Bacillus cereus for promoting color change of strawberry fruit appearance and application

Abstract

The present application relates to a bacillus cereus for promoting the appearance color change of strawberry fruits and an application, the bacillus cereus is preserved in China Microbial Culture Collection Center, the address of preservation is: No. 1, Beichen West Road, Chaoyang District, Beijing, China Institute of Microbiology, Chinese Academy of Sciences, the preservation number is CGMCC No. 27829, the preservation date is July 7, 2023, and the classification and naming is bacillus cereus.The bacillus cereus and the bacterial agent are non-toxic to plants, friendly to the environment and do not produce harmful substances during fermentation, production, processing and use, and have no toxic side effects.The bacillus cereus bacterial agent can promote the appearance color change of strawberry fruits, can effectively prevent and control strawberry leaf spot and receptacle calyx wilt, strawberry anthracnose and strawberry wilt and other fungal diseases caused by new pestalosphaeria, colletotrichum and fusarium oxysporum, reduce the safety hidden danger of agricultural products while having good biological control effect.

Description

Technical Field

[0001] This invention relates to a biocontrol strain, a biocontrol agent, and their applications, particularly to a Bacillus cereus strain that promotes changes in the appearance and color of strawberry fruits and its applications. Background Technology

[0002] strawberry( Fragaria × ananassa Strawberries (Duch.) are perennial herbaceous plants belonging to the Rosaceae family. Strawberries are beloved for their unique flavor and rich nutritional value. In recent years, the strawberry industry has developed rapidly, with 200-300 new strawberry varieties developed and introduced from abroad. Hebei, Shandong, Liaoning, and Jiangsu provinces have become major strawberry cultivation areas in my country. With the promotion and application of greenhouse cultivation technology, my country has become a major strawberry producer and exporter in the world. However, strawberries are easily infected by fungal diseases during greenhouse cultivation, leading to reduced yields, a problem that limits the development of the strawberry industry. Furthermore, the appearance quality of strawberry fruits is also very important during cultivation, directly affecting their economic value.

[0003] In northern my country, strawberries are typically cultivated in greenhouses. It takes about 25-30 days for strawberry fruit to transition from green to ripe red. Currently, most greenhouse-grown strawberries reach maturity in mid-to-late November before being harvested and sold in the market. Accelerating the ripening and market entry of strawberries is crucial for maximizing economic value. Strawberry ripening generally occurs in four stages: green ripening, white ripening, color changing, and red ripening. Each stage involves the consumption of substances and the accumulation of new substances. Accelerating the ripening process requires starting from the green fruit stage to increase the degradation rate of relevant substances in the fruit, speeding up the accumulation of beneficial substances, and shortening the stage of color change in the strawberry fruit from the outset.

[0004] The color of strawberries at different stages is mainly determined by the content of substances such as chlorophyll, proanthocyanidins, anthocyanins, and quinacrine. During the green fruit stage, the fruit contains more chlorophyll, so the skin is mostly green. As the fruit gradually ripens, chlorophyll breaks down, and the proanthocyanidin content increases, causing the strawberry surface to turn from green to white, reaching the white-ripe stage. Afterward, the fruit enters the color-changing stage, where the color is mainly determined by the content of anthocyanins and quinacrine. The large-scale synthesis and accumulation of anthocyanins and quinacrine causes the fruit to gradually turn from white to red. During the red-ripe stage, the anthocyanin content gradually increases, and the fruit eventually turns red.

[0005] Strawberry leaf spot is a disease caused by *Neoplasmosis* (…). Neopestalotiopsis clavisporaThis is a new disease caused by *Neoplasmosis polychaete*. In addition, *Neoplasmosis polychaete* can also infect strawberry leaves, receptacles, and calyxes, severely impacting strawberry yield. Strawberry leaves infected with *Neoplasmosis polychaete* develop brown spots, with the leaves around the spots turning pale green and developing a yellow halo. The center of the spots dries out, turning from brown to grayish-white, and then small black dots appear on the spots—the conidiophores of the pathogen. The receptacles and calyxes wilt and turn black, subsequently producing white mycelium in the affected areas. Small black dots appear inside the white mycelium—the conidiophores of the pathogen. The strawberry roots also turn brown; when the strawberry root system is cut, the interior is entirely brown. This disease mainly occurs during the flowering and fruiting period and the vigorous growth period of strawberries. Currently, the control of strawberry diseases caused by *Neoplasmosis polychaete* is mainly based on chemical control, using agents such as mancozeb, difenoconazole, metalaxyl, carbendazim, chlorothalonil, and pyraclostrobin.

[0006] Strawberry anthracnose is a disease caused by Colletotrichum Strawberry spp. is a major disease affecting strawberry production. It can infect various organs of the strawberry plant, including roots, petioles, leaves, flowers, fruits, and runners, causing a range of symptoms such as root rot, leaf spot, fruit rot, and decreased reproductive capacity. Current research indicates that... Colletotrichum There are three main species: C. fragariae , C. acutatum and C. gloeosporioides Furthermore, this fungus overwinters in the soil as mycelium and sclerotia. The following year, the mycelium produces conidia that can directly infect strawberries. This overwintering form of the disease is detrimental to field control of strawberry anthracnose, posing a serious threat to strawberry yield and quality. In greenhouse cultivation, chemical agents are mainly used to control strawberry diseases. Currently, widely used chemical agents include prochloraz, fluazinam, mancozeb, and difenoconazole.

[0007] Strawberry wilt is a disease caused by Fusarium oxysporum strawberry-specific strain (Fusarium oxysporum). Fusarium oxysporium f.sp. fragariae Fusarium wilt is a soil-borne disease caused by chloropicrin. It is very common in strawberry-growing areas both domestically and internationally, and its severity in damaging strawberry crops is increasing year by year, seriously affecting both yield and quality. The pathogen infects the root system of strawberry plants, causing vascular blockage and releasing toxins within the plant. This severely hinders the transport of nutrients and normal physiological metabolism, and can occur throughout the entire growth cycle of strawberries. Currently, chemical control remains the primary method for controlling Fusarium wilt. Before planting, soil disinfection is carried out through irrigation or spraying with chemical agents. The most common chemical agents on the market include chloropicrin and methylphenidate, which are low-cost, fast-acting, and highly effective.

[0008] Currently, chemical agents are mainly used to control strawberry field diseases. However, long-term use of chemical agents leads to drug resistance in pathogens, damages the ecological environment, harms human health, and hinders the sustainable development of the strawberry industry. Therefore, there is an urgent need for a biocontrol agent that can control strawberry field diseases.

[0009] Biological control of plant diseases involves selecting beneficial microorganisms or their metabolites with biocontrol effects. These microorganisms compete with pathogens for nutrients and growth space, secrete antimicrobial substances, induce systemic resistance, and promote plant growth, thereby inhibiting the growth and reproduction of pathogens and achieving significant control over plant diseases. Bacillus is a dominant strain of bacteria found in soil or plant roots with strong antibacterial activity and resistance. It is environmentally friendly and has a strong, sustained effect, making it promising for widespread application in agricultural production. With increasing public concern about food safety and higher demands for food quality, there is an urgent need for safe and healthy biocontrol agents to control field diseases in strawberries and improve the appearance and quality of strawberry fruits.

[0010] Currently, there are some problems regarding the application of Bacillus inoculants in strawberries. These include issues with fermentation methods and field application techniques. Therefore, how to prepare Bacillus inoculants and, by adjusting field application methods, obtain biocontrol agents with good control effects and good stability against strawberry leaf spot and receptacle wilt caused by *Pseudomonas aeruginosa*, strawberry anthracnose caused by *Anthracnose sianna*, and strawberry wilt caused by *Fusarium oxysporum* is a pressing issue to be addressed in strawberry cultivation. Summary of the Invention

[0011] To overcome the differences in the existing technologies, the purpose of this invention is to propose a Bacillus cereus agent that promotes changes in the appearance and color of strawberry fruits and its application. The Bacillus cereus agent and its inoculant are non-toxic to plants, environmentally friendly, and produce no harmful substances or toxic side effects during fermentation, production, processing, and use. Furthermore, the fermentation method and application method of this inoculant improve the control effect of Bacillus cereus on the aforementioned strawberry field diseases and significantly promote changes in the appearance and color of strawberry fruits.

[0012] This invention proposes a Bacillus cereus strain for improving the appearance and color of strawberry fruits. The Bacillus cereus strain is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; accession number CGMCC No. 27829; deposit date July 7, 2023; and classified as Bacillus cereus (…). Bacillus cereus ).

[0013] The culture characteristics of the *Bacillus cereus* were as follows: on LB agar plates, the colonies were milky white with a rough, wrinkled surface; under a microscope, they were rod-shaped; and they were Gram-positive. 16S Constructing a phylogenetic tree of Bacillus cereus using rDNA sequences, see Figure 1 and Figure 2 .

[0014] This invention proposes a method for preparing a biocontrol agent of Bacillus cereus to improve the appearance and color of strawberry fruits. The key technical points include the following steps: (1) Activated Bacillus cereus Bacillus cereus strains were activated and cultured on LB medium at a temperature of 25-30℃ for 3 days. The LB medium formulation was as follows: 10 g / L tryptone, 5 g / L yeast extract, 5 g / L sodium chloride, 15 g / L agar powder, pH 7.2-7.4. (2) Preparation of biocontrol agent stock solution Preparation of stock solution: Activated Bacillus cereus was inoculated into LB medium and cultured at 25°C with shaking at 140 rpm for 12 hours to prepare a stock solution with a concentration of 10. 8 cfu / ml-10 9 The stock solution contains cfu / ml; the LB culture medium formula is: 10g / L tryptone, 5g / L yeast extract, 5g / L sodium chloride, pH=7.2-7.4; (3) Fermentation culture The above mother liquor was added to the fermentation medium for fermentation and culture. The fermentation medium formula is as follows: 5 g / L tryptone, 5 g / L soybean meal, 2.5 g / L beef extract, 2.5 g / L yeast extract, 5 g / L sucrose, pH 7.0-7.5; The inoculum size of the yeast broth was 0.6% of the volume of the liquid fermentation medium. The fermentation temperature was 25℃, the aeration rate was 3.0 L / min, the pH was 7.0-7.5, and the culture was carried out with shaking at 140 rpm for 2 days to obtain the biocontrol agent stock solution. The concentration of the biocontrol agent stock solution was 1.5-2.0 × 10⁻⁶. 10 cfu / ml.

[0015] (4) Preparation of biocontrol agents The original solution (1.5-2.0×10) 10 Dilute (cfu / ml) 30-50 times to obtain a biocontrol agent (0.3-0.6×10⁻⁶). 9 (cfu / ml).

[0016] This invention proposes a method for applying a biocontrol agent of Bacillus cereus. The key technical point is that the biocontrol agent is used to improve the appearance and color of strawberry fruits. The application method for improving the appearance and color of strawberry fruits involves spraying a 30-fold diluted Bacillus cereus biocontrol agent (0.5-0.6 × 10⁻⁶) at the green fruit stage, white fruit stage, and color-changing stage of the strawberry fruit, respectively. 9 Each fruit was sprayed with 3 ml of Bacillus cereus biocontrol agent (cfu / ml), only once. After that, the fruit was bagged to keep it moist. The fruit was removed after it changed color. The treatment time was 48-60 hours. During the entire treatment period, the strawberry fruit did not leave the strawberry plant. The strawberry plant was planted in a greenhouse (temperature 25℃, humidity 75%, normal light conditions).

[0017] The biocontrol agent of Bacillus cereus is used to reduce the decrease in chlorophyll content of strawberry fruits during the green fruit stage, increase the proanthocyanidin content, and promote the fruit to enter the white fruit stage earlier.

[0018] The biocontrol agent of Bacillus cereus is used to promote the growth of Cy3G (anthocyanin 3-O-glucoside) and Pg3G (anthocyanin 3-O-glucoside) in strawberry fruit during the white fruit stage. O Synthesis of β-glucan accelerates the fruit's transition to the color-changing stage.

[0019] The biocontrol agent of Bacillus cereus is used to increase the content of Cy3G (anthocyanin 3-) in strawberry fruits during the color-changing stage. O β-glucoside) and Pg3G (anthocyanin 3- O It increases the content of β-glucan and accelerates fruit ripening.

[0020] This invention also proposes a method for applying a biocontrol agent of Bacillus cereus, the key technical point of which is: the biocontrol agent of Bacillus cereus is used to control diseases including those caused by *Neoplasmosis* (…). Neopestalotiopsis clavispora Strawberry leaf spot and strawberry receptacle wilt caused by *Anthracnose* (Siamese anthracnose) Colletotrichum siamense Strawberry anthracnose is caused by Fusarium oxysporum (Fusarium oxysporum). Fusarium oxysporium f. sp. fragariae Strawberry wilt disease caused by )

[0021] The biocontrol agent of Bacillus cereus is used to control *Pseudomonas neoformans* (…). Neopestalotiopsis clavispora Strawberry leaf spot and strawberry receptacle and calyx wilt caused by *Anthracnose* (Siamese anthracnose) Colletotrichum siamense The method for controlling strawberry anthracnose caused by [unspecified substance] is as follows: At the initial stage of disease on strawberry plants, use foliar spraying to apply a 50-fold diluted biocontrol agent (0.3-0.4 × 10⁻⁶) per square meter. 9Apply 600-800 ml of cfu / ml solution every 4 days for a total of 5 applications. After each application, the greenhouse should be sealed for 20-24 hours, maintaining a temperature of 25℃-27℃.

[0022] The biocontrol agent of Bacillus cereus is used to control Fusarium oxysporum (…). Fusarium oxysporium f.sp. fragariae To control strawberry wilt caused by [unspecified disease], a root drenching method is used. Apply a 50-fold diluted biocontrol agent (0.3-0.4 × 10⁻⁶) to each strawberry seedling. 9 Apply 300-500 ml of cfu / ml solution every 10 days for a total of 6 applications.

[0023] The strawberry leaf spot and receptacle wilt caused by the fungus *Neoprothiolane* are new and highly damaging field diseases discovered in 2021. The biocontrol agent *Bacillus cereus* has a significant control effect on these diseases.

[0024] Compared with the prior art, the beneficial effects of this invention are: (1) The biocontrol strain Bacillus cereus is derived from the rhizosphere soil of strawberries. It is safe for the plant itself, the environment, and humans and animals. It does not produce harmful substances or have any toxic side effects during fermentation, production, processing and use.

[0025] (2) Spraying strawberry fruits with biocontrol strains after fermentation can promote the change in the appearance and color of strawberry fruits, which involves the synthesis and degradation of chlorophyll, proanthocyanidins, anthocyanins and anthocyanins.

[0026] (3) Regarding the prevention and control of strawberry leaf spot, anthracnose and wilt in the field, Bacillus cereus inoculants have good control effects, especially for the new disease of receptacle and calyx wilt caused by Neospora cereus, and have broad application prospects. Attached Figure Description

[0027] Figure 1 Morphological analysis of Bacillus cereus, where A shows the growth status of Bacillus cereus on LB agar plates; B shows the shape of Bacillus cereus under a scanning electron microscope. Figure 2 Molecular biological identification results of Bacillus cereus (phylogenetic tree analysis of Bacillus cereus); Figure 3Results of the experiment on the inhibition of the growth of *Plasmodium necrophorum* by *Bacillus cereus*: A shows the growth of *CK* *Plasmodium necrophorum* after 2 days of culture; B shows the growth of *CK* *Plasmodium necrophorum* after 5 days of culture; C shows the inhibition of *Plasmodium necrophorum* growth by *Bacillus cereus* after 2 days of culture; D shows the inhibition of *Plasmodium necrophorum* growth by *Bacillus cereus* after 5 days of culture. Figure 4 Results of Bacillus cereus control of strawberry diseases caused by *Neoproterozoa*: E represents the phenotypic symptoms of strawberry diseases caused by *Neoproterozoa* controlled by Bacillus cereus. a: CK: Symptoms of strawberry plants sprayed with water alone on days 0 and 5; b: BCFS: Symptoms of strawberry plants sprayed with Bacillus cereus biocontrol agent only on days 0 and 5; c: N. clavispora To illustrate the symptoms of strawberry plants treated with a suspension of *Pseudomonas cephalothorax* spores on days 0 and 5, d:BCFS+ N. clavispora Symptoms of strawberry plants inoculated with *Neoprothiolane spores* suspension 24 hours after spraying with *Bacillus cereus* biocontrol agent on days 0 and 5; F represents the field disease index analysis results of *Bacillus cereus* biocontrol agent controlling strawberry leaf spot disease caused by *Neoprothiolane spores*; G represents the field disease index analysis results of *Bacillus cereus* biocontrol agent controlling strawberry receptacle and calyx wilt disease caused by *Neoprothiolane spores*.

[0028] Figure 5 Results of the experiment on the inhibition of Siamese anthrax growth by Bacillus cereus: A is the CK-Siamese anthrax fungus cultured for 2 days, B is the CK-Siamese anthrax fungus cultured for 5 days, C is the inhibition of Siamese anthrax fungus growth by Bacillus cereus after 2 days of culture, and D is the inhibition of Siamese anthrax fungus growth by Bacillus cereus after 5 days of culture. Figure 6 Results of an experiment using Bacillus cereus to control strawberry anthracnose. E represents the incidence of strawberry anthracnose under Bacillus cereus control. a: CK: Symptoms of strawberry plants sprayed with water alone on days 0, 3, and 6; b: BCFS: Symptoms of strawberry plants sprayed with Bacillus cereus biocontrol agent only on days 0, 3, and 6; c: Symptoms of strawberry plants sprayed with only Bacillus cereus biocontrol agent only on days 0, 3, and 6. C. siamense Symptoms on strawberry plants after spraying with spore suspension on days 0, 3, and 6, d:BCFS+ C. siamense Inoculate strawberry plants 24 hours after spraying with Bacillus cereus biocontrol agent. C. siamense Symptoms of spore suspension on day 0, day 3, and day 6; F represents the results of field disease index analysis.

[0029] Figure 7Results of an indoor experiment on the control of Fusarium oxysporum by Bacillus cereus: A represents the control of Fusarium oxysporum fungi (CK) after 5 days of culture; B represents the inhibition of Fusarium oxysporum fungal growth by Bacillus cereus after 5 days of culture. Figure 8 Results of the Bacillus cereus control experiment on strawberry wilt: CF represents the disease incidence 5 days after Bacillus cereus control of strawberry wilt: C is the water control, D is the treatment with Bacillus cereus biocontrol agent alone, E is the treatment with Fusarium oxysporum spore suspension 24 hours after Bacillus cereus biocontrol agent spraying, and F is the treatment with Fusarium oxysporum spore suspension alone; G is the field disease index analysis, where CK is the treatment with water alone; BCFS is the treatment with Bacillus cereus biocontrol agent alone. Fusarium oxysporium For spraying Fusarium oxysporum spore suspension alone; BCFS+ Fusarium oxysporium Inoculate strawberry plants with a suspension of Fusarium oxysporum spores 24 hours after spraying them with a Bacillus cereus biocontrol agent. Figure 9 The effects of Bacillus cereus biocontrol agent on improving strawberry fruit color at different stages: CK-0h represents the color of strawberry fruit treated with water at 0h; CK-60h represents the color of strawberry fruit treated with water at 60h; BCFS-0h represents the color of strawberry fruit treated with Bacillus cereus biocontrol agent at 0h; and BCFS-60h represents the color of strawberry fruit treated with Bacillus cereus biocontrol agent at 60h.

[0030] Figure 10 The effect of Bacillus cereus biocontrol agent on improving the content of related substances in strawberry fruits at different stages: where A represents the chlorophyll content of strawberry fruits at different stages; B represents the proanthocyanidin content of strawberry fruits at different stages; and C represents the Cy3g (anthocyanin 3-) content of strawberry fruits at different stages. O β-glucoside content; D represents the content of Pg3G (anthocyanin 3-glucoside) in strawberry fruit at different stages .... O β-glucoside content. Detailed Implementation Example 1

[0031] (1) Activation of biocontrol bacteria The biocontrol strain is *Bacillus cereus*, which is deposited at the China Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 27829, deposited on July 7, 2023, and classified as *Bacillus cereus*. Bacillus cereus ).

[0032] In this embodiment, Bacillus cereus was labeled as Bce-2-102, which was isolated from strawberry rhizosphere soil. On LB agar plates, Bce-2-102 colonies were milky white with a rough, wrinkled surface. Under a microscope, they were rod-shaped and Gram-positive. Based on morphological observation and 16S rDNA sequencing, Bacillus cereus... B. cereus (BD18-S14) 100% similar, named Bacillus cereus ( Bacillus cereus )(See Figure 1-2 ).

[0033] The cells were activated and cultured in LB medium at 25°C for 3 days. The LB medium formulation was as follows: 10 g / L tryptone, 5 g / L yeast extract, 5 g / L sodium chloride, 15 g / L agar powder, pH 7.2-7.4. (2) Preparation of biocontrol agent stock solution Activated Bacillus cereus Bce-2-102 was picked and inoculated into a 100ml Erlenmeyer flask containing 50ml of LB liquid culture medium. The flask was incubated at 25℃ with shaking at 140rpm for 12 hours to prepare a Bacillus cereus culture medium (10...). 8 cfu / ml-10 9 The concentration of cfu / ml is the mother liquor.

[0034] (3) Fermentation culture The fermentation medium formula for the biocontrol agent is as follows: tryptone 5 g / L, soybean meal 5 g / L, beef extract 2.5 g / L, yeast extract 2.5 g / L, sucrose 5 g / L, pH 7.0-7.5; the inoculum size of the yeast broth is 0.6% of the liquid fermentation medium volume, fermented at 25℃, pH=7.0-7.5, aeration rate of 3 L / min, and 140 rpm for 2 days to obtain the biocontrol agent stock solution (1.5-2.0 × 10⁻⁶). 10 (cfu / ml).

[0035] (4) Preparation of biocontrol agents Dilute the stock solution of the biocontrol agent 30-50 times to obtain a biocontrol agent (0.3-0.6×10⁻⁶). 9 (cfu / ml).

[0036] Example 2: Indoor plate test of Bacillus cereus inhibiting Polychaete cereus; (1) Test materials Bacillus cereus culture medium, Pseudomonas neoplasm (2) Experimental treatment Set up a control group and a treatment group, with 3 replicates in each group.

[0037] The control group was cultured with *Neoplasmosis polychaete* alone, while the treatment group was treated with *Bacillus cereus* to inhibit fungal growth. The concentration of *Bacillus cereus* culture medium was 10. 8 cfu / ml-10 9 cfu / ml; (3) Test results See results Figure 3 Bacillus cereus inhibited the growth of Polychaete nephrons by 79.48%, and compared with the control group, the growth of Polychaete nephrons in the treatment group was significantly inhibited.

[0038] Example 3: Evaluation of the effectiveness of field control of strawberry leaf spot and receptacle wilt caused by *Pseudomonas aeruginosa* (1) Test materials 'Yanli' strawberry plants, biocontrol agents: *Pseudomonas aeruginosa* and *Bacillus cereus*. (2) Experimental treatment Design four different treatments, with 10 replicates for each treatment.

[0039] Treatment 1: Spray only with water as a blank control; Treatment 2: Spray with Bacillus cereus biocontrol agent (0.4×10⁻⁶). 9 (cfu / ml) Treatment 3: Spray with a suspension of *Neoproterozoa* spores at a concentration of 1×10⁻⁶. 6 spores / ml; Treatment 4: First spray with Bacillus cereus biocontrol agent (0.4×10⁻⁶). 9 (cfu / ml), and the spraying concentration after 24 hours is 1×10 6 spores / ml of *Pseudomonas aeruginosa* spore suspension; Each treatment consisted of 10 strawberry plants. The disease incidence was investigated 7 days after inoculation, and the disease index was calculated according to the following grading criteria.

[0040] (3) Disease grading standards are classified into levels 0-3 as follows: 0 = No obvious disease symptoms; 1 = Mild infection, small spots are visible (diameter ≤ 3 mm); 2 = Moderate infection, with medium-sized spots visible (3-5 mm in diameter); 3 = Severe infection, with large spots visible (diameter > 5 mm).

[0041] (4) Test results See results Figure 4The disease index of treatment 4 was significantly lower than that of treatment 3. The Bce-2-102 biocontrol agent achieved a control efficacy of 78.56% against strawberry leaf spot caused by *Neoprothiolane neoplasm*, and 64.59% against strawberry receptacle and calyx wilt caused by the same fungus. Bce-2-102 biocontrol agent also showed good control effects against strawberry leaf spot and receptacle and calyx wilt caused by *Neoprothiolane neoplasm*.

[0042] Example 4: Indoor plate test of Bacillus cereus inhibiting anthrax bacteria from Siam; (1) Test materials Bacillus cereus culture medium, Anthrax bacillus sicca (2) Experimental treatment Set up a control group and a treatment group, with 3 replicates in each group.

[0043] The control group was cultured with *Anthrax sicca* alone, while the treatment group was treated with *Bacillus cereus* to inhibit fungal growth. The concentration of *Bacillus cereus* culture medium was 10. 8 cfu / ml-10 9 cfu / ml; (3) Test results See results Figure 5 Bacillus cereus inhibited the growth of Bacillus anthracis in the treatment group by 76.67%, and the growth of Bacillus anthracis in the treatment group was significantly inhibited compared with the control group.

[0044] Example 5: Evaluation of the effectiveness of field control of strawberry anthracnose caused by *Anthracnose sirenus* (1) Test materials 'Yanli' strawberry plants, anthracnose fungus, and Bacillus cereus biocontrol agents (2) Experimental treatment Design four different treatments, with 10 replicates for each treatment.

[0045] Treatment 1: Spray only with water as a blank control; Treatment 2: Spray with Bacillus cereus biocontrol agent (0.4×10⁻⁶). 9 (cfu / ml) Treatment 3: First spray with Bacillus cereus biocontrol agent (0.4×10⁻⁶). 9 (cfu / ml), and the spraying concentration after 24 hours is 1×10 6 Siamese anthrax spore suspension (spores / ml); Treatment 4: Spray with a suspension of Siamese anthrax spores at a concentration of 1×10⁻⁶. 6 spores / ml; Each treatment consisted of 10 strawberry plants. The disease incidence was investigated 7 days after inoculation, and the disease index was calculated according to the following grading criteria.

[0046] (3) Disease grading standards are classified into levels 0-3 as follows: 0 = No obvious disease symptoms; 1 = Mild infection, small spots are visible (diameter ≤ 3 mm); 2 = Moderate infection, with medium-sized spots visible (3-5 mm in diameter); 3 = Severe infection, with large spots visible (diameter > 5 mm).

[0047] (4) Test results See results Figure 6 The disease index of treatment 3 was significantly lower than that of treatment 4, and the efficacy of Bce-2-102 biocontrol agent reached 69.86%. Bce-2-102 biocontrol agent of Bacillus cereus has a good control effect on strawberry anthracnose caused by Anthracnose sicca.

[0048] Example 6: Indoor plate test of Bacillus cereus inhibiting Fusarium oxysporum; (1) Test materials Bacillus cereus culture medium, Fusarium oxysporum (2) Experimental treatment Set up a control group and a treatment group, with 3 replicates in each group.

[0049] The control group was cultured with Fusarium oxysporum alone, while the treatment group was treated with Bacillus cereus to inhibit fungal growth. The concentration of Bacillus cereus culture medium was 10. 8 cfu / ml-10 9 cfu / ml; (3) Test results See results Figure 7 Bacillus cereus inhibited the growth of Fusarium oxysporum by 80.21% in the treated group, and the growth of Fusarium oxysporum was significantly inhibited compared with the control group.

[0050] Example 7: Evaluation of the effectiveness of field control of strawberry wilt caused by Fusarium oxysporum (1) Test materials 'Yanli' strawberry plants, Fusarium oxysporum fungi, Bacillus cereus biocontrol agents; (2) Experimental treatment Design four different treatments, with 10 replicates for each treatment.

[0051] Treatment 1: Spray only with water as a blank control; Treatment 2: Spray with Bacillus cereus biocontrol agent (0.4×10⁻⁶). 9(cfu / ml) Treatment 3: First spray with Bacillus cereus biocontrol agent (0.4×10⁻⁶). 9 (cfu / ml), and the spraying concentration after 24 hours is 1×10 6 spores / ml of Fusarium oxysporum spore suspension; Treatment 4: Spray with a suspension of Fusarium oxysporum spores at a concentration of 1×10⁻⁶. 6 spores / ml; Each treatment consisted of 10 strawberry plants. The disease incidence was investigated 7 days after inoculation, and the disease index was calculated according to the following grading criteria.

[0052] (3) Disease grading standards are classified into levels 0-3 as follows: 0 = No obvious disease symptoms; 1 = Mild infection, small spots are visible (diameter ≤ 3 mm); 2 = Moderate infection, with medium-sized spots visible (3-5 mm in diameter); 3 = Severe infection, with large spots visible (diameter > 5 mm).

[0053] (4) Test results See results Figure 8 The disease index of treatment 3 was significantly lower than that of treatment 4, and the efficacy of Bce-2-102 biocontrol agent reached 64.59%. Bce-2-102 biocontrol agent of Bacillus cereus has a good control effect on strawberry wilt caused by Fusarium oxysporum.

[0054] Example 8: Evaluation of the effect of Bacillus cereus biocontrol agent on promoting changes in the appearance and color of strawberry fruit. (1) Test materials 'Yanli' strawberry fruits at the green, white, and color-changing stages; Bacillus cereus biocontrol agent; self-sealing bags; 'Yanli' strawberry seedlings were purchased from the market.

[0055] (2) Experimental treatment Treatment 1: Strawberry fruits at different stages were sprayed with water separately and then sealed in self-sealing bags to retain moisture for 60 hours as a blank control. Treatment 2: Spray strawberry fruits with Bacillus cereus biocontrol agent at all stages (diluted 30 times, 0.6×10⁻⁶). 9 (cfu / ml), put it in a resealable bag to keep it moist for 60 hours, then remove the resealable bag; (3) Test results The test results are shown in Figure 9 and Figure 10 After spraying strawberry fruits with Bacillus cereus inoculant during the green fruit stage, the chlorophyll content in the fruits decreased, while the proanthocyanidin content increased. Cy3G (anthocyanin 3-), a substance associated with fruit color changes, was also observed.O β-glucoside) and Pg3G (anthocyanin 3- O β-glucoside (Pg3G) was not detected in the fruit, and the fruit entered the white fruit stage prematurely; After spraying strawberry fruits with Bacillus cereus inoculant during the white fruit stage, the chlorophyll and proanthocyanidin contents decreased, and the Cy3G (anthocyanin 3-) content in the fruit decreased. O β-glucoside) and Pg3G (anthocyanin 3- O β-glucosides begin to be synthesized, accelerating the fruit's transition to the color-changing stage; After spraying strawberry fruits with Bacillus cereus inoculant during the ripening stage, the Cy3G (anthocyanin 3-) content in the fruit increased. O β-glucoside) and Pg3G (anthocyanin 3- O The levels of β-glucan were significantly increased, resulting in widespread reddening of the fruit and accelerated ripening. These results indicate that the Bacillus cereus biocontrol agent is beneficial for improving the appearance and color of strawberry fruits and has broad application prospects.

Claims

1. A Bacillus cereus that promotes changes in the appearance and color of strawberry fruit ( Bacillus cereus Bce-2-102, characterized in that, The Bacillus cereus Bce-2-102 is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 27829.

2. A method for preparing a Bacillus cereus biocontrol agent Bce-2-102 that promotes color change in strawberry fruit as described in claim 1, characterized in that: The preparation method of the Bacillus cereus Bce-2-102 biocontrol agent includes the following steps: (1) Activated Bacillus cereus Bce-2-102 Bacillus cereus Bce-2-102 was activated and cultured on LB medium at a temperature of 25-30℃ for 3 days. The LB medium formulation was as follows: 10 g / L tryptone, 5 g / L yeast extract, 5 g / L sodium chloride, 15 g / L agar powder, pH=7.2-7.

4. (2) Preparation of Bacillus cereus Bce-2-102 bacterial inoculum stock solution Preparation of stock solution: Activated Bacillus cereus Bce-2-102 was inoculated into LB medium and cultured at 25°C with shaking at 140 rpm for 12 hours to prepare a stock solution with a concentration of 10. 8 cfu / mL-10 9 The stock solution was prepared at cfu / mL; the LB culture medium was formulated as follows: 10 g / L tryptone, 5 g / L yeast extract, 5 g / L sodium chloride, pH=7.2-7.

4. (3) Preparation of Bacillus cereus Bce-2-102 inoculum stock solution The above mother liquor was added to the fermentation medium for fermentation and culture. The fermentation medium formulation was as follows: 5 g / L tryptone, 5 g / L soybean meal, 2.5 g / L beef extract, 2.5 g / L yeast extract, 5 g / L sucrose, pH 7.0-7.5; the fermentation temperature was 25℃, the aeration rate was 3.0 L / min, pH=7.0-7.5, and the culture was carried out with shaking at 140 rpm for 2 days to obtain the biocontrol agent stock solution. The concentration of the Bacillus cereus Bce-2-102 agent stock solution was 1.5 × 10⁻⁶. 10 cfu / mL -2.0×10 10 cfu / mL; (4) Preparation of Bacillus cereus Bce-2-102 biocontrol agent The Bacillus cereus Bce-2-102 bacterial agent stock solution was diluted 30-50 times to obtain Bacillus cereus Bce-2-102 biocontrol agent.

3. The application of the biocontrol agent prepared by the method for preparing Bacillus cereus Bce-2-102 biocontrol agent that promotes color change in strawberry fruit according to claim 2, characterized in that: The Bacillus cereus Bce-2-102 biocontrol agent is used to promote the color change of strawberry fruit. The application method is as follows: Apply Bacillus cereus Bce-2-102 biocontrol agent evenly to strawberry fruits during the green fruit stage, white fruit stage, and color-changing stage, with a bacterial count of 0.5 × 10⁻⁶. 9 cfu / mL -0.6×10 9 CFU / mL, spray each fruit with 3mL of Bacillus cereus Bce-2-102 biocontrol agent, spray once, and then cover the fruit with bags to keep it moist for 48h-60h. Remove the fruit after it changes color. During the entire treatment period, the strawberry fruit did not leave the strawberry plant. The strawberry plants were grown in a greenhouse with a temperature of 25℃, humidity of 75%, and normal light conditions. The Bacillus cereus Bce-2-102 biocontrol agent is used to reduce the chlorophyll content of strawberry fruits during the green fruit stage, increase the proanthocyanidin content, and promote the fruit to enter the white fruit stage earlier. The Bacillus cereus Bce-2-102 biocontrol agent is used to promote the production of anthocyanins 3- in strawberry fruit during the white fruit stage. O -Glucoside Cy3G and Anthocyanin 3- O β-glucoside Pg3G synthesis accelerates the fruit's entry into the color-changing stage; The Bacillus cereus Bce-2-102 biocontrol agent is used to increase anthocyanin 3- in strawberry fruits during the color-changing stage. O -Glucoside Cy3G and Anthocyanin 3- O -Increases the content of glucosinolate Pg3G, accelerating fruit ripening.

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