Bacillus sorola, application of bacillus sorola and tobacco disease prevention and control method

Abstract

The invention relates to a bacillus sorola, application of the bacillus sorola and a method for preventing and treating tobacco diseases. The invention relates to a strain of bacillus soronorrhoea, which is bacillus soronorrhoea with the number of MC417, is preserved in the China Center for Type Culture Collection (CCTCC), and has the preservation number of CCTCC NO: M20242589. The invention also discloses a fermentation product, which is produced by fermentation culture of the bacillus sorola. The microbial agent comprises one or more of the bacillus sorola, a fermentation culture of the bacillus sorola or a fermentation product of the bacillus sorola. According to the application, a new strain of bacillus sorola MC417 obtained by separation has a relatively strong inhibition effect on three plant pathogenic fungi, i.e., colletotrichum nicotianae, phytophthora parasitica var nicotianae and fusarium root rot, and has a growth promoting effect in a tobacco seedling stage.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial technology, in particular to a strain of Bacillus sonorensis, application thereof and a method for preventing tobacco diseases. BACKGROUND

[0002] Flue-cured tobacco is the most widely cultivated tobacco type in the world and is one of the important economic crops in China. The cultivation area of flue-cured tobacco in China has reached 1.4 million hectares, and the yield has been maintained at 1700 kg / hm 2 , ranking first in the world. Due to the limitation of arable land area and the drive of economic benefits, continuous cropping of tobacco is serious in China. Continuous cropping for many years will promote the transformation of soil microorganisms from high-fertility "bacterial type" to low-fertility "fungal type", and aggravate the occurrence of soil-borne diseases.

[0003] Tobacco black shank, tobacco fusarium root rot and tobacco anthracnose are common and serious fungal diseases in flue-cured tobacco production, which have the characteristics of wide distribution, difficult prevention and control, and high composite incidence rate. The composite infection of these diseases poses a serious threat to the growth and yield of tobacco, and effective comprehensive prevention and control strategies are urgently needed. Tobacco black shank is caused by the pathogenic variety of Phytophthora parasitica, which is an oomycete disease. The initial infection source is mycelium or chlamydospore attached to plant disease residues. These spores can be transmitted by rainwater and irrigation water, usually invade the plant from the tobacco stem base, and produce a large number of zoospores, which rapidly spread in the field and are the important re-infection source of the disease. Tobacco fusarium root rot is caused by fusarium, especially fusarium oxysporum. The initial infection sources include dormant chlamydospores, mycelium or conidia in soil and disease residues. Fusarium has strong sporulation capacity and diverse transmission routes. After the pathogen invades through the root wound, it forms conidia, which are then spread by wind, rain, disease residues or agricultural operations. In addition, the pathogen can also be transmitted through the air, invade the plant vascular system and damage the transport tissue, further aggravating the spread of the disease. Tobacco anthracnose is a common leaf disease of tobacco, which can occur at all growth stages, especially at the seedling stage. Conidia are transmitted to leaves or stems by wind and rain, germinate to form appressoria, penetrate the epidermis to form irregular primary hyphae, and then invade adjacent cells through secondary hyphae. Within a few days, water-stained small spots gradually expand to about 3 mm, and finally form dark brown necrotic spots, causing leaf wilting.

[0004] Biological control is one of the important directions of plant disease prevention and control, which has attracted widespread attention due to its abundant resources, wide selection range and environmental friendliness. Bacillus (Bacillus) is a kind of gram-positive, endospore-forming, rod-shaped bacteria, which has the characteristics of strong adaptability, strong antagonistic ability, high safety and no pollution to the environment, and is widely used in the biological control of plant diseases and insect pests. Bacillus sp.) has become the focus of research due to its high activity and excellent environmental adaptability. In the biological control of tobacco diseases, Bacillus velezensis YC11 shows significant control effect on black shank, bacterial wilt, root rot and root knot nematode (Li Yanyan et al., 2022); Bacillus subtilis GUMT323 can effectively antagonize tobacco black shank (Ding Haixia, 2018); Bacillus amyloliquefaciens B011 has good inhibitory effect on tobacco bacterial wilt and black shank (Zhou Xiangping et al., 2015). Bacillus sonorensis (Bacillus sonorensis) Bacillus sonorensis ) is a salt-tolerant strain isolated from the soil of the Sonoran Desert, which can survive in extreme environments and has wide application potential (Palmisano et al., 2001). There is no research report on its prevention and control of tobacco anthracnose, tobacco black shank and tobacco fusarium root rot. SUMMARY

[0005] To solve the technical problems existing in the prior art, one of the purposes of the present application is to provide a Bacillus sonorensis for tobacco, which can efficiently inhibit pathogenic fungi and reduce the incidence of diseases.

[0006] To achieve the above-mentioned purposes, the technical solutions of the present application are as follows: The present application provides a Bacillus sonorensis, which is Bacillus sonorensis (Bacillus sonorensis) Bacillus sonorensis ) with the number of MC417, which is preserved in China Center for Type Culture Collection, with the preservation number of CCTCC NO: M20242589.

[0007] The present application provides a fermentation product produced by the fermentation culture of the above-mentioned Bacillus sonorensis. The fermentation culture medium can be prepared as follows: yeast powder 5.0 g, peptone 10.0 g, glucose 12.0 g, NaCl 5.0 g, K2HPO43H2O 0.1 g, corn starch 20.0 g, soybean meal powder 5.0 g, KH2PO40.7 g, MgSO40.2 g, distilled water 1000 mL, pH=7.0, 121℃ high pressure steam sterilization for 30 min.

[0008] The present application provides a microbial agent containing one or more of the above-mentioned Bacillus sonorensis, its fermentation culture or its fermentation product.

[0009] Preferably, the microbial agent can survive in an environment with a salt concentration of 15% and pH of 10.

[0010] The present application provides the above-mentioned Bacillus sonorensis, the above-mentioned fermentation product or the above-mentioned microbial agent for use in salt-tolerant, tobacco disease control and growth promotion.

[0011] Preferably, the tobacco disease includes one or more of tobacco anthracnose caused by Colletotrichum gloeosporioides, tobacco black shank caused by Phytophthora nicotianae or tobacco fusarium root rot caused by Fusarium oxysporum.

[0012] The application provides a method for preventing and treating a tobacco disease, wherein the above-mentioned Bacillus sonorensis, the above-mentioned fermentation product or the above-mentioned microbial agent is applied to tobacco.

[0013] The application has the following beneficial effects: The application separates a new Bacillus sonorensis MC417 which has a strong inhibitory effect on three plant pathogenic fungi, namely tobacco anthracnose fungus, tobacco black shank fungus and tobacco fusarium root rot fungus.

[0014] The application is applied to the biological prevention and treatment of tobacco anthracnose caused by Colletotrichum gloeosporioides, tobacco black shank caused by Phytophthora nicotianae and tobacco fusarium root rot caused by Fusarium oxysporum, and significantly reduces the incidence of diseases. Meanwhile, the application has the function of promoting indole acetic acid production, and can promote the agronomic traits of tobacco seedling stage, such as plant height, biomass and leaf area. The application has a very good application prospect in biological microbial agents and disease prevention and growth promotion.

[0015] The prevention and treatment method of the application can effectively prevent and treat tobacco anthracnose caused by Colletotrichum gloeosporioides, tobacco black shank caused by Phytophthora nicotianae and tobacco fusarium root rot caused by Fusarium oxysporum. Due to the secondary metabolism of the rhizosphere caused by the continuous cropping of flue-cured tobacco, the phenomenon of coexistence of multiple pathogenic fungi is caused, and it is generally difficult for a general strain to simultaneously produce antagonism to multiple pathogenic fungi, resulting in that the strain cannot produce a prevention and treatment effect. The prevention and treatment method of the application is aimed at tobacco diseases, and uses Bacillus sonorensis MC417 to efficiently inhibit pathogenic fungi and promote tobacco growth. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 It is a colony morphology of Bacillus sonorensis MC417 of the application.

[0017] Figure 2 It is an antagonistic effect diagram of Bacillus sonorensis MC417 of the application on three kinds of pathogenic fungi.

[0018] Figure 3 It is a phylogenetic tree of Bacillus sonorensis MC417 and related strains of the application based on 16S rRNA sequences.

[0019] Figure 4 It is a phylogenetic tree of three housekeeping gene sequences of Bacillus sonorensis MC417 of the application.

[0020] Figure 5 It is a test result diagram of the salt and alkali tolerance of Bacillus sonorensis MC417 of the application.

[0021] Figure 6 Growth of Bacillus sonorensis MC417 in different salt concentration medium at pH = 10.0.

[0022] Figure 7 Prevention effect of Bacillus sonorensis MC417 on Phytophthora parasitica in detached leaves.

[0023] Figure 8 Prevention effect of Bacillus sonorensis MC417 on Colletotrichum gloeosporioides in detached leaves.

[0024] Figure 9 Prevention effect of Bacillus sonorensis MC417 on Fusarium oxysporum in detached leaves.

[0025] Figure 10 Prevention effect of Bacillus sonorensis MC417 on three kinds of pathogens in detached leaves.

[0026] Figure 11 Comparison of growth promotion effect of Bacillus sonorensis MC417 on Nicotiana benthamiana. DETAILED DESCRIPTION

[0027] The preferred embodiments of the present application will be described in detail below with reference to the examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present application. Those skilled in the art can make various modifications and replacements to the present application without departing from the spirit and principles of the present application.

[0028] The application of Bacillus sonorensis MC417, fermentation product or bacterial agent of the present application in tobacco disease prevention and growth promotion. The tobacco diseases include one or more of tobacco anthracnose caused by Colletotrichum gloeosporioides, tobacco black shank caused by Phytophthora parasitica or tobacco fusarium root rot caused by Fusarium oxysporum. The planting environment of tobacco is different from other crops, and multiple pathogenic fungi coexist in the continuous cropping state. It is generally difficult for a bacterial strain to simultaneously produce antagonism to multiple pathogenic fungi and achieve prevention effect. The present application has good prevention effect on the above-mentioned tobacco diseases, and the prevention effect on tobacco fusarium reaches more than 95.6%.

[0029] Example 1 Isolation, purification and identification of strain MC417 1.1 Isolation and purification of strain MC417 In 2024, rhizosphere soil samples were collected from a continuously cropped tobacco field in Sanying Town, Eryuan County, Dali Bai Autonomous Prefecture, Yunnan Province. Approximately 10g of soil sample was weighed and added to an Erlenmeyer flask containing glass beads and 90mL of sterile physiological saline. The flask was shaken thoroughly at 180 rpm for 30 minutes and allowed to stand for 10 minutes to obtain a soil suspension. 1mL of the supernatant was then diluted 10 mL with sterile physiological saline. -1 10 -2 10 -3 10 -4 and 10 -5 Five gradients, take 10 -2 10 -3 10 -4 10 -5 100 μL of each dilution was evenly spread on LB solid medium (5 g / L yeast extract, 10 g / L peptone, 10 g / L NaCl, 18 g / L agar), with 3 replicates for each gradient. After incubation at 28 °C for 2 days, strain MC417 was isolated. Figure 1 A total of 118 rhizosphere bacteria strains were isolated.

[0030] 1.2 Screening of antagonistic strains Tobacco anthrax bacteria ( Colletotrichum nicotianae ), Tobacco Phytophthora ( Phytophthora nicotianae Fusarium oxysporum, the pathogen causing tobacco root rot, is... Fusarium oxysporum Three pathogenic fungi (including *[names of fungi]*, *[names of fungi]*, and *[names of fungi]* were used as target fungi. The biocontrol effect of the isolated rhizosphere bacteria was tested using a confrontation culture method. First, the pathogenic fungi were activated on PDA plates. Pathogen fungal discs were then created using a 5mm punch and inoculated into the center of the PDA plate. 118 isolated rhizosphere bacteria were then inoculated at a distance of 2.5cm from the fungal discs. The plates were incubated at 25℃ for 4-7 days. The colony diameters of the pathogens inoculated and uninoculated treatments were measured. The inhibition rate was calculated using the following formula: .

[0031] From 118 strains, 30 strains with inhibitory effects against three pathogens were initially screened. After further screening, five strains with inhibition rates greater than 40% were selected and named MC417, MC124, MC130, MC211, and MC420, respectively. Among them, MC417 showed the highest inhibition rate against the three pathogens (e.g., Figure 2 As shown in the figure, MC417 was selected for subsequent experiments. This strain MC417 was deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M20242589, address: Wuhan University, Wuhan, China, on November 18, 2024.

[0032] 1.3 Strain Identification 1.3.1 16S rRNA Sequence analysis Strain MC417 was inoculated in LB solid medium for 24h, 0.2ml sterilized PCR tube was taken, 10μL ddH2O was added, a single colony was picked up with a sterile toothpick and stirred in the PCR tube, 27F: 5'-AGAGTTTGATCCTGGCTCAG-3'(SEQ ID NO.2) and 1492R: 5'-GGTTACCTTGTTACGACTT-3'(SEQ ID NO.3) primers were used to amplify 16S rDNA sequence. The PCR reaction system and conditions were carried out according to Table 1 to Table 2. After PCR amplification, the product was detected by 1% agarose gel electrophoresis, and there was obvious characteristic band.

[0033] Table 1 16S rRNA PCR reaction system used

[0034] Table 2 16S rRNA PCR reaction conditions used

[0035] The PCR amplification product was sent to Beijing Qianke Biotechnology Co., Ltd. for sequencing, and the obtained sequence was analyzed by Blast and compared with the sequence in the gene library. The results showed that the strain was most similar to B-2 and KTT-24, with a homology of 99.86%, and was similar to B-2 and KTT-24. Bacillus sonorensis strain B-2 and Bacillus sonorensis strain KTT-24, with a homology of 99.86%, and was similar to B-2 and KTT-24. Bacillus sonorensis strainBacillus sonorensis ), and the strain was named as Bacillus sonorensis MC417 ( Bacillus sonorensis Bacillus sonorensis MC417 and its related strains were based on the phylogenetic tree of 16S rRNA rRNA sequence as shown in Figure 3 .

[0036] 1.3.2 Amplification and phylogenetic analysis of housekeeping genes To further clarify the relationship between the strain and its related strains, three housekeeping genes, gyrA, gyrB and rpoB, were amplified and sequenced. The primers and amplification conditions are shown in Tables 3 and 4. The sequences of the three housekeeping genes were concatenated in the order of gyrA-gyrB-rpoB, and the phylogenetic tree was constructed using the Neighbor-Joining method with MEGA-X software. The results are shown in Figure 4 , the similarities of the housekeeping genes gyrA, gyrB and rpoB of strain MC417 and its Bacillus sonorensis NBRC 101234 standard strain reached 98%.

[0037] Table 3 PCR primers used in this experiment

[0038] Table 4 PCR reaction conditions for amplifying housekeeping genes

[0039] Example 2 Determination of salt tolerance of the strain First, strain MC417 was activated in LB liquid medium, and then the activated strain was inoculated into LB liquid medium containing 0, 5%, 9%, 13%, 15%, and 17% NaCl (w / v) at a inoculation amount of 1%, pH 10.0, 28°C, 150 rpm shaking culture for 48 h, and finally the OD 600 value was determined. At the same time, LB medium without inoculation was used as a control (CK), and each treatment was repeated three times.

[0040] The results showed that strain MC417 exhibited certain tolerance to alkaline high-salt environment. At pH 10.0, when the salt concentration of the medium was less than 15%, the growth of the strain was not significantly inhibited; when the salt concentration reached 15%, the growth of the strain was inhibited to a certain extent, and the growth rate decreased by 34.4% ( Figure 5 ); when the salt concentration was further increased to 17%, the growth of the strain was significantly inhibited, and the growth rate decreased by 91.9%. In addition, it was found that the strain NaCl exhibited good growth adaptability in the environment of pH 10.0 and salt concentration of 15% ( Figure 6 ).

[0041] Identification of IAA production function of Example 3 Prepare reagents according to the following proportions: 1) IAA detection medium: Proteose peptone 5.0 g, yeast extract 1.5 g, beef extract 1.5 g, NaCl 5.0 g, tryptophan 0.5 g, dissolved in deionized water and diluted to 1000 mL, pH 7.0, 121 ℃ high pressure steam sterilization for 20 min.

[0042] 2) IAA detection reagent Solution I (10 mM phosphoric acid): 1 mL of phosphoric acid is dissolved in 13.48 mL of deionized water to obtain 1 M phosphoric acid, which is diluted by 100 times to obtain 10 mM phosphoric acid.

[0043] 3) IAA detection reagent Solution II (Salkowskis colorimetric solution): Mix 1 mL of 0.5 M FeCl3 and 50 mL of 35% HCIO4.

[0044] Inoculate the MC417 strain in LB liquid medium and shake culture (28°C, 150 rpm) for 12 h, inoculate in IAA detection medium at 1% inoculation amount, and shake culture under the same conditions for 7 d. Centrifuge the bacterial liquid at 12000 rpm for 5 min, take 500 μL of supernatant in a 2 mL centrifuge tube, add 25 μL of 10 mM phosphoric acid and 1 mL of Salkowskis colorimetric solution, mix, and react at room temperature for 25 min. Measure the absorbance at 530 nm, and calculate the IAA production according to the standard curve. The results show that the amount of IAA produced by MC417 is 7.01 μg / mL.

[0045] Example 4: Strain MC417 for controlling tobacco diseases 1. Preparation of MC417 bacterial agent Inoculate the activated Bacillus sonorensis MC417 into LB medium for culture; the inoculation amount is 1.2%, the culture temperature is 28°C, the rotation speed is 200 rpm, and the culture time is 24 h to obtain MC417 liquid bacterial agent, and adjust the OD 600 = 1.0 × (1 × 10 8 CFU mL -1 ).

[0046] 2. Application of MC417 bacterial agent The experiment was carried out by leaf infection in vitro, and was divided into two groups with 6 replicates in each group. The specific treatment methods are as follows: CK: No bacterial agent treatment; MC417: Inoculated with MC417 bacterial agent.

[0047] Nicotiana benthamiana and K326 were used as test samples. Five layers of sterile filter paper were placed in a culture dish (130 x 130 mm), and the filter paper was soaked with 10 mL of sterile water. The leaves of N. benthamiana and K326 grown to the six-leaf stage were washed clean with sterile water and placed on the wet filter paper. The MC417 strain fermentation liquid was uniformly sprayed on the leaves, and the medicament was fully absorbed by incubating at 22°C. After the leaves were dried, the =3 mm pathogenic fungus cake was attached to the surface of the leaves (N. benthamiana and K326 were inoculated with P. nicotianae, P. nicotianae, and F. oxysporum, respectively), and incubated at 22°C for 3 days. Sterile water was used as a negative control. Five replicates were set for each treatment, and one leaf was placed in each replicate. The lesion area was measured after the control was completely diseased, and the experiment was repeated three times. Photographs were taken under bright field and ultraviolet light (365 nm; Analytik Jena US, Upland, CA, USA) 72 h after inoculation, and the lesion area size was measured using the software APS Assess (APS Press, USA). The control effect was calculated according to the formula: , Ca represents the lesion area of the control group, and Ta represents the lesion area of the treatment group.

[0048] The results of tobacco in vitro leaf disease control showed that the three kinds of pathogens could infect tobacco in vitro leaves and produce lesions ( Figure 7 , Figure 8 , Figure 9 ). As shown in Table 5, the lesion area produced by F. oxysporum was the largest, 2.04 ± 0.63 cm 2 , the lesion area produced after treatment with strain MC417 was 0.57 ± 0.29 cm 2 , which was significantly lower than that of the CK treatment, and the control effect was the best, 95.6%; the lesion area produced by P. nicotianae was 1.89 ± 0.52 cm 2 , the lesion area produced after treatment with strain MC417 was 0.49 ± 0.35 cm 2 , and the control effect was 65.5%; the lesion area produced by P. nicotianae was 1.47 ± 0.57 cm 2 , the lesion area produced after treatment with strain MC417 was 0.36 ± 0.21 cm 2 , and the control effect was 82.0% (Table 5). Figure 10 .

[0049] Table 5 Lesion area of three kinds of pathogens (cm 2 )

[0050] Note: The same column Indicates significant difference (p<0.05), Indicates extremely significant difference (p<0.01), Indicates extremely significant difference (p<0.001).

[0051] The application carries out pathogen infection and prevention test on ex vivo leaves, which can reflect the real effect, can observe whether the leaves have adverse reactions, etc., and provides more reliable basis for field application.

[0052] Example 5 Strain MC417 promotes tobacco seedling growth 1. Preparation of MC417 microbial agent The same as Example 4.

[0053] 2. Application of MC417 microbial agent The experiment is carried out by potting, which is divided into two groups, each group has 6 repeats, and the specific treatment method is as follows: CK: without any microbial agent treatment; MC417: inoculated with MC417 microbial agent treatment.

[0054] The Nicotiana benthamiana seeds with full particles are soaked in sterile water for 1 h, washed, soaked in 3% sodium hypochlorite for 30 min, washed, soaked in 70% ethanol for 10 min, and then washed with sterile water for 3 times. The seeds are soaked in sterile water at room temperature for 10-12 h. Two layers of sterile filter paper are laid in a large culture dish, the seeds are arranged on the filter paper, and 20 mL of sterilized distilled water is used to soak the filter paper. Cover the culture dish with sterile gauze. Several small holes are punched on the surface of the culture dish covered with plastic wrap, and the culture dish is incubated at 28°C until the seeds are white. About 30 mL of prepared bacterial suspension is placed in the culture dish, and the Nicotiana benthamiana with uniform germination is soaked for 5 h. The fermentation medium without inoculation of bacterial liquid is used as a blank control.

[0055] The seeds are transplanted into the cultivation substrate (nutrient soil: vermiculite = 1:1, volume ratio), and the bacterial liquid is added by root irrigation, 50 mL of bacterial liquid is added per pot, and the fermentation medium without inoculation of bacterial liquid is used as a blank control. Water 50 mL every two days after sowing, and 10 days later, supplement MS nutrient solution (purchased from Qingdao Haibo Biotechnology Co., Ltd., 4.74 g is weighed and dissolved in 1000 ml of distilled water, and autoclaved at 121°C for 30 minutes).

[0056] The samples are collected and the growth-promoting ability of MC417 microbial agent on Nicotiana benthamiana seedlings is detected on the 30th day after sowing. The height, leaf length and width, and dry weight of tobacco seedlings are determined.

[0057] The results show that the strain MC417 has good growth-promoting effect on Nicotiana benthamiana seedlings (such as Figure 11The plant height of tobacco seedlings was significantly increased by 4.3%, the maximum leaf length was significantly increased by 5.7%, the maximum leaf width was increased by 3.4%, the maximum leaf area was significantly increased by 9.3%, and the dry weight was significantly increased by 6.7%, as shown in Table 6.

[0058] Table 6 Promoting effect of strain MC417 on Nicotiana benthamiana

[0059] Note: In the same column Indicates a significant difference (p<0.05), Indicates a very significant difference (p<0.01), Indicates a very significant difference (p<0.001).

[0060] The above only describes the preferred embodiments of the present application, but those skilled in the art can make various changes or modifications to the present application on the basis of the present application, and these equivalent forms also fall within the scope defined by the claims of the present application.

Claims

1. A Bacillus sonorensis strain characterized in that, It is Bacillus sonorensis (Bacillus sonorensis) Bacillus sonorensis The number is MC417, and it is preserved in China Center for Type Culture Collection, and the preservation number is CCTCC NO: M20242589.

2. A fermentation product, characterized in that, produced by fermenting a culture of the Bacillus sonorensis of claim 1.

3. An inoculant characterized in that, comprising one or more of the Bacillus sonorensis of claim 1, a fermenting culture thereof, or a fermentation product thereof.

4. Use of the Bacillus sonorensis of claim 1, the fermentation product of claim 2, or the inoculant of claim 3 for salt-alkali tolerance, tobacco disease control, and growth promotion.

5. Use according to claim 4, characterized in that, The tobacco disease comprises one or more of tobacco anthracnose caused by Colletotrichum gloeosporioides, tobacco black shank caused by Phytophthora nicotianae, or tobacco fusarium root rot caused by Fusarium oxysporum.

6. A method for controlling tobacco diseases, characterized by, The Bacillus sonorensis of claim 1, the fermentation product of claim 2, or the inoculant of claim 3 is applied to tobacco.

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