Streptomyces rochei and application thereof in increasing crop yield

Abstract

The invention relates to the field of microorganisms, in particular to streptomyces rochei and application thereof in increasing crop yield. Specifically, the preservation number of the streptomyces rochei is CGMCC (China General Microbiological Culture Collection Center) C28006. The streptomyces rochei provided by the invention can reduce the aromatization degree and condensation degree of lignite humic acid in lignite, so that the lignite humic acid is degraded into micromolecular biochemical humic acid, and the biochemical humic acid contains more oxygen-containing and nitrogen-containing functional groups and can promote the increase of crop yield; meanwhile, the disease resistance of crops can also be improved.

Description

Technical Field

[0001] The invention relates to the field of microorganisms, and in particular to Streptomyces rocherii and application thereof in increasing crop yield. Background Art

[0002] As the most important part of the ecosystem, humic acid is widely present in nature, such as soil, water, peat, lignite and sediment. Humic acid is a more stable organic matter formed by the biodegradation and abiotic degradation and condensation of animal, plant and microbial residues. Humic acid is the main source of carbon in the biosphere, accounting for about 70% of soil organic matter and 25% of groundwater solvent organic carbon. From the perspective of energy absorption and biogeology, humic acid is more difficult for microorganisms to use than other carbon sources. This is because 14 C age in soil has a long span, about 250 to 3000 years. In addition, the stability of humic acid depends on its structure composed of a heterogeneous and complex molecular mixture, consisting of many carbon rings connected by aromatic, oxygen and nitrogen. In addition, there are many functional groups connected to the carbon rings, including amines, alcohols, ethers, phenyls, aldehydes, ketones, carboxylic acids, esters, amides and quinones.

[0003] Lignite is a low-rank coal. When used as an industrial energy source, it produces harmful gases such as sulfur and nitrogen oxides, which seriously harm the environment. However, the use of lignite by microorganisms can have the opposite effect. Lignite contains a variety of aromatic and aliphatic compounds that can serve as nutrients for the growth of microorganisms.

[0004] Although numerous studies have shown that the interaction between microorganisms and humus is a green way to promote agricultural development, there is still a lack of ready-made technology. Summary of the invention

[0005] The purpose of the present invention is to provide a strain of Streptomyces rocheri (Streptomyces L1) and its application in increasing crop yield. The Streptomyces rocheri provided by the present invention can reduce the aromatization degree and condensation degree of lignite humic acid in lignite, and degrade the lignite humic acid into small molecular biochemical humic acid, which contains more oxygen-containing and nitrogen-containing functional groups and can promote the increase of crop yield.

[0006] The first object of the present invention is to provide a Streptomyces rocherii, the preservation number of the Streptomyces rocherii is CGMCC28006.

[0007] The second object of the present invention is to provide a composition comprising the above-mentioned Streptomyces rocheri and / or its fermentation product.

[0008] The third object of the present invention is to provide the use of the Streptomyces rocheri or the composition as described above in degrading humic acid.

[0009] In a preferred embodiment, the humic acid is derived from lignite.

[0010] The fourth object of the present invention is to provide the use of the Streptomyces rocheri or the composition as described above in the preparation of biological fertilizer.

[0011] A fifth object of the present invention is to provide a method for preparing a biofertilizer, the method comprising: fermenting the above-mentioned Streptomyces rocherii in a culture medium containing humic acid to obtain the biofertilizer.

[0012] In a preferred embodiment, the humic acid is derived from lignite.

[0013] A sixth object of the present invention is to provide the biological fertilizer as described above.

[0014] A seventh object of the present invention is to provide a fertilizer composition, which contains the biological fertilizer as described above.

[0015] The eighth object of the present invention is to provide the use of the Streptomyces rocheri described above, the composition described above, the biological fertilizer described above or the fertilizer composition described above in increasing crop yield, improving crop disease resistance and improving soil compaction.

[0016] In the process of utilizing lignite humic acid, the peroxidase synthesis gene katE and the esterase synthesis genes pdgA and fabG were significantly upregulated by Streptomyces L1. These two enzymes are the key enzymes for breaking macromolecular humic acid into small molecular humic acid. Streptomyces L1 first broke the side chains of the aromatic rings, causing the cross-linkers between the aromatic rings to disappear. Subsequently, the broken cross-linkers were converted into hydrocarbons, and the simple aromatic structure was converted into aromatic acids. Esterase hydrolyzed aliphatic compounds, causing them to decompose into fatty acids and fatty alcohols. Among them, peroxidation is the main mechanism for breaking the functional groups of lignite. The side chains of the aromatic rings are broken and oxidized to form simple aromatic acids. Therefore, the biodissolution of lignite starts with the breaking of the side chains of the aromatic rings. Without the side chain structure, it is difficult to biodissolve lignite. Hard coal and bituminous coal are more difficult to biodissolve because they have fewer side chains in their aromatic ring structures.

[0017] Under the catalytic action of Streptomyces L1 peroxidase and esterase, the aromatic ring side chain of humic acid breaks, which reduces the aromatization degree and condensation degree of macromolecular humic acid and degrades it into small molecular biochemical humic acid. These biochemical humic acids contain more oxygen-containing and nitrogen-containing functional groups, which can promote the increase of crop yields. As recorded in the examples of this application, the corn yield can be increased by an average of 381kg / ha, with an increase rate of 4.0%. Therefore, the biochemical humic acid produced after lignite degradation can promote the growth of crops.

[0018] In addition, the application of lignite humic acid fermentation liquid as a new biological fertilizer can increase the leaf area of ​​individual crops, reduce the disease rate, reduce sunburn of fruits in high temperature seasons, and enhance disease resistance. It has a particularly significant effect on soil compaction.

[0019] Other features and advantages of the present invention will be described in detail in the following detailed description.

[0020] Biological Deposit

[0021] The Streptomyces rochei provided by the present invention was deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration on July 25, 2023, with a deposit number of CGMCC28006 and a deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences (abbreviated as CGMCC), referred to as Streptomyces L1. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein:

[0023] Figure 1 NJ phylogenetic tree based on 16S rRNA gene sequences.

[0024] Figure 2 The bacterial cells in GA medium and humic acid fermentation broth were observed under a scanning electron microscope at a magnification of 500 times.

[0025] Figure 3 It is the UV-visible spectrum and Fourier transform infrared spectrum of lignite humic acid in the wavelength range of 190nm-700nm after 7d degradation under the action of Streptomyces L1.

[0026] Figure 4 The decomposition metabolites in lignite humic acid fermentation broth and the secondary metabolites of Streptomyces rocheri L1 were detected by GC-MS.

[0027] Figure 5 Effects of lignite humic acid fermentation broth of Streptomyces L1 on corn rhizomes and yield.

[0028] Figure 6 Effect of lignite humic acid fermentation broth of Streptomyces L1 on kiwifruit yield. DETAILED DESCRIPTION

[0029] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0030] The first object of the present invention is to provide a Streptomyces rocherii, the preservation number of the Streptomyces rocherii is CGMCC28006.

[0031] The Streptomyces rocherii provided by the present invention was isolated from a soil sample collected from a potato field in Zhouzhi County, Xi'an City (108°37'E, 34°32'N), and was deposited in the General Microbiology Center of the China Microbiological Culture Collection on July 25, 2023. Its deposit number is CGMCC28006, and its deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences (abbreviated as CGMCC), referred to as Streptomyces L1.

[0032] The Streptomyces rocherii CGMCC28006 provided by the invention has a round appearance, abundant, velvety, grey aerial filaments, colourless basal filaments, and sometimes a slightly reddish hue after transfer.

[0033] The Streptomyces rocherii CGMCC28006 provided by the present invention can be obtained in large quantities by culturing. For example, a large amount of Streptomyces rocherii CGMCC28006 can be obtained by inoculating it into a culture medium for culturing.

[0034] The culture medium can be a culture medium conventionally used for actinomycetes. In one embodiment, the culture medium is GA culture medium. In another embodiment, the culture medium is MSM culture medium.

[0035] The culture conditions may be conventional conditions for culturing actinomycetes. In a preferred embodiment, the culture temperature is 25-40°C (for example, 25, 30, 35, 40°C); in another preferred embodiment, the culture time is 1-10 days (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days).

[0036] In some embodiments, an inhibitor for inhibiting the growth of bacteria and fungi may be added to the culture medium, for example, ampicillin and / or fluconazole. The concentration of the inhibitor added may be adjusted by those skilled in the art according to actual conditions.

[0037] The second object of the present invention is to provide a composition comprising the above-mentioned Streptomyces rocheri and / or its fermentation product.

[0038] The Streptomyces rocherii contained in the composition may be live cells and / or dead cells. In some embodiments, the composition contains live cells of Streptomyces rocherii, for example, the live cells of Streptomyces rocherii are prepared into seeds, wherein the live cells of Streptomyces rocherii can be propagated in a culture medium to obtain a target product. In some embodiments, the composition contains dead cells of Streptomyces rocherii, for example, the product obtained after the cells are inactivated after the fermentation is completed. In some embodiments, the composition contains live cells and dead cells of Streptomyces rocherii, for example, the product after the fermentation is completed includes both live cells and dead cells of Streptomyces rocherii at the end of the fermentation.

[0039] In some embodiments, the composition does not contain any bacteria of Streptomyces rocherii, for example, the fermentation clear liquid obtained by separating the bacteria after the fermentation is completed. It can be understood that the "does not contain" mentioned here means substantially not containing, and the bacteria residues caused by technical factors during the separation process of the bacteria are not included.

[0040] In some embodiments, the composition is in liquid form (stock solution or concentrated solution), and in some embodiments, the composition is in solid form. Those skilled in the art can select a suitable form according to the intended purpose. It is understood that when preparing compositions of different forms, auxiliary materials that stabilize the corresponding forms should also be added.

[0041] The third object of the present invention is to provide the use of the Streptomyces rocheri or the composition as described above in degrading humic acid.

[0042] The Streptomyces rocherii provided by the invention can grow and reproduce with humic acid as the only carbon source, so that the aromatization degree and condensation degree of macromolecular humic acid are reduced and degraded into small molecular biochemical humic acid.

[0043] In a preferred embodiment, the humic acid contains an aromatic ring, and in a further preferred embodiment, the aromatic ring contains a side chain structure. Streptomyces L1 first breaks the side chain of the aromatic ring, causing the cross-linking agent between the aromatic rings to disappear. Subsequently, the broken cross-linking agent is converted into hydrocarbons, and the simple aromatic structure is converted into aromatic acids. Peroxidation is the main mechanism for breaking the lignite functional group, and the side chain of the aromatic ring is broken and oxidized to form a simple directional acid.

[0044] In a preferred embodiment, the humic acid is derived from lignite. Lignite is a low-rank coal. When used as an industrial energy source, the harmful gases such as sulfur and nitrogen oxides produced will seriously harm the environment. However, as shown in Example 4, lignite humic acid has almost no toxic effect on Streptomyces L1.

[0045] The fourth object of the present invention is to provide the use of the Streptomyces rocheri or the composition as described above in the preparation of biological fertilizer.

[0046] The Streptomyces L1 provided by the present invention contains more oxygen-containing and nitrogen-containing functional groups after humic acid degradation, which can promote the increase of crop yields, and can therefore be used as a biofertilizer. As recorded in the examples of the present application, the corn yield can be increased by an average of 381 kg / ha, with a yield increase rate of 4.0%, and the kiwifruit yield increase rate of 24.7%. Therefore, the biochemical humic acid produced after lignite degradation can be used as a biofertilizer to promote the growth of crops.

[0047] A fifth object of the present invention is to provide a method for preparing a biofertilizer, the method comprising: fermenting the above-mentioned Streptomyces rocherii in a culture medium containing humic acid to obtain the biofertilizer.

[0048] The Streptomyces loucherii provided by the present invention can grow and reproduce in an environment with humic acid as the only carbon source, so that the aromatization degree and condensation degree of macromolecular humic acid are reduced, and degraded into small molecular biochemical humic acid. In a preferred embodiment, the humic acid contains an aromatic ring, and in a further preferred embodiment, the aromatic ring contains a side chain structure. In a preferred embodiment, the humic acid is derived from lignite.

[0049] In a preferred embodiment, lignite humic acid is prepared by the following method:

[0050] Dissolve low-rank lignite in NaOH solution, stir at 80-100°C and let stand, take the supernatant and adjust the pH to 1-3, bath at 60-80°C, then separate the solid and liquid and discard the supernatant (HAs is insoluble in acidic medium). In a further preferred embodiment, in order to remove inorganic pollutants, HAs is purified by adding HCl / HF solution, stirring to separate the solid and liquid and remove the precipitate, to obtain purified HAs, which can be dried for later use.

[0051] The fermentation conditions can be conventional conditions for culturing actinomycetes. In a preferred embodiment, the fermentation temperature is 25-40°C (for example, 25, 30, 35, 40°C); in another preferred embodiment, the fermentation time is 1-10 days (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days).

[0052] A sixth object of the present invention is to provide the biological fertilizer as described above.

[0053] A seventh object of the present invention is to provide a fertilizer composition, which contains the biological fertilizer as described above.

[0054] In the present invention, each component in the fertilizer composition can exist in an independent form or in the form of a mixture. In the case of fertilizer combination, the fertilizer composition contains at least two fertilizers, one of which is the biological fertilizer provided by the present invention, and the other can be determined according to specific circumstances. In a specific embodiment, the fertilizer composition contains the biological fertilizer and chemical fertilizer. In some embodiments, the two fertilizers are mixed together and applied together. In some embodiments, the two fertilizers are applied in sequence. Those skilled in the art can select a specific application method according to the intended purpose. In a specific embodiment, the chemical fertilizer is nitrogen, phosphorus and potassium fertilizer.

[0055] The eighth object of the present invention is to provide the use of the Streptomyces rocheri described above, the composition described above, the biological fertilizer described above or the fertilizer composition described above in increasing crop yield, improving crop disease resistance and improving soil compaction.

[0056] As shown in the examples, the yield of crops using the biofertilizer provided by the present invention is significantly increased, and pests and diseases are significantly reduced, and the compaction of the soil is also improved. Specifically, the corn yield is increased by an average of more than 5 kg, and the average yield increase rate of kiwifruit reaches 24.7%. This shows that the Streptomyces L1 provided by the present invention has the ability to decompose complex aromatic hydrocarbons in the lignite matrix and the fermentation liquid can promote agricultural production.

[0057] Example 1 Isolation, identification and preservation of strains

[0058] 5 g of soil (soil samples collected from potato fields in Zhouzhi County, Xi'an (108°37'E, 34°32'N)) was transferred to a 25 ml sterile conical flask, 10 ml of sterile water was added for mechanical stirring, 1 ml was diluted 10 times in 0.9% NaCl solution and 50 μl was applied to Casein Starch Agar (CSA) medium (soluble starch 10 g, casein 0.3 g, KNO3 2 g, NaCl 2 g, K2HPO4 2 g, MgSO4-7H2O 0.05 g, CaCO3 0.02 g, FeSO4-7H2O 0.01 g, agar 18 g, distilled water to 1 L, pH 7.2). In order to inhibit the growth of bacteria and fungi, ampicillin (20 μg / ml) and fluconazole (25 μg / ml) were added to the culture medium. After incubation in a 28°C incubator for 48 hours, a single colony was selected and transferred to GA medium (soluble starch 20.0g, KNO3 1.0g, K2HPO4 0.5g, NaCl0.5g, MgSO4-7H2O 0.5g, FeSO4-7H2O 0.01g, agar 18g, distilled water to 1L) for purification and culture. Five actinomycetes were isolated, and actinomycete L1 was finally selected as the strain for this study through biological activity assay and growth curve assay. Actinomycete L1 has a round appearance, with abundant aerial filaments, velvety, gray, and colorless basal filaments, sometimes with a slightly reddish hue after transfer.

[0059] The NJ phylogenetic tree based on 16S rRNA gene sequences showed that Actinomycetes L1 and Streptomyces rocheiNRRL B-2410T formed a monophyletic branch ( Figure 1 ). Therefore, the result of identification of actinomycetes L1 is Streptomyces loucheri. It was deposited in the General Microbiological Center of China Microorganism Culture Collection Administration on July 25, 2023, with the deposit number CGMCC28006, and the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences (abbreviated as CGMCC), referred to as Streptomyces L1.

[0060] Example 2 Preparation of lignite humic acid

[0061] Low-rank lignite (low-rank lignite in Jixi, Heilongjiang) was dissolved in 0.1 mol / L NaOH solution (extract: NaOH = 1:10), stirred on a 90°C magnetic stirrer for 4 h, allowed to stand for 2 h, centrifuged at 3 000 rpm for 15 min, the precipitate was discarded, the supernatant was adjusted to pH 2 with 0.1 mol / L HCl, bathed in 70°C water for 1 h, and centrifuged at 3 000 rpm for 15 min, and the supernatant was discarded (HAs is insoluble in acidic media). In order to remove inorganic pollutants, HAs was purified by adding 0.2 L of 5% HCl / HF solution 1:1 (v / v), stirred for 2 h, and then centrifuged to remove the precipitate. The purified HAs was washed twice with deionized water and dried at 60°C for use.

[0062] Example 3 Degradation of humic acid by Streptomyces L1

[0063] In order to investigate the degradation of HA and its effect on crops, strain L1 was used to ferment HAs. Specifically, L1 cultures grown for 3 days in MNGA medium (10 g / L glucose, 5 g / L peptone, 3 g / L beef extract, 3 g / L dry yeast, 3 g / L NaCl and 3 g / LCaCO3, pH = 7) were harvested, washed and resuspended in MSM medium (10 g / L (NH4)2SO4, 1.1 g / L NaCl, 1.1 g / L KCl, 4.4 g / L Na2HPO4, 3.4 g / L KH2PO4, 0.5 g / L MgSO4, 0.01 g / L FeSO4, pH = 7), and HAs were fermented in MSM liquid medium by inoculating 3% (v / v) of strain L1, with 5 g / L HAs as the only carbon resource. Uninoculated flasks were used as controls. The flasks were cultured in the dark at 150 rpm and 30 ° C for 7 days. All treatments were used in triplicate, including the control.

[0064] Example 4 Toxic effect of humic acid solution on Streptomyces L1 during degradation

[0065] In order to better observe whether the humic acid solution has a toxic effect on Streptomyces L1 during the degradation process, the bacterial suspension strain was collected for SEM observation. The preparation method of the SEM sample is as follows: the collected microbial cell samples were fixed with 2.5% glutaraldehyde solution for 12 hours, the fixative was poured off, and then washed three times with 0.1M phosphate buffer (pH 7.4), each wash for 15 minutes. The samples were transferred to 1% O S O4 in 0.1 M phosphate buffer (pH 7.4) and place at room temperature for 1-2 hours. Carefully pour out the O SAfter the O4 waste liquid, the sample was washed three times in 0.1M phosphate buffer (pH 7.4), each time for 15 minutes. The sample was dehydrated with the following five concentration gradients of ethanol aqueous solution (30%, 50%, 70%, 80%, 90%), each concentration for 15 minutes. It was then treated with anhydrous ethanol twice, each time for 20 minutes, and dehydrated with isoamyl acetate for 15 minutes. The sample was dried in a critical point dryer, gold-plated for 30 seconds, and observed and photographed using a TESCAN CLARA field emission scanning electron microscope.

[0066] After 40 h of degradation, the bacteria in the GA medium and the humic acid fermentation broth were collected and the morphology of the bacteria was observed under a scanning electron microscope at a magnification of 500 times ( Figure 2 ). The surface of the bacteria in the GA medium is uneven and wrinkled (A). The morphological characteristics of the bacteria in the humic acid fermentation liquid are similar to those in the GA medium, but one side of the bacteria is slightly broken and a honeycomb structure appears (B). By comparing the amount of bacteria in the fermentation bottle, it was found that the biomass of Streptomyces L1 strains was roughly the same during the same incubation time, which shows that the humic acid solution has no obvious toxic effect on Streptomyces L1.

[0067] Example 5 Chemical Analysis of Biosolubilization Products

[0068] (1) Analysis of organic elements before and after humic acid fermentation, UV-visible spectroscopy, and Fourier transform infrared spectroscopy

[0069] Take part of the lignite humic acid fermentation liquid to extract (method is the same as in Example 2) and dry the humic acid, and then use an element analyzer (VARIO EL III) to detect the change of the content of organic elements (C, H, O, N) in organic compounds together with the control group. The sample uses helium as carrier gas and purge gas, fully burns under high temperature conditions, and is detected by a thermal conductivity detector after gas separation. The remaining humic acid fermentation liquid and the control group are characterized by ultraviolet-visible light spectrum and Fourier infrared spectrum. Ultraviolet-visible light spectrum analysis uses UV-1800 (Shimadzu, Japan) analyzer, scanning wavelength range 190-700nm, scanning spacing 1nm. The absorbance ratio at 465nm and 665nm is recorded as E465 / E665. Fourier infrared spectrum can identify the changes of specific organic functional groups in the degradation process of potassium humate. The humic acid fermentation liquid sample to be tested is dripped onto the window of the liquid pool, and the liquid layer thickness is generally selected to be 0.01-1mm. After the liquid film is formed, the sample can be tested on the machine. Sample at 4000cm -1 -400cm -1 FTIR analysis was performed on the area.

[0070] a) Organic element analysis

[0071] Table 2 shows the elemental composition and atomic ratio of lignite humic acid and biochemical humic acid. The element contents of C, H, O, and N in the degraded biochemical humic acid are 32.36%, 3.52%, 36.49%, and 0.85%, respectively. Compared with lignite humic acid, the C content decreased by 3.09%, the H content increased by 0.29%, the O content increased by 2.22%, and the N content increased by 0.02%. The increase in H and O content can be used to determine that oxidation and hydrolysis reactions may be involved in the biodegradation process. The H / C and O / C of biochemical humic acid are higher than those of lignite humic acid, indicating that the nitrogen and oxygen-containing functional groups in the molecule have increased. It can be seen that the molecular structure of lignite humic acid has changed after being used by Streptomyces rocheri.

[0072] Table 2

[0073]

[0074] b) UV-Vis spectroscopy analysis

[0075] After 7 days of degradation of lignite humic acid by Streptomyces L1, UV-visible spectroscopy was performed in the wavelength range of 190nm-700nm. Figure 3 (Left) It can be seen that the blank control and the experimental treatment group both have a strong absorption peak at a wavelength of 210nm, and the absorbance of the control group is greater than that of the experimental group. The absorption peak at 210nm is the characteristic peak of the aromatic ring. The change in absorbance at this wavelength is caused by the π→π* transition of electrons in the π-π conjugated structure of the molecule.

[0076] Humic acid has specific absorption peaks at wavelengths of 465nm and 665nm. The absorbance at 465nm is recorded as E4, and the absorbance at 465nm is recorded as E6. E4 / E6 is usually used as an important indicator of the condensation degree and aromatization degree of humic acid. This value is closely related to the molecular size or condensation degree of humic acid. The lower the E4 / E6 value, the higher the condensation degree and aromatization degree of the humic acid molecule, and the larger the molecular weight. In this study, the E4 / E6 value of biochemical humic acid was 3.17, and the E4 / E6 value of lignite humic acid was 4.25. The results show that under the catalysis of Streptomyces rocheri, long-chain lignite humic acid continuously generates a large amount of small-molecule biochemical humic acid, and the condensation degree and aromatization degree of lignite humic acid are reduced.

[0077] c) Fourier transform infrared spectroscopy

[0078] Fourier transform infrared spectroscopy analysis of lignite humic acid fermentation broth after degradation by Streptomyces L1 and blank control without added strains Figure 3 (right) The main infrared absorption band in biosolidified coal is attributed to hydroxyl stretching (3000–3450 cm -1), carboxyl (3300–2500cm -1 ), cycloalkanes (2925cm -1 ), carbonyl (1600cm-1600cm -1 ), ether bonds (1300–1000 cm -1 ), and side chain aromatic rings (1000–500 cm -1 The blank control and humic acid after 7 days of degradation were both at 3249 cm -1 and 1636cm -1 There is a strong absorption wave at 3249cm, and the absorbance changes only slightly. -1 The observation point of -OH is 1636cm -1 The observation point of C=O is shown in Figure 1, indicating that the -OH and C=O of the side chain of lignite humic acid after degradation are slightly affected by Streptomyces L1. 1000–500 cm -1 The absorption waves in the wavelength range are caused by the vibration of CH and CO bonds of the side chain aromatic rings. Lignite humic acid and biochemical humic acid have an absorption wavelength of 589 cm -1 A strong absorption wave appeared at the wavelength, but the absorbance of lignite humic acid around this wavelength was weaker than that of biochemical humic acid, indicating that the aromatic rings in the lignite humic acid solution were less than those in the biochemical humic acid fermentation liquid after degradation. This shows that during the biodissolution of lignite humic acid, Streptomyces L1 has the ability to decompose the side chains of the aromatic rings, causing the cross-linking agent in the lignite humic acid to disappear, and finally releasing these aromatic substances in the humic acid into the fermentation liquid.

[0079] (2) Gas chromatography-mass spectrometry (GC-MS) analysis

[0080] In order to identify the products of humic acid degradation, gas chromatography (GC) was used to separate the components, and then the components were identified by mass spectrometry (MS). The degradation products in the fermentation broth were extracted with an equal amount of ethyl acetate, and the combined organic phase was evaporated by rotary evaporator under vacuum at 45 ° C to reduce the volume in the evaporating bottle to 1 ml, and then the concentrate was diluted with 10 ml of ethyl acetate and analyzed by GC-MS. GCMS analysis was performed using Agilent 7890A, 6890N, Thermo Trace GC Ultra (GC)-Thermo DSQ II (MS), equipped with a DB-5MS column (film thickness 30ml×0.25mm ID×0.25μm, Permenex, Inc., Torrance, CA, USA). The mass spectrometer was operated in electron impact ionization mode with an electron energy of 70eV. The injection volume was 5μL, and the sample was injected in a splitless manner, with helium as the carrier, and the flow rate was maintained at 1.5ml / min. The column was heated from 120°C to 280°C at a rate of 10°C / min and maintained for 30 min. The temperatures of the injector and analyzer (detector) were set to 270°C and 280°C, respectively. The mass spectrometer scan range was 50-1000 m / z, and the injection volume was 1 μL. Metabolites were confirmed and searched using the GC-MS library (NIST2014).

[0081] GC-MS detected the decomposition metabolites in the lignite humic acid fermentation broth such as Figure 4 (A) As shown. More than 35 components were detected in the mass spectrum, of which 19 had relatively high abundance, including aromatic compounds and aliphatic compounds such as phenols, ethers, esters, ketones, alcohols, acids, phenols, and alkanes. The molecular formulas of these compounds are between C6-C35, which is much smaller than the typical lignite humic acid structure (C661).

[0082] The results of GC-MS identification analysis are consistent with the results of UV-Vis and FT-IR analysis. The results of UV-visible spectroscopy analysis show that the condensation degree and aromatization degree of lignite humic acid are reduced under the action of Streptomyces L1. The results of FT-IR analysis show that the aromatic ring side chain structure of lignite humic acid is decomposed by Streptomyces L1, so that the aromatic substances are released into the fermentation broth. In the GC-MS identification results, unsaturated aromatic compounds and derivatives such as aromatic phenols, aromatic esters and aromatic ethers are also identified, and aromatic esters occupy a large proportion. At the same time, GC-MS also identified some low molecular weight compounds such as 3-methyl-4-oxopentanoic acid, which also occupies a large proportion in the fermentation broth, which is consistent with the conclusion that the condensation degree of lignite humic acid is reduced in UV-Vis.

[0083] In addition, some secondary metabolites were detected from the fermentation broth of Streptomyces L1 Figure 4(B), including antibiotics and growth hormones. In particular, the present application also discovered an important antibiotic, Borrelidin (C 28 H 43 NO6), which was first isolated from the metabolites of Salmonella rosenbergii by Berger et al. in 1949. Borrelidin has a broad spectrum of antibacterial and antifungal activities. Carotenoids (C 40 H 56 ) and sphenococcin (C 18 H 26 N 10 O3). Carotenoids increase the photosynthetic capacity and antioxidant capacity of plants, promote the production of ABA (abscisic acid), and regulate the development of plant roots and stems. These antibiotics can be very good at controlling plant pathogens, including fungi and pests.

[0084] Example 6 Application of humic acid fermentation liquid in agricultural production

[0085] Germination and field experiments were conducted to analyze the effects of HA degradation on crop growth and yield. Corn seeds (Jinkeyu 3306) were sown in plastic pots under greenhouse controlled conditions for about 10 days. There was one control group and three treatment groups, and each experimental group was repeated three times. The control group was given 20 mL of sterile water every day, and the treatment groups were added with 20 mL of HAs solution without L1 (I), 20 mL of L1 fermentation broth in MNGA medium without HAs (II), and 20 mL of L1 fermentation broth in MSM medium with 5 g / L HAs as the sole carbon source (III). After 10 days, the root and stem lengths of corn seedlings were measured. Subsequently, the effects of HA degradation on corn yield were verified by field application experiments. The experimental group design for field application was the same as that for seedling germination experiments. The area of ​​each experimental group was 0.06 ha, and the application rate of fermentation broth in each treatment group was 330 L / ha. Fertilization was applied once during the seedling stage and once during the fruiting period. The kiwifruit variety tested is "Cui Xiang", which is 10 years old, with an annual yield of 1,500 kg / mu and uniform and strong tree growth. The experiment is designed with two treatments. Treatment 1 is the control group: 40 kg / mu of nitrogen, phosphorus and potassium (15-15-15) compound fertilizer and 6 L / mu of lignite humic acid solution are applied during the young fruit stage, and 48 kg / mu of nitrogen, phosphorus and potassium (20-10-20) compound fertilizer and 10 L / mu of lignite humic acid solution are applied during the fruit expansion stage. Treatment 2 is the experimental group: 40 kg / mu of nitrogen, phosphorus and potassium (15-15-15) compound fertilizer and 6 L / mu of humic acid fermentation liquid are applied during the young fruit stage, and 48 kg / mu of nitrogen, phosphorus and potassium (20-10-20) compound fertilizer and 10 L / mu of humic acid fermentation liquid are applied during the fruit expansion stage. The demonstration area is 6.7 mu, the row spacing is 2m×2m, and a total of 754 plants are planted. The demonstration area covers 3 mu and the control area covers 2 mu. Every 15 trees were taken as one replicate treatment and then divided into 3 groups. The average value of each group was taken to calculate the yield. Other field management measures were the same as those in the field.

[0086] All statistical analyses were performed using GraphPad Prism statistical software (GraphPad Software, La Jolla, CA) and mean ± SD. Data were analyzed by one-way analysis of variance (ANOVA). When one-way ANOVA showed significant differences (P < 0.05), t-test analysis was used.

[0087] The length of roots and stems of 10-day-old maize seedlings in the control group and each treatment group was examined. Compared with the roots (8.86 cm) and stems (10.37 cm) of the control group, the roots (21.56 cm) and stems (20.48 cm) after application of HA fermentation liquid (Ⅲ) increased significantly, and the roots (13.83 cm) and stems (13.54 cm) also showed a certain increase after application of L1 fermentation liquid (Ⅱ) ( Figure 5A). However, the root length (10.23 cm) and the aboveground length (11.51 cm) of the HAs solution without S. rochei L1 (Ⅰ) did not change significantly compared with the control group. The yield of the control group was 8,359 kg / ha. Compared with the control group, the yield of the treatment group increased by 68 kg / ha, 136 kg / ha and 381 kg / ha, with an increase of 0.4%, 1.5% and 4.0% respectively ( Figure 5 B). The results of variance analysis showed that the yield difference between treatment II and the control group was significant (P<0.05), and the yield difference between treatment III and the control group was extremely significant (P<0.01). However, there was no significant difference in yield between treatment I and the control group. The above results show that the degradation of HAs can promote the growth of corn rhizomes and has a significant effect on increasing corn yield. The growth characteristics and yield of corn indicate that when HAs are biodegraded into small molecular compounds, they are more easily absorbed and utilized by corn. According to the results of transcriptome analysis, genes related to oxidoreductase synthesis and TCA process were significantly upregulated. The results show that after releasing aromatic substances into the fermentation broth, Streptomyces L1 uses them as a carbon source for growth and reproduction, which may reduce the toxicity associated with the accumulation of aromatic substances.

[0088] When lignite humic acid fermentation liquid was used as a new type of biological fertilizer and chemical fertilizer in proportion as a growth agent for kiwifruit trees, a detailed investigation and record of the growth status of kiwifruit trees during their growth period was conducted. The yield results of the five groups of kiwifruit were statistically analyzed ( Figure 6 ), the average yield of the control group was 11.2kg / plant to 13.2kg / plant, and the average yield of the treatment group was 14.3kg / plant to 15.7kg / plant. In the demonstration field, the number of fruits per plant increased by 21, the weight of each fruit increased by 5.5g, and the average yield per plant increased by 3kg. Compared with the control field, the yield increased by 326.7kg, and the yield increase rate was 24.7%, reaching an extremely significant level. Compared with treatment 1, the average leaf weight of treatment 2 increased by 25.7g, the diseased plant rate decreased by 4.2%, the number of fruits per plant increased by 5.5g, and the average yield per plant in the plot increased by 3kg. There was a difference in yield between the treatment group applied with lignite humic acid fermentation liquid and the control group not applied with lignite humic acid fermentation liquid, among which the yield difference of Ⅰ to Ⅳ was extremely significant (p<0.01), and the difference of group V was extremely significant (p<0.001). This shows that the biochemical humic acid produced after degradation by Streptomyces rocheri has a significant effect on increasing kiwifruit yield and plays a positive role in promoting agricultural production.

[0089] Therefore, the above experiments can prove that biochemical humic acid is more easily absorbed and utilized by crops, which increases the weight of crops and enhances the photosynthesis of leaves. It also improves the resistance of crops to diseases and pests, and ultimately effectively improves the yield and quality of crops.

[0090] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A Streptomyces rochei, characterized in that The deposit number of the Streptomyces rocheri is CGMCC28006.

2. A composition, characterized in that The composition contains the Streptomyces rocherii and / or its fermentation product as claimed in claim 1.

3. Use of the Streptomyces rocherii described in claim 1 or the composition described in claim 2 in degrading humic acid.

4. The use according to claim 3, wherein: The humic acid is derived from lignite.

5. Use of the Streptomyces rocherii described in claim 1 or the composition described in claim 2 in the preparation of biological fertilizer.

6. A method for preparing a biological fertilizer, characterized in that: The method comprises: fermenting the Streptomyces rocherii described in claim 1 in a culture medium containing humic acid to obtain the biological fertilizer.

7. The method according to claim 6, wherein: The humic acid is derived from lignite.

8. The biological fertilizer prepared by the method according to claim 6 or 7.

9. A fertilizer composition, characterized in that The fertilizer composition contains the biological fertilizer according to claim 8.

10. Use of the Streptomyces rocherii described in claim 1, the composition described in claim 2, the biological fertilizer described in claim 8, or the fertilizer composition described in claim 9 in increasing crop yield, improving crop disease resistance, and improving soil compaction.

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