Biological organic liquid fertilizer and production method thereof

Abstract

The invention provides a biological organic liquid fertilizer and a production method thereof, relates to the technical field of microorganisms, and aims to solve the problems that an existing method for preventing and treating fusarium wilt of cucumbers mainly tends to chemical prevention and treatment, and long-term excessive use of chemical agents not only can cause drug resistance of diseases, but also can threaten human health and environment. The invention provides Penicillium oxalicum S-1 which is preserved in Institute of Microbiology, Chinese Academy of Sciences, the preservation address is Yard 1, Beichen West Road, Chaoyang District, Beijing, and the Penicillium oxalicum S-1 has a remarkable antagonistic effect on fusarium oxysporum. The biological organic liquid fertilizer prepared by effectively combining the penicillium oxalicum and the biogas slurry organic fertilizer containing humic acid can effectively prevent and treat cucumber fusarium wilt, promote cucumber growth and improve the enzyme activity of cucumber planting soil.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, in particular to a biological organic liquid fertilizer and a production method thereof. Background Art

[0002] Cucumber (Cucumis sativus L.) is an important vegetable crop, also known as cucumber. It occupies an important position in production and supply. However, with the production needs, high-density planting of cucumbers and frequent continuous cropping of land have led to an increasing incidence of cucumber wilt year by year. Cucumber wilt is a common soil-borne disease in cucumber cultivation, also known as vine cutting disease and wilt disease. In recent years, this disease has become one of the most serious fungal soil-borne diseases in cucumber production. Cucumber wilt has a serious impact on cucumber yield and is known as the "cancer" of plants.

[0003] Cucumber wilt is caused by infection by Fusarium oxysporumf.sp.cucumerinum Owen. It may occur in all growth stages of cucumber, especially in the flowering and fruiting period. It mainly harms the root and stem of the plant. The typical symptoms of the disease are plant wilt and slow growth. In the early stage of the disease, the cucumber leaves wilt and droop at noon, which seems to be a symptom of water shortage, and return to normal in the morning and evening. As time goes by, the number of wilted leaves increases, gradually extending from the root to the top, and finally spread throughout the plant, and cannot return to normal in the morning and evening until the cucumber plant wilts and dies. After the plant is infected, the color of the vascular bundle at the base of the stem turns yellow-brown, and the epidermis cracks longitudinally, and usually a small amount of amber colloid flows out. When the moisture content of the surrounding environment becomes high, a white or pink mold layer will appear at the base of the stem, which is the conidia and mycelium of the pathogen.

[0004] At present, the prevention and control methods of cucumber wilt generally adopt a comprehensive method of agricultural prevention, chemical prevention and biological prevention. Agricultural prevention is one of the commonly used methods in the integrated prevention and control of diseases. In order to reduce the occurrence of wilt, it is mainly through improving the cultivation and management conditions of crops, using reasonable supporting cultivation measures, breeding disease-resistant varieties, and reasonable rotation and intercropping. Chemical control is the most direct method for preventing and controlling cucumber wilt. With the development of technology, more advanced chemical agents are put into production. Chemical fungicides such as carbendazim, Junduqing, mancozeb, and kresoxim-methyl are currently the most commonly used and effective agents for preventing and controlling wilt. Chemical control has the advantages of rapidity and high efficiency, but it also has disadvantages such as high residue, high pollution, and easy to produce drug resistance. Biological control has the advantages of safety and environmental friendliness, but the existing technology lacks effective strains for cucumber wilt.

[0005] The application of biological organic fertilizer can not only improve soil conditions, reduce the incidence of pests and diseases, and protect the ecological environment, but is also an inevitable trend to promote sustainable agricultural development and build modern ecological agriculture. Summary of the invention

[0006] The technical problems to be solved by the present invention are:

[0007] Existing methods for the prevention and control of cucumber wilt mainly tend to rely on chemical control. Long-term excessive use of chemical agents will not only make the disease resistant, but also pose a threat to human health and the environment.

[0008] The present invention adopts the following technical solutions to solve the above technical problems:

[0009] The invention provides a Penicillium oxalicum strain, which is Penicillium oxalicum S-1, and is preserved in the Institute of Microbiology, Chinese Academy of Sciences, at Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

[0010] The invention provides a bacterial agent with the Penicillium oxalicum S-1 as an active ingredient.

[0011] The invention provides application of the Penicillium oxalicum S-1 in antagonizing Fusarium oxysporum.

[0012] The invention provides application of the Penicillium oxalicum S-1 or the Penicillium oxalicum S-1 bacterial agent in preventing and controlling cucumber wilt.

[0013] The invention provides application of the Penicillium oxalicum S-1 or the Penicillium oxalicum S-1 bacterial agent in preparing a product for preventing and controlling cucumber wilt disease.

[0014] The invention provides a biological organic liquid fertilizer which takes a liquid organic fertilizer containing humic acid as a base liquid and takes the Penicillium oxalicum S-1 as an active ingredient.

[0015] The present invention provides a method for preparing the biological organic liquid fertilizer, comprising the following steps:

[0016] Step 1, preparation of the humic acid-containing liquid organic fertilizer:

[0017] (1) Solid-liquid separation: filter the biogas slurry and remove the residue;

[0018] (2) Acidification: Adjust the pH of the filtered biogas slurry to neutral;

[0019] (3) Flocculation: Polyaluminium chloride and polyacrylamide are used as flocculants to carry out flocculation reaction to remove impurities in the biogas slurry;

[0020] (4) Separation: Use centrifugal separation to remove the flocculent material produced by the flocculation reaction;

[0021] (5) Aeration: Add biological deodorant to the centrifuged biogas slurry, and then place it in an aeration device for aeration;

[0022] (6) Mixing: adding potassium humate and potassium hydroxide to the biogas slurry for chelating reaction, centrifuging again to obtain the supernatant, and finally adding ammonium dihydrogen phosphate and urea to the supernatant to obtain a mixed solution;

[0023] (7) Filtration: Further solid-liquid separation of the mixed solution to remove solid impurities to obtain a pure, uniform liquid organic fertilizer containing humic acid;

[0024] Step 2: injecting Penicillium oxalicum S-1 into liquid organic fertilizer containing humic acid to obtain the biological organic liquid fertilizer.

[0025] Furthermore, the aeration time in step 1 (5) is 8 hours.

[0026] The invention provides a method for preventing and treating cucumber wilt disease, comprising mixing the Penicillium oxalicum S-1 bacterial agent or the biological organic liquid fertilizer with soil, and then transplanting cucumber seedlings into the mixed soil for cultivation.

[0027] Furthermore, the usage amount of the Penicillium oxalicum S-1 agent or the biological organic liquid fertilizer is 25 to 35 mL / kg of soil.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] The present invention separates and screens Penicillium oxalicum from soil, which has a strong antagonistic effect on the pathogen of cucumber wilt, and has a significant effect on preventing and controlling cucumber wilt. The present invention effectively combines Penicillium oxalicum with biogas slurry organic fertilizer containing humic acid to prepare a biological organic liquid fertilizer, which can effectively prevent and control cucumber wilt, promote cucumber growth, and increase the enzyme activity of cucumber planting soil. The present invention provides a technical reference for the green prevention and control of cucumber wilt, and also lays a foundation for the large-scale production and promotion and application of functional biological organic liquid fertilizers. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The present invention is a flow chart of preparing liquid organic fertilizer containing humic acid in an embodiment of the present invention;

[0031] Figure 2 The antibacterial effects of different strains in the embodiments of the present invention on Fusarium oxysporum;

[0032] Figure 3 The colony morphology of the S-1 antagonistic strain in the embodiment of the present invention;

[0033] Figure 4The figure is a result diagram of the number of live bacteria in different periods of the biogas slurry biological organic fertilizer in the embodiment of the present invention;

[0034] Figure 5 This is a graph showing the effects of different treatments on soil enzyme activity in an embodiment of the present invention;

[0035] Figure 6 This is a phylogenetic tree constructed using the neighbor-joining method in the examples of the present invention. DETAILED DESCRIPTION

[0036] In order to enable those skilled in the art to better understand the scheme of the present invention, exemplary implementations or embodiments of the present invention will be described below in conjunction with the accompanying drawings. Obviously, the described implementations or embodiments are only implementations or embodiments of a part of the present invention, not all of them. Based on the implementations or embodiments of the present invention, all other implementations or embodiments obtained by ordinary technicians in the field without creative work should fall within the scope of protection of the present invention.

[0037] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, specific embodiments of the present invention are described in detail below with reference to the accompanying drawings.

[0038] Example 1

[0039] Soil samples were collected from Shuikouzi Village, Siziwang Banner, Ulanqab City, Inner Mongolia (41°59′N, 111°58′E), Huhewenduergacha, Shadege Sumu, Urad Front Banner, Bayannur City, Inner Mongolia (40°81′N, 109°59′E), and Jirengaole Town, Ulannur Sumu, Xiwuzhumuqin Banner, Xilinguole League, Inner Mongolia (44°47′N, 116°95′E), and stored in a −80°C refrigerator for later use.

[0040] 1.1 Isolation of bacterial strains

[0041] Preparation of potato dextrose agar (PDA) culture medium: take 200g peeled potatoes, cut them into small pieces, put them into boiling water and cook until they can be easily crushed with a glass rod, add 20g glucose and 15-20g agar powder, use distilled water to make up to 1000mL, and sterilize them under high pressure conditions at 121℃.

[0042] Weigh 10g of soil sample and mix with 90mL of sterile water, shake on a shaker at 28℃ and 180r / min for 30min, and after the soil sample is fully suspended, take the soil suspension and dilute it 10 times continuously, and apply 100μL of each gradient dilution on the PDA plate. Repeat each treatment 3 times, and incubate the plate upside down in a 28℃ incubator for 3d. Observe the growth of the colony, pick up single colonies with obvious differences in shape, size, color, etc. with an inoculation loop, purify them by streaking on the PDA plate, and number them to obtain purified colonies S-1, S-2, S-3, S-4, and S-5.

[0043] 1.2 Screening of antagonistic bacteria

[0044] 1.2.1 Screening of strains with antagonistic effects on cucumber wilt pathogens

[0045] Taking the cucumber wilt pathogen as the target bacteria, the 9 mm diameter Fusarium oxysporum (provided by the Plant Pathology Laboratory of the College of Horticulture and Plant Protection of Inner Mongolia Agricultural University) and the purified candidate strain cakes S-1 to S-5 were symmetrically inoculated on the PDA plate, and the plate inoculated with only the cucumber wilt pathogen was used as the blank control. Three replicates were set for each treatment. The plate was inverted and cultured in a 28°C incubator for 5 days. The diameter of the inhibition zone was measured using the cross method, and the mycelium growth inhibition rate was calculated according to formula (1). The results are shown in Table 1 and Figure 2 shown.

[0046]

[0047] Table 1 Inhibition rate of different strains on Fusarium oxysporum hyphae

[0048]

[0049] Note: The data in the table are mean ± standard error. Different lowercase letters represent significant differences in the inhibition rate of different fungi against Fusarium oxysporum (P<0.05).

[0050] The results showed that strain S-1 had the best mycelium growth inhibition rate, followed by strain S-2, and the inhibition rates of the other three strains did not reach 50%.

[0051] 1.2.2 Screening of strains that inhibit spore germination of cucumber wilt pathogen

[0052] The bacterial cakes of Fusarium oxysporum and antagonistic bacteria S-1 to S-5 were inoculated into 250 mL Erlenmeyer flasks containing PDB culture medium, and cultured in a shaking incubator at 28°C and 180 r / min for 7 days. Mycelium was filtered out using sterile gauze. The spore count was determined using a hemocytometer, and the concentration of the bacterial suspension was adjusted to 10 with sterile water. 6 cfu / mL.

[0053] The spore suspension of Fusarium oxysporum was mixed with the spore suspension of two antagonistic bacteria in equal volumes, and after thorough mixing, 30 μL was dripped onto a concave glass slide, which was placed in a culture dish lined with moist filter paper and cultured at 28°C in the dark. Each treatment was repeated three times, and the spore germination inhibition rate was calculated according to formula (2). The results are shown in Table 2.

[0054]

[0055] Table 2 Inhibition rate of different strains against Fusarium oxysporum spores

[0056]

[0057]

[0058] Note: The data in the table are mean ± standard error. Different lowercase letters represent significant differences in the inhibition rate of different fungi against Fusarium oxysporum (P<0.05).

[0059] The results showed that strain S-1 had the best inhibitory effect, with an inhibition rate of 81.30%, followed by strain S-2, with an inhibition rate of 54.23%. The inhibition rates of other tested strains on the spore germination of Fusarium oxysporum ranged from 5.79% to 25.59%.

[0060] The S-1 strain of the present invention is isolated from Shuikouzi Village, Siziwang Banner, Ulanqab City, Inner Mongolia (41°59′N, 111°58′E).

[0061] 1.3 Identification of antagonistic bacteria

[0062] 1.3.1 Morphological characteristics identification

[0063] The purified antagonistic bacterial strains were inoculated on PDA plates and cultured at 28°C in the dark for 3 days. During the culture process, the growth rate, colony size, texture, color and other morphological characteristics of the strains were observed. Figure 3 As shown, the strain has velvety plaques, the colonies are flat or nearly flat, and the surface color of the colonies is white at first, and gradually deepens to dark green over time. According to the "Handbook of Fungal Identification", the colonies of Sphaeropsidales change from white to green, and the surface is dry, so the strain S-1 was preliminarily identified as Sphaeropsidales.

[0064] 1.3.2 Molecular Biological Identification

[0065] After activating the strain with PDA medium, the genomic DNA of the antagonistic bacteria was extracted using the Ezup column fungal genomic DNA extraction kit. The universal fungal primers ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) were used for PCR amplification.

[0066] The 25μL PCR reaction system is: 2×PCR Taq Master Mix 12.5μL, upstream and downstream primers (10μM) 1ΜL each, DNA template 1μL, double distilled water 9.5μL.

[0067] PCR amplification conditions: pre-denaturation at 95°C for 5 min, 94°C for 30 s, 57°C for 30 s, and 72°C for 90 s for 30 cycles; after repair and extension at 72°C for 10 min, the resulting product was stored at 4°C.

[0068] The PCR amplification products were subjected to 1% agarose electrophoresis at 150V and 100mA for 20min, and then recovered and purified using the SanPrep column DNA gel recovery kit and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The obtained sequences were proofread and then compared with the GenBank nucleic acid sequence database using BLAST homology. Phylogenetic analysis was performed based on the rDNA-ITS gene sequences of each strain, and a phylogenetic tree was constructed using the NJ method (neighbor-joining method), as shown in Figure 2. Figure 6 shown.

[0069] It can be seen that strain S-1 is most closely related to Penicillium oxalicum. Combining morphological and molecular identification, it can be determined that strain S-1 is Penicillium oxalicum.

[0070] 1.4 Preservation

[0071] Penicillium oxalicum S-1 was preserved in the General Microbiology Center of China Microbiological Culture Collection Administration, with the preservation address at No. 1 Beichen West Road, Chaoyang District, Beijing. The preservation date was September 4, 2024, the preservation number was CGMCC No.41497, and the classification name was Penicillium oxalicum.

[0072] Example 2

[0073] Preparation of biological organic liquid fertilizer

[0074] The biogas slurry stock solution provided by Baotou Jiuyuan Animal Husbandry Co., Ltd. was used to detect the basic physical and chemical properties of the biogas slurry stock solution using the following method.

[0075] Nitrogen content determination method: alkaline diffusion method;

[0076] Phosphorus content determination method: Vanadium-molybdenum yellow colorimetric method;

[0077] Potassium content determination method: Tetraphenylboric acid weight method;

[0078] Organic matter determination method: potassium dichromate volumetric method;

[0079] pH value determination method: Mettler FE28 benchtop pH meter was used for determination.

[0080] The results of basic physical and chemical properties of biogas slurry are shown in Table 3.

[0081] Table 3 Basic physical and chemical indicators of biogas slurry

[0082]

[0083] The results showed that the organic matter content of the biogas slurry was only 3.74 g / L, and the content of macroelements was 1.63 g / L, which was significantly lower than the liquid product standard of water-soluble fertilizer containing humic acid (macroelements) in Table 1 (Standard "Water-soluble Fertilizer Containing Humic Acid" (NY1106-2010)). Based on the comparison and calculation of the relevant indicators in Tables 3 and 4, potassium humate, potassium hydroxide, diammonium phosphate and urea were added to the biogas slurry, and the specific application amounts are shown in Table 5.

[0084] Table 4 Liquid product standards for water-soluble fertilizers containing humic acid (macroelements)

[0085]

[0086] Table 5 Addition amount of each substance

[0087]

[0088] like Figure 1 As shown in the figure, the preparation process of liquid organic fertilizer containing humic acid is as follows:

[0089] (1) Solid-liquid separation: Filter the biogas slurry with gauze, remove the filter residue, and take the liquid part to obtain biogas slurry.

[0090] (2) Acidification: Take the separated biogas slurry and add sodium dihydrogen phosphate solution to adjust the pH of the biogas slurry to about 8;

[0091] (3) Flocculation: A CL-2 magnetic heating stirrer is used for chemical flocculation reaction. Polyaluminium chloride (PAC) and polyacrylamide (PAM) are selected as flocculants. The flocculation is carried out in two steps. First, polyaluminium chloride solution is added to the biogas slurry at 35-40°C and under stirring conditions, and then polyacrylamide solution is added to the biogas slurry until flocs are produced. The flocculant can strongly adsorb the colloidal particles in the liquid, causing the impurities in the liquid to aggregate to form floccules with a larger volume fraction, thereby removing the impurities.

[0092] (4) Separation: The flocculent substances produced by flocculation were removed by centrifugation at 24°C and 4000 r / min for 5 min.

[0093] (5) Aeration: Add biological deodorant to the centrifuged biogas slurry, and then place it in an aeration device for aeration for 8 hours. Aeration is the process of contact between liquid and gas, thereby achieving the purpose of fermentation and material exchange.

[0094] (6) Mixing: Add potassium humate and potassium hydroxide to the biogas slurry, add water to make the concentration of potassium humate and potassium hydroxide 20%, put the biogas slurry into a 60°C water bath for 1.5 hours to carry out chelating reaction, take out after the reaction is completed and cool to room temperature, centrifuge again at 24°C and 4000 r / min to obtain supernatant, and finally add ammonium dihydrogen phosphate and urea to the supernatant to obtain a mixed solution.

[0095] (7) Filtration: The mixed liquid is further subjected to solid-liquid separation to remove solid impurities and obtain pure and uniform liquid organic fertilizer containing humic acid.

[0096] The nutritional components of humic acid liquid organic fertilizer are shown in Table 6. All indicators meet the standard requirements of humic acid liquid organic fertilizer.

[0097] Table 6 Basic physical and chemical indicators of liquid organic fertilizer containing humic acid

[0098]

[0099] The selected antagonistic bacteria S-1 was injected into the liquid organic fertilizer containing humic acid to make the final concentration of the bacterial solution 10 8 cfu / mL order of magnitude, and the biological organic liquid fertilizer was obtained. The survival of functional bacteria in the biological organic liquid fertilizer was determined by the dilution coating plate method. The results showed that the number of functional live bacteria in the biological organic liquid fertilizer was 2.67×10 8 cfu / mL.

[0100] The dilution plate method was used to determine the number of viable bacteria in the biological organic liquid fertilizer at different stages. The results are as follows: Figure 4As shown in the figure, during the period of 30 days to 90 days, the number of live bacteria in the biological organic liquid fertilizer increased, and then began to gradually decrease, but within 6 months, the number of live bacteria in the biological organic liquid fertilizer remained at 10 8 It is of the same order of magnitude and has no pollutants or mildew, which meets the requirements of the "Microbial Fertilizer" (NY227-94) standard.

[0101] Example 3

[0102] Effect of biological organic liquid fertilizer on the prevention and control of cucumber wilt

[0103] 3.1 Preparation of diseased soil

[0104] The test soil was taken from the teaching farm of Inner Mongolia Agricultural University. The test soil was sieved through a 3 mm pore size sieve and placed in an oven for high temperature sterilization to obtain sterilized soil. The suspension of Fusarium oxysporum was inoculated into the sterilized soil at a rate of 50 mL / kg and the diseased soil was obtained after 7 days.

[0105] 3.2 Preparation of S-1 bacterial solution

[0106] The bacterial cake of antagonistic bacteria S-1 was inoculated into a 250 mL Erlenmeyer flask filled with PDB culture medium, and cultured in a shaking incubator at 28 °C and 180 r / min for 7 days. The mycelium was filtered out with sterile gauze to obtain S-1 bacterial solution (10 8 cfu / mL).

[0107] 3.3 Experimental design

[0108] The experiment set up 5 treatments, each treatment was repeated with 15 pots, and each pot contained 1 kg of test soil, as shown below:

[0109] Treatment I: Sterilized soil

[0110] Treatment II: Diseased soil

[0111] Treatment III: diseased soil + liquid organic fertilizer containing humic acid (30 mL)

[0112] Treatment IV: diseased soil + S-1 bacterial solution (10 8 cfu / mL, 30mL)

[0113] Treatment V: Diseased soil + biological organic liquid fertilizer

[0114] 3.4 Measurement items and methods

[0115] Use 75% alcohol to disinfect the surface of cucumber seeds for 1-2 minutes, then soak them in warm water at 50-60℃ for 10 minutes, then soak them in water at 30℃ for 4-6 hours, and place them in a 30℃ constant temperature incubator to moisturize and germinate. After the cucumber seeds germinate, they are sown in plug trays for cultivation. When the cucumber seedlings grow to the stage of two leaves and one heart, healthy cucumber seedlings with the same growth are selected and transplanted into 5 treated flower pots and placed in a greenhouse for cultivation. On the 30th day after the cucumber seedlings were transplanted, the incidence of cucumber was investigated according to the cucumber wilt disease grading standard shown in Table 7. The incidence rate, disease index and control effect were calculated according to formulas (3), (4) and (5). The results are shown in Table 8.

[0116]

[0117] Table 7 Disease classification standards for cucumber wilt

[0118]

[0119] Table 8 Control effect of biological organic liquid fertilizer on cucumber wilt

[0120]

[0121] Note: The data in the table are mean ± standard error. Different lowercase letters represent significant differences in the incidence, disease index, and control effect of different treatments (P<0.05).

[0122] The results in Table 8 show that the incidence of cucumber wilt disease was significantly lower than that of treatment II when antagonistic bacteria S-1, humic acid-containing liquid organic fertilizer or biological organic liquid fertilizer were applied alone (P < 0.05). Biological organic liquid fertilizer significantly reduced the incidence and disease index of cucumber wilt disease, among which treatment VI had the lowest incidence and disease index, which were 54.17% and 60.42 lower than those of treatment II, respectively, and the control effect could reach 76.50%.

[0123] 3.4.1 Determination of cucumber growth indicators

[0124] Cucumber growth indexes were measured 20 days and 30 days after transplanting. Plant height is the distance from the base of the cucumber stem to the top of the growth point; stem thickness is the diameter of the cucumber stem base. Leaf length (the distance from the base of the leaf to the tip of the leaf) and leaf width (the width of the upper shoulder of the leaf) were measured, and the leaf area y was calculated according to the formula y = 0.743x (x = leaf length × leaf width), where 0.743 is the correction factor for leaf area. The growth index results of cucumber 20 days after transplanting are shown in Table 9, and the growth index results of cucumber 30 days after transplanting are shown in Table 10.

[0125] Table 9 Effects of different treatments on growth indicators of cucumber transplanted 20 days

[0126]

[0127]

[0128] Note: The data in the table are mean ± standard error. Different lowercase letters represent significant differences in the growth indicators of cucumbers among different treatments (P<0.05).

[0129] As shown in Table 9, 20 days after cucumber transplanting, the plant height, stem diameter and leaf area of ​​treatment V were greater than those of other treatments, and compared with treatment II, the plant height, stem diameter and leaf area increased significantly by 24.07%, 17.69% and 53.58% respectively (P < 0.05). Then the order of plant height, stem diameter and leaf area from large to small was treatment IV > treatment I > treatment III > treatment II.

[0130] Table 10 Effects of different treatments on growth indicators of cucumber transplanted for 30 days

[0131]

[0132] Note: The data in the table are mean ± standard error. Different lowercase letters represent significant differences in the growth indicators of cucumbers among different treatments (P<0.05).

[0133] As shown in Table 10, 30 days after cucumber transplanting, the indexes of treatment II were still the lowest. The application of biological organic liquid fertilizer significantly improved the growth indexes of cucumber (P < 0.05). Among them, the plant height and stem diameter of treatment V were significantly (P < 0.05) greater than those of other treatments, and the leaf area was also greater than that of other treatments (P < 0.05).

[0134] 3.4.2 Determination of cucumber physiological indicators

[0135] Malondialdehyde (MDA) content determination

[0136] Thiobarbituric acid method was used. Weigh 0.5 g of cucumber leaves, add 5 mL of 5% TCA solution and grind. Centrifuge the grinding solution at 3000 r / min for 10 min. Take 2 mL of supernatant and mix it with 2 mL of 0.67% TBA solution. Boil it in a boiling water bath for 30 min and immediately put it in cold water. After cooling, centrifuge it again at 3000 r / min for 15 min. Take the supernatant and measure the absorbance at 450 nm, 532 nm and 600 nm. Calculate the MDA content according to formula (6).

[0137]

[0138] In the formula, A 450 , A 532 , A 600 Respectively represent the absorbance values ​​at 450nm, 532nm, and 600nm wavelengths; Vt is the total volume of the extract (mL); V s is the volume of the extract used for determination (mL); FW is the fresh weight of the sample (g).

[0139] Superoxide dismutase (SOD) activity assay

[0140] The nitroblue tetrazolium photoreduction method was used. Weigh 0.5 g of cucumber leaves and place them in a mortar. Add 2 mL of pH 7.8, 0.05 mol / L phosphate buffer. Grind in an ice bath and pour into a centrifuge tube. Make up to 10 mL. Centrifuge at 4°C and 10,000 r / min for 15 min. The supernatant is the enzyme solution to be tested. Take a test tube and add 1.5 mL of phosphate buffer, 0.3 mL of Met solution, 0.3 mL of NBT solution, 0.25 mL of distilled water, 0.3 mL of riboflavin solution, and 0.1 mL of enzyme solution. After mixing, place one control tube in a dark place as a blank. The other tubes reacted under 4000 lx light for 20 min. The absorbance was measured at a wavelength of 560 nm. The SOD activity was calculated according to formula (7).

[0141]

[0142] Wherein, A0 is the absorbance value of the control tube; As is the absorbance value of the sample measurement tube; Vt is the total volume of the sample extract (mL); Vs is the crude enzyme solution taken during the measurement (mL); t is the illumination time of the color development reaction (min); FW is the fresh weight of the sample (g).

[0143] Peroxidase (POD) activity assay

[0144] The guaiacol method was used. Weigh 0.5 g of cucumber leaves and place them in a mortar. Add an appropriate amount of distilled water, a small amount of quartz sand, and calcium carbonate and grind them in an ice bath. Centrifuge at 4°C and 5000 r / min for 15 min. The supernatant is the enzyme solution to be tested. Take a test tube and add 6.9 mL of distilled water, 1 mL of 0.18% H2O2, 1 mL of 0.1% guaiacol solution, and 1 mL of enzyme solution. Boiled enzyme solution was used as a control. After adding the enzyme solution, place it in a 37°C water bath for 10 min and then quickly transfer it to an ice bath. Add 0.2 mL of 5% metaphosphoric acid to terminate the reaction. Measure the absorbance at a wavelength of 470 nm and calculate the POD activity according to formula (8).

[0145]

[0146] Wherein, △A470 is the change of absorbance during the reaction time; Vt is the total volume of the extracted enzyme solution (mL); W is the fresh weight of the sample (g); Vs is the volume of the enzyme solution taken during the determination (mL); and t is the reaction time (min).

[0147] Catalase (CAT) activity assay

[0148] The ultraviolet absorption method was used. Weigh 0.5 g of cucumber leaves and place them in a mortar. Add 5.0 mL of pH 7.0, 0.05 mol / L phosphate buffer and grind them in an ice bath. Pour the mixture into a centrifuge tube and centrifuge at 4°C and 15,000 r / min for 15 min. The supernatant is the enzyme solution to be tested. Take a test tube and add 1 mL of pH 7.0, 0.05 mol / L Tris-Hcl solution, 0.1 mL of enzyme solution, boiled enzyme solution as a control, 1.7 mL of distilled water, and add 0.2 mL of 0.2 mol / L H2O2 after 3 min of water bath at 25°C. Measure the absorbance at a wavelength of 240 nm. Read the results every 30 seconds for a total of 3 min. Calculate the CAT activity according to formula (9).

[0149]

[0150] In the formula, A S0 A is the absorbance of the control tube; S1 , A S2 , A S3 is the absorbance of the sample tube; Vt is the total volume of the extracted enzyme solution (mL); Vs is the volume of the enzyme solution taken during the determination (mL); t is the reaction time (min); FW is the fresh weight of the sample (g).

[0151] Phenylalanine ammonia lyase (PAL) activity assay

[0152] Weigh 1g of cucumber leaves in a mortar, add 5ml of pH 8.8, 0.1mlo / L boric acid buffer (containing 5mmol / L mercaptoethanol) and 0.5g of polyvinyl pyrrolidone, grind in an ice bath, dilute to 10mL, and filter with gauze. Centrifuge at 4℃ and 10000r / min for 15min, and the supernatant is the enzyme solution to be tested. Take a test tube and add 1mL of 0.02mol / L L-phenylalanine solution, 2mL of pH8.8 boric acid buffer, and 1mL of enzyme solution. Shake well and place in a water bath at 30℃ for 1h. Finally, add 0.2mL of 6mol / L HCl solution to terminate the reaction. Measure the absorbance at a wavelength of 290nm and calculate the PAL activity according to formula (10).

[0153]

[0154] In the formula, A 290 V is the absorbance value at a wavelength of 290 nm; t is the total volume of the extracted enzyme solution (mL); V is the total volume of the reaction solution (mL); V s is the volume of enzyme solution taken during the determination (mL); FW is the fresh weight of the sample (g); t is the reaction time (h).

[0155] Polyphenol oxidase (PPO) activity assay

[0156] Weigh 0.5 g of cucumber leaves in a mortar, add 5 mL of pH 6.5, 0.05 mol / L phosphate buffer and grind in an ice bath. Centrifuge at 4°C, 10000 r / min for 10 min, and the supernatant is the enzyme solution to be tested. Take a test tube and add 0.3 mL of enzyme solution, 1 mL of 0.1 mol / L catechol solution, and 2 mL of pH 7.0, 0.05 mol / L phosphate buffer. Keep warm at 37°C for 10 min and then quickly put on ice. Finally, add 3 mL of 20% trichloroacetic acid solution to terminate the reaction. Measure the absorbance at a wavelength of 525 nm and calculate the PPO activity according to formula (11).

[0157]

[0158] In the formula, △A 525 V is the absorbance change during the reaction time; t is the total volume of the extracted enzyme solution (mL); W is the fresh weight of the sample (g); V s is the volume of enzyme solution taken during the determination; t is the reaction time (min).

[0159] The measurement results of cucumber physiological indicators are shown in Table 11.

[0160] Table 11 Effects of different treatments on malondialdehyde content and defense enzyme activity in cucumber leaves

[0161]

[0162]

[0163] Note: The data in the table are mean ± standard error. Different lowercase letters represent significant differences in malondialdehyde content and defense enzyme activity among different treatments (P<0.05).

[0164] From the results in Table 11, it can be seen that the malondialdehyde content of treatment II and treatment III is significantly higher than that of other treatments (P < 0.05). Among them, the malondialdehyde content of treatment II is the highest, which is 33.69nmol / g, and the malondialdehyde content of treatment I is the lowest, which is 20.42nmol / g, which is significantly lower than that of other treatments (P < 0.05). The malondialdehyde content of treatment V is slightly higher than that of treatment I, which is 22.63nmol / g, which is significantly lower than that of other treatments. It can be seen that the application of biological organic liquid fertilizer can significantly reduce the malondialdehyde content of diseased plants.

[0165] The activities of superoxide dismutase (SOD) and catalase (CAT) in treatment V were significantly higher than those in other treatments (P < 0.05), and their activities were 4.54 and 5.42 times of those in treatment II, respectively. The activities of peroxidase (POD), phenylalanine ammonia lyase (PAL) and polyphenol oxidase (PPO) in treatments I and V were significantly higher than those in other treatments (P < 0.05). The activities of the three enzymes in treatment II were the lowest.

[0166] 3.4.3 Determination of enzyme activity in cucumber rhizosphere soil

[0167] Soil sucrase (S-SC) activity: The activity of soil sucrase (S-SC) was determined using a soil sucrase (S-SC) activity detection kit. The result was expressed as 1 mg of reducing sugar produced per gram of soil sample per day, in units of mg / d / g.

[0168] Soil urease (S-UE) activity: The activity was determined using a soil urease (S-UE) activity detection kit, and the result was expressed as 1 mg NH3-N produced per gram of soil sample per day, in units of mg / d / g.

[0169] Soil alkaline phosphatase (S-AKP / ALP) activity: The activity was determined using a soil alkaline phosphatase (S-AKP / ALP) activity detection kit, and the result was expressed as 1 mg of phenol produced per gram of soil sample per day, in units of mg / d / g.

[0170] Soil catalase (S-CAT) activity: The activity was determined using a soil catalase (S-CAT) activity detection kit, and the result was expressed as 1 mg H2O2 catalyzed per gram of air-dried soil sample per day, in units of mg / d / g.

[0171] The results of cucumber rhizosphere soil enzyme activity are as follows Figure 5 As shown in the figure, the activities of soil sucrase, urease, alkaline phosphatase and catalase in treatment V were higher than those in other treatments. The enzyme activities were 1.60, 1.50, 1.63 and 1.39 times of those in treatment I, and 2.50, 2.61, 2.55 and 1.99 times of those in treatment II. The activities of the four soil enzymes in treatment IV, where S-1 bacteria were applied alone, were higher than those in treatment II, and all of them could enhance soil enzyme activities to a certain extent.

[0172] Although the present invention is disclosed as above, the protection scope of the present invention is not limited thereto. Those skilled in the art may make various changes and modifications without departing from the spirit and scope of the present invention, and these changes and modifications will fall within the protection scope of the present invention.

[0173] ITS Sequence

[0174]

Claims

1. A Penicillium oxalicum strain, characterized in that The strain is Penicillium oxalicum S-1, which is deposited in the Institute of Microbiology, Chinese Academy of Sciences, and the deposit address is No. 1, Beichen West Road, Chaoyang District, Beijing.

2. A bacterial agent with the Penicillium oxalicum S-1 as claimed in claim 1 as an active ingredient.

3. Use of Penicillium oxalicum S-1 according to claim 1 in antagonizing Fusarium oxysporum.

4. Use of the Penicillium oxalicum S-1 according to claim 1 or the bacterial agent according to claim 2 in preventing and treating cucumber wilt.

5. Use of the Penicillium oxalicum S-1 according to claim 1 or the bacterial agent according to claim 2 in the preparation of a product for preventing and treating cucumber wilt.

6. A biological organic liquid fertilizer with a humic acid-containing liquid organic fertilizer as a base liquid and the Penicillium oxalicum S-1 described in claim 1 as an active ingredient.

7. The method for preparing the biological organic liquid fertilizer according to claim 6, characterized in that: The steps include: Step 1, preparation of the humic acid-containing liquid organic fertilizer: (1) Solid-liquid separation: filter the biogas slurry and remove the residue; (2) Acidification: Adjust the pH of the filtered biogas slurry to neutral; (3) Flocculation: Polyaluminium chloride and polyacrylamide are used as flocculants to carry out flocculation reaction to remove impurities in the biogas slurry; (4) Separation: Use centrifugal separation to remove the flocculent material produced by the flocculation reaction; (5) Aeration: Add biological deodorant to the centrifuged biogas slurry, and then place it in an aeration device for aeration; (6) Mixing: adding potassium humate and potassium hydroxide to the biogas slurry for chelating reaction, centrifuging again to obtain the supernatant, and finally adding ammonium dihydrogen phosphate and urea to the supernatant to obtain a mixed solution; (7) Filtration: Further solid-liquid separation of the mixed solution to remove solid impurities to obtain a pure, uniform liquid organic fertilizer containing humic acid; Step 2: injecting Penicillium oxalicum S-1 into liquid organic fertilizer containing humic acid to obtain the biological organic liquid fertilizer.

8. The method for preparing the biological organic liquid fertilizer according to claim 7, characterized in that: The aeration time in step 1 (5) is 8 hours.

9. A method for preventing and treating cucumber wilt, characterized in that: The microbial agent according to claim 2 or the biological organic liquid fertilizer according to claim 6 is mixed with soil, and then the cucumber seedlings are transplanted into the mixed soil for cultivation.

10. The method for preventing and controlling cucumber wilt according to claim 9, characterized in that: The usage amount of the microbial agent described in claim 2 or the biological organic liquid fertilizer described in claim 6 is 25 to 35 mL / kg of soil.

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