Application of bacillus cereus BCS1 in improving resistance of plant induction system

Abstract

The invention discloses an application of a bacillus cereus BCS1 strain in improving the resistance of a plant induction system. Proved by an adjacent inoculation co-culture method, the BCS1 and the rhizoctonia solani Rs7 strain do not show a direct antagonistic or competitive effect. However, after the hydroponic system is inoculated with the BCS1, the sheath blight resistance of the rice is remarkably enhanced, specifically, the expression quantity of disease-resistant related genes in rice plants and the active oxygen level of roots are remarkably improved, and the disease spot area is remarkably reduced. Meanwhile, the roots of the rice in the BCS1 treatment group grow well, and the root length is remarkably increased. The result shows that the BCS1 can significantly enhance the induced resistance of rice, and realizes the dual functions of disease prevention and growth promotion. The application range of the bacillus cereus in the field of agricultural disease control is further widened, a new way is opened up for research and development of a green and efficient plant disease biological control strategy, and the bacillus cereus has great application potential in the fields of plant protection and green agriculture and is expected to promote green sustainable development of the industry.

Description

Technical Field

[0001] The invention belongs to the technical field of biological control, and in particular, the invention relates to application of Bacillus cereus BCS1 in improving plant induced systemic resistance. Background Art

[0002] The field of plant disease prevention and control has long relied on chemical pesticides, but the environmental pollution, health risks and pathogen resistance caused by them have become increasingly prominent. In rice production, sheath blight caused by Rhizoctonia solani has become one of the most destructive soil-borne diseases. The pathogen is widely spread in the soil-water-plant system through sclerotia and spores, continuously infecting key parts of rice such as leaf sheaths and stems from the seedling stage to the heading stage, causing leaf death, plant lodging and grain yield reduction. Pathogenic strains that are resistant to conventional chemical agents have generally appeared in major rice-growing areas in my country, and existing rice varieties generally lack effective resistance, making the prevention and control of this disease difficult.

[0003] Induced systemic resistance (ISR) has become an important development direction for green agriculture due to its unique advantages. As a plant defense mechanism activated by beneficial microorganisms, ISR is broad-spectrum, durable, and stable. It can enhance the resistance of plants to multiple pathogens at the same time, and pathogens are difficult to develop resistance. This systemic defense response can be transmitted to untreated parts through signals in the plant body. Many ISR-inducing strains can also promote plant growth at the same time, achieving the dual benefits of "disease prevention and growth promotion".

[0004] At present, microbial ISR-inducing strains represented by Bacillus have shown great potential in agricultural applications. Patent CN1221659C discloses that Bacillus cereus 98-I strain has good broad-spectrum, high antibacterial activity, and has the effect of promoting growth and seed germination of various plants, and has an inducing resistance effect on plants, but only the activity of four enzymes (catalase, phenylalanine aminotransferase, polyphenol oxidase, β-1,3 glucanase) induced by Bacillus cereus 98-I in three plants, watermelon, cucumber, and green pepper, has been determined to be improved to varying degrees. However, not all Bacillus cereus can induce plant systemic resistance, and its effect is closely related to strain characteristics, functional genes and metabolites. Bacillus cereus BCS1 is a new strain isolated by the inventors in the early stage that can effectively degrade pyrethroids and exhibit significant environmental stress resistance (Huang Y, Yang L, Pan K, et al. Heavy metal-tolerant bacteria Bacillus cereus BCS1 degradespyrethroid in a soil-plant system[J]. Journal of Hazardous Materials, 2024, 461. DOI: 10.1016 / j.jhazmat.2023.132594, GenBank sequence accession number is OR418499). Therefore, exploring whether Bacillus cereus BCS1 can induce plants, especially rice, to produce induced systemic resistance is of great significance for broadening the application scope of Bacillus cereus in the field of agricultural disease control. Summary of the invention

[0005] The purpose of the present invention is to overcome the above-mentioned defects and shortcomings in the prior art and provide an application of Bacillus cereus BCS1 in enhancing plant induced systemic resistance and / or promoting plant growth.

[0006] The second object of the present invention is to provide a method for enhancing plant-induced systemic resistance to control plant diseases.

[0007] The above-mentioned object of the present invention is achieved through the following technical solutions:

[0008] The present invention adopts the adjacent inoculation co-cultivation method and uses Bacillus cereus as the research material. It is found that Bacillus cereus BCS1 has no antagonistic or competitive effect on Rhizoctonia solani Rs7. However, after BCS1 treats rice for one day, disease-resistant related genes in rice leaves are detected by real-time fluorescence quantitative PCR (Real-Time quantitative PCR, RT-qPCR). It is found that Bacillus subtilis treatment can activate the expression of disease-resistant related genes through the jasmonic acid (Jasmonic acid, JA) pathway, and the level of reactive oxygen species (Reactive oxygen species, ROS) in the rice roots is also significantly increased. After treating rice leaves with Bacillus cereus BCS1 and inoculating Rhizoctonia solani Rs7, it was found that rice showed obvious resistance to sheath blight, and the area of ​​lesions decreased significantly. Transcriptome sequencing (RNA-seq) analysis showed that Bacillus cereus BCS1 promoted the synthesis of pathogenesis-related protein 1 (PR1) through the mitogen-activated protein kinase (MAPK) pathway and the JA disease resistance pathway, which enabled rice to induce systemic resistance. In addition, the rice roots also showed a significant effect of promoting growth, achieving the dual benefits of "disease prevention and growth promotion".

[0009] The present invention provides application of Bacillus cereus BCS1 in enhancing plant induced systemic resistance and / or promoting plant growth.

[0010] The ISR induction characteristics of the BCS1 strain have significant advantages: on the one hand, it does not rely on direct antibacterial effects, avoiding the selection pressure of pathogens; on the other hand, the defense response it activates is more persistent and systematic, can be used in conjunction with other prevention and control methods, and has a significant growth-promoting effect on rice roots, achieving the dual benefits of "disease prevention-growth promotion". At the same time, combined with the BCS1 strain's ability to resist environmental stress and effectively degrade pyrethroids, its application scenarios can be significantly broadened. For example, the BCS1 strain can be used to "prevent disease and promote growth" under environmental stress.

[0011] Furthermore, the enhancing of plant induced systemic resistance is to increase the expression level of plant disease resistance genes, increase the expression level of jasmonic acid pathway related genes, increase the level of ROS in plants and / or reduce the area of ​​plant leaf lesions.

[0012] Furthermore, the disease resistance genes include OsNH1-1 and OsPR1a.

[0013] Furthermore, the jasmonic acid pathway related genes include OsLOX and OsAOS2.

[0014] Furthermore, the promoting plant growth is promoting plant root growth.

[0015] The present invention also provides a method for enhancing plant induced systemic resistance to prevent and control plant diseases, the method comprising applying a bacterial suspension of a fermentation culture solution of Bacillus cereus BCS1 to the plants.

[0016] Furthermore, the application method includes spraying, root irrigation or root soaking.

[0017] Furthermore, the fermentation culture broth bacterial suspension is obtained by resuspending Bacillus cereus BCS1 fermentation culture broth in rice nutrient solution.

[0018] The specific method is as follows: the activated cultured Bacillus cereus BCS1 is resuspended in rice nutrient solution until the OD of the bacterial solution reaches 600 The value reaches about 1.0; then, the obtained bacterial suspension is added to the rice nutrient solution. When adding, the volume of the nutrient solution is accurately calculated to ensure that after adding the bacterial suspension, the OD value of the entire nutrient solution is 600 The value was finally adjusted to 0.001.

[0019] Furthermore, the plant disease is rice sheath blight.

[0020] Furthermore, the plant is rice.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The present invention discloses the application of Bacillus cereus in improving plant induced systemic resistance. The present invention adopts the adjacent inoculation co-cultivation method, and finds that Bacillus cereus BCS1 has no antagonistic or competitive effect on Rhizoctonia solani Rs7, but after BCS1 treats rice, it can specifically regulate the expression of genes related to the rice JA signaling pathway and the expression of disease resistance-related genes, improve the ROS level in the rice root, and the rice root grows significantly. After the rice leaves are treated with Bacillus cereus BCS1 and inoculated with Rhizoctonia solani Rs7, the rice shows obvious resistance to sheath blight, and the area of ​​lesions is significantly reduced, indicating that the treatment of BCS1 can enable rice to produce the ability to induce systemic resistance, and achieve the dual benefits of "disease prevention-growth promotion" for rice. The present invention broadens the application scope of Bacillus cereus in the field of agricultural disease prevention and control, and at the same time provides a new path for the development of green and efficient plant disease biological control strategies, and has broad application prospects in the fields of plant protection and green agriculture. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Bacillus cereus BCS1 and Rhizoctonia solani Rs7 are co-cultured in close proximity. Note: BC stands for BCS1; green * stands for the inoculation site of BCS1; red * stands for the inoculation site of Rs7; the red dotted line is the growth range of Rs7.

[0024] Figure 2 This is the effect of Bacillus cereus BCS1 treatment on the expression of resistance-related genes induced in rice leaves.

[0025] Figure 3 These are the results of ROS detection in the root cap of rice roots treated with Bacillus cereus BCS1.

[0026] Figure 4 These are the results of ROS detection in the elongation zone of rice roots treated with Bacillus cereus BCS1.

[0027] Figure 5 Bacillus cereus BCS1 induces in vitro resistance to rice sheath blight.

[0028] Figure 6 Bacillus cereus BCS1 induces resistance to rice sheath blight in vivo.

[0029] Figure 7 Analysis of rice transcriptome sequencing (RNA-seq) results under the treatment of Bacillus cereus BCS1. Note: Green represents activation, and red boxes represent significant upregulation.

[0030] Figure 8 Bacillus cereus BCS1 promotes the growth of rice roots. DETAILED DESCRIPTION

[0031] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0032] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0033] Source reference of Bacillus cereus BCS1: Huang Y, Yang L, Pan K, et al. Heavy metal-tolerant bacteria Bacillus cereus BCS1 degrades pyrethroidin as oil-plant system[J]. Journal of Hazardous Materials, 2024, 461. DOI: 10.1016 / j.jhazmat.2023.132594, GenBank sequence accession number is OR418499.

[0034] Rhizoctonia solani (Rs7) was purchased from Guangdong Microbiological Culture Collection Center with the culture collection number of GDMCC3.700 (other numbers: CGMCC3.7376).

[0035] Example 1 Co-cultivation of Bacillus and Rice Sheath Blight Pathogen

[0036] Co-cultivation method of Bacillus and rice sheath blight pathogen (Rhizoctonia solani Rs7) by adjacent inoculation:

[0037] Use a 0.5 cm diameter puncher to punch holes on the culture plate of Rhizoctonia solani Rs7, and take the agar block at the edge of the colony and inoculate it in the center of the PDA plate. Pick Bacillus cereus BCS1 from the plate and inoculate it into 50 mL LB liquid medium, and culture it at 160 rpm and 30°C for 4-5 hours until the OD value of the bacterial solution reaches 0. 600 Then, the bacterial solution was diluted with fresh liquid LB to the initial OD 600 0.004, and continue to culture until OD 600 The concentration of BCS1 was 0.4, and 1 μL of bacterial solution was added to a PDA plate 1 cm away from the agar block of pathogenic fungi for co-culture to observe the interaction between BCS1 and pathogenic fungi.

[0038] The results of the co-culture experiment of Bacillus and Rs7 are shown in Figure 1 As shown, compared with CK, although Bacillus cereus BCS1 has a certain degree of inhibition on the growth of Rhizoctonia solani Rs7, the hyphae of Rs7 can still cover the surface of the colony and grow, indicating that Bacillus cereus BCS1 will not have a direct inhibitory effect on the growth of Rs7.

[0039] Example 2 Effect of Bacillus treatment on rice disease resistance

[0040] 1. Effect of Bacillus treatment on the expression of resistance-related genes in rice

[0041] A single colony of Bacillus cereus BCS1 was picked from the plate and placed in 50 mL LB and cultured at 160 rpm and 30 °C for 4-5 hours until the bacterial solution concentration OD 600 The bacterial solution was then diluted with LB to the initial OD 600 0.004, and continue to culture until OD 600 Centrifuge at 4°C, 5000 rpm for 10 min, discard the supernatant, resuspend with sterile water, continue centrifugation at 4°C, 5000 rpm for 10 min, and resuspend with rice nutrient solution to OD 600The bacterial suspension was then added to the rice nutrient solution. When adding, the volume of the nutrient solution was accurately calculated to ensure that the OD of the entire nutrient solution was 600 The value was finally adjusted to 0.001. The rice was soaked in nutrient solution and cultured at 25°C, 75% humidity, 12 hours of light, and 12 hours of darkness. After 24 hours of treatment, the rice leaves were sampled. The sample RNA was extracted and then reverse transcribed. RT-qPCR was used to detect the transcription level of disease resistance-related genes in rice leaves, and the expression of genes was analyzed by relative quantitative analysis.

[0042] Quantitative results such as Figure 2 As shown in the figure, a total of 4 rice disease resistance-related genes were detected in the experiment, namely OsNH1-1, OsPR1a, OsLOX, OsAOS2, and 1 internal reference gene UBQ5-2. Among them, OsPR1a and OsNH1-1 are downstream genes for induced resistance, OsLOX and OsAOS2 are marker genes for JA pathway disease resistance, and the induced resistance genes in rice leaves treated with Bacillus cereus BCS1 were significantly upregulated compared with CK, which preliminarily indicates that it may have induced disease resistance to rice through the JA pathway.

[0043] 2. Effect of Bacillus cereus BCS1 treatment on ROS levels in rice

[0044] Because ROS in rice can act as local and systemic signal molecules to induce plant disease resistance, in order to explore the relationship between reactive oxygen species in rice and induced resistance, the reactive oxygen species were measured in the roots of rice inoculated with Bacillus cereus BCS1.

[0045] The DCFH-DA kit was diluted with buffer to a concentration of 10 μM. After treating rice with the method in Example 2 for 24 hours, the roots of the rice were sampled. The samples of each treatment group were immersed in 10 μM DC-FDA, and vacuum infiltrated at a pressure of 60 kpa for 5 minutes, and incubated in the dark at room temperature for 10 minutes. After the incubation is completed, the samples were washed five times with double distilled water. The positive control reagent was added to the positive control group, and the color was developed after waiting for 20-30 minutes. The sample was placed on a polylysine slide, excess water was absorbed with absorbent paper, an appropriate amount of anti-fluorescence quencher was added to the center of the sample, and the slide was sealed with nail polish. After the slide was dry, the sample was observed and photographed under a confocal microscope with a laser beam with an excitation wavelength of 488 nm. The average fluorescence intensity of the photographed rice roots was quantified using imagine J.

[0046] The ROS detection results of rice root cap are as follows Figure 3 As shown, compared with CK, the mean fluorescence intensity of the Bacillus cereus BCS1-treated group was significantly enhanced.

[0047] The results of ROS detection in the elongation zone of rice roots are as follows Figure 4 As shown, compared with CK, the average fluorescence intensity of the Bacillus cereus BCS1 treatment group was significantly enhanced, indicating that BCS1 can effectively increase the ROS level in rice roots, thereby enhancing the immune resistance of rice and inducing plants to produce induced systemic resistance.

[0048] Example 3 Determination of rice resistance to sheath blight

[0049] In order to determine whether Bacillus cereus BCS1 can improve the resistance of rice to sheath blight, the rice was treated with the method of Example 2 and then inoculated with pathogens in vitro and in vivo (the pathogen was Rhizoctonia solani Rs7).

[0050] (1) 24 hours after the Bacillus liquid treatment, samples were taken from rice leaves in each treatment group and placed flat in a moisturizing box. Mechanical damage was caused on the surface of the leaves with a needle. A 0.5 cm diameter agar block of pathogens was uniformly placed at the wound. The moisturizing box was placed in a cool, lighted place for 5-6 days. The disease condition of the leaves was observed, the leaves were photographed, and the size of the rice lesions was quantified using Imagine J.

[0051] (2) The rice treated with Bacillus liquid for 24 hours was inoculated with live pathogens. Rice leaves with similar leaf sizes and growth conditions were selected, and mechanical damage was caused to the rice leaves with a needle. Then, a 0.5 cm diameter pathogen agar block was uniformly placed at the wound site. The agar block was fixed on the rice leaf with transparent glue and cultured at 28°C, 90% humidity, 12 hours of light and 12 hours of darkness. After one week of culture, the diseased leaves were observed and photographed, and the rice lesion area was quantified using imagine J.

[0052] The results are as follows Figure 5 and Figure 6 As shown in the figure, compared with CK, the lesion area of ​​rice leaves treated with Bacillus cereus BCS1 was significantly reduced, indicating that the disease resistance of rice was significantly improved after treatment with BCS1. This shows that BCS1 can induce induced systemic resistance in plants.

[0053] Example 4 RNA-seq analysis of rice induced resistance-related pathways and expression differences of related genes

[0054] In order to explore the changes in disease resistance-related genes of rice after inoculation with Bacillus cereus BCS1, the rice treated with bacteria was sent for RNA-seq. The culture method of Bacillus is as in Example 2. One day after the rice leaves were inoculated with Bacillus, a plate of Rhizoctonia solani Rs7 cultured for 7 days was taken, and 10 mL of sterile water was injected into each plate. The fungal hyphae were gently scraped with a flat brush and mixed evenly with sterile water. After the fungal mixture was collected, it was filtered on medical gauze to make a mycelium suspension, and the suspension was immediately sprayed on the rice leaves of each treatment group. The rice treated with Rhizoctonia solani Rs7 was cultured at 28 ° C, 90% humidity, 12 hours of light, and 12 hours of darkness for 3 days. The rice leaves inoculated with Bacillus cereus BCS1 and treated with Rhizoctonia solani Rs7 were taken for transcriptome sequencing.

[0055] The results are as follows Figure 7 As shown in the figure, the transcriptome results show that after rice is inoculated with Bacillus cereus BCS1, the pattern recognition receptor (Fls2) on the surface of plant cells recognizes the bacterial flagellar protein Flg22 and activates MKK4 / 5 in the MAPK pathway. MKK4 / 5 then activates the downstream MPK3 / 6, phosphorylating it (+p) and acting on DNA, inducing PR1 gene expression; at the same time, this pathway is associated with the jasmonic acid signaling pathway. JA generates JA-Ile through JAR1, binds to COI1, promotes JAZ ubiquitination (+u) degradation in the nucleus, releases MYC2, and MYC2 acts on DNA at the same time, inducing PR1 expression to resist pathogens. The transcriptome results show that after BCS1 treatment is applied to rice roots, the synthesis of PR1 is promoted through the synergistic effect of the MAPK signaling pathway and the JA-mediated plant disease resistance signaling pathway, so that rice can play an immune and disease-resistant role.

[0056] The bar graph shows the logarithm of genes related to JA signaling pathway and MAPK signaling pathway in the BCS1 treatment group compared with the CK group after inoculation of pathogens in rice. 2 (FoldChange) value. 2 When the (Fold Change) value is greater than 1, it indicates that the corresponding gene in the BCS1 treatment group is significantly upregulated compared with the CK group, indicating that BCS1 can significantly enhance the induced systemic resistance of plants.

[0057] Example 5 Bacillus cereus BCS1 promotes the growth of rice roots

[0058] In order to explore whether BCS1 treatment has a growth-promoting effect on rice, BCS1 fermentation liquid was added to sterile water to soak rice seeds, and then they were cultivated on sterile plates. The specific method is: sterilize rice seeds, remove the seed husks, put the brown rice in a sterilized triangular bottle, add 75% ethanol to submerge the seeds, and pour out the ethanol after disinfection for 2 minutes. Add 1% NaClO, 160rpm, 25℃

[0059] Sterilize by shaking for 30-40 minutes. Wash with sterile water in a clean bench for 5 times, and then soak the seeds in sterile water at 37°C in the dark for 12-14 hours.

[0060] A single colony of Bacillus cereus BCS1 was picked from the plate and placed in 50 mL LB at 160 rpm.

[0061] Culture at 30℃ for 4-5 hours until the bacterial solution concentration OD 600 The bacterial solution was then diluted with LB to the initial OD 600 0.004, and continue to culture until OD 600 0.4, and the OD 600 = 0.4 fresh bacterial liquid was inoculated into a triangular bottle soaked in sterile water, and cultured at 80rpm and 37℃ for 8 hours, then rice seeds were sown on the plate in the clean bench, and the orientation of the seed embryos should be consistent when placed for easy observation, and finally sealed with sealing film. The plate was tilted and placed in a constant temperature light box at 28℃, 12 hours of darkness and 12 hours of light for culture, and the growth of rice was observed after 4-5 days of culture.

[0062] Rice growth results Figure 8 As shown, compared with CK, the roots of rice treated with BCS1 grew significantly and the number of lateral roots increased significantly, indicating that BCS1 can not only activate rice-induced systemic resistance, but also promote rice root growth, achieving the dual benefits of "disease prevention-growth promotion".

Claims

1. Application of Bacillus cereus BCS1 in enhancing plant-induced systemic resistance and / or promoting plant growth.

2. The use according to claim 1, characterized in that: The enhancing of plant induced systemic resistance is to increase the expression level of plant disease resistance genes, increase the expression level of jasmonic acid pathway related genes, increase the level of ROS in plants and / or reduce the area of ​​plant leaf lesions.

3. The use according to claim 2, characterized in that: The disease resistance genes include OsNH1-1 and OsPR1a.

4. The use according to claim 2, characterized in that: The jasmonic acid pathway related genes include OsLOX and OsAOS2.

5. The use according to claim 1, characterized in that: The promoting plant growth is promoting the growth of plant roots.

6. A method for enhancing plant-induced systemic resistance to control plant diseases, characterized in that: The method comprises applying a fermentation culture suspension of Bacillus cereus BCS1 to plants.

7. The method according to claim 6, characterized in that The application methods include spraying, root irrigation or root soaking.

8. The method according to claim 6, characterized in that The fermentation culture liquid bacterial suspension is obtained by resuspending the fermentation culture liquid of Bacillus cereus BCS1 in rice nutrient solution.

9. The method according to claim 6, characterized in that The plant disease is rice sheath blight.

10. The use according to claim 1 or the method according to claim 6, characterized in that: The plant is rice.

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