Endophytic strain of wheat, Bacillus halotolerans YB-1576, biocontrol agent and its application

Abstract

The present application discloses a salt-tolerant Bacillus endophytic strain of wheat, Bacillus sp. YB-1576, a biocontrol agent thereof, and its application, aiming to solve the technical problem of the lack of effective biological control measures for current wheat basal rot disease. In the present application, a salt-tolerant Bacillus endophytic strain of wheat, Bacillus sp. Halotolerant Bacillus ) YB-1576, with a preservation number of CGMCC No. 24705, is isolated and screened from wheat. This strain can induce the systemic disease resistance of wheat plants, has a good control effect on wheat basal rot disease, and can promote the growth and development of wheat at the same time; the biocontrol agent prepared by using the strain of the present application can replace or reduce the use of chemical pesticides, improve food and ecological environment safety, and has good economic and social benefits.

Description

Technical Field

[0001] The present invention relates to the technical field of biological control, and particularly relates to a salt-tolerant Bacillus endophytic strain YB-1576, a biocontrol agent and their applications. Background Art

[0002] Wheat is the main food source for humans and livestock worldwide. Currently, the yield and quality of wheat are affected by various fungal diseases; among them, wheat basal stalk rot caused by Fusarium pseudograminearum is one of the most destructive diseases of wheat worldwide. It not only causes a significant reduction or even complete loss of wheat yield, but also contaminates various harmful mycotoxins (such as deoxynivalenol, etc.) in wheat grains, posing a serious threat to food safety and the health and safety of humans and livestock.

[0003] At present, the prevention and control measures for wheat basal stalk rot mainly include cultivating resistant varieties and applying chemical agents. Cultivating and planting disease-resistant varieties is the most economical, safe and effective measure for controlling wheat basal stalk rot; however, due to the scarcity of natural disease-resistant germplasm resources and the relatively complex inheritance of resistance to wheat basal stalk rot, it is difficult to obtain disease-resistant varieties of basal stalk rot through traditional breeding at present. Therefore, applying chemical agents is still the main measure for controlling basal stalk rot at present; but long-term application of chemical agents will lead to an increase in the drug resistance of Fusarium pseudograminearum, increasing the difficulty of prevention and control. In addition, the occurrence frequency of wheat basal stalk rot in wheat production areas in China has increased significantly, showing a trend of spreading and expanding, seriously affecting the sustainability of safe and high-quality wheat production in China.

[0004] In recent years, there has been increasing attention to ecological environmental protection globally, advocating green, environmentally friendly and sustainable agriculture, and biological control of crop diseases has received more and more attention. Biocontrol agents can reduce and avoid the negative impacts brought by chemical agents, thereby achieving the prevention and control of the occurrence of wheat stalk rot and the damage to the ecological environment, and then ensuring the safe and high-quality production of wheat. Therefore, the selection and breeding of highly effective antagonistic strains against Fusarium pseudograminearum, and the preparation and application methods of the agents are problems that need to be urgently solved by those skilled in the art.

[0005] The information disclosed in this background art section is only used to deepen the understanding of the background art of the present disclosure, and should not be regarded as an admission or any form of implication that this information constitutes the prior art known to those skilled in the art. Summary of the Invention

[0006] The inventor isolated a salt-tolerant Bacillus endophytic strain ( Bacillus halotolerans ) YB-1576 from wheat, and through research, it was found that its metabolites can not only antagonize Fusarium graminearum ( Fusarium pseudograminearum, it can grow under the condition of (FPS), and can also induce the expression of defense enzymes in wheat tissues, improve the resistance of wheat to basal stalk rot, and thus have a good control effect on wheat basal stalk rot. In addition, the fermentation broth of the salt-tolerant Bacillus sp. YB-1576 can promote the growth and development of wheat seedlings.

[0007] According to one aspect disclosed in the present application, a wheat endophyte YB-1576 is screened and obtained, and its taxonomic name is Bacillus halotolerans , and it was deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on April 18, 2022 (Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Zip Code: 100101), and its deposit number is CGMCC No. 24705.

[0008] According to another aspect disclosed in the present application, a biocontrol agent containing the salt-tolerant Bacillus sp. YB-1576 or / and its metabolites is provided.

[0009] According to another aspect disclosed in the present application, a preparation method of a biocontrol agent is provided, including the following steps:

[0010] Inoculate the salt-tolerant Bacillus sp. YB-1576 into an LB liquid medium, and culture it at 28°C and 180 rpm until the OD600 nm reaches 0.8 - 0.9, and the obtained fermentation broth is the biocontrol agent.

[0011] In some embodiments disclosed in the present application, the LB liquid medium contains: tryptone 8 - 12 g / L, yeast extract 4 - 6 g / L, sodium chloride 8 - 12 g / L, pH 7.0.

[0012] According to another aspect disclosed in the present application, the salt-tolerant Bacillus sp. YB-1576 or the biocontrol agent is applied to the control of wheat basal stalk rot.

[0013] According to another aspect disclosed in the present application, the salt-tolerant Bacillus sp. YB-1576 or the biocontrol agent is applied to promoting the growth of wheat.

[0014] According to another aspect disclosed in the present application, the salt-tolerant Bacillus sp. YB-1576 or the biocontrol agent is applied to improving the activity of wheat defense enzymes.

[0015] One or more technical solutions provided in the embodiments of the present application have at least the following technical effects or advantages:

[0016] 1. There has been no research report on using salt-tolerant Bacillus spores to control wheat basal stalk rot disease; there are differences in the phenotypic characteristics among salt-tolerant Bacillus spore strains, showing individual strain specificity; the salt-tolerant Bacillus spore strain YB-1576 of the present application is an endophyte isolated from wheat. This strain can not only antagonize the growth of Fusarium graminearum, but also induce the expression of defense enzymes in wheat tissues, improve the resistance of wheat to basal stalk rot disease, and thus has a good control effect on wheat basal stalk rot disease; in addition, the fermentation broth of salt-tolerant Bacillus spore YB-1576 can improve the growth and development of wheat, showing a significant growth-promoting effect.

[0017] 2. The biocontrol agent prepared using the salt-tolerant Bacillus spore YB-1576 of the present application can prevent the occurrence of wheat basal stalk rot disease, promote the growth of wheat, reduce the use of chemical pesticides, ensure the safe and high-quality production of wheat, and has good social and economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is a morphological characteristic and molecular identification diagram of strain YB-1576 in an embodiment of the present application; among them, (A) colony of YB-1576 on NA medium; (B) Gram staining result of strain YB-1576; (C) confrontation result of strain YB-1576 and Fusarium pseudograminearum on a plate.

[0019] Figure 2 It is a phylogenetic tree diagram of strain YB-1576 based on the 16S rDNA gene sequence in an embodiment of the present application. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0020] In the following embodiments, the instrument equipment involved is all conventional instrument equipment unless otherwise specified; the biochemical reagents and raw materials involved are all commercially available conventional products unless otherwise specified; the detection, testing, experimental methods, etc. involved are all conventional methods unless otherwise specified.

[0021] In order to better understand the technical solution of the present application, the above technical solution will be described in detail below in conjunction with the accompanying drawings of the specification and specific embodiments.

[0022] Example 1: Obtaining of Strain and Species Identification

[0023] An endophyte was isolated from wheat (isolated from wheat in May 2021 by Xie Xia, variety: Zhengmai 366), named YB-1576. The colony on NA medium is ivory white, opaque, and the surface is smooth; Gram staining is positive, and the bacterial cells are rod-shaped ( Figure 1 A, B), and it has a significant antagonistic effect on Fusarium pseudograminearum that causes wheat basal stalk rot disease ( Figure 1C). The genomic DNA of strain YB-1576 was extracted as a template, and the 16S rRNA gene of strain YB-1576 was amplified using the universal primers 27F (5’-AGAGTTTGATCCTGGCTCAG–3’) and 1492R (5’-TACGGCTACCTTGTTACGACTT-3’) for bacterial 16S rDNA sequences. Then, the full-length nucleic acid sequence was obtained by sequencing and analyzed by online alignment with the NCBI NR database. The results showed that the similarity with the 16S rDNA sequence of strain YB-1576 reached over 99%.

[0024] The 16S rDNA sequence obtained by sequencing of YB-1576 was aligned with the 16S rDNA sequences of known Bacillus strains downloaded from the NCBI database using the MUSCLE software in Mega 7.0 software. Then, a phylogenetic tree of strain YB-1576 was constructed using the NJ method, with the number of bootstrap replicates being 1000, to construct the phylogenetic tree ( Figure 2 ), indicating that strain YB-1576 and the known Bacillus halotolerans could cluster in the same branch.

[0025] In summary, strain YB-1576 was identified as Bacillus halotolerans .

[0026] Example 2: Biocontrol agent based on strain YB-1576

[0027] Strain YB-1576 was streaked and inoculated on an LB solid medium for activation, and then a single colony was picked and inoculated into an LB liquid medium (formula: yeast extract 5 g / L, tryptone 10 g / L, sodium chloride 10 g / L, initial pH adjusted to 7.0). The fermentation broth when cultured at 28 °C and 180 rpm until the OD600 nm reached 0.8 - 0.9 was the biocontrol agent of strain YB-1576.

[0028] Example 3: Experiment on promoting the growth of wheat seedlings by the biocontrol agent of YB-1576

[0029] Plump and uniform wheat seeds were selected, rinsed with sterile water, disinfected with 75% ethanol for 30 s first, then rinsed 3 times with sterile distilled water, and placed in a petri dish lined with filter paper moistened with sterile water. They were cultured at 25 °C with humidity until the wheat showed white tips. Then, they were treated with 10 7 、10 8 and 10 9cfu / mL YB-1576 biocontrol agent was used to soak the seeds for 2 h, and sterile distilled water was used as a control. Each treatment was repeated 3 times. The soaked wheat seeds were evenly placed in a culture dish with a diameter of 9 cm and a filter paper moistened with bacterial water, with 20 seeds in each dish. After being placed at 25 ℃ and 12 h / 12 h light and dark alternating conditions for moisturizing for 10 days, samples were taken to measure the fresh weight and plant height of the wheat seedlings. The statistical method for the growth promotion index of wheat seedlings is as follows: the seedlings in the dish were separated from the filter paper, rinsed with sterile water and dried, and measured and weighed; the results showed that 10 7 and 10 8 cfu / mL YB-1576 biocontrol agent can significantly promote the growth of wheat seedlings and make them strong.

[0030] Table 1 Statistical analysis of YB-1576 biocontrol agent on wheat seedling growth promotion indexes

[0031] .

[0032] Example 4: Verification test of the biocontrol agent YB-1576 on wheat stem rot

[0033] Mix the cooked wheat grains and sand in a volume ratio of 3:1 in a 500ml flask, shake well and sterilize to obtain the wheat grain sand medium. Activate the Pseudofusorium graminearum on the PDA medium, then use a 1ml sterilized blue pipette tip to pick up 5 pieces of Pseudofusorium graminearum hyphae and place them in the sterilized wheat grain sand medium and culture them at 28°C for 7 days. Mix the cultured wheat grain sand medium containing Pseudofusorium graminearum with sterilized fine soil at a mass ratio of 5% and put them into a 10cm×10cm pot. The control group is sterilized soil. Select full and uniform wheat seeds, rinse them with sterile water, disinfect them with 75% ethanol for 30s, then rinse them 3 times with sterile distilled water, place them in a culture dish covered with filter paper moistened with sterile water, and culture them at 25°C to keep the wheat white. Adjust the cultured YB-1576 biocontrol agent to a concentration of 10 8 cfu / mL and then soak the wheat seeds, while the control group was soaked in LB medium.

[0034] The experimental treatment settings are as follows:

[0035] (1) Wheat seeds soaked with YB-1576 biocontrol agent were planted in sterile soil free of F. graminearum;

[0036] (2) Unsoaked wheat seeds were planted in sterile soil free of F. graminearum;

[0037] (3) Wheat seeds soaked with YB-1576 biocontrol agent were planted in sterile soil containing F. graminearum;

[0038] (4)The non-soaked wheat seeds were planted in sterile soil containing *Fusarium pseudograminearum*.

[0039] After culturing the wheat seedlings in a greenhouse under the conditions of 25 °C and a 12 h / 12 h light / dark cycle for 20 d, the wheat seedlings were rinsed clean with clear water, and the fresh weight and length of the above-ground part and the fresh weight and length of the underground part of the seedlings were respectively counted.

[0040] The incidence of basal stalk rot was investigated, and the disease grading criteria were as follows:

[0041] Grade 0 was asymptomatic; Grade 1 was slight browning of the root / stem (less than 25%);

[0042] Grade 2 was moderate browning (25% to 50%);

[0043] Grade 3 was browning of 50% to 75%;

[0044] Grade 4 was severe browning (more than 75%), and the plant tissue was completely rotten or dead.

[0045] The disease index and control effect were calculated. The disease index = ∑(disease grade × number of diseased plants at this disease grade) / total number of plants investigated; the control effect = (disease index of the control group - disease index of the treatment group) / disease index of the control group × 100%.

[0046] Table 2 Control effect of the biocontrol agent YB-1576 on basal stalk rot of wheat seedlings

[0047] 。

[0048] Table 3 Effects of the biocontrol agent YB-1576 on growth parameters of wheat seedlings

[0049] 。

[0050] The results showed that the biocontrol agent YB-1576 could significantly reduce the incidence of basal stalk rot of wheat seedlings. The incidence in the control group was 90.31 ± 1.43%, and after treatment with the biocontrol agent YB-1576, it was 14.89 ± 2.73%, and the control effect was 69.73 ± 0.86% (Table 2); in addition, the biocontrol agent YB-1576 could increase the fresh weight and length of the above-ground part and the fresh weight and length of the underground part of wheat seedlings (Table 3); the biocontrol agent YB-1576 could significantly promote the growth of wheat seedlings and had a good control effect on basal stalk rot of wheat seedlings.

[0051] Example 5: Verification test on the induction of the expression of defense-related enzymes in wheat seedlings by the biocontrol agent YB-1576

[0052] On the basis of Example 4, 20 days after planting, wheat leaves treated differently were quickly frozen in liquid nitrogen and then stored at -80 °C. First, 1 g of wheat seedling leaves was ground in a pre-cooled mortar and then transferred to a new 1.5 mL centrifuge tube, and 1 mL of extraction buffer was added. After centrifugation at 8000×g for 10 min, the supernatant was transferred to another new 1.5 mL centrifuge tube for detecting enzyme activity. The determination of enzyme activity was carried out using the kits of LOX (product number: BC0325), PAL (product number: BC0215), CAT (product number: BC0205), PPO (product number: BC0195) and POD (product number: BC0095) from Solarbio Science & Technology Co., Ltd. The results are shown in Table 4. The results indicate that the biocontrol agent YB-1576 can improve the activity of defense enzymes in wheat seedlings, especially the induction of defense enzyme activity is more significant in the cooperation with Fusarium pseudograminearum.

[0053] Table 4 Effects of the biocontrol agent YB-1576 on the activity of defense enzymes in wheat

[0054] 。

[0055] Although some preferred embodiments of the present application have been described, those skilled in the art can make additional changes and modifications once they know the basic creative concept. Therefore, the appended claims are intended to be construed to include the preferred embodiments and all changes and modifications falling within the scope of the present application.

[0056] Obviously, those skilled in the art can make various changes and modifications to the present application without departing from the spirit and scope of the invention of the present application. Thus, if these modifications and variations fall within the scope of the claims of the present application and their equivalent technologies, the present application is also intended to include these modifications and variations.

Claims

1. A wheat endophytic strain, Bacillus halotolerans ( Bacillus halotolerans ), YB-1576, characterized in that Its preservation number is CGMCC No. 24705.

2. A biocontrol agent, characterized in that, Contains the halotolerant Bacillus sp. YB-1576 described in claim 1.

3. The preparation method of the biocontrol agent according to claim 2, characterized in that, Comprises the following steps: Inoculate the halotolerant Bacillus sp. YB-1576 described in claim 1 into LB liquid medium, and culture at 28°C and 180 rpm until the OD600 nm reaches 0.8 - 0.

9. The obtained fermentation broth is the biocontrol agent.

4. The preparation method according to claim 3, wherein The LB liquid medium comprises: tryptone 8 - 12 g / L, yeast extract 4 - 6 g / L, sodium chloride 8 - 12 g / L, pH 7.

0.

5. Use of the halotolerant Bacillus sp. YB-1576 described in claim 1 or the biocontrol agent described in claim 2 in the prevention and control of wheat basal rot.

6. Use of the halotolerant Bacillus sp. YB-1576 described in claim 1 or the biocontrol agent described in claim 2 in promoting wheat growth.

7. Use of the halotolerant Bacillus sp. YB-1576 described in claim 1 or the biocontrol agent described in claim 2 in enhancing the activity of wheat defense enzymes, wherein the wheat defense enzymes are at least one of LOX, PAL, CAT, PPO, and POD.

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