Multifunctional biocontrol and growth-promoting streptomyces LG94 and application thereof

Abstract

The application provides a multifunctional biocontrol growth-promoting Streptomyces LG94 and application thereof, and belongs to the technical field of biotechnology. The preservation number of the Streptomyces LG94 is GDMCC NO: 65017. The Streptomyces LG94 is isolated from deep-sea sediments of hippocampus, can inhibit the growth of various pathogenic fungi such as white-roted Discula annulata, banana wilt Fusarium oxysporum f. sp. cubense race 4, pitaya wilt Fusarium oxysporum f. sp. alisatiae, Fusarium venenatum, Fusarium reticulatum and citrus green mold, has the effects of producing iron carrier and secreting plant hormone indole acetic acid (IAA) to promote plant growth, and can be used for developing a multifunctional biological control agent.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to a multifunctional biocontrol and growth-promoting Streptomyces LG94 and its applications. Background Technology

[0002] Plant cultivation faces increasing biotic and abiotic stresses, leading to slower plant growth and reproduction, and resulting in economic losses. Chemical products are commonly used as pesticides or fertilizers to increase crop yields. However, the overuse of agricultural chemicals not only causes adverse environmental problems such as soil pollution, pesticide tolerance, and dysbiosis, but also poses potential hazards to human health. Utilizing microorganisms to promote plant growth and enhance plant tolerance to environmental stresses can provide an alternative approach for safe agricultural production.

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

[0004] Therefore, screening microbial resources with biocontrol and life-promoting properties from deep-sea sediments and exploring potential multifunctional biological control agents has profound significance for modern agriculture. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a multifunctional biocontrol and growth-promoting Streptomyces LG94 and its applications. The Streptomyces LG94 has a relatively broad antibacterial spectrum and also has the functions of producing siderophores and plant hormone IAA.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides a Streptomyces strain LG94, which is classified as Streptomyces albidoflavus. The Streptomyces strain LG94 was deposited on August 16, 2024, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC NO: 65017.

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

[0009] Preferably, the plant pathogenic fungi include *Scutellaria baicalensis*, *Fusarium oxysporum* race 4 (the causal agent of banana wilt), *Fusarium solani* (the causal agent of dragon fruit canker), *Fusarium solani*, *Fusarium solani*, or *Green mold* (the causal agent of citrus).

[0010] This invention provides the application of the aforementioned Streptomyces LG94 in promoting plant growth.

[0011] This invention provides the application of the aforementioned Streptomyces LG94 in siderogenic carriers.

[0012] This invention provides the application of the aforementioned Streptomyces LG94 in the production of plant hormone IAA.

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

[0014] This invention isolates a Streptomyces strain LG94 from deep-sea sediments of seahorses. It can inhibit the growth of various pathogenic fungi, such as white rot scutellarin, Fusarium oxysporum race 4 (the causal agent of banana wilt), dragon fruit canker fungus, Fusarium odorata, Fusarium tumefaciens, and citrus green mold. It also has the effect of promoting plant growth by producing siderophores and secreting the plant hormone indoleacetic acid (IAA). It can be used to develop multifunctional biocontrol agents.

[0015] Biological Preservation Instructions

[0016] Streptomyces LG94, classified as Streptomyces albidoflavus, was deposited on August 16, 2024, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC NO: 65017. Attached Figure Description

[0017] Figure 1 This image shows the colony morphology of Streptomyces LG94 on 2216E medium; where A is the front view and B is the back view.

[0018] Figure 2 The images show the morphology of a single colony and spore of Streptomyces LG94 under an optical microscope; where A represents a single colony and B represents a spore.

[0019] Figure 3 Phylogenetic analysis diagram of Streptomyces LG94 based on 16S rRNA;

[0020] Figure 4 This diagram illustrates the antagonistic effect of Streptomyces LG94 on plant pathogens.

[0021] Figure 5 The graph shows the inhibition rate of Streptomyces LG94 against plant pathogens.

[0022] Figure 6This is a graph showing the detection characteristics of hydrolytic enzymes in Streptomyces LG94 cells;

[0023] Figure 7 Graphs showing the effects of Streptomyces LG94 on phosphorus solubilization, potassium solubilization, iron carrier production, and indoleacetic acid production.

[0024] Figure 8 This is the IAA standard curve. Detailed Implementation

[0025] This invention provides a Streptomyces strain LG94, taxonomically named Streptomyces albidoflavus. This Streptomyces strain LG94 was deposited on August 16, 2024, at the Guangdong Provincial Microbial Culture Collection Center, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC NO: 65017. This strain originates from deep-sea sediments and grows well in 2216E 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] This invention provides the application of Streptomyces LG94 in inhibiting plant pathogenic fungi. In this invention, the plant pathogenic fungi are preferably *Streptomyces leucovorum*, *Fusarium oxysporum* race 4 (the wilt pathogen of banana), *Dracaena cochinchinensis*, *Fusarium oxysporum*, *Fusarium oxysporum*, or *Phyllostachys citrus*, more preferably *Streptomyces leucovorum* or *Phyllostachys citrus*, and even more preferably *Streptomyces leucovorum*.

[0031] This invention provides the application of Streptomyces LG94 in promoting plant growth. Its growth-promoting effect is manifested in the production of protease and chitinase to inhibit the growth of harmful fungi, as well as the production of lipase to help plants decompose macromolecular nutrients and help plants absorb them. It also has the function of producing siderophores and plant hormone IAA, thus promoting plant growth in multiple ways.

[0032] This invention provides the application of the aforementioned Streptomyces LG94 on siderogenic carriers, which can generate orange or yellow halos on CAS detection media.

[0033] This invention provides the application of the aforementioned Streptomyces LG94 in the production of plant hormone IAA, which can turn Salkowski reagent red.

[0034] Experimental materials:

[0035] Sediment samples: The deep-sea sediments of the seahorse originated from (111.0557°E, 17.6227°N).

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

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

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

[0039] Example 1: Isolation and Identification of Streptomyces LG94

[0040] 1.1 Sample Source: Deep-sea sediments from seahorses

[0041] Weigh 1.0 g of dried deep-sea sediment into a centrifuge tube containing 9.0 mL of sterile water, vortex until homogeneous, and dilute to different gradients. Take 200 μL of each gradient, resulting in a dilution of 10. -2 and 10 -3 The sample solution was spread on humic acid solid medium and incubated at 28°C until the colony count remained unchanged. Single colonies were picked and purified by the three-zone streak method. The purified strains were transferred to cryovials containing 30% glycerol and stored at -80°C.

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

[0043] Streptomyces LG94 was isolated from deep-sea sediments from seahorses. This strain grew well in 2216E medium, producing circular colonies with concentric rings and a concave center. The mycelium was well-developed, with aerial hyphae being grayish-brown and substrate hyphae being lilac-brown (see...). Figure 1 Under an optical microscope, the colony is surrounded by spores, which are round, oblong, or peanut-shaped (see...). Figure 2 ).

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

[0045] Bacterial DNA was extracted using Chelex-100 resin, and PCR amplification was performed using universal primers 27F and 1522R. The PCR amplified fragments were detected using 1% agarose gel electrophoresis (110V, 30 min). Successfully amplified products were sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. Sequences were assembled using ContigExpress software, and BLAST homology search was performed. The 16S rRNA gene sequence of the strain with the highest homology was selected as the reference. Multiple sequence alignment was performed using Clustal W in MEGA 7.0 software. A phylogenetic tree was constructed using Neighbor-Joining (NJ), and the bootstrap value was tested 1000 times to determine the confidence value of each branch to analyze the phylogenetic position of the strains.

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

[0047] >LG94 Streptomyces albidoflavus DSM 40455

[0048]

[0049] PCR amplification yielded the 16S rRNA gene sequence of strain LG94. Sequencing results, compared with those obtained from NCBI, showed that strain LG94 exhibited greater than 98.71% similarity to strains of the genus *Streptomyces* within the actinomycete family. It showed 99.64% homology with *Streptomyces daghestanicus* NRRL B-5418(T) and *Streptomyces violascens* ISP 5183(T)), 99.36% homology with *Streptomyces koyangensis* VK-A60(T)), and the highest homology of 99.71% with *Streptomyces albidoflavus* DSM40455(T) (see [link to PCR analysis]). Figure 3 The 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* race 4 (fusarium wilt of banana), *Neoscytalidium dimidiatum*, *Fusarium redolens*, *Fusarium f. f. fujikuroi*, and *Penicillium digitatum* (citrus green mold) was assessed using the plate confrontation method. *Streptomyces* LG94 was cross-inoculated at a distance of 2.6 cm from the center of a PDA plate using the spot inoculation method. After incubation at 28°C for one day, mycelial blocks were punched from the edge of pathogen colonies on the PDA plate using an 8 mm diameter sterile punch and inoculated upside down into the center of the plate. Plates inoculated only with pathogens served as the control group, with three replicates per group. After incubation at 28℃ until the control group's PDA plates were fully covered, the diameter of the pathogen colonies was measured using the cross-cross method, and the inhibition rate was calculated. The calculation formula is as follows:

[0052] Inhibition rate (%) = (Coronavirus colony diameter in control group - Coronavirus colony diameter in treatment group) / Coronavirus colony diameter in control group × 100%

[0053] Plate confrontation experiments showed that Streptomyces LG94 exhibited varying degrees of antagonistic activity against *Streptomyces glabripennis*, *Fusarium oxysporum* race 4 (the causal agent of banana wilt), *Fusarium oxysporum* (the causal agent of dragon fruit canker), *Fusarium oxysporum*, *Fusarium oxysporum* var. *truncatum*, and *Aureobasidium aeruginosa* (the causal agent of citrus green mold), demonstrating broad-spectrum antibacterial activity (see...). Figure 4 Among them, the inhibitory effect on white rot scutellaria was the strongest, with an inhibition rate of 66.46±1.41%, followed by citrus green mold, with an inhibition rate of 53.64±1.94% (see...). Figure 5 ).

[0054] Example 3: Study on the growth-promoting characteristics of Streptomyces LG94

[0055] 3.1 Detection of cell wall hydrolytic enzyme characteristics:

[0056] Protease production capacity: Inoculate 8 mm diameter Streptomyces LG94 bacterial blocks into skim milk powder medium (10.0 g skim milk powder, 20.0 g agar, 1000 mL H2O) and incubate at 28 °C for 5 days. The appearance of a clear zone around the colony indicates a positive result.

[0057] β-1,3-glucanase production capacity: 8 mm diameter Streptomyces LG94 bacterial blocks were inoculated into β-1,3-glucan medium (10.0 g β-1,3-glucan, 100 mg aniline blue, 10 mL of compound salt stock solution, 20.0 g agar, 1000 mL H2O) and incubated at 28℃ for 7 days. A clear zone around the colony indicated a positive result.

[0058] Chitinase production capacity: 8 mm diameter Streptomyces LG94 bacterial blocks were inoculated into chitin medium (250 mL 2% chitin colloid, 10 mL compound salt stock solution, 20.0 g agar, 1000 mL H2O) and incubated at 28℃ for 7 days. The appearance of a clear zone around the colony indicated a positive result.

[0059] Cellulase production capacity: 8 mm diameter Streptomyces LG94 bacterial blocks were inoculated onto sodium carboxymethyl cellulose medium (10.0 g sodium carboxymethyl cellulose, 10 mL compound salt stock solution, 20.0 g agar, 1000 mL H2O). After incubation at 28 °C for 7 days, staining with Congo red showed that an orange-yellow transparent zone around the colony was positive.

[0060] Lipase production capacity: 8 mm diameter Streptomyces LG94 bacterial blocks were inoculated onto Tween medium (1% substrate (Tween 20, Tween 60, and Tween 80), 1.0 g tryptone, 5.0 g NaCl, 0.1 g CaCl2·2H2O, 20.0 g agar, and 1000 mL H2O), and incubated at 28°C for 5 days. A positive result was indicated by the appearance of a precipitate zone around the colony.

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

[0062] The detection results of the characteristics of cellular hydrolytic enzymes are as follows: Figure 6 As shown, *Streptomyces LG94* produced clear zones on skim milk medium, β-1,3-glucan medium, and chitin medium, and exhibited distinct precipitation zones on Tween 20, 60, and 80 medium, but did not produce clear zones on sodium carboxylate medium. This indicates that *Streptomyces LG94* can secrete proteases, β-1,3-glucanases, chitinases, and lipases, but cannot secrete cellulase.

[0063] 3.2 Other growth-promoting properties

[0064] Phosphate solubilization: 8mm diameter Streptomyces LG94 bacterial blocks were inoculated into inorganic phosphorus bacteria culture medium (10.0g glucose, 0.5g ammonium sulfate, 0.3g sodium chloride, 0.3g magnesium sulfate, 0.03g manganese sulfate, 0.3g potassium sulfate, 0.03g ferrous sulfate, 5.0g calcium phosphate, 0.025g BPB, 20.0g agar, 1000mL H2O) and organic phosphorus bacteria culture medium (10.0g glucose, 0.5g ammonium sulfate, 0.3g sodium chloride, 0.3g magnesium sulfate, 0.03g manganese sulfate, 0.3g potassium sulfate, 0.03g ferrous sulfate, 5.0g calcium phosphate, 0.2g lecithin, 0.025g BPB, 20.0g agar, 1000mL H2O). The cultures were incubated at 28℃ for 7 days. A positive result was indicated by the appearance of a clear zone around the colony.

[0065] Potassium solubilization: Inoculate 8mm diameter Streptomyces LG94 bacterial blocks onto silicate bacterial culture medium (5.0g sucrose, 5.0g glucose, 0.5g ammonium sulfate, 0.5g yeast extract, 0.3g magnesium sulfate, 2.0g disodium hydrogen phosphate, 0.03g ferrous sulfate, 0.03g manganese sulfate, 2.0g potassium feldspar, 20.0g agar, 1000mL H2O), and incubate at 28℃ for 7 days. A positive result is indicated by the appearance of a clear zone around the colony.

[0066] Iron-producing carrier: 8mm diameter *Streptomyces* LG94 colony blocks were inoculated onto CAS detection medium (containing 0.0605g chromamarone (CAS)), 0.0729g hexadecyltrimethylammonium bromide (HDTMA), 2.645mg ferric chloride hexahydrate, 0.295g sodium dihydrogen phosphate dihydrate, 1.214g disodium hydrogen phosphate dodecahydrate, 0.125g ammonium chloride, 0.0375g potassium dihydrogen phosphate, 0.0625g sodium chloride, 20.0g agar, and 1000mL H2O) and incubated at 28℃ for 7 days. A positive result was indicated by an orange or yellow halo around the colony.

[0067] IAA production: 1% of the seed culture of *Streptomyces LG94* was inoculated into LB liquid medium containing 0.5 mg / mL L-tryptophan and cultured at 28°C with shaking at 200 rpm for 6 days. The fermentation broth was centrifuged at 12000 rpm for 10 min. The supernatant was mixed with an equal volume of Salkowski's reagent (50 mL of 35% HClO4 and 1 mL of 0.5 mol / L FeCl3 solution) and incubated at room temperature in the dark for 30 min. A red color indicated the secretion of IAA. Finally, the absorbance at 530 nm (OD530) was measured, and the concentration of IAA in the supernatant was calculated using the regression equation of the standard curve.

[0068] Other results of the test for growth-promoting properties, such as Figure 7 As shown, *Streptomyces LG94* produces an orange clear zone on CAS medium, but no clear zone appears on inorganic phosphorus bacteria medium, organic phosphorus bacteria medium, or silicate bacteria medium. This indicates that *Streptomyces LG94* has the ability to produce siderophores, but cannot solubilize phosphorus or potassium. Furthermore, the Salkowski reagent turns red, indicating that *Streptomyces LG94* can produce the plant hormone IAA. The IAA produced, quantified using the IAA standard curve, is 13.59 ± 0.30 μg / mL (see...). Figure 8 ).

[0069] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of *Streptomyces microcyticum* LG94 in inhibiting plant pathogenic fungi, characterized in that, The plant pathogenic fungi include white rot scutellaria, Fusarium oxysporum race 4 (the causal agent of banana wilt), dragon fruit canker fungus, Fusarium odorata, Fusarium tumefaciens, or citrus green mold fungus. The microysperm (Streptomyces microcytogenes) Streptomyces albidoflavus LG94 was deposited on August 16, 2024, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC NO: 65017.

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