Bacterium Humibacillusxanthopallidus and application thereof
By screening and identifying the IAA function of bacteria Humibacillus xanthopallidus 2212CXJCT4-6, the problem of insufficient research on vegetarian proliferation bacteria was solved, and a new microbial fertilizer was developed to significantly improve the growth performance of vegetarian, including buds, root growth and germination rate. index.
Patent Information
- Application Number
- CN202411205368.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-08-30
- Publication Date
- 2024-12-13
AI Technical Summary
Currently, there are few researches on probiotics in cabbage, and agriculture has challenges in reducing the use of inorganic fertilizers and PGP microorganisms, and new probiotics are needed to achieve sustainable cultivation.
The bacteria Humibacillus xanthopallidus 2212CXJCT4-6 were screened and identified. This strain has the function of producing IAA and can promote the growth of cabbage hearts. It was developed as a new microbial fertilizer or bacterial agent through its application.
Humibacillus xanthopallidus 2212CXJCT4-6 significantly promotes the bud and root growth of cabbage hearts, improves the germination rate, enhances the plant height, fresh ground weight, maximum leaf width and root length of cabbage hearts, and provides a new microbial fertilizer solution.
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Figure CN119120273A_ABST
Abstract
Description
Technical Field
[0001] The present application relates to the field of microbial technology, and in particular to a bacterium Humibacillus xanthopallidus and an application thereof. Background Art
[0002] Chinese cabbage (Brassica parachinensis L.) is a plant in the genus Brassica of the family Cruciferae, which produces scapes. It is also known as Guangdong cauliflower, cauliflower, etc. The root system of Chinese cabbage is shallow, with many fibrous roots and strong regeneration ability. The stem is shortened before bolting, and the scape is round, yellow-green or green. The leaves are broadly oval or elliptical, green or yellow-green, with obvious veins. The petiole is narrow and has shallow grooves. Chinese cabbage has a racemose inflorescence, with a yellow, cross-shaped corolla. The fruit is a long silique, which is yellow-brown when mature. The seeds are nearly round, brown or dark brown.
[0003] Chinese cabbage originated in southern my country and is one of the main plants in southern China. Chinese cabbage is rich in calcium, iron, crude fiber, vitamins, carotene and other nutrients, and has the effects of preventing anemia, enhancing immunity, preventing iron deficiency anemia, lowering cholesterol, clearing away heat and detoxifying, relieving restlessness and thirst, and diuresis and laxative.
[0004] At present, research on Chinese cabbage has found that Bacillus polymyxa and Bacillus amyloliquefaciens can antagonize anthracnose; disease-resistant varieties react quickly to anthracnose infection, and their ability to self-regulate and restore to normal state and resist membrane lipid peroxidation is stronger than that of susceptible varieties; mineral nutrients such as nitrogen, potassium, and silicon can improve the disease resistance of Chinese cabbage. However, there are few reports on growth-promoting bacteria in Chinese cabbage. The current trend in agriculture is to reduce the input of inorganic fertilizers and the use of PGP microorganisms to achieve sustainability, so it is necessary to develop and research growth-promoting bacteria.
[0005] Humibacillus xanthopallidus was screened from Chinese cabbage base soil and was first isolated from a rice field soil sample in Japan. It is a Gram-positive, catalase-positive, rod-shaped bacterium, but there is no report publicly showing that Humibacillus xanthopallidus has a growth-promoting function on plants. Summary of the invention
[0006] The purpose of the present application is to overcome the deficiencies of the above-mentioned prior art and provide a bacterium Humibacillusxanthopallidus and its application. The bacterium Humibacillus xanthopallidus of the present application has the function of producing IAA and can promote the growth of Chinese cabbage. The bacterium Humibacillus xanthopallidus with growth-promoting function is expected to become a new type of microbial fertilizer or preparation.
[0007] The present application provides a bacterium Humibacillus xanthopallidus, wherein the bacterium Humibacillus xanthopallidus is Humibacillus xanthopallidus 2212CXJCT4-6, which has been deposited in the Guangdong Provincial Microbiological Culture Collection Center on January 24, 2024, with the classification name Humibacillus xanthopallidus, the deposit number is GDMCC No: 64310, and the deposit address is Building 59, No. 100 Xianlie Middle Road, Guangzhou.
[0008] The colony morphology of Humibacillus xanthopallidus 2212CXJCT4-6 screened in the present application is round, raised, yellow and opaque, with a smooth surface, and is a Gram-positive bacterium.
[0009] The 16S rDNA sequence of Humibacillus xanthopallidus 2212CXJCT4-6 is shown in SEQ ID NO:1.
[0010] The Humibacillus xanthopallidus 2212CXJCT4-6 with growth-promoting function screened in the present application has the function of producing IAA, which can promote the growth of plants, specifically promote the growth of buds and roots and improve the germination rate, and further improve the plant height, aboveground fresh weight, maximum leaf width, root length, and root weight of the plant. Therefore, the Humibacillusxanthopallidus 2212CXJCT4-6 screened in the present application is expected to become a new type of microbial fertilizer or strain to promote plant growth.
[0011] The screening process of Humibacillus xanthopallidus 2212CXJCT4-6 comprises the following steps:
[0012] The cauliflower soil collected from the cauliflower planting base in Futian, Huizhou City, Guangdong Province was mixed and stored in a 4°C refrigerator. A sterilized conical flask with glass beads was added with 45 ml of sterile water and 5 g of the rhizosphere soil sample. The mixture was shaken at 160 rpm for 30 min. 0.5 ml of the supernatant was gradually diluted into a test tube containing 4.5 ml of sterile water to prepare 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 of soil suspension, take 10 -4 and 10 -5 100 μl of the diluted bacterial solution was spread on a NA solid medium plate, and then placed in a constant temperature incubator for 2 days. Single colonies with different morphologies were selected and streaked onto a new NA solid medium plate for purification, and the purification was continued for more than 3 generations until a pure strain was isolated, and the Humibacillusxanthopallidus 2212CXJCT4-6 of the present application was obtained by screening.
[0013] The present application also provides a microbial agent, which includes the above-mentioned Humibacillusxanthopallidus 2212CXJCT4-6.
[0014] As a preferred embodiment of the microbial agent of the present application, the total concentration of live bacteria of Humibacillusxanthopallidus 2212CXJCT4-6 in the microbial agent is 10 8 cfu / g.
[0015] The Humibacillus xanthopallidus 2212CXJCT4-6 screened in the present application has a growth-promoting effect. In the technical scheme of the present application, when the Humibacillus xanthopallidus2212CXJCT4-6 in the microbial agent adopts the above total live bacteria concentration, it helps to promote plant growth, specifically by improving the bud growth and root growth of the plant and increasing the germination rate, and further increasing the plant height, aboveground fresh weight, maximum leaf width, root length, and root weight of the plant.
[0016] As a preferred embodiment of the microbial agent described in the present application, the preparation method of the microbial agent comprises the following steps:
[0017] A single colony of the Humibacillus xanthopallidus 2212CXJCT4-6 was picked and inoculated into a liquid culture medium for cultivation to obtain a bacterial liquid of Humibacillus xanthopallidus 2212CXJCT4-6, i.e., a microbial agent.
[0018] As a preferred embodiment of the microbial agent described in the present application, the liquid culture medium includes LB liquid culture medium.
[0019] The liquid culture medium used in the present application includes LB liquid culture medium, but is not limited to LB liquid culture medium. The liquid culture medium may also include liquid culture medium commonly used in the art, as long as it can achieve the effect of culturing bacteria.
[0020] As a preferred embodiment of the microbial agent described in the present application, the culture conditions are: the culture temperature is 28°C-30°C, and the culture time is 24h-48h.
[0021] The present application also provides the use of the above-mentioned bacterium Humibacillus xanthopallidus or the above-mentioned microbial agent in promoting plant growth.
[0022] As a preferred embodiment of the application described in the present application, the plant includes Chinese cabbage.
[0023] The Chinese cabbage includes Futian Chinese cabbage. The types of Chinese cabbage are not limited to the above types, but also include common types of Chinese cabbage in the art. The Humibacillus xanthopallidus2212CXJCT4-6 screened in this application can be used to promote the growth of Chinese cabbage.
[0024] The present application also provides the use of the above-mentioned bacterium Humibacillus xanthopallidus in the preparation of a microbial agent or microbial fertilizer for promoting the growth of Chinese cabbage.
[0025] The present application also provides a method for promoting plant growth, which comprises adding the above-mentioned bacterium Humibacillus xanthopallidus or the above-mentioned microbial agent.
[0026] As a preferred embodiment of the method for promoting plant growth described in the present application, the plant includes Chinese flowering cabbage.
[0027] In the technical scheme of the present application, compared with other soil probiotics (Microbacterium laevaniformans), the Humibacillus xanthopallidus 2212CXJCT4-6 with growth-promoting function obtained by screening in the present application helps to promote the growth of Chinese cabbage, which is specifically manifested in that the plant height, aboveground fresh weight, maximum leaf width, root length and root weight of the Chinese cabbage are better, indicating that the Humibacillus xanthopallidus2212CXJCT4-6 is expected to become a new type of microbial fertilizer or preparation.
[0028] Compared with the prior art, this application has the following beneficial effects:
[0029] The present application provides a bacterium Humibacillus xanthopallidus and an application thereof. The bacterium Humibacillus xanthopallidus with a growth-promoting function screened and obtained in the present application has the effects of high IAA production, etc., which helps to promote the growth of Chinese cabbage, specifically by promoting the growth of buds and roots and improving the germination rate, further improving the plant height, above-ground fresh weight, maximum leaf width, root length, and root weight of Chinese cabbage, and laying a theoretical foundation for microbial fertilizer. BRIEF DESCRIPTION OF THE DRAWINGS
[0030] Figure 1 The colony morphology of Humibacillus xanthopallidus 2212CXJCT4-6 strain is shown;
[0031] Figure 2 The gel electrophoresis diagram of the 16S rDNA amplified fragment of Humibacillus xanthopallidus 2212CXJCT4-6 strain;
[0032] Figure 3 Phylogenetic tree of Humibacillusxanthopallidus 2212CXJCT4-6 strain constructed based on 16S rRNA gene sequence using the neighbor-joining method. DETAILED DESCRIPTION
[0033] In order to better illustrate the purpose, technical solutions and advantages of the present application, the present application will be further described below in conjunction with the accompanying drawings and specific embodiments.
[0034] In the following examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all commercially available unless otherwise specified.
[0035] In the following examples, Microbacterium laevaniformans (GDAAS 02931) was derived from the National Agricultural Environment Microbial Germplasm Resource Bank (Guangdong).
[0036] NA solid medium: peptone 10.0 g, beef extract powder 3.0 g, NaCl 5.0 g, agar 18.0 g, distilled water 1000 mL, pH 7.3±0.2, sterilization at 121℃ for 15 min.
[0037] LB liquid medium: peptone 10.0 g, NaCl 10.0 g, yeast extract powder 5 g, distilled water 1000 mL, pH 7.0, sterilization at 121 °C for 30 min.
[0038] PKO inorganic phosphorus medium: glucose 10.0 g, Ca3(PO4)2 5.0 g, (NH4)2SO4 0.50 g, NaCl 0.20 g, KCl 0.20 g, MgSO4·7H20 0.30 g, MnSO4 0.03 g, FeSO4·7H20 0.03 g, yeast extract 0.50 g, distilled water 1000 mL, pH 6.8-7.0, sterilization at 121°C for 30 min.
[0039] King's liquid medium: 20.0 g of tryptone, 2.0 g of dipotassium hydrogen phosphate, 1.5 g of magnesium sulfate, 0.1 g of tryptophan, 15 mL of glycerol, pH 7.0, sterilized at 121°C for 30 min.
[0040] Example 1: Screening of Humibacillus xanthopallidus 2212CXJCT4-6 strain
[0041] The cauliflower soil collected from the cauliflower planting base in Futian, Huizhou City, Guangdong Province was mixed and stored in a 4°C refrigerator. A sterilized conical flask with glass beads was added with 45 ml of sterile water and 5 g of the rhizosphere soil sample. The mixture was shaken at 160 rpm for 30 min. 0.5 ml of the supernatant was gradually diluted into a test tube containing 4.5 ml of sterile water to prepare 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 of soil suspension, take 10 -4 and 10 -5 100ul of the diluted bacterial solution was spread on a NA solid medium plate, and then placed in a constant temperature incubator for 2 days. Single colonies with different morphologies were selected and streaked on a new NA solid medium plate for purification. Purification was continued for more than 3 generations until a pure strain was isolated. The purified strains were classified and labeled, frozen in glycerol tubes, and stored in a -80℃ refrigerator for later use.
[0042] The purified rhizospheric bacteria of Chinese flowering cabbage were subjected to IAA determination, and the bacteria with better growth-promoting effect were obtained through potted plant verification.
[0043] A single colony was picked from the purified rhizosphere bacteria and inoculated into 5 mL of LB liquid culture medium, and cultured at 28° C. for 24 h to obtain a bacterial culture solution.
[0044] The bacterial DNA was extracted using a bacterial DNA extraction kit (purchased from Ark Biosafety Technology Co., Ltd.), and PCR amplification was performed using bacterial 16S rRNA universal primers. The universal primers were 27F (5'-AGAGTTTGATCCTGGCTCAG-3', SEQID NO: 2) and 1492R (5'-GGTTACCTTGTTACGACTT-3', SEQ ID NO: 3). The extracted bacterial DNA was used as a template and PCR amplification was performed under the action of 2×PCR Mix (the PCR amplification program is shown in Table 1) to obtain the bacterial PCR product; the bacterial PCR amplification product was detected by 1% agarose gel electrophoresis, purified and recovered, and then subjected to first generation sequencing to obtain the nucleic acid sequence of the PCR product.
[0045] The nucleic acid sequences obtained by sequencing were compared in the EzBioCloud database (https: / / www.ezbiocloud.net / identify), and the comparison results identified it as Humibacillus xanthopallidus.
[0046] The above strain was named Humibacillus xanthopallidus 2212CXJCT4-6, and was deposited in Guangdong Provincial Microbiological Culture Collection on January 24, 2024. The classification name is Humibacillus xanthopallidus, the collection number is GDMCC No: 64310, and the collection address is Building 59, No. 100 Xianlie Middle Road, Guangzhou.
[0047] The colony morphology of Humibacillus xanthopallidus 2212CXJCT4-6 screened in this application is round, raised, yellow and opaque, with a smooth surface, and is a Gram-positive bacterium. Figure 1 shown.
[0048] The 16S rDNA sequence of Humibacillus xanthopallidus 2212CXJCT4-6 is shown in SEQ ID NO: 1. The gel electrophoresis diagram thereof is shown in Figure 2 shown.
[0049] SEQ ID NO: 1:
[0050]
[0051] The phylogenetic tree of Humibacillusxanthopallidus 2212CXJCT4-6 constructed based on 16S rRNA gene sequences using the neighbor-joining method is shown in Figure 3 shown.
[0052] Table 1 PCR amplification program
[0053]
[0054] Example 2: Determination of IAA content in Humibacillus xanthopallidus 2212CXJCT4-6
[0055] The Humibacillus xanthopallidus 2212CXJCT4-6 obtained by the above screening was inoculated on a NA solid plate and cultured at 28°C for 24 hours. A single colony was picked and added to King's liquid medium containing 100 mg / L tryptophan, and King's liquid medium containing 100 mg / L tryptophan was used as a blank control. The plate was placed in the dark for 35 minutes, and the absorbance at 530 nm was measured. The IAA content was calculated using the IAA standard curve.
[0056] The results are shown in Table 2.
[0057] Table 2
[0058] Group IAA(mg / L) CK 6.18 Humibacillus xanthopallidus 2212CXJCT4-6 15.50
[0059] It can be seen from Table 2 that compared with the control group (CK group), the Humibacillusxanthopallidus2212CXJCT4-6 screened in this application has a growth-promoting function and an IAA-producing function, which can significantly promote the growth of Chinese cabbage.
[0060] Example 3: Experiment on the determination of Humibacillus xanthopallidus 2212CXJCT4-6 in the cultivation of Chinese cabbage
[0061] The Humibacillus xanthopallidus 2212CXJCT4-6 obtained by the above screening was inoculated on a NA solid plate and cultured at 28°C for 24 hours. A single colony was picked and inoculated into 5ml LB and cultured for 24 hours. The obtained bacterial culture was diluted 1000 times for standby use. 5mL of the bacterial culture dilution was added to a culture dish. Each culture dish contained 10 seeds of Futian Chinese cabbage. The culture dishes containing Chinese cabbage seeds were cultured at 30°C in the dark for 3 days. The sprout length, root length and germination rate of the Chinese cabbage seeds in different treatment groups were counted. Each treatment was repeated 3 times, and the LB culture medium was diluted 1000 times as a control.
[0062] The data processing of Chinese cabbage sprout length, root length and germination rate was statistically performed using SPSS25.0 software, and the results are shown in Table 3.
[0063] Table 3
[0064]
[0065] Note: Different lowercase letters represent differences in physiological parameters among different treatments (P<0.05).
[0066] It can be seen from Table 3 that compared with the control group (CK group), the Humibacillusxanthopallidus2212CXJCT4-6 of the present application has a growth-promoting function, which is specifically manifested in that it can promote the growth of Chinese cabbage sprouts and roots and increase the germination rate. It can significantly promote the growth of Futian Chinese cabbage and further increase the resources of Futian Chinese cabbage growth-promoting bacteria.
[0067] Example 4, potted plant experiment
[0068] The bacterial culture obtained in Example 3 and the control bacteria (Microbacterium laevaniformans) were diluted 10 times and 1000 times for later use; the sprouted Chinese cabbage seeds obtained in the germination experiment were used for potted plant experiments.
[0069] The roots of the germinated seeds of Futian Chinese cabbage were soaked in a 1000-fold diluted bacterial culture for 15 minutes, transplanted to a planting cup, and cultured at 25-30°C with a light / dark cycle of 12h / 12h. The roots were irrigated with a 10-fold diluted bacterial culture in the second and third weeks after transplantation. The seedlings were harvested 26 days after transplantation, and the fresh weight of the aboveground part of the Chinese cabbage, plant height, maximum leaf width, root length and root weight were counted. The culture medium was used as the control group.
[0070] The data of seedling harvest were statistically analyzed using SPSS25 software, and the differences among multiple groups were compared using one-way analysis of variance, and multiple comparisons were performed using the Duncan method. The results are shown in Table 4.
[0071] Table 4
[0072]
[0073]
[0074] Note: Different lowercase letters represent differences in physiological parameters among different treatments (P<0.05).
[0075] As shown in Table 4, compared with other soil probiotics (Microbacterium laevaniformans) and the control group (CK), the Humibacillus xanthopallidus2212CXJCT4-6 with growth-promoting function obtained by the present application can be applied to the growth of Chinese cabbage, and has a better effect of promoting the growth of Chinese cabbage, which is specifically manifested in the plant height (23.9±2.7cm), aboveground fresh weight (6.49±0.46g), maximum leaf width (6.8±0.4cm), root length (31.95±1.45cm), and root weight (2.22±0.52g) of the Chinese cabbage. This shows that the Humibacillus xanthopallidus 2212CXJCT4-6 obtained by the present application has a better growth-promoting function, which is beneficial to the growth of plants such as Futian Chinese cabbage.
[0076] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present application rather than to limit the scope of protection of the present application. Although the present application has been described in detail with reference to the preferred embodiments, ordinary technicians in this field should understand that the technical solution of the present application can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present application.
Claims
1. A bacterium Humibacillus xanthopallidus, characterized in that The bacterium Humibacillusxanthopallidus is Humibacillus xanthopallidus 2212CXJCT4-6, which was deposited in the Guangdong Provincial Microbiological Culture Collection Center on January 24, 2024, with the deposit number GDMCC No: 64310, and the deposit address is Building 59, No. 100 Xianlie Middle Road, Guangzhou.
2. A microbial agent, characterized in that: The microbial agent includes the Humibacillus xanthopallidus 2212CXJCT4-6 as claimed in claim 1.
3. The microbial agent according to claim 2, characterized in that The total concentration of live bacteria of Humibacillusxanthopallidus 2212CXJCT4-6 in the microbial agent is 10 8 cfu / g.
4. The microbial agent according to claim 2, characterized in that The preparation method of the microbial agent comprises the following steps: A single colony of the Humibacillus xanthopallidus 2212CXJCT4-6 was picked and inoculated into a liquid culture medium for cultivation to obtain a bacterial liquid of Humibacillus xanthopallidus 2212CXJCT4-6, i.e., a microbial agent.
5. The microbial agent according to claim 4, characterized in that The liquid culture medium includes LB liquid culture medium.
6. The microbial agent according to claim 4, characterized in that The culture conditions are as follows: the culture temperature is 28°C-30°C, and the culture time is 24h-48h.
7. Use of the bacterium Humibacillus xanthopallidus according to claim 1 or the microbial agent according to any one of claims 2 to 6 in promoting plant growth.
8. Use of the bacterium Humibacillus xanthopallidus according to claim 1 in the preparation of a microbial agent or microbial fertilizer for promoting the growth of flowering cabbage.
9. A method for promoting plant growth, characterized in that: The method comprises adding the bacterium Humibacillus xanthopallidus according to claim 1 or the microbial agent according to any one of claims 2 to 6.
10. The method for promoting plant growth according to claim 9, characterized in that: The plants include Chinese cabbage.