A kind of Bacillus saffron GX-H6 strain and its application

Abstract

The present invention discloses a Bacillus safensis GX-H6 strain and its application. The Bacillus safensis GX-H6 strain was deposited in the General Microbiology Center of the China National Committee for the Preservation of Microorganisms on June 16, 2022, with a deposit number of CGMCC No. 25106. The Bacillus safensis GX-H6 of the present invention has a very good antagonistic effect on the pathogens of rice bacterial leaf streak, rice bacterial leaf blight, citrus canker and various plant pathogenic fungi. The strain and its fermentation liquid can be used to prepare biocontrol agents, are easy to industrialize, have low production costs, have good market prospects, and also have important industrial application value.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial application, and in particular to a Bacillus saffron GX-H6 strain and an application thereof. Background Art

[0002] Bacterial leaf streak of rice is a major bacterial disease of rice, widely distributed in Asia and has gradually become the fourth most common rice disease in my country. Currently, all commonly cultivated rice varieties in my country, as well as varieties resistant to bacterial leaf blight, are susceptible to bacterial leaf streak of rice. Infected rice typically suffers a yield reduction of 5% to 10% in mild cases, while severe cases can result in yield reductions of over 20%. First discovered in Oka Prefecture, Japan in 1884, bacterial leaf blight of rice is a major bacterial disease of rice and currently occurs in rice-growing areas across Asia, Africa, the Americas, and Australia. The disease is most severe in Asia, particularly in South and Southeast Asia, where rice is grown year-round. It also occurs in most areas of my country. The disease can reduce rice yields by 30% to 30%, with severe cases resulting in yield reductions of over 50%.

[0003] The pathogen of rice bacterial leaf streak is Xanthomonas oryzae pv. oryzicola (Xoc), and the pathogen of rice bacterial leaf blight is Xanthomonas oryzae pv. oryzae (Xoo). These two pathogens are different pathogenic variants of the Xanthomonas species. Currently, chemical agents containing organocopper compounds are primarily used to control rice bacterial leaf streak in production. However, the extensive use of chemical agents can lead to the development of drug resistance in pathogens, while residual pesticides can also damage the ecological environment and even threaten food safety. Therefore, microbial-based biocontrol technologies—the use of beneficial microorganisms to kill or reduce the number of pathogens to control plant diseases—are gaining attention and are considered one of the key development directions for future crop bacterial disease control. Consequently, the use of biocontrol agents to control rice bacterial leaf streak and rice bacterial leaf blight has attracted considerable attention.

[0004] Citrus, a widely cultivated cash crop in southern my country, is susceptible to citrus canker, caused by the pathogen Xanthomonas citri subsp. citri. Affected plants experience leaf and fruit drop, weakening, and stunted growth. In severe cases, all leaves fall out, and the entire plant dies, causing significant losses to production. Currently, copper-containing agents or antibiotics are the primary treatments for this disease. However, excessive use of these agents can lead to a range of environmental and health concerns. Therefore, the design and development of new, environmentally friendly methods for controlling plant diseases is of great importance.

[0005] Fungi belong to the kingdom Fungi, phylum Fungi. There are more than 8,000 pathogenic fungi that attack plants, and they are the most important type of pathogens in plant diseases. Diseases caused by plant pathogenic fungi account for about 70-80% of plant diseases. For example: Neoscytalidium novaehollandiae, the pathogen that causes pitaya canker, harms the stems and fruits of pitaya, causing them to discolor and rot; Botrytis cinere, the pathogen that causes cantaloupe gray mold, mainly affects the fruits, causing them to become water-soaked and soft and rotten; Fusarium oxysporum f.sp.cubense (Foc) race 4, the pathogen that causes banana wilt, is a devastating disease that can cause severe diseased plants to die, and severely diseased fields may even have no harvest; Colletotrichum spp., the pathogen that causes mango anthracnose, is gloeosporioides), mango anthracnose mainly harms the tender leaves, tender shoots, inflorescences and young fruits of seedlings and mature plants, causing most of the leaves to wither and die. Black spots will form on the fruits after infection. If the fruit stalk is partially infected, the fruits will fall off quickly. Rice blast (Magnaporthe oryzae) is one of the important diseases of rice, which can cause a significant reduction in yield. In severe cases, the yield reduction can reach 40% to 50%, or even a complete loss of harvest. It occurs evenly in rice-growing areas around the world. This disease occurs in all regions, most of which occur in leaves and nodes, and can cause varying degrees of yield reduction after occurrence. In particular, neck blast or node blast occurs early and is severe, which can cause white ears and even complete crop failure. At present, the prevention and control measures for plant fungal diseases are still mainly chemical pesticides, which will bring a series of environmental and health problems. Therefore, it is of great significance to design and develop new methods for environmentally friendly plant disease prevention and control.

[0006] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art. Summary of the Invention

[0007] In view of this, the present invention provides a Bacillus safensis GX-H6 strain, which can be used to prevent and control rice bacterial leaf streak disease, rice bacterial leaf blight, citrus canker and plant diseases caused by various plant pathogenic fungi.

[0008] The purpose of the present invention is achieved through the following technical solutions:

[0009] The present invention discloses a Bacillus safensis GX-H6 strain, which is deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms with a deposit number of CGMCC No. 25106 and a deposit date of June 16, 2022.

[0010] The present invention provides a fermentation product of the Bacillus safensis GX-H6 strain.

[0011] The present invention provides a biocontrol agent comprising the Bacillus safensis GX-H6 strain and / or a fermentation product thereof.

[0012] The present invention provides a method for preparing a biocontrol agent comprising the Bacillus safensis GX-H6 strain and / or a fermentation product thereof, comprising the following steps:

[0013] (1) Seed liquid culture: inoculating the Bacillus safensis GX-H6 strain into a liquid culture medium and fermenting to obtain a seed liquid;

[0014] (2) Expanding the culture: inoculating 1% of the seed solution by volume into a liquid culture medium, and fermenting to obtain a fermentation liquid;

[0015] (3) preparing the biocontrol agent: adding a surfactant to the fermentation broth and mixing uniformly to obtain the biocontrol agent;

[0016] Wherein, the fermentation culture conditions of step (1) and step (2) are: 26-30°C, rotation speed 200 rpm, and culture for 18-22 hours;

[0017] Wherein, the liquid culture medium described in step (1) and step (2) is NB liquid culture medium; the surfactant described in step (3) is Tween 80, and the amount of the surfactant added is 1% by volume of the fermentation liquid.

[0018] The present invention also provides the use of the Bacillus safensis GX-H6 strain or its fermentation product or the above-mentioned biocontrol agent in preventing and controlling rice bacterial leaf streak disease, rice bacterial leaf blight, citrus canker or plant diseases caused by plant pathogenic fungi; the plant diseases are pitaya canker, melon gray mold, banana wilt, mango anthracnose or rice blast.

[0019] The present invention also provides the use of the Bacillus safensis GX-H6 strain or its fermentation product in the preparation of a biocontrol agent for preventing and treating rice bacterial leaf streak disease, rice bacterial leaf blight or plant diseases caused by plant pathogenic fungi; the plant diseases are pitaya canker, melon gray mold, banana wilt, mango anthracnose or rice blast.

[0020] The present invention also provides a method for using the above-mentioned biocontrol agent in preventing rice bacterial leaf streak disease and / or rice bacterial leaf blight, comprising the following steps: spraying the biocontrol agent on rice leaves until all the leaves are soaked; or, using a needle-free syringe to pressure-infiltrate the biocontrol agent into the rice leaves.

[0021] The present invention also provides a method for using the above-mentioned biocontrol agent in treating rice bacterial leaf streak disease and / or rice bacterial leaf blight, comprising the following steps: spraying the biocontrol agent on rice leaves until all the leaves are soaked; or, injecting the biocontrol agent into rice leaves at the early stage of the disease using a syringe; or, soaking the rice leaves at the early stage of the disease in the biocontrol agent for 20 to 30 seconds.

[0022] The present invention also provides the use of the biocontrol agent in treating citrus canker, comprising the following steps: diluting the biocontrol agent by 25 to 80 times and spraying the diseased plants.

[0023] Compared with the prior art, the present invention has the following beneficial technical effects:

[0024] (1) The present invention is the first to isolate and screen from nature a new strain of Bacillus safensis GX-H6 that can be used to prevent and control rice bacterial leaf streak disease, rice white leaf blight, citrus canker and plant diseases caused by various plant pathogenic fungi, further broadening the use of microorganisms to prevent and control rice bacterial leaf streak disease, rice white leaf blight, citrus canker and plant pathogenic fungi.

[0025] (2) The Bacillus safensis GX-H6 of the present invention has a very good antagonistic effect on the pathogens of rice bacterial leaf streak, rice white leaf blight, citrus canker and various plant pathogenic fungi. The strain and its fermentation liquid can be used to prepare biocontrol agents, are easy to industrialize, have low production costs, have good market prospects, and also have important industrial application value.

[0026] Preservation Information

[0027] Bacillus safensis GX-H6, the deposit number is CGMCC No. 25106, the deposit date is June 16, 2022, the depository is the General Microbiology Center of the China Culture Collection Administration, and the deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate exemplary embodiments, features, and aspects of the invention and, together with the description, serve to explain the principles of the invention.

[0029] Figure 1 Phylogenetic tree analysis of the 16S rDNA sequence of Bacillus safensis GX-H6;

[0030] Figure 2 The colony morphology of Bacillus safensis GX-H6 is shown in Figure 2.

[0031] Figure 3 The plate antagonistic results of Bacillus safensis GX-H6 against the pathogen of rice bacterial leaf streak;

[0032] Figure 4 The plate antagonistic results of Bacillus safensis GX-H6 against the pathogen of rice bacterial blight;

[0033] Figure 5 The results of the prevention and treatment of rice bacterial leaf streak by Bacillus safensis GX-H6 on the 7th day;

[0034] Figure 6 The results of the 14th day of the prevention and treatment of rice bacterial leaf streak by Bacillus safensis GX-H6;

[0035] Figure 7 The results of the prevention and treatment of rice bacterial blight by Bacillus safensis GX-H6 on the 7th day;

[0036] Figure 8 The results of the 14th day of the prevention and treatment of rice bacterial blight by Bacillus safensis GX-H6;

[0037] Figure 9The plate antagonism results of Bacillus safensis GX-H6 against the pathogen of citrus canker;

[0038] Figure 10 These are the results of the antagonistic spectrum test of Bacillus safensis GX-H6 against plant pathogenic fungi. DETAILED DESCRIPTION

[0039] The present invention will be further described in detail below with reference to specific embodiments, which are intended to explain the present invention rather than to limit it.

[0040] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources. Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0041] The formula of the NB liquid culture medium used in the examples of the present invention is: polypeptone 5 g / L, beef extract 3 g / L, yeast powder 1 g / L, sucrose 10 g / L, pH 7.0; sterilization conditions: 121°C, 100 kPa, 20 min.

[0042] The formula of the NA medium used in the examples of the present invention is: 1% agar is added to the NB liquid medium.

[0043] Example 1: Isolation, screening and identification of Bacillus safensis GX-H6

[0044] 1. Separation and screening

[0045] 1.1 Sampling: On October 20, 2020, in Nanning, Guangxi Zhuang Autonomous Region, researcher Li Ruifang collected samples from the rhizosphere of a peanut field.

[0046] 1.2 Separation and screening

[0047] 1.2.1 Preparation of primary screening plates: Culture indicator bacteria Xoc (Xanthomolias oryzae pv.oryzicola, Xoc) and Xoo (Xanthomonas oryzae pv.oryzae, Xoo) for 20-24 hours until the logarithmic growth phase. Add each to melted NA medium, mix thoroughly, pour into a culture dish, and solidify to prepare the primary screening plates.

[0048] 1.2.2 Primary screening: Take 1 g of soil sample collected from the peanut rhizosphere, add 10 mL of physiological saline and shake thoroughly to form a soil suspension. After gradient dilution, take 100 μL and spread it on the above-mentioned primary screening plate. Incubate at 28°C for 2-3 days. Pick colonies with inhibition zones and purify them into pure cultures. Then, number and preserve them for subsequent experiments.

[0049] 1.2.3 Rescreening: Use the water agar double-layer diffusion method to rescreen. Inoculate the strains screened above into NB liquid medium, culture at 28°C for 24 hours, and adjust the OD600 to 1.0 with physiological saline. This is used as the test bacterial solution.

[0050] 10 mL of 1% (w / v) water agar medium was added to a sterile plate. After the water agar solidified, a sterile Oxford cup was placed. The indicator bacteria Xoc (Xanthomolias oryzae pv. oryzicola, Xoc) and Xoo (Xanthomonas oryzae pv. oryzae, Xoo) were added to the melted NA medium, mixed thoroughly, and poured into the plate. After the medium solidified, the Oxford cup was removed with tweezers. 40 μL of the test bacteria solution was added to the resulting wells. The wells were incubated at 28°C for 48-72 hours, and the diameters were measured. The experiment was repeated three times, and the average value was calculated. The strain with the strongest antibacterial ability was identified and designated GX-H6.

[0051] 2. Identification

[0052] 2.1 Biochemical characteristics of strain GX-H6 were tested and identified. The results are shown in Table 1:

[0053] Table 1 Biochemical characteristics test results

[0054]

[0055]

[0056] In Table 1, -: negative reaction; +: positive reaction; W+: weak positive reaction.

[0057] 2.2 Molecular biological identification of strain GX-H6

[0058] 2.2.1 Extraction of DNA from strain GX-H6

[0059] 2.2.2 PCR amplification was performed using the universal primers 27F 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO. 1) and 1492R 5'-TACGGCTACCTTGTTACGACTT-3' (SEQ ID NO. 2) for bacterial 16S rRNA and the degenerate primers F 5'-GAAGTCATCATGACCGTTCTGCAYGCNGGNGGNAARTTYGA-3' (SEQ ID NO. 3) and R 5'-AGCAGGGTACGGATGTGCGAGCCRTCNACRTCNGCRTCNGTCAT-3' (SEQ ID NO. 4) for the housekeeping gene gyrB, using total DNA from GX-H6 as a template.

[0060] In the above primer sequences, N = A / T / G / C, Y = C / T, and R = A / G;

[0061] PCR reaction conditions were: initial denaturation at 95°C for 5 minutes, followed by 30 cycles of denaturation at 95°C for 30 seconds, annealing at 48°C for 30 seconds, and extension at 72°C for 1 minute; and a final extension at 72°C for 10 minutes. PCR products were verified by 1.5% agarose gel electrophoresis. Once the correct bands were detected, the PCR products were purified and sequenced.

[0062] 2.2.3 The sequencing results are shown in SEQ ID NO.5. SEQ ID NO.5 was subjected to BLAST analysis on the NCBI website. GX-H6 had the highest similarity with Bacillus sabdariffa, which was 99.93%. The reference sequence downloaded from GenBank was used to construct a phylogenetic tree using the Neighbor-Joining method (NJ) of MEGA7.0, as shown in the following figure: Figure 1 As shown, the reliability of the constructed phylogenetic tree was estimated by bootstrap test, with 1000 repetitions. The sequencing result of the housekeeping gene gyrB is shown in SEQ ID NO.6. BLAST analysis was performed on the NCBI website, and the similarity between GX-H6 and Bacillus safensis was 97.14%. The strain was finally identified as Bacillus safensis and named Bacillus safensis GX-H6. Its colony morphology is shown in Figure 2 As shown, on June 16, 2022, the strain was sent to the General Microbiology Center of China Culture Collection Administration for Microorganisms for preservation, with the preservation number CGMCC No.25106.

[0063] Example 2 Antagonistic test of Bacillus safensis GX-H6 against pathogens of rice bacterial leaf streak and rice bacterial leaf blight on plate

[0064] 1. Preparation of fermentation broth:

[0065] 1.1 Prepare NB liquid culture medium;

[0066] 1.2 Seed liquid culture: The Bacillus safensis GX-H6 strain was inoculated into NB liquid culture medium and fermented to obtain seed liquid. The fermentation culture conditions were: 28° C., 200 rpm, and culture for 20 h.

[0067] 1.3 Expansion culture: 1% of the seed solution was inoculated into NB liquid culture medium by volume, and fermentation culture was performed to obtain fermentation broth. The fermentation culture conditions were: 28° C., 200 rpm, and culture for 20 h.

[0068] 2. Antagonistic test of Bacillus safensis GX-H6 against the pathogen of rice bacterial leaf streak on plate

[0069] 2.1 Test Method: The water agar double-layer diffusion method was used. First, 10 mL of 1% (w / v) water agar medium was added to a sterile plate. After the water agar medium solidified, a sterile Oxford cup was placed in the plate. The indicator bacteria Xoc (Xanthomolias oryzae pv. oryzicola, Xoc) was added to the melted NA medium, mixed thoroughly, and poured into the plate. After the NA medium solidified, the Oxford cup was removed with tweezers. 40 μL of the fermentation broth was added to the resulting wells. The wells were incubated at 28°C for 60 h, and the diameters were measured. The test was repeated three times, and the average value was calculated.

[0070] 2.2 Test results

[0071] like Figure 3 As shown, Bacillus safensis GX-H6 has a significant antagonistic effect on the pathogen of rice bacterial leaf streak, with an average inhibition zone diameter of 3.69 cm. The pathogen of rice bacterial leaf streak cannot grow around Bacillus safensis GX-H6.

[0072] 3. Antagonistic test of Bacillus safensis GX-H6 against the pathogen of rice bacterial blight on plate

[0073] 3.1 Test Method: The water agar double-layer diffusion method was used for testing. First, 10 mL of 1% (w / v) water agar medium was added to a sterile plate. After the water agar medium solidified, a sterile Oxford cup was placed in the plate. The indicator bacteria Xoo (Xanthomonas oryzae pv. oryzae, Xoo) was added to the melted NA medium, mixed thoroughly, and poured into the plate. After the NA medium solidified, the Oxford cup was removed with tweezers. 40 μL of the above-mentioned fermentation broth was added to the formed wells. The wells were incubated at 28°C for 60 h, and the diameters were measured. The test was repeated three times, and the average value was calculated.

[0074] 3.2 Test results

[0075] like Figure 4 As shown, Bacillus safensis GX-H6 has a significant antagonistic effect on the pathogen of rice bacterial blight, with an average inhibition zone diameter of 5.79 cm. The rice bacterial blight pathogen cannot grow around Bacillus safensis GX-H6.

[0076] Example 3 Prevention and Treatment of Rice Bacterial Leaf Streak with Bacillus safensis GX-H6

[0077] 1. Preparation of biocontrol agents

[0078] 1% volume of Tween 80 was added to the fermentation liquid prepared in Example 2, and the mixture was uniformly mixed to obtain a biocontrol agent having a viable bacterial count concentration of 10 8 ~10 9 CFU / mL.

[0079] 2. Prevention of Rice Bacterial Leaf Streak with Bacillus safensis GX-H6

[0080] 2.1 Test method

[0081] 4-7 week old Oryza sativa L. spp. japonica rice was used as the test material. The prepared biocontrol agent was infiltrated into the rice leaves with a needleless syringe. After 3 hours, the bacterial solution of pathogen Xanthomolias oryzae pv. oryzicola, Xoc (viable bacteria concentration of 10 8 ~10 9 CFU / mL) was infiltrated into the treated rice leaves at the same position, and a total of 20 leaves were treated. Rice leaves not treated with biocontrol agents were used as the control group, and the experiment was repeated three times.

[0082] Leaves were collected and observed on the 7th and 14th days respectively, and the length of water-soaked lesions was measured. The results were averaged and the control effect was calculated according to the formula: control effect = (lesion length of control group - lesion length of treatment group) / lesion length of control group × 100%.

[0083] 2.2 Test results

[0084] like Figure 5 and Figure 6 As shown, on the 7th and 14th days after treatment, the length of the lesions on the leaves of the prevention treatment was significantly shorter than that of the control group, and the prevention efficacy of the prevention group was 51.64% and 40.91%, respectively.

[0085] 3. Treatment of Rice Bacterial Leaf Streak with Bacillus safensis GX-H6

[0086] 3.1 Test method

[0087] 4-7 week old Nipponbare rice was used as the test material, and the bacterial solution of pathogen Xanthomolias oryzaepv.oryzicola, Xoc (live bacteria concentration of 10 8 ~10 9 CFU / mL) was infiltrated into rice leaves with a needle-free syringe. After 3 h, the prepared biocontrol agent was infiltrated into the position treated with the pathogen solution with a needle-free syringe. A total of 20 leaves were treated, and the results were averaged. The experiment was repeated three times.

[0088] Rice leaves that had not been treated with biocontrol agents were used as the control group. Leaves were collected on the 7th and 14th days to observe and measure the length of water-soaked lesions. The control effect was calculated according to the formula: control effect = (control lesion length - treatment lesion length) / control lesion length × 100%.

[0089] 3.2 Test results

[0090] like Figure 5 and Figure 6 As shown, on the 7th and 14th days after treatment, the length of the lesions on the leaves treated was significantly shorter than that of the control group, with the effects of the treatment group being 42.69% and 36.36% respectively.

[0091] Example 4 Prevention and Treatment of Rice Bacterial Blight with Bacillus safensis GX-H6

[0092] 1. Prevention of Rice Bacterial Blight with Bacillus safensis GX-H6

[0093] 1.1 Test method

[0094] 4-7 week old Nipponbare rice was used as the test material. Sterile scissors were dipped in the biocontrol agent prepared in Example 3 and cut the rice leaves 1-2 cm from the tip. After 3 h, the bacterial solution of pathogen Xanthomonas oryzae pv.oryzae, Xoo (live bacteria concentration of 10 8 ~10 9 CFU / mL) were inoculated into the wounds of the treated rice leaves, and a total of 20 leaves were treated. Rice leaves not treated with biocontrol agents were used as the control group, and the experiment was repeated three times.

[0095] Leaves were collected on the 7th and 14th days respectively to observe and measure the length of yellowing spots. The results were averaged and the control effect was calculated according to the formula: control effect = (spot length of control group - spot length of treatment group) / spot length of control group × 100%.

[0096] 1.2 Test results

[0097] like Figure 7 and Figure 8 As shown, on the 7th and 14th days after treatment, the length of the lesions on the leaves of the prevention treatment was significantly shorter than that of the control group, and the prevention efficacy of the prevention group was 87.18% and 56.63%, respectively.

[0098] 2. Treatment of Rice Bacterial Blight with Bacillus safensis GX-H6

[0099] 2.1 Test method

[0100] 4-7 week old Nipponbare rice was used as the test material, and sterile scissors were used to dip the bacterial solution of pathogen Xanthomonasoryzae pv.oryzae, Xoo (live bacteria concentration was 10 8 ~10 9 CFU / mL), the rice leaves were cut at 1-2 cm from the tip, and 3 hours later, the wounds of the rice leaves were soaked in the biocontrol agent prepared in Example 3 for 25 seconds. A total of 20 leaves were treated, and rice leaves not treated with the biocontrol agent were used as controls. The experiment was repeated three times.

[0101] Leaves were collected on the 7th and 14th days respectively to observe and measure the length of yellowing spots. The results were averaged and the control effect was calculated according to the formula: control effect = (spot length of control group - spot length of treatment group) / spot length of control group × 100%.

[0102] 2.2 Test results

[0103] like Figure 7 and Figure 8As shown, on the 7th and 14th days after treatment, the length of the lesions on the leaves caused by the treatment was significantly shorter than that of the control group, and the effects of the treatment group were 82.05% and 50.81%, respectively.

[0104] Example 5 Preliminary Experiment on the Control Effect of Bacillus safensis GX-H6 on Rice Bacterial Leaf Streak

[0105] 1. Preparation of biocontrol agent: Bacillus safensis GX-H strain was inoculated into NB liquid medium, cultured at 28°C to the logarithmic phase, adjusted to OD600 of 1.0, and transferred to 1 L of NB liquid medium at a 10% inoculum for expansion culture. After shaking culture at 28°C and 200 rpm for 36-48 hours, 1% Tween was added and set aside.

[0106] 2. Field trials

[0107] 2.1 Trial Site and Rice Varieties: The trial site was located in the late rice fields of Shangling Village, Shibu Town, Nanning City, Guangxi Province. Rice has been cultivated in this area for many years, and bacterial leaf streak has been a significant problem over the years. Rice was grown in and around the trial site, and the rice variety tested was "Changliang Youxiang Jiujiu."

[0108] 2.2 Test method:

[0109] The field experiment was carried out from the tillering stage to the booting stage of rice. Five treatments were set up in the experiment: water control (CK), biocontrol agent 500ml / mu, biocontrol agent 750ml / mu, biocontrol agent 1500ml / mu, and 20% thiophanate-methyl suspension concentrate 125ml / mu. Each treatment was replicated three times, and each plot was 15m2. 2 , use 60kg of water per mu and spray the leaves evenly.

[0110] The pesticide was applied three times. The first application was on September 17, 2021, at the end of rice tillering; the second application was on September 24, 2021, at the rice heading stage; the third application was on October 1, 2021, at the end of rice heading stage.

[0111] 3. Test results

[0112] When the pesticide was applied for the first time, sporadic bacterial leaf streak occurred in the field, which was in the early stage of the disease. Therefore, this experiment was investigated twice. The average disease index of each agent treatment was calculated on September 17, 2021 (before the first drug application) and October 11, 2021 (10 days after the third drug application), and the control effect was statistically analyzed. The results are shown in Table 2; the control effect of the biocontrol agent at 500mL / mu was 65.0%, the control effect of 750mL / mu was 68.6%, and the control effect of 1500mL / mu was 72.8%, which was similar to the control effect of the control agent 20% thiophanate-methyl suspension at 125mL / mu (76.0%).

[0113] Table 2 Preliminary results of field trials on the efficacy of Bacillus safensis GX-H6 against rice bacterial leaf streak

[0114]

[0115] Example 6 Preliminary Experiment on the Field Efficacy of Bacillus safensis GX-H6 against Citrus Canker

[0116] 1. Preparation of biocontrol agent: Bacillus safensis GX-H strain was inoculated into NB liquid medium, cultured at 28°C to the logarithmic phase, adjusted to OD600 of 1.0, and transferred to 1 L of NB liquid medium at a 10% inoculum for expansion culture. After shaking culture at 28°C and 200 rpm for 36-48 hours, 1% Tween was added and set aside.

[0117] 2. Field trials

[0118] 2.1 Test site and citrus varieties: The test site was set up in Shuangding Citrus Garden, Longan, Nanning City, Guangxi. Citrus has been planted in the test site for many years. Citrus canker has occurred seriously in previous years. Citrus was planted in and around the test site. The citrus variety tested was "Wogan".

[0119] 2.2 Test method:

[0120] The experiment set up five treatments: clean water control (CK), biocontrol agent diluted 25 times, biocontrol agent diluted 50 times, diluted 80 times, 10 billion CFU / g Bacillus subtilis wettable powder diluted 1000 times, each treatment was repeated 3 times, and each plot was 15m 2 , use 80kg of water per mu and spray the leaves evenly.

[0121] The pesticide was applied three times. The first application was on May 16, 2022, which was the early stage of the disease; the second application was on May 26, 2022, and the third application was on June 2, 2022.

[0122] 3. Test results

[0123] This trial was conducted once, with disease indexes measured on June 9, 2022 (7 days after the third application), and statistical analysis of control efficacy was conducted. The results, shown in Table 3, show that the biocontrol agent achieved a control efficacy of 82.0% at a 25-fold dilution, 75.5% at a 50-fold dilution, and 71.7% at an 80-fold dilution. This compares to 76.0% for the control agent, a 1000-fold dilution of 10 billion CFU / g of Bacillus subtilis WP. The 25-fold dilution significantly increased the control efficacy compared to the 50-fold and 80-fold dilutions, as well as the 1000-fold dilution of 10 billion CFU / g of Bacillus subtilis WP.

[0124] Table 3 Preliminary test results of the field efficacy of Bacillus safensis GX-H6 against citrus canker

[0125]

[0126] Note: Different uppercase and lowercase letters indicate significant differences at the 0.01 and 0.05 levels, respectively.

[0127] Example 7 Antagonistic test of Bacillus safensis GX-H6 against pathogens of citrus canker on plate

[0128] Referring to the method of Example 2, a plate antagonism test was conducted on the pathogen of citrus canker. The test results are as follows: Figure 9 As shown, Bacillus safensis GX-H6 has a significant antagonistic effect on the pathogen of citrus canker, with an average inhibition zone diameter of 33.6 mm. The pathogen of citrus canker cannot grow around Bacillus safensis GX-H6.

[0129] Example 8 Antagonistic spectrum test of Bacillus safensis GX-H6 against plant pathogenic fungi

[0130] The antagonistic activity of Bacillus safensis GX-H6 against various plant pathogens was tested using a plate standoff assay. The pathogens included Neoscytalidium novaehollandiae, the pathogen of pitaya canker; Colletotrichum plurivorum, the pathogen of pitaya anthracnose; Botrytis cinere, the pathogen of melon gray mold; Fusarium oxysporum f.sp.cubense (Foc) race 4, the pathogen of banana wilt; Colletotrichum gloeosporioides, the pathogen of mango anthracnose; and Magnaporthe oryzae, the pathogen of rice blast. The pathogens were activated on PDA plates, and the resulting cakes were transferred to fresh PDA plates. Adjust the OD600 of a culture of Bacillus safensis GX-H6 to 1.0. Streak lines approximately 35 mm apart around the perimeter of the bacterial cake. Use an unstreaked plate as a blank control. Incubate at 28°C for 3-5 days, photograph, and record the results. Repeat three times.

[0131] The results are as follows Figure 10 As shown, in Figure 10 Among them, 1: Neoscytalidium novaehollandiae, 2: the pathogen of melon gray mold, 3: Fusarium oxysporum f.sp.cubense (Foc) race 4, 4: the pathogen of mango anthracnose, 5: the pathogen of rice blast. Figure 10 It can be seen that Bacillus safensis GX-H6 has a strong antagonistic effect on the pathogen of melon gray mold, and also has a certain antagonistic effect on the pathogens of new dark columnar spores, Fusarium oxysporum f.sp.cubense (Foc) race 4, mango anthracnose and rice blast (Magnaporthe oryzae).

[0132] The foregoing descriptions of specific exemplary embodiments of the present invention are for purposes of illustration and description. These descriptions are not intended to limit the invention to the precise forms disclosed, and it is apparent that many variations and modifications are possible in light of the foregoing teachings. The exemplary embodiments have been selected and described for the purpose of explaining the specific principles of the invention and their practical application, thereby enabling those skilled in the art to realize and utilize a variety of exemplary embodiments of the invention and various options and modifications. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A Bacillus sabdariffa ( Bacillus safensis ) GX-H6 strain, characterized in that The strain is deposited in the General Microbiology Center of China Culture Collection Administration, with the deposit number CGMCC No.25106 and the deposit date being June 16, 2022.

2. A method comprising the Bacillus sabdariffa of claim 1 ( Bacillus safensis ) Biocontrol agent for GX-H6 strain.

3. The method for preparing the biocontrol agent according to claim 2, wherein The following steps are involved: (1) Seed liquid culture: the Bacillus sabdariffa ( Bacillus safensis ) The GX-H6 strain was inoculated into a liquid culture medium and fermented to obtain a seed solution; (2) Expanding the culture: inoculate 1% of the seed solution into the liquid culture medium according to the volume percentage, and ferment to obtain the fermentation liquid; (3) Preparation of biocontrol agents: Add surfactant to the fermentation liquid and mix well to obtain the biocontrol agent.

4. The preparation method according to claim 3, characterized in that The fermentation culture conditions in step (1) and step (2) are: 26-30°C, rotation speed 200 rpm, and culture for 18-22 hours.

5. The preparation method according to claim 3, wherein: The liquid culture medium in step (1) and step (2) is NB liquid culture medium; the surfactant in step (3) is Tween 80, and the amount of the surfactant added is 1% by volume of the fermentation liquid.

6. The Bacillus sabdariffa of claim 1 ( Bacillus safensis ) Application of GX-H6 strain in the prevention and control of rice bacterial leaf streak disease, rice white leaf blight, and citrus canker.

7. The Bacillus sabdariffa of claim 1 ( Bacillus safensis ) GX-H6 strain inhibits the new dark columnar spore ( Neoscytalidium novaehollandiae )、Botrytis cinerea( Botrytis cinere ), Fusarium oxysporum Cuban type ( Fusarium oxysporum f.sp.cubense,Foc ) No. 4 small species, disc long spore spore ( Colletotrichum gloeosporioides ) or the rice blast pathogen ( Magnaporthe oryzae ) in the application.

8. The Bacillus sabdariffa of claim 1 ( Bacillus safensis ) Application of GX-H6 strain in the preparation of biocontrol agents for preventing and controlling rice bacterial leaf streak disease, rice white leaf blight, and citrus canker.

9. The Bacillus sabdariffa of claim 1 ( Bacillus safensis ) GX-H6 strain is used to prepare the new dark columnar spores ( Neoscytalidium novaehollandiae )、Botrytis cinerea( Botrytis cinere ), Fusarium oxysporum Cuban type ( Fusarium oxysporum f.sp.cubense,Foc ) No. 4 small species, disc long spore spore ( Colletotrichum gloeosporioides ) or the rice blast pathogen ( Magnaporthe oryzae ) in the application of biocontrol agents.

10. A method for using the biocontrol agent prepared by the preparation method according to any one of claims 3 to 5 in preventing and treating rice bacterial leaf streak disease and / or rice bacterial leaf blight, characterized in that: The application method for preventing rice bacterial leaf streak disease and / or rice bacterial leaf blight is: spraying the biocontrol agent on the rice leaves until all the leaves are wet; or, using a needle-free syringe to pressurize the biocontrol agent into the rice leaves; The application method in treating rice bacterial leaf streak disease and / or rice bacterial leaf blight is: spraying the biocontrol agent on the rice leaves until all the leaves are soaked; or, using a syringe to inject the biocontrol agent into the rice leaves at the early stage of the disease; or, soaking the rice leaves at the early stage of the disease in the biocontrol agent for 20 to 30 seconds.

11. A method for using the biocontrol agent prepared by the preparation method according to any one of claims 3 to 5 in treating citrus canker, comprising the following steps: Dilute the biocontrol agent 25 to 80 times and spray it on the diseased plants.

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