Bacillus subtilis for preventing and controlling fungal diseases of Pinellia ternata and its application

Abstract

The present invention provides a strain of Bacillus subtilis for preventing and treating fungal diseases of Pinellia ternata and its application, belonging to the field of microbial technology. The present invention provides a strain of Bacillus subtilis WBT6, with a deposit number of CCTCC NO: M20232028. The present invention further provides a bacterial agent prepared by Bacillus subtilis WBT6, and the application of Bacillus subtilis WBT6 and its bacterial agent in preventing and treating fungal diseases of Pinellia ternata and inhibiting a variety of pathogenic fungi of traditional Chinese medicine, which is of great significance to the healthy development of the Pinellia ternata industry.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a Bacillus subtilis strain for preventing and treating fungal diseases of Pinellia ternata and application thereof. Background Art

[0002] Pinellia ternata belongs to the genus Pinellia of the Araceae family. It was recorded as early as in the "Shennong Bencao Jing" and has a history of medicinal use of more than 2,000 years. Pinellia ternata is used as medicine with underground tubers, which have the functions of drying dampness and resolving phlegm, relieving adverse reactions and stopping vomiting. However, in recent years, the problem of continuous cropping of Pinellia ternata has become prominent, pests and diseases have become more frequent, and fungal diseases have occurred in a variety of ways, among which white rot is more serious. At present, there are relatively few studies on the prevention and control of fungal diseases in Pinellia ternata, and there is no registered drug. Farmers often face the dilemma of no drugs available and no guidance. The indiscriminate use of various agents, on the one hand, leads to an increased risk of pathogen resistance and cannot effectively prevent and control diseases. On the other hand, it seriously affects the quality and yield of Pinellia ternata and also damages the ecological environment. Therefore, it is urgent to carry out research on the prevention and control of Pinellia ternata fungal diseases, explore effective prevention and control methods, and ensure the yield and quality of Pinellia ternata medicinal materials.

[0003] Biocontrol bacteria are beneficial microorganisms, whose strains can kill pathogens and reduce their numbers, thereby controlling the occurrence of diseases. Soil microorganisms, crop rhizosphere microorganisms, plant endophytes, etc. are the main sources of biocontrol bacteria resources. For example, the main types of Bacillus include Bacillus subtilis and Bacillus amyloliquefaciens, which have a wide range of antibacterial effects. Due to their outstanding advantages, they have significant effects on the prevention and treatment of various plant diseases and are widely used in the field of biocontrol. Therefore, screening biocontrol bacteria that are beneficial to Pinellia fungal diseases is of great significance to the healthy development of the Pinellia industry. Summary of the invention

[0004] In order to solve the problems existing in the prior art, the purpose of the present invention is to provide a Bacillus subtilis WBT6 and a bacterial agent thereof, wherein the Bacillus subtilis WBT6 has a preservation number of CCTCCNO: M20232028 and has a significant inhibitory effect on a variety of pathogenic fungi of traditional Chinese medicines.

[0005] The present invention also aims to provide a method for preventing and treating fungal diseases of Pinellia ternata, which can effectively prevent or treat fungal diseases of Pinellia ternata.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The invention provides a Bacillus subtilis WBT6, and the deposit number of the Bacillus subtilis WBT6 is CCTCCNO: M20232028.

[0008] The present invention also provides a bacterial agent prepared from the above-mentioned Bacillus subtilis WBT6.

[0009] Preferably, the bacterial agent is the fermentation broth of Bacillus subtilis WBT6.

[0010] Preferably, the method for preparing the fermentation broth comprises: inoculating Bacillus subtilis WBT6 into a fermentation medium to obtain a fermentation broth; the fermentation medium comprises: 8-12 g / L peptone, 4-6 g / L yeast extract powder, 8-12 g / L NaCl, 4-6 g / L glucose, and pH=6.5-7.5.

[0011] Preferably, the culture conditions are: constant temperature culture at 30-37°C and 200-250 r / min for 36-72 h.

[0012] The present invention also provides the use of the Bacillus subtilis WBT6 or its bacterial agent in preventing and controlling fungal diseases of Pinellia ternata.

[0013] Preferably, the pathogenic fungi of the fungal diseases of Pinellia ternata include Sclerotinia pinellia, Sclerotinia pinellia, and Coriolus pinellia.

[0014] The present invention also provides a method for preventing and controlling fungal diseases of Pinellia ternata, wherein Bacillus subtilis WBT6 or a bacterial agent thereof is used for prevention and control during the growth of Pinellia ternata.

[0015] The present invention also provides the use of Bacillus subtilis WBT6 or its bacterial agent in inhibiting pathogenic fungi of traditional Chinese medicine.

[0016] Preferably, the pathogenic fungi of traditional Chinese medicine include pathogenic fungi of the genus Sclerotium of Coptis chinensis, Fusarium oxysporum of tobacco, Fusarium oxysporum of polygonatum, Fusarium acuminate of Ophiopogon japonicus, Alternaria japonica, Botrytis cinerea of ​​Panax notoginseng, Subsporangium of Bupleurum chinense, Botrytis cinerea of ​​Paris polyphylla and Botrytis cinerea of ​​Fritillaria hupehensis.

[0017] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0018] The present invention obtains a Bacillus subtilis WBT6 for the first time, and prepares a bacterial agent using the strain, which can inhibit a variety of pathogenic fungi of traditional Chinese medicines, can effectively prevent or treat fungal diseases of Pinellia ternata, and is of great significance to the healthy development of the Pinellia ternata industry.

[0019] Biological Deposit Description

[0020] The biocontrol bacterium WBT6 (Bacillus subtilis WBT6) is classified and named Bacillus subtilis (Bacillussubtilis), the preservation unit: China Center for Type Culture Collection (CCTCC for short), the preservation unit address: Wuhan University, Wuhan, China, the preservation number: CCTCC NO: M20232028, the preservation date is October 25, 2023. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 :The plate photos of the isolates with obvious antibacterial effect on the pathogenic fungus QJ3;

[0022] Figure 2 : Inhibition rate of 9 isolated strains against QJ3;

[0023] Figure 3 : The inhibition rate of 9 isolated strains against BXHP2;

[0024] Figure 4 : Inhibition rate of 9 isolated strains against QJFY2;

[0025] Figure 5 :The inhibitory effect of biocontrol bacteria WBT6 on three Pinellia pathogenic fungi;

[0026] Figure 6 : Colony morphology of biocontrol bacteria WBT6;

[0027] Figure 7 : Phylogenetic tree of WBT6 based on 16S rDNA sequence;

[0028] Figure 8 :In vitro control effect of biocontrol bacteria WBT6 on Pinellia ternata pathogenic fungus QJ3;

[0029] Fig. 9 : Safety test of biocontrol bacteria WBT6 on Pinellia ternata. DETAILED DESCRIPTION

[0030] The present invention provides a Bacillus subtilis WBT6, and the deposit number of the Bacillus subtilis WBT6 is CCTCC NO: M20232028. The Bacillus subtilis WBT6 of the present invention is separated and purified from the soil of a Pinellia ternata planting base, and is proved to belong to Bacillus subtilis by identification. It was deposited in the China Center for Type Culture Collection on October 25, 2023, and the depository address is: Wuhan University, Wuhan, China.

[0031] The present invention also provides a bacterial agent, which is prepared by Bacillus subtilis WBT6. The bacterial agent of the present invention is preferably a fermentation broth of Bacillus subtilis WBT6. The preparation method of the fermentation broth of Bacillus subtilis WBT6 is that Bacillus subtilis WBT6 is inoculated in a fermentation medium to obtain a fermentation broth. The fermentation medium includes: peptone 8-12g / L, yeast extract powder 4-6g / L, NaCl 8-12g / L, glucose 4-6g / L, pH=6.5-7.5; the fermentation medium preferably includes peptone 9-11g / L, yeast extract powder 4-6g / L, NaCl 9-11g / L, glucose 4-6g / L, more preferably includes peptone 10g / L, yeast extract powder 5g / L, NaCl 10g / L, glucose 5g / L; the pH of the culture medium is preferably 6.8-7.2, more preferably 7. The culture conditions are: constant temperature culture at 30-37°C and 200-250r / min for 36-72h; the culture temperature is preferably 32-37°C, and the culture time is preferably 45-60h. The fermentation medium of the present invention is prepared with water, and the water is preferably deionized water. As an optional embodiment, the present invention picks a fresh single colony of WBT6 cultured on a NA plate, inoculates it in a conical flask of NB culture medium for expansion culture, and then transfers it to a conical flask containing fermentation medium (liquid) according to 10% (v / v) of the bacterial liquid to obtain a WBT6 fermentation liquid.

[0032] The present invention also provides the use of the above-mentioned Bacillus subtilis WBT6 or its bacterial agent in preventing and treating fungal diseases of Pinellia ternata, wherein the pathogenic fungi in the fungal diseases of Pinellia ternata include Sclerotium rolfsii (QJ3), Sclerotinia sclerotiorum (BXHP2), and Choanephora cucurbitarum (QJFY2). The antibacterial rates of the Bacillus subtilis WBT6 of the present invention on the pathogenic fungi QJ3, BXHP2 and QJFY2 of Pinellia ternata can reach 81.54%, 80.95% and 74.59%, respectively.

[0033] The present invention also provides a method for preventing and controlling fungal diseases of Pinellia ternata: during the growth of Pinellia ternata, Bacillus subtilis WBT6 or its bacterial agent is used for prevention and control. As an optional embodiment, the present invention irrigates the fungal disease site with the bacterial agent of Bacillus subtilis WBT6.

[0034] The present invention also provides the use of Bacillus subtilis WBT6 or its bacterial agent in inhibiting pathogenic fungi of traditional Chinese medicine, wherein the pathogenic fungi of traditional Chinese medicine include Sclerotium coptidis (LC1), Fusarium oxysporum of tobacco (QK-4), Fusarium hyssopus (HYTJ), Fusarium acuminata (MD1), Alternaria nicotianae (YCCX1), Botrytis cinerea of ​​Panax notoginseng (ZJSYB1), Subsporium glomerulosa (CHYB), Botrytis cinerea of ​​Paris (ES8) and Botrytis cinerea of ​​Fritillaria hupehensis (BM XTX2), the bacteriostasis rate of the Bacillus subtilis WBT6 of the present invention to LC1 can reach more than 70%, the bacteriostasis rate to QK-4 can reach more than 57%, the bacteriostasis rate to HYTJ can reach more than 67%, the bacteriostasis rate to MD1 can reach more than 63%, the bacteriostasis rate to YCCX1 can reach more than 77%, the bacteriostasis rate to ZJSYB1 can reach more than 65%, the bacteriostasis rate to CHYB can reach more than 74%, the bacteriostasis rate to ES8 can reach more than 73%, and the bacteriostasis rate to BMXTX2 can reach more than 95%. The Bacillus subtilis WBT6 of the present invention or its bacterial agent has a high bacteriostasis rate and can be used for the prevention and treatment of fungal diseases of traditional Chinese medicines.

[0035] The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0036] In the following embodiments, unless otherwise specified, all of them are conventional methods.

[0037] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0038] Example 1

[0039] 1. Separation and screening

[0040] The soil samples were collected from the Pinellia ternata planting base in Zhugentan Town, Qianjiang City. The soil was collected using the five-point sampling method. The finely ground soil samples were placed in a centrifuge tube filled with sterile water and fully shaken to make a concentration of 10 -1 The soil suspension was diluted step by step to 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , and obtain soil suspensions of different concentrations. Use a pipette to take 20 μL of 10 -5 and 10 -6The soil suspension was spread on the NA medium plate and then placed in an incubator at 30°C for 48 hours to obtain isolated strains with different morphologies. Single colonies with different morphologies were selected and streaked on the NA plate using the three-zone streak method and cultured for 24 hours. A total of 106 bacterial strains were isolated and all isolated strains were stored in a refrigerator at 4°C for future use.

[0041] The confrontation culture method was used to screen out biocontrol bacteria with antibacterial effects. The steps of the confrontation culture method are: use an inoculation loop to inoculate the isolated strain (bacterial solution OD 600 =1.0) were spread along the center (i.e., the longest diameter) of a PDA plate (diameter = 9 cm), and after culturing at 28°C for 24 hours, a round bacterial cake was punched on a plate of Sclerotium solani (QJ3), the pathogen of Pinellia ternata, which had been activated for 2 days, using a 5mm diameter puncher. Two bacterial cakes were placed on the edges of the longest diameter of the PDA plate, and the isolated strain was inoculated in the middle. The plate without the isolated strain was used as the blank control. Each treatment group was repeated 3 times. After culturing at 28°C until the mycelia of the two bacterial cakes of the blank control were in contact, the colony radius of different treatments was recorded, and the inhibition rate was calculated: inhibition rate (%) = (R2-R1) / R2×100%, R2 is the colony radius of the blank control group, and R1 is the colony radius of the treatment group, and the strain with better antibacterial effect was screened out.

[0042] The plate photo of the isolate with obvious antibacterial effect on the pathogenic fungus QJ3 is shown in Figure 1 In the figure, QJ3 represents the blank control group, T-6, T-9, T-12, T-14, T-16, T-21, T-40, T-47, and T-54 represent the isolated strains with corresponding numbers, and 9 isolated strains were screened to have an inhibitory effect on QJ3.

[0043] The inhibition rates of the nine isolated strains against QJ3 are as follows Figure 2 The results showed that the inhibition rate of 9 isolated strains was greater than 60%.

[0044] The antibacterial rates of 9 isolated strains against B. sclerotiorum (BXHP2), the pathogen of Pinellia ternata wilt, and Q. julibrissin (QJFY2), the pathogen of Pinellia ternata wilt, were determined by confrontation culture method. Figure 3 The inhibition rates of the 9 isolated strains against QJFY2 are shown in Figure 4 shown.

[0045] Combination Figure 2 to Figure 4 The best antibacterial effect of biocontrol bacteria T-6 was obtained. The antibacterial rate of biocontrol bacteria T-6 against QJ3 reached 81.54%, the antibacterial rate of biocontrol bacteria T-6 against BXHP2 reached 80.95%, and the antibacterial rate of biocontrol bacteria T-6 against QJFY2 reached 74.59%. Figure 5 shown.

[0046] T-6 was renamed WBT6 as the final selected strain.

[0047] 2. Identification

[0048] (1) Morphological identification

[0049] The selected biocontrol bacteria WBT6 was streaked onto LB medium plates using the three-zone streak method and cultured at 37°C for 24 hours. The growth of the colonies on the plates was observed and recorded. Figure 6 The colonies are milky white, round, smooth, slightly convex, and sticky.

[0050] (2) Physiological and biochemical identification

[0051] Physiological and biochemical identification experiments were carried out on the biocontrol bacteria WBT6, including catalase assay, oxidase assay, starch hydrolysis assay, methyl red MR assay, VP experiment, gelatin liquefaction assay, nitrate reduction assay, glucose fermentation, hydrogen sulfide production assay, citrate utilization, lecithinase activity assay, malonate utilization, cellulose decomposition, galactose utilization, arabinose utilization, mannitol utilization, D-fructose utilization, and D-xylose utilization. The physiological and biochemical results are shown in Table 1.

[0052] Table 1 Physiological and biochemical results of biocontrol bacteria WBT6

[0053]

[0054]

[0055] In the table, “+” indicates positive; “W” indicates weakly positive; and “-” indicates negative.

[0056] (3) Molecular biological identification

[0057] The genomic DNA of the biocontrol bacterium WBT6 was extracted using a DNA extraction kit, and the 16SrDNA sequence was amplified using the primer pair 27F / 1492R. The PCR amplification system was: Mix 25 μL, genomic DNA 2 μL, upstream and downstream primers 2 μL each, ddH 2 Figure 7 shown.

[0058] Table 2 Primer sequences for 16S rDNA gene amplification

[0059]

[0060] The phylogenetic tree of 16S rDNA showed that the biocontrol bacterium WBT6 and Bacillus subtilis were clustered in one branch. In summary, the results of morphological, physiological and biochemical identification and molecular biological identification showed that the biocontrol bacterium WBT6 was Bacillus subtilis.

[0061] The strain WBT6 (Bacillus subtilis WBT6) was deposited in the China Center for Type Culture Collection on October 25, 2023. The address of the depository is: Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: M20232028.

[0062] 3. Determination of antibacterial spectrum

[0063] The confrontation culture method (same as step 1) was used to determine the inhibitory effect of the biocontrol bacteria WBT6 on LC1, QK-4, HYTJ, MD1, YCCX1, ZJSYB1, CHYB1, ES8, and BMXTX2, and the antibacterial spectrum of WBT6 was preliminarily screened.

[0064] Table 3 Determination of the antibacterial spectrum of biocontrol bacteria WBT6

[0065]

[0066] It can be seen from Table 3 that, except for QK-4, the inhibition rate of WBT6 against pathogenic fungi is greater than 60%, indicating that the biocontrol bacteria has a wide antibacterial spectrum and a wide range of applications.

[0067] 4. In vitro protective efficacy determination

[0068] Use cleaned Pinellia leaves for in vitro efficacy determination:

[0069] (1) Preparation of WBT6 fermentation broth: Prepare a fermentation medium with a formula of 10 g / L peptone, 5 g / L yeast extract powder, 10 g / L NaCl, 5 g / L glucose, and pH = 7. Pick a fresh single colony of WBT6 cultured on a NA plate for 24 hours, inoculate it into a conical flask containing 150 mL NB medium, and culture it at 37°C and 220 r / min for 24 hours. Transfer the WBT6 bacterial solution to a conical flask containing a fermentation medium at a volume of 10% (v / v), and culture it at 37°C and 220 r / min for 48 hours to obtain WBT6 fermentation broth.

[0070] (2) Determination of the effect of WBT6 fermentation liquid on the prevention of Pinellia tuber Sclerotium rot: Use clean Pinellia tuber leaves, place them in a sterilized glass dish covered with sterile filter paper, stab them, and inoculate 10 μL of WBT6 fermentation liquid with a pipette, and let it stand for 24 hours. Inoculate fresh fungus cakes (6 mm in diameter) of the pathogenic fungus QJ3 at the puncture site, and measure the diameter of the lesions after 4 days. The blank control was inoculated with 10 μL of NA medium, and each treatment was repeated three times.

[0071] (3) Determination of the effect of WBT6 fermentation broth on the treatment of Pinellia tuber Scabies: Use clean Pinellia tuber leaves, place them in a sterilized glass dish covered with sterile filter paper, puncture them, and inoculate fresh fungus cakes (6 mm in diameter) of the pathogenic fungus QJ3. After 24 hours, inoculate 10 μL of WBT6 fermentation broth with a pipette, and measure the diameter of the lesions after 4 days. The blank control was inoculated with 10 μL of NA medium, and each treatment was repeated three times.

[0072] Three replicates were used for each treatment, and the diameter of the lesions was measured, the control effect was calculated, and photos were taken for record.

[0073] Disease index = 100 × ∑ (number of diseased leaves at each level × representative value of each level) / (total number of leaves investigated × 4);

[0074] Control effect (%) = (control disease index - treatment disease index) / control disease index × 100.

[0075] In vitro efficacy of biocontrol strain WBT6 against Pinellia ternata pathogen QJ3 Figure 8 The results showed that the diameter of the lesions in the treatment group treated with CK was (2.00±0.12) cm, and the diameter of the lesions after WBT6 treatment was (0.7±0.07) cm, with a control effect of 65%; the diameter of the lesions in the prevention group treated with CK was (3.5±0.70) cm, and the diameter of the lesions after WBT6 treatment was (1.05±0.22) cm, with a control effect of 70%.

[0076] 5. Safety test

[0077] Preparation of WBT6 fermentation broth: Prepare fermentation medium with the formula of 10 g / L peptone, 5 g / L yeast extract powder, 10 g / L NaCl, 5 g / L glucose, pH = 7. Pick a fresh single colony of WBT6 cultured on a NA plate for 24 hours, inoculate it into a conical flask containing 150 mL NB medium, and culture it at 37°C, 220 r / min for 24 hours. Transfer the WBT6 bacterial solution to a conical flask containing fermentation medium at a volume of 10% (v / v), and culture it at 37°C, 220 r / min for 48 hours to obtain WBT6 fermentation broth.

[0078] Two tubers of the same diameter were placed in each planting box. They were cultured in the greenhouse for 25 days. After the growth was stable, 100 mL of WBT6 fermentation liquid was sprinkled on the 1st, 3rd and 7th day. The blank control was sprinkled with sterile water. Each treatment was repeated 5 times. The growth was observed on the 15th day, and the photos were recorded. The results are as follows Fig. 9 As shown (the three Pinellia plants in each group in the figure represent three replicate groups in each group of five replicate treatments). The average plant height of Pinellia in the CK group was (10.84±1.26) cm, and the average weight of rhizomes and plant buds was (2.56±0.80) g; the average plant height of Pinellia after WBT6 treatment was (10.96±1.65) cm, and the average weight of rhizomes and plant buds was (2.76±0.92) g. The results showed that after 16 days of irrigation with the fermentation liquid of the biocontrol fungus WBT6, there was no effect on the growth of Pinellia compared with the blank control, and it can be safely used for the prevention and control of Pinellia fungal diseases.

[0079] In summary, the biocontrol bacterium WBT6 has a good antibacterial effect on Sclerotium pinellia, Sclerotinia pinellia, and Sclerotinia scoparia, and has a good in vitro control effect on Sclerotium rolfsii of pinellia, and has a broad-spectrum antibacterial activity. Therefore, it has the potential to be developed into a biocontrol bacterium.

[0080] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A Bacillus subtilis WBT6, characterized in that The deposit number of the Bacillus subtilis WBT6 is CCTCC NO: M20232028.

2. A bacterial agent, characterized in that Prepared by the Bacillus subtilis WBT6 described in claim 1.

3. The bacterial agent according to claim 2, characterized in that The bacterial agent is the fermentation broth of Bacillus subtilis WBT6.

4. The bacterial agent according to claim 3, characterized in that The method for preparing the fermentation liquid comprises: inoculating Bacillus subtilis WBT6 into a fermentation medium to obtain the fermentation liquid; the fermentation medium comprises: 8-12 g / L of peptone, 4-6 g / L of yeast extract powder, 8-12 g / L of NaCl, 4-6 g / L of glucose, and pH=6.5-7.

5.

5. The bacterial agent according to claim 4, characterized in that The culture conditions are: constant temperature culture at 30-37°C and 200-250r / min for 36-72h.

6. Use of the Bacillus subtilis WBT6 according to claim 1 or the bacterial agent according to any one of claims 2 to 5 in preventing and controlling fungal diseases of Pinellia ternata.

7. The use according to claim 6, characterized in that: The pathogenic fungi of the pinellia fungal diseases include Sclerotium pinellia, Sclerotinia pinellia, and Sclerotiorum pinellia.

8. A method for preventing and treating fungal diseases of Pinellia ternata, characterized in that: During the growth of Pinellia ternata, the Bacillus subtilis WBT6 described in claim 1 or the bacterial agent described in any one of claims 2 to 5 is used for prevention and treatment.

9. Use of the Bacillus subtilis WBT6 according to claim 1 or the bacterial agent according to any one of claims 2 to 5 in inhibiting pathogenic fungi of traditional Chinese medicines.

10. The use according to claim 9, characterized in that: The pathogenic fungi of traditional Chinese medicines include pathogenic fungi of the genus Coptis chinensis, Fusarium oxysporum of tobacco, Fusarium oxysporum of polygonatum, Fusarium acuminate of ophiopogon, Alternaria japonica, Botrytis cinerea of ​​Panax notoginseng, Subsporangium glomerata of Bupleurum chinense, Botrytis cinerea of ​​Paris polyphylla and Botrytis cinerea of ​​Fritillaria hupehensis.

Images