Bacillus amyloliquefaciens strain for preventing and treating wheat foot rot and application thereof

Abstract

The application discloses a bacillus amyloliquefaciens strain for preventing and treating wheat stem base rot and application thereof. The strain XJTC-29 for promoting wheat growth and preventing and treating plant diseases is screened out from wheat rhizosphere soil, and is identified as bacillus amyloliquefaciens through molecular biology and morphology identification, and the microbial preservation number is CGMCC No.34774. Test proves that the bacillus amyloliquefaciens can obviously promote wheat plant height, root length and fresh weight, and the prevention and treatment effects on wheat stem base rot caused by fusarium graminearum, fusarium pseudograminearum and fusarium graminearum are 54.62%, 60.29% and 55.73% respectively. The bacillus amyloliquefaciens strain provided by the application exhibits a double function of obvious bacteriostasis and growth promotion in the prevention and treatment of wheat stem base rot, and has the potential to develop a biocontrol agent for preventing and treating wheat stem base rot.

Description

Technical Field

[0001] This invention relates to biocontrol strains for controlling plant diseases obtained from soil, and particularly to Bacillus amyloliquefaciens isolated from soil. Bacillus amyloliquefaciens Bacillus amyloliquefaciens and its application in the prevention and control of wheat diseases or the promotion of wheat growth belong to the field of Bacillus amyloliquefaciens and its application. Background Technology

[0002] Wheat is one of the important food crops, with Fusarium graminearum (… F. pseudo graminearum Fusarium graminearum ( ), F. graminearum ), Fusarium oxysporum ( F. culmorum Wheat stem base rot ( ) is the dominant species Fusariumcrown rot This is a major reason for the reduction in wheat production in Xinjiang, with a reduction rate of 30%-70%. Although the use of chemical agents can control the spread of diseases in the short term, long-term use of pesticides is not only costly and pollutes the soil and damages the ecology, but also increases the resistance of pathogens. Therefore, screening for highly effective antagonistic bacteria can reduce the amount of pesticides used and thus mitigate the negative impact on the ecosystem.

[0003] In recent years, extensive 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 crucial area for plant-microbe interaction, and screening for antagonistic bacteria within this region is essential for biological control and promoting plant growth. Generally, a serial dilution method is used to isolate bacteria from the rhizosphere soil. The isolated bacteria are then cultured on the same plate as the target pathogen using a plate confrontation method to observe whether the bacteria can inhibit the growth of the pathogen, thereby screening for bacteria with antagonistic effects. Subsequently, the mechanism of action of the antagonistic bacteria is studied, and their control effects on plant diseases and their growth-promoting effects are evaluated through greenhouse field trials. Bacillus is an important antagonistic bacterium, and researchers have conducted extensive studies on its use in controlling plant diseases caused by Fusarium. Li Zhong et al. (Li Zhong, Zhang Yi, Wu Xiaomao et al. Screening, identification and characteristic study 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 diseased fields. The antibacterial substance produced by the strain in liquid culture had a strong inhibitory effect on a variety of pathogens, and the inhibition rate of root rot of Polygonum multiflorum 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 showed a strong antagonistic effect against Fusarium graminearum. Further research revealed that the lipopeptides produced by the antagonistic bacteria could affect the germination of conidia of the pathogen. The control effect of the antagonistic bacteria was as high as 79%-88% in the control of wheat scab, which was comparable to that of the chemical fungicide 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 );

[0006] The second purpose of the present application is to provide a microbial preparation prepared from the bacillus amyloliquefaciens strain.

[0007] 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.

[0008] In order to achieve the above-mentioned purposes, the main technical solutions adopted by the present application include:

[0009] One aspect of the present application is to provide a bacillus amyloliquefaciens (XJTC-29) with a microbial preservation number of CGMCC No. 34774, a classification name of 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 Bacterial Preservation Management Committee, and the preservation address is No. 3, Beichen West Road, Chaoyang District, Beijing, China Institute of Microbiology.

[0010] The colony and bacterial body form 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 bacterial morphology in rod shape on NA medium.

[0011] Another aspect of the present application is to provide a microbial preparation prepared from the bacillus amyloliquefaciens XJTC-29.

[0012] The bacillus amyloliquefaciens XJTC-29 provided by the present application can be prepared into various conventional microbial preparations by those skilled in the art according to the conventional preparation method of microbial preparations, which are all technical means familiar to those skilled in the art.

[0013] 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.

[0014] In a preferred specific embodiment, the pathogenic bacteria of the plant disease are preferably fusarium graminearum.​F. graminearum ) Fusarium pseudograminearum or (Fusarium graminearum) F. pseudograminearum ) Fusarium pseudograminearum or (Fusarium graminearum) F. culmorum ) Fusarium pseudograminearum or (Fusarium graminearum)

[0015] Another aspect of the present application is to apply the Bacillus amyloliquefaciens XJTC-29 or the microbial preparation of Bacillus amyloliquefaciens XJTC-29 to promote the growth of wheat; wherein the promotion of the growth of wheat preferably refers to increasing the chlorophyll content, plant height, root length and fresh weight of wheat, wherein the fresh weight refers to the weight of the whole plant of wheat.

[0016] The present application screens a strain XJTC-29 capable of preventing and treating wheat diseases and promoting the growth of wheat from the rhizosphere soil of wheat, which is identified by morphology and molecular biology as a Bacillus amyloliquefaciens strain. B. amyloliquefaciens It is proved by tests that the strain XJTC-29 has obvious prevention and treatment effect on the wheat stem base rot caused by Fusarium pseudograminearum (Fusarium graminearum) or (Fusarium graminearum), and can significantly increase the chlorophyll content, plant height, root length and fresh weight of wheat. F. graminearum F. pseudograminearum F. culmorum The present application provides excellent strain resources for the biological control of wheat stem base rot, and provides a theoretical basis and application guidance for the development and application of the biocontrol preparation for wheat stem base rot. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 Plate inhibition of the Bacillus amyloliquefaciens XJTC-29 on the pathogenic bacteria of wheat stem base rot.

[0018] Figure 2 Colony morphology of the Bacillus amyloliquefaciens XJTC-29.

[0019] Figure 3 Gram staining result of the Bacillus amyloliquefaciens XJTC-29.

[0020] Figure 4 16s rDNA phylogenetic tree of the Bacillus amyloliquefaciens XJTC-29.

[0021] Figure 5 Promoting effect of the Bacillus amyloliquefaciens XJTC-29 on wheat.

[0022] Figure 6 Prevention and treatment effect of the Bacillus amyloliquefaciens XJTC-29 on wheat stem base rot. DETAILED DESCRIPTION

[0023] ​​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.

[0024] Example 1 Isolation, purification and identification of Bacillus amyloliquefaciens strain XJTC-29

[0025] 1. Test materials

[0026] Soil samples: Wheat rhizosphere soil samples were collected from four regions of Xinjiang, namely, Taicheng, Aletai, Bobo and Yili. In order to ensure the integrity of the rhizosphere microorganisms, the samples were stored in a low-temperature environment before the test, and the storage time should not exceed 72 h.

[0027] Test pathogenic fungi: Fusarium graminearum (Schwein.) (Snyder et F. culmorum ), Fusarium culmorum (Schwein.) (W.Gams) Wiedemann (2001), Fusarium pseudograminearum (Biltsh et F. graminearum ) and Fusarium pseudograminearum (Biltsh et F. pseudograminearum ) were stored in the Plant Protection Institute of Xinjiang Uygur Autonomous Region Academy of Agricultural Sciences.

[0028] 2. Test methods

[0029] 2.1 Screening and purification method of Bacillus amyloliquefaciens strain XJTC-29

[0030] The spore solution of Fusarium graminearum (Schwein.) (Snyder et F. culmorum ) was diluted to 1×10 6 CFU / mL and spread on PDA plates; the collected rhizosphere soil samples were prepared 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 the PDA was dried, an equal volume of soil suspension was superimposed, 3 repeats were set for each dilution concentration, and the formation of inhibition zones was observed after 2-3 days; the strains producing obvious inhibition zones were purified in time and stored in 50% glycerol tubes (-80℃).

[0031] The above-mentioned preserved strains were further screened for antagonistic bacteria by using plate confrontation method: the antagonistic bacteria were confronted with Fusarium culmorum (Schwein.) (W.Gams) Wiedemann (2001), and the strains without antagonistic effect were removed; the remaining strains were further confronted with Fusarium pseudograminearum (Biltsh et F. graminearum F. pseudograminearum The radius of the antagonistic bacteria to different pathogenic fungi was measured, and 3 repeats were set for each test group.

[0032] ​The formula for calculating the inhibition rate is: inhibition rate (%) = (control colony radius - treatment colony radius / control colony radius - 2.5 mm) x 100.

[0033] 2.2 Identification of Bacillus amyloliquefaciens strain XJTC-29

[0034] 2.2.1 Morphological, physiological and biochemical identification method of strain XJTC-29

[0035] After antagonistic bacteria were cultured in nutrient agar medium (NA) at 37°C in a constant temperature incubator for 48 h, the surface morphology, edge characteristics of the antagonistic bacteria were observed and the colony diameter was measured according to the "Bergey's Manual of Determinative Bacteriology".

[0036] 2.2.2 Molecular biological identification of strain XJTC-29

[0037] 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 x 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 after detection 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.

[0038] 3 Test results

[0039] 3.1 Screening and purification results of Bacillus amyloliquefaciens strain XJTC-29

[0040] The final screening of the dominant antagonistic bacteria XJTC-29 (see Figure 1 ) with significant inhibitory effect on the three pathogenic fungi was carried 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.

[0041] 3.2 Morphological, physiological and biochemical identification results of strain XJTC-29

[0042] The strain XJTC-29 showed irregular colony shape on NA medium, with rough colony surface and milk-white color (Fig. 2), and was stained purple by Gram staining (Fig. 3), which indicated that it was a Gram-positive bacterium. The bacterial morphology was rod-shaped. Based on the above results, the strain XJTC-29 was preliminarily identified as Bacillus amyloliquefaciens. Figure 2 Figure 3

[0043] 3.3 Molecular biology identification results of strain XJTC-29

[0044] The 16s rDNA sequencing of the strain XJTC-29 obtained a 1413 bp sequence fragment (SEQ ID No. 3) :

[0045] ​​

[0046] The NCBI Blast comparison is the highest (100%) with Bacillus amyloliquefaciens (KF933607), and the XJTC-29 is found in the same system evolution branch after the construction of the adjacent method ( Bacillus amyloliquefaciens ). Bacillus amyloliquefaciens The XJTC-29 strain is finally identified as Bacillus amyloliquefaciens combined with morphological observation and gram staining. Figure 4

[0047] Test Example 1: Bacillus amyloliquefaciens strain XJTC-29 potting growth promotion test

[0048] 1. Test method

[0049] The planted wheat variety is Xindong 20, and the small and healthy wheat seeds are selected, germinated, and sown in a small flower pot, 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, and then the supernatant is discarded after centrifugation at 8000 rpm for 10 min, and the bacterial precipitate is reserved. The bacterial suspension is prepared by diluting the bacterial body with sterile water, and the OD is 1. 600

[0050] After 5 days of wheat planting, 10 mL of 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 wheat are recorded after 21 days.

[0051] 2. Test results

[0052] The test results are shown in Table 1. Figure 5 Compared with CK, the chlorophyll content of Bacillus amyloliquefaciens XJTC-29 treatment group is 40.66±0.47, and the chlorophyll content is significantly increased by 13.97%; the wheat treated by Bacillus amyloliquefaciens XJTC-29 has a height of 37.37±1.22 cm, and the 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.

[0053] The test results show that the wheat treated by Bacillus amyloliquefaciens XJTC-29 bacterial suspension has a significant increase in chlorophyll content in the leaves, and the root length, plant height and fresh weight are significantly increased.

[0054] Test Example 2: Bacillus amyloliquefaciens strain XJTC-29 potting prevention test

[0055] 1. Test method​​

[0056] The wheat variety planted was Xindong 20. Healthy wheat seeds of similar size were selected, germinated, and sown in small flowerpots, 12 seeds per pot. Each treatment was replicated three times. The antagonistic bacteria were cultured on NB medium for 2 days. After centrifugation at 8000 rpm for 10 min, the supernatant was discarded, and the bacterial pellet was retained. The bacterial cells were diluted with sterile water. OD 600 =1 Prepare an antagonistic bacterial suspension for later use.

[0057] The pathogenic fungal strain was inoculated into CMC medium and cultured at 28℃ with shaking at 180 rpm for 7 days. After filtering to remove mycelia, the spore suspension was diluted to a concentration of 1×10⁻⁶. 6 ,spare.

[0058] Experimental Groups:

[0059] Treatment group: After 6 days of wheat growth, inoculate with 10 mL of Bacillus amyloliquefaciens XJTC-29 suspension; after 7 days of wheat growth, inoculate with 10 mL of Fusarium oxysporum (…). F. culmorum Fusarium graminearum ( ), F. graminearum ), Fusarium graminearum ( F. pseudograminearum ) spore liquid, these three treatment groups are described as follows: F.c +29, F.g +29 and F.p +29;

[0060] Positive control group: After wheat growth for 7 days, only 10 mL of Fusarium oxysporum (Flavorholia fulva) was inoculated. F.culmorum Fusarium graminearum ( ), F. graminearum ), Fusarium graminearum ( F.pseudograminearum The spore liquid, and the three positive control groups are respectively described as follows: F.c, F.g and F.p .

[0061] Negative control group: Six days after wheat growth, only 10 mL of Bacillus amyloliquefaciens XJTC-29 bacterial suspension was inoculated. This negative control group is referred to as XJTC-29.

[0062] CK: Untreated wheat.

[0063] Twenty-one days after inoculation, the disease index and control effect were calculated and statistically analyzed according to the wheat stem base rot survey grading standards, disease index calculation formula, and control efficacy calculation formula in Table 1.

[0064] Table 1 Grading Standards for Wheat Stem Base Rot Survey

[0065]

[0066] The formula for calculating the disease index is:

[0067] Disease index = ∑ (disease index of each level × corresponding level) / total number of investigation × highest disease level

[0068] The formula for calculating the control effect is:

[0069]

[0070] 2 Test results

[0071] The results of the pot experiment in the greenhouse are shown in Table 2 and Figure 6 It is shown that the wheat plants in the negative control group inoculated with Bacillus amyloliquefaciens XJTC-29 bacterial suspension had no disease symptoms. The wheat plants in the three positive control groups 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 base, and some wheat stem bases rotted and died on the 26th day. Compared with the positive control groups, the disease index of the wheat plants in 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 wheat plants in the XJTC-29 treatment group against F. graminearum was reduced by 30.74, and the control effect reached 54.62%; F.p the disease index of the wheat plants in the XJTC-29 treatment group against P. graminicola was reduced by 35.49, and the control effect reached 60.29%; F.g the disease index of the wheat plants in the XJTC-29 treatment group against F. graminearum was reduced by 31.60, and the control effect reached 55.73%.

[0072] Table 2 Control effect of antagonistic bacteria on wheat

[0073]

[0074] 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, The microbial preservation number thereof is: CGMCC No. 34774.

2. A microbial preparation prepared from the Bacillus amyloliquefaciens XJTC-29 of claim 1.

3. The application of Bacillus amyloliquefaciens XJTC-29 in the prevention of wheat diseases; the pathogenic bacteria of the wheat diseases are Fusarium graminearum ( F. graminearum ), Fusarium pseudograminearum ( F. pseudograminearum ) or Fusarium culmorum ( F. culmorum ).​​​​​​ 4. The use of the microbial preparation according to claim 2 for the control of wheat diseases, the pathogenic fungi of which are Fusarium graminearum (Schwein) S. Ito ( F. graminearum ), Fusarium pseudograminearum (Buller) Blancham ( F. pseudograminearum ) or Fusarium culmorum (W. G. Smith) Wiedeman ( F. culmorum ).​​​ 5. The Bacillus amyloliquefaciens XJTC-29 of claim 1 for use in promoting the growth of wheat; the promotion of the growth of wheat is to increase the content of chlorophyll in leaves, root length, plant height or fresh weight; the fresh weight refers to the weight of the whole wheat plant.

6. The microbial preparation of claim 2 for use in promoting the growth of wheat; the promotion of the growth of wheat is to increase the content of chlorophyll in leaves, root length, plant height or fresh weight; the fresh weight refers to the weight of the whole wheat plant.

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