Bacillus amyloliquefaciens strain for preventing and treating wheat stem rot and application of bacillus amyloliquefaciens strain

Abstract

The invention discloses a bacillus amyloliquefaciens strain for preventing and treating wheat stem rot and application of the bacillus amyloliquefaciens strain. A strain XJTC-29 capable of promoting wheat growth and preventing and treating plant diseases is screened out by separating a strain from wheat rhizosphere soil, and is determined as bacillus amyloliquefaciens through molecular biology and morphological identification, and the microbial preservation number of the bacillus amyloliquefaciens is CGMCC (China General Microbiological Culture Collection Center) No.34774. Tests prove that the bacillus amyloliquefaciens can obviously promote the plant height, the root length and the fresh weight of wheat, and the prevention and treatment effects on wheat stem rot caused by fusarium flavum, fusarium pseudograminearum and fusarium graminearum are 54.62%, 60.29% and 55.73% respectively. The bacillus amyloliquefaciens strain provided by the invention shows obvious dual functions of inhibiting bacteria and promoting growth in prevention and control of wheat stem rot, and has the potential of developing a biocontrol preparation for preventing and controlling wheat stem rot.

Description

Technical Field

[0001] The present invention relates to a biocontrol strain for preventing and controlling plant diseases isolated from soil, and in particular to a biocontrol strain of Bacillus amyloliquefaciens isolated from soil. Bacillus amyloliquefaciens ) and its application in preventing and controlling wheat diseases or promoting wheat growth, belonging to the field of Bacillus amyloliquefaciens and its application. Background Art

[0002] Wheat is one of the important food crops. F. pseudo graminearum ), Fusarium graminearum ( F. graminearum )、Yellow Fusarium ( F. culmorum ) is the dominant species of wheat stem rot ( Fusariumcrown rot ) is a major cause of wheat yield decline in Xinjiang, with yield reductions reaching 30%-70%. While chemical pesticides can control the spread of diseases in the short term, long-term use of pesticides is not only costly, pollutes the soil and damages the ecosystem, but also increases pathogen resistance. Therefore, screening for highly effective antagonistic bacteria can reduce pesticide use and mitigate negative impacts on the ecosystem.

[0003] In recent years, a lot of research has been conducted both domestically and internationally on the screening and application of antagonistic bacteria against wheat stem rot. The rhizosphere soil is a key area for interaction between plants and microorganisms, and screening antagonistic bacteria in this area is crucial for biological control and promoting plant growth. Bacteria in the rhizosphere soil are generally isolated using the gradient dilution method, and the isolated bacteria are cultured on the same plate with the target pathogen using the plate confrontation method to observe whether the bacteria can inhibit the growth of the pathogen, thereby screening out bacteria with antagonistic effects. The mechanism of action of the antagonistic bacteria is then studied, and greenhouse field trials are conducted to evaluate their effects on controlling plant diseases and promoting plant growth. As an important antagonistic bacterium, researchers have conducted extensive research on its use in preventing and controlling plant diseases caused by Fusarium. Li Zhong et al. (Li Zhong, Zhang Yi, Wu Xiaomao, et al. Screening, identification and characteristics of biocontrol bacteria for soil-borne plant diseases [J]. Henan Agricultural Sciences, 2013, 42(4): 103-106.) screened a strain of Bacillus subtilis from the rhizosphere soil of healthy plants in a diseased field. The antibacterial substances produced by the liquid culture of this strain have a strong inhibitory effect on a variety of pathogens, and the inhibition rate against Polygonum multiflorum root rot reached 96.14%. Lin Guoqiang et al. (Lin Guoqiang, Liao Yucai, Gong Andong et al. Screening and identification of antagonistic bacteria against Fusarium graminearum [J]. Journal of Huazhong Agricultural University, 2013, 32(3): 28-32.) isolated a strain of Bacillus amyloliquefaciens from soil, which has a strong antagonistic effect against Fusarium graminearum. Further research found that the lipopeptides produced by the antagonistic bacteria can affect the germination of the pathogen's conidia, and the control effect in preventing and controlling wheat fusarium fusarium is as high as 79%-88%, which is comparable to the chemical agent carbendazim.

[0004] Providing a Bacillus amyloliquefaciens with the functions of preventing and treating wheat basal stem rot and promoting growth, which has important application value for the prevention and treatment of wheat basal stem rot. SUMMARY

[0005] One of the purposes of the present application is to provide a Bacillus amyloliquefaciens capable of effectively preventing and treating wheat basal stem rot or promoting wheat growth. Bacillus amyloliquefaciens The second purpose of the present application is to provide a microbial preparation prepared from the Bacillus amyloliquefaciens strain. The third purpose of the present application is to apply the Bacillus amyloliquefaciens strain or the microbial preparation prepared therefrom to the prevention and treatment of wheat diseases or the promotion of wheat growth.

[0006] In order to achieve the above-mentioned purposes, the main technical solutions adopted by the present application include: One aspect of the present application is to provide a Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) XJTC-29, whose microbial preservation number is CGMCC No. 34774; its classification name is Bacillus amyloliquefaciens. Bacillus amyloliquefaciens Bacillus amyloliquefaciens The preservation time is June 5, 2025; the preservation unit is the General Microbiological Center of the Chinese Microorganism Strain Preservation Management Committee; and the preservation address is No. 3, Beichen West Road, Chaoyang District, Beijing, China Institute of Microbiology, Chinese Academy of Sciences.

[0007] The colony and cell morphology of the Bacillus amyloliquefaciens in the present application are as follows: irregular colony shape, rough colony surface, milk white color, purple gram staining, determined as gram-positive bacteria, and rod-shaped bacteria morphology on NA medium.

[0008] Another aspect of the present application is to provide a microbial preparation prepared from the Bacillus amyloliquefaciens XJTC-29.

[0009] The skilled person in the art can prepare the Bacillus amyloliquefaciens XJTC-29 provided by the present application into various conventional microbial preparations according to the conventional preparation method of microbial preparations, which are all technical means familiar to the skilled person in the art.

[0010] Another aspect of the present application is to apply the Bacillus amyloliquefaciens XJTC-29 or the Bacillus amyloliquefaciens XJTC-29 microbial preparation to the prevention and treatment of plant diseases.

[0011] In a preferred specific embodiment, the pathogenic bacteria of the plant disease are preferably Fusarium graminearum (Fusarium graminearum) F. graminearum and Pseudofusarium graminearum (Pseudofusarium graminearum).​​F. pseudograminearum ) or Fusarium oxysporum ( F. culmorum ); The wheat disease is preferably wheat stem rot. Another aspect of the present invention is to apply the Bacillus amyloliquefaciens XJTC-29 or the Bacillus amyloliquefaciens XJTC-29 microbial preparation to promote wheat growth; wherein, the promotion of wheat growth is preferably to increase the chlorophyll content of wheat leaves, wheat plant height, root length and fresh weight, wherein the fresh weight refers to the weight of the entire wheat plant.

[0012] The present invention screened out a strain XJTC-29 from wheat rhizosphere soil that can prevent and control wheat diseases and promote wheat growth. Morphological and molecular biological identification showed that the strain XJTC-29 is a Bacillus amyloliquefaciens strain ( B. amyloliquefaciens ), the experiment proved that the strain XJTC-29 can be used to kill Fusarium graminearum ( F. graminearum ), Pseudomonas graminearum ( F. pseudograminearum ) or Fusarium oxysporum ( F. culmorum ) has obvious preventive and control effects on wheat base rot caused by , and can significantly increase the chlorophyll content, plant height, root length and fresh weight of wheat; the present invention provides excellent bacterial strain resources for the biological control of wheat base rot, and provides a theoretical basis and application guidance for the development and application of biological control agents for wheat base rot. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The results show that Bacillus amyloliquefaciens XJTC-29 can inhibit wheat stem rot pathogens on plate.

[0014] Figure 2 This is the colony morphology of Bacillus amyloliquefaciens XJTC-29.

[0015] Figure 3 This is the Gram staining result of Bacillus amyloliquefaciens XJTC-29.

[0016] Figure 4 Phylogenetic tree of Bacillus amyloliquefaciens XJTC-29 based on 16S rDNA.

[0017] Figure 5 The growth-promoting effect of Bacillus amyloliquefaciens XJTC-29 on wheat.

[0018] Figure 6 The control effect of Bacillus amyloliquefaciens XJTC-29 on wheat stem rot. DETAILED DESCRIPTION

[0019] The present application will be further described in conjunction with specific embodiments. The advantages and features of the present application will become more apparent with the description. However, it should be understood that the described embodiments are only exemplary, and do not constitute any limitation on the scope of the present application. Those skilled in the art should understand that the details and forms of the technical solutions of the present application can be modified or replaced without departing from the spirit and scope of the present application, and such modifications or replacements all fall within the protection scope of the present application.

[0020] Example 1 Isolation, purification and identification of Bacillus amyloliquefaciens strain XJTC-29 1 Test materials Soil samples: wheat rhizosphere soil samples were collected from four regions of Xinjiang Tacheng, Altai, Bobo and Yili. In order to ensure the integrity of rhizosphere microorganisms, the samples were kept in low temperature environment before the test, and the sample storage time should not exceed 72 h.

[0021] Test pathogenic fungi: Fusarium graminearum (Schwein.) S. Ito (Fg) F. culmorum ), Fusarium pseudograminearum (Fp) F. graminearum ), and Fusarium pseudograminearum (Fp) F. pseudograminearum ) were stored in the Institute of Plant Protection, Xinjiang Academy of Agricultural Sciences.

[0022] 2 Test method 2.1 Screening and purification method of Bacillus amyloliquefaciens strain XJTC-29 The spore solution of Fusarium graminearum (Schwein.) S. Ito (Fg) F. culmorum ) was diluted to 1×10 6 CFU / mL and spread on PDA plates. The collected rhizosphere soil samples were made into a suspension, and the soil suspension was diluted to 1×10 -2 , 1×10 -3 and 1×10 -4 three concentration gradients by 10-fold gradient dilution method. After the fungal spore solution on PDA was dried, equal volume of soil suspension was overlaid, 3 replicates were set for each dilution concentration, and the formation of inhibition zone was observed after 2-3 days. The strains producing obvious inhibition zone were purified in time and stored in 50% glycerol tubes (-80℃).

[0023] The above-mentioned strains were further screened for antagonistic bacteria by plate confrontation method: the antagonistic bacteria were confronted with Fusarium graminearum (Schwein.) S. Ito (Fg) F. graminearum ), and the strains without antagonistic effect were removed. The remaining strains were further confronted with Fusarium pseudograminearum (Fp) F. pseudograminearum The radius of the antagonistic bacteria to different pathogenic fungi was measured, and 3 replicates were set for each test.

[0024] The formula for calculating the inhibition rate is: inhibition rate (%) = (control colony radius - treatment colony radius / control colony radius - 2.5 mm) × 100.

[0025] 2.2 Identification of Bacillus amyloliquefaciens strain XJTC-29 2.2.1 Morphological, physiological and biochemical identification method of strain XJTC-29 After antagonistic bacteria were cultured in nutrient agar medium (NA) at 37°C for 48 h, the surface morphology, edge characteristics and colony diameter of the antagonistic bacteria were observed according to the “Bergey's Manual of Determinative Bacteriology”.

[0026] 2.2.2 Molecular biological identification of strain XJTC-29 16S rDNA is a highly conserved region in the bacterial genome. The DNA of the strain was extracted using a kit, and the primer set 27-F (5'-AGAGTT TGATCCTGGCTCAG-3' (SEQ ID No. 1)) and 1492 - R (5'-AAGGAGGTGATCCAGCC-3' (SEQ ID No. 2)) were used to amplify the 16S rDNA gene sequence. The 20 μL system was as follows: dd H2O 6 μL, 2 × Taq Plus Master Mix II (Dye Plus) 10 μL, 27-F 1 μL, 1492 - R 1 μL, DNA template 2 μL. The amplification program was as follows: denaturation at 95°C for 15 s, annealing at 60°C for 20 s, extension at 72°C for 100 s, 35 cycles, and the PCR product with a positive band was sequenced by 1% agarose gel electrophoresis. The sequencing results were analyzed by NCBI Blast comparison, and the phylogenetic tree was constructed by the neighbor-joining method using MEGA12 software to determine the taxonomic status of XJTC-29.

[0027] 3 Test results 3.1 Screening and purification results of Bacillus amyloliquefaciens strain XJTC-29 The dominant antagonistic bacteria XJTC-29 (see Figure 1 ) with significant inhibitory effect on the three pathogenic fungi were finally screened out. The inhibition rates of XJTC-29 on F. graminearum, F. pseudograminearum and F. graminearum were 71.23%, 72.23% and 65.13%, respectively. The dominant antagonistic bacteria XJTC-29 had stable antagonistic effect on the three pathogenic fungi, and the inhibition rates were all above 65%. The inhibition rate on F. pseudograminearum was the highest, followed by F. graminearum, and F. graminearum was the weakest.

[0028] 3.2 Morphological, physiological and biochemical identification results of strain XJTC-29 The strain XJTC-29 showed irregular colony shape, rough colony surface and milk white color (see Figure 2 ) on NA medium, and the gram staining was purpleFigure 3 ), and was determined as a gram-positive bacterium with bacillary morphology. Based on the above results, the strain XJTC-29 was preliminarily identified as Bacillus amyloliquefaciens.

[0029] 3.3 Molecular biological identification results of the strain XJTC-29 The 16s rDNA sequencing of the strain XJTC-29 obtained a 1413 bp sequence fragment (SEQ ID No. 3):

[0030] The NCBI Blast comparison is the highest (100%) with Bacillus amyloliquefaciens (KF933607), and the neighbor-joining tree shows that XJTC-29 is in the same evolutionary branch as Bacillus amyloliquefaciens (KF933607). Bacillus amyloliquefaciens Bacillus amyloliquefaciens The NCBI Blast comparison is the highest (100%) with Bacillus amyloliquefaciens (KF933607), and the neighbor-joining tree shows that XJTC-29 is in the same evolutionary branch as Bacillus amyloliquefaciens (KF933607). Figure 4

[0031] Test Example 1: Pot-cultivation growth promotion test of Bacillus amyloliquefaciens strain XJTC-29 1. Test method The wheat variety planted is Xindong 20, and the seeds of the same size and healthy are selected, germinated, and sown in small flower pots, 12 seeds per pot, and 3 repetitions are set for each treatment in the test. The antagonistic bacteria are shaken in NB medium for 2 days, centrifuged at 8000 rpm for 10 min, the supernatant is discarded, the bacterial precipitate is reserved, the bacterial body is diluted with sterile water, OD 600 = 1 to prepare the bacterial suspension, and standby.

[0032] After 5 days of wheat planting, 10 mL of the prepared bacterial suspension is inoculated per pot, and sterile water is inoculated as a control CK, 3 repetitions for each treatment, and the root length, stem length, chlorophyll content, and fresh weight of the wheat are recorded after 21 days.

[0033] 2. Test results The test results are shown in Table 1. Figure 5 Compared with CK, the chlorophyll content of the wheat treated by Bacillus amyloliquefaciens XJTC-29 is 40.66±0.47, and the chlorophyll content is significantly increased by 13.97%; the plant height of the wheat treated by Bacillus amyloliquefaciens XJTC-29 is 37.37±1.22 cm, and the plant height is significantly increased by 21.81% compared with CK; the root length of the wheat treated by Bacillus amyloliquefaciens XJTC-29 is 18.20±1.46, and the root length is significantly increased by 35.38% compared with CK; the fresh weight of the wheat treated by Bacillus amyloliquefaciens XJTC-29 is 1.10±0.03, and the fresh weight is significantly increased by 31.81% compared with CK.

[0034] The test results show that the chlorophyll content of the wheat treated by the bacterial suspension of Bacillus amyloliquefaciens XJTC-29 is significantly increased, and the root length, plant height, and fresh weight are significantly increased.

[0035] Test Example 2: Pot-cultivation control efficiency test of Bacillus amyloliquefaciens strain XJTC-29 1. Test method ​​The wheat variety used was Xindong 20. Healthy wheat seeds of similar size were germinated and sown in small pots, with 12 seeds per pot. Each treatment was replicated three times. Antagonistic bacteria were cultured on NB medium for 2 days. Centrifuged at 8000 rpm for 10 minutes, the supernatant was discarded, and the bacterial pellet was retained. The bacterial cells were diluted with sterile water and the OD value was calculated. 600 =1 to prepare antagonistic bacterial suspension for later use.

[0036] The pathogenic bacteria cake was inoculated into CMC medium and cultured at 28°C and 180 rpm / min for 7 days. After the hyphae were removed by filtration, the spore suspension was diluted to a concentration of 1×10 6 ,spare.

[0037] Trial groups: Treatment group: After wheat was grown for 6 days, 10 mL of Bacillus amyloliquefaciens XJTC-29 suspension was inoculated; after wheat was grown for 7 days, 10 mL of Fusarium oxysporum ( F. culmorum ), Fusarium graminearum ( F. graminearum ), Pseudomonas graminearum ( F. pseudograminearum ) spore liquid, the three treatment groups are expressed as F.c +29, F.g +29 and F.p +29; Positive control group: After wheat was grown for 7 days, 10 mL of Fusarium oxysporum was inoculated. F.culmorum ), Fusarium graminearum ( F. graminearum ), Pseudomonas graminearum ( F.pseudograminearum ) spore liquid, the three positive control groups are expressed as F.c, F.g and F.p .

[0038] Negative control group: After wheat had grown for 6 days, only 10 mL of Bacillus amyloliquefaciens XJTC-29 suspension was inoculated. This negative control group was expressed as XJTC-29. CK: wheat without any treatment.

[0039] 21 days after inoculation, the disease index and control effect were calculated according to the wheat stem rot investigation and grading standards, disease index calculation formula, and control effect calculation formula in Table 1.

[0040] Table 1 Survey and grading standards for wheat stem rot

[0041] The disease index calculation formula is: Disease index = ∑ (disease index at each level × corresponding level) / total number of surveys × highest disease level The formula for calculating the prevention effect is:

[0042] 2 Test results Pot experiments were conducted in a greenhouse, and the results are shown in Table 2 and Figure 6 It is shown that the negative control group of wheat plants inoculated with Bacillus amyloliquefaciens XJTC-29 bacterial suspension had no disease symptoms. The three positive control groups of wheat plants inoculated with Fusarium graminearum, Pseudocercospora graminicola and F. graminearum (respectively F.c, F.g and F.p ) showed yellowing of leaves and severe browning of stem bases, and some wheat stem bases rotted and died on the 26th day. Compared with the positive control groups, the disease index of the three XJTC-29 treatment groups (respectively F.c +29, F.g +29 and F.p +29) had significant differences, among which, F.c The disease index of the +29 treatment group against F. graminearum was reduced by 30.74, with a control effect of 54.62%; F.p The disease index of the +29 treatment group against P. graminicola was reduced by 35.49, with a control effect of 60.29%; F.g The disease index of the +29 treatment group against F. graminearum was reduced by 31.60, with a control effect of 55.73%.

[0043] Table 2 Antagonistic bacteria control effect on wheat

[0044] The results show that Bacillus amyloliquefaciens XJTC-29 has no pathogenicity to wheat and can prevent and control multiple pathogens of wheat stem base rot.

Claims

1. A strain of Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ) XJTC-29, characterized in that, Its microbial preservation number is: CGMCC No.34774.

2. A microbial preparation prepared by the Bacillus amyloliquefaciens XJTC-29 according to claim 1.

3. Use of the Bacillus amyloliquefaciens XJTC-29 according to claim 1 in preventing and controlling wheat diseases; the pathogen of wheat diseases is Fusarium graminearum ( F. graminearum ), Pseudomonas graminearum ( F. pseudograminearum ) or Fusarium oxysporum ( F. culmorum ).

4. Use of the microbial preparation according to claim 2 in the prevention and treatment of wheat diseases, wherein the pathogen of the wheat disease is Fusarium graminearum ( F. graminearum ), Pseudomonas graminearum ( F. pseudograminearum ) or Fusarium oxysporum ( F. culmorum ).

5. Use of the Bacillus amyloliquefaciens XJTC-29 according to claim 1 in promoting wheat growth.

6. Use of the microbial preparation according to claim 2 in promoting wheat growth.

7. The use according to claim 5 or 6, characterized in that The promoting wheat growth includes increasing wheat plant height, wheat root length or wheat fresh weight; wherein the fresh weight refers to the weight of the entire wheat plant.

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