A kind of Paecilomyces lilacinus, bacterial agent and preparation method thereof

Abstract

The invention discloses a Paecilomyces lilacinus, a bacterial agent and a preparation method thereof, wherein the Paecilomyces lilacinus PM0327-3 has a preservation number of GDMCC No: 62375. The Paecilomyces lilacinus PM0327-3 has the ability of resistance to metabolite antagonism, high temperature resistance, acid and alkali resistance, and has good application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial fertilizers, and in particular relates to a Paecilomyces lilacinus, a bacterial agent and a preparation method thereof. Background Art

[0002] The technology of using natural microorganisms to prepare microbial agents and fertilizers is a green and environmentally friendly biological treatment technology that has developed rapidly internationally in the middle and late last century. Through the comprehensive application of multiple disciplines, it uses mixed microbial communities to decompose multiphase organic matter in a specific environment, and ferments organic solid waste into humic acid. This technology is used to fertilize and improve soil, and is economical, practical, and does not produce secondary pollution, so it has received widespread attention. However, due to the seemingly simple preparation of microbial agents and fertilizers, individual farmers or small and micro-enterprises in China are currently mass-producing them, and generally, in the production process, microbial strains and raw materials are not screened or conditioned, and they are directly piled up for fermentation. As a result, the fermentation cycle is long, the production efficiency is low, the number of miscellaneous bacteria is large, and even a large number of harmful pathogens are included. The target beneficial bacteria are unclear and the target beneficial bacteria have a low survival rate during use or on the shelf, which seriously affects the quality of the product and the safety of users.

[0003] Paecilomyces lilacinus is a common and effective parasite in the soil. It has control effects on a variety of nematodes, including root-knot nematodes, cyst nematodes, golden nematodes, and heterodermal nematodes. It is the most promising biocontrol agent with the characteristics of high efficiency, broad spectrum, long-term, safety, no pollution, and no residue. Applying Paecilomyces lilacinus around the root system of plants can promote the growth of plant roots and plant nutritional organs, while having a good inhibitory and killing effect on nematodes in the soil and improving the disease resistance of crops. Therefore, developing it into a safe and environmentally friendly microbial agent and fertilizer has broad application prospects and good economic and social benefits. However, the current Paecilomyces lilacinus agent has the disadvantages of long fermentation cycle, short shelf life, and easy antagonism when used in combination with other strains.

[0004] In addition, the prior art lacks a simple and effective method for screening agricultural beneficial bacteria with high activity and strong anti-antagonistic effects. Summary of the invention

[0005] In order to solve the defects of the prior art such as long fermentation cycle of bacterial agents, low production efficiency, large number of miscellaneous bacteria, low shelf survival rate, etc., the present invention provides a Paecilomyces lilacinus, a bacterial agent thereof and a preparation method thereof.

[0006] A first aspect of the present invention provides a Paecilomyces lilacinus PM0327-3, wherein the deposit number of the Paecilomyces lilacinus PM0327-3 is GDMCC No:62375.

[0007] The (Paecilomyces lilacinus) PM0327-3 has the ability of resistance to metabolite antagonism, high temperature resistance, acid and alkali resistance, and has good application prospects.

[0008] The second aspect of the present invention provides a fermentation product obtained by fermenting the Paecilomyces lilacinus PM0327-3 as described in the first aspect of the present invention.

[0009] The third aspect of the present invention provides the use of Paecilomyces lilacinus PM0327-3 as described in the first aspect of the present invention or the fermentation product as described in the second aspect of the present invention in a microbial fertilizer.

[0010] The fourth aspect of the present invention provides a microbial fertilizer, which comprises the Paecilomyces lilacinus PM0327-3 described in the first aspect of the present invention and / or the fermentation product described in the second aspect of the present invention.

[0011] Optionally, the microbial fertilizer includes any one of a microbial inoculant (bacterial agent), a compound microbial fertilizer and a biological organic fertilizer.

[0012] The fifth aspect of the present invention provides a method for preparing the microbial fertilizer according to the fourth aspect of the present invention, comprising the following steps:

[0013] Paecilomyces lilacinus PM0327-3 seed liquid is added and deep fermentation is performed to obtain a fermentation liquid, and the fermentation liquid is further subjected to solid-state fermentation and drying to obtain the microbial fertilizer.

[0014] A sixth aspect of the present invention provides a metabolite concentrate mixture, which consists of a filtrate concentrate of a submerged fermentation broth of Paecilomyces lilacinus, a filtrate concentrate of a submerged fermentation broth of Bacillus licheniformis, and a filtrate concentrate of a submerged fermentation broth of Bacillus subtilis.

[0015] The present application does not limit Paecilomyces lilacinus, Bacillus licheniformis, and Bacillus subtilis. In some preferred embodiments, the Paecilomyces lilacinus is Paecilomyces lilacinus CICC 40276; the Bacillus licheniformis is Bacillus licheniformis CICC 10037; and the Bacillus subtilis is Bacillus subtilis CMCC 63501.

[0016] Optionally, the submerged fermentation broth of Paecilomyces lilacinus is a fermentation broth of Paecilomyces lilacinus when the number of viable cells is ≥ 1.5 billion CFU / ml;

[0017] The Bacillus licheniformis deep fermentation broth is a fermentation broth obtained by deep fermentation of Bacillus licheniformis until the viable count is ≥ 6 billion CFU / ml;

[0018] The Bacillus subtilis deep fermentation broth is a fermentation broth obtained when the Bacillus subtilis is deep fermented to a viable bacterial count of ≥7.5 billion CFU / ml.

[0019] Optionally, the filtrate concentrate of the submerged fermentation broth of Paecilomyces lilacinus is a liquid in which the filtrate of the fermentation broth of Paecilomyces lilacinus is concentrated more than 3 times;

[0020] The filtrate concentrate of the Bacillus licheniformis deep fermentation broth is a liquid obtained by concentrating the filtrate of the Bacillus licheniformis fermentation broth more than three times;

[0021] The filtrate concentrate of the Bacillus subtilis deep fermentation broth is a liquid obtained by concentrating the filtrate of the Bacillus subtilis fermentation broth more than three times.

[0022] The seventh aspect of the present invention provides a method for preparing the metabolite concentrate mixture according to the sixth aspect of the present invention, the preparation method comprising the following steps:

[0023] The submerged fermentation liquid of Paecilomyces lilacinus is obtained by submerged fermentation, filtered, and the filtrate is concentrated;

[0024] Submerged fermentation to obtain Bacillus subtilis submerged fermentation liquid, filtering, and concentrating the filtrate;

[0025] Submerged fermentation to obtain Bacillus licheniformis submerged fermentation liquid, filtering, and concentrating the filtrate;

[0026] The three concentrated solutions obtained by concentrating the filtrate are mixed to obtain the metabolite concentrated solution mixture.

[0027] The eighth aspect of the present invention provides the use of the metabolite concentrate mixture as described in the sixth aspect of the present invention or the metabolite concentrate mixture obtained by the method according to the seventh aspect of the present invention in screening metabolite-resistant antagonistic strains, or in preparing a product for screening metabolite-resistant antagonistic strains.

[0028] The ninth aspect of the present invention provides a method for selecting a strain, wherein the selection method uses the metabolite concentrate mixture as described in the sixth aspect of the present invention or the metabolite concentrate mixture obtained by the method according to the seventh aspect of the present invention for selection.

[0029] Optionally, the breeding method comprises the following steps:

[0030] (1) The starting strain is mixed with the metabolite concentrate mixture, treated at 35-70°C for 5-40 min, and plated to screen for strains that are resistant to metabolite antagonism and temperature.

[0031] Optionally, the breeding method further comprises the following steps:

[0032] (2) The strains resistant to metabolite antagonism and temperature-resistant are further screened on an acid-resistant medium and / or an alkali-resistant medium to obtain strains resistant to metabolite antagonism, temperature-resistant, and acid-resistant and / or alkali-resistant.

[0033] Optionally, the starting strain is selected from any one of Paecilomyces lilacinus, Bacillus licheniformis and Bacillus subtilis.

[0034] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention. Unless otherwise specified, the reagents and raw materials used in the present invention are commercially available.

[0035] The positive and progressive effects of the present invention are:

[0036] 1. The Paecilomyces lilacinus strain PM0327-3 provided in the present application is not antagonized by the fermentation metabolites of Paecilomyces lilacinus, Bacillus subtilis, and Bacillus licheniformis and is resistant to temperature, acid, and alkali.

[0037] 2. The Paecilomyces lilacinus PM0327-3 provided in the present application has high production activity, and the number of colonies produced by submerged fermentation + solid-state fermentation is more than 40% higher than that of the control strain Paecilomyces lilacinus CICC40276.

[0038] 3. The Paecilomyces lilacinus PM0327-3 provided in the present application has a high recovery rate, and the survival rate (recovery rate) after one year of storage under commercial shelf conditions is nearly 20% higher than that of the control.

[0039] 4. The Paecilomyces lilacinus PM0327-3 provided in the present application is not antagonistic to the mixture of metabolites of Paecilomyces lilacinus, Bacillus subtilis and Bacillus licheniformis. When used in combination with Bacillus subtilis and / or Bacillus licheniformis, it is beneficial to form a coexisting dominant bacterial community with Bacillus subtilis and / or Bacillus licheniformis, which is beneficial to the rapid and directional creation of an artificially required dominant microecological environment that is beneficial to the growth of desired plants, hinders and inhibits the growth and reproduction of pathogenic microorganisms, decomposes and degrades chemical substances in the soil, increases soil aggregate structure, and increases soil permeability. The dominant microecological environment will produce a large amount of antibacterial and bactericidal substances, prevent and control soil-borne diseases, reduce plant diseases, promote plant growth, be green and environmentally friendly, and increase production and reduce carbon emissions.

[0040] 5. The metabolite concentrate mixture provided in the present application contains the filtrate concentrate of the fermentation broth of three bacteria, Paecilomyces lilacinus, Bacillus licheniformis and Bacillus subtilis, which can be used to breed strains resistant to metabolites of Paecilomyces lilacinus, Bacillus licheniformis and Bacillus subtilis.

[0041] 6. The strain selection method provided in the present application can select strains that are resistant to metabolite antagonism and high temperature by using a mixture of metabolite concentrates and high temperature screening. Further selection using acid-resistant and alkali-resistant culture media can improve the acid and alkali resistance of the strains.

[0042] Biomaterial Deposit Information

[0043] Paecilomyces lilacinus PM0327-3 was deposited in the Guangdong Microbial Culture Collection Center (GDMCC) on April 12, 2022. The storage address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Postal Code: 510070, the storage number is: GDMCC No: 62375, the culture name is PM0327-3, and the classification name is Paecilomyces lilacinus. DETAILED DESCRIPTION

[0044] For the purpose of the present invention, unless otherwise specified, the terms used in this application have the following meanings:

[0045] The term "submerged fermentation", also known as liquid fermentation, refers to a fermentation method in which the bacteria are inoculated into a liquid fermentation medium, and sterile air is continuously introduced and stirred.

[0046] The term "solid-state fermentation" refers to a biological reaction process of one or more microorganisms carried out in a solid matrix with a certain humidity in the absence of or very little free water.

[0047] The term "microbial fertilizer" refers to products containing specific living microorganisms, which are used in agricultural production. Through the life activities of the microorganisms contained therein, they increase the supply of plant nutrients or promote plant growth, increase yields, and improve the quality of agricultural products and the agricultural ecological environment. Currently, microbial fertilizers include microbial inoculants (bacterial agents), compound microbial fertilizers and biological organic fertilizers.

[0048] Among them, microbial inoculants (microbial agents) refer to live bacterial products made by directly using one or more target microorganisms after industrial production and proliferation, or by concentrating, drying or adsorbing on a carrier, including:

[0049] Single inoculant: a microbial inoculant made from one microbial species;

[0050] Composite inoculant: a microbial inoculant made from two or more microbial strains that are not mutually antagonistic;

[0051] Organic material decomposition agent: a microbial inoculant that can accelerate the decomposition and decomposition of various organic materials (including crop straw, livestock and poultry manure, domestic garbage and urban sludge, etc.);

[0052] Bioremediation agent: a microbial inoculant that can reduce the concentration, toxicity or render harmless harmful substances in the environment through the growth and metabolic activities of microorganisms.

[0053] Compound microbial fertilizer refers to a live bacterial product made by combining the target microorganisms with nutrients after industrial production and proliferation.

[0054] Bio-organic fertilizer refers to a live bacterial product made by multiplying the target microorganisms through industrial production and compounding them with organic materials mainly derived from animal and plant residues (such as livestock and poultry manure, crop straw, etc.) and treated harmlessly.

[0055] The term "CFU" refers to colony forming units (CFU), which refers to each colony formed after incubation at a certain temperature and time on an agar plate.

[0056] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples.

[0057] In one embodiment, in the bacterial agent provided by the present invention, the content of Paecilomyces lilacinus PM0327-3 is ≥12 billion CFU / g, for example, 12.5-18 billion CFU / g, preferably 13.5-15 billion CFU / g.

[0058] In one embodiment, the preparation method of the bacterial agent provided by the present invention comprises the following steps: adding Paecilomyces lilacinus PM0327-3 seed liquid and performing deep fermentation to obtain a fermentation liquid, and the fermentation liquid is further subjected to solid-state fermentation and drying to obtain the bacterial agent.

[0059] Optionally, the culture medium used in submerged fermentation (i.e., liquid fermentation) includes the following components:

[0060]

[0061]

[0062] pH 6.0-7.0.

[0063] Optionally, the conditions of the submerged fermentation (i.e., liquid fermentation) meet at least one of the following conditions:

[0064] The inoculation rate of seed liquid is 5-10%;

[0065] The number of viable bacteria in the seed solution is ≥ 1 billion CFU / ml, for example, 1-2 billion CFU / ml;

[0066] The culture temperature is 25-32°C;

[0067] The culture period is 120-160 hours;

[0068] The ventilation volume is (0.8-1.2):1v / vm;

[0069] Stirring speed 200-500rpm.

[0070] Optionally, the solid-state fermentation is carried out in a solid-state fermentation medium, and the solid-state fermentation medium comprises the following components:

[0071]

[0072] Optionally, the solid-state fermentation conditions satisfy at least one of the following:

[0073] The inoculation rate is 5-10%;

[0074] First, culture at a temperature of 25-32° C. and a relative humidity of 80-95% for 4-6 days; then adjust the temperature to 32-35° C. and culture for 3-5 days.

[0075] Optionally, the drying conditions are:

[0076] Air supply temperature: 25-32℃;

[0077] Relative humidity: ≤35%, for example 10-20%;

[0078] The air flow drying time is 5-7 days.

[0079] In one embodiment, the metabolite concentrate mixture provided by the present invention includes the filtrate concentrate of the submerged fermentation broth of Paecilomyces lilacinus, the filtrate concentrate of the submerged fermentation broth of Bacillus licheniformis, and the filtrate concentrate of the submerged fermentation broth of Bacillus subtilis.

[0080] In some preferred embodiments, the Bacillus licheniformis is Bacillus licheniformis CICC 10037; the Paecilomyces lilacinus is Paecilomyces lilacinus CICC 40276; and the Bacillus subtilis is Bacillus subtilis CMCC63501.

[0081] Optionally, the submerged fermentation broth of Paecilomyces lilacinus is a fermentation broth when the number of viable bacteria is ≥1.5 billion CFU / ml; preferably, the fermentation broth when the number of viable bacteria is ≥2 billion CFU / ml, or the fermentation broth when the number of viable bacteria is ≥3 billion CFU / ml, the fermentation broth when the number of viable bacteria is 1.5-6 billion CFU / ml, the fermentation broth when the number of viable bacteria is 2-5 billion CFU / ml, or the fermentation broth when the number of viable bacteria is 3-5 billion CFU / ml.

[0082] Optionally, the Bacillus licheniformis deep fermentation broth is a fermentation broth in which Bacillus licheniformis is deep fermented to a viable count of ≥6 billion CFU / ml; preferably a fermentation broth in which the viable count is ≥9 billion CFU / ml or a fermentation broth in which the viable count is ≥12 billion CFU / ml; preferably a fermentation broth in which the viable count is 6-18 billion CFU / ml, a fermentation broth in which the viable count is 9-15 billion CFU / ml or a fermentation broth in which the viable count is 12-15 billion CFU / ml.

[0083] Optionally, the Bacillus subtilis submerged fermentation broth is a fermentation broth in which Bacillus subtilis is submerged and fermented to a viable count of ≥7.5 billion CFU / ml; preferably, a fermentation broth with a viable count of ≥10 billion CFU / ml, a fermentation broth with a viable count of ≥12 billion CFU / ml, or a fermentation broth with a viable count of ≥15 billion CFU / ml; preferably, a fermentation broth with a viable count of 7.5-20 billion CFU / ml, a fermentation broth with a viable count of 12-18 billion CFU / ml, or a fermentation broth with a viable count of 15-18 billion CFU / ml.

[0084] Optionally, the filtrate concentrate of the submerged fermentation broth of Paecilomyces lilacinus is a liquid in which the filtrate of the fermentation broth of Paecilomyces lilacinus is concentrated more than 3 times, preferably more than 4 times or more than 6 times; preferably 3-10 times, 4-8 times, 4-9 times or 5-8 times.

[0085] Optionally, the filtrate concentrate of the Bacillus licheniformis deep fermentation broth is a liquid in which the filtrate of the Bacillus licheniformis fermentation broth is concentrated more than 3 times, preferably more than 4 times or more than 6 times; preferably 3-10 times, 4-8 times, 4-9 times or 5-8 times.

[0086] Optionally, the filtrate concentrate of the Bacillus subtilis deep fermentation broth is a liquid in which the filtrate of the Bacillus subtilis fermentation broth is concentrated more than 3 times, preferably more than 4 times or more than 6 times; preferably 3-10 times, 4-8 times, 4-9 times or 5-8 times.

[0087] In one embodiment, the strain selection method provided in the present application comprises the following steps:

[0088] (1) The starting strain is mixed with the metabolite concentrate mixture, treated at 35-70°C for 5-40 min, and plated to screen for strains that are resistant to metabolite antagonism and heat resistance.

[0089] Optionally, the breeding method further comprises the following steps:

[0090] (2) The strains resistant to metabolite antagonism and heat resistance obtained by screening are further screened on an acid-resistant medium and / or an alkali-resistant medium to obtain strains resistant to metabolite antagonism, heat resistance, acid resistance and / or alkali resistance.

[0091] Optionally, the pH of the acid-resistant culture is 3-6; preferably the pH is 4-5.

[0092] The pH value of the alkali-resistant culture medium is 8-11, preferably 9-10.

[0093] The following is combined with the specific operating method of the present invention to illustrate the pressure selection and screening of the acid- and alkali-resistant Paecilomyces lilacinus strain that is not antagonized by the fermentation metabolites of the above-mentioned Bacillus subtilis and Bacillus licheniformis, and the industrial production and application of the strain, and further elaborate on the present invention. The provided embodiments are only used to further illustrate the present invention and do not constitute any limitation to the scope of the present invention.

[0094] Example 1

[0095] Metabolite concentrate mixture preparation:

[0096] 1. Preparation of the filtrate concentrate of the deep fermentation broth of Paecilomyces lilacinus:

[0097] 1.1. Production strain: Paecilomyces lilacinus CICC 40276;

[0098] 1.2. Fermentation culture in fermenter:

[0099] 1.2.1, Fermentation medium (wt%): 6.0 potatoes, 3.0 corn steep liquor, 3.0 soybean powder, 2.0 glucose, 1.5 corn flour, 1.5 corn starch, 3.0 rice flour, the balance is water, pH value 6.5.

[0100] 1.2.2. Fermentation culture conditions: inoculation volume 10%, fermentation temperature 28±1°C, fermentation cycle 150h, intermediate process flow feeding to ensure that the reducing sugar is 1.1±0.2%, rotation speed 350rpm, ventilation volume (0.8-1.2): 1v / vm, ​​control the dissolved oxygen of the fermentation liquid in the fermenter between 60-70%, control pH 6.8-7.2, and measure the viable bacteria count of the fermentation liquid at 3 billion CFU / ml.

[0101] 1.3. Preparation of fermentation broth filtrate concentrate: The fermentation broth was filtered and the filtrate was concentrated 6 times to obtain a fermentation broth filtrate concentrate, which was then stored in a refrigerator at 4-6°C.

[0102] 2. Preparation of filtrate concentrate from deep fermentation of Bacillus licheniformis:

[0103] 2.1. Production strain: Bacillus licheniformis CICC 10037;

[0104] 2.2. Fermentation culture in fermenter:

[0105] 2.2.1. Fermentation medium (wt%): 2% glucose, 1.0% corn syrup, 0.5% yeast extract, 3.0% soybean meal, 0.5% beef extract, 2.0% corn starch, 0.3% sodium chloride, 0.2% potassium dihydrogen phosphate, 0.3% dipotassium hydrogen phosphate, 0.05% magnesium sulfate, 0.15% ammonium sulfate, 0.03% manganese sulfate, 0.3% light calcium carbonate, the balance is water, and the pH is adjusted to 7.2.

[0106] 2.2.2. Fermentation culture conditions: inoculation volume 5%, fermentation temperature 32°C, cycle 84h, intermediate process flow feeding to ensure that the reducing sugar is 1.1±0.2%, rotation speed 450rpm, ventilation volume (1.0±0.2):1v / vm, ​​control the dissolved oxygen of the fermentation liquid in the fermenter to 60±5%, control the pH to 7.5±1.0, and the viable bacterial count of the fermentation liquid is 12 billion CFU / ml.

[0107] 2.3. Preparation of fermentation broth filtrate concentrate: The fermentation broth was filtered through a ceramic membrane and the filtrate was concentrated 6 times to obtain a fermentation broth filtrate concentrate, which was then stored in a refrigerator at 4-6°C.

[0108] 3. Preparation of Bacillus subtilis submerged fermentation broth filtrate concentrate:

[0109] 3.1. Production strain: Bacillus subtilis CMCC 63501;

[0110] 3.2. Fermentation culture in fermenter:

[0111] 3.2.1. Fermentation medium (wt%): 2% glucose, 1.5% soy peptone, 2.0% Angel yeast powder, 2.0% soybean meal, 2.0% beef extract, 2.0% corn starch, 0.3% sodium chloride, 0.2% potassium dihydrogen phosphate, 0.05% magnesium sulfate, 0.15% ammonium sulfate, 0.03% manganese sulfate, 0.3% light calcium carbonate, and the balance is water. The pH is adjusted to 7.2.

[0112] 3.2.2 Fermentation culture conditions: inoculation volume 5%, fermentation temperature 35°C, cycle 84h, intermediate process flow feed to ensure that reducing sugar is 1.1±0.2%, rotation speed 500rp, ventilation volume (1.0±0.2):1v / vm, ​​control the dissolved oxygen of the fermentation liquid in the fermenter to 60±5%, control pH7.5±1.0, and measure the viable bacterial count of the fermentation liquid at 15 billion CFU / ml.

[0113] 3.3. Preparation of fermentation broth filtrate concentrate: The fermentation broth was filtered through a ceramic membrane and the filtrate was concentrated 6 times to obtain a fermentation broth filtrate concentrate, which was then stored in a refrigerator at 4-6°C.

[0114] 4. Preparation of metabolite concentrate mixture:

[0115] The above-mentioned concentrated solutions of the filtrate of the fermentation broth of Bacillus subtilis, the concentrated solutions of the filtrate of the fermentation broth of Bacillus licheniformis and the concentrated solutions of the filtrate of the fermentation broth of Paecilomyces lilacinus, which were concentrated 6 times, were mixed evenly in equal proportions and filtered through a sterilizing filter to obtain a metabolite concentrated solution mixture.

[0116] Example 2

[0117] Breeding of dominant Paecilomyces lilacinus:

[0118] 1. Using Paecilomyces lilacinus CICC 40276 as the starting strain, a Paecilomyces lilacinus strain that is not antagonized by the metabolite concentrate mixture prepared in Example 1 and is temperature-resistant, acid-resistant and alkali-resistant was selected.

[0119] 1.1. Preparation of Paecilomyces lilacinus CICC 40276 slant: Prepare a PDA medium slant; inoculate the slant with Paecilomyces lilacinus CICC 40276, and culture the inoculated slant at 29° C. for 180 h.

[0120] 1.2. Preparation of Paecilomyces lilacinus CICC 40276 slant culture liquid: Take 5 ml of the above metabolite concentrate mixture, add it to the above mature Paecilomyces lilacinus slant, scrape and wash the bacterial moss, and fully disperse the bacterial moss into single colonies.

[0121] 1.3. Preparation of thermotolerant bacterial solution: Take 1 ml of the above slant bacterial solution and add 9 ml of metabolite concentrate to form a 10 -1 The diluted samples were stored in a 35°C constant temperature water bath for 30 min.

[0122] 1.4. Plate separation and breeding of strains:

[0123] 1.4.1. Plate culture medium: Prepare PDA culture medium into double-plate plates.

[0124] 1.4.2. Selection of strains tolerant to metabolite concentrate mixtures: 10 -1 Samples were taken from the dilution tubes at 0, 5, 10, 15, 20, 25 and 30 minutes, and further diluted with the metabolite concentrate mixture to obtain 10 -2 Up to 10 -9 of samples.

[0125] 1.4.3. Dilution of the mixture of tolerant metabolite concentrates: Take 10 of the above dilutions -3 Up to 10 -9 1 ml of the sample was spread on a double-plate and cultured at 29°C for 180 h.

[0126] 1.4.4. Breeding of thermotolerant and antagonistic plate colonies:

[0127]

[0128] 1.4.5. Selection of acid-resistant and alkali-resistant plate colonies:

[0129] 1.4.5.1. Select the above-mentioned antagonist-resistant and 35°C temperature-resistant for 10 minutes (dilution 10 -5 ), 15 minutes (dilution 10 -4 ) and 20 minutes (dilution 10 -4) were further selected for acid-resistant colonies and alkali-resistant colonies:

[0130] ① Acid-resistant culture medium plate: Adjust the pH of PDA culture medium to 4.5 and make a separate double-disc plate.

[0131] ② Alkali-resistant culture medium plate: Adjust the pH of the PDA culture medium to pH 9.5 and prepare a separate double-disc plate.

[0132] 1.4.5.2. Spread the three strains T10, T15 and T20 on acid-resistant medium plates and culture them at 29℃ for 180h, pick one acid-resistant strain from each and label them as ST10, ST15 and ST20. Spread the three strains ST10, ST15 and ST20 on alkali-resistant medium plates and culture them at 29℃ for 180h, pick one acid-resistant and alkali-resistant strain from each and label them as NST10, NST15 and NST20.

[0133] 2. Strains preserved in sand tubes:

[0134] The three strains of Paecilomyces lilacinus, NST10, NST15 and NST20, were made into bacterial suspensions and added to sand tubes, which were marked with the corresponding bacterial numbers and time. The sand tubes were vacuum dried, wrapped with kraft paper, placed in a desiccator and then stored in a refrigerator at 4-6°C.

[0135] Example 3

[0136] Stability test:

[0137] 1. Take one sand tube from each of the three Paecilomyces lilacinus strains NST10, NST15 and NST20 that have been stored in sand tubes for 60 days, and use the starting strain Paecilomyces lilacinus CICC 40276 as a control to perform shake flask fermentation to verify their stability.

[0138] 1.1. Shake flask seed culture medium (wt%): 15 potatoes, 5 corn steep liquor, 5 soybean powder, 3 glucose, the balance is water, pH natural.

[0139] In a 250ml Erlenmeyer flask, fill it with 25ml of the scooped agar and inoculate it. Oscillate it on a shaker at 200r / min and culture it at 28±1℃ for 4-5 days. The number of live bacteria in the liquid shake flask fermentation is about 2 billion CFU / ml.

[0140] 1.2. Shake flask fermentation medium (wt%): 8 parts potato, 4 parts corn syrup, 3 parts soybean powder, 2 parts glucose, 1 part corn flour, 2 parts corn starch, the balance being water, and the pH was adjusted to 6.5.

[0141] The first group of shake flask fermentation conditions: 40 ml shake flask fermentation medium, 5 ml metabolite concentrate mixture, inoculation volume 5 ml, shake on a shaker at 200 r / min, culture at 28±1°C for 120 h, 3 shake flasks per group.

[0142] The second group of shake flask fermentation conditions: add 45 ml shake flask fermentation medium to a 500 ml Erlenmeyer flask, inoculate 5 ml, shake on a shaker at 200 r / min, and culture at 28±1°C for 120 h, with 3 shake flasks per group.

[0143] 2. The average data of 3 shake bottles in each group are as follows:

[0144]

[0145] From the above results, it can be seen that after 60 days of storage in sand tubes, the number of shake flask culture colonies of the three strains of Paecilomyces lilacinus, NST10, NST15 and NST20, was higher than that of the starting strain CICC40276 in the control group. It can be seen that NST10, NST15 and NST20 have high stability, and their growth is almost not antagonized by the fermentation products of Bacillus subtilis, Bacillus licheniformis and Paecilomyces lilacinus, while the growth activity of the control strain CICC 40276 in the medium containing the metabolite concentrate mixture is significantly lower than that in the medium without the metabolite concentrate mixture. Among them, NST20 performed the best.

[0146] Example 4

[0147] 1. Production verification:

[0148] Take one sandy soil tube of Paecilomyces lilacinus strains NST10, NST15 and NST20 that have undergone stability tests, and use the starting strain Paecilomyces lilacinus CICC 40276 as the control strain for production verification.

[0149] 1.1. Fermentation tank culture: The culture medium is (wt%): 6 potatoes, 3 corn steep liquor, 3 soybean powder, 2 glucose, 1.5 corn flour, 1.5 corn starch, 3 rice flour, the balance is water, pH value 6.5. Seed liquid (the number of viable bacteria in the seed liquid is 1 billion CFU / ml) inoculation amount 10%, 28±1℃, culture for 120 hours, ventilation volume 1:1v / vm, ​​stirring speed 200rpm. The colony count reaches 3 billion CFU / ml.

[0150] 1.2 Solid fermentation medium (wt%): 58.0% rice, 5.0% bran, 6.0% rice bran, 3.0% soybean powder, and the balance is water. Rice is soaked in a mixture of the concentrated solution of the fermentation filtrate of the three strains heated to 60°C (metabolite concentrated solution mixture) for 1 hour, picked up and dried, and bran, soybean powder, rice bran, and water are added and mixed and stirred evenly. Sterilize.

[0151] 1.3. Cultivation conditions for solid-state fermentation: inoculation volume 10%, temperature 28±1°C, relative humidity 90±2%, culture for 5 days (add brown sugar water from the third to fifth day to keep the gauze on the culture plate moist), adjust the temperature to 33±1°C, continue to culture for 4 days, and most of the mycelium turns light purple.

[0152] 1.4. Drying air: Drying at room temperature with air flow: temperature 30±1℃, relative humidity 20%, air flow drying for 6 days. Make the moisture content of solid culture ≤5%. After drying, the solid culture is sieved through a 30-mesh sieve to obtain the sifted material, Paecilomyces lilacinus spore powder (microbial agent); the sieved residue is crushed and sieved through a 30-mesh sieve.

[0153] 1.5. Solid-state fermentation production data:

[0154] strains CICC 40276 control NST10 NST15 NST20 Colony count of sieved material (billion CFU / g) 98 130 125 138 Colony count of sieve residue (100 million CFU / g) 5.2 8.3 7.6 8.8 Moisture (%) 3.4 3.3 3.2 3.3

[0155] From the above results, it can be seen that the colony counts of the sieved and sieved residues of NST10, NST15 and NST20 are higher than those of the starting strain CICC 40276 in the control group. It can be seen that NST10, NST15 and NST20 still have high production activity under the conditions of the metabolite concentrate mixture, and the colony counts are increased by more than 27% compared with the control group CICC 40276. In addition, NST20 has the highest colony count, and the colony count has increased by more than 40% compared with the control group.

[0156] Example 5

[0157] Product shelf stability test:

[0158] 1. The products of each strain number of Paecilomyces lilacinus (sieved materials) produced in Example 4 were packaged in aluminum-plastic vacuum packaging, with 1000 g per bag, and stored at a temperature of 20° C., a relative humidity of 60%, and no direct sunlight. Data detection was performed every 3 months for a period of 12 months.

[0159] 2. Shelf stability data are as follows:

[0160]

[0161] From the above results, it can be seen that after 12 months of testing, the bacterial survival rate (i.e., recovery rate) of NST10, NST15, and NST20 was higher than that of the starting bacteria CICC 40276 in the control group, which was more than 10% higher than that of the control group. It can be seen that NST10, NST15, and NST20 have high shelf stability. Under the same conditions, the number of live bacteria in NST20 decreased the slowest, and the bacterial survival rate was nearly 20% higher than that of the control group.

[0162] The best performing Paecilomyces lilacinus NST20 strain was named Paecilomyces lilacinus PM0327-3 and sent to Guangdong Microbial Culture Collection Center (Institute of Microbiology, Guangdong Academy of Sciences) for preservation.

[0163] The above description is only a preferred embodiment of the present invention and is not intended to limit the scope of implementation of the present invention. Therefore, any changes or modifications made according to the claims and description of the present invention should fall within the scope of the patent of the present invention.

Claims

1. A species of Paecilomyces lilacinus ( Paecilomyces lilacinus ) PM0327-3, characterized in that, The Paecilomyces lilacinus ( Paecilomyces lilacinus )The deposit number of PM0327-3 is GDMCC No: 62375.

2. The Paecilomyces lilacinus according to claim 1 ( Paecilomyces lilacinus )Application of PM0327-3 in microbial fertilizer.

3. A microbial fertilizer, characterized in that: The microbial fertilizer comprises the Paecilomyces lilacinus described in claim 1 ( Paecilomyces lilacinus )PM0327-3.

4. A method for preparing the microbial fertilizer according to claim 3, characterized in that: The preparation method comprises the following steps: Add Paecilomyces lilacinus ( Paecilomyces lilacinus ) PM0327-3 seed liquid and perform deep fermentation to obtain fermentation liquid, and the fermentation liquid is further subjected to solid-state fermentation and drying to obtain the microbial fertilizer.

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