Streptomyces carteus and application thereof

Abstract

The invention relates to streptomyces carteti and application thereof, and belongs to the technical field of microorganisms. The Streptomyces katrae 30213 disclosed by the invention is preserved in the Guangdong Microbial Culture Collection Center, the preservation number is GDMCC No: 64315, and the preservation time is January 24, 2024. The Streptomyces katrae 30213 disclosed by the invention has the advantages that the Streptomyces katrae 30213 is preserved in the Guangdong Microbial Culture Collection Center; according to the invention, a plurality of white gourd rhizosphere microorganisms are screened in healthy and diseased white gourd rhizosphere soil through a continuous dilution method, and a strain of Streptomyces katrae 30213 is found through single colony morphological observation and molecular biological identification, so that the strain of Streptomyces katrae 30213 is obtained through screening. A pot experiment shows that the Streptomyces katrae 30213 disclosed by the invention has an effect of promoting the growth of a white gourd plant, and can be applied to preparation of a preparation for promoting the growth of the white gourd.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to Streptomyces catera and applications thereof. Background Art

[0002] Winter melon (Benincasa hispida Cogn.) is an annual vine herb belonging to the genus Wax melon of the Cucurbitaceae family. Winter melon is rich in nutrients, including a variety of essential nutrients for the human body and various vitamins. In addition, winter melon has the advantages of easy cultivation, high yield, and resistance to storage and transportation. It has become the preferred vegetable variety to help industrial revitalization in various places in recent years. Current research on winter melon has found that Trichoderma, Bacillus, Trichoderma harzianum, etc. can antagonize wilt disease; disease-resistant varieties respond quickly to wilt infection, which is positively correlated with the activity of the plant's defense enzymes. However, there are few reports on winter melon growth-promoting bacteria. The current trend in agriculture is focused on reducing the input of inorganic fertilizers and the use of growth-promoting microorganisms to achieve sustainability. Therefore, it is necessary to develop and research growth-promoting bacteria. Summary of the invention

[0003] The object of the present invention is to overcome the shortcomings of the prior art and provide a Streptomyces katrae having the function of promoting the growth of cucurbitaceae plants and an application thereof.

[0004] To achieve the above object, the technical solution adopted by the present invention is:

[0005] In a first aspect, the present invention provides a Streptomyces katrae, wherein the Streptomyces katrae is Streptomyces katrae 30213, which is deposited in Guangdong Provincial Microbiological Culture Collection Center with a deposit number of GDMCC No: 64315 and a deposit date of January 24, 2024.

[0006] As a preferred embodiment of the Streptomyces katrae of the present invention, the 16S rRNA nucleic acid sequence of the Streptomyces katrae 30213 is shown as SEQ ID NO.1.

[0007] In a second aspect, the present application provides a culture of Streptomyces katrae obtained by fermenting the above-mentioned Streptomyces katrae 30213.

[0008] In a third aspect, the present application provides a method for preparing the above-mentioned Streptomyces catera culture, comprising the following steps: inoculating the activated Streptomyces catera into a liquid culture medium, and culturing at 28-32° C. for 48-96 hours to obtain a Streptomyces catera culture.

[0009] As a preferred embodiment of the preparation method of the present invention, the preparation method further comprises the following steps: culturing at 28-32°C for 48-96 hours and then filtering with a single-layer gauze, and the obtained filtrate is the culture of Streptomyces catera.

[0010] As a preferred embodiment of the preparation method of the present invention, the activated Streptomyces catera is mainly prepared by the following method: inoculating Streptomyces catera strains into a solid culture medium, culturing at 28-32° C. for 5-7 days, and obtaining the activated Streptomyces catera.

[0011] As a preferred embodiment of the preparation method of the present invention, the liquid culture medium is Go's liquid culture medium.

[0012] As a preferred embodiment of the preparation method of the present invention, the formula of the Gao liquid culture medium is 0.5g / LNaCl, 0.01g / L FeSO4, 1.0g / L KNO3, 0.5g / L K2HPO4, 0.5g / LMgSO4, and the pH is adjusted to 7.2-7.4.

[0013] In a fourth aspect, the present invention provides the use of the above-mentioned Streptomyces catera and its culture in the preparation of a preparation for promoting the growth of Cucurbitaceae plants.

[0014] As a preferred embodiment of the application of the present invention, the Cucurbitaceae plant includes at least one of wax gourd, bottle gourd, cucumber, gourd, pumpkin, watermelon and melon.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] The invention obtains a plurality of wax gourd rhizosphere microorganisms by screening in rhizosphere soil of healthy and diseased wax gourds through a continuous dilution method, finds a strain of Streptomyces katrae 30213 obtained through single colony morphology observation and molecular biological identification, finds through potted plant experiments that the Streptomyces katrae 30213 has an effect of promoting the growth of wax gourd plants, and can be used in the preparation of a preparation for promoting the growth of wax gourd. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 is a phylogenetic tree of Streptomyces catera 30213 in Example 1 of the present invention;

[0018] Figure 2 This is the IAA standard curve in Example 1 of the present invention. DETAILED DESCRIPTION

[0019] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0020] Unless otherwise specified, the "Streptomyces katrae 30213", "Streptomyces katrae 30213" and "30213" mentioned in the following Examples and Effect Examples are all "Streptomyces katrae 30213".

[0021] Unless otherwise specified, other materials, reagents, etc. used in the following embodiments and effect examples can be obtained from commercial sources.

[0022] Gao's liquid medium: 0.5g / L NaCl, 0.01g / L FeSO4, 1.0g / L KNO3, 0.5g / LK2HPO4, 0.5g / L MgSO4, adjust the pH to 7.2-7.4 after adding water, sterilize under high pressure at 121℃ for 30min, and cool it for use after sterilization.

[0023] Gao's solid medium: Add 20g / L agar to Gao's liquid medium, sterilize it under high pressure at 121℃ for 30min after it is completely dissolved, and cool it for later use after sterilization.

[0024] King medium: tryptone 20g, K2HPO4 2.0g, MgSO4 1.5g, tryptophan 0.1g, glycerol 15mL, pH adjusted to 7.0, distilled water 1L.

[0025] Salkowski's reagent is mainly composed of 0.5M FeCl3 and 35v / v% HClO4, and the volume ratio of FeCl3 to HClO4 is 1:50.

[0026] The bacterial genomic DNA extraction kit was purchased from Tiangen Biochemical Technology Co., Ltd. with the catalog number DP302.

[0027] The sequencing of PCR products was commissioned to Tianyi Gene Biotechnology Co., Ltd.

[0028] Example 1

[0029] This embodiment provides a method for separating and purifying Streptomyces catera, comprising the following steps:

[0030] 1. Isolation of Streptomyces catera

[0031] Healthy and diseased wax gourd rhizosphere soils collected in Sanshui District, Foshan City, Guangdong Province were mixed and stored in a 4°C refrigerator. A sterilized conical flask with glass beads was added with 90 mL of sterile water and 10 g of wax gourd rhizosphere soil sample. The mixture was shaken at 150 rpm for 30 min and allowed to stand for 20-30 s. 1 mL of the supernatant was added to a test tube containing 9 mL of sterile water and diluted 10 times 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 the Gao's solid medium and placed in a constant temperature incubator at 30°C for 5-7 days. Single colonies with different morphological characteristics were selected and inoculated into the Gao's solid medium, numbered, and placed in a constant temperature incubator at 28°C for 5-7 days. Streptomyces numbered 30213 was initially isolated.

[0032] 2. Purification of Streptomyces catera

[0033] The Streptomyces 30213 separated in step 1 is purified to obtain purified Streptomyces 30213.

[0034] 3 Identification of Streptomyces catera

[0035] A single colony was picked from the purified Streptomyces 30213 and inoculated into 5 mL of Goethe's liquid medium, and cultured at 28° C. for 72 h to obtain a 30213 bacterial liquid.

[0036] The total DNA of 30213 was extracted by a bacterial DNA extraction kit, and the diluted total DNA of 30213 was used as a template, and the universal primer 16S rRNA for bacterial molecular identification was used to perform PCR amplification under the action of PCR mix. The PCR amplification program is shown in Table 1, and the PCR product of 30213 was obtained;

[0037] 2 μL of the obtained PCR product of 30213 was taken and identified as specific amplification by agarose gel electrophoresis, and the remaining PCR product of 30213 was subjected to first generation sequencing to obtain the nucleic acid sequence of the PCR product;

[0038] The nucleic acid sequences of the PCR products are spliced ​​and the technical error fragments at both ends are removed to obtain the processed nucleic acid sequence. The processed 16S rRNA nucleic acid sequence is shown in SEQ ID NO.1. Since the first-generation sequencing technology can only sequence DNA sequences within 1000bp each time, and the PCR product fragment of the amplified 16S rRNA is about 1500bp, the nucleic acid sequences obtained need to be spliced ​​after sequencing by the first-generation sequencing technology, which can be performed using software such as ChromasPro and DNAMAN.

[0039] The processed nucleic acid sequences were searched for homology in the EzBioCloud database (https: / / www.ezbiocloud.net / ), and the model strain sequences with high homology were downloaded. The phylogenetic tree was constructed using MEGA11 (see Figure 1 ),according to Figure 1 30213 can be identified as Streptomyces katrae.

[0040] Streptomyces katrae 30213 was deposited in Guangdong Microbiological Culture Collection Center on January 24, 2024, with a deposit number of GDMCC No: 64315, and the address of the depository unit is Building 59, No. 100, Xianlie Middle Road, Guangzhou City, Guangdong Province. Unless otherwise specified in the following examples and comparative examples, the Streptomyces katrae used are all Streptomyces katrae 30213.

[0041] Table 1 PCR amplification program

[0042]

[0043] Example 2

[0044] This embodiment provides a culture of Streptomyces catera and a preparation method thereof, wherein the preparation method of Streptomyces catera comprises the following steps:

[0045] (1) Pick a single colony of 30213 and inoculate it into Gao's solid medium, and culture it at 30°C for 5-7 days to obtain activated 30213;

[0046] (2) Use a sterile punch to punch out 6 mycelium blocks from the densely growing areas of the aerial mycelium of the activated 30213 obtained in step (1), inoculate them into 50 mL of Goethe's liquid medium, culture them at 28°C and 180 rpm for 7 days, and filter them with a single layer of gauze. The filtrate obtained is the 30213 fermentation broth (1×10 8 cfu / mL), that is, 30213 cultures.

[0047] Effect Example 1

[0048] To determine the production of indoleacetic acid (IAA) by Streptomyces catera 30213, the specific steps are as follows:

[0049] The activated Streptomyces catera 30213 was inoculated into King medium and cultured at 180 rpm for 72 h at 28 ° C. King medium without tryptophan was used as a negative control. After the culture was completed, centrifugation was performed at 8000 g for 10 min. 1 mL of the supernatant was taken into a test tube, and 2 mL of Salkowski's reagent was added. The reaction was carried out in the dark for 20 min. Compared with the control, if pink appeared in the test tube, IAA was determined to be produced. The IAA concentration was calculated according to the IAA standard curve by measuring the absorbance value of OD530. The results are shown in Table 2.

[0050] Weigh 0.1g IAA, dissolve it with a small amount of 95v / v% ethanol, and dilute it to 100mL with ddH2O to prepare a stock solution with a concentration of 1000mg / L, and store it at 4℃. Dilute the IAA stock solution to prepare IAA standard solutions with concentrations of 0, 5, 10, 20, 40, 80, and 100μg / mL. Similarly, take 1mL of the standard solution and 2mL of Salkowski's reagent to react in the dark for 20min, measure the absorbance value of OD530, and draw the IAA standard curve. Set 3 replicates. The standard curve is shown in Figure 2 .

[0051] Table 2 IAA production determination results of Streptomyces catera 30213

[0052] strain IAA value (μg / mL) 30213 13.50±0.767

[0053] As shown in Table 2, the IAA production value of 30213 was 13.50 μg / L, indicating that S. catera 30213 has the ability to produce IAA.

[0054] Effect Example 2

[0055] In order to verify the effect of Streptomyces catlae 30213 in promoting the growth of cucurbitaceae plants, a potted experiment was conducted using wax gourd as a representative of cucurbitaceae plants, as follows:

[0056] The wax gourd seeds were disinfected with 1% v / v sodium hypochlorite for 15 min, then rinsed several times with sterile water, soaked in sterile water for 2-3 h, and transferred to a sterile seedling tray. After the wax gourd grew to 4 leaves, the wax gourd seedlings with good and consistent growth were selected and moved to pots for 2 days, and 30 mL of the 30213 fermentation liquid obtained in Example 2 was irrigated to the roots of the wax gourd seedlings. After culturing for 30 days at 25-30 ° C and 14 h / day of light, the fresh weight and plant height, underground root weight and root length were counted, and the results are shown in Table 2. Irrigation water was used as the control group, and each treatment group was repeated 6 times.

[0057] Table 2 Statistical results of aboveground fresh weight, plant height, root length and root weight of wax gourd plants in different treatment groups

[0058]

[0059] As shown in Table 2, after the wax gourd seedlings were treated with 30213 culture for root irrigation, the above-ground fresh weight and root weight of the wax gourd plants were significantly increased, indicating that 30213 has the effect of promoting the growth of wax gourd plants. Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. Although the present invention is described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A Streptomyces catera, characterized in that The Streptomyces katrae is Streptomyces katrae 30213, which is deposited in Guangdong Provincial Microbiological Culture Collection Center with a deposit number of GDMCC No: 64315 and a deposit date of January 24, 2024.

2. The Streptomyces catera according to claim 1, characterized in that The 16S rRNA nucleic acid sequence of Streptomyces katrae 30213 is shown in SEQ ID NO.

1.

3. A culture of Streptomyces catera, characterized in that Obtained by fermentation of Streptomyces katrae 30213 as described in any one of claims 1-2.

4. The method for preparing a culture of Streptomyces catera according to claim 3, characterized in that: The method comprises the following steps: inoculating the activated Streptomyces catera into a liquid culture medium, and culturing at 28-32°C for 48-96h to obtain a Streptomyces catera culture.

5. The preparation method according to claim 4, characterized in that: The activated Streptomyces catera is mainly prepared by the following method: inoculating Streptomyces catera strains into a solid culture medium, and culturing at 28-32° C. for 5-7 days to obtain the activated Streptomyces catera.

6. The preparation method according to claim 4, characterized in that: The liquid culture medium is Go's liquid culture medium.

7. The preparation method according to claim 6, characterized in that: The formula of the Gao's liquid culture medium is 0.5g / LNaCl, 0.01g / L FeSO4, 1.0g / L KNO3, 0.5g / L K2HPO4, 0.5g / L MgSO4, and the pH is adjusted to 7.2-7.

4.

8. Use of the Streptomyces catera according to claim 1 or the culture of Streptomyces catera according to claim 3 in the preparation of a preparation for promoting the growth of Cucurbitaceae plants.

9. The use according to claim 8, characterized in that The cucurbitaceae plant includes at least one of wax gourd, bottle gourd, cucumber, gourd, pumpkin, watermelon and musk melon.

Images