Multifunctional biocontrol growth-promoting streptomyces LG94 and application thereof

Abstract

The invention provides multifunctional biocontrol growth-promoting streptomyces LG94 and application thereof, and belongs to the technical field of biology. The preservation number of the streptomyces sp. LG94 disclosed by the invention is GDMCC (China General Microbiological Culture Collection Center) NO: 65017. The streptomyces LG94 is separated from deep-sea sediments of hippocampus, and can inhibit the growth of various pathogenic fungi, such as coniothyrium albidum, fusarium oxysporum No.4 microspecies of banana fusarium wilt, pitaya canker pathogen, fusarium aromaticum, fusarium nigrum and citrus green mold pathogen; the strain has the effects of producing siderophores and secreting plant hormone indoleacetic acid (IAA) to promote plant growth, and can be used for developing a multifunctional biological control agent.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a multifunctional biocontrol and growth-promoting Streptomyces LG94 and an application thereof. Background Art

[0002] Plant cultivation faces an increasing number of biotic and abiotic stresses, which slow down plant growth and reproduction and cause economic losses. Chemical products are often used as pesticides or fertilizers to increase crop yields. However, the misuse of agrochemicals not only causes adverse environmental problems such as soil pollution, pesticide tolerance, and native flora imbalance, but also poses a potential hazard to human health. Using microbial bacteria to promote plant growth and improve plant tolerance to environmental stress can provide an alternative method for safe agricultural production.

[0003] Plant growth promoting bacteria (PGPB) can promote plant growth by: (1) inhibiting the growth of plant pathogens (bacteriocins, antibiotics and hydrolases), (2) promoting the acquisition of phosphorus and iron (phosphate solubilization, production of siderophores) and (3) regulating plant hormone levels (indoleacetic acid-IAA, cytokinins and ethylene). Therefore, obtaining effective multifunctional PGPB is the key to the sustainable development of new agriculture. The deep sea is an extreme environment, which is mainly characterized by high pressure, low temperature and poor nutrient levels. These harsh conditions affect the gene expression and metabolism of microorganisms and are an important source for the development of new biocontrol agents.

[0004] Therefore, screening bacterial species with biocontrol and life-promoting properties from deep-sea sediments and exploring potential multifunctional biological control agents have far-reaching significance for modern agriculture. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a multifunctional biocontrol and growth-promoting Streptomyces LG94 and its application, wherein the Streptomyces LG94 has a relatively broad antibacterial spectrum and has the function of producing siderophores and the plant hormone IAA.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a strain of Streptomyces LG94, and the classification name of the Streptomyces LG94 is Streptomyces albidoflavus; the Streptomyces LG94 was deposited in the Guangdong Provincial Microbiological Culture Collection Center on August 16, 2024, and the preservation address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the preservation number is: GDMCC NO: 65017.

[0008] The present invention provides application of the Streptomyces LG94 in inhibiting plant pathogenic fungi.

[0009] Preferably, the plant pathogenic fungi include white rot shield mold, banana wilt Fusarium oxysporum race 4, pitaya canker pathogen, fragrant Fusarium, vine black Fusarium or citrus green mold pathogen.

[0010] The present invention provides application of the Streptomyces LG94 in promoting plant growth.

[0011] The present invention provides application of the Streptomyces LG94 in producing siderophore.

[0012] The invention provides application of the Streptomyces LG94 in producing the plant hormone IAA.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] The present invention isolates a strain of Streptomyces LG94 from deep-sea sediments of seahorses, which can inhibit the growth of multiple pathogenic fungi, such as white rot shield mold, banana wilt Fusarium oxysporum race 4, pitaya canker pathogen, fragrant Fusarium, Fujinoku Fusarium and citrus green mold pathogen, and has the effects of promoting plant growth by producing iron carriers and secreting plant hormone indoleacetic acid (IAA), and can be used to develop multifunctional biological control agents.

[0015] Biological Deposit Description

[0016] Streptomyces LG94, classified as Streptomyces albidoflavus, was deposited in the Guangdong Provincial Microbiological Culture Collection Center on August 16, 2024. The collection address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the collection number is: GDMCC NO: 65017. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The colony morphology of Streptomyces LG94 on 2216E medium; A is the front side and B is the back side;

[0018] Figure 2 The single colony and spore morphology of Streptomyces LG94 under an optical microscope; A is a single colony, and B is a spore;

[0019] Figure 3 This is a phylogenetic analysis diagram of Streptomyces LG94 based on 16S rRNA;

[0020] Figure 4 This is a diagram showing the antagonistic effect of Streptomyces LG94 on plant pathogens;

[0021] Figure 5 This is the inhibition rate of Streptomyces LG94 against plant pathogens;

[0022] Figure 6This is a test diagram of the hydrolase characteristics of Streptomyces LG94 cells;

[0023] Figure 7 This is a diagram showing the effects of Streptomyces LG94 in solubilizing phosphate, potassium, producing siderophores and producing indoleacetic acid;

[0024] Figure 8 This is the IAA standard curve. DETAILED DESCRIPTION

[0025] The present invention provides a strain of Streptomyces LG94, the classification name of the Streptomyces LG94 is Streptomyces albidoflavus; the Streptomyces LG94 was deposited in the Guangdong Provincial Microbiological Culture Collection Center on August 16, 2024, and the deposit address is 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou, and the deposit number is: GDMCC NO: 65017. The strain is derived from deep-sea sediments and can grow well in 2216E culture medium.

[0026] The 16S rRNA gene sequence of Streptomyces LG94 is as follows:

[0027] SEQ ID NO: 1

[0028] >LG94 Streptomyces albidoflavus DSM 40455

[0029]

[0030] The present invention provides an application of the Streptomyces LG94 in inhibiting plant pathogenic fungi. In the present invention, the plant pathogenic fungi are preferably white rot shield mold, banana wilt Fusarium oxysporum race 4, pitaya canker pathogen, fragrant Fusarium, vine black Fusarium or citrus green mold pathogen, further preferably white rot shield mold or citrus green mold pathogen, and further preferably white rot shield mold.

[0031] The present invention provides an application of the Streptomyces LG94 in promoting plant growth. The growth-promoting effect is reflected in the production of protease and chitinase to inhibit the growth of harmful fungi, and the production of lipase to help plants decompose macromolecular nutrients to help plants absorb them. It also has the function of producing iron carriers and plant hormone IAA, thereby promoting plant growth in many aspects.

[0032] The present invention provides the use of the Streptomyces LG94 in producing siderophores, which can generate an orange or yellow halo on a CAS detection culture medium.

[0033] The invention provides the use of the streptomyces LG94 in producing the plant hormone IAA, which can make the color of the Salkowski reagent turn red.

[0034] Experimental Materials:

[0035] Sediment samples: The deep-sea sediments of Hippocampus were sourced from (111.0557°E, 17.6227°N)

[0036] Plant pathogens: Fusarium redolens TR-11 (preservation number CGMCC NO: 3.17397) described in the embodiments of the present invention is from the China General Microbiological Culture Collection Center, Fusarium fujikuroi D69-2 (preservation number ACCC 39250), Fusarium solani SM1180 (preservation number ACCC 37381), Neoscytalidium dimidiatum (ACCC 39211), Fusarium oxysporum f.sp. Cubense tropical race 4 (preservation number ACCC 38875) and Coniothyrium diplodiella IVF134 (preservation number ACCC 36140) are from the China Agricultural Microbiological Culture Collection Center.

[0037] Reagents: Chelex-100 resin was purchased from Bio-Rad, USA; 2×EasyTaq SuperMix, primers, DNAMarker, EB nucleic acid dye and other biological reagents were purchased from Shanghai Sangon Biotechnology Co., Ltd.; synthetic culture medium was purchased from Haibo Biotechnology Co., Ltd.; HClO4 was purchased from Chengdu Cologne Chemicals Co., Ltd.; other chemical reagents were purchased from Shanghai MacLean Biochemical Technology Co., Ltd.

[0038] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0039] Example 1 Isolation and Identification of Streptomyces LG94

[0040] 1.1 Sample source: deep-sea sediments of seahorses

[0041] Weigh 1.0 g of dried deep-sea sediment into a centrifuge tube containing 9.0 mL of sterile water, vortex evenly, and dilute to different gradients. Take 200 μL of each dilution of 10 -2 and 10 -3 The sample solution was spread on humic acid solid culture medium and cultured at 28°C until the number of colonies remained unchanged. A single colony was picked and the strain was purified by the three-zone streak method. The purified strain was transferred to a cryopreservation tube containing 30% glycerol and stored in a -80°C refrigerator.

[0042] 1.2 Morphological observation: Streptomyces LG94 was transferred to 2216E solid culture medium and cultured at 28°C for 7 days. The colony color and growth morphological characteristics were recorded. In addition, the morphology of spores was observed using an optical microscope.

[0043] Streptomyces LG94 was isolated from deep-sea sediments of a seahorse. The strain grew well in 2216E medium. The colony was round with concentric rings and a depression in the middle. The mycelium was well developed, the aerial mycelium was gray-camel, and the base mycelium was lilac brown (see Figure 1 Under an optical microscope, the colonies are covered with spores, which are round, oblong or peanut-shaped (see Figure 2 ).

[0044] 1.3 Phylogenetic analysis of 16S rRNA of Streptomyces LG94

[0045] The DNA of bacteria was extracted by Chelex-100 resin, and the DNA was amplified by PCR using universal primers 27F and 1522R. The PCR amplified fragments were detected by 1% agarose gel (voltage 110V, time 30min), and the qualified amplified products were sequenced by Shanghai Sangon Biotechnology Co., Ltd. The sequences were spliced ​​by ContigExpress software and searched by BLAST homology. The 16S rRNA gene sequence of the strain with the highest homology was selected as the reference object, and the sequence was multiple aligned by Clustal W in MEGA7.0 software. The phylogenetic tree was constructed by the neighbor-joining method (NJ), and the confidence value of each branch was tested 1000 times by bootstrap value to analyze the phylogenetic status of the strain.

[0046] The 16S rRNA gene sequence is as follows:

[0047] >LG94 Streptomyces albidoflavus DSM 40455

[0048]

[0049] The 16S rRNA gene sequence of strain LG94 was obtained by PCR amplification. The sequencing results were compared with NCBI and found that strain LG94 had a similarity of more than 98.71% with the strains of Streptomyces in actinomycetes, 99.64% with Streptomyces daghestanicus NRRL B-5418 (T) and Streptomyces violascens ISP 5183 (T), 99.36% with Streptomyces koyangensis VK-A60 (T), and the highest homology with Streptomyces albidoflavus DSM40455 (T) was 99.71% (see Figure 3 ), strain LG94 was preliminarily identified as Streptomyces albidoflavus.

[0050] Example 2 Antagonistic effect of Streptomyces LG94 on plant pathogens

[0051] The antagonistic activity of Streptomyces LG94 against Coniothyrium diplodiella, Fusarium oxysporum f.sp. Cubense tropical race 4, Neoscytalidium dimidiatum, Fusarium redolens, Fusarium fujikuroi and Penicillium digitatum was detected by plate confrontation method. Streptomyces LG94 was cross-inoculated 2.6 cm from the center of the PDA plate by dot-grafting method. After constant temperature cultivation at 28℃ for 1 day, bacterial blocks were punched from the edge of the pathogen colony on the PDA plate with a sterile puncher with a diameter of 8 mm and inoculated inverted in the center of the plate. The plate inoculated with only the pathogen was used as the control group, and each group was repeated 3 times. After the control group was cultured at 28°C until the PDA plate was fully grown, the diameter of the pathogen colony was measured using the cross method to calculate the inhibition rate. The calculation formula is as follows:

[0052] Inhibition rate (%) = (the colony diameter of the plant pathogen in the control group - the colony diameter of the plant pathogen in the treatment group) / the colony diameter of the plant pathogen in the control group × 100%

[0053] Plate confrontation experiments showed that Streptomyces LG94 had different degrees of antagonism against white rot shield mold, banana wilt Fusarium oxysporum race 4, pitaya canker pathogen, fragrant Fusarium, Fuji black Fusarium and citrus green mold pathogen, showing a broad-spectrum antibacterial activity (see Figure 4 ), among which the strongest inhibitory effect was on white rot shield fungus, with an inhibition rate of 66.46±1.41%, followed by citrus green mold pathogen, with an inhibition rate of 53.64±1.94% (see Figure 5 ).

[0054] Example 3 Study on the Growth-Promoting Properties of Streptomyces LG94

[0055] 3.1 Detection of cell wall hydrolase characteristics:

[0056] Protease production ability: Inoculate a Streptomyces LG94 bacterial block with a diameter of 8 mm into a skim milk powder culture medium (skim milk powder 10.0 g, agar 20.0 g, H2O 1000 mL), and culture at 28°C for 5 days. The appearance of a transparent circle around the colony is positive.

[0057] β-1,3-glucanase production ability: Inoculate a Streptomyces LG94 bacterial block with a diameter of 8 mm into a β-1,3-glucan medium (β-1,3-glucan 10.0 g, aniline blue 100 mg, complex salt mother solution 10 mL, agar 20.0 g, H2O 1000 mL), and culture at 28°C for 7 days. The appearance of a transparent circle around the colony is positive.

[0058] Chitinase production ability: Inoculate a Streptomyces LG94 bacterial block with a diameter of 8 mm into a chitin medium (250 mL 2% chitin colloid, 10 mL complex salt mother solution, 20.0 g agar, 1000 mL H2O), and culture at 28°C for 7 days. The appearance of a transparent circle around the colony is positive.

[0059] Cellulase production capacity: Inoculate a Streptomyces LG94 bacterial block with a diameter of 8 mm into a sodium carboxymethyl cellulose medium (10.0 g sodium carboxymethyl cellulose, 10 mL of complex salt mother solution, 20.0 g agar, 1000 mL of H2O), culture at 28°C for 7 days, and stain with Congo red. The appearance of an orange-yellow transparent circle around the colony is positive.

[0060] Lipase production ability: Inoculate a Streptomyces LG94 colony with a diameter of 8 mm into Tween medium (substrate 1% (Tween 20, Tween 60 and Tween 80), tryptone 1.0 g, NaCl 5.0 g, CaCl2·2H2O0.1 g, agar 20.0 g, H2O 1000 mL), and culture at 28°C for 5 days. The appearance of a precipitation zone around the colony is positive.

[0061] Note: Composite salt mother liquor KNO3 1.0 g, K2HPO4 0.5 g, MgSO4·7H2O 0.5 g, NaCl 0.5 g, NH4NO3 0.1 g, FeSO4 0.01 g, ZnSO4·7H2O 0.001 g, MnCl2·H2O 0.001 g, H2O 10 mL; The bacterial block was prepared in Example 2.

[0062] The results of the cell hydrolase characteristics test are as follows Figure 6 As shown in the figure, Streptomyces LG94 can produce transparent circles on skim milk medium, β-1,3-glucan medium and chitin medium, and obvious precipitation circles appear on Tween 20, 60, and 80 medium, but no transparent circle appears on sodium carboxycellulose medium. This shows that Streptomyces LG94 can secrete protease, β-1,3-glucanase, chitinase and lipase, but cannot secrete cellulase.

[0063] 3.2 Other growth-promoting properties

[0064] Phosphorus solubilization: Inoculate 8 mm diameter Streptomyces LG94 colonies into inorganic phosphorus bacterial culture medium (10.0 g glucose, 0.5 g ammonium sulfate, 0.3 g sodium chloride, 0.3 g magnesium sulfate, 0.03 g manganese sulfate, 0.3 g potassium sulfate, 0.03 g ferrous sulfate, 5.0 g calcium phosphate, 0.025 g BPB, 20.0 g agar, 1000 mL H2O) and organophosphorus bacterial culture medium (10.0 g glucose, 0.5 g ammonium sulfate, 0.3 g sodium chloride, 0.3 g magnesium sulfate, 0.3 g manganese sulfate, 0.3 g potassium sulfate, 0.03 g ferrous sulfate, 5.0 g calcium phosphate, 0.2 g lecithin, 0.025 g BPB, 20.0 g agar, 1000 mL H2O), respectively, and culture at 28°C for 7 days. The appearance of transparent circles around the colonies is positive.

[0065] Potassium dissolution: inoculate a Streptomyces LG94 colony with a diameter of 8 mm into a silicate bacterial culture medium (5.0 g sucrose, 5.0 g glucose, 0.5 g ammonium sulfate, 0.5 g yeast powder, 0.3 g magnesium sulfate, 2.0 g disodium hydrogen phosphate, 0.03 g ferrous sulfate, 0.03 g manganese sulfate, 2.0 g potassium feldspar, 20.0 g agar, 1000 mL H2O), culture at 28°C for 7 days, and the appearance of a transparent zone around the colony is positive.

[0066] Iron carrier production: Inoculate a Streptomyces LG94 bacterial block with a diameter of 8 mm in CAS detection medium (chrome azurol (CAS) 0.0605 g, hexadecyltrimethylammonium bromide (HDTMA) 0.0729 g, ferric chloride hexahydrate 2.645 mg, sodium dihydrogen phosphate dihydrate 0.295 g, disodium hydrogen phosphate dodecahydrate 1.214 g, ammonium chloride 0.125 g, potassium dihydrogen phosphate 0.0375 g, sodium chloride 0.0625 g, agar 20.0 g, H2O 1000 mL), and incubate at 28°C for 7 days. The appearance of an orange or yellow halo around the colony is positive.

[0067] IAA production: The seed liquid of Streptomyces LG94 was inoculated into LB liquid medium containing 0.5 mg / mL L-tryptophan at a 1% inoculum, and cultured at 28°C with shaking at 200 r / min for 6 days. The fermentation broth was centrifuged at 12000 r / min for 10 minutes, and the fermentation supernatant was mixed with an equal volume of Salkowski reagent (50 mL 35% HClO4 and 1 mL 0.5 mol / L FeCl3 solution), and incubated at room temperature in the dark for 30 minutes. The color turned red, indicating that IAA can be secreted. Finally, the absorbance at 530 nm (OD530) was measured, and the concentration of IAA in the supernatant was calculated according to the standard curve regression equation.

[0068] Other growth-promoting properties such as Figure 7 As shown in the figure, Streptomyces LG94 can produce orange transparent circles on CAS medium, but no transparent circles appear on inorganic phosphorus bacteria medium, organic phosphorus bacteria medium and silicate bacteria medium. This shows that Streptomyces LG94 has the ability to produce iron carriers, but cannot dissolve phosphorus and potassium. In addition, the color of Salkowski reagent turns red, indicating that Streptomyces LG94 can produce plant hormone IAA. The IAA produced by quantification using the IAA standard curve is 13.59±0.30μg / mL (see Figure 8 ).

[0069] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A strain of Streptomyces LG94, characterized in that: The classification name of the Streptomyces LG94 is Streptomyces albidoflavus; the Streptomyces LG94 was deposited in the Guangdong Provincial Microbiological Culture Collection Center on August 16, 2024, and the preservation address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the preservation number is: GDMCC NO: 65017.

2. Use of the Streptomyces LG94 described in claim 1 in inhibiting plant pathogenic fungi.

3. The use according to claim 2, characterized in that: The plant pathogenic fungi include white rot shield mold, banana wilt Fusarium oxysporum race 4, pitaya canker pathogen, fragrant Fusarium, vine black Fusarium or citrus green mold pathogen.

4. Use of the Streptomyces LG94 described in claim 1 in promoting plant growth.

5. Use of the Streptomyces LG94 described in claim 1 in producing siderophores.

6. Use of the Streptomyces LG94 described in claim 1 in producing the plant hormone IAA.

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