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, and in particular to a Sonora Desert Bacillus, its application, and a method for preventing and controlling tobacco diseases. Background Art

[0002] Flue-cured tobacco is the most widely cultivated type of tobacco in the world and one of my country's important economic crops. The cultivated area of ​​flue-cured tobacco in my country has reached 1.4 million hectares, and the yield remains at 1,700 kg / hm2. 2 China ranks first in the world in terms of both cultivated area and yield. Driven by limited arable land and economic efficiency, my country has a serious problem of continuous tobacco cropping. This long-term continuous cropping can cause soil microorganisms to shift from high-fertility "bacterial" to low-fertility "fungal" microorganisms, exacerbating 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. They are characterized by widespread distribution, difficulty in prevention and control, and high combined morbidity. The combined infection of these diseases poses a serious threat to tobacco growth and yield, and an effective integrated prevention and control strategy is urgently needed. Tobacco black shank is caused by the tobacco pathogenic variant of Phytophthora parasiticus and is an oomycete disease. Its primary source of infection is mycelium or chlamydospores attached to diseased plant debris. These spores can be spread by rainwater and irrigation water, usually invading the plant from the base of the tobacco stem and producing large numbers of zoospores that spread rapidly in the field, representing an important source of secondary infection for the disease. Tobacco Fusarium root rot is caused by the genus Fusarium, especially Fusarium oxysporum. Its primary sources of infection include dormant chlamydospores, mycelium, or conidia in the soil and diseased debris. Fusarium has a strong spore-producing ability and diverse transmission pathways. The pathogen forms conidia after invading through root wounds, and then spreads through wind and rain, diseased debris, or agricultural operations. In addition, the pathogen can also be spread through the air, infecting the plant's vascular system, destroying transport tissues, and further exacerbating the spread of the disease. Tobacco anthracnose is a common leaf disease of tobacco that can occur at all growth stages, especially the seedling stage, which is most vulnerable. Conidia are spread to leaves or stems by wind and rain. After germination, they form appressoriums, penetrate the epidermis to produce irregular primary hyphae, and then invade adjacent cells through secondary hyphae. Within a few days, small water-soaked spots gradually expand to about 3 mm, eventually forming dark brown necrotic spots, causing the leaves to wilt.

[0004] Biological control is a key area of ​​plant disease prevention and control, attracting widespread attention due to its abundant resources, wide selection, and environmentally friendly properties. Bacillus sp., with its high activity and excellent environmental adaptability, has become a research focus. In tobacco disease biological control, Bacillus velez YC11 demonstrates significant efficacy against black shank, bacterial wilt, root rot, and root-knot nematodes (Li Yanyan et al., 2022); Bacillus subtilis GUMT323 effectively antagonizes tobacco black shank pathogen (Ding Haixia, 2018); and Bacillus amyloliquefaciens B011 exhibits significant inhibitory activity against tobacco bacterial wilt and black shank pathogens (Zhou Xiangping et al., 2015). Bacillus sonorensis, a salt-tolerant strain isolated from Sonoran Desert soil, is capable of surviving in extreme environments and has broad potential for application (Palmisano et al., 2001). There are currently no research reports on its use in preventing and controlling tobacco anthrax, tobacco black shank and tobacco Fusarium root rot. Summary of the Invention

[0005] In order to solve the technical problems existing in the prior art, one of the purposes of this application is to provide a Sonora Desert Bacillus strain for tobacco, which can effectively inhibit pathogenic fungi and reduce the incidence of diseases.

[0006] To achieve the above objectives, the technical solutions of this application are as follows:

[0007] The present application provides a Sonora Desert Bacillus, which is Sonora Desert Bacillus (Bacillus sonorensis) numbered MC417, deposited in the China Center for Type Culture Collection, with a deposit number of CCTCCNO: M20242589.

[0008] The present application provides a fermentation product produced by fermentation culture of the Sonora Desert Bacillus. The fermentation broth can be prepared as follows: 5.0 g yeast powder, 10.0 g peptone, 12.0 g glucose, 5.0 g NaCl, 0.1 g K2HPO43H2O, 20.0 g corn starch, 5.0 g soybean meal, 0.7 g KH2PO4, 0.2 g MgSO4, 1000 mL distilled water, pH = 7.0, and autoclaved at 121°C for 30 min.

[0009] The present application provides a bacterial agent comprising one or more of the above-mentioned Sonora Desert Bacillus, its fermentation culture or its fermentation product.

[0010] Preferably, the bacterial agent can survive in an environment with a salt concentration of 15% and a pH of 10.

[0011] The present application provides the use of the Sonora Desert Bacillus, the fermentation product or the bacterial agent in salt-alkali tolerance, tobacco disease prevention and control, and growth promotion.

[0012] Preferably, the tobacco diseases include one or more of tobacco anthracnose caused by Colletotrichum anthracnose, tobacco black shank caused by Phytophthora spp., or tobacco Fusarium root rot caused by Fusarium oxysporum.

[0013] The present application provides a method for preventing and controlling tobacco diseases, wherein the Sonora Desert Bacillus, the fermentation product or the bacterial agent is applied to tobacco.

[0014] The beneficial effects of this application are:

[0015] The present application isolated a new Sonora Desert Bacillus MC417, which has a strong inhibitory effect on three plant pathogenic fungi: tobacco anthracnose, tobacco black shank pathogen and tobacco Fusarium root rot pathogen.

[0016] This application is applied to the biological control of tobacco anthracnose caused by Colletotrichum spp., tobacco black shank caused by Phytophthora spp., and tobacco Fusarium root rot caused by Fusarium oxysporum, significantly reducing the incidence of the diseases. It also has the growth-promoting function of producing indoleacetic acid, which can improve agronomic traits such as plant height, biomass, and leaf area in the tobacco seedling stage. It has excellent application prospects in biological microbial agents and disease prevention and growth promotion.

[0017] The control method of the present application can effectively prevent and treat tobacco anthrax caused by anthrax bacteria, tobacco black shank caused by Phytophthora, and tobacco Fusarium root rot caused by Fusarium oxysporum. Continuous tobacco cultivation causes allelopathic effects in the rhizosphere secondary metabolism, leading to the coexistence of multiple pathogenic fungi. Conventional strains have difficulty antagonizing multiple pathogens simultaneously, resulting in their inability to produce a control effect. The control method of the present application targets tobacco diseases, utilizing Sonora Desert Bacillus MC417 to effectively inhibit pathogenic fungi while promoting tobacco growth. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the colony morphology of the Sonora Desert Bacillus MC417 of the present invention.

[0019] Figure 2 This is a diagram showing the antagonistic effect of Sonora Desert Bacillus MC417 on three pathogens.

[0020] Figure 3 This is a phylogenetic tree of Sonora Desert Bacillus MC417 and its related strains based on 16S rRNA sequences.

[0021] Figure 4 This is a phylogenetic tree of the concatenation of three housekeeping gene sequences of Sonora Desert Bacillus MC417 of the present invention.

[0022] Figure 5 This is a graph showing the salt-alkali tolerance test results of Sonora Desert Bacillus MC417 of the present invention.

[0023] Figure 6 The figure shows the growth of Sonora Desert Bacillus MC417 of the present invention in culture medium with pH=10.0 and different salt concentrations.

[0024] Figure 7 This is a diagram showing the control effect of Sonora Desert Bacillus MC417 on tobacco black shank pathogens in detached leaves.

[0025] Figure 8 This is a diagram showing the control effect of Sonora Desert Bacillus MC417 on tobacco anthracnose pathogens in detached leaves.

[0026] Figure 9 This is a diagram showing the control effect of Sonora Desert Bacillus MC417 on tobacco Fusarium root rot pathogens in detached leaves.

[0027] Figure 10 This is the result of Sonora Desert Bacillus MC417 controlling the lesion area of ​​three pathogens in detached leaves.

[0028] Figure 11 This is a comparison of the growth-promoting effects of Sonora Desert Bacillus MC417 on Nicotiana benthamiana. DETAILED DESCRIPTION

[0029] 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 provided only for the purpose of illustration and are not intended to limit the scope of the present application. Those skilled in the art may make various modifications and substitutions to the present application without departing from the purpose and spirit of the present application.

[0030] The present application discloses the use of Sonora Desert Bacillus MC417, fermentation products, or microbial agents for tobacco disease prevention and control and growth promotion. Tobacco diseases include one or more of tobacco anthracnose caused by Colletotrichum anthracnose, tobacco black shank caused by Phytophthora, or tobacco Fusarium root rot caused by Fusarium oxysporum. The tobacco growing environment differs from that of other crops. During continuous cropping, multiple pathogenic fungi coexist. It is difficult for general strains to simultaneously antagonize multiple pathogenic fungi, thereby achieving a control effect. The present application has a good control effect on the aforementioned tobacco diseases, with a control effect on tobacco Fusarium exceeding 95.6%.

[0031] Example 1 Isolation, purification and identification of strain MC417

[0032] 1.1 Isolation and purification of strain MC417

[0033] In 2024, rhizosphere soil was collected from the continuous tobacco fields in Sanying Town, Eryuan County, Dali Bai Autonomous Prefecture, Yunnan Province. About 10 g of soil sample was weighed and added to a triangular flask containing glass beads and 90 mL of sterile saline. The soil suspension was fully shaken at 180 rpm for 30 min and allowed to stand for 10 min to obtain a soil suspension. 1 mL of the supernatant was taken and diluted with sterile saline for 10 min. -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 (yeast powder 5 g / L, peptone 10 g / L, NaCl 10 g / L, agar 18 g / L), and each gradient was repeated 3 times. After constant temperature culture at 28 ° C for 2 days, the strain MC417 ( Figure 1 ), and a total of 118 rhizospheric bacteria were isolated.

[0034] 1.2 Screening of antagonistic strains

[0035] The biocontrol efficacy of isolated rhizospheric bacteria was tested using a standoff culture method against three pathogenic fungi: Colletotrichum nicotianae, Phytophthoranicotianae, and Fusarium oxysporum, the causative agent of tobacco Fusarium root rot. The pathogens were first activated on a PDA plate. A 5mm borer was used to create a bacterial cake, which was then inoculated onto the center of the plate. The 118 isolated rhizospheric bacteria were then inoculated 2.5 cm from the cake and incubated at 25°C for 4-7 days. The colony diameters of the pathogens in the treatments with and without rhizospheric bacteria inoculation were measured, and the inhibition rate was calculated using the following formula:

[0036]

[0037] 30 strains with inhibitory effects on the three pathogens were selected from 118 strains through preliminary screening. Then, 5 strains with inhibition rates greater than 40% were selected through secondary screening and named MC417, MC124, MC130, MC211 and MC420. Among them, MC417 had the highest inhibition rate against the three pathogens (e.g. Figure 2MC417 was selected for subsequent experiments. The strain MC417 was deposited with the China Center for Type Culture Collection (CCTCC No. M20242589) at Wuhan University, Wuhan, China, on November 18, 2024.

[0038] 1.3 Strain identification

[0039] 1.3.1 16S rRNA sequence analysis

[0040] Strain MC417 was inoculated into LB solid medium and cultured for 24 hours. 10 μL of ddH₂O was added to a 0.2 ml sterile PCR tube. A single colony was picked with a sterile toothpick and transferred to the PCR tube, stirring thoroughly. The 16S rDNA sequence was amplified using primers 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO. 2) and 1492R: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO. 3). The PCR reaction system and conditions were as shown in Tables 1 and 2. After PCR amplification, the product was analyzed by 1% agarose gel electrophoresis, revealing distinct characteristic bands.

[0041] Table 1 PCR reaction system used for 16S rRNA

[0042] Reagents Dosage (μL) MIX 12.5 27F 1 1492R 1 DNA 1 <![CDATA[ddH2O]]> 9.5

[0043] Table 2 PCR reaction conditions used for 16S rRNA

[0044]

[0045] The PCR amplification product was sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The obtained sequence was compared with the sequence in the gene library by Blast analysis. The results showed that the strain had the highest similarity with Bacillus sonorensis strain B-2 and Bacillus sonorensis strain KTT-24, with 99.86% homology. It also had a high similarity with Bacillus sonorensis strain KLH-26, with 99.72% homology. Based on the above identification results of strain MC417 (SEQ ID NO: 1:

[0046] Figure 3 shown.

[0047] 1.3.2 Housekeeping gene sequence amplification and phylogenetic analysis

[0048] To further clarify the relationship between the strain and its closely related strains, the 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 from beginning to end in the order gyrA-gyrB-rpoB, and the phylogenetic tree was constructed using the Neighbor-Joining method using MEGA-X software. The results are shown in Tables 3 and 4. Figure 4 As shown, the housekeeping genes gyrA, gyrB, and rpoB of strain MC417 are 98% similar to those of the standard strain Bacillus sonorensis NBRC 101234.

[0049] Table 3 PCR primers used in this experiment

[0050]

[0051] Table 4 PCR reaction conditions used to amplify housekeeping genes

[0052]

[0053] Example 2 Determination of salt tolerance of strains

[0054] First, the strain MC417 was activated in LB liquid medium. Then, the activated strain was inoculated into LB liquid medium containing 0, 5, 9, 13, 15, and 17% NaCl (w / v), pH 10.0 at a 1% inoculum size. The culture was shaken at 28°C and 150 rpm for 48 h. Finally, the OD 600 At the same time, LB medium without inoculation was used as the control (CK), and each treatment was repeated 3 times.

[0055] The results showed that strain MC417 showed a certain tolerance to alkaline high-salt environment. Under the condition of pH 10.0, when the salt concentration of the culture medium was lower 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, the study also found that the strain NaCl showed good growth adaptability in an environment with a pH of 10.0 and a salt concentration of 15% ( Figure 6 ).

[0056] Example 3 Identification of IAA production function

[0057] Prepare the reagents according to the following ratio:

[0058] 1) IAA assay medium: 5.0 g of peptone, 1.5 g of yeast extract, 1.5 g of beef extract, 5.0 g of NaCl, and 0.5 g of tryptophan were dissolved in deionized water and diluted to 1000 mL, pH 7.0, and sterilized by high-pressure steam at 121°C for 20 min.

[0059] 2) IAA detection reagent Solution I (10 mM phosphoric acid): 1 mL of phosphoric acid was dissolved in 13.48 ml of deionized water to obtain 1 M phosphoric acid, which was then diluted 100-fold to obtain 10 mM phosphoric acid.

[0060] 3) IAA detection reagent Solution II (Salkowskis colorimetric solution): Mix 1 mL of 0.5 M FeCl 3 and 50 mL of 35% HClO 4.

[0061] The MC417 strain was inoculated into LB liquid medium and incubated on a shaker (28°C, 150 rpm) for 12 hours. A 1% inoculum was then inoculated into IAA assay medium. After 7 days of incubation under the same shaking conditions, the culture was centrifuged at 12,000 rpm for 5 minutes. 500 μL of the supernatant was transferred to a 2 mL centrifuge tube, and 25 μL of 10 mM phosphoric acid and 1 mL of Salkowskis colorimetric solution were added. After mixing, the mixture was incubated at room temperature in the dark for 25 minutes. The absorbance was measured at 530 nm and the IAA yield was calculated using a standard curve. The results showed that MC417 produced 7.01 μg / mL of IAA.

[0062] Example 4: Control of tobacco diseases by strain MC417

[0063] 1. Preparation of MC417 inoculant

[0064] The activated Sonora Desert Bacillus MC417 was inoculated into LB medium for cultivation; the inoculation amount was 1.2%, the culture temperature was 28°C, the rotation speed was 200 rpm, and the culture time was 24 h to obtain the MC417 liquid bacterial agent, and the bacterial liquid OD was adjusted. 600 =1.0×(1×10 8 CFU mL -1 ).

[0065] 2. Application of MC417 microbial agent

[0066] The experiment was conducted by in vitro leaf infection and divided into two groups, with 6 replicates in each group. The specific treatment methods are as follows:

[0067] CK: no bacterial treatment;

[0068] MC417: Inoculated with MC417 bacterial agent.

[0069] Nicotiana benthamiana and Yunyan 87 were selected as test samples. Five layers of sterile filter paper were spread in a culture dish (130×130mm) and the filter paper was soaked with 10mL of sterile water. The leaves of Nicotiana benthamiana and Yunyan 87 that had grown to the six-leaf stage were cleaned with sterile water and placed on the moistened filter paper. The fermentation liquid of MC417 strain was evenly sprayed on the leaves and incubated at 22℃ to allow the agent to be fully absorbed. After the leaves were dried, a 3mm pathogenic fungus cake was pasted on the surface of the leaves (Nicotiana benthamiana and Yunyan 87 were inoculated with tobacco anthracnose, tobacco phytophthora and tobacco fusarium, respectively), and incubated at 22℃ for 3 days. Sterile water was used as the negative control. Five replicates were set for each treatment, with one leaf placed in each replicate. The lesion area was measured after the control was fully diseased. The experiment was repeated 3 times. 72 hours after inoculation, photos were taken under bright field and UV light (365 nm; Analytik Jena US, Upland, CA, USA), and the lesion area was measured using the software APS Assess (APS Press, USA). The control effect was calculated according to the formula:

[0070] Control effect (%) = (Ca-Ta) / Ca×100%,

[0071] Ca represents the lesion area of ​​the control group, and Ta represents the lesion area of ​​the treatment group.

[0072] The results of tobacco detached leaf disease control showed that all three pathogens could infect tobacco detached leaves and produce lesions ( Figure 7 、 Figure 8 、 Figure 9 As shown in Table 5, the lesion area produced by tobacco Fusarium oxysporum was the largest, at 2.04 ± 0.63 cm. 2 The lesion area after treatment with strain MC417 was 0.57±0.29cm 2 , which was significantly lower than that of CK treatment, and had the best control effect among the three pathogens, at 95.6%; the lesion area produced by tobacco anthracnose was 1.89±0.52cm 2 The lesion area after treatment with strain MC417 was 0.49 ± 0.35 cm 2 The control effect was 65.5%; the lesion area of ​​tobacco blight was 1.47±0.57cm 2 The lesion area after treatment with strain MC417 was 0.36±0.21cm 2 , the control effect was 82.0% ( Figure 10 ).

[0073] Table 5. Area of ​​lesions of three pathogens (cm 2 )

[0074]

[0075] Note: In the same column, * indicates significant difference (p<0.05), ** indicates extremely significant difference (p<0.01), and *** indicates extremely significant difference (p<0.001).

[0076] This application conducted pathogen infection and control tests on detached leaves, which can reflect the real effect and observe whether the leaves have adverse reactions, etc., providing a more reliable basis for field application.

[0077] Example 5: Strain MC417 promotes tobacco seedling growth

[0078] 1. Preparation of MC417 inoculant

[0079] Same as Example 4.

[0080] 2. Application of MC417 microbial agent

[0081] The experiment was conducted in potted plants and divided into two groups, with 6 replicates in each group. The specific treatment methods are as follows:

[0082] CK: no bacterial treatment;

[0083] MC417: Inoculated with MC417 bacterial agent.

[0084] Select full-grained Nicotiana benthamiana seeds and soak them in sterile water for 1 hour. After washing, soak them in 3% sodium hypochlorite for 30 minutes. After washing, soak them in 70% ethanol for 10 minutes. Then wash them 3 times with sterile water and soak them in sterile water at room temperature for 10-12 hours. Place two layers of sterile filter paper in a large culture dish, arrange the seeds on them, soak the filter paper with 20mL of sterilized distilled water, and cover with a layer of sterile gauze. Cover the entire culture dish with a layer of plastic wrap, poke several small holes on its surface, and culture at 28°C until the seeds turn white. Use about 30mL of the prepared bacterial suspension in the culture dish, put the Nicotiana benthamiana with the same white color, and soak the seeds for 5 hours. At the same time, soak the seeds in fermentation medium without inoculation of bacterial liquid as a blank control.

[0085] The seeds were transplanted into a cultivation medium (nutrient soil: vermiculite = 1:1, volume ratio) and the bacterial solution was added by root irrigation. 50 mL of bacterial solution was added to each pot. A blank control was also used for root irrigation with fermentation medium without bacterial solution. Water was applied every two days after sowing. After 10 days, MS nutrient solution (purchased from Qingdao Haibo Biotechnology Co., Ltd., 4.74 g was weighed, dissolved in 1000 mL of distilled water, and autoclaved at 121°C for 30 minutes) was added.

[0086] Samples were collected 30 days after sowing to test the growth-promoting ability of MC417 on Nicotiana benthamiana seedlings, and the plant height, leaf length and width, and dry weight of tobacco seedlings were measured.

[0087] The results showed that strain MC417 had a good growth-promoting effect on Nicotiana benthamiana seedlings (e.g. Figure 11 As shown in Table 6), the height of tobacco seedlings increased significantly by 4.3%, the maximum leaf length increased significantly by 5.7%, the maximum leaf width increased by 3.4%, the maximum leaf area increased significantly by 9.3%, and the dry weight increased significantly by 6.7%.

[0088] Table 6 Growth-promoting effect of strain MC417 on Nicotiana benthamiana

[0089]

[0090] Note: In the same column, * indicates significant difference (p<0.05), ** indicates extremely significant difference (p<0.01), and *** indicates extremely significant difference (p<0.001).

[0091] The above is only a preferred embodiment of the present application. However, based on the present application, those skilled in the art can make various changes or modifications to the present application, and these equivalent forms also fall within the scope defined by the claims of the present application.

Claims

1. A Sonora Desert Bacillus, characterized in that The strain is Bacillus sonorensis, numbered MC417, and deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: M20242589.

2. A fermentation product, characterized in that The product is produced by fermentation culture of the Sonora Desert Bacillus according to claim 1.

3. A bacterial agent, characterized in that The method comprises one or more of the Sonora Desert Bacillus, its fermentation culture or its fermentation product according to claim 1.

4. Use of the Sonora Desert Bacillus according to claim 1, the fermentation product according to claim 2, or the bacterial agent according to claim 3 in salt-alkali tolerance, tobacco disease prevention and control, and growth promotion.

5. The use according to claim 4, characterized in that The tobacco diseases include one or more of tobacco anthracnose caused by Colletotrichum anthracnose, tobacco black shank disease caused by Phytophthora spp., or tobacco Fusarium root rot caused by Fusarium oxysporum.

6. A method for preventing and treating tobacco diseases, characterized in that: The Sonora Desert Bacillus according to claim 1, the fermentation product according to claim 2, or the bacterial agent according to claim 3 is applied to tobacco.

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