replaces them with strains with chemotaxis in the subsequent ranking in A;

[0031] Using Ralstonia solanacearum as the target, the spray plate confrontation method was used to screen antagonistic bacteria. Ralstonia solanacearum was inoculated into liquid modified TTC medium and cultured at 30°C and 150rpm / min for 24 hours for standby use. The purified antagonistic strain was inoculated in the center of the modified TTC medium by the dot grafting method. After dark culture at 30°C for 12 hours, the fermentation liquid of Ralstonia solanacearum was evenly sprayed on the culture dish and placed in a 30°C incubator for culture for 24 hours. The diameter of the inhibition zone was measured by the cross method. The same volume of sterile water was inoculated as a control. The chemotaxis detection method of antagonistic bacteria uses the bacterial histidine kinase gene CheA as a target for molecular diagnosis to screen out chemotactic bacteria. Primers P4P5-F (5'-GGIMGIGGIGTIGGIATGGAYGTIGT-3') and P4P5-R (5'-CCRTCICCIARIATIGTIGCICC-3'). The PCR reaction system is 25μL, which contains 12.5μL of KOD polymerase (One TM PCR Master Mix), 2 μL primers (1 μL each for forward and reverse primers), 1 μL DNA template and 9.5 μL sterile ddH2O. PCR program: 95℃ pre-denaturation for 10 min, 94℃ denaturation for 20 s; 55℃ annealing for 60 s, 68℃ extension for 5 s, 30 cycles; 72℃ extension for 5 min. ddH2O was used as negative control. The bright bands were observed by gel imaging system.

[0032] The experiment showed that using the bacterial histidine kinase gene CheA as a target for molecular diagnosis, a specific band of about 500bp in length could be amplified, of which 12 strains were negative for CheA gene amplification, accounting for 48%, and 13 strains were positive, accounting for 52%. The three strains with the strongest antibacterial effect were Y364, Y362 and Y366.

[0033] Table 1 Antibacterial effect and chemotaxis of antagonistic bacteria on tobacco solanacearum

[0034]

[0035] Note: + indicates positive, - indicates no positive

[0036] S5 used the compatibility between strains as the screening criterion, obtained different solutions S, and selected the S with the largest N value as the optimal combination of compound functional bacterial agents.

[0037] The cross-streaking method was used to determine whether there was compatibility between the two strains. A square culture dish was used to prepare a 50% TSA plate medium. The antagonistic bacterial strains were inoculated on the medium in vertical cross-streaking patterns. The culture medium was repeated three times and placed in a 30°C incubator for 24 hours. If no growth occurred at the intersection of the two bacteria, it indicated that the bacteria were incompatible. Otherwise, the two bacteria were compatible ( Figure 4 ).

[0038] The compatibility data of antagonistic bacteria are presented in the form of heat map grids, such as Figure 5 As shown. Overall, most strains are compatible with each other, and 79.52% of all pairwise strain combinations can be recombined into bacterial communities. Y835 has the best compatibility, being compatible with all other strains, while Y365 is only incompatible with one strain, Y364, followed by Y832, Y836, and Y361; the strain with the worst compatibility is Y391, with only 12 strains compatible with it, followed by Y878, Y343, and Y900, which are only compatible with 15 strains.

[0039] Test results: The top three strains with the highest antagonistic diameters are Y364, Y362, and Y366. First, we determined that the composite bacterial community S0 is Y364, Y362, and Y366, and the sum of their antagonistic diameters N0 is 78.7. According to the results in Table 1, strains Y366 and Y362 have no chemotaxis and are not considered as candidate strains. Add strains Y831 and Y832 that are ranked first in antagonistic diameter and have chemotaxis. At this time, S is Y364, Y831, and Y832. Figure 4It was found that Y831 and Y832 are incompatible. First, assume that Y831 is a necessary strain, and screen the strain Y835, which ranks high in antagonistic diameter, has chemotaxis and is compatible with Y831 and Y364, to replace Y832, and obtain S1 of Y364, Y831, and Y835, and the sum of the antagonistic diameters of the three N1 is 73.0. Assume that Y832 is a necessary strain, and similarly, screen the strain Y878, which ranks high in antagonistic diameter, has chemotaxis and is compatible with Y832 and Y364, to replace Y831, so that S2 is Y364, Y832, and Y878, and the sum of the antagonistic diameters of the three N2 is 75.3. Since N2 is greater than N1, Y364, Y832, and Y878 are selected to form a composite bacterial community.

[0040] S6 strain identification.

[0041] The bacterial 16S rDNA sequence was amplified by colony PCR. The 16S RNA sequences of each strain were sequenced for full multiple sequence alignment. After removing the differential sequences at both ends, the Kimura2-parameter model in the Neighbor-Joining method was used to run 1000 bootstraps to construct the phylogenetic tree of the strain.

[0042] The results of 16S rDNA sequencing showed that the gene fragments of strains Y364 and Y832 were 1433bp and 1516bp, respectively. BLAST on the NCBI website found that strains with high 16S rDNA homology of strains Y364 and Y832 both belonged to Pseudomonas spp., with similarities of 100% and 99% to Pseudomonas nicosulfuronedens and Pseudomonasdelhiensis, respectively. The gene fragment size of strain Y878 was 1447bp, with a similarity of 100% to Bacillus spp. strains. Phylogenetic tree analysis results ( Figure 6 ) showed that strain Y364 and strain Y832 were closely clustered with Pseudomonas nicosulfuronedens (NR_180597.1) and Pseudomonas delhiensis (NR_043731.1), respectively, and strain Y878 was closely clustered with Bacillus altitudinis (NR_042337.1). The confidence values ​​between the major branches of the evolutionary tree were all high, so Y878 was identified as Bacillus altitudinis.

[0043] The experiment first uses high-throughput screening and sorting to quickly obtain targeted strains from a large number of strains, greatly shortening the screening time. When compounding, by comprehensively considering the functional size, multi-function and compatibility of the strains, it can ensure that the selected strain combination is functionally optimal and avoid the combination of incompatible strains, which helps to ensure the stability and synergy of the compound bacterial flora, thereby achieving the best effect in practical applications.

[0044] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A composite bacterial flora for preventing and treating bacterial wilt, characterized in that: The composite bacterial flora is a combination of Pseudomonas Y364, Pseudomonas Y832 and Bacillus altitudinis Y878; wherein, Pseudomonas Y364 is Pseudomonas nicosulfuronedens, with a preservation number of CGMCC No.30249; Pseudomonas Y832 is Pseudomonas delhiensis, with a preservation number of CGMCC No.30247; and Bacillus altitudinis Y878 is Bacillus altitudinis, with a preservation number of 30246.

2. The composite bacterial flora for preventing and treating bacterial wilt according to claim 1, characterized in that: The total cell number ratio of Pseudomonas Y364, Pseudomonas Y832 and Bacillus subtilis Y878 in each milliliter of the composite bacterial flora is 1:1:1.

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