Strain of streptomyces avermitilis 11-7 and application thereof

Abstract

The application discloses a strain of Streptomyces clyindrosporus 11-7 and application thereof, and belongs to the technical field of microorganisms. The strain of Streptomyces clyindrosporus 11-7 disclosed by the application has a preservation number of CCTCC NO: M 2025222. The strain of Streptomyces clyindrosporus 11-7 disclosed by the application has the best inhibitory activity on Colletotrichum chlorophyti, Colletotrichum plurivorum and Rigidoporus lignosus, and the inhibitory rates are 96.75%, 95.89% and 92.94% respectively. The volatile substances produced by the strain of Streptomyces clyindrosporus 11-7 have inhibitory effects on various pathogenic bacteria. The strain of Streptomyces clyindrosporus 11-7 can fix nitrogen, produce ammonia and produce indole acetic acid.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial technology, more particularly to a strain of Streptomyces scabies 11-7 and its application. BACKGROUND

[0002] Natural rubber is an important agricultural industry in the hot region of China, and is also an important strategic material of the country. There are 91 diseases reported on rubber trees in China, among which anthracnose and root disease are two diseases that seriously harm rubber trees and have become an important factor restricting the development of the rubber industry. Rubber tree anthracnose is an important leaf disease caused by infection of fungi of the genus Colletotrichum Corda. The disease can occur in different leaf ages and different growth stages of rubber trees, causing serious losses to natural rubber production. The incidence rate of rubber tree root disease in rubber plantations in China is more than 2%, and the white root disease of rubber tree caused by the fungus Rigidoprus lignosus (Klotzsch) Imaz. is a serious disease of rubber trees worldwide and a plant quarantine disease in China. The disease has been reported to harm young rubber trees in many rubber-producing areas, causing rapid death of rubber trees.

[0003] Moringa oleifera Lam. belongs to the family Moringaceae and is a kind of fast-growing small tree. In 2012, Moringa oleifera Lam. was approved by the State Health Department as a new resource food. Its roots, leaves, bark, seeds, flowers and pods are all edible, with high calcium, high protein, high fiber and low lipid characteristics, and rich in vitamins, minerals, polypeptides, polysaccharide nutrients, polyphenols, flavonoids and other bioactive ingredients, with the functions of lowering blood sugar, lowering blood lipids, anti-tumor, anti-inflammatory, antibacterial and the like. The main diseases reported for Moringa oleifera Lam. include fruit rot and leaf spot, among which Moringa oleifera Lam. fruit rot is one of the most common and serious diseases. According to reports, the average incidence of fruit rot in Moringa oleifera Lam. planting areas in Yunnan Province is 4% to 72%, and the fruit pod damage rate is more than 90% in the heavy rainfall months of July and August, and the whole fruit pod cracks and exposes the seeds, resulting in a reduction in seed yield. Colletotrichum chlorophyti has been confirmed to not only infect fruit pods to cause Moringa oleifera Lam. fruit rot, but also infect leaves to cause leaf spot, causing serious harm to Moringa oleifera Lam. production.

[0004] At present, the prevention and treatment of plant diseases is still mainly based on chemical control; but long-term use of chemical agents will cause serious drug resistance problems, and there is certain drug damage and pollution to crops and the environment. Therefore, it is imperative to find a relatively efficient and safe control measure. The use of microbial resources or their metabolites for biological control has become a major trend under the trend of green agriculture. Most of the bioactive substances found in microorganisms are produced by Streptomyces. The active substances produced by Streptomyces can be used as agricultural fungicides, insecticides, herbicides and plant growth regulators, and have important position in the green prevention and control of crop diseases and insect pests due to their high efficiency, low toxicity and environmental friendliness.

[0005] Using microorganisms that produce volatile fungistatic 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 penetration and high efficiency of diffusion in air and soil voids, which can quickly expand the action range of volatile substances and improve the inhibition rate of target microorganisms, and have important significance for crop food safety. At present, there is no report on the use of Streptomyces and the volatile substances produced by Streptomyces to prevent and control Moringa oleifera fruit rot and leaf spot.

[0006] Therefore, providing a strain of Streptomyces xanthocidicus 11-7 and its application is a problem that those skilled in the art urgently need to solve. SUMMARY

[0007] Therefore, the present application provides a strain of Streptomyces xanthocidicus 11-7 and its application.

[0008] In order to achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0009] A strain of Streptomyces xanthocidicus 11-7, the preservation number of which is CCTCC NO: M 2025222, has been preserved in the China Center for Type Culture Collection (CCTCC) located in Wuhan, China, and the preservation date is February 14, 2025, and the classification and naming is Streptomyces xanthocidicus 11-7.

[0010] Further, the application of the Streptomyces lydicus 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 the Streptomyces lydicus 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 the Streptomyces lydicus 11-7 in nitrogen fixation.

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

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

[0015] Compared with the prior art, the application provides a strain of Streptomyces cichorius 11-7 and an application thereof. The Streptomyces cichorius 11-7 has inhibitory effects on 14 kinds of pathogenic bacteria, i.e., 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, and the inhibitory activity on Colletotrichum chlorophyti, Colletotrichum plurivorum and Rigidoporus lignosus is the best, and the inhibition rates are 96.75%, 95.89% and 92.94%, respectively. The volatile substances produced by the Streptomyces cichorius 11-7 have inhibitory effects on a plurality of pathogenic bacteria, and the inhibitory effect on Colletotrichum chlorophyti is relatively significant. The Streptomyces cichorius 11-7 can fix nitrogen, produce ammonia and produce indole acetic acid. BRIEF DESCRIPTION OF DRAWINGS

[0016] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced. Obviously, the drawings in the following description are only embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative effort on the basis of the provided drawings.

[0017] Figure 1 The drawing is an antagonistic effect of the 11-7 strain of the present application on Rigidoporus lignosus; A: antagonistic effect of the 11-7 strain; B: control;

[0018] Figure 2 The drawing is an antagonistic effect of the 11-7 strain of the present application on Colletotrichum chlorophyti; A: antagonistic effect of the 11-7 strain; B: control;

[0019] Figure 3 Figure 11-7 strain volatile substances on the inhibition of Colletotrichum chlorophyti; A: 11-7 strain of wheat culture volatile substances inhibition; B: control;

[0020] Figure 4 Figure 11-7 strain and GenBank database related strains of phylogenetic analysis;

[0021] Figure 5 Figure 11-7 strain in Ashby nitrogen-free medium after continuous transfer 5 times the growth conditions;

[0022] Figure 6 Figure 11-7 strain ammonia production capacity determination;

[0023] Figure 7 Figure 11-7 strain indole acetic acid production capacity determination. DETAILED DESCRIPTION

[0024] The technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the scope of the present application.

[0025] Tested strains (14 pathogenic fungi): 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, preserved by the Plant Protection and Microorganism Utilization Research Center of the Yunnan Institute of Tropical Crops.

[0026] Culture medium: soil separation 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); confrontation experiment medium was potato agar medium PDA (potato 200 g, glucose 20 g, agar 15-20 g, distilled water 1000 ml); ISP1-7 medium was purchased from Qingdao Haibo Biotechnology Co., Ltd.

[0027] Main reagents and instruments: PCR amplification instrument (USA ABI); Bacteria Genomic DNA Kit purchased from Beijing Kangwei Century Company; DYY-6C electrophoresis instrument (Beijing Liuyi); CLIMACELL incubator (USA 3M); BSD-400 Boxun shaking incubator.

[0028] Example 1 Actinomycete separation

[0029] The soil sample collected from Pingyuan 1 team of Yingjiang farm in Yunnan was naturally dried and finely ground with a mortar. 2.5 g of the dried soil sample was placed in a flask containing 25 ml of water, shaken thoroughly, and left for 10 min. The actinomycetes were isolated by gradient dilution method using Gao's No. I medium. After 4-5 days of culture at 28°C, the actinomycetes were preliminarily identified according to the colony morphology, color, aerial mycelium, and pigment production, and numbered. The isolated actinomycetes were purified using Gao's No. I medium, and the purified actinomycetes were stored on Gao's No. I slant medium for future use.

[0030] Example 2: Screening of antagonistic actinomycetes

[0031] The isolated actinomycetes were subjected to preliminary screening of antibacterial activity by plate confrontation method. Rigidoporus lignosus, the pathogen of rubber tree white root rot, was selected as the indicator strain for preliminary screening of activity. The strains with good antibacterial activity were further stored at -80°C. The antibacterial spectrum was determined by inoculating the actinomycetes at equal intervals around the PDA plate, culturing at 28°C for 4 days, and then inoculating 14 kinds of pathogenic fungi of the above-mentioned test strains in the form of 5 mm diameter discs in the center of the PDA plate after the actinomycetes grew slightly. The plates without actinomycetes were used as controls, and the experiment was repeated three times. The plates were cultured at 28°C, and the pathogenic fungi were observed after the control group grew completely. The diameter of the pathogenic fungi was measured, and the inhibition rate was calculated. Inhibition rate = [(control colony diameter - treatment colony diameter) / control colony diameter] x 100%.

[0032] A strain of actinomycetes with strong inhibitory effect on Rigidoporus lignosus was screened from the isolated actinomycetes, numbered 11-7. The inhibition rate of 11-7 strain on Rigidoporus lignosus was 92.94% ( Figure 1 ), significantly higher than that of other strains. Moreover, 11-7 strain had broad-spectrum antibacterial activity, with obvious antagonistic activity against 14 kinds of pathogenic fungi on rubber and pricklyash, with an inhibition rate of more than 60% (Table 1). The antibacterial activity against Colletotrichum chlorophyti, the fruit rot pathogen of pricklyash, was the best, with an inhibition rate of 96.75%, which could almost completely inhibit the growth of the pathogenic fungus ( Figure 2 ). The second was Colletotrichum plurivorum, with an inhibition rate of 95.89%.

[0033] Table 1: Antagonistic effect of 11-7 strain on 14 kinds of pathogenic fungi

[0034]

[0035]

[0036] Example 311-7 strain volatile substance activity assay

[0037] (1) The antibacterial activity was determined by plate pairing method. After 11-7 strain was streaked on PDA plates for 4 days, fresh test pathogenic fungi with a diameter of 5 mm were inoculated in the center of another PDA plate. The plate containing the pathogenic fungus cake was inverted and covered on the 11-7 strain inoculated plate, and the two plates were wrapped tightly with a sealing film. The plates were incubated at 28°C. At the same time, blank PDA medium and pathogenic fungi pairing were set as controls. The controls and treatments were repeated three times. After the pathogenic fungi in the control group grew throughout the plate, the colony diameter was measured by cross method and the inhibition rate was calculated. Inhibition rate = [(control colony diameter - treatment colony diameter) / control colony diameter] x 100%.

[0038] The volatile substances produced by 11-7 strain on PDA medium had different degrees of inhibition on the 14 test pathogenic fungi (Table 2). Among them, the inhibition 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 that of other pathogenic fungi.

[0039] Table 2 Inhibition of 11-7 strain volatile substances on 14 pathogenic fungi

[0040]

[0041]

[0042] (2) Agar-plate assay for antibacterial activity. The 11-7 strain was inoculated on a liquid Gause's medium I and incubated at 28°C with 140 rpm shaking for 5 days to obtain the 11-7 bacterial solution. 5 mL of the bacterial solution was inoculated on the prepared Gause's medium and incubated at 28°C for 4 days to obtain the Gause's medium culture of the 11-7 strain. The Gause's medium was prepared according 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. A fresh pathogenic fungus was inoculated on the PDA plate in the center with a diameter of 5 mm. The culture dish containing the pathogenic fungus cake was inverted and covered on the plate containing the 11-7 Gause's medium culture, and the two dishes were wrapped tightly with a sealing film. The culture was incubated at 28°C. The Gause's medium without the 11-7 bacterial solution was used as a blank control, and the experiment was repeated three times. When the pathogenic fungus in the control group grew full plate, the colony diameter was measured by cross method and the inhibition rate was calculated. Inhibition rate = [(control colony diameter - treatment colony diameter) / control colony diameter] x 100%.

[0043] The antibacterial effect of the volatile substances of the 11-7 strain on C. chlorophyti was verified using the Gause's medium, and it was found that the volatile substances produced by the 11-7 strain Gause's medium culture could completely inhibit the growth of C. chlorophyti mycelium, and the inhibition rate was 100%. Figure 3

[0044] Example 4 Identification of the strain

[0045] (1) Morphological characteristics: The sterilized cover glass was inserted into the Gause's medium I inoculated with the actinomycete 11-7 at an angle of 45 degrees, and after incubation at 28°C for 10-30 days, the cover glass was taken out and observed under a light microscope for the shape of the intramural mycelium and aerial mycelium, and the presence or absence of spore chains, etc.

[0046] The 11-7 strain has typical Streptomyces characteristics. On the Gause's medium I, it has developed aerial mycelium and intramural mycelium, spore chains on the aerial mycelium, and intramural mycelium that is not easy to break.

[0047] ​(2) Culture characteristics and physiological and biochemical characteristics: The target actinomycetes were cultured at 28°C for 10-25 d, and the color of subterranean mycelium, the color of aerial mycelium, and the presence or absence of soluble pigment were observed and recorded according to the method of Xu et al. (Xu L, Li WJ, Liu ZH, et al. Actinomycetes systematics-principles, methods and practice[M]. Beijing: Science Press, 2007, 40-45).

[0048] Culture characteristics: The 11-7 strain was gray on Gause I medium, with slightly convex in the middle of the colony, concentric rings, irregular edges, or diffuse, and developed aerial mycelium, which was thicker than the vegetative mycelium. The 11-7 strain grew well on 9 different media, with white aerial mycelium (ISP1, ISP5, ISP6), gray aerial mycelium (ISP2, ISP3, ISP4, ISP7, PDA, Gause I medium), black subterranean mycelium (PDA), white subterranean mycelium (ISP1, ISP3, ISP4, ISP5, Gause I medium), gray-black subterranean mycelium (ISP2), brown subterranean mycelium (ISP6), and light gray subterranean mycelium (ISP7), and no soluble pigment was produced (Table 3).

[0049] Table 3 Culture characteristics of the 11-7 strain

[0050]

[0051] Physiological and biochemical characteristics: The 11-7 strain could produce oxidase, catalase, urease, and lipase, liquefy gelatin, coagulate and peptonize milk, and reduce nitrate. It could not hydrolyze starch (produce amylase) and decompose cellulose (not produce cellulase), and could not produce H2S. The MR test was negative (the strain did not produce or produced little acid substances during metabolism), and the v-p test was negative (the strain did not decompose glucose indirectly into acetyl methyl carbinol). The tolerance range of NaCl was 1-3%, and the tolerance range of pH was 4-10. The 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, and tyrosine, but could not utilize arginine (Table 4).

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

[0053]

[0054]

[0055] Note: +: positive reaction; -: negative reaction.

[0056] (3) Phylogenetic characteristics: The 16S rRNA gene was amplified by PCR using 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), and its sequence was determined. By comparison through the BLAST program, the most similar and representative strains were selected from the GenBank database, and a phylogenetic tree was constructed by the Neighbor-Joining method of MEGA 6.0 to determine the classification status of the actinomycete.

[0057] The 16S rRNA gene sequence of the 11-7 strain was extracted and amplified by PCR to obtain the 16S rRNA gene sequence, which was sequenced by Shenzhen Huada Gene Research Institute to obtain a sequence of 1545 bp, as shown in SEQ ID NO. 3.

[0058]

[0059] The obtained sequence was subjected to BLAST comparison on NCBI, and strains with higher homology and effective publication were selected for phylogenetic analysis, and the phylogenetic tree of 16S rRNA gene sequence was constructed by MEGA6.0 maximum likelihood method. Figure 4 The results showed that the 11-7 strain was clustered with Streptomyces xanthocidicus, and the sequence similarity was 99.72%. Combined with morphological characteristics, physiological and biochemical characteristics and phylogenetic analysis results, the 11-7 strain was identified as Streptomyces xanthocidicus.

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

[0061] Example 511-7 strain growth-promoting characteristics

[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 15d, with 3 replicates for each treatment. If colonies grew on the medium, they were transferred to a new Ashby nitrogen-free plate, and the transfer was repeated 5 times. After 5 times, the Ashby nitrogen-free plate still had colonies of biocontrol Streptomyces 11-7, indicating that the strain had nitrogen fixation ability.

[0064] After 5 times of continuous transfer of the 11-7 strain on Ashby nitrogen-free medium, the strain still grew well Figure 5 , indicating that the strain had nitrogen fixation ability and could convert inorganic nitrogen source into organic nitrogen source that was easy to absorb and utilize.

[0065] (2) Ammonia production ability detection

[0066] The activated 11-7 strain was inoculated into ISP2 liquid medium and cultured at 140r / min and 28°C for 5d, and then used as seed liquid to inoculate into proteose peptone water (10g / L) and cultured at 160r / min and 28°C for 15d. The culture medium without 11-7 strain inoculation was used as control, and each treatment was repeated 3 times. After the culture, centrifugation was performed at 12000r / min for 5min, and the supernatant was added with 1mL Nessler's reagent. The solution was observed, and if orange precipitate appeared, it indicated that the strain had ammonia production ability; the more orange precipitate, the stronger the ammonia production ability.

[0067] After 1 mL of Nessler's reagent was added to the supernatant of the 11-7 strain, the supernatant produced a yellow-brown precipitate relative to the blank control Figure 6 , indicating that the 11-7 strain had ammonia-producing ability and could cause deamination of amino acids to generate ammonia and various acids.

[0068] (3) Indoleacetic acid-producing ability detection

[0069] The activated 11-7 strain was inoculated into Gause's No. I liquid medium containing tryptophan (0.5 g / L), and the medium without inoculation of the 11-7 strain was used as a control, and the culture was carried out at 140 r / min and 28°C for 5 d, with 3 replicates for each treatment. After centrifugation at 10,000 r / min for 10 min, 1 mL of the supernatant was taken, an equal volume of Salkowski's color reagent was added, and the mixture was mixed well, and then it was placed in the dark for 30 min. If the solution turned pink, it indicated that the strain had the ability to produce indoleacetic acid, and the deeper the color, the stronger the ability of the strain to produce indoleacetic acid.

[0070] After an equal amount of Salkowski's color reagent was added to the supernatant of the 11-7 strain, the supernatant turned pink relative to the blank control Figure 7 , indicating that the strain could produce a certain amount of indoleacetic acid.

[0071] The above description of the disclosed embodiments enables a person skilled in the art to implement or use the present application. 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 application. Therefore, the present application will not be limited to these embodiments shown herein, but will conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A strain of Streptomyces chrysosporium ( Streptomyces xanthocidicus )11-7, characterized in that, The preservation number of which is CCTCC NO: M 2025222.

2. The strain of Streptomyces lydicus 11-7 of claim 1 for use in inhibiting Rigidoporus lignosus 、 Phellinus noxius 、 Colletotrichum siamense 、 Colletotrichum mengdingense 、 Colletotrichum brevisporum 、 Colletotrichum fructicola 、 Colletotrichum wanningense 、 Colletotrichum bannaense 、 Colletotrichumjinpingense 、 Bipolarissetariae 、 Corynesporacassiicola 、 Colletotrichumchlorophyti 、 Colletotrichumplurivorum and Colletotrichumkarstii .

3. Use according to claim 2, characterized in that, The Streptomyces scabies 11-7 inhibits 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 .

4. The application of the strain Streptomyces c. 11-7 in fixing nitrogen according to claim 1.

5. The application of the strain Streptomyces c. 11-7 in producing ammonia according to claim 1.

6. The application of the strain Streptomyces c. 11-7 in producing indole acetic acid according to claim 1.

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