Penicillium oxalicum TWSF1 and application thereof

Abstract

The invention discloses penicillium oxalicum TWSF1 and application thereof, and belongs to the technical field of agricultural microorganisms. The preservation number of the penicillium oxalicum TWSF1 is CGMCC (China General Microbiological Culture Collection Center) No.41553, and the penicillium oxalicum TWSF1 is preserved in China General Microbiological Culture Collection Center on October 25, 2024. The strain is separated from alpine soil in Branchiomega in the autonomous region of Tibet, can well grow under the condition of 4-35 DEG C, normally grow under the condition that the salt concentration is 5%, can grow under the condition that the salt concentration is 10% and normally grow under the condition that the pH is 10, and has high inorganic phosphorus dissolving capacity; the strain has better environmental adaptability in saline-alkali soil in northern cold regions in China, can effectively repair saline-alkali soil, and has higher application prospect and economic value.

Description

Technical Field

[0001] The invention relates to the technical field of agricultural microorganisms, and specifically to a strain of Penicillium oxalicum TWSF1 and an application thereof. Background Art

[0002] Saline-alkali land is a type of salt accumulation, which means that the salt contained in the soil affects the normal growth of crops. The salt content of light saline-alkali land is less than 3 / 1000; the salt content of heavy saline-alkali land exceeds 6 / 1000. The pH value of light saline-alkali land is 7.1-8.5, the pH value of moderate saline-alkali land is 8.5-9.5, and the pH value of heavy saline-alkali land is above 9.5. There are about 99.13 million hectares of saline-alkali land in my country, which are mainly divided into the saline-alkali area in the northwest inland, the saline-alkali area in the Songnen Plain in the northeast, the coastal saline-alkali area, the saline-alkali area in the Huanghuaihai Plain, and the saline-alkali area in the upper and middle reaches of the Yellow River.

[0003] At present, my country has achieved initial success in screening salt-alkali tolerant microorganisms, developing efficient microbial preparation technology, and applying microbial agents to improve saline-alkali soil. However, my country's saline-alkali areas are mainly in the northern region, with a long low temperature period. The activity of commonly used functional microorganisms decreases in the low temperature season, and their functionality is greatly reduced. The present invention screened a strain of Penicillium oxalicum TWSF1 from alpine soil, which has a wide growth temperature range and can grow under conditions of 4-35°C. It has the functions of decomposing inorganic phosphorus and salt-alkali resistance, and is more suitable for the treatment of saline-alkali land in high latitudes. Summary of the invention

[0004] The invention discloses a strain of Penicillium oxalicum TWSF1 and application thereof, so as to solve the deficiencies of the prior art.

[0005] In order to achieve the above-mentioned invention object, the technical solution adopted by the present invention is:

[0006] A strain of Penicillium oxalicum TWSF1, the Penicillium oxalicum TWSF1 (Penicillium oxalicum) was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on October 25, 2024, with the deposit number CGMCC No.41553.

[0007] The solid fermentation product of Penicillium oxalicum TWSF1 is prepared by the Penicillium oxalicum TWSF1.

[0008] The method for preparing the solid fermentation product of Penicillium oxalicum TWSF1 comprises the following steps: Step 1: inoculate Penicillium oxalicum TWSF1 into PDA solid culture medium and activate for 72 hours, and then rinse with sterile water to obtain a Penicillium oxalicum spore suspension; Step 2: Take the activated Penicillium oxalicum spore suspension in step 1 and evenly inoculate it into the fermentation solid culture medium at an inoculation rate of 1%-5%. The fermentation temperature is 20-30°C and the fermentation time is 72-120h. During the fermentation, the fermentation solid culture medium is stirred evenly every 24h. When the fermentation is completed, the Penicillium oxalicum TWSF1 solid fermentation product is obtained.

[0009] Furthermore, the fermentation solid culture medium in step 2 contains the following raw materials counted by weight: 15-30 parts of bran, 5-15 parts of bran, 5-15 parts of desaponified tea meal powder, 0.5-2.0 parts of KH2PO4, 0.5-2.0 parts of Na2HPO4, 0.01-0.1 parts of MgSO4·7H2O, 0.01-0.1 parts of CaCO3, and 50 parts of deionized water.

[0010] Furthermore, the solid fermentation product of Penicillium oxalicum TWSF1 is dried, crushed, and solid diluted to obtain a microbial agent, that is, the microbial agent, with an effective viable count of ≥0.2×10 8 cfu / g.

[0011] Furthermore, the bio-organic fertilizer or compound microbial fertilizer prepared by the solid fermentation product of Penicillium oxalicum TWSF1 through secondary solid fermentation and mixing is the microbial fertilizer.

[0012] Furthermore, the Penicillium oxalicum TWSF1 is used to dissolve inorganic phosphorus at 4-35°C.

[0013] Furthermore, the Penicillium oxalicum TWSF1 and / or the solid fermentation product of the Penicillium oxalicum TWSF1 and / or the microbial agent and / or the microbial fertilizer are used to promote rapeseed growth and reduce the pH of saline-alkali soil under the condition of 4-35°C.

[0014] Furthermore, the application scenario is application in improving saline-alkali land.

[0015] Beneficial effects of the invention: In view of the problem that saline-alkali lands in my country are mainly distributed in the northern high-latitude regions and have a long low-temperature season, and conventional functional microorganisms have low effectiveness under low-temperature conditions, the invention screens out a strain of Penicillium oxalicum TWSF1 with phosphate-solubilizing and salt-alkali tolerance functions within a large temperature range, in order to assist in the efficient restoration of saline-alkali lands. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a diagram showing the phosphorus solubilization effect of strain TWSF1;

[0017] Figure 2is the 16S rRNA phylogenetic tree of strain TWSF1;

[0018] Figure 3 The height of rapeseed plants under different treatments;

[0019] Figure 4 is the fresh weight of aboveground part of rapeseed in different treatments;

[0020] Figure 5 The available phosphorus content in soils of different treatments;

[0021] Figure 6 Soil pH values ​​for different treatments.

[0022] Biological deposit information

[0023] TWSF1, classified as Penicillium oxalicum, is deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, with the deposit address at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is October 25, 2024, and the deposit number is CGMCC NO.41553. DETAILED DESCRIPTION

[0024] The following examples are provided for better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified.

[0025] It should be noted that the details of the present invention are known to those skilled in the art. The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer, and the experimental steps not described in detail refer to pathophysiological experiments, online databases, etc.

[0026] Unless otherwise specified, the raw materials used in the following examples are as follows:

[0027] PDA solid culture medium (1L): 200g potatoes (cooked and filtered), 20g glucose, 15-20g agar, 1L deionized water, sterilized at 115℃ for 30min.

[0028] Phosphate-dissolving solid culture medium (1 L): glucose 10 g, (NH4)2SO4 0.5 g, yeast extract 0.5 g, NaCl 0.3 g, KCl 0.3 g, MgSO4·7H2O 0.3 g, FeSO4·7H2O 0.03 g, MnSO4·4H2O 0.03 g, Ca3(PO4)2 5 g, agar powder 20 g, dilute to 1 L with deionized water, sterilize at 115°C for 30 min.

[0029] Example 1

[0030] A strain of Penicillium oxalicum TWSF1, the Penicillium oxalicum TWSF1 (Penicillium oxalicum) was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on October 25, 2024, with the deposit number CGMCC No.41553.

[0031] Isolation, screening and identification of Penicillium oxalicum TWSF1:

[0032] (1) Isolation of strains

[0033] Soil samples were collected from the high-altitude mountainous areas of Qamdo, Tibet Autonomous Region. 10 g of soil sample was added to a conical flask containing 100 ml of sterile saline. After standing for 20 min, it was shaken on a shaker at 30 °C and 180 rpm for 30 min. 1 ml of the sample was added to 9 ml of sterile saline and then diluted 10 2 , 10 3 , 10 4 Times, take 100 μl of the above soil suspension and evenly spread it on the PDA solid culture medium plate, culture it in a 10℃ incubator for 48 h, pick up a single bacterial colony with a sterile toothpick, streak it, purify it, and save it for later use.

[0034] (2) Screening of functional strains

[0035] The strain stored at 4°C was inoculated into PDA solid medium and cultured at 28°C for 72h. The bacterial block was inoculated into phosphate-dissolving solid medium and cultured upside down in a 28°C incubator. After 7 days of culture, colonies with phosphate-dissolving rings and different morphologies were selected. The strain TWSF1 was tested to have the ability to dissolve inorganic phosphorus ( Figure 1 ).

[0036] The strain stored at 4°C was inoculated into PDA solid medium, cultured at 28°C for 72h, and the spore powder was scraped and inoculated into PDA liquid medium supplemented with 0%, 2%, 5%, 8%, and 10% NaCl, and fermented on a shaking table for 48h. It was found that strain TWSF1 could grow normally when the salt concentration was lower than 5%, and could still grow at a salt concentration of 10%, but was inhibited.

[0037] The strain stored at 4°C was inoculated into PDA solid medium, cultured at 28°C for 72 hours, and the spore powder was scraped and inoculated into PDA liquid medium with pH values ​​of 7.0, 8.0, 9.0, 10.0, and 11.0, and fermented on a shaking platform for 48 hours. It was found that strain TWSF1 could grow normally at a pH value below 10.0.

[0038] (3) Strain identification

[0039] After the strain TWSF1 was cultured on PDA solid medium at 30℃ for 48h, its colonies were dark green, velvety, and had dense spores. The phylogenetic tree constructed by ITS rRNA gene sequence comparison analysis showed that the strain TWSF1 had the highest homology with Penicillium oxalicum. The phylogenetic tree constructed by combining the colony morphology characteristics and ITS rRNA gene sequence ( Figure 2 ) comparison analysis results, strain TWSF1 was identified as Penicillium oxalicum.

[0040] The solid fermentation product of Penicillium oxalicum TWSF1 is prepared by Penicillium oxalicum. The preparation method of the solid fermentation product of Penicillium oxalicum TWSF1 comprises the following steps: Step 1: inoculate Penicillium oxalicum TWSF1 into PDA solid culture medium and activate for 72 hours, and then rinse with sterile water to obtain a Penicillium oxalicum spore suspension; Step 2: Take the activated oxalic acid Penicillium spore suspension in step 1 and evenly inoculate it into the fermentation solid culture medium with an inoculation amount of 1%, the fermentation temperature is 20°C, the fermentation time is 72h, and the fermentation solid culture medium is evenly stirred every 24h during the fermentation. After the fermentation is completed, the solid fermentation product of Penicillium oxalic acid TWSF1 is obtained. The fermentation solid culture medium contains the following raw materials counted by weight: 15 parts of bran, 5 parts of bran, 5 parts of desaponified tea meal powder, 0.5 parts of KH2PO4, 0.5 parts of Na2HPO4, 0.01 parts of MgSO4·7H2O, 0.01 parts of CaCO3, and 50 parts of deionized water.

[0041] The solid fermentation product of Penicillium oxalicum TWSF1 is dried, crushed, and solid diluted to obtain a microbial agent, i.e., the microbial agent, with an effective viable count of ≥ 0.2×10 8 cfu / g.

[0042] The bio-organic fertilizer or compound microbial fertilizer prepared by solid fermentation of Penicillium oxalicum TWSF1 through secondary solid fermentation and mixing is the microbial fertilizer.

[0043] The application of Penicillium oxalicum TWSF1 in dissolving inorganic phosphorus at 4° C. is in improving saline-alkali land.

[0044] The application of the Penicillium oxalicum TWSF1 and / or the solid fermentation product of the Penicillium oxalicum TWSF1 and / or the microbial agent and / or the microbial fertilizer in promoting rapeseed growth and reducing the pH of saline-alkali soil at 4°C, and the application scenario is in improving saline-alkali land.

[0045] Example 2

[0046] The content of this embodiment is basically the same as that of embodiment 1, except that the method for preparing the solid fermentation product of Penicillium oxalicum TWSF1 is prepared by Penicillium oxalicum TWSF1.

[0047] The solid fermentation product of Penicillium oxalicum TWSF1 is prepared by Penicillium oxalicum. The preparation method of the solid fermentation product of Penicillium oxalicum TWSF1 comprises the following steps: Step 1: inoculate Penicillium oxalicum TWSF1 into PDA solid culture medium and activate for 72 hours, and then rinse with sterile water to obtain a Penicillium oxalicum spore suspension; Step 2: Take the activated oxalic acid Penicillium spore suspension in step 1 and evenly inoculate it into the fermentation solid culture medium with an inoculation amount of 3%, the fermentation temperature is 25°C, the fermentation time is 96 hours, and the fermentation solid culture medium is evenly stirred every 24 hours during the fermentation. After the fermentation is completed, the solid fermentation product of Penicillium oxalic acid TWSF1 is obtained. The fermentation solid culture medium contains the following raw materials counted by weight: 23 parts of bran, 10 parts of bran, 10 parts of desaponified tea meal powder, 1.3 parts of KH2PO4, 1.3 parts of Na2HPO4, 0.05 parts of MgSO4·7H2O, 0.05 parts of CaCO3, and 50 parts of deionized water.

[0048] The solid fermentation product of Penicillium oxalicum TWSF1 is dried, crushed, and solid diluted to obtain a microbial agent, i.e., the microbial agent, with an effective viable count of ≥ 0.2×10 8 cfu / g.

[0049] The bio-organic fertilizer or compound microbial fertilizer prepared by solid fermentation of Penicillium oxalicum TWSF1 through secondary solid fermentation and mixing is the microbial fertilizer.

[0050] The application of Penicillium oxalicum TWSF1 in dissolving inorganic phosphorus at 20° C. is in improving saline-alkali land.

[0051] The application of the Penicillium oxalicum TWSF1 and / or the solid fermentation product of the Penicillium oxalicum TWSF1 and / or the microbial agent and / or the microbial fertilizer in promoting rapeseed growth and reducing the pH of saline-alkali soil at 20°C, and the application scenario is in improving saline-alkali land.

[0052] Example 3

[0053] The content of this embodiment is basically the same as that of embodiment 1, except that the method for preparing the solid fermentation product of Penicillium oxalicum TWSF1 is prepared by Penicillium oxalicum TWSF1.

[0054] The solid fermentation product of Penicillium oxalicum TWSF1 is prepared by Penicillium oxalicum. The preparation method of the solid fermentation product of Penicillium oxalicum TWSF1 comprises the following steps:

[0055] Step 1: inoculate Penicillium oxalicum TWSF1 into PDA solid culture medium and activate for 72 hours, and then rinse with sterile water to obtain a Penicillium oxalicum spore suspension;

[0056] Step 2: Take the activated oxalic acid Penicillium spore suspension in step 1 and evenly inoculate it into the fermentation solid culture medium with an inoculation amount of 5%, the fermentation temperature is 30°C, the fermentation time is 120h, and the fermentation solid culture medium is evenly stirred every 24h during the fermentation. When the fermentation is completed, the solid fermentation product of Penicillium oxalic acid TWSF1 is obtained. The fermentation solid culture medium contains the following raw materials counted by weight: 30 parts of bran, 15 parts of bran, 15 parts of desaponified tea meal powder, 2.0 parts of KH2PO4, 2.0 parts of Na2HPO4, 0.1 parts of MgSO4·7H2O, 0.1 parts of CaCO3, and 50 parts of deionized water.

[0057] The solid fermentation product of Penicillium oxalicum TWSF1 is dried, crushed, and solid diluted to obtain a microbial agent, i.e., the microbial agent, with an effective viable count of ≥ 0.2×10 8 cfu / g.

[0058] The bio-organic fertilizer or compound microbial fertilizer prepared by solid fermentation of Penicillium oxalicum TWSF1 through secondary solid fermentation and mixing is the microbial fertilizer.

[0059] The application of Penicillium oxalicum TWSF1 in dissolving inorganic phosphorus at 35° C. is in improving saline-alkali land.

[0060] The Penicillium oxalicum TWSF1 and / or the solid fermentation product of the Penicillium oxalicum TWSF1 and / or the microbial agent and / or the microbial fertilizer are used to promote rapeseed growth and reduce the pH of saline-alkali soil at 35°C, and the application scenario is to improve saline-alkali land.

[0061] The following is a test of microbial agents to reduce soil pH and promote rapeseed growth

[0062] The potted plant test site is the open-air platform of the factory area of ​​Hunan Tewos Ecological Co., Ltd. in Suining County, Hunan Province. The test time is from October 2023 to March 2024.

[0063] Sow rapeseed seeds in seedling cups, and when the rapeseed seedlings grow 3 true leaves, select rapeseed seedlings with relatively consistent growth potential and transplant them into plastic pots (one rapeseed seedling in each pot) filled with soil (the soil salt content is adjusted to 0.3% and pH 8.0 by sodium carbonate, calcium hydrogen phosphate, etc., 6 kg of soil per pot, and base fertilizer is mixed). After transplanting, water the plastic pots and water them regularly afterwards.

[0064] The rapeseed seedlings transplanted into the plastic pots were set up with four treatments, namely T1 (control treatment, clean water), T2 (addition of 0.1% organic fertilizer), T3 (addition of the TWSF1 microbial agent prepared in Example 2), and T4 (addition of the TWSF1 microbial agent prepared in Example 2 and 0.1% organic fertilizer). Each treatment was repeated 5 times. After the transplanted rapeseed seedlings grew stably for one week, the microbial agent was irrigated with water to the roots, 6 ml per pot. After completion, routine maintenance was performed. In March of the second year, rapeseed seedlings and rhizosphere soil samples were collected to measure the rapeseed plant height ( Figure 3 ), rapeseed aboveground fresh weight ( Figure 4 ), soil available phosphorus content ( Figure 5 ), soil pH( Figure 6 ), and compare the rapeseed growth conditions and soil nutrient changes of each treatment.

[0065] The experiment showed that adding microbial agents and organic fertilizers could increase the effective phosphorus content in the soil, promote rapeseed growth, and reduce soil pH. The combined application of microbial agents prepared by Penicillium oxalicum TWSF1 and microbial fertilizers had better effects.

[0066] Components not described in detail herein are prior art.

[0067] Although the specific embodiments of the present invention are described in detail above, the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in this field without departing from the purpose of the present invention, and modifications or deformations without creative labor are still within the protection scope of the present invention.

Claims

1. A strain of Penicillium oxalicum TWSF1, characterized in that: The Penicillium oxalicum TWSF1 (Penicillium oxalicum) was deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration on October 25, 2024, with the deposit number CGMCC No.41553.

2. The solid fermentation product of Penicillium oxalicum TWSF1 prepared by Penicillium oxalicum TWSF1 according to claim 1.

3. The method for preparing the solid fermentation product of Penicillium oxalicum TWSF1 according to claim 2, characterized in that: The following steps are involved: Step 1: inoculate Penicillium oxalicum TWSF1 into PDA solid culture medium and activate for 72 hours, and then rinse with sterile water to obtain a Penicillium oxalicum spore suspension; Step 2: Take the activated Penicillium oxalicum spore suspension in step 1 and evenly inoculate it into the fermentation solid culture medium at an inoculation rate of 1%-5%. The fermentation temperature is 20-30°C and the fermentation time is 72-120h. During the fermentation, the fermentation solid culture medium is stirred evenly every 24h. When the fermentation is completed, the Penicillium oxalicum TWSF1 solid fermentation product is obtained.

4. The solid fermentation product of Penicillium oxalicum TWSF1 according to claim 3, characterized in that: The fermentation solid culture medium in step 2 comprises the following raw materials counted by weight: 15-30 parts of bran, 5-15 parts of bran, 5-15 parts of desaponified tea meal powder, 0.5-2.0 parts of KH2PO4, 0.5-2.0 parts of Na2HPO4, 0.01-0.1 parts of MgSO4·7H2O, 0.01-0.1 parts of CaCO3, and 50 parts of deionized water.

5. A microbial agent containing the solid fermentation product of Penicillium oxalicum TWSF1 according to claim 2 or 3, characterized in that: The solid fermentation product of Penicillium oxalicum TWSF1 is dried, crushed, and solid diluted to obtain a microbial agent, i.e., the microbial agent, with an effective viable count of ≥0.2×10 8 cfu / g.

6. A microbial fertilizer containing the microbial agent according to claim 5, characterized in that: The biological organic fertilizer or compound microbial fertilizer prepared by the solid fermentation product of Penicillium oxalicum TWSF1 through secondary solid fermentation and mixing is the microbial fertilizer.

7. Use of the Penicillium oxalicum TWSF1 according to claim 1 in dissolving inorganic phosphorus at 4-35°C.

8. Use of Penicillium oxalicum TWSF1 according to claim 1 and / or the solid fermentation product of Penicillium oxalicum TWSF1 according to claim 2 and / or the microbial agent according to claim 5 and / or the microbial fertilizer according to claim 6 in promoting rapeseed growth and lowering the pH of saline-alkali soil under 4-35°C.

9. The use according to claim 7 or 8, characterized in that: The application scenario is application in improving saline-alkali land.

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