Atrophylus bacillus strains and their use in the control of plant diseases and in the promotion of plant growth

Abstract

This invention discloses a strain of *Bacillus atrophicus* and its application in controlling plant diseases and promoting plant growth. This invention isolates endophytic bacteria from chickpea plants and ultimately screens out an endophytic strain, YB-14, which can effectively control plant diseases and promote plant growth. Molecular biological and morphological identification confirms that strain YB-14 belongs to the taxonomic group *Bacillus atrophicus* (…). Bacillus atrophaeus The strain YB-14, with its microbial preservation number CGMCC No. 29008, has been shown in experiments to have good control effects on chickpea wilt and potato aerial stem rot. It can promote plant emergence and improve plant resistance to salt and alkali stress, showing promising application prospects in plant disease control, promoting plant growth, and improving plant resistance to salt and alkali stress.

Description

Technical Field

[0001] This invention relates to endophytic bacteria isolated from plants, and more particularly to Bacillus atrophus isolated from chickpea plants. Bacillus atrophaeus Bacillus spp. and its application in preventing and controlling plant diseases and promoting plant growth belong to the field of Bacillus spp. isolated from plants and their application. Background Technology

[0002] Chickpeas Cicer arietinum Chickpea (L.) is a type of edible legume, a cultivated species of the genus Chickpea in the tribe Vicia of the legume family. It is the world's second most cultivated legume after common bean, which has the largest planting area in the world. It is relatively drought resistant. Chickpea is a mixed legume crop that is both food and medicine. While it is rich in a variety of nutrients needed by the human body, it also has potential value in drug preparation and other aspects.

[0003] Chickpea wilt is one of the most devastating diseases in chickpea production. It is caused by *Fusarium oxysporum* (…). Fusarium oxysporum This condition is caused by [a specific disease / condition], which mainly damages the roots and underground stems of plants, leading to slow growth in the seedling stage, discoloration and sunken areas of the roots and underground stems, brownish-yellow spots on the stems, yellowing of leaves, weakened and stunted plant growth, root rot, wilting above ground, and in severe cases, even death of the entire plant. It mainly occurs in the seedling stage and seriously affects crop yield.

[0004] Traditional control methods are insufficient to effectively control wilt disease during the chickpea growing season, and the disease is more severe in chickpea varieties with higher yields and wider planting areas. While chemical pesticides offer some control, pesticide residues can harm human health and pollute the environment, contradicting the principles of green agriculture. Microorganisms have a strong inhibitory effect on fungal diseases; therefore, developing microbial agents for controlling chickpea wilt is of significant scientific and practical value.

[0005] Studies have shown that soil bacteria are a class of microorganisms with great development potential. They can inhibit pathogens, enhance plant defense capabilities, improve soil structure, and promote plant growth. Currently, there are many Bacillus atrophus species... Bacillus atrophaeus The development and utilization of Bacillus atrophicus has revealed that the spores produced by Bacillus atrophicus have stronger resistance to stress compared to other Bacillus species. This makes Bacillus atrophicus easier to colonize or survive than general biocontrol bacteria, thus giving it greater potential for the development and utilization of biological pesticides. Summary of the Invention

[0006] One of the objectives of this invention is to provide a strain of Bacillus atrophus isolated from plants; A second objective of this invention is to provide a microbial preparation or compost containing the aforementioned Bacillus atrophus; The third objective of this invention is to apply the aforementioned Bacillus atrophus or microbial preparations or compost containing the aforementioned Bacillus atrophus to the prevention and control of plant diseases, the promotion of plant growth, the improvement of plant resistance to adverse environmental stress, or the improvement of soil physical and chemical properties.

[0007] To achieve the above objectives, the main technical solutions adopted by the present invention include: One aspect of the present invention is to provide a strain of Bacillus atrophus ( Bacillus atrophaeus YB-14, its microbial preservation number is CGMCC No. 29008; its classification name is: Bacillus atrophus. Bacillus atrophaeus The deposit date is November 14, 2023; the depositary institution is the China General Microbiological Culture Collection Center; the depositary address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0008] The colony and cell morphology of Bacillus atrophus described in this invention are as follows: the colony is nearly round with raised folds in the middle, and it is sticky when picked up. In the early stage of growth, it is nearly pale yellowish-white and transparent, and it turns yellowish-white when mature.

[0009] Another aspect of the present invention is to apply the aforementioned Bacillus atrophus to the prevention and control of plant diseases, to promote plant growth, to improve the plant's resistance to adverse environmental stress, or to improve the physical and chemical properties of soil.

[0010] In a preferred embodiment of the present invention, the *Bacillus atrophicus* YB-14 is applied to the prevention and control of plant diseases; wherein the pathogen of the plant disease is *Fusarium oxysporum*, and it has a significant control effect on chickpea wilt caused by *Fusarium oxysporum*; in addition, the *Bacillus atrophicus* YB-14 of the present invention can effectively antagonize *Pectinobacter carotenoides* subsp. *Brazilianus*, and has a significant control effect on potato aerial stem rot caused by *Pectinobacter carotenoides* subsp. *Brazilianus*.

[0011] In a preferred embodiment of the present invention, the Bacillus atrophus YB-14 is applied to promote plant growth; wherein, the promotion of plant growth is preferably achieved by increasing the germination rate of plant seeds.

[0012] In a preferred embodiment of the present invention, the Bacillus atrophus YB-14 is applied to improve the plant's resistance to abiotic stress, wherein the abiotic stress is preferably salt stress or alkali stress.

[0013] In a preferred embodiment of the present invention, the *Bacillus atrophicus* YB-14 is applied to improve the physical and chemical properties of soil. Because the *Bacillus atrophicus* YB-14 provided by the present invention has excellent salt and alkali tolerance, applying microbial preparations or compost made from *Bacillus atrophicus* YB14 to saline-alkali soil can enrich the microbial diversity of the saline-alkali soil, improve the saline-alkali soil ecosystem, and thus effectively improve the physical and chemical properties of the saline-alkali soil, including: regulating soil pH, increasing soil porosity, or enriching the types of soil microbial communities.

[0014] Another aspect of the present invention is to provide a microbial preparation or compost containing the aforementioned Bacillus atrophus YB-14.

[0015] Those skilled in the art can prepare various conventional microbial preparations or composts from the Bacillus atrophicus YB-14 provided by this invention according to conventional preparation methods for microbial preparations. These are all technical means well known to those skilled in the art. Correspondingly, various conventional microbial preparations obtained from Bacillus atrophicus YB-14 can be used for the prevention and control of plant diseases, promoting plant growth and improving the plant's resistance to adverse abiotic stress, or improving the physical and chemical properties of soil.

[0016] This invention isolates endophytic bacteria from chickpea plants and ultimately screens out a strain YB-14 that can prevent and control plant diseases, promote plant growth, and enhance its resistance to abiotic stress. Molecular biological and morphological identification confirms that strain YB-14 is *Bacillus atrophus* (…). Bacillus atrophaeus Experiments have shown that strain YB-14 has significant control effects on chickpea wilt caused by Fusarium oxysporum or potato aerial stem rot caused by Bacillus carotenoides subsp. Brazil. In addition, it can increase the germination rate of plants, grow under salt and alkali stress, and effectively improve the physical and chemical properties or soil structure of saline-alkali soil. Attached Figure Description

[0017] Figure 1 This is a colony morphology diagram of Bacillus atrophus YB-14; Figure 2 The plate inhibition effect of Bacillus atrophus YB-14 on the pathogen of chickpea wilt; Figure 3 The plate inhibition effect of Bacillus atrophus YB-14 on the pathogen of potato aerial stem rot; Figure 4 The effect of Bacillus atrophus YB-14 on the germination of different varieties of chickpea seeds; Figure 5 The growth of Bacillus atrophus YB-14 under different salt concentrations; Figure 6The growth of Bacillus atrophus YB-14 under different alkali concentrations. Detailed Implementation

[0018] The present invention will be further described below with reference to specific embodiments or test examples, and the advantages and features of the present invention will become clearer with the description. However, it should be understood that the embodiments or test examples are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but such modifications or substitutions all fall within the protection scope of the present invention.

[0019] Example 1: Isolation and Identification of Bacillus atrophus YB-14 1. Strain Isolation Chickpea plant samples were cleaned and dried using a dilution-spreading method. Several 1 cm segments of tissue were cut from the plant samples and soaked in 75% alcohol for approximately 2 minutes. After drying, the samples were soaked in 5% sodium hypochlorite solution for approximately 2 minutes, then soaked again in 75% alcohol for 1 minute, followed by rinsing 2-3 times in sterile water. The extracted chickpea tissue was dried, ground evenly in a sterile mortar, and 1 mL of the liquid was evenly spread onto LB agar plates. This process was repeated 10 times. The treated LB plates were then inverted and incubated at 28 °C for 24-48 h. One endophytic bacterium was selected and named YB-14. It was purified by continuous streaking on LB agar, and strain YB-14 was stored in a -81 °C ultra-low temperature freezer with 70% glycerol.

[0020] 2. Identification of strain YB-14 After culturing strain YB-14 on LB solid medium at 25 ℃ for 24 h, the colonies were nearly round with raised wrinkles in the center, sticky when picked up, and nearly yellowish-white and transparent in the early stages of growth, turning yellowish-white when mature. Single colonies of the strain to be identified were picked and cultured in liquid LB medium. After 3 days of culture, 1 mL of YB-14 fermentation broth was transferred to a 1.5 mL centrifuge tube and centrifuged at 12000 rpm for 1 min to collect the bacterial cells. Bacterial DNA was extracted using a bacterial genomic DNA kit purchased from Tiangen Biotech Co., Ltd. Detailed procedures can be found in the product manual. 16S rDNA universal primers and... gyrA Gene amplification primers gyrA-F and gyrA-R target the 16S rDNA of the strain and gyrAGene fragments were amplified by PCR using conventional methods. After passing 1% agarose gel electrophoresis, the PCR products were sequenced, and the sequences were analyzed using NCBI online BLAST, combined with morphological characteristics (…). Figure 1 ) was identified as Bacillus atrophus ( Bacillus atrophaeus ).

[0021] Experimental Example 1: Plate confrontation test of Bacillus atrophus YB-14 1. Experimental Methods The pathogen used in this invention is Fusarium oxysporum (Fusarium oxysporum) Fusarium oxysporum ) and *Carotene Soft Rot Pectinobacter brasiliensis* subsp. *carotene* ( Pectobacterium carotovorum subsp. brasiliense The sample was preserved and purified by the College of Horticulture and Plant Protection of Inner Mongolia Agricultural University.

[0022] The plate confrontation method was used for the experiment. Two pathogenic fungi were activated and cultured for 5 days. Fungal discs with a diameter of 6 mm were made by punching holes in areas rich in colonies. These discs were then inverted in the center of PDA agar plates. Simultaneously, single colonies of isolated endophytic bacteria were inoculated 2.5 cm from the center of the plate. Four inoculations were made per plate. PDA plates containing only fungal discs served as controls. The experiment was repeated three times. The plates were incubated at 25 °C until the fungal hyphae in the control group completely covered the culture medium. The presence of inhibition zones on the control group plates was observed, and the diameter of the inhibition zones was measured. In a clean bench, 60 μL of the activated *Pectinobacter carotenoides* subsp. *brassica* culture was inoculated onto an LB agar plate and spread evenly using a spreader. *Bacillus atrophus* YB-14 was then inoculated at a 2.5 cm perimeter using an inoculation loop. This process was repeated three times. An LB agar plate inoculated only with the pathogen served as a control. After inoculation, the plates were sealed with sealing film and incubated upside down in a 28°C incubator. Colony growth was observed daily, and the inhibition rate was assessed after 5 days.

[0023] 2. Experimental Results The results of the fungal plate confrontation test are shown in Table 1 and Figure 2 As shown, Bacillus atrophicus YB-14 exhibited significant inhibitory effects on two strains of Fusarium oxysporum on agar plates, with an average inhibition rate of 65.54%. The results of the bacterial plate confrontation test are shown in Table 2 and... Figure 3 As shown, Bacillus atrophus YB-14 exhibited an inhibition radius of 2.13 cm against Bacillus carotenoidis subsp. Brazil, demonstrating a good inhibitory effect on this species.

[0024] Table 1. Plate inhibition effect of Bacillus atrophus YB-14 on Fusarium oxysporum, the pathogen of chickpea wilt.

[0025] Table 2. Plate inhibition effect of Bacillus atrophus YB-14 on Bacillus carotenoidis soft rot.

[0026] Experiment Example 2: Field control efficacy of Bacillus atrophus YB-14 and its effect on the emergence of different chickpea varieties. 1. Experimental Methods The field efficacy of isolated and purified Bacillus atrophicus YB-14 was determined. Activated Bacillus atrophicus YB-14 was used to create 6 mm diameter mycelial cakes by punching holes at single colony sites. These cakes were placed in sterilized liquid LB medium (5 g yeast extract, 10 g tryptone, 10 g sodium chloride, 1000 mL distilled water) and cultured at 100 r / min for 3 days in a constant temperature shaker to prepare Bacillus atrophicus YB14 fermentation broth. The concentration of the fermentation broth was adjusted to OD600=1 using distilled water. Different varieties of chickpea seeds were sterilized and then immersed in the 10-fold diluted fermentation broth for 30 min, with an equal volume of sterile water as a control. Germination of different chickpea varieties was recorded 14 days after sowing, and disease incidence was investigated 14 days after seed emergence.

[0027] 2. Experimental Results The results of the field efficacy trials are shown in Table 3. Although the effects of Bacillus atrophus YB-14 on the germination and control efficacy of different chickpea varieties varied, all showed an effect of promoting seedling emergence. Figure 4 Furthermore, the field control efficacy against chickpea wilt reached an average of 41.67%.

[0028] Table 3. Field efficacy determination of Bacillus atrophus YB-14

[0029] Experimental Example 3: Control efficacy of Bacillus atrophus YB-14 against potato aerial stem rot in potted plants. 1. Experimental Methods The fermentation broth of Bacillus atrophus YB14 was prepared in the same way as in Experiment 2. The concentration of the fermentation broth was adjusted to OD600=1 using distilled water and then set aside. Potato tissue culture seedlings (Xisen No. 6 variety) with uniform growth were selected and transplanted into sterilized substrate. After hardening off for 7 days in an artificial climate chamber (25±2℃, light 12h / d, humidity 70%), pot experiments were conducted.

[0030] Treatment 1: Select vigorous potato tissue culture seedlings, and slightly damage the lateral roots of the seedlings with a sterilized toothpick. Each treatment was repeated 3 times, with 18 seedlings per repeat. 20 mL of fermentation broth of Bacillus carotenoidis subsp. Brazili was inoculated into flower pots. The plants were then cultured under 16 h light (21℃) / 8 h darkness (16℃) conditions, with regular watering.

[0031] Treatment 2: Select vigorous potato tissue culture seedlings, and slightly damage the lateral roots of the seedlings with a sterile toothpick. Each treatment was repeated 3 times, with 18 seedlings per repeat. An equal amount of sterile water was used as a control. The seedlings were cultured in a 16-hour light (21°C) / 8-hour dark (16°C) environment, and watered regularly.

[0032] Treatment 3: Potato plants were drenched with a 10-fold dilution of 20 mL of Bacillus atrophicus YB14 fermentation broth 3 days in advance. Root damage treatment was performed 3 days later. Each treatment was repeated 3 times, with 18 plants per repeat. The plants were then inoculated with 20 mL of Bacillus carotenoidis subsp. Brazil fermentation broth and cultured under 16 h light (21℃) / 8 h darkness (16℃) conditions, with regular watering.

[0033] Disease index = ∑(Number of diseased plants at each level × Representative value at each level) / Total number of diseased plants × Highest representative value × 100 Prevention and control efficacy (%) = [(Control disease index - Treatment disease index) / Control disease index] × 100 Table 4. Indoor Grading Standards for Potato Aerial Stem Base Rot

[0034] 2. Experimental Results According to the results of the pot disease resistance test for potato aerial stem rot in Table 5, the application of Bacillus atrophicus YB14 fermentation broth resulted in a control efficacy of 38.14±0.42% against potato aerial stem rot. Therefore, it can be seen that Bacillus atrophicus YB14 has a good control effect on potato aerial stem rot.

[0035] Table 5. Indoor control efficacy against potato aerial stem base rot.

[0036] Test Example 4: Salt Tolerance Test of Bacillus atrophus YB-14 1. Experimental Methods The salt tolerance of isolated and purified Bacillus atrophicus YB-14 was determined using the plate method. Activated Bacillus atrophicus YB-14 was spotted onto LB agar plates with salt concentrations of 9% and 10%, and incubated at 25°C for 48–72 h. The size and diameter of the colonies were observed, and the salt tolerance of Bacillus atrophicus YB-14 was determined based on the colony diameter.

[0037] 2. Experimental Results The results of the salt tolerance test are shown in Table 6 and Figure 5 As shown, Bacillus atrophus YB-14 was inoculated onto LB medium with salt concentrations of 9% and 10%, respectively, and the colony diameter was measured.

[0038] Table 6. Salt tolerance test of Bacillus atrophus YB-14

[0039] According to Table 6 and Figure 5 The experimental results show that Bacillus atrophus YB-14 can grow well on LB medium with salt concentrations of 9% and 10%.

[0040] Test Example 5: Alkali Resistance Test of Bacillus atrophus YB-14 1. Experimental Methods The alkali resistance of isolated and purified Bacillus atrophicus YB-14 was determined using the plate method. Activated Bacillus atrophicus YB-14 was spotted onto LB agar plates with pH values ​​of 9, 10, and 11, and incubated at 25 °C for 48–72 h. The size and diameter of the colonies were observed, and the alkali resistance of Bacillus atrophicus YB-14 was determined based on the colony diameter.

[0041] 2. Experimental Results Table 7. Alkali resistance test of Bacillus atrophus YB-14

[0042] The results of the alkali resistance test are shown in Table 7 and Figure 6 As shown, Bacillus atrophicus YB-14 was inoculated onto LB medium with pH values ​​of 9, 10, and 11, and the colony diameter was measured. The experimental results show that Bacillus atrophicus YB-14 can grow well on LB medium with pH values ​​of 9, 10, and 11.

Claims

1. A strain of Bacillus atrophus ( Bacillus atrophaeus YB-14, characterized in that, Its microbial preservation number is: CGMCC No.29008.

2. A microbial preparation or compost containing Bacillus atrophus YB-14 as described in claim 1.

3. The application of Bacillus atrophus YB-14 as described in claim 1 in the prevention and control of plant diseases, or the application of the microbial preparation or compost as described in claim 2 in the prevention and control of plant diseases.

4. The application according to claim 3, characterized in that, The plant disease mentioned is chickpea wilt, and the pathogen of chickpea wilt is Fusarium oxysporum.

5. The application according to claim 3, characterized in that, The plant disease mentioned is potato aerial stem rot, and the pathogen causing potato aerial stem rot is potato aerial stem rot.

6. The application of Bacillus atrophus YB-14 as described in claim 1 in promoting plant growth, or the application of the microbial preparation or compost as described in claim 2 in promoting plant growth.

7. The application according to claim 6, characterized in that, The promotion of plant growth is to increase the germination rate of plant seeds; the plant is chickpea.

8. The application of Bacillus atrophus YB-14 as described in claim 1 in improving the plant's resistance to abiotic stress, or the application of the microbial preparation or compost as described in claim 2 in improving the plant's resistance to abiotic stress.

9. The application according to claim 8, characterized in that, The aforementioned abiotic stresses mainly include salt stress or alkali stress; the aforementioned plant is chickpea.

10. The application of Bacillus atrophus YB-14 as described in claim 1 in improving soil physical and chemical properties, or the application of the microbial preparation or compost as described in claim 2 in improving soil physical and chemical properties.

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