Streptomyces flavus 11-7 and application thereof

Abstract

The invention discloses streptomyces fulvus 11-7 and application thereof, and belongs to the technical field of microorganisms. The invention discloses a streptomyces fulvus strain 11-7, and the preservation number of the streptomyces fulvus strain 11-7 is CCTCC (China Center for Type Culture Collection) NO: M 2025222. The streptomyces fulvus 11-7 disclosed by the invention has the optimal bacteriostatic activity on Colletotrichum chlorophyti, Colletotrichum plurivorin and Rigidoporus ligosus, and the bacteriostatic rates of the streptomyces fulvus 11-7 are respectively 96.75%, 95.89% and 92.94%. The streptomyces fulvus 11-7 disclosed by the invention has the advantages that the streptomyces fulvus 11-7 can be used for preparing the antibacterial agent for preventing and treating the pathogenic bacteria of the Colletotrichum chlorophyti, the Colletotrichum plurivorin and the Rigidoporus ligosus; volatile substances generated by the streptomyces fulvus 11-7 have an inhibition effect on a plurality of pathogenic bacteria. The streptomyces fulvus 11-7 can be used for fixing nitrogen, producing ammonia and producing indoleacetic acid.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and more specifically to a Streptomyces xanthocidicus 11-7 and its applications. Background Art

[0004] Currently, the prevention and control of plant diseases still mainly rely on chemical control; however, long-term and large-scale use of chemical agents will lead to serious problems of drug resistance, and there are certain phytotoxicity and pollution to crops and their environment. Therefore, it is imperative to find a relatively efficient and safe prevention and control measure. Biological control using microbial resources or their metabolites has become the main trend under the green agriculture trend. Currently, most of the bioactive substances discovered from microorganisms are produced by Streptomyces. The active substances produced by Streptomyces can be used as agricultural fungicides, insecticides, herbicides, and plant growth regulators, and they play an important role in the green prevention and control of crop diseases, pests, and weeds due to their characteristics of high efficiency, low toxicity, and environmental friendliness.

[0005] Using microorganisms that produce volatile antibacterial substances to control plant diseases is a prevention and control method that has emerged in recent years. These volatile substances are a class of small molecular weight organic and inorganic compounds, with good cell membrane penetrability and high-efficiency diffusion ability in air and soil pores, which can quickly expand the action range of volatile substances and improve the inhibition rate against target microorganisms, and are of great significance for crop food safety. Currently, there is no report on the prevention and control of Moringa oleifera fruit rot and leaf spot using Streptomyces and its volatile substances.

[0006] Therefore, providing a Streptomyces xanthocidicus 11-7 and its applications is an urgent problem to be solved by those skilled in the art. Summary of the Invention

[0007] In view of this, the present invention provides a Streptomyces xanthocidicus 11-7 and its applications.

[0008] In order to achieve the above object, the present invention adopts the following technical scheme:

[0009] A strain of Streptomyces xanthocidicus 11-7, with the preservation number of CCTCC NO: M2025222, has been preserved in the China Center for Type Culture Collection, abbreviated as CCTCC, address: Wuhan University, Wuhan, China, and the preservation date is February 14, 2025, and the taxonomic name is Streptomyces sp. 11-7.

[0010] Further, the application of a strain of Streptomyces flavidofuscus 11-7 in inhibiting Rigidoporus lignosus, Phellinus noxius, Colletotrichum siamense, Colletotrichum mengdingense, Colletotrichum brevisporum, Colletotrichum fructicola, Colletotrichum wanningense, Colletotrichum bannaense, Colletotrichum jinpingense, Bipolaris setariae, Corynespora cassiicola, Colletotrichum chlorophyti, Colletotrichum plurivorum and Colletotrichum karstii.

[0011] Further, the application of the volatile substances produced by a strain of Streptomyces flavidofuscus 11-7 in inhibiting Rigidoporus lignosus, Phellinus noxius, Colletotrichum siamense, Colletotrichum mengdingense, Colletotrichum brevisporum, Colletotrichum fructicola, Colletotrichum wanningense, Colletotrichum bannaense, Colletotrichum jinpingense, Bipolaris setariae, Corynespora cassiicola, Colletotrichum chlorophyti, Colletotrichum plurivorum and Colletotrichum karstii.

[0012] Further, the application of a strain of Streptomyces flavidofuscus 11-7 in nitrogen fixation.

[0013] Further, the application of a strain of Streptomyces flavidofuscus 11-7 in ammonia production.

[0014] Further, the application of a strain of Streptomyces flavidofuscus 11-7 in indole-3-acetic acid production.

[0015] As can be seen from the above technical solutions, compared with the prior art, the present invention discloses a Streptomyces flavidus 11-7 and its application. Streptomyces flavidus 11-7 has inhibitory effects on 14 pathogenic bacteria, namely Rigidoporus lignosus, Phellinus noxius, Colletotrichum siamense, Colletotrichum mengdingense, Colletotrichum brevisporum, Colletotrichum fructicola, Colletotrichum wanningense, Colletotrichum bannaense, Colletotrichum jinpingense, Bipolaris setariae, Corynespora cassiicola, Colletotrichum chlorophyti, Colletotrichum plurivorum and Colletotrichum karstii. It has the best antibacterial activities against Colletotrichum chlorophyti, Colletotrichum plurivorum and Rigidoporus lignosus, and the antibacterial rates are 96.75%, 95.89% and 92.94% respectively. The volatile substances produced by Streptomyces flavidus 11-7 have inhibitory effects on various pathogenic bacteria, especially significant inhibitory effect on Colletotrichum chlorophyti of Moringa oleifera. Streptomyces flavidus 11-7 can fix nitrogen, produce ammonia and produce indole acetic acid. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained according to the provided drawings.

[0017] Figure 1 The drawings show the antagonistic effect of the 11-7 strain of the present invention against Rigidoporus lignosus, the white root rot pathogen of rubber; A: the antagonistic effect of the 11-7 strain; B: the control;

[0018] Figure 2 The drawings show the antagonistic effect of the 11-7 strain of the present invention against Colletotrichum chlorophyti, the anthracnose pathogen of Moringa oleifera; A: the antagonistic effect of the 11-7 strain; B: the control;

[0019] Figure 3 The attached figure shows the inhibitory effect of the volatile substances of strain 11-7 of the present invention on Colletotrichum chlorophyti of Cymbidium: A: the inhibitory effect of the volatile substances of the wheat grain culture of strain 11-7; B: control.

[0020] Figure 4 The attached figure shows the phylogenetic analysis of strain 11-7 of the present invention and related strains in the GenBank database.

[0021] Figure 5 The attached figure shows the growth status of strain 11-7 of the present invention after continuous subculture on Ashby nitrogen-free medium for 5 times.

[0022] Figure 6 The attached figure shows the determination of the ammonia-producing ability of strain 11-7 of the present invention.

[0023] Figure 7 The attached figure shows the determination of the indole-3-acetic acid-producing ability of strain 11-7 of the present invention. Detailed implementation manners

[0024] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the attached figures in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0025] Test strains (14 pathogenic bacteria): Rigidoporus lignosus, Phellinus noxius, Colletotrichum siamense, Colletotrichum mengdingense, Colletotrichum brevisporum, Colletotrichum fructicola, Colletotrichum wanningense, Colletotrichum bannaense, Colletotrichum jinpingense, Bipolaris setariae, Corynespora cassiicola, Colletotrichum chlorophyti, Colletotrichum plurivorum, Colletotrichum karstii were preserved by the Research Center for Plant Protection and Microbial Utilization, Yunnan Institute of Tropical Crops Science.

[0026] Media: The soil isolation medium was Gao's No. 1 medium (soluble starch 20 g, KNO3 1 g, K2HPO4 0.5 g, MgSO4·7H2O 0.5 g, NaCl 0.5 g, FeSO4·7H2O 0.01 g, distilled water 1000 ml, agar 20 g, pH 7.4 - 7.6); the confrontation experiment medium was potato dextrose agar medium PDA (potato 200 g, glucose 20 g, agar 15 - 20 g, distilled water 1000 ml); ISP1 - 7 media were all purchased from Qingdao Haibo Biotechnology Co., Ltd.

[0027] Main reagents and instruments: PCR amplification instrument (ABI, USA); Bacteria Genomic DNA Kit was purchased from Beijing ComWin Biotech Co., Ltd.; DYY - 6C electrophoresis apparatus (Beijing Liuyi); CLIMACELL incubator (3M, USA); BSD - 400 Boxun shaking incubator.

[0028] Example 1 Isolation of Actinomycetes

[0029] The soil sample collected from Team 1 of the plain in Yingjiang Farm, Yunnan was air-dried naturally and ground finely with a mortar. 2.5 g of the dried soil sample was weighed and placed in a triangular flask containing 25 ml of water. After shaking well, it was allowed to stand for 10 min, and the actinomycetes were isolated by the plate gradient dilution method using Gao's No. 1 medium. After culturing at 28 °C for 4 - 5 d, according to the characteristics such as colony morphology, color, presence or absence of aerial hyphae, and pigment production, it was preliminarily identified as actinomycetes, and the isolated actinomycetes were numbered. The isolated actinomycetes were purified using Gao's No. 1 medium, and the purified actinomycetes were stored for later use on the slant medium of Gao's No. 1.

[0030] Example 2 Screening of antagonistic actinomycetes

[0031] The primary screening of the antibacterial activity of the isolated actinomycetes was carried out by the plate confrontation method. Rigidoporus lignosus, the white root pathogen of rubber tree, was selected as the primary screening activity indicator bacterium, and the strains with good antibacterial activity were further stored at -80 °C. And the antibacterial spectrum was determined. Actinomycetes were inoculated at equal distances around the PDA plate. After culturing at 28 °C for 4 d, when the actinomycetes slightly grew hyphae, 14 pathogen discs (diameter 5 mm) of the above-mentioned test strains were each inoculated in the center of the PDA plate. The plate without inoculating actinomycetes was used as a control, and the experiment was repeated three times. It was cultured at 28 °C, and after the pathogen in the control group covered the whole plate, it was observed, the diameter of the pathogen was measured, and the inhibition rate was calculated. Inhibition rate = [(control colony diameter - treated colony diameter) / control colony diameter] × 100%.

[0032] An actinomycete with strong inhibitory effect on the white root pathogen of rubber tree was screened from the isolated actinomycetes, numbered 11 - 7. The inhibition rate of strain 11 - 7 against the white root pathogen was 92.94% ( Figure 1 ), significantly higher than other strains. And strain 11 - 7 had a broad-spectrum antibacterial activity, and had obvious antagonistic activities against 14 test pathogenic fungi on rubber and moringa, and the inhibition rates were all above 60% (Table 1). It had the best antibacterial activity against the fruit rot pathogen of moringa, Colletotrichum chlorophyti, with an inhibition rate of 96.75%, almost completely inhibiting the growth of the pathogen ( Figure 2 ); followed by the leaf spot pathogen of moringa, Colletotrichum plurivorum, with an inhibition rate of 95.89%.

[0033] Table 1 Antagonistic effects of strain 11 - 7 against 14 pathogenic fungi

[0034]

[0035]

[0036] Determination of the Activity of Volatile Substances Produced by Strain 11-7 in Example 3

[0037] (1) The antibacterial activity was determined by the method of placing two Petri dishes face to face. After the 11-7 strain was streaked and cultured on a PDA plate for 4 days, a fresh test pathogen with a diameter of 5 mm growing on the edge of the colony was inoculated in the center of another PDA plate. The plate containing the pathogen disc was inverted and covered above the plate inoculated with the 11-7 strain, and the two dishes were tightly wrapped with sealing film and cultured at 28 °C. At the same time, a blank PDA medium and the pathogen were set as a control by placing them face to face, and both the control and the treatment were repeated three times. After the pathogen in the control group covered the whole dish, the colony diameter was measured by the cross method and the inhibition rate was calculated. Inhibition rate = [(control colony diameter - treatment colony diameter) / control colony diameter] × 100%.

[0038] The volatile substances produced by the 11-7 strain on the PDA medium had inhibitory effects on the 14 tested pathogenic fungi to varying degrees (Table 2). Among them, the inhibitory effect on Colletotrichum chlorophyti was the best, with an inhibition rate of 94.74%; followed by Colletotrichum plurivorum, with an inhibition rate of 73.13%, which was significantly higher than other pathogenic fungi.

[0039] Table 2 Inhibitory Effects of Volatile Substances Produced by Strain 11-7 on 14 Pathogenic Fungi

[0040]

[0041]

[0042] (2) Determination of antibacterial activity by the method of sandwiching wheat grain medium plates. Pick a single colony of strain 11-7 and inoculate it on liquid Czapek's No. 1 medium. Incubate it at 28 °C with shaking at 140 rpm for 5 days to obtain the 11-7 bacterial solution. Take 5 mL of the bacterial solution and inoculate it into the prepared wheat grain medium, and incubate it at 28 °C for 4 days to obtain the wheat grain culture of strain 11-7. The preparation of the wheat grain medium refers to the method of Wu, Y., Yuan, J., E, Y., Raza, W., Shen, Q., and Huang, Q. Effects of volatile organic compounds from Streptomyces albulus NJZJSA2 on growth of two fungal pathogens. Journal of basic microbiology, 2015, 55(9), 1104-1117. Inoculate a fresh pathogen with a diameter of 5 mm growing at the edge of the colony in the center of the PDA plate. Invert and cover the petri dish containing the pathogen cake above the petri dish containing the 11-7 wheat grain culture, wrap the two dishes tightly with sealing film, and incubate at 28 °C. Use the wheat grain medium without inoculating the 11-7 bacterial solution as the blank control, and repeat the experiment three times. After the pathogen in the control group has grown over the entire dish, measure the colony diameter by the cross method and calculate the inhibition rate. Inhibition rate = [(control colony diameter - treated colony diameter) / control colony diameter] × 100%.

[0043] The antibacterial effect of the volatile substances of strain 11-7 on Colletotrichum chlorophyti was verified using wheat grain medium, and it was found that the volatile substances produced by the wheat grain culture of strain 11-7 could completely inhibit the growth of the hyphae of Colletotrichum chlorophyti, and the inhibition rate was 100% ( Figure 3 ).

[0044] Example 4 Identification of the Strain

[0045] (1) Morphological characteristics: Using the method of inserting cover slips, insert the sterilized cover slips into Czapek's No. 1 medium inoculated with actinomycete 11-7 at a 45-degree angle. After incubating at 28 °C for 10-30 days, take out the cover slips and observe the shape of the substrate mycelium and aerial mycelium, and the presence or absence of spore chains under an optical microscope.

[0046] Strain 11-7 has typical Streptomyces characteristics. On Czapek's No. 1 medium, it has well-developed aerial mycelium and substrate mycelium. Spore chains grow on the aerial mycelium, and the substrate mycelium is not easily broken.

[0047] (2) Cultural characteristics and physiological and biochemical characteristics: Referring to the method of Xu Lihua et al. (Xu Lihua, Li Wenjun, Liu Zhiheng, et al. Actinomycete Systematics - Principles, Methods and Practices [M]. Beijing: Science Press, 2007, 40 - 45), the target actinomycetes were cultured at 28 °C for 10 - 25 days, and the color of substrate mycelium, aerial mycelium, and the presence or absence of soluble pigments were observed and recorded.

[0048] Cultural characteristics: Strain 11 - 7 was gray on Gao's No. 1 medium, with a slightly raised center of the colony, showing concentric rings, irregular edges, or a diffused shape. The aerial mycelium was well - developed and was thicker than the vegetative mycelium. Strain 11 - 7 grew well on 9 different media. The aerial mycelium was white (ISP1, ISP5, ISP6), gray (ISP2, ISP3, ISP4, ISP7, PDA, Gao's No. 1 medium), the substrate mycelium was black (PDA), white (ISP1, ISP3, ISP4, ISP5, Gao's No. 1 medium), gray - black (ISP2), brown (ISP6), light gray (ISP7), and no soluble pigments were produced (Table 3).

[0049] Table 3 Cultural characteristics of strain 11 - 7

[0050]

[0051] Physiological and biochemical characteristics: Strain 11 - 7 could produce oxidase, catalase, urease, and lipase, could liquefy gelatin, coagulate and peptonize milk, and reduce nitrate. It did not hydrolyze starch (did not produce amylase) and decompose cellulose (did not produce cellulase), and could not produce H2S. The MR test was negative (the strain did not produce acidic substances or produced a small amount of acidic substances during metabolism), and the v - p test was negative (the strain did not indirectly decompose glucose into acetoin). The tolerance range of NaCl was 1 - 3%, and the tolerance range of pH was 4 - 10. Strain 11 - 7 could utilize glucose, maltose, sucrose, lactose, and fructose; the nitrogen source utilization test showed that the strain could utilize alanine, glycine, aspartic acid, tyrosine, and could not utilize arginine (Table 4).

[0052] Table 4 Physiological and biochemical characteristics of strain 11 - 7

[0053]

[0054]

[0055] Note: +: Positive reaction; -: Negative reaction.

[0056] (3) Phylogenetic characteristics: Universal primers Primer 16S-27-F (5'-AGAGTTTGATCCTGGCTCAG-3'; SEQ ID NO.1) and Primer 16S-1492-R (5'-GGTTACCTTGTTACGACTT-3'; SEQ ID NO.2) of the 16S rRNA gene were used for PCR amplification, and its sequence was determined. Through comparison by the BLAST program, the strain with the highest similarity and representativeness was selected from the GenBank database, and a phylogenetic tree was constructed by the Neighbor-Joining method of MEGA 6.0 to determine the taxonomic status of the actinomycetes.

[0057] Genomic DNA was extracted from strain 11-7, and the 16S rRNA gene sequence was obtained by PCR amplification. The 1545bp sequence was sent to BGI-Shenzhen for sequencing, as shown in SEQ ID NO.3.

[0058]

[0059] The obtained sequences were subjected to BLAST alignment on NCBI, and strains with high homology and effective publication were selected for phylogenetic analysis. The maximum likelihood method of MEGA6.0 was used to construct a phylogenetic tree of the 16S rRNA gene sequences( Figure 4 ). The results showed that strain 11-7 clustered in the same branch with Streptomyces xanthocidicus, and the sequence similarity was 99.72%. Combining the morphological characteristics, physiological and biochemical characteristics and the results of phylogenetic analysis, strain 11-7 was identified as Streptomyces xanthocidicus.

[0060] The preservation number of strain 11-7 is CCTCC NO: M 2025222, which has been preserved in the China Center for Type Culture Collection, abbreviated as CCTCC, address: Wuhan University, Wuhan, China. The preservation date is February 14, 2025, and the taxonomic name is Streptomyces sp.11-7.

[0061] Example 5 Study on the growth-promoting characteristics of strain 11-7

[0062] (1) Determination of nitrogen fixation ability

[0063] The activated biocontrol Streptomyces 11-7 was inoculated into Ashby nitrogen-free medium and cultured at 28 °C for 15 d. Each treatment was repeated 3 times. If colonies grew on the medium, they were transferred to a new Ashby nitrogen-free plate and subcultured 5 times continuously. After 5 subcultures, colonies of biocontrol Streptomyces 11-7 still appeared on the Ashby nitrogen-free plate, indicating that the strain has nitrogen fixation ability.

[0064] After strain 11-7 was continuously subcultured 5 times on Ashby nitrogen-free medium, the strain still grew well( Figure 5 ), indicating that the strain has nitrogen fixation ability and can convert inorganic nitrogen sources into organic nitrogen sources that are easily absorbed and utilized.

[0065] (2) Detection of ammonia production ability

[0066] The activated strain 11-7 was inoculated into ISP2 liquid medium and cultured at 28 °C at 140 r / min for 5 d. This was used as the seed liquid and inoculated into peptone water (10 g / L) and cultured at 28 °C at 160 r / min for 15 d. The medium without inoculating strain 11-7 was used as the control, and each treatment was repeated 3 times. After the culture was completed, it was centrifuged at 12,000 r / min for 5 min, and 1 mL of Nessler's reagent was added to the supernatant to observe the change of the solution. If an orange precipitate appeared, it indicated that the strain had ammonia production ability; the more orange precipitate, the stronger the ammonia production ability.

[0067] After adding 1 mL of Nessler's reagent to the supernatant of strain 11-7, compared with the blank control, a yellowish-brown precipitate was formed in the supernatant ( Figure 6 ), indicating that strain 11-7 has the ability to produce ammonia, can cause the deamination reaction of amino acids, and generate ammonia and various acids.

[0068] (3) Detection of indole-3-acetic acid production ability

[0069] The activated strain 11-7 was inoculated into Gause's No. 1 liquid medium containing tryptophan (0.5 g / L). The medium without inoculating strain 11-7 was used as the control. The cultures were incubated at 28 °C with a shaking speed of 140 r / min for 5 days, and each treatment was repeated 3 times. After centrifugation at 10000 r / min for 10 min, 1 mL of the supernatant was taken out, and an equal volume of Salkowski's reagent was added and mixed well. The mixture was allowed to stand in the dark for 30 min. If the solution turned pink, it indicated that the strain had the ability to produce indole-3-acetic acid, and the darker the color, the stronger the ability of the strain to produce indole-3-acetic acid.

[0070] After adding an equal amount of Salkowski's reagent to the supernatant of strain 11-7, it was found that compared with the blank control, the supernatant turned pink ( Figure 7 ), indicating that the strain can produce a certain amount of indole-3-acetic acid.

[0071] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A strain of Streptomyces xanthocidicus 11-7, characterized in that: Its deposit number is CCTCC NO: M 2025222, and its classification name is Streptomyces sp.11-7.

2. Use of the Streptomyces flavus 11-7 according to claim 1 in inhibiting Rigidoporus lignosus, Phellinus noxius, Colletotrichum siamense, Colletotrichum mengdingense, Colletotrichum brevisporum, Colletotrichum fructicola, Colletotrichum wanningense, Colletotrichum bannaense, Colletotrichum jinpingense, Bipolaris setariae, Corynespora cassiicola, Colletotrichum chlorophyti, Colletotrichum plurivorum and Colletotrichum karstii.

3. Use of the volatile substances produced by the Streptomyces flavus 11-7 according to claim 1 in inhibiting Rigidoporus lignosus, Phellinus noxius, Colletotrichum siamense, Colletotrichum mengdingense, Colletotrichum brevisporum, Colletotrichum fructicola, Colletotrichum wanningense, Colletotrichum bannaense, Colletotrichum jinpingense, Bipolarissetariae, Corynespora cassiicola, Colletotrichum chlorophyti, Colletotrichum mplurivorum and Colletotrichum karstii.

4. Use of the Streptomyces flavus 11-7 according to claim 1 in nitrogen fixation.

5. Use of the Streptomyces flavus 11-7 according to claim 1 in producing ammonia.

6. Use of the Streptomyces flavus 11-7 according to claim 1 in producing indoleacetic acid.

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