Bacillus anthracis and uses thereof

Abstract

The present application discloses a kind of anthracnose fungi and its application, belong to microbial technical field.The anthracnose fungi of the present application is isolated from the stems and leaves of Adenostoma pinnatum collected in Dongchu Island, Rongcheng City, Shandong Province, and was preserved in China Center for Type Culture Collection on August 29, 2022, with the preservation number CCTCC NO.M 20221345.The fungus has a significant control effect on barnyard grass and a significant inhibitory effect on Botrytis cinerea, so the fungus can be prepared into a microbial agent for the control of weeds and related plant diseases in the field.In addition, the anthracnose fungi described in the present application also has salt and alkali tolerance characteristics, so it can also be applied in saline-alkali soil to improve the saline-alkali soil and control weeds and plant diseases in the saline-alkali soil.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial technology, and particularly relates to a Colletotrichum fungus and application thereof. BACKGROUND

[0002] Echinochloa crusgalli is one of the most serious malignant weeds in rice fields, which has strong growth and reproduction ability, can cause a substantial reduction in rice yield, and even absolute loss. The prevention and control methods of Echinochloa crusgalli mainly include chemical control, physical control and biological control. Although the chemical control has low cost and quick effect, its long-term, excessive and unreasonable use not only causes the significant increase of Echinochloa crusgalli resistance, but also leads to ecological problems such as pesticide residues and biodiversity reduction.

[0003] Botrytis cinerea is the pathogen of gray mold, which is listed as one of the world's top ten plant fungal diseases. Botrytis cinerea can infect more than 200 economic crops, causing huge economic losses every year. Similar to the prevention and control of Echinochloa crusgalli, although chemical fungicides can effectively prevent and control Botrytis cinerea, due to its strong genetic variability, Botrytis cinerea is easy to develop resistance to chemical fungicides with relatively single action target.

[0004] In view of the low efficiency of physical control and the problems such as resistance and ecology caused by chemical control, the use of microorganisms to control weeds and plant diseases has become an important direction for the development of green pesticides in the future. Microbial pesticides have the advantages of good degradation, high safety and low resistance, and therefore have important significance for the development of environment-friendly ecological agriculture, and also meet the development trend of green production of agricultural products in China. So far, some strains of bacteria such as Bacillus and Streptomyces, and fungi such as Trichiderma, Phytophthora and Fusarium have been developed into commercial herbicides or fungicides. SUMMARY

[0005] The technical solutions of the present application are as follows:

[0006] The present application provides a Colletotrichum fungus DC22, which is isolated from the stems and leaves of Tamarix austrotimoriensis collected in Dongchu Island, Rongcheng City, Shandong Province, and preserved in China Center for Type Culture Collection on August 29, 2022, with the preservation number CCTCC NO.M20221345.

[0007] The above-mentioned Colletotrichum fungus is proved to have weed control effect, especially significant inhibition effect on Echinochloa crusgalli seedlings. Therefore, the present application provides the application of the above-mentioned Colletotrichum fungus in weed control. The weeds include but are not limited to Echinochloa crusgalli and other field malignant weeds.

[0008] The above-mentioned Colletotrichum fungi has been proved to have a plant disease control effect, especially for a plant disease caused by Botrytis cinerea. Therefore, the present application provides an application of the above-mentioned Colletotrichum fungi in controlling a plant disease caused by Botrytis cinerea. In the application, the control purpose can be diagnostic or non-diagnostic. Specifically, the present application provides an application of the above-mentioned Colletotrichum fungi in preparing a microbial preparation for controlling a plant disease caused by Botrytis cinerea.

[0009] The present application provides a microbial preparation comprising the above-mentioned Colletotrichum fungi. The microbial preparation can be used for controlling field weeds such as Echinochloa crus-galli or a plant disease caused by Botrytis cinerea.

[0010] The present application provides a fermentation product of the above-mentioned Colletotrichum fungi. The fermentation product is an ethyl acetate extract of a fermentation medium of the Colletotrichum fungi. The fermentation product has a significant inhibitory effect on Echinochloa crus-galli seedlings. Therefore, the present application provides an application of the fermentation product in controlling Echinochloa crus-galli. The fermentation product has a significant inhibitory effect on Botrytis cinerea. Therefore, the present application provides an application of the fermentation product in controlling a plant disease caused by Botrytis cinerea.

[0011] In a specific application process, the fermentation product can be prepared into a preparation with a concentration of 50 μg / mL to effectively control Echinochloa crus-galli. The fermentation product can be prepared into a preparation with a concentration of 200 mg / mL to effectively control Botrytis cinerea.

[0012] The present application provides a preparation method of the above-mentioned fermentation product, as follows:

[0013] The above-mentioned Colletotrichum fungi is inoculated into a culture medium and fermented under suitable conditions. The fermentation medium is extracted by using an organic solvent, and the extract is distilled under reduced pressure to obtain the fermentation product.

[0014] In the preparation method of the fermentation product, the Colletotrichum fungi can be preferably activated before inoculation. The activation step is as follows: the Colletotrichum fungi is inoculated on the surface of a PDA plate and cultured at 28℃ until the colonies cover the surface of the plate to achieve the activation of the Colletotrichum fungi.

[0015] In the preparation method of the fermentation product, the fermentation conditions are preferably as follows: static fermentation at 28℃ for 30 d.

[0016] In the preparation method of the fermentation product, the organic solvent is selected from one or more of ethyl acetate, dichloromethane and acetone; and the ethyl acetate is preferred.

[0017] The medium in the preparation method of the fermentation product is selected from a solid medium, and can be selected from the following formula: 70 g of rice, 0.15 g of corn syrup, 0.45 g of peptone and 150 mL of artificial seawater. In actual application, the amount of each component can be reduced or increased according to the formula. Of course, the amount ratio of each component can also be reduced or increased within a reasonable range, without affecting the use performance of the medium.

[0018] The above Colletotrichum has been proved to have salt and alkali tolerance, and therefore, the application of the above Colletotrichum in the improvement of saline-alkali soil is provided. In addition, the application of the above Colletotrichum in the prevention and treatment of Echinochloa crus-galli or plant diseases caused by Botrytis cinerea is also provided.

[0019] The beneficial effects of the present application are as follows:

[0020] The present application provides a Colletotrichum which has a significant prevention and treatment effect on Echinochloa crus-galli and a significant inhibitory effect on Botrytis cinerea, and therefore, the Colletotrichum can be prepared into a microbial agent for the prevention and treatment of weeds and related plant diseases in the field. In addition, the Colletotrichum has salt and alkali tolerance, and therefore, it can also be applied to saline-alkali soil to improve the saline-alkali soil and prevent and treat weeds and plant diseases in the saline-alkali soil. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 It is a photo of the front and back of the PDA plate of strain DC22;

[0022] Figure 2 It is a phylogenetic tree of strain DC22;

[0023] Figure 3 It is the inhibitory effect of the fermentation product of strain DC22 on the growth of Echinochloa crus-galli seedlings;

[0024] Figure 4 It is the effect of the fermentation product of strain DC22 on the growth of rice seedlings;

[0025] Figure 5 It is the inhibitory effect of the fermentation product of strain DC22 on the growth of multi-drug resistant Botrytis cinerea;

[0026] Figure 6 It is a growth chart of strain DC22 under different salt concentrations; the lower bar chart is the colony diameter under different salt concentrations. DETAILED DESCRIPTION

[0027] Strain separation and identification:

[0028] The strain of the application is isolated from the stems and leaves of Tamarix austromongolica collected in Dongchudi Island, Rongcheng City, Shandong Province, and the specific steps are as follows: the stems and leaves of Tamarix austromongolica are washed clean with distilled water, cut into 2 cm small sections, placed on filter paper to absorb surface moisture, and naturally air-dried; the stem and leaf sections are soaked in 4% sodium hypochlorite for 1 min, rinsed with sterile water for 2-3 times, and the surface moisture is absorbed with sterile gauze to complete surface sterilization; the stem and leaf sections are further separated into 0.5 cm sections with sterile scissors, and the cut surfaces are inserted into PDA double-antibiotic culture medium prepared with artificial seawater (3% sea salt is prepared to obtain artificial seawater), 3-4 pieces are planted in each dish; the plates are placed in an incubator at 28℃ and cultured upside down, and observed once every 12 h. When mycelium grows on the cut surface, the tip is picked to a PDA plate, and repeated streaking is performed until single colonies with consistent morphology are obtained, which are transferred to a strain preservation tube and stored at -20℃ for use, and numbered as DC22.

[0029] The strain DC22 is inoculated onto the surface of a PDA plate prepared with artificial seawater, and cultured at 28℃ for 5 d, and the photos of the colonies on the front and back surfaces are as shown in Figure 1 It can be seen that the mycelium of the strain DC22 can cover the entire plate after 5 d of culture, which indicates that the strain has salt and alkali tolerance characteristics. The morphological characteristics are that the mycelium in the center of the colony is grayish white, the edge is yellowish brown, and brown conidia are scattered; the back of the colony is light yellow.

[0030] The rDNA gene sequence determination result (ITS-5.8S-ITS2 region) of the strain is as follows:

[0031]

[0032] The sequence information is compared with the corresponding sequence information of known strains in the Genbank database, and the phylogenetic tree is constructed, and the result is as shown in Figure 2 Finally, the strain DC22 is identified as Colletotrichum aciculare. The strain is preserved in China Center for Type Culture Collection, Wuhan University, China on August 29, 2022, and the preservation number is CCTCC NO.M 20221345.

[0033] Unless otherwise specified, other terms used in the application generally have the meanings commonly understood by those of ordinary skill in the art. The application is described in further detail below in conjunction with specific examples and by reference to the data. The following examples are only intended to illustrate the application, and in no way limit the scope of the application.

[0034] Example 1

[0035] Preparation of fermentation products:

[0036] A small amount of strain DC22 preserved in PDA medium slant was inoculated onto the surface of PDA (glucose 20 g, agar 20 g, sea salt 3%, prepared with 1000 mL of 0.2 g / mL potato extract) prepared with artificial seawater, and cultured at 28°C until the colonies covered the surface of the plate. The strain DC22 on the surface of the above plate (about 2 cm x 2 cm) was inoculated into sterilized solid medium and incubated at 28°C for 30 days. The solid medium was prepared with 70 g of rice, 0.15 g of corn syrup, 0.45 g of peptone, and 150 mL of artificial seawater. The fermentation medium was extracted twice with ultrasonic extraction with ethyl acetate (EtOAc) for 15 min each time, and the combined extract was distilled under reduced pressure to obtain the ethyl acetate extract of the fermentation medium of strain DC22, i.e., the fermentation product. The strain was inactivated when the fermentation medium was extracted with ethyl acetate.

[0037] Meanwhile, the above fermentation medium was extracted with n-butanol (n-BuOH) and water (H2O) respectively to obtain the n-butanol extract and water extract of the fermentation medium of strain DC22, and the extraction method was the same as that of ethyl acetate.

[0038] Example 2

[0039] Herbicidal activity determination:

[0040] The herbicidal activity determination method used the agar mixed powder method established by Luo Xiaoyong et al. (Journal of Qingdao Agricultural University, 2007, 24: 267-270), which can quickly determine the herbicidal activity of plant extracts or microbial fermentation products. This method has the advantages of small sample consumption, simple operation, good repeatability, and easy observation of the growth status of the indicator plants. Wang Zhaofu et al. (J. Agric. Food Chem. 2019, 67, 14102-14109; Nat. Prod. Res. 2020, 34, 889-892) used the agar mixed powder method to quickly determine the herbicidal activity of several microbial fermentation products using barnyard grass as the test weed, to demonstrate the feasibility and accuracy of the above test method.

[0041] The specific steps of this method are as follows:

[0042] The seeds of Echinochloa crus-galli and Oryza sativa were treated with 0.2% sodium hypochlorite for 15 min, and then washed with sterile water for several times and soaked for 4-6 h. Then, the seeds were placed on the surface of sterile wet filter paper and germinated in dark for 12 h. The fermentation product (ethyl acetate extract) of strain DC22 was dissolved in 50% dimethyl sulfoxide aqueous solution to prepare a sample solution with a concentration of 50 μg / mL. 1 mL of the sample solution was mixed with 99 mL of sterile agar aqueous solution to prepare 3 samples, which were condensed in 3 small beakers with a volume of 25 mL, respectively. Five small holes were punched on the surface of the condensed agar with a pair of sharp forceps, and the radicles of the germinated Echinochloa crus-galli or Oryza sativa seedlings with a length of 4-5 mm were inserted into the holes, respectively. Five seedlings were placed in each beaker, and the experiment was repeated for 3 times. The beakers were covered with tin foil and cultured in an artificial climate chamber in dark for 2 d. The artificial climate chamber was set to automatically cycle 14 h light (25°C) and 10 h dark (20°C), and the relative humidity in the chamber was 60%. The length of the radicle was measured after 2 d, and the growth inhibition rate of the Echinochloa crus-galli or Oryza sativa seedlings was calculated.

[0043] The calculation formula is as follows:

[0044] Growth amount = radicle length after treatment - radicle length before treatment;

[0045] Inhibition rate (%) = [(control average growth amount - treatment average growth amount) / control average growth amount] x 100%.

[0046] The test results are shown in Tables 1 and 2. Figure 3 and Figure 4

[0047] When the concentration of the fermentation product of strain DC22 was 50 μg / mL, the growth inhibition rate of the radicle of the Echinochloa crus-galli seedlings, which is a malignant weed in rice field, was 75.8% ( Figure 3 ), and the growth of the Oryza sativa seedlings was not affected ( Figure 4 ). This indicates that the fermentation product of the strain can effectively prevent and control Echinochloa crus-galli and is safe for Oryza sativa.

[0048] Example 3

[0049] Bacteriostatic activity determination:

[0050] The bacteriostatic activity determination method is filter paper method, and the specific steps are as follows:

[0051] ​The multi-drug resistant Botrytis cinerea was isolated from a grape picking garden in Laixi, Qingdao, and it was resistant to the commonly used chemical fungicides of Botrytis cinerea, boscalid and azoxystrobin. The fermentation product of strain DC22 was dissolved with methanol to prepare a sample solution with a concentration of 200 mg / mL for standby. A 5 mm or so fungus cake was punched on the edge of the colony of the test plant pathogen with a puncher, and it was placed on the surface of a 9 cm PDA plate together with a 5 mm sterile filter paper. Both were 25 mm away from the edge of the plate and on the same straight line. 5 μL of sample solution was taken to the filter paper (the amount of sample was 1 mg), and an equal volume of methanol was added as a control. After incubation at 28°C for 5 days, it was observed whether the fermentation product could inhibit the growth of the test plant pathogen.

[0052] The test results are shown in Table 1. Figure 5 As shown in Table 1, the ethyl acetate extract of the fermentation medium of strain DC22 could significantly inhibit the growth of multi-drug resistant Botrytis cinerea, and the inhibition zone diameter was 27 mm; while the n-butanol extract and water extract of the fermentation medium of strain DC22 had no inhibitory activity. This indicates that the active ingredients of strain DC22 for inhibiting multi-drug resistant Botrytis cinerea are mainly concentrated in the ethyl acetate extract, which is a moderately polar component.

[0053] In summary, strain DC22 has the potential to develop into a multi-functional microbial pesticide (preparation) for weed control and plant disease prevention.

[0054] Example 4

[0055] Salt and alkali tolerance determination:

[0056] According to the method of preparing PDA medium with artificial seawater as shown in Example 1, PDA plates with 1%, 3%, 5% and 10% seawater concentrations were prepared in turn, and the conventional PDA medium prepared with distilled water was used as a control. A small amount of strain DC22 stored in the PDA medium slant was picked and inoculated on the surface of the PDA plate, and incubated at 28°C until the colony covered the surface of the plate. A 5 mm fungus cake was punched on the edge of the colony with a puncher, and inoculated on the surface of the PDA plate with different seawater concentrations, and incubated at 28°C for 5 days. The mycelial growth of strain DC22 was observed, and the colony diameter was measured by cross-intersection method.

[0057] The results are shown in Table 2. Figure 6

[0058] ​The strain DC22 can grow normally on PDA prepared with 1% and 3% sea salt, and the growth rate and colony diameter are significantly greater than those of the control; even at a 5% sea salt concentration, the strain DC22 can grow normally; but at a 10% extreme sea salt concentration, the growth of the strain DC22 is obviously inhibited. The salinity of saline-alkali soil environment is generally in the range of 1% to 2%, and the strain DC22 has strong adaptability in this salinity range, and can even fully utilize this saline-alkali environment to accelerate its own growth. The above results show that the strain DC22 has strong colonization ability in the saline-alkali soil environment. Therefore, it can be applied to the soil improvement of saline-alkali soil, and has important value.

[0059] The above merely describes preferred embodiments of the present application, but is not intended to limit the present application in other forms, and any person skilled in the art can modify or change the above disclosed technical content into equivalent embodiments with equivalent changes. However, any simple modification, equivalent change and modification made to the above embodiments without departing from the technical solution of the present application, and in accordance with the technical essence of the present application, still belongs to the protection scope of the technical solution of the present application.

Claims

1. A fungus of the genus Colletotrichum (Colletotrichum sp.) characterized in that, Colletotrichum aciculare The strain was deposited in China Center for Type Culture Collection on August 29, 2022, and the deposit number is CCTCC NO. M 20221345. ​ 2. Use of the Colletotrichum fungus of claim 1 in controlling Euphorbia heterophylla and / or plant diseases caused by Botrytis cinerea.

3. A microbial preparation, characterized in that, A composition comprising the Colletotrichum fungus of claim 1.

4. A fermentation product of the Colletotrichum fungus of claim 1. A method for preparing the fermentation product, comprising the following steps: inoculating the Colletotrichum fungus into a culture medium, and statically fermenting at 28℃ for 30 days; extracting the fermentation culture medium with an organic solvent, and distilling the extract under reduced pressure to obtain the fermentation product. The organic solvent is selected from one or more of ethyl acetate, dichloromethane, and acetone.

5. A preparation containing the fermentation product according to claim 4, characterized in that, The concentration of the fermentation product is selected from 50 μg / mL or 200 mg / mL.

6. Use of the Colletotrichum fungus of claim 1 in improving saline soil.

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