Bacillus amyloliquefaciens for preventing and controlling oat anthracnose and German mildew leaf spot and its application

Abstract

The invention discloses a Bacillus amyloliquefaciens strain for preventing and controlling oat anthracnose and German mildew leaf spot and its application, and relates to the technical field of biological control. The Bacillus is strain SAF-1, and its deposit number is CGMCC No. 27187. The SAF-1 bacterial suspension of the invention is used to carry out indoor prevention and control effect determination on oat anthracnose, and the prevention and control effect is 75.27%.

Description

Technical Field

[0001] The invention relates to the technical field of biological control, and more particularly to a Bacillus amyloliquefaciens strain for controlling oat anthracnose and German mildew leaf spot and its application. Background Art

[0002] Colletotrichum graminicola is an important pathogen in agricultural production. It has a wide host range and can infect corn, sorghum, wheat, oats and other gramineous crops, causing significant losses to agricultural production. The pathogen infects corn leaves, causing leaf death, and corn stalks to cause stem rot symptoms; infecting oat leaves will cause spindle-shaped dark brown necrotic spots, seriously affecting the photosynthetic efficiency of the leaves, and causing nearly 30% yield loss in severe cases; it can infect sorghum at all growth stages, causing seedling death, leaf spots, stem rot and ear rot, and can cause 30-50% loss of sorghum grain and stalk yield.

[0003] Drechslera avenacea is an important pathogen that can cause oat leaf spot disease in oat production. The pathogen invades the leaves or sheaths, causing purple spots with yellow halos in the field; in severe cases, the spots merge into pieces, and the entire leaf dries up from the tip downwards.

[0004] At present, the prevention and control of crop anthracnose and German mildew leaf spot caused by Colletotrichum graminearum mainly rely on chemical control. Studies have shown that carbendazim, imidaclostrobin and azoxystrobin are all highly toxic to Colletotrichum graminearum and have good field control effects. However, the single, excessive and repeated use of chemical pesticides will cause fungicide residues in farmland, which will cause a series of food safety and environmental pollution problems, and will also increase the selection pressure on pathogens, making pathogens resistant; therefore, the single use of chemical pesticides for control does not meet the needs of green development of modern agriculture. The application of disease-resistant varieties is an important measure to prevent and control anthracnose. However, there is a differentiation of pathogenicity in Colletotrichum graminearum, and the pathogenicity of different strains varies greatly. A single disease-resistant material cannot show broad-spectrum resistance to all physiological subspecies; and the period of hybrid breeding is long, and it takes several years or even more than ten years to obtain disease-resistant materials with various excellent traits.

[0005] Grain anthracnose mainly invades plant tissues by forming infection structures called appressorium. When conidia fall on the surface of plant tissues, they germinate and grow germ tubes. The top of the germ tube swells, melanin is deposited in the swollen cells, and the glycerol content inside increases, forming mature appressorium. The appressorium grows invasion spikes, which pierce the host epidermis through mechanical pressure and enter the plant tissue.

[0006] Plant endophytes are an important microbial resource. Plant endophytes have a variety of disease resistance mechanisms such as antibiosis and inducing plant disease resistance. They can also improve the ability of plants to resist and tolerate diseases by promoting growth and providing plants with necessary nutrients such as phosphorus and potassium solubilization. The present invention takes oat anthracnose as the prevention and control target, and screens oat endophytes from different locations to screen microbial strains with disease resistance and growth promotion effects, thereby providing important microbial resources for the prevention and control of oat anthracnose and the development and application of special microbial fertilizers for oat fields.

[0007] Therefore, a Bacillus amyloliquefaciens strain for preventing and controlling oat anthrax and its application are problems that those skilled in the art need to solve urgently. Summary of the invention

[0008] In view of this, the present invention provides a Bacillus amyloliquefaciens strain for preventing and controlling oat anthracnose and German mildew leaf spot and its application.

[0009] In order to achieve the above object, the present invention adopts the following technical solution:

[0010] A Bacillus amyloliquefaciens strain for preventing and controlling oat anthracnose and German mildew leaf spot, the Bacillus is strain SAF-1, which is isolated and screened from the inner part of oat leaf tissue, has a significant inhibitory effect on oat anthracnose, and is identified as Bacillus amyloliquefaciens by morphological characteristics, physiological and biochemical and molecular identification analysis. It was deposited in the General Microbiology Center (CGMCC) of the China Microbiological Culture Collection on April 23, 2023, and the deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, and the registration number of the deposit center is CGMCC No. 27187.

[0011] Application of Bacillus amyloliquefaciens strain SAF-1 in controlling oat anthracnose and German mildew leaf spot.

[0012] A method for preparing a suspension of Bacillus amyloliquefaciens for preventing and controlling oat anthracnose and German mildew leaf spot comprises the following steps:

[0013] The strain SAF-1 is subjected to liquid aerobic fermentation to obtain a fermentation broth, the fermentation broth is centrifuged to remove the supernatant, and a bacterial precipitate is obtained; sterile water is added to the bacterial precipitate to obtain a bacterial suspension;

[0014] The concentration of bacterial suspension was 1×10 7 cfu / mL.

[0015] A method for preparing a Bacillus amyloliquefaciens seed coating agent for preventing and controlling oat anthracnose and German mildew leaf spot comprises the following steps:

[0016] The strain SAF-1 was subjected to liquid aerobic fermentation to obtain a fermentation broth, and the fermentation broth was centrifuged to remove the supernatant to obtain a bacterial precipitate; the bacterial precipitate was adsorbed with clay, and 3% sodium carboxymethyl cellulose (CMC) was added to prepare a seed coating agent;

[0017] The concentration of the seed coating agent was 1×10 9 cfu / g.

[0018] A method for preventing and controlling anthracnose and German mildew leaf spot of oats comprises mixing the seed coating agent with 2% of the seed weight; fully soaking the seeds with water of about 5% of the seed weight before mixing the seeds, and then adding 2% of the seed coating agent and stirring evenly; spraying the SAF-1 bacterial suspension 3 times during the heading and flowering period of the oats, once every 10 days.

[0019] It can be seen from the above technical solution that, compared with the prior art, the beneficial effects of the present invention are:

[0020] The SAF-1 bacterial suspension of the present invention is used to carry out indoor control effect test on oat anthracnose and German mildew leaf spot, and the control effects are 75.27% and 68.45% respectively.

[0021] The SAF-1 bacterial suspension and seed coating agent of the present invention are used for seed dressing and spraying treatment respectively, and the control effect of oat anthracnose in two test sites of Wuchuan and Jining is 71.77% and 77.38% respectively; in addition, the strain also has a good control effect on oat leaf spot caused by Drechslera avenae, and the control effect in two test sites of Wuchuan and Jining is 67.51% and 77.60% respectively; the control effect of strain SAF-1 is better than that of commercially available Bacillus subtilis bacterial agent. The strain has a good control effect on oat anthracnose and has a good development and application prospect. Compared with chemical agents, SAF-1 bacterial agent has the advantages of not easy to produce drug resistance, safety for humans and animals, and no agricultural residue pollution. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0023] Figure 1 Plate confrontation test of strain SAF-1 with Colletotrichum graminearum and M. germanica;

[0024] Figure 2 Indoor control effect test of strain SAF-1;

[0025] Figure 3 Pictures of the effect of strain SAF-1 on promoting oat growth;

[0026] Figure 4 Colony morphology and microscopic morphology of strain SAF-1;

[0027] Figure 5 Normal appressorium formed by conidia of Colletotrichum graminearum that was not treated with strain SAF-1;

[0028] Figure 6 Abnormal appressorium formation of Colletotrichum graminearum treated with strain SAF-1. DETAILED DESCRIPTION

[0029] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] Experimental materials: Unless otherwise specified, the materials used in the following examples can be purchased from conventional biochemical reagent companies.

[0031] The oat anthracnose bacteria and Drechslera avenae in the following examples were isolated by the inventor (Plant Pathology Laboratory, College of Horticulture and Plant Protection, Inner Mongolia Agricultural University) from diseased oat leaves in Pingdiquan Town, Chahar Right Front Banner, Ulanqab City, Inner Mongolia. The obtained pathogens were verified by Koch's postulates to be pathogenic to oats; they were identified as Colletotrichum graminicola and Drechslera avenae by morphological, physiological and biochemical identification and molecular identification.

[0032] Example 1 Isolation of endogenous antagonistic bacteria

[0033] The strain SAF-1 was isolated from oat plants growing vigorously in Huize County, Qujing City, Yunnan Province. 1g of oat leaf tissue was fully surface sterilized, cut into small pieces and placed in a sterile mortar, and 1mL of sterile water was added to fully grind into a homogenate; the homogenate was gradiently diluted to 10 -5 , take 100 μL of each dilution and spread it evenly on LB solid medium plates. Repeat 3 plates for each concentration and culture at 28℃ / dark conditions until a single colony grows.

[0034] Example 2 Determination of antagonistic activity of strain SAF-1

[0035] Antagonistic bacteria were screened by plate confrontation method. Colletotrichum gramineum was inoculated on PDA solid culture medium and cultured at 25℃ / dark conditions until the colony diameter was about 6cm. A bacterial cake with a diameter of 0.5cm was punched on the edge of the colony with a hole puncher and inoculated in the center of a new PDA culture medium. Endophytic bacteria to be screened were inoculated 3cm away from the bacterial cake around the culture dish, with 4 inoculation points per dish. The culture dish without the strain to be screened was used as a control and cultured at 25℃ / dark conditions for 5 days. The colony diameter of Colletotrichum gramineum was measured by the cross method, and the inhibition rate was calculated. The test results showed that the inhibition rate of strain SAF-1 was 79.03%; strain SAF-1 had obvious inhibitory activity against Colletotrichum gramineum.

[0036] The determination method for German leaf spot pathogen is the same as above.

[0037] Indoor plate confrontation test showed that strain SAF-1 had significant inhibitory effect on Colletotrichum graminearum and Pseudomonas aeruginosa, two major pathogens that cause oat leaf spot diseases (see Figure 1 ).

[0038] Example 3 Indoor bioassay and field control efficacy assay of strain SAF-1

[0039] The in vitro bioassay of strain SAF-1 was performed by the detached leaf method. The SAF-1 bacterial suspension cultured under shaking conditions at 28°C / dark conditions was centrifuged, the bacterial precipitate was collected, and the concentration was adjusted to 1×10 7 cfu / mL, and prepare a bacterial suspension. Collect fresh oat leaves in the field and cut them into 15 cm lengths. Add 10 mL of sterile water to the plates of Fusarium graminearum and Germania leaf spot pathogens that have been cultured for 5 days, use a sterile applicator to gently scrape up the conidia, and filter with 3 layers of sterile oil mirror paper. The filtrate is a spore suspension, and the concentration of the spore suspension is adjusted to 1×10 with sterile water. 5 cfu / mL as inoculum. Spray the bacterial suspension evenly on fresh oat leaves, inoculate 10μL spore suspension after drying, place the inoculated leaves in a fresh-keeping box covered with moist filter paper, and culture them at 25℃, 18h light / 8h dark for 4 days, measure the lesion diameter, and calculate the inhibition rate; spray sterile water and then inoculate with spore suspension as the control; spray sterile water without inoculation as the blank control. Inhibition rate = (control lesion diameter - treatment lesion diameter) × 100% / control lesion diameter.

[0040] The results of indoor bioassays showed that the length of anthracnose lesions after treatment with strain SAF-1 was 0.23 cm, while the length of lesions in the control was 0.93 cm. Under in vitro conditions, the control effect of strain SAF-1 on anthracnose of oats was 75.27%; the control effect on German mildew leaf spot was 68.45%. In addition, 4 days after inoculation, the control leaves had turned green, while the leaves treated with strain SAF-1 remained green (see Figure 2 , Figure 2 A and Figure 2 B is the control group, Figure 2 C and Figure 2 D is the experimental group; Figure 2 A and 2C were inoculated with oat anthracnose, Figure 2 The results showed that the strain SAF-1 could effectively control the infection and spread of Colletotrichum thunbergii and Colletotrichum thunbergii in vitro.

[0041] Oats were coated with SAF-1 seed coating agent, and the control group was blank treated. 20 days after sowing, the height and biomass of oats were measured. The results are shown in Table 1.

[0042] Table 1: Oat plant height and biomass

[0043]

[0044] The results in Table 1 show that after SAF-1 treatment, the plant height, plant fresh weight and plant dry weight of oats were significantly higher than those of the control, indicating that SAF-1 can promote plant growth (see Figure 3 , the left is the control group, and the right is the experimental group).

[0045] The field control efficacy was tested at the Wuchuan County test site and the Jining test site, which were divided into a blank control group (CK), SAF-1 group, methyl thiophanate group, and Bacillus subtilis group.

[0046] Field control effect of strain SAF-1 was determined. The suspension of SAF-1 cultured under shaking conditions at 28°C / dark conditions was centrifuged, the bacterial precipitate was collected, adsorbed with clay, and 3% CMC by weight of clay was added to prepare a seed coating agent. The number of viable bacteria was 1×10 9 cfu / g. The bacterial precipitate was directly diluted with sterile water to 1×10 7 cfu / mL bacterial suspension. Before sowing, soak the seeds with 5% water of seed weight, add 2% seed dressing agent and mix evenly, dry and sow. At the beginning of heading and flowering period, spray the bacterial suspension evenly 3 times with an interval of 10 days; and investigate the disease index of oat anthracnose 10 days after the last spray, and investigate the control effect.

[0047]

[0048]

[0049] The determination method of the Bacillus subtilis agent (Biovo, Inc., USA, pesticide PD20160669) group was the same as that of the SAF-1 group. The dosage of 70% methyl thiophanate wettable powder was 60 g / mu and 25 kg of water, and the mixture was sprayed evenly. The results are shown in Table 2.

[0050] Table 2: Field control effect of strain SAF-1

[0051]

[0052] The results in Table 2 show that the disease indexes of oat leaf spot and anthracnose in the control field of Wuchuan County were 60.00 and 64.44, respectively, and the disease indexes of leaf spot and anthracnose in the SAF-1 treatment were 19.49 and 18.19, respectively; the control effects were 67.51% and 71.77%, respectively. In the Jining test site, the disease indexes of oat leaf spot and anthracnose in the control field were 74.73 and 69.77, respectively, and the disease indexes of oat leaf spot and anthracnose in the SAF-1 treatment oat field were 16.74 and 15.78, respectively, and the control effects were 77.60% and 77.38%, respectively. The above results show that strain SAF-1 can effectively prevent and control the occurrence of oat anthracnose German mildew leaf spot.

[0053] Example 4 Identification of strain SAF-1

[0054] Morphological identification

[0055] The colonies of strain SAF-1 are pale yellow and waxy, with raised and irregularly wrinkled surfaces and irregular colony edges. The bacteria are rod-shaped, with a size of 2.23-4.78μm×0.27-0.43μm; in the late stage of culture, the bacteria shorten and swell to form spores, which are oval or nearly round and formed in the center of the bacteria. The spore size is 1.28-1.65μm×0.62-1.14μm (see Figure 4 , left is the early stage, right is the late stage).

[0056] Physiological and biochemical identification

[0057] According to Fang Zhongda's version of "Methods for Plant Pathology Research", the SAF-1 strain was subjected to physiological and biochemical identification. The physiological and biochemical indicators included carbon source utilization, nitrogen source utilization, Gram reaction, methyl red reaction, VP reaction, citrate utilization, malonate utilization, starch hydrolysis, catalase reaction, and nitrate reduction reaction, a total of 10 physiological and biochemical reaction indicators.

[0058] The results showed that the Gram staining reaction, VP reaction, citric acid reaction, malonate reaction, starch hydrolysis, catalase reaction and nitrate reduction reaction of strain SAR-1 were all positive; the methyl red reaction was negative; the carbon sources that could be utilized included D-fructose, D-mannose, D-sorbitol, xylose, maltose, glucose, sucrose and i-inositol; the nitrogen sources that could be utilized included potassium nitrate, sodium nitrate and histidine; and the nitrogen source that could not be utilized was L-hydroxyproline.

[0059] Table 3: Physiological and biochemical indicators of SAF-1

[0060]

[0061] Molecular identification

[0062] DNA of strain SAF-1 was extracted, and primer pairs 27F / 1492R and gyr-F / gyr-R were used to amplify 16s rDNA and housekeeping genes, respectively. After gel excision, purification and sequencing, the PCR products were compared by Blast in the NCBI database. The 16s rDNA sequence of strain SAF-1 had a similarity of 99.88% with that of Bacillus amyloliquefaciens MW559233.1; the housekeeping gene gyr sequence had a similarity of 99.77% with that of Bacillus amyloliquefaciens KU987462.1. The above results showed that, combined with the results of morphological, physiological and biochemical, and molecular identification, strain SAF-1 belonged to Bacillus amyloliquefaciens.

[0063] Example 5 Strain SAF-1 is able to inhibit spore germination and appressorium formation

[0064] 5 ml of sterile water was taken and placed in a culture dish where Colletotrichum graminearum was grown on PDA medium for 12 to 18 days. The mixture was gently scraped with a sterile applicator to prepare 1×10 5 cfu / ml spore suspension for later use. Centrifuge the bacterial suspension of strain SAF-1 cultured in LB liquid medium, collect the precipitate and prepare it into 1×10 8 cfu / ml bacterial suspension. Add the strain SAF-1 bacterial suspension to the spore suspension to make the bacterial concentration in the spore suspension 1×10 4 , 5×10 4 , 1×10 5 , 5×10 5 , 1×10 6cfu / ml. Drop 10μl of the spore suspension containing the bacteria on a hydrophobic glass slide and place it in a culture dish covered with moist filter paper. Culture it in a dark constant temperature incubator at 23℃. The treatment without adding the bacteria of strain SAF-1 was used as the control. Observe at 10h and 30h of culture, and calculate the spore germination rate and appressorium formation rate. Observe 10 fields of view under a 10× eyepiece for each replicate.

[0065] The test results showed that the germination rate of conidia of Colletotrichum spp. treated with the strain SAF-1 was significantly reduced. The conidia that could germinate had swollen tips of germ tubes, but no melanin deposition, and could not form normal appressorium; the diameter of the swollen structure was 27.65 μm, 6.71 times that of normal appressorium; some of the swollen structures at the tips of germ tubes redifferentiated into germ tubes and continued to elongate (see Figure 5 , 6). For bacterial treatment, linear regression analysis was performed on the inhibition of spore germination rate and appressorium formation rate to obtain the virulence regression equation.

[0066] The inhibitory concentration of SAF-1 on spore germination (EC 50 value) is 1.14×10 5 cfu / ml; EC for appressorium formation 50 1.99×10 4 cfu / ml. This showed that strain SAF-1 could inhibit the infection of Colletotrichum graminearum by inhibiting the germination of conidia and the formation of appressorium.

[0067] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A strain of Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ), It is characterized in that The Bacillus is strain SAF-1, and its deposit number is CGMCC No. 27187.

2. The Bacillus amyloliquefaciens of claim 1 is used to prevent and treat Colletotrichum graminearum ( Colletotrichum graminicola ) and caused by German mold ( Drechslera avenacea ) caused by oat leaf spot.

3. The method for preparing the Bacillus amyloliquefaciens cell suspension according to claim 1, It is characterized in that The following steps are involved: The strain SAF-1 is subjected to liquid aerobic fermentation to obtain a fermentation broth, the fermentation broth is centrifuged to remove the supernatant, and a bacterial precipitate is obtained; sterile water is added to the bacterial precipitate to obtain a bacterial suspension; The concentration of bacterial suspension was 1×10 7 cfu / mL.

4. A method for preparing the Bacillus amyloliquefaciens seed coating agent according to claim 1, It is characterized in that The following steps are involved: The strain SAF-1 was subjected to liquid aerobic fermentation to obtain a fermentation broth, and the fermentation broth was centrifuged to remove the supernatant to obtain a bacterial precipitate; the bacterial precipitate was adsorbed with clay, and 3% sodium carboxymethyl cellulose (CMC) was added to prepare a seed coating agent; The concentration of the seed coating agent was 1×10 9 cfu / g.

5. A method for preventing and controlling oat anthracnose caused by Colletotrichum graminearum and oat leaf spot caused by Moraxella gracilis, It is characterized in that The seed dressing agent of claim 4 is mixed with seeds at 2% of the seed weight; before mixing, the seeds are fully soaked with water of about 5% of the seed weight, and then 2% of the seed dressing agent is added and stirred evenly; when the oats are heading and flowering, the SAF-1 bacterial suspension is sprayed 3 times, once every 10 days.

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