A strain of Streptomyces rocherii and its application

Abstract

The present invention relates to a strain of Streptomyces rochei for preventing and controlling bacterial diseases of the genus Pectinobacterium and having the function of promoting plant growth and its application. The Streptomyces rochei 26B provided by the present invention is deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration Committee with a number of CGMCC No. 24171. The strain is isolated from the rhizosphere soil of cabbage under water shortage stress, has a strong inhibitory effect on a variety of pathogenic bacteria and pathogenic fungi, has a highly effective prevention and control effect on bacterial diseases of the genus Pectinobacterium, and promotes plant seed germination and root elongation. The strain has a short fermentation culture time and a low production cost, is more advantageous than other bacterial agents, is conducive to its application in the field of plant disease prevention and control, and has a good application prospect.

Description

Technical Field

[0001] The invention belongs to the field of agricultural, forestry and horticultural crop plant protection, and particularly relates to a streptomyces strain having a strong inhibitory effect on pectinobacterium bacterial diseases and a growth-promoting function and a metabolite thereof. Technical Background

[0002] Pectobacterium spp. bacteria can infect a variety of crops and cause soft rot. For example, Pectobacterium carotovorum can cause disease in a variety of crops such as cabbage, carrot, konjac, and potato. Once it occurs, it is difficult to control. Cabbage soft rot, konjac soft rot, and potato black leg disease have become key diseases that restrict the production of cabbage, konjac, and potato. Once they occur, the yield will be reduced by 10% to 20% in mild cases, and more than 50% in severe cases, or even a total crop failure. In the years when the disease is prevalent, the incidence rate is as high as 30% to 40% (Li Lin, 2018; Yang Xianli, 2010; Wang Wei, 2019; Li Jianjun et al., 2010). Soil moisture is one of the main factors affecting the occurrence and spread of bacterial diseases. Excessive watering or heavy rains cause waterlogging on the ground, and excessive local air humidity is conducive to the invasion and spread of pathogens, which ultimately causes a large-scale outbreak of the disease.

[0003] There are currently few biological control agents available for bacterial soft rot. The control effect of agricultural streptomycin is poor. Copper ion preparations can easily cause bacteria to produce drug-resistant gene clusters. In production, oxadinomycin, kasugamycin and sinoprotinin are mostly used for control. Zhao Chenrui (2015) reported that oxadinomycin and sinoprotinin had good field control effects on cabbage soft rot, with control effects reaching 87.26% and 79.91%, respectively. Deng Huaqiang (2022) reported that potassium permanganate was often used to treat seeds in production, and quinoline wettable powder was sprayed later, or kasugamycin and other agents were used to control potato black leg disease. Yan Jiahui (2020) reported that the use of 6% kasugamycin wettable powder for seed potato dressing (25g preparation / 100Kg seed potato) could achieve a field control effect of 66.88% on potato black leg disease. Wuningmycin is a fermentation metabolite of Streptomyces ahygroscopicus var. wuzhouensis, and its main active component is a macrolide tetraene antibiotic. Zhongshengmycin is a N-glycoside secondary metabolite produced by Streptomyces lavendulae var. hainanensis, and belongs to IV-glycoside alkali-modifying substances. It has an antibacterial effect by inhibiting the protein synthesis of pathogens. Kasugamycin is a metabolite produced by Streptomyces microaureus, an aminoglycoside antibiotic, and its main active ingredient is [5-amino-2-methyl-6-(2,3,4,5,6-hydroxycyclohexyloxy)tetrahydropyran-3-yl]amino-α-imidoacetic acid, which mainly inhibits pathogens by interfering with the protein synthesis of pathogens. As we all know, the metabolites and action mechanisms produced by different actinomycetes, or even different strains of the same species, are not exactly the same, and the control effects on different types of pathogenic bacteria can also vary greatly. Obtaining new strains for controlling plant bacterial diseases is of great significance to enriching the resources of biocontrol bacteria.

[0004] At present, there are not many actinomycete resources for the prevention and control of bacterial soft rot. Dai Xuefeng et al. (2021) isolated antagonistic actinomycetes against Pectobacterium carotovorum subsp.carotovorum, but only determined the control effect of actinomycetes on soft rot bacteria and isolated konjac tubers, and did not identify the species of actinomycetes; Li Wei et al. (2017) reported that the fermentation liquid of Streptomyces rochei XL-6 had a strong antagonistic effect on eggplant bacterial wilt (Ralstonia solanacearum), but did not test its disease prevention effect; He Fei et al. (2015) isolated the antagonistic actinomycete Streptomyces rocheiD74, which had a strong antagonistic effect on Pectobacterium chrysanthemi, a konjac soft rot pathogen, with an antagonistic circle diameter of 11.0-18.3 mm. In the potted experiment, the control effect of D74 mixed with pathogens was only 17.0%. Zamanian et al. (2005) reported the inhibitory effect of S. rochei on Erwinia carotovora subsp. carotovora, but the highest antibacterial activity was achieved after 10-11 days of liquid culture; Osman et al. (2011) reported the antibacterial activity of S. rochei against medical pathogens; Boortseva et al. (2019) reported the antibacterial effect of Streptomyces plicatus on plant pathogenic bacteria Clavibatermichiganensis and Xanthomonas campestris, with the diameters of the inhibition zones being 22.7-30.0 mm and 16.2-30.0 mm, respectively. At present, S. plicatus has been classified as S. rochei (Komaki, 2019), and there are no reports on the preventive and control effects of S. rochei and S. plicatus on diseases such as cabbage soft rot, potato black leg disease, and ginger blight.

[0005] In July 2021, the inventor isolated a strain of Streptomyces 26B from the rhizosphere soil of Shangzhuang cabbage in Haidian District, Beijing, my country, which was identified as Streptomyces rochei. This strain has a strong inhibitory effect on a variety of pathogenic bacteria such as Pectobacterium carotovorum subsp. brasiliensis, P. atrosepticum, P. carotovorum subsp. brasiliensis, P. polaris, Fusarium oxysporum, Fusarium solani, Phytophythora capsici, Colletotrichum gloeosporioides and other pathogenic fungi, and the disease prevention effect on cabbage soft rot is 89.8%-96.0%. In addition to its disease prevention effect, this strain also has a growth-promoting effect, and is a strain with great application potential. Summary of the invention

[0006] The purpose of the present invention is to provide a strain of Streptomyces rocherii which has the effect of preventing and controlling bacterial diseases of the genus Pectinobacterium and promoting plant growth and its application.

[0007] The Streptomyces rochei provided by the present invention is specifically Streptomyces rochei 26B, which was deposited in the General Microbiology Center of China Culture Collection Administration (CGMCC for short, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing) on ​​December 21, 2021, with a deposit number of CGMCC No. 24171.

[0008] The present invention also protects a bacterial agent whose active ingredient is the Streptomyces rochei 26B.

[0009] The microbial agent may further include an adsorption carrier; the adsorption carrier may be at least one of diatomaceous earth, peat, vermiculite, rice husk powder, straw powder, perlite and calcium carbonate.

[0010] More specifically, in one embodiment of the present invention, the adsorption carrier is specifically formed by mixing a mixture of diatomaceous earth and calcium carbonate (mass ratio 2:1) in a volume ratio of 1:1.

[0011] Furthermore, the bacterial agent is formed by mixing the fermentation broth of the Streptomyces rochei 26B, or the bacterial mud obtained by centrifuging the fermentation broth, with the adsorption carrier in a ratio of 1L:1Kg to 1L:4Kg (such as 1L:1Kg). In one embodiment of the present invention (Example 5), a large amount of fermentation culture broth is used when preparing the fermentation broth of the Streptomyces rochei 26B, and the number of viable bacteria of the Streptomyces rochei 26B in the fermentation broth of the Streptomyces rochei 26B is 2 to 6 billion / mL (such as 4 billion / mL) of the fermentation broth. The solvent of the large-scale fermentation culture liquid is water, and the solutes and concentrations are as follows: 1.2% soybean cake powder, 1.5% starch, 0.8% glucose, 0.5% peptone, 0.25% ammonium sulfate, 0.03% magnesium sulfate, 0.025% potassium dihydrogen phosphate, 0.04% sodium chloride, 0.8% calcium carbonate; wherein % represents the mass percentage. Accordingly, in the prepared bacterial agent, the viable count of Streptomyces rocherii 26B is 2 to 6 billion / g (such as 4 billion / g); the water content of the bacterial agent is within 5 to 8%.

[0012] The preparation method of the bacterial agent also belongs to the protection scope of the present invention.

[0013] The preparation method of the bacterial agent may specifically include the following steps:

[0014] (1) culturing the Streptomyces rochei 26B to obtain a fermentation broth of the Streptomyces rochei 26B; in the fermentation broth, the number of viable cells of the Streptomyces rochei 26B is 2 to 6 billion / mL (e.g., 4 billion / mL);

[0015] (2) The fermentation liquid or the centrifuged bacterial sludge obtained in step (1) and the adsorption carrier are mixed in a ratio of 1L:1Kg to 1L:4Kg (e.g., 1L:1Kg) to obtain the bacterial agent.

[0016] In step (1), the solvent of the culture medium for culturing the Streptomyces rochei 26B is water, and the solutes and their concentrations are as follows: 1.0-1.5% soybean cake powder, 1.2-1.5% starch, 0.6-1.0% glucose, 0.4-0.6% peptone, 0.2-0.3% ammonium sulfate, 0.02-0.03% magnesium sulfate, 0.015-0.03% potassium dihydrogen phosphate, 0.02-0.04% sodium chloride, and 0.6-1.0% calcium carbonate; wherein % represents the mass percentage.

[0017] More specifically, the solvent of the culture medium is water, and the solutes and concentrations are as follows: 1.2% soybean cake powder, 1.5% starch, 0.8% glucose, 0.5% peptone, 0.25% ammonium sulfate, 0.03% magnesium sulfate, 0.025% potassium dihydrogen phosphate, 0.04% sodium chloride, and 0.8% calcium carbonate; wherein % represents the mass percentage.

[0018] The fermentation broth of Streptomyces rochei 26B also belongs to the protection scope of the present invention.

[0019] The invention also provides a product for preventing and controlling plant bacterial diseases.

[0020] The use of the Streptomyces rochei 26B or the bacterial agent or the fermentation broth in the following (1) or (2) also falls within the protection scope of the present invention:

[0021] (1) Prevent and control plant diseases caused by bacterial pathogens, combined infection of bacteria and fungi, or combined infection of bacteria and nematodes.

[0022] (2) Prepare products for preventing and controlling diseases described in (1).

[0023] The CGMCC No. 24171 of Streptomyces rochei 26B provided by the present invention has the following advantages:

[0024] 1. The strain is isolated from the plant rhizosphere, is more adaptable to the plant rhizosphere environment, and grows rapidly.

[0025] 2. This strain was isolated from the rhizosphere soil under water stress and is more tolerant to soil drought and water shortage environments. It not only has a high efficacy in preventing and controlling bacterial soft rot caused by pathogenic bacteria, but also has better disease prevention effects under conditions of very low soil moisture content, which is conducive to its survival and function in adverse environments. It is a potential biocontrol bacterium.

[0026] 3. The strain has a short fermentation cycle, low cost, and is easy to mass produce and commercialize.

[0027] The Streptomyces rochei 26B CGMCC No. 24171 provided by the present invention is a strain of Streptomyces that has antagonistic effects on various pathogens isolated by the inventor from the rhizosphere soil of Shangzhuang cabbage in Haidian District, Beijing, my country in July 2021. The strain is characterized in that the strain 26B is white in the early stage of growth on ISP2 medium (yeast extract 4g, malt extract 10g, anhydrous glucose 4g, agar 18g, distilled water 1L, pH 7.3), turns to mouse gray after spore production, and the aerial hyphae are lush. The colony bulges on the surface of the culture medium, loose, porous, rough, dry, and wrinkled; the center of the back of the colony is yellow-brown, the edge is yellow, and dark red pigments are produced on the culture medium around the colony. A large number of oval spores are produced and the spore surface is smooth. The strain has a strong inhibitory effect on a variety of pathogenic bacteria and fungi, promotes seed germination and root growth, and is a very potential strain. The strain was identified as Streptomyces rochei, named 26B. The sterilized fermentation filtrate of the strain was mixed with pathogenic bacteria at 10% (V / V) to completely inhibit the colonization of pathogenic bacteria in the roots of cabbage. Whether in soils with low water content (14%, W / W) or high water content (20%, W / W), the disease prevention effect was very high, reaching 89.8% and 96.0% respectively, which was higher than other strains. When the 26B strain was fermented and cultured, the growth rate was fast and the fermentation could be completed within 4 days. After the fermentation, the number of live bacteria could reach 2 to 6 billion / mL, and the production cost was low. Therefore, 26B is a very potential degradation bacterium, and thus the present invention was proposed.

[0028] Collection Instructions

[0029] Bacterial species name: Streptomyces rocherii

[0030] Latin name: Streptomyces rochei

[0031] Strain ID: 26B

[0032] Depository: China National Microbiological Culture Collection Administration General Microbiology Center

[0033] Abbreviation of depository institution: CGMCC

[0034] Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0035] Date of deposit: December 21, 2021

[0036] CGMCC Registration Number: CGMCC No.24171 DETAILED DESCRIPTION

[0037] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0038] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0039] Example 1: Isolation and screening of Streptomyces rochei

[0040] (I) Isolation and antagonism of Streptomyces rocherii 26B strain

[0041] (1) Isolation and screening of Streptomyces rocherii 26B strain

[0042] The Streptomyces rochei 26B of the present invention is obtained by diluting and applying the soil from the rhizosphere of Chinese cabbage to which Bacillus is applied and subjected to water shortage stress, and is separated and obtained as follows:

[0043] From Shangzhuang, Haidian District, Beijing, rhizosphere soil was collected from the rhizosphere of cabbage treated with Bacillus B006 and subjected to water shortage stress by multi-point sampling method, with 2-3g for each treatment. Actinomycetes were isolated by dilution coating method. Nalidixic acid and cycloheximide were added to ISP2 medium and Gao's No. 1 medium at final concentrations of 20mg / L and 50mg / L, respectively, to inhibit the growth of bacteria and fungi. After 7-14 days of culture at 28℃, actinomycetes with different colony morphology were selected for streaking purification, and pure strains were stored in 30% glycerol tubes and frozen at -80℃. The Oxford cup method was then used to determine the antibacterial activity of different actinomycete fermentation broths. The activated single actinomycete colonies were selected and transferred to 100mL fermentation medium, and cultured at 28℃ and 180r / min for 5 days. The fermentation broth was centrifuged at 4°C and 5000 r / min for 10 min, and the supernatant was filtered through a 0.22 μm microporous filter membrane to obtain a sterilized fermentation filtrate. 9 CFU / mL) of Chinese cabbage soft rot pathogen Pectobacterium carotovorum subsp.brasiliensis (Pcb) was mixed with 100mL LB medium and poured into a plate to prepare a medium plate containing pathogens. 200μL of sterilized fermentation filtrate was added to an Oxford cup. An equal amount of sterile water was used as a control. Each treatment was repeated 3 times. After culturing at 28℃ for 2 days, the formation of inhibition zones was observed. The strains with strong antagonistic activity against Chinese cabbage soft rot pathogens were screened out according to the size of the inhibition zones.

[0044] The components and proportions of the ISP2 medium are: 4 g yeast extract, 10 g malt extract, 4 g anhydrous glucose, 18 g agar, 1 L distilled water, pH 7.3. The components and proportions of Gao's No. 1 medium are: 20.0 g soluble starch, 1.0 g KNO3, 0.5 g K2HPO4, 0.5 g MgSO4·7H2O, 0.5 g NaCl, 0.01 g FeSO4·7H2O, 18.0 g agar, 1 L distilled water, pH 7.2-7.3.

[0045] After streaking, 38 actinomycetes were obtained. The antagonistic activity of the sterilized fermentation filtrate of the isolated strains against soft rot bacteria was determined by the Oxford cup method, and actinomycete 26B was obtained, which had the best antibacterial effect, with an inhibition zone diameter of 22.44±0.22mm.

[0046] (2) Inhibitory effects of fermentation filtrates of Streptomyces 26B at different culture times on various pathogenic bacteria

[0047] The Streptomyces 26B cells were inoculated into 50 mL ISP2 seed culture medium (4 g yeast extract, 10 g malt extract, 4 g anhydrous glucose, 1 L distilled water, pH 7.3), and cultured at 28°C and 180 r / min for 18 h; then the seed solution was transferred to 100 mL fermentation medium (1.2% soybean meal, 1.5% starch, 8.0 g glucose, 5.0 g peptone, 2.5 g ammonium sulfate, 0.3 g magnesium sulfate, 0.25 g potassium dihydrogen phosphate, 0.4 g sodium chloride, 8.0 g calcium carbonate, 1 L distilled water, pH 7.3) at an inoculum volume of 2% (V / V), and continued to culture with shaking. At 3, 4, 5, and 6 days of culture, 50 mL of fermentation liquid was taken from each treatment shake flask and centrifuged at 4°C and 5000 r / min for 10 min. The supernatant was retained for the determination of antibacterial activity and pH of the fermentation liquid.

[0048] The Oxford cup method was used to determine the antibacterial activity of the fermentation filtrates of 26B strain at different times against four pathogenic bacteria. The results (Table 1) showed that the antibacterial effect of the fermentation broth of Streptomyces 26B cultured for 4 days was higher than that at 3 days. The diameters of the inhibition zones against Pectobacterium carotovorum subsp. brasiliensis, P. atrosepticum, P. carotovorum subsp. brasiliensis, P. polaris and Pectobacterium sp. were 23.34 mm, 17.80 mm, 16.83 mm, 22.37 mm and 14.25 mm, respectively. The antibacterial effect of the fermentation broth cultured for 5 days and 6 days was not significantly different from that at 4 days (Table 1). The results of pH measurement of the fermentation broth of strain 26B showed that the initial pH of the fermentation broth was 7.42±0.01, and the pH of the fermentation filtrate was basically 6.84±0.05 during the 3-6 days of culture, indicating that the antibacterial activity of the fermentation broth of strain 26B was not caused by the change of pH value. From the perspective of industrial production, the 4th day of fermentation culture can be regarded as the fermentation end point.

[0049] Table 1 Inhibitory effects of the sterilized filtrates of Streptomyces 26B fermentation at different culture times on plant pathogenic bacteria

[0050]

[0051] Note: Different letters after the same row of data indicate significant differences (p<0.05).

[0052] (3) Inhibitory effect of Streptomyces 26B fermentation filtrate on various pathogenic fungi

[0053] The fermentation liquid of Streptomyces 26B cultured for 4 days was prepared according to the method in Example 2. A 5 mm mycelium block of the pathogenic fungus to be tested was inoculated on a potato glucose medium (200 g peeled potato, 20 g glucose, 18 g agar, 1 L distilled water) plate, and after culturing at 26°C for 3 days, an Oxford cup was placed at the four corners of the PDA plate 2 cm away from the fungal colony, and 200 μL of the fermentation sterilization filtrate was added. Each treatment was repeated 5 times, and after culturing at 26°C for 3-4 days, the diameter of the inhibition zone (mm) was measured. The measurement results show (Table 2) that the fermentation sterilization filtrate of Streptomyces 26B cultured for 4 days has a good inhibitory effect on the four pathogenic fungi, among which the inhibitory effect on cucumber wilt fungus, bean root rot fungus, and pepper phytophthora is higher, and the inhibitory effect on gloeosporium anthracnose is weaker.

[0054] Table 2 Inhibitory effects of the fermentation filtrates of Streptomyces 26B at different culture times on plant pathogenic fungi

[0055]

[0056] (II) Identification of Streptomyces 26B strain

[0057] The genomic DNA of strain 26B was extracted, and the 16S rDNA sequence was amplified with universal primers 27F and 1492R and sequenced.

[0058] 27F: 5′-AGA GTT TGA TCC TGG CTC AG-3′; 1492R: 5′-TAC GGC TAC CTT GTT ACGACT T-3′. The sequence of 16S rDNA of strain 26B has been submitted to GenBank, with the accession number OM214460. In view of the above molecular biological characteristics identification results, strain 26B isolated and purified in step one was identified as Streptomyces rochei. The Streptomyces rochei was deposited in the General Microbiology Center of China National Microbiological Culture Collection (CGMCC for short, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing) on ​​December 21, 2021, with the deposit number CGMCC No. 24171.

[0059] Example 2: Fermentation filtrate of Streptomyces 26B inhibits colonization of soft rot pathogens on cabbage roots

[0060] Cabbage seed germination preparation: Select cabbage seeds with full seeds and sterilize them in 3% NaClO solution for 1 minute, rinse them with sterile water 3 times, and dry them on sterile filter paper. Then transfer the sterilized cabbage seeds to a culture dish covered with sterile filter paper and place them in a 25°C incubator. When the seedling radicle grows to 2.5 cm, select cabbage seedlings with consistent growth for testing.

[0061] The experiment was divided into three treatments: T1: the roots of cabbage seedlings were treated with 20 mL of soft rot fungus Pcb with kanamycin resistance and green fluorescence marker. R Bacterial suspension (5×10 6 CFU / mL) soaking treatment; T2: the roots of cabbage seedlings were soaked in 20 mL of 10% (V / V) Streptomyces 26B sterilized fermentation filtrate R Bacterial suspension (5×10 6 CFU / mL) soaking treatment; CK was used as the control and soaked in the same amount of sterile water. The roots of the cabbage seedlings were uniformly soaked in the above bacterial suspension for 5 seconds, transferred to sterile dry filter paper to remove excess water, and then transferred to a 15 cm culture dish covered with three layers of sterile wet filter paper, with 10 seedlings per dish and 3 replicates per treatment. They were cultured in a 25°C artificial climate chamber. After 48 hours, the cabbage seedlings with different treatments were taken, 100 mg of radicles were cut, ground and diluted, of which T1 was diluted 10 4The different gradient grinding solutions were then spread on LB solid medium (tryptone 10 g, NaCl 10 g, yeast extract 5 g, agar 18 g, distilled water 1 L, pH 7.0-7.2) plates containing kanamycin, and cultured at 28°C for 24 h. The plates were then observed under a stereofluorescence microscope to count the number of pathogens. Each treatment was repeated 3 times.

[0062] Pcb R The count results showed that the pathogen content in the cabbage roots treated with T1 was (2.3±0.79)×10 9 CFU / g, no pathogens were detected from the cabbage roots treated with T2 and CK, indicating that the fermentation filtrate of Streptomyces 26B strain had a strong antibacterial effect.

[0063] Example 3: Growth-promoting effect of Streptomyces 26B fermentation filtrate on Chinese cabbage

[0064] Select cabbage seeds with full grains and sterilize them in 3% NaClO solution for 1 min. Rinse with sterile water 3 times, dry on sterile filter paper and set aside. Soak the cabbage seeds in 50 mL of 10-fold diluted Streptomyces 26B sterilization fermentation filtrate for 3 hours. After soaking, air dry naturally and place them in a culture dish covered with 3 layers of sterile moist filter paper. Place 30 seeds evenly in each culture dish, repeat 6 times, and use the same amount of sterile water as a control. All culture dishes were placed in an incubator at 25°C. After 3 days, the germination rate, germination potential and germination index were counted. After 4 days, 20 germinated cabbage seedlings were randomly selected from each plate to measure their radicle length and fresh weight.

[0065] The results showed (Table 3) that compared with normal growth of cabbage, the germination potential, germination index, root length and fresh weight of cabbage seeds soaked in 10-fold diluted Streptomyces 26B sterilized fermentation filtrate were significantly higher than those of the control group (p < 0.05), indicating that the fermentation filtrate of strain 26B has a promoting effect on cabbage seed germination and root elongation.

[0066] Table 3 Effects of 10-fold diluted strain 26B fermentation filtrate on cabbage seed germination

[0067]

[0068] Note: Different letters after the numbers in the same column indicate significant differences (p<0.05).

[0069] Example 4: Preparation of Streptomyces rochei 26B bacterial agent

[0070] 1. The bacterial lawn of Streptomyces rochei 26B CGMCC No. 24171 obtained in Example 1 was transplanted into ISP2 culture medium and cultured in a shaking incubator at 28-30° C. for 18 h to obtain a seed solution.

[0071] The formula of ISP2 culture medium is: 4g yeast extract, 10g malt extract, 4g anhydrous glucose, 1L distilled water, pH 7.3.

[0072] 2. Inoculate the seed solution obtained in step 1 into a large amount of culture fermentation medium at a ratio of 5% (volume ratio), with a culture temperature of 28-30° C., a rotation speed of 150-180 rpm, an initial pH of 7.0-7.2, and a culture time of 4 days. After the fermentation is completed, a fermentation liquid is obtained, in which the number of viable bacteria of Streptomyces rochei 26B CGMCC No. 24171 can reach 3 billion / mL.

[0073] Among them, the formula of the large-scale fermentation culture medium is: 1.2% soybean cake powder, 1.5% starch, 0.8% glucose, 0.5% peptone, 0.25% ammonium sulfate, 0.03% magnesium sulfate, 0.025% potassium dihydrogen phosphate, 0.04% sodium chloride, 0.8% calcium carbonate, pH 7.2.

[0074] 3. The bacterial sludge obtained by centrifuging the fermentation liquid of Streptomyces rochei obtained in step 2 is evenly mixed with the adsorption carrier, diatomaceous earth and calcium carbonate (mass ratio 2:1), in a volume-mass ratio of 1:1 (i.e. 1L:1Kg), to obtain a bacterial agent. The centrifugal speed is 10,000 rpm, and the centrifugal time is 20 minutes; the number of viable bacteria of Streptomyces rochei 26B CGMCC No.24171 in the bacterial agent can reach 6 billion / g, and the water content is within 5%.

[0075] Example 5: Disease prevention effect of Streptomyces 26B fermentation liquid on Chinese cabbage soft rot in soils with different water contents

[0076] Cabbage seeds were evenly sown in pots filled with moist sterilized soil and cultured in an artificial climate box at 25°C. Five seedlings were sown in each pot. After the three true leaves were unfolded, the cabbage soft rot pathogen Pcb and 26B fermentation filtrate were inoculated by watering. The soil moisture content of T1-1, T1-2, and CK1 was set to 14%, and the soil moisture content of T2-1, T2-2, and CK2 was set to 20%. T1-1 and T2-1 were treated with pathogen inoculation, and 10 mL of Pcb bacterial suspension was poured into each pot. The concentration of Pcb bacterial suspension was 5×10 6CFU / mL; T1-2 and T2-2 were inoculated with a mixture of 26B fermentation broth and Pcb bacterial suspension, and each pot was irrigated with 10 mL; the volume ratio of the mixture of 26B fermentation broth and Pcb bacterial suspension was 1:10, and the concentration of Pcb in the mixture was 5×10 6 CFU / mL; CK1 and CK2 were used as controls and inoculated with the same amount of sterile water. Each treatment was repeated 3 times, each repeat was 3 pots, and each pot had 5 cabbage seedlings. In order to maintain the soil moisture content, the number and amount of watering need to be controlled. The amount of watering for potted seedlings treated with soil moisture content of 14% and 20% was 12mL and 15mL respectively, and watering was done twice a day. The disease situation was investigated after 15 days. Disease grading standard: Grade 0-no disease; Grade 1-1 to 2 leaves on the lower part of the plant have a small number of lesions; Grade 2-3 or more leaves on the lower part of the plant have lesions, and other infected parts are slightly heavier; Grade 3-all leaves are infected, and other infected parts are heavier; Grade 4-all leaves are infected, the plant is tilted as a whole, the leaves droop and cannot return to normal, but it is still alive; Grade 5-the whole plant dies and falls to the ground.

[0077] The test results showed that the fermentation filtrate of Streptomyces 26B significantly inhibited the occurrence of soft rot of cabbage (Table 4). Compared with T2-1 with a soil moisture content of 20%, the incidence and disease index of soft rot of cabbage in T1-1 with a soil moisture content of 14% were significantly reduced, and its relative prevention efficiency was 87.0%; on this basis, the incidence and disease index of soft rot of cabbage in T1-2 with the addition of 26B sterilization fermentation filtrate were even lower, and the relative prevention efficiency reached 96.0%. This result shows that the incidence of soft rot of cabbage is affected by soil moisture, and the application of fermentation filtrate of Streptomyces 26B at a lower soil moisture content can further reduce the incidence and disease index of cabbage seedlings.

[0078] Table 4 The disease prevention effect of Streptomyces 26B fermentation filtrate on Chinese cabbage soft rot in soils with different water contents

[0079]

[0080] Note: Different letters after the numbers in the same column indicate significant differences (p < 0.05); * represents that the blank control was not inoculated with soft rot bacteria and did not develop the disease, so no disease data survey was conducted.

Claims

1. Streptomyces rochei 26B, deposited in the General Microbiological Center of China Microorganism Culture Collection Administration, with the deposit number of CGMCC No.24171.

2. A bacterial agent, characterized in that The active ingredient of the bacterial agent is the Streptomyces rochei 26B described in claim 1.

3. The bacterial agent according to claim 2, characterized in that The bacterial agent is a liquid bacterial agent, and the number of viable bacteria of Streptomyces rochei 26B in the liquid bacterial agent is 3 to 6 billion / mL.

4. The bacterial agent according to claim 2, characterized in that The bacterial agent is a solid bacterial agent, which is prepared by mixing bacterial sludge obtained by centrifuging the fermentation liquid of Streptomyces rochei 26B and an adsorption carrier in a ratio of 1L:1Kg.

5. The bacterial agent according to claim 4, characterized in that The preparation method of the solid bacterial agent comprises the following steps: Cultivating Streptomyces rochei 26B to obtain a fermentation broth of Streptomyces rochei 26B; The bacterial sludge obtained by centrifuging the fermentation liquid and the adsorption carrier are mixed in a ratio of 1L:1Kg to obtain the bacterial agent; The culture medium solvent for culturing Streptomyces rochei 26B is water, and the culture medium solutes and concentrations are as follows: 1.0-1.5% soybean cake powder, 1.2-1.5% starch, 0.6-1.0% glucose, 0.4-0.6% peptone, 0.2-0.3% ammonium sulfate, 0.02-0.03% magnesium sulfate, 0.015-0.03% potassium dihydrogen phosphate, 0.02-0.04% sodium chloride, and 0.6-1.0% calcium carbonate; wherein % represents the mass percentage content.

6. Use of the Streptomyces rochei 26B according to claim 1 and the bacterial agent according to any one of claims 2 to 4 in the prevention and treatment of soft rot of cabbage.

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