Streptomyces vinegaticus and application thereof

Abstract

The invention discloses streptomyces vinegaticus and application thereof. The preservation number of the streptomyces vinegaticus is CCTCC (China Center for Type Culture Collection) NO: M 20232450. The streptomyces vinegaticus W143 obtained from soil is found to have growth promoting functions of producing indoleacetic acid and siderophore, dissolving phosphorus, degrading lignin and the like for the first time, and then the streptomyces vinegaticus W143 is found to be capable of effectively promoting rooting of root-cut grafted seedlings and seedlings, and can become a good bacterium source for developing microbial agents such as rooting agents and the like; and a new technology is provided for promoting rapid rooting of the root-cut grafted seedlings.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a Portuguese Streptomyces and an application thereof. Background Art

[0002] Plant roots play a key role in absorbing water, inorganic salts and a small amount of organic matter. These nutrients are essential for plant growth and development. The health of the root system is directly related to the growth and development of the plant. Therefore, ensuring the good development of the root system is of great significance to improving the quality of seedlings and achieving high efficiency and high yield of crops.

[0003] Grafting technology has been widely used in factory-based plant seedling cultivation. Common grafting methods include insert grafting, approach grafting and stick grafting. Since 1997, researchers have gradually proposed the "double root-breaking grafting method", which involves the technology of "root breaking of the rootstock, oblique insertion or stick grafting of the scion". Compared with traditional grafting methods, the double root-breaking grafting method has a faster grafting speed and higher work efficiency. However, after the root is broken, the rootstock faces insufficient nutrient and water supply due to the lack of root system, which leads to the death of seedlings, directly affecting the seedling recovery and survival rate of the grafted seedlings. Therefore, the rooting ability of the rootstock is one of the important factors affecting the survival of root-breaking grafted seedlings.

[0004] Researchers have extensively explored the endogenous and exogenous factors that affect root growth. Endogenous factors mainly include the affinity between the scion and the rootstock and the growth vitality of the rootstock, which directly affect the ability to take root early after grafting. Exogenous factors, such as the temperature, light conditions, air humidity, seedling medium used, and growth regulators applied in the healing environment after grafting, also play an important role in the rapid rooting and seedling retardation process of the rootstock. The combined effect of these factors can effectively improve the survival rate and seedling quality of grafted seedlings.

[0005] Streptomyces is an important member of the actinomycetes. It can effectively utilize nutrients in the rhizosphere through hyphae, thereby successfully colonizing the roots of plants. As one of the most abundant bacterial communities in the microbial ecosystem, Streptomyces has a strong proliferation ability and can produce antibiotics and plant hormones (including auxins, cytokines and gibberellins). In addition, Streptomyces is rich in siderophores and can secrete enzymes such as cellulase and chitinase. Through effects such as phosphate solubilization and nitrogen fixation, they can promote plant growth and reduce the stress of the external environment on plants. Streptomyces is widely used in the field of microbial agents and biological control, opening up new directions for agricultural development.

[0006] However, there is no literature report on the application of Streptomyces Lusitanus in the rapid rooting technology of root-cut grafting seedlings. Therefore, exploring the rooting potential of Streptomyces Lusitanus in root-cut grafting seedlings has important theoretical significance and application prospects. Summary of the invention

[0007] Based on the above-mentioned prior art, the present invention provides a Portuguese Streptomyces and an application thereof. The present invention obtains a Portuguese Streptomyces W143 from the soil, and finds that the strain has growth-promoting functions such as producing indoleacetic acid, siderophore, solubilizing phosphate, and degrading lignin. It is then found that the strain can effectively promote the rooting of root-cut grafted seedlings and seedlings, and can become a good source of bacteria for developing microbial agents such as rooting agents, thereby providing a new technology for promoting the rapid rooting of root-cut grafted seedlings.

[0008] The technical solution adopted to achieve the above-mentioned purpose of the present invention is:

[0009] The applicant carried out screening of beneficial bacteria and obtained an actinomycete that promotes rapid rooting of root-cut grafted seedlings. Through morphological characteristics and 16S rRNA gene sequencing analysis, the strain was identified as Streptomyces lusitanus W143, named Streptomyces lusitanus W143, and was deposited in the China Center for Type Culture Collection (address: Wuhan University, Luojia Mountain, Wuchang District, Wuhan City, Hubei Province) on December 4, 2023, with the deposit number of CCTCC NO: M 20232450.

[0010] Application of Streptomyces Portugalensis W143 in promoting plant growth.

[0011] Application of Portuguese Streptomyces W143 in promoting rapid rooting of vegetable seedlings.

[0012] Portuguese Streptomyces W143 has the characteristics of dissolving organic and inorganic phosphorus, producing siderophore, degrading lignin and generating indoleacetic acid. These characteristics promote the root growth of vegetable seedlings such as watermelon seedlings, cucumber seedlings and watermelon grafted seedlings. Specifically, it helps the rapid growth of the radicle, and can quickly form new roots after the rootstock is broken, promote the formation of roots / root tufts, and increase the length and number of roots.

[0013] A microbial agent, wherein the active ingredients of the microbial agent are Portuguese Streptomyces W143 and its fermentation products.

[0014] Furthermore, the fermentation product is the fermentation liquid of Streptomyces portugalensis W143 or the volatile gas substances produced therefrom.

[0015] Compared with the prior art, the beneficial effects and advantages of the present invention are:

[0016] 1. Portuguese Streptomyces W143 can effectively decompose organic phosphorus and inorganic phosphorus, releasing nutrients that can be absorbed by plants, indirectly improving the absorption and utilization rate of nutrients by plants. Compared with relying solely on chemical fertilizers, it has a significant effect in promoting plant growth.

[0017] 2. Using Portuguese Streptomyces W143 to promote root growth can improve soil structure and increase the organic matter content of soil or seedling medium by decomposing lignin and other organic matter. This biological improvement helps to enhance the water retention and aeration of soil or medium, ultimately improving fertility and facilitating healthy plant growth.

[0018] 3. Compared with conventional fertilizers and chemical growth regulators, microbial agents greatly reduce the demand for chemical drugs. This not only reduces the risk of environmental pollution, but also reduces the potential harm to humans and animals, and achieves safe production of crops.

[0019] 4. Portuguese Streptomyces W143 can also stimulate the diversification and growth of root systems. It can quickly form new roots after the rootstock is rooted. The strong root system can help the root-cut grafted seedlings survive and develop under adverse conditions such as low temperature and disease. This not only improves the resistance of the root-cut grafted seedlings, but also enhances their ability to adapt to adversity.

[0020] 5. The present invention reports for the first time the application of Streptomyces Lusitanus in the rapid rooting of grafted seedlings with broken roots, providing a new source of bacteria for the development of microbial agents such as rooting agents. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the microscopic morphology of Portuguese Streptomyces W143 (1000X).

[0022] Figure 2 The colony morphology of Streptomyces Portugalensis W143. The left picture is the front view of the ISP-2 culture medium plate, and the right picture is the back view of the ISP-2 culture medium plate.

[0023] Figure 3 This is the experimental result of the characteristics of Portuguese Streptomyces W143 in phosphate solubilization, siderophore production, and lignin degradation. Figure 3 -A is the experimental result diagram of decomposition of organic phosphorus. Figure 3 -B is the experimental result diagram of decomposition of inorganic phosphorus, Figure 3 -C is the experimental result of siderophore production, Figure 3 -D is the experimental result diagram of lignin degradation.

[0024] Figure 4 This is the experimental result of the indoleacetic acid production characteristics of Portuguese Streptomyces W143.

[0025] Figure 5 This is a picture showing the actual effect of the volatile gases produced by the culture of Portuguese Streptomyces W143 on the growth-promoting effect of Arabidopsis thaliana.

[0026] Figure 6 This is a real-life effect diagram showing the effect of the fermentation products of Portuguese Streptomyces W143 on promoting the root growth of cucumber seedlings.

[0027] Figure 7 This is a real-life effect diagram showing the effect of the fermentation products of Portuguese Streptomyces W143 on promoting the root growth of watermelon grafted seedlings. DETAILED DESCRIPTION

[0028] The present invention is described in detail below with reference to specific embodiments.

[0029] Unless otherwise specified, the sources of the raw materials used in the following examples are as follows:

[0030] Bacteria: Streptomyces murinus JKTJ-3 (Mihong Ge, Xiang Cai, Dehuan Wang, et al. Efficacy of Streptomyces murinus JKTJ-3 in Suppression of Pythium Damping-Off of Watermelon. Microorganisms, 2023, 11, 1360.

[0031] Example 1: Isolation and identification of Streptomyces portugalensis W143

[0032] 1. In May 2021, during the research on root-promoting melon seedling substrates, a group of Streptomyces were isolated from the watermelon planting soil in Wuhu Agricultural Ecological Park, Huangpi District, Wuhan City. A Streptomyces strain was found that promoted the root growth and development of watermelon seedlings. The pure culture W143 was finally obtained by plate streaking purification method. The pure culture W143 was observed under a microscope, and the results are as follows: Figure 1 As shown. Figure 1 It can be seen that the spores of strain W143 are straight and the spores are oval.

[0033] 2. Strain W143 was cultured in ISP-2 medium, and then the colony morphology of strain W143 was photographed. The results are as follows: Figure 2 As shown. Figure 2 It can be seen that strain W143 grows well on ISP-2 medium, with brown-yellow mycelium and no soluble pigment.

[0034] 3. The physiological and biochemical indicators of strain W143 were determined. The results are shown in Table 1:

[0035] Table 1 Physiological and biochemical characteristics of strain W143

[0036]

[0037]

[0038] Note: +: positive reaction; -: negative reaction; W: weak positive reaction

[0039] 4. The 16S rRNA gene of strain W143 was amplified by PCR and sequenced. The obtained DNA fragment was connected to the T vector using a T vector kit, and positive clones were screened. Subsequently, these clones were sent to Wuhan Tianyi Huayu Gene Technology Co., Ltd. for sequence determination, and the obtained sequence is shown in SEQ ID NO: 1. The target fragment obtained according to the primer selection was compared by sequence similarity comparison using EzbioCloud, and the results showed that strain W143 and Streptomyces Lusitanus NBRC 13464 T The similarity between them was as high as 99.1%. Based on the analysis results of morphological observation, physiological and biochemical characteristics, 16S rRNA gene sequencing and sequence comparison, strain W143 was finally confirmed to be classified as Streptomyces Lusitanus.

[0040] 5. The applicant has deposited it with the China Center for Type Culture Collection on December 4, 2023, and the classification name is Streptomyces Lusitanus W143, and the deposit number is CCTCC NO: M 20232450. Example 2 Streptomyces Lusitanus W143 has the characteristics of solubilizing phosphate, degrading lignin, producing siderophore and indoleacetic acid

[0041] 1. Inoculate the Portuguese Streptomyces W143 strain into the Montgena organic phosphorus and inorganic phosphorus culture media respectively, place them in a biochemical incubator at 28°C for 7 days, and judge their phosphorus solubilization ability based on whether a transparent circle appears on the edge of the colony.

[0042] 2. Inoculate the Portuguese Streptomyces W143 strain onto a universal CAS test plate and culture it in a biochemical incubator at 28°C for 2 to 7 days in the dark. Determine its ability to produce siderophores by observing whether a clear orange-yellow siderophore chelation ring is formed on the plate.

[0043] 3. Inoculate the Portuguese Streptomyces W143 strain onto a universal aniline blue-PDA plate and culture it in a biochemical incubator at 28°C in the dark for 2 to 7 days. Evaluate its lignin degradation ability by detecting whether the blue color at the edge of the colony fades.

[0044] 4. Inoculate the Portuguese Streptomyces W143 strain into PDB liquid culture medium (add 200 mg / L L-tryptophan) and culture for 24 hours, then filter the culture solution with a bacterial filter (pore size of 0.22 μm) to obtain W143 sterile filtrate. Add an equal volume of Salkowski colorimetric reagent to the W143 sterile filtrate, quickly place it in the dark for 30 minutes, and observe whether there is a color reaction. If it shows red, it is preliminarily judged that it has the ability to produce indoleacetic acid, and the strength of the indoleacetic acid synthesis ability is preliminarily judged based on the depth of red.

[0045] 5. The above experimental results are as follows Figure 3 , Figure 4 As shown by Figure 3 -A and Figure 3 -B shows that transparent circles appeared at the edge of the colony of Streptomyces Portugale W143 in both the organic phosphorus and inorganic phosphorus medium of Monkina, indicating that Streptomyces Portugale W143 has the ability to solubilize phosphorus. Figure 3 -C shows that an obvious orange-yellow siderophore chelation ring appeared on the CAS test plate, indicating that Portuguese Streptomyces W143 has the ability to produce siderophores. Figure 3 -D shows that the blue color at the edge of the colony of Streptomyces portugalensis W143 on the aniline blue-PDA plate faded significantly, indicating that Streptomyces portugalensis W143 has the ability to degrade lignin. Figure 4 It can be seen that when the sterile filtrate of W143 is mixed with the Salkowski colorimetric reagent, a red color reaction occurs, indicating that W143 has the ability to produce indoleacetic acid.

[0046] Example 3 Growth-promoting ability of volatile gases produced by culture of Streptomyces portugalensis W143 on Arabidopsis thaliana

[0047] 1. Take an appropriate amount of Arabidopsis seeds in a 1.5ml EP tube, add 2% (v / v) effective chlorine sodium hypochlorite solution to disinfect for 8 minutes, then disinfect with 75% (v / v) ethanol solution for 5 minutes, and then wash with sterile water 5 times, each time for 1 minute. Add the disinfected seeds to 0.2% (w / v) water agar medium and place in a 4°C refrigerator overnight.

[0048] 2. Pour 1 / 2MS culture medium into one compartment of a two-compartment culture dish and ISP-2 culture medium into the other compartment. After the culture medium in the two compartments solidifies, spot 7 Arabidopsis seeds on the 1 / 2MS culture medium and inoculate 2.5 cm long Portuguese Streptomyces W143 on the ISP-2 culture medium.

[0049] 3. Seal the two-grid culture dish with sealing film and place it in a light incubator at 20°C with day and night alternation (16h / 8h). At the same time, use blank and S.murinus JKTJ-3 as controls and repeat the experiment 3 times.

[0050] 4. After 20 days of cultivation, the seedlings germinated from the experimental group and the control group were treated as follows:

[0051] 1) Take photos of the germinating seedlings of Arabidopsis thaliana in the experimental group and the control group. Figure 5 As shown by Figure 5 It can be seen that the volatile gases produced by the culture of Portuguese Streptomyces W143 have the most obvious effect on promoting the growth of seedlings germinated from Arabidopsis seeds, and the root length and leaf number are significantly higher than those of the two control groups.

[0052] 2) The fresh weight, root length and number of lateral roots of the seedlings germinated from the Arabidopsis seeds in the experimental group and the control group were counted. The results are shown in Table 2 below:

[0053] Table 2 Growth-promoting effects of volatile gases produced by the culture of Streptomyces portugalensis W143 on Arabidopsis thaliana

[0054]

[0055] As can be seen from Table 2, the volatile gases produced by the culture of Portuguese Streptomyces W143 have the most significant effect on promoting the growth of Arabidopsis thaliana. The number of lateral roots, root length and fresh weight of Arabidopsis thaliana seedlings promoted by the volatile gases produced by the culture of Portuguese Streptomyces W143 are significantly higher than those of Arabidopsis thaliana seedlings promoted by the volatile gases produced by the culture of Streptomyces murinus JKTJ-3, and the difference with Arabidopsis thaliana seedlings without growth promotion is more significant.

[0056] Example 4 Cultures of Streptomyces portugalensis W143 and Streptomyces muridarum JKTJ-3 and preparation of fermentation products

[0057] 1. Plate culture: Each culture dish uses PDA medium to make a plate. The formula of PDA medium is as follows: 200 grams of fresh peeled potatoes, 20 grams of glucose, 13 grams of agar powder and 1000 milliliters of tap water. On the prepared PDA plate, use aseptic operation to streak inoculated Portuguese Streptomyces W143 and mouse gray Streptomyces JKTJ-3 respectively, and then culture at 28°C for 7 days to produce a large number of spores. Add 10 ml of sterile water to each culture dish to wash the spores on the culture medium, and gently shake the culture dish to ensure that the spores are effectively collected. Then, dilute the washed spore suspension to 10 8 CFU / ml concentration to prepare for the next fermentation.

[0058] 2. Fermentation culture: PDB liquid culture medium was used for fermentation culture. The formula of PDB liquid culture medium is as follows: 200 g fresh peeled potatoes, 20 g glucose and 1000 ml tap water. 1 ml of 10 8 CFU / ml of the spore suspension of Streptomyces prausnitzii W143 and the spore suspension of Streptomyces muridarum JKTJ-3, and then cultured with shaking at 220 rpm at 28°C for 4 days to obtain the fermentation broth containing Streptomyces prausnitzii W143 and the fermentation broth containing Streptomyces muridarum JKTJ-3, respectively, for use.

[0059] Example 5 Effect of the fermentation product of Streptomyces Portugalensis W143 on the root growth of cucumber seedlings

[0060] 1. The fermentation broth containing Portuguese Streptomyces W143 and mouse gray Streptomyces JKTJ-3 prepared in Example 4 was diluted 10 times to obtain fermentation broth W143 and fermentation broth JKTJ-3. The fermentation broth W143 and fermentation broth JKTJ-3 were respectively mixed with a common seedling medium (mixed by peat and perlite in a volume ratio of 3:1) at a volume ratio of 1:60 to obtain W143 seedling medium and JKTJ-3 seedling medium, respectively.

[0061] 2. Fill the W143 seedling medium into a 50-hole plug tray for later use, then sow the plump and uniform cucumber seeds into the plug tray filled with the W143 seedling medium, and then place it in the seedling greenhouse for cultivation. Ordinary seedling medium and JKTJ-3 seedling medium were used as controls. The experiment was repeated 3 times, and 50 grafted seedlings were treated in each treatment group in each experiment.

[0062] 3. 35 days after sowing (seedling stage), the cucumber seedlings in the experimental group and the control group were treated as follows:

[0063] 1) Take photos of the seedlings in the experimental group and the control group. Figure 6 .Depend on Figure 6 It can be seen that according to the growth of the root system of cucumber seedlings in the seedling stage, the seedling medium containing the fermentation product of Portuguese Streptomyces W143 has the most obvious promoting effect on the root growth of cucumber seedlings, and the root volume is significantly higher than that of the two control groups.

[0064] 2) The underground fresh weight, underground dry weight, root length, root volume and root activity of the cucumber seedlings in the experimental group and the control group were statistically analyzed. The results are shown in Table 3 below:

[0065] Table 3 Effects of Streptomyces W143 on root growth of cucumber seedlings

[0066]

[0067] As shown in Table 3, compared with the control group CK, Portuguese Streptomyces W143 significantly increased the underground fresh weight, underground dry weight, root length, root volume and root activity of cucumber seedlings, with increases of 40%, 40.8%, 17.6%, 29.9% and 9.6%, respectively. Compared with the control group JKTJ-3, Portuguese Streptomyces W143 also significantly increased the underground fresh weight, underground dry weight, root length, root volume and root activity of cucumber seedlings, with increases of 26.5%, 31.5%, 8.27%, 25.6% and 6.9%, respectively. In terms of underground fresh weight and root length, the control group JKTJ-3 significantly increased by 10.7% and 8.6% compared with the control group CK, respectively. These results show that Portuguese Streptomyces W143 has obvious advantages in promoting the root growth of cucumber seedlings.

[0068] Example 6 Effect of the fermentation product of Portuguese Streptomyces W143 on the root growth of watermelon root-cut grafted seedlings 1. The fermentation broth containing Portuguese Streptomyces W143 and mouse gray Streptomyces JKTJ-3 prepared in Example 4 was diluted 10 times to obtain fermentation broth W143 and fermentation broth JKTJ-3. The fermentation broth W143 and fermentation broth JKTJ-3 were respectively mixed with ordinary seedling medium (mixed by peat and perlite in a volume ratio of 3:1) at a volume ratio of 1:60 to obtain W143 seedling medium and JKTJ-3 seedling medium, respectively.

[0069] 2. Fill the W143 seedling medium into a 50-hole plug tray for later use. Take pumpkin rootstock seedlings and watermelon scion seedlings suitable for grafting, and graft them using the root cutting method. Insert the grafted seedlings into the plug tray containing the W143 seedling medium, and then place them in a healing room (temperature 30℃, light intensity increased from 1000lx-3000lx-5000lx day by day) for 4 days. Ordinary seedling medium and JKTJ-3 seedling medium were used as controls. Repeat the experiment 3 times, and 50 grafted seedlings were treated in each treatment group in each experiment.

[0070] 3. During the healing period (5th day) and seedling period (45th day) after grafting, the grafted seedlings in the experimental group and the control group were treated as follows:

[0071] 1) Take photos of the grafted seedlings in the experimental group and the control group. Figure 7 .Depend on Figure 7 It can be seen that according to the growth of the root system of the grafted seedlings in the healing period and the seedling stage, the seedling medium containing the fermentation product of Portuguese Streptomyces W143 has the most obvious promoting effect on the root growth of the grafted seedlings, and the number and length of new roots are significantly higher than those of the two control groups.

[0072] 2) The number and length of new roots of the grafted seedlings in the experimental group and the control group were statistically analyzed. The results are shown in Table 4 below:

[0073] Table 4 Growth-promoting effect of Streptomyces Portugalensis W143 on root-cut grafting

[0074]

[0075] As shown in Table 4, during the graft healing period, compared with the control group CK, Portuguese Streptomyces W143 significantly increased the number of new roots of grafted seedlings, with an increase of 191.3%. Compared with the control group JKTJ-3, Portuguese Streptomyces W143 significantly increased the number of new roots of grafted seedlings, with an increase of 103.0%. Further analysis showed that compared with the grafted seedlings of the control group CK, the root length of the new roots of the grafted seedlings of the experimental group W143 and the control group JKTJ-3 increased by 996.2% and 626.9%, respectively. In the seedling stage, Portuguese Streptomyces W143 also significantly increased the root volume, underground fresh weight and underground dry weight of the grafted seedlings, compared with the control group CK, the increases were 88.6%, 93.3% and 68.5%, respectively, and the grafted seedling rate increased by 15.7%. Compared with the control group JKTJ-3, the increases were 33.0%, 26.7% and 33.0% respectively, and the grafting seedling rate increased by 6.5%. Further variance analysis showed that the experimental group W143 was significantly better than the control group JKTJ-3 and the control group CK in all indicators. These results show that Portuguese Streptomyces W143 has obvious advantages in promoting root growth after root cutting and grafting of watermelon, thereby effectively improving the grafting seedling rate.

Claims

1. Streptomyces lusitanus W143, characterized in that It is deposited in China Center for Type Culture Collection with the deposit number CCTCC NO: M 20232450.

2. Use of the Streptomyces portugalensis W143 according to claim 1 in promoting plant growth.

3. Use of the Portuguese Streptomyces W143 described in claim 1 in promoting rapid rooting of vegetable seedlings.

4. The use of Streptomyces portugalensis W143 according to claim 3, characterized in that: The vegetable seedlings include seedlings and grafted seedlings.

5. A microbial agent, characterized in that: The active ingredient of the microbial agent is the Portuguese Streptomyces W143 and its fermentation product as described in claim 1.

6. The microbial agent according to claim 5, characterized in that: The fermentation product is the fermentation liquid of Streptomyces portugalensis W143 according to claim 1 or the volatile gas substances produced therefrom.

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