A streptomyces portugaliae and its use

Abstract

The application discloses Streptomyces portugensis and application thereof, wherein the preservation number of the Streptomyces portugensis is CCTCC NO: M 20232450. The Streptomyces portugensis W143 obtained from soil is found to have the functions of producing indole acetic acid, siderophore, dissolving phosphorus and degrading lignin for the first time, and then it is found that the strain can effectively promote the root formation of grafted seedlings with broken roots and seedling seedlings, and can become a good bacterial source for developing rooting agents and other microbial agents, and provides a new technology for promoting the rapid root formation of grafted seedlings with broken roots.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a type of Streptomyces lucida and its applications. Background Technology

[0002] Plant roots play a crucial role in absorbing water, inorganic salts, and small amounts of organic matter, all of which are essential for plant growth and development. The health of the root system directly affects plant growth and development; therefore, ensuring healthy root development is of great significance for improving seedling quality and achieving high-efficiency, high-yield crops.

[0003] Grafting technology is widely used in the industrialized seedling production of plants. Common grafting methods include cleft grafting, approach grafting, and mat grafting. Since 1997, researchers have gradually developed the "double-root-severance grafting method," which involves "root severance of the rootstock and oblique insertion or mat grafting of the scion." Compared to traditional grafting methods, double-root-severance grafting offers faster grafting speed and higher work efficiency. However, after root severance, the rootstock lacks a root system, leading to insufficient nutrient and water supply, which in turn causes seedling death, directly affecting the recovery and survival rate of grafted seedlings. Therefore, the rooting ability of the rootstock is one of the important factors affecting the survival rate of root-severance grafted seedlings.

[0004] Researchers have extensively explored the endogenous and exogenous factors influencing root growth. Endogenous factors mainly include the compatibility between the scion and rootstock, as well as the rootstock's growth vigor, which directly affect the early rooting ability after grafting. Exogenous factors, such as temperature, light conditions, air humidity, the seedling substrate used, and the applied growth regulators in the healing environment after grafting, also play a significant role in the rapid rooting and recovery 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, capable of effectively utilizing nutrients in the rhizosphere through its hyphae to successfully colonize plant roots. As one of the most abundant microbial communities in the ecosystem, Streptomyces possesses a strong proliferative capacity, producing antibiotics and plant hormones (including auxins, cytokinins, and gibberellins). Furthermore, Streptomyces is rich in iron cells and can secrete enzymes such as cellulase and chitinase. Through phosphorus solubilization and nitrogen fixation, they can promote plant growth and alleviate environmental stress on plants. Streptomyces is widely used in microbial inoculants and biological control, opening up new avenues for agricultural development.

[0006] However, there are currently no reports in the literature regarding the application of Streptomyces lusitanus in the rapid rooting technique of root-severed grafting. Therefore, exploring the rooting potential of Streptomyces lusitanus in root-severed grafting has significant theoretical implications and promising application prospects. Summary of the Invention

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

[0008] The technical solution adopted to achieve the above-mentioned objectives of this invention is as follows:

[0009] The applicant conducted screening for beneficial bacteria and obtained an actinomycete that promotes rapid rooting of grafted seedlings with severed roots. Based on morphological characteristics and 16S rRNA gene sequencing analysis, the strain was identified as *Streptomyces lusitanus* W143 and named *Streptomyces lusitanus* W143. It was deposited on December 4, 2023, at the China Center for Type Culture Collection (address: Wuhan University, Luojia Mountain, Wuchang District, Wuhan, Hubei Province), with accession number CCTCC NO: M 20232450.

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

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

[0012] Streptomyces cerevisiae W143 possesses the properties of dissolving organic and inorganic phosphorus, producing ferrophosphate, degrading lignin, and generating indoleacetic acid. These properties promote the root growth of vegetable seedlings such as watermelon seedlings, cucumber seedlings, and grafted watermelon seedlings. Specifically, it helps the rapid growth of the radicle and enables the rapid formation of new roots after the rootstock is severed, promoting the formation of roots / root balls and increasing the length and number of roots.

[0013] A microbial inoculant, wherein the active ingredient of the microbial inoculant is Streptomyces lucida W143 and its fermentation products.

[0014] Furthermore, the fermentation product is the fermentation broth of Streptomyces lucida W143 or the volatile gaseous substances produced therefrom.

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

[0016] 1. Streptomyces cerevisiae W143 can effectively decompose organic 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, its effect on promoting plant growth is significant.

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

[0018] 3. Compared with conventional fertilizers and chemical growth regulators, microbial inoculants greatly reduce the need for chemical reagents. This not only reduces the risk of environmental pollution but also reduces potential harm to humans and livestock, thus ensuring safe crop production.

[0019] 4. Streptomyces cerevisiae W143 can also stimulate root diversification and growth. After the rootstock is cut, new roots can be formed rapidly. 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. This invention is the first to report the application of Streptomyces Lusitanus Portugal in the rapid rooting of grafted seedlings with severed roots, providing a new source of microorganisms for the development of rooting agents and other microbial agents. Attached Figure Description

[0021] Figure 1 Microscopic morphology (1000X) of Streptomyces lucida W143.

[0022] Figure 2 The images show the colony morphology of Streptomyces Portugueseum W143. The left image is a front view of the ISP-2 medium plate, and the right image is a back view of the ISP-2 medium plate.

[0023] Figure 3 The figure shows the experimental results of the phosphorus-solubilizing, heparin-producing, and lignin-degrading characteristics of Streptomyces lucida Portuguesei W143. Figure 3 -A shows the experimental results of decomposing organophosphates. Figure 3 -B shows the experimental results of decomposing inorganic phosphorus. Figure 3 -C represents the experimental results of heptaphilic production. Figure 3 -D is the experimental results of lignin degradation.

[0024] Figure 4 Figure showing the experimental results of indoleacetic acid production characteristics of Streptomyces cerevisiae W143 in Portugal.

[0025] Figure 5 This image shows the effect of volatile gases produced by a culture of Streptomyces lucida W143 on the growth-promoting effect on Arabidopsis thaliana.

[0026] Figure 6 This is a photograph showing the effect of Streptomyces cerevisiae W143 fermentation product on the root growth of cucumber seedlings.

[0027] Figure 7 This is a photograph showing the effect of fermentation products of Streptomyces cerevisiae W143 on promoting root growth in watermelon seedlings grafted with root pruning. Detailed Implementation

[0028] The present invention will now be described in detail with reference to specific embodiments.

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

[0030] Strain: 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 lucida W143

[0032] 1. In May 2021, during a study on root-promoting substrates for cucurbit seedlings, a batch of *Streptomyces* strains were isolated from watermelon planting soil in the Wuhu Agricultural Ecological Park, Huangpi District, Wuhan City. One strain of *Streptomyces* was found to promote root growth and development in watermelon seedlings. A pure culture, designated W143, was obtained using the streak plate purification method. The pure culture W143 was observed under a microscope, and the results are as follows: Figure 1 As shown. From Figure 1 It can be seen that the spore hyphae of strain W143 are straight, and the spores are elliptical.

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

[0034] 3. The physiological and biochemical parameters of strain W143 were determined, and 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 ligated into a T-vector using a T-vector kit, and positive clones were screened. These clones were then sent to Wuhan Tianyi Huayu Gene Technology Co., Ltd. for sequencing, and the resulting sequence is shown in SEQ ID NO:1. The target fragment obtained according to the primers was selected, and sequence similarity was compared using EzbioCloud. The results showed that strain W143 is similar to *Streptomyces lusitanus* NBRC 13464. T The similarity between them was as high as 99.1%. Based on the analysis results of comprehensive morphological observation, physiological and biochemical characteristic tests, and 16S rRNA gene sequencing and sequence alignment, strain W143 was finally confirmed to be classified as Streptomyces Lusitanus.

[0040] 5. The applicant deposited the specimen with the China Center for Type Culture Collection (CCTCC) on December 4, 2023, classifying it as *Streptomyces lusitanus* W143, with accession number CCTCC NO: M 20232450. Example 2: *Streptomyces lusitanus* W143 exhibits properties of phosphorus solubilization, lignin degradation, and the production of heparin and indoleacetic acid.

[0041] 1. Inoculate Streptomyces Portugueseum W143 strain into Monkina organic phosphorus and inorganic phosphorus media, respectively, and incubate them in a biochemical incubator at 28°C for 7 days. The phosphorus solubilizing ability is judged by whether a clear zone appears at the edge of the colony.

[0042] 2. Inoculate Streptomyces Portugueseum W143 strain onto a universal CAS detection plate and incubate in the dark at 28°C for 2 to 7 days. Determine its siderophore production capacity by observing whether a distinct orange-yellow siderophore chelation zone forms on the plate.

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

[0044] 4. Inoculate Streptomyces Portugueseum strain W143 into PDB liquid medium (with 200 mg / L L-tryptophan added) and culture for 24 h. Filter the culture medium through a bacterial filter (0.22 μm pore size) to obtain sterile W143 filtrate. Add an equal volume of Salkowski colorimetric reagent to the sterile W143 filtrate, and incubate in the dark for 30 min. Observe whether a color reaction occurs. If a red color is observed, it is preliminarily determined that the strain has the ability to produce indoleacetic acid. The intensity of the red color is used to preliminarily determine the strength of the indoleacetic acid synthesis ability.

[0045] 5. The above experimental results are as follows: Figure 3 , Figure 4 As shown, by Figure 3 -A and Figure 3 -B indicates that, regardless of whether it was in the Monkina organic or inorganic phosphorus medium, the colonies of *Streptomyces lucida* W143 exhibited a clear zone at their edges, indicating that *Streptomyces lucida* W143 possesses phosphate-solubilizing ability. From... Figure 3 As indicated by -C, a distinct orange-yellow siderophore chelation zone appeared on the CAS test plate, suggesting that *Streptomyces lucida* W143 possesses the ability to produce siderophores. Figure 3 As indicated by -D, on the aniline blue-PDA plate, the blue color at the edge of *Streptomyces cerevisiae* W143 colonies faded significantly, indicating that *Streptomyces cerevisiae* W143 possesses the ability to degrade lignin. From... Figure 4 It can be seen that when W143 sterile filtrate is mixed with Salkowski colorimetric reagent, a red color reaction occurs, indicating that W143 has the ability to produce indoleacetic acid.

[0046] Example 3: The growth-promoting effect of volatile gases produced by Streptomyces Portugal W143 culture on Arabidopsis thaliana.

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

[0048] 2. Pour 1 / 2 MS medium into one compartment of a two-compartment petri dish and ISP-2 medium into the other compartment. After the medium in both compartments has solidified, place 7 Arabidopsis thaliana seeds on the 1 / 2 MS medium and streak 2.5 cm long Streptomyces lucida W143 on the ISP-2 medium.

[0049] 3. Seal the bipartite culture dishes with sealing film and place them in a 20℃, day / night alternating light incubator (16h / 8h). Simultaneously, use a blank control and *Streptomyces murinus* JKTJ-3 as controls, repeating the experiment three times.

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

[0051] 1) Photographs were taken of the germinated Arabidopsis seedlings from the experimental and control groups. The results are as follows: Figure 5 As shown, by Figure 5 It can be seen that the volatile gases produced by the Streptomyces Portugali W143 culture have the most significant effect on promoting the growth of Arabidopsis seedlings, with root length and leaf number significantly higher than those of the two control groups.

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

[0053] Table 2. Growth-promoting effects of volatile gases produced by Streptomyces lucida W143 cultures on Arabidopsis thaliana.

[0054]

[0055] As shown in Table 2, the volatile gases produced by the Streptomyces lucida W143 culture had 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 induced by the volatile gases produced by the Streptomyces lucida W143 culture were significantly higher than those induced by the volatile gases produced by the Streptomyces oryzae JKTJ-3 culture, and the difference was even more significant compared with Arabidopsis thaliana seedlings without the stimulation of any strain.

[0056] Example 4: Preparation of cultures and fermentation products of *Streptomyces lucida* W143 and *Streptomyces oryzae* JKTJ-3

[0057] 1. Plate Culture: Each petri dish was prepared using PDA medium, with the following formulation: 200g fresh peeled potatoes, 20g glucose, 13g agar powder, and 1000ml tap water. Under aseptic conditions, *Streptomyces lucida* W143 and *Streptomyces griseus* JKTJ-3 were streaked onto the prepared PDA plates and incubated at 28°C for 7 days to produce abundant spores. 10ml of sterile water was added to each petri dish to wash the spores from the medium, and the dishes were gently agitated to ensure effective spore collection. The washed spore suspension was then diluted to 10... 8 The concentration of CFU / ml is used to prepare for the next stage of fermentation.

[0058] 2. Fermentation Culture: Fermentation was carried out using PDB liquid medium. The PDB liquid medium formula is as follows: 200g fresh peeled potatoes, 20g glucose, and 1000ml tap water. 1ml of 10% glucose solution was inoculated into 100ml of PDB liquid medium. 8 CFU / ml of Streptomyces lucida W143 spore suspension and Streptomyces ursevieria JKTJ-3 spore suspension were cultured at 28°C and shaken at 220 rpm for 4 days to obtain fermentation broth containing Streptomyces lucida W143 and Streptomyces ursevieria JKTJ-3, respectively, for later use.

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

[0060] 1. The fermentation broth containing *Streptomyces lucida* W143 and *Streptomyces oryzae* JKTJ-3 prepared in Example 4 was diluted 10 times to obtain fermentation broth W143 and fermentation broth JKTJ-3. Fermentation broth W143 and fermentation broth JKTJ-3 were respectively mixed with ordinary seedling substrate (made by mixing peat moss and perlite in a volume ratio of 3:1) at a volume ratio of 1:60 to obtain seedling substrate W143 and seedling substrate JKTJ-3, respectively.

[0061] 2. Fill 50-cell seedling trays with W143 seedling substrate and set aside. Then, sow plump and uniform cucumber seeds into the trays filled with W143 seedling substrate, and then place them in a seedling greenhouse for cultivation. Ordinary seedling substrate and JKTJ-3 seedling substrate were used as controls. The experiment was repeated 3 times, with 50 grafted seedlings treated in each treatment group each time.

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

[0063] 1) Photographs were taken of the seedlings in the experimental and control groups. The results are shown in the figure. Figure 6 .Depend on Figure 6 It can be seen from the growth of the root system of cucumber seedlings in the seedling stage that the seedling substrate containing Streptomyces cerevisiae W143 fermentation product 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 fresh weight of the underground part, dry weight of the underground part, root length, root volume, and root activity of 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] Table 3 shows that, compared with the control group (CK), *Streptomyces lucida* 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, *Streptomyces lucida* 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. Regarding underground fresh weight and root length, compared with the control group (CK), the JKTJ-3 group showed significant increases of 10.7% and 8.6%, respectively. These results indicate that *Streptomyces lucida* W143 has a significant advantage in promoting root growth in cucumber seedlings.

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

[0069] 2. Fill 50-cell seedling trays with W143 seedling substrate for later use. Take suitable pumpkin rootstock seedlings and watermelon scion seedlings for grafting, and perform grafting using the root-cutting grafting method. Insert the grafted seedlings into the trays containing W143 seedling substrate, and then place them in a healing chamber (temperature 30℃, light intensity gradually increasing from 1000lx to 3000lx to 5000lx) for 4 days. Use ordinary seedling substrate and JKTJ-3 seedling substrate as controls. Repeat the experiment 3 times, with 50 grafted seedlings treated in each treatment group each time.

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

[0071] 1) Photographs were taken of the grafted seedlings in the experimental and control groups, and the results are shown below. Figure 7 .Depend on Figure 7 It can be seen from the growth of the root system of grafted seedlings during the healing period and the seedling stage that the seedling substrate containing Streptomyces lucida W143 fermentation product has the most obvious promoting effect on the root growth of 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 and control groups were counted, and the results are shown in Table 4 below:

[0073] Table 4. Growth-promoting effect of Streptomyces lucida W143 on root grafting.

[0074]

[0075] Table 4 shows that during the grafting healing period, compared with the control group (CK), *Streptomyces cerevisiae* W143 significantly increased the number of new roots in grafted seedlings, with an increase of 191.3%. Compared with the control group (JKTJ-3), *Streptomyces cerevisiae* W143 significantly increased the number of new roots in grafted seedlings, with an increase of 103.0%. Further analysis showed that compared with the grafted seedlings in the control group (CK), the root length of new roots in the experimental group (W143) and the control group (JKTJ-3) increased by 996.2% and 626.9%, respectively. During the seedling stage, *Streptomyces cerevisiae* W143 also significantly increased the root volume, underground fresh weight, and underground dry weight of grafted seedlings, with increases of 88.6%, 93.3%, and 68.5%, respectively, compared with the control group (CK), and the grafting seedling success rate increased by 15.7%. Compared to the control group JKTJ-3, the increases were 33.0%, 26.7%, and 33.0%, respectively, and the grafted seedling survival rate increased by 6.5%. Further analysis of variance showed that the experimental group W143 was significantly superior to both the control group JKTJ-3 and the control group CK in all indicators. These results indicate that *Streptomyces lucida* W143 has a significant advantage in promoting root growth after watermelon root pruning grafting, thus effectively improving the grafted seedling survival rate.

Claims

1. A strain of Streptomyces cerevisiae ( Streptomyces lusitanus W143, characterized in that, It is deposited at the China Center for Type Culture Collection, with accession number CCTCC NO: M 20232450.

2. The application of the Portuguese Streptomyces W143 as described in claim 1 in promoting rapid rooting of vegetable root-cut grafted seedlings.

3. A microbial inoculant, characterized in that: The microbial agent contains the Portuguese Streptomyces W143 and its fermentation products as active ingredients as described in claim 1.

4. The microbial agent according to claim 3, characterized in that: The fermentation product is the fermentation broth of Streptomyces lucida W143 as described in claim 1, or the volatile substances produced therefrom.

5. The application of the microbial agent according to claim 3 or 4 in promoting rapid rooting of vegetable root-cut grafted seedlings.

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