Penicillium oxalicum strain g6 and application thereof

Abstract

The present application relates to the field of microorganisms, in particular to a strain of Penicillium oxalicum G6 and application thereof.The Penicillium oxalicum G6 provided in the present application has a preservation number of CGMCC No.41386.The Penicillium oxalicum G6 provided in the present application can rapidly start the composting temperature rising process under a low-temperature environment, prolong the high-temperature period, and improve the high-temperature peak of composting, and the produced composting product is highly harmless and non-toxic.At the same time, the strain has a strong cellulose degradation function, and the strain also has a growth promoting effect, and compared with commercial fertilizers, can effectively improve the dry weight of the aboveground part and the underground part of plants.It can be seen that the strain provided in the present application has multiple functions, and is a probiotic with excellent properties.

Description

Technical Field

[0001] This invention relates to the field of microbiology, and in particular to a strain of Penicillium oxalate G6 and its applications. Background Technology

[0002] In recent years, the rapid development of the agricultural economy has led to a serious accumulation of solid waste. Straw from farmland cultivation and livestock manure from animal husbandry are important components of China's agricultural solid waste. The annual output of livestock manure reaches approximately 3.8 billion tons, while the annual output of straw reaches approximately 700 million tons. Therefore, the resource-based treatment of agricultural solid waste is a problem that needs to be addressed.

[0003] Aerobic composting is a common method for turning solid waste such as livestock and poultry manure, agricultural straw, and household garbage into valuable resources. Under conditions of sufficient oxygen, suitable moisture content, C / N ratio, and temperature, a series of biochemical reactions occur through the metabolism of microorganisms such as bacteria, fungi, and actinomycetes in the livestock and poultry manure. These reactions break down large organic molecules in the raw materials into smaller ones, ultimately transforming them into humus, thus reducing the volume and rendering the agricultural waste harmless. Composting technology has become one of the most important technologies for utilizing agricultural waste resources due to its low cost, high safety, and applicability to agriculture. However, the Xinjiang Uygur Autonomous Region, located in the northwest border region of my country, experiences low temperatures in autumn and winter, making it difficult to start the composting process and reach the required composting temperature. Furthermore, the high content of recalcitrant cellulose in agricultural straw prolongs the composting process and results in incomplete material degradation, significantly reducing composting efficiency and limiting the application and promotion of composting technology.

[0004] Microbial degradation of cellulose offers advantages such as environmental friendliness, high efficiency, and low cost. Although researchers both domestically and internationally have conducted a series of studies on the aerobic composting process and the role of microbial agents, research on microorganisms used to promote cellulose degradation in low-temperature composting environments is still in its early stages. Therefore, screening out cold-resistant bacteria with cellulose degradation capabilities and preparing mature microbial agents is of great significance for accelerating the aerobic composting process in low-temperature environments. Summary of the Invention

[0005] The purpose of this invention is to provide a Penicillium oxalate strain G6 and its applications to solve the problems existing in the prior art. The Penicillium oxalate strain G6 provided by this invention can rapidly initiate the composting heating process under low-temperature conditions, prolong the high-temperature period, and increase the peak temperature of composting. At the same time, this strain also has the ability to degrade cellulose.

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

[0007] This invention provides a strain of Penicillium oxalicum G6, with the preservation number CGMCC No. 41386. During the composting heating period, this strain can rapidly increase the compost temperature, exhibiting high biological activity and adaptability at this stage.

[0008] This invention provides a method for culturing the above-mentioned Penicillium oxalate G6, comprising the following steps:

[0009] The activated Penicillium oxalate G6 was inoculated into the fermentation medium and fermented to obtain a fermentation broth containing Penicillium oxalate G6.

[0010] Preferably, the fermentation medium comprises the following components at the following concentrations: CMC-Na 5.0 g / L, peptone 5.0 g / L, yeast extract 1.0 g / L, KH2PO4 0.5 g / L and MgSO4 0.25 g / L.

[0011] Preferably, the initial pH of the fermentation culture is 6.5-7.0, the temperature is 0℃-25℃, the rotation speed is 150r / min, and the time is 48h.

[0012] This invention provides the application of the above-mentioned Penicillium oxalate G6 in the preparation of low-temperature composting fermentation agents.

[0013] This invention provides a low-temperature composting fermentation agent, the effective component of which includes the aforementioned Penicillium oxalate G6.

[0014] More preferably, the effective viable bacteria count of the low-temperature composting fermentation agent is ≥1×10⁻⁶. 8 CFU / mL.

[0015] This invention provides the application of the aforementioned low-temperature composting fermentation agent in the preparation of compost.

[0016] Preferably, it includes the following steps:

[0017] After inoculating the fertilizer to be fermented with the above-mentioned low-temperature composting fermentation agent, aerobic fermentation is carried out to obtain compost.

[0018] More preferably, the effective viable bacteria count of the low-temperature composting fermentation agent is ≥1×10⁻⁶. 8 CFU / mL, inoculum amount is 1% of the mass of the fertilizer to be fermented.

[0019] More preferably, the aerobic fermentation is low-temperature aerobic fermentation; the initial temperature of the aerobic fermentation is 20°C.

[0020] More preferably, the present invention provides the application of the above-mentioned Penicillium oxalate G6 or the above-mentioned low-temperature composting fermentation agent in promoting composting maturity.

[0021] More preferably, the compost is low-temperature aerobic compost of cow dung and cotton stalks.

[0022] Furthermore, the present invention provides the application of the above-mentioned Penicillium oxalicum G6 in the degradation of agricultural straw.

[0023] More preferably, the present invention provides the application of the above-mentioned Penicillium oxalate G6 in cellulase production.

[0024] This invention provides the application of the above-mentioned Penicillium oxalate G6 in the preparation of plant growth-promoting inoculants or microbial fertilizers.

[0025] More preferably, the plant is cotton.

[0026] This invention provides a microbial agent or microbial fertilizer that promotes plant growth, wherein the effective ingredient of the microbial agent or microbial fertilizer includes the aforementioned Penicillium oxalate G6.

[0027] More preferably, the plant is cotton.

[0028] Furthermore, the present invention also provides the application of the above-mentioned plant growth promoting agents or microbial fertilizers in promoting plant growth.

[0029] More preferably, the plant is cotton.

[0030] Furthermore, the present invention also provides a method for promoting plant growth, comprising the following steps:

[0031] Apply the above-mentioned microbial agents or microbial fertilizers that promote plant growth during the seedling stage.

[0032] More preferably, the plant is cotton.

[0033] The present invention discloses the following technical effects:

[0034] 1. The *Penicillium oxalate* G6 provided by this invention can rapidly initiate the composting heating process under low-temperature conditions, prolong the high-temperature period of composting, increase the peak high-temperature period of composting, and the resulting compost products are highly harmless and non-toxic. Chinese patent document CN117660252 (application number: 202311683194.5) discloses a low-temperature composite microbial agent, comprising: 3 parts of low-temperature cellulose-degrading bacteria, 1 part of low-temperature starch-degrading bacteria, and 1 part of low-temperature sugar-degrading bacteria. Compared to this invention, the advantages and differences of this invention regarding the microbial strain are: the microbial agent provided by this invention contains only one type of microorganism. In comparison, the preparation process is easier, and it can produce the same degree of effect in initiating composting heating and prolonging the high-temperature period under low-temperature conditions. Moreover, the preparation of the microbial agent containing this strain is simple and convenient.

[0035] 2. The *Penicillium oxalate* G6 provided by this invention has a strong cellulose degradation function. Chinese patent document CN111909852A (application number: 202010698524.8) discloses a cellulose-degrading bacterium, named *Talaromyces amestolkiae*, strain P5, which has a good degradation effect on crop straw. Compared with this invention, the advantages and differences of the strain provided by this invention are: *Penicillium oxalate* G6 can survive at 0℃-25℃, and the degradation rate of wheat straw measured after one week of cultivation at 15℃ is 73.3%, which is 3 times higher than the wheat straw degradation rate measured by *Talaromyces amestolkiae* P5 at 25℃ on day 25.

[0036] 3. By inoculating the compost with a fermentation broth containing Penicillium oxalate G6, the degradation of materials can be accelerated, resulting in a high degree of harmlessness and non-toxicity in the compost. Chinese patent document CN115725447A (application number: 202211001055.5) discloses a Bacillus velezensis SSF6 strain, which can reduce the biotoxicity of straw compost. Compared to this invention, the advantages and differences of the strain provided in this invention are: the compost extract of this invention with added Penicillium oxalate G6 can achieve a seed germination index of 225%, while the germination index of the straw compost extract with added Bacillus velezensis SSF6 in this invention is only 108%. Therefore, the compost of this invention has a higher degree of non-toxicity.

[0037] 4. The Penicillium oxalate G6 provided by this invention also has a growth-promoting effect, and compared with commercial fertilizers, it can effectively increase the dry weight of the above-ground and underground parts of plants.

[0038] In summary, the Penicillium oxalate G6 provided by this invention has multiple functions and is a high-performance probiotic. Attached Figure Description

[0039] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0040] Figure 1 This is a colony diagram of the growth of strain G6;

[0041] Figure 2 The diagrams show the cellulose degradation zones of strain G6 at 25℃ (A), 15℃ (B), 10℃ (C), and 0℃ (D).

[0042] Figure 3Phylogenetic tree of 16S rDNA from strain G6;

[0043] Figure 4 The curve shows the temperature change over composting time; where Environment represents ambient temperature; CK represents the control group; and T represents the experimental group.

[0044] Figure 5 The curve shows the change in pH over composting time; where CK is the control group and T is the experimental group.

[0045] Figure 6 The curves show the change of EC with composting time; where CK is the control group and T is the experimental group.

[0046] Figure 7 The curves show the change in GI over composting time; where CK is the control group and T is the experimental group.

[0047] Figure 8 The curves show the change in the proportion of cellulose with composting time; where CK is the control group and T is the experimental group.

[0048] Figure 9 The changes in EC values ​​at different depths in soil under different treatments are shown; where CK represents 100% chemical fertilizer; M1 represents 100% T; and M2 represents 100% commercial organic fertilizer.

[0049] Figure 10 The changes in aboveground and belowground dry matter accumulation during the cotton seedling stage under different treatments were shown; where CK was 100% chemical fertilizer, M1 was 100% T, and M2 was 100% commercial organic fertilizer.

[0050] Information on the preservation of biological materials

[0051] Penicillium oxalicum G6 is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, on June 27, 2024, with accession number CGMCC No. 41386. Detailed Implementation

[0052] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0053] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0054] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0055] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0056] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0057] Materials preparation:

[0058] 1. Experimental reagents and equipment: The main experimental reagents are detailed in Table 1, and the equipment is detailed in Table 2.

[0059] Table 1 Main Reagents

[0060] NaCl Analytical Pure Zinc chloride Analytical Pure Sodium carboxymethyl cellulose Analytical Pure Congo Red Indicator dipotassium hydrogen phosphate Analytical Pure Magnesium sulfate heptahydrate Analytical Pure Potassium dihydrogen phosphate Analytical Pure manganese sulfate Analytical Pure Sodium phosphate Analytical Pure Ammonium sulfate Analytical Pure Ferrous sulfate heptahydrate Analytical Pure Potassium sodium tartrate Chemically pure potassium nitrate Analytical Pure 3,5-Dinitrosalicylic acid Chemically pure

[0061] Table 2 Main Equipment and Instruments

[0062] Clean bench ZHJH-C1112B China pH meter PHSJ-4F China Autoclave MJ-54A USA Electronic balance SQP Germany Ultrapure water system MILL-QBIOCCEL USA pipette Eppendorf Germany Refrigerated high-speed centrifuge Frcsco17 USA Ultraviolet spectrophotometer Evolution201 USA Electric heating constant temperature water bath DK-80 China -80℃ refrigerator DW-86L388J China constant temperature incubator HWS-80 China -20℃ refrigerator BCD-271TMCM China Constant temperature shaking table HZQ-F160A China Flow cytometer CytoFLEX USA Forced air drying oven DHG-9030A China

[0063] 2. Prepare the culture medium:

[0064] (1) PDA medium: 1L potato extract, 16.0g agar, 20.0g glucose, sterilized at 115℃ for 15min.

[0065] (2) LB solid medium: 5.0g yeast extract, 10.0g tryptone, 10.0g NaCl, 14.0g agar, diluted with water to 1L, pH 7.0, sterilized at 121℃ for 15min.

[0066] (3) Beef extract peptone medium: 5.0g beef extract, 10.0g peptone, 10.0g NaCl, 20.0g agar, add water to make up to 1L, pH 7.2-7.4, sterilize at 121℃ for 15min to obtain the medium.

[0067] (4) Sodium carboxymethyl cellulose medium: CMC-Na 10.0g, KH2PO4 1.5g, NaH2PO4·7H2O 2.5g, MgSO4·7H2O 0.3g, FeSO4·7H2O 0.005g, ZnCl2 0.0017g, MnSO4 0.0016g, (NH4)2SO4 1.5g, add water to a final volume of 1L, pH 7.0, sterilize at 121℃ for 15min.

[0068] (5) Liquid fermentation medium: CMC-Na 5.0g, peptone 5.0g, yeast extract 1.0g, KH2PO4 0.5g, MgSO4 0.25g, add water to make up to 1L, pH 6.5-7.0, sterilize at 121℃ for 15min to obtain the medium.

[0069] Example 1: Screening of strains

[0070] 1. Sampling: Samples of livestock and poultry manure and straw compost from Xinjiang Uygur Autonomous Region were collected during the heating period and stored in sterile sealed bags. After sealing, the bags were placed in a 4°C refrigerator.

[0071] 2. Screening of cellulose-degrading bacteria

[0072] The sample was soaked in sterile water and shaken on a shaker for 6 hours to obtain the sample extract. The sample extract was then diluted with sterile water, and a 10⁻⁶ solution was taken. -4 10 -5 10 -6 10 -7 The diluted solution was spread onto PDA medium and LB solid medium and incubated at 0℃, 10℃, 15℃ and 25℃ for 48 h, respectively. Bacteria that could grow at all four temperatures were isolated and their species were preliminarily determined.

[0073] The obtained fungi were inoculated onto PDA medium, and the bacteria onto LB solid medium. After incubation at 25°C for 48 hours, the best-growing strains were selected for streak plating to obtain numerous single colonies. This streaking process was repeated until pure cultures of each strain were obtained. The purified strains were then inoculated onto beef extract peptone medium and cultured for 2 days, during which colony morphology was observed. Finally, the cultures were inoculated onto slant agar and stored at 4°C for later use.

[0074] Single colonies of the strain with good growth were transferred onto sodium carboxymethyl cellulose agar plates and incubated upside down in incubators at 0℃, 10℃, 15℃, and 25℃ for 10 days, 8 days, 6 days, and 4 days, respectively. Congo red staining solution was slowly poured into the plates, and staining was carried out for 30 min, followed by destaining with 0.9% (w / v) sodium chloride solution for 25 min. The clear zones appearing on the plates were observed. The strain with the clearest clear zone and the largest ratio of clear zone diameter to colony diameter (D / d) was named strain G6, and the other strains were named strains X1 and X3. The results for each strain are shown in Table 3. A photograph of the growth colonies of strain G6 after 48 h of inverted incubation at 25℃ is shown. Figure 1 The degradation zones of strain G6 at 25℃ (A), 15℃ (B), 10℃ (C), and 0℃ (D) are shown in the following images. Figure 2 As shown.

[0075] Table 3. Determination results of degradation zone diameter (D / d) of strain G6 on sodium carboxymethyl cellulose medium.

[0076]

[0077] Depend on Figure 1 It can be seen that the colonies of strain G6 have a fluffy or cottony texture, are light green in color, and are round or oval in shape. (From Table 3 and...) Figure 2 Records show that the degradation zone diameter of strain G6 ranged from 2.11 to 5.52 cm, with the largest diameter observed at 15°C. The colony diameter of G6 ranged from 0.71 to 4.15 cm, again with the largest diameter observed at 15°C. Compared to strains X1 and X3, strain G6 exhibited the highest D / d values ​​at both 25°C and 10°C, indicating that its growth and metabolic activities were more active at these temperatures, demonstrating strong adaptability.

[0078] The G6 strain was purified by streaking for three generations and stored in glycerol at -80°C for later use.

[0079] Example 2: Construction of a phylogenetic tree

[0080] Figure 3 As shown, sequences in GenBank with high homology to the obtained sequences all originate from the genus *Penicillium*.

[0081] Example 3: Degradation of filter paper strips

[0082] Filter paper treatment: Soak the filter paper in 1% (V / V) dilute acetic acid for 24 hours. After confirming the absence of starch by testing with iodine solution, wash it with 2% (W / V) baking soda solution until neutral, and then air dry for later use.

[0083] Filter paper culture medium: 1×6cm filter paper strip, 1.0g KH2PO4, 0.1g NaCl, 0.3g MgSO47H2O, 2.5g NaNO3, 0.01g FeCl3, 0.1g CaCl2, diluted to 1L with water, sterilized at 121℃ for 15min.

[0084] The G6 strain was inoculated into PD medium and cultured at 25℃ and 150 rpm for 48 h. 5 mL of the inoculum was then transferred to a 250 mL Erlenmeyer flask containing 50 mL of filter paper culture medium, with 5 mL of sterile water as a control. The flasks were cultured at 15℃ and 150 rpm with shaking for 5 days. The filter paper deterioration and weight loss (degradation rate) were observed and calculated, as shown in Table 4.

[0085] Table 4 Degradation results of filter paper strips

[0086]

[0087]

[0088] Note: "+" indicates that the filter paper edge is swollen; "++" indicates that the filter paper is neatly swollen and curved; "++++" indicates that the filter paper is irregular and basically pasty; "++++" indicates that the filter paper is pasty.

[0089] As shown in Table 4, the degradation rate of strain G6 was the highest at 15℃, reaching 72.67%, and the filter paper became pasty.

[0090] Based on the comprehensive examples 1-3, strain G6 belongs to the Penicillium genus. Therefore, the inventors named it Penicillium oxalicum G6, which is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, on June 27, 2024, with accession number CGMCC No. 41386.

[0091] Example 4: Degradation of wheat straw by Penicillium oxalate G6

[0092] Cut the wheat straw into 2cm×3cm pieces, dry them, and then grind them to 80 mesh.

[0093] Penicillium oxalicum G6 was inoculated into liquid fermentation medium and cultured at 25℃ and 150 rpm for 48 h to obtain a bacterial suspension. The viable count of this suspension was 1 × 10⁻⁶. 8 CFU / mL. The obtained bacterial culture was inoculated into 100 mL of liquid fermentation medium containing 2% (w / v) straw cellulose at an inoculum ratio of 10%. The culture was incubated at 0℃, 10℃, 15℃, and 25℃ with constant temperature shaking at 150 rpm. After one week of incubation, the fermentation broth was filtered through filter paper, rinsed with distilled water, and dried in a drying oven at 85℃ until constant weight. The residue was weighed, and the straw degradation rate was calculated using the weight loss method. The formula for calculating the straw degradation rate is: Straw degradation rate = (Initial straw weight (g) - Degraded straw weight (g)) / Initial weight (g) × 100%. The straw degradation rate of this strain is shown in Table 5.

[0094] Table 5 Results of wheat straw degradation

[0095]

[0096] As shown in Table 5, the straw degradation rate of Penicillium oxalate G6 ranges from 12.45% to 73.65%, with the highest straw degradation rate of 73.65% at a temperature of 15℃.

[0097] Example 5: Determination of the enzyme activity of Penicillium oxalate G6 during wheat straw degradation.

[0098] The enzyme activity of Penicillium oxalate G6 during straw degradation at 0℃, 10℃, 15℃ and 25℃ was determined. The enzyme activity was measured using fermentation broth from days 1 to 7 of straw degradation in Example 4.

[0099] Enzyme activity definition: Using the dinitrosalicylic acid colorimetric method, one unit of enzyme activity (U) is defined as the amount of enzyme that breaks down a substrate to produce 1 μL of glucose per minute. The calculation formula is as follows:

[0100]

[0101] In the formula, ρ is OD 540 The glucose concentration (mg / mL) corresponding to the absorbance value, where N is the solution dilution factor and T is the reaction time.

[0102] CMC enzyme activity assay: Straw degradation fermentation broth samples cultured at different time points were centrifuged at 4℃ and 8000r / min for 15min. 1.0mL of the supernatant (crude enzyme solution) was taken and 1.0mL of 0.2% (w / v) sodium carboxymethyl cellulose (CMC-Na) solution was added. The mixture was incubated in a 50℃ water bath for 1h. Then, 2.0mL of DNS solution was added and the reaction was stopped by incubating in a 100℃ water bath for 10min. After cooling to room temperature, the absorbance was measured at 540nm.

[0103] FPA enzyme activity assay: Cut Xinhua filter paper into strips of 1.0cm × 6.0cm and place them in a test tube. Take 1.0mL of the crude enzyme solution prepared during the CMC enzyme activity assay, add 2.0mL of acetate buffer, and incubate at 50℃ for 50min. Then, add 2.0mL of the termination reaction solution (DNS solution), mix and shake well, and develop the color in a 100℃ water bath for 5min. Remove and allow to cool naturally at room temperature. Measure the absorbance at 540nm according to the DNS method.

[0104] The results of enzyme activity determination of the strain at 0℃, 10℃, 15℃ and 25℃ during straw degradation are shown in Table 6.

[0105] Table 6. Results of enzyme activity determination of Penicillium oxalicum G6 at different temperatures and times.

[0106]

[0107] As shown in Table 6, from a temporal perspective, the CMC and FPA enzyme activities of this strain exhibited a trend of first increasing and then decreasing at 10℃, 15℃, and 25℃, with peak activity occurring between days 4 and 6. However, at 0℃, the enzyme activity generally showed a decreasing trend, with the peak occurring on day 2. From a temperature perspective, although 0℃ is an extremely low temperature, in the early stages of the experiment, the CMC and FPA enzyme activities produced by the strain were significantly higher than those of other treatments. This may be because the metabolic activity of the strain slows down at low temperatures, allowing the initially accumulated enzymes to be retained at higher concentrations, resulting in higher enzyme activity in a short period.

[0108] Example 6: Application of Penicillium oxalate G6 in aerobic composting

[0109] Preparation of Penicillium oxalate G6 fermentation broth: Penicillium oxalate G6 was inoculated into liquid fermentation medium at an inoculation rate of 5% (V / V), with an initial pH of 6.5-7.0. The fermentation conditions were: 0℃-25℃, 150 r / min for 48 h, yielding the Penicillium oxalate G6 fermentation broth. This fermentation broth was then diluted to obtain an effective viable count of 1×10⁻⁶ cells / day. 8 CFU / mL Penicillium oxalate G6 fermentation broth.

[0110] The composting experiment was conducted at the open-air experimental field of Xi'an University of Technology. The outdoor ambient temperature was around 20℃, and the ventilation was excellent. The total dry weight of the compost mixture was 15 kg, with a dry weight ratio of cow dung to cotton stalks of 3:1. The cow dung and cotton stalks were mixed thoroughly, and 1% (inoculum weight / compost weight) of Penicillium oxalate G6 fermentation broth was added (the effective viable count of this fermentation broth was 1×10⁻⁶). 8 The mixture (CFU / mL) was thoroughly mixed with a carbon-to-nitrogen ratio of 25:1 and the moisture content was maintained at around 60 wt.%, which was designated as the experimental group (T). A natural fermentation composting experiment was set up as the control group (CK), and the compost was turned over at irregular intervals to prevent insufficient oxygen from affecting the quality of the compost.

[0111] The composting process included the initial stage, heating stage, high-temperature stage, cooling stage, and maturation stage, lasting 23 days. Samples were taken on days 0, 2, 5, 9, and 23, and parameters such as temperature, pH, EC, and seed germination index (GI) were measured simultaneously. Temperature, pH, and EC were measured using a thermometer, pH meter, and conductivity meter, respectively. For the seed germination index (GI): 5.0 g of sample was weighed and mixed with deionized water at a ratio of 1:10. The mixture was shaken on a shaker at 180 rpm for 20 minutes, then allowed to stand for 10 minutes. 5 ml of the extract was transferred to a petri dish lined with filter paper, and 25 cabbage seeds were evenly placed inside. The mixture was incubated at a constant temperature for 48 hours. Distilled water was used as the control group (CK). The seed germination index (GI) was calculated using the following formula:

[0112]

[0113] In the formula, M1 is the average germination rate of the treated seeds, R1 is the average root length of the treated seeds, M2 is the average germination rate of the control seeds, and R2 is the average root length of the control seeds.

[0114] Temperature changes in different composting treatments, such as Figure 4 As shown in the figure, during the composting process, the ambient temperature was relatively low, consistently around 20℃. The T treatment, inoculated with *Penicillium oxalicum* G6, reached a compost temperature above 50℃ after the second day, peaking at 65.1℃ on the fourth day, and maintained above 50℃ for 9 days. The CK treatment, however, maintained above 50℃ for 7 days, with a maximum temperature below 60℃, 7.9℃ lower than the maximum temperature of the T treatment. In a low-temperature environment, the addition of *Penicillium oxalicum* G6 can accelerate the initiation of the compost's heating process, increase the peak compost temperature, and promote the compost maturation process.

[0115] pH changes in different composting treatments, such as Figure 5As shown, in the early stages of composting, sufficient oxygen and nutrients led to increased microbial activity and the production of large amounts of ammonium nitrogen. The pH rise rate in treatment T was higher than that in treatment CK. In the later stages, continuous microbial metabolism released ammonia, causing the pH to decrease. Ultimately, the pH values ​​of treatments CK and T dropped to 8.09 and 8.12, respectively. This demonstrates that the strain provided by this invention has a significant advantage in regulating pH during the composting process. This indicates that the strain performs well in promoting rapid initial fermentation and optimizing the final pH value, effectively improving compost quality.

[0116] Changes in EC values ​​of different treatments in composting are as follows Figure 6 As shown. In the early stages of composting, the EC value increases due to mineralization, but in the later stages, as small molecules are continuously converted into large humic molecules, the EC value decreases. Figure 6 It is known that the strain provided by this invention can effectively promote the mineralization process and effectively convert small molecules into humic substances in the later stage of fermentation, thereby optimizing the electrical conductivity of compost.

[0117] Seed germination index survey results as follows Figure 7 As shown, the GI of the T treatment reached over 80% on the second day alone, and reached the decomposition standard earlier than the CK treatment. Ultimately, the GI value of the T treatment reached as high as 255%, while the CK treatment was only 99%. Therefore, it is evident that the strain provided by this invention can significantly improve the seed germination index of plants.

[0118] The cellulose content in compost accounts for, for example Figure 8 As shown in the figure, the cellulose content was 29.2% at the beginning of the composting process. At the end of composting, the cellulose content in the CK and T treatments decreased to 28.5% and 23.4%, respectively, with degradation rates of 2.4% and 19.9%, respectively. This indicates that the T treatment is more conducive to the degradation of cellulose in the compost, thus improving the quality of the compost.

[0119] Example 7: Changes in EC values ​​at different depths in soils under different treatments

[0120] Preparation of Penicillium oxalate G6 fermentation broth: Penicillium oxalate G6 was inoculated into liquid fermentation medium at an inoculation rate of 5% (V / V), with an initial pH of 6.5-7.0. The fermentation conditions were: 0℃-25℃, 150 r / min for 48 h, yielding the Penicillium oxalate G6 fermentation broth. This fermentation broth was then diluted to obtain an effective viable count of 1×10⁻⁶ cells / day. 8 CFU / mL Penicillium oxalate G6 fermentation broth.

[0121] Before cotton sowing, different base fertilizers were applied to the soil at depths of 0-60cm, all providing a nitrogen content of 375 kg / ha. Other management practices remained consistent and adapted to local conditions. Based on the base fertilizer, three treatments were applied: CK (100% chemical fertilizer), M1 (100% T), and M2 (100% commercial organic fertilizer). Specifically, the CK treatment involved applying inorganic chemical fertilizer with a nitrogen content of 46% at different depths, with a total application rate of 815.2 kg / ha, applied every 10 days for a total of three applications. The M1 treatment involved applying compost fermented from Penicillium oxalate G6 fermentation liquid with a nitrogen content of 2% at different depths, with a total application rate of 18750 kg / ha, applied every 10 days for a total of three applications. The M2 treatment involved applying agricultural commercial organic fertilizer with a nitrogen content of 10% at different depths, with a total application rate of 3750 kg / ha, applied every 10 days for a total of three applications.

[0122] Cotton was sown 7 days after the application of base fertilizer. Thirty days after sowing, the cotton entered the seedling stage. The EC values ​​of the soil at different depths (0-5cm, 5-10cm, 10-20cm, 20-30cm, 30-40cm, and 40-60cm) during the cotton seedling stage are shown below. Figure 9 As shown, with increasing soil depth, the CK treatment increased the soil EC value, while the M1 and M2 treatments decreased the soil EC value. The EC value of the M1 treatment was as low as 0.12 mS·cm at a soil depth of 40-60 cm. -1 Therefore, the finished composted fertilizer containing Penicillium oxalate G6 inoculant provided by this invention has the significant advantage of reducing soil electrical conductivity, effectively improving soil salinity accumulation, and enhancing soil quality.

[0123] Example 8: Changes in aboveground and belowground dry matter accumulation during cotton seedling stage under different treatments

[0124] Preparation of Penicillium oxalate G6 fermentation broth: Penicillium oxalate G6 was inoculated into liquid fermentation medium at an inoculation rate of 5% (V / V), with an initial pH of 6.5-7.0. The fermentation conditions were: 0℃-25℃, 150 r / min for 48 h, yielding the Penicillium oxalate G6 fermentation broth. This fermentation broth was then diluted to obtain an effective viable count of 1×10⁻⁶ cells / day. 8 CFU / mL Penicillium oxalate G6 fermentation broth.

[0125] Before cotton sowing, different base fertilizers were applied to the soil at depths of 0-60cm, all providing a nitrogen content of 375 kg / ha. Other management practices remained consistent and adapted to local conditions. Based on the base fertilizer, three treatments were applied: CK (100% chemical fertilizer), M1 (100% T), and M2 (100% commercial organic fertilizer). Specifically, the CK treatment involved applying inorganic chemical fertilizer with a nitrogen content of 46% at different depths, with a total application rate of 815.2 kg / ha, applied every 10 days for a total of three applications. The M1 treatment involved applying compost fermented from Penicillium oxalate G6 fermentation liquid with a nitrogen content of 2% at different depths, with a total application rate of 18750 kg / ha, applied every 10 days for a total of three applications. The M2 treatment involved applying agricultural commercial organic fertilizer with a nitrogen content of 10% at different depths, with a total application rate of 3750 kg / ha, applied every 10 days for a total of three applications.

[0126] Cotton was sown 7 days after the application of base fertilizer. Thirty days after sowing, the cotton entered the seedling stage. The cumulative dry matter (aboveground and belowground parts) of the cotton seedlings was investigated, and the results are as follows: Figure 10 As shown, the cumulative dry matter content of cotton seedlings under treatments M1 and M2 was higher than that under control (CK). The cumulative dry matter content of the underground and aboveground parts of cotton seedlings under treatment M1 exceeded that under treatment M2 by 0.16 g and 0.64 g, respectively. Therefore, the finished composted fertilizer containing Penicillium oxalate G6 inoculant provided by this invention can effectively promote plant growth.

[0127] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. Penicillium oxalate ( Penicillium oxalicum The application of G6 in the preparation of low-temperature composting fermentation agents is characterized by, The preservation number of Penicillium oxalate G6 is CGMCC No. 41386.

2. The application according to claim 1, characterized in that, The method for culturing Penicillium oxalate G6 includes the following steps: The activated Penicillium oxalate G6 was inoculated into the fermentation medium and fermented to obtain a fermentation broth containing Penicillium oxalate G6.

3. The application according to claim 2, characterized in that, The fermentation medium comprises the following components at the following concentrations: CMC-Na 5.0 g / L, peptone 5.0 g / L, yeast extract 1.0 g / L, KH2PO4 0.5 g / L and MgSO4 0.25 g / L.

4. The application according to claim 2, characterized in that, The initial pH of the fermentation culture was 6.5-7.0, the temperature was 0℃-25℃, the rotation speed was 150r / min, and the time was 48h.

5. A low-temperature composting fermentation agent, characterized in that, The effective component of the low-temperature composting fermentation agent includes *Penicillium oxalicum* G6 as described in claim 1; the effective viable count of the low-temperature composting fermentation agent is ≥1×10⁻⁶. 8 CFU / mL.

6. The application of the low-temperature composting fermentation agent according to claim 5 in the preparation of compost, characterized in that, The compost is low-temperature aerobic compost of cow dung and cotton stalks.

7. The application of Penicillium oxalate G6 in the preparation of microbial agents or microbial fertilizers that promote cotton growth, characterized in that, The preservation number of Penicillium oxalate G6 is CGMCC No. 41386.

8. A microbial agent or fertilizer for promoting cotton growth, characterized in that, The active ingredient of the microbial agent or microbial fertilizer includes Penicillium oxalate G6 in the application described in claim 1.

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