Auxotrophic mutant of aureobasidium pullulans cy28 and application thereof

Abstract

This invention relates to a budding short-stem mold Cy28 and its applications, belonging to the field of microbial technology. This strain can reduce the Cd content in soil and produce more pullulan in Cd-containing environments. When applied to lettuce cultivation, it can significantly increase the dry weight of edible lettuce tissues and reduce the Cd content in edible lettuce tissues and roots, bringing the Cd content in edible lettuce tissues to edible standards, both in cadmium-free and cadmium-containing environments. Furthermore, it can increase the vitamin C, chlorophyll, soluble protein, and IAA content of edible lettuce tissues. Simultaneously, it can significantly increase beneficial microorganisms in the soil under cadmium conditions. Pseudarthrobacter The content of these elements helps to improve the soil.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a budding short-stemmed mold Cy28 and its applications. Background Technology

[0002] Cadmium (Cd) is one of the most toxic heavy metals and is listed as a core pollutant in China's heavy metal pollution control efforts. The excessive use of chemical fertilizers and pesticides, the discharge of cadmium-containing industrial wastewater from mining and smelting, and the direct application of untreated urban sewage to farmland are all major causes of cadmium pollution in farmland. Cadmium pollution in farmland inhibits soil microbial activity, leading to reduced crop yields. Food crops, vegetables, fruits, and forage grasses grown in mildly or moderately cadmium-contaminated soil accumulate cadmium, which can then enter the human body through the food chain, posing a serious threat to human health.

[0003] In-situ fixation technology is an important means of remediating heavy metal pollution in soil, especially suitable for large areas of farmland with light to moderate cadmium contamination. Its core principle is to change the chemical form of cadmium by adding passivating agents (such as converting it into insoluble compounds), thereby reducing the bioavailability and mobility of cadmium. However, the continuous addition of organic and inorganic passivating agents can have harmful effects on soil properties, structure, and ecosystems.

[0004] In-situ microbial immobilization technology can confine functional microorganisms (such as degrading and mineralizing bacteria) to a carrier or specific area, thereby allowing them to exert their metabolic activity in polluted environments. This is a cutting-edge technique in environmental remediation. Currently, most reports on existing technologies that can simultaneously achieve heavy metal degradation and plant growth promotion involve combining multiple microorganisms or combining microorganisms with certain carriers to achieve multiple functions in soil. Reports on single-strain microorganisms that combine plant growth promotion, heavy metal degradation, and soil improvement functions are relatively rare. Therefore, developing a multifunctional microbial strain is an urgent problem to be solved in the field of agricultural microbiology. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a budding short-stemmed mold Cy28 and its applications.

[0006] The technical solution of this invention is as follows:

[0007] A budding short-stalked mold Cy28, the budding short-stalked mold ( Aureobasidium Cy28 sp. was deposited on March 31, 2025 at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with accession number CCTCC NO: M2025660.

[0008] The ITS rDNA sequence of the budding short-stalked dermatophyte Cy28 is shown in SEQ ID NO.1.

[0009] The cultivation method of the budding short-stalked mold Cy28 includes the following steps:

[0010] (1) Inoculate the budding short-stalked fungus Cy28 onto PDA solid medium and activate it at 28~30℃ to obtain the activated strain;

[0011] (2) Inoculate the activated strain from step (1) into PDB liquid culture medium and culture it under shaking conditions at 160~180 rpm and 28~30℃ to obtain Cy28 bacterial culture.

[0012] The application of the aforementioned budding short-stem mold Cy28 is for the production of pullulan polysaccharides.

[0013] Preferably, the budding short-stem mold Cy28 produces pullulan polysaccharide under cadmium conditions.

[0014] The application of the aforementioned budding short-stalked mold Cy28 is used to reduce the cadmium content in soil.

[0015] The application of the aforementioned short-stemmed budding mold Cy28 is in lettuce cultivation.

[0016] Preferably, the budding short-stem mold Cy28 is used to promote lettuce growth, reduce the cadmium content in the edible tissues and roots of lettuce, and increase the vitamin C content, chlorophyll content, soluble protein content, and indoleacetic acid content in the edible tissues of lettuce.

[0017] Preferably, the budding short-stem mold Cy28 is used for lettuce cultivation in a cadmium environment.

[0018] The application of the aforementioned budding short-stalked mold Cy28 is for soil improvement, whereby the improved soil increases the concentration of cadmium-contaminated soil. Pseudarthrobacter Content of microorganisms (Pseudorobacterium).

[0019] A live bacterial preparation using the aforementioned budding short-stalked sclerotium Cy28 as the active ingredient.

[0020] The beneficial effects of this invention are:

[0021] This invention provides a budding short-stem mold, Cy28, which can reduce the Cd content in soil and produce more pullulan in Cd-containing environments. When applied to lettuce cultivation, it significantly increases the dry weight of edible lettuce tissues and reduces the Cd content in both cadmium-free and cadmium-containing environments, bringing the Cd content in edible lettuce tissues to edible standards. Furthermore, it increases the vitamin C, chlorophyll, soluble protein, and IAA content in edible lettuce tissues. Simultaneously, it significantly increases beneficial microorganisms in the soil under cadmium conditions. Pseudarthrobacter The content of these elements helps to improve the soil. Attached Figure Description

[0022] Figure 1 This is a colony morphology diagram of strain Cy28;

[0023] Figure 2 This is a growth curve of strain Cy28;

[0024] Figure 3 The graph shows the change in polysaccharide production of strain Cy28 over time.

[0025] Figure 4 FTIR spectra of polysaccharides produced by strain Cy28 and pullulan polysaccharide standards;

[0026] Figure 5 The ability of strain Cy28 to remove Cd from soil filtrate;

[0027] Figure 6 Comparison of pullulan production capacity of strain Cy28 under Cd-containing and Cd-free environments;

[0028] Figure 7 Dry weight of the edible tissues of lettuce;

[0029] Figure 8 The cadmium content in the edible tissues of lettuce;

[0030] Figure 9 The cadmium content in lettuce roots;

[0031] Figure 10 The vitamin C content in the edible tissues of lettuce;

[0032] Figure 11 This refers to the chlorophyll content in the edible tissues of lettuce.

[0033] Figure 12 This refers to the soluble protein content in the edible tissues of lettuce.

[0034] Figure 13 The IAA content in the edible tissues of lettuce;

[0035] Figure 14 In the soil around the roots of lettuce Pseudarthrobacter The relative content of microorganisms. Detailed Implementation

[0036] The following description is based on specific embodiments:

[0037] Example 1: Isolation, screening and identification of Cy28, a budding short-stalked fungus.

[0038] Fresh grapes were purchased from markets in Shandong Province, China. The grape skins were placed in a mortar and ground thoroughly with an appropriate amount of physiological saline. Then, a gradient dilution method was used to dilute the grapes to a concentration of 10... -3 10 -4 10 -5 The sample suspension was spread onto PDA solid medium containing 200 μg / mL chloramphenicol and incubated upside down at 28°C for 48 h until single colonies grew. Single colonies were picked and cultured, ultimately resulting in the isolation of a bacterial strain. The morphology of this strain on PDA solid medium is as follows: Figure 1 As shown, the strain is black with irregular circular edges and a smooth surface, and it is named "Cy28".

[0039] The ITS rDNA gene sequence of strain Cy28 was sequenced by Sangon Biotech (Shanghai) Co., Ltd., and the sequencing results are shown in SEQ ID NO.1. The obtained ITS rRNA sequence was compared with existing gene sequences in the NCBI database. The results showed that strain Cy28 is similar to... Aureobasidium pullulans With a similarity of over 99%, and based on the physiological characteristics of the strain, this strain was identified as *Bratrichia coli*. Aureobasidium pullulans ).

[0040] Aureobasidium pullulans ( Aureobasidium Cy28 sp. was deposited on March 31, 2025 at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with accession number CCTCC NO: M 2025660.

[0041] Example 2: Preparation of Cy28 budding short-stalked fungal agent

[0042] Follow these steps:

[0043] (1) The budding short-stalked fungus Cy28 was inoculated onto PDA solid medium and activated at 28°C for 48 h to obtain the activated strain;

[0044] (2) The activated strain was inoculated into PDB liquid medium and cultured with shaking at 180 rpm and 28 ℃ for 72 h to obtain Cy28 bacterial culture;

[0045] (3) Transfer the Cy28 budding short-stalked fungal culture to a sterile bottle, centrifuge at 5000 rpm for 5 min, collect the cells, wash the cells with deionized water and resuspend them, and adjust the concentration of the culture to 5 × 10⁻⁶. 8 cfu / mL, yielding Cy28 bacterial agent.

[0046] Example 3: The ability of *Berberis brevis* Cy28 to produce pullulan polysaccharide

[0047] Follow these steps:

[0048] The activated strain from Example 2 was inoculated into PDB liquid medium and cultured with shaking at 180 rpm and 28°C for 120 h. Samples were taken every 12 h during this period, and the absorbance (OD) of the samples was measured at 600 nm using a spectrophotometer. 600 Then, a growth curve was plotted with time on the x-axis and absorbance on the y-axis. The results are as follows: Figure 2 As shown: OD of strain Cy28 at approximately 60 hours of culture. 600 The value is the highest, and the bacterial cells reach their maximum biomass.

[0049] The activated strain from Example 2 was inoculated into PDB liquid medium and cultured with shaking at 180 rpm and 28°C for 120 h. Samples were taken every 12 h, centrifuged at 10000 rpm for 5 min, and the supernatant was collected. Polysaccharides were extracted from the supernatant using alcohol extraction. A curve was then plotted with time on the x-axis and polysaccharide content on the y-axis. The results are shown below. Figure 3 As shown, the polysaccharide production of strain Cy28 tends to stabilize around 60 hours of cultivation, and reaches its highest level of 3.74 g / L around 84 hours.

[0050] The obtained polysaccharide was analyzed by FTIR (Fourier Transform Infrared Spectroscopy), and the results are as follows: Figure 4 As shown: The polysaccharide extracted from strain Cy28 is pullulan polysaccharide.

[0051] Example 4: Cadmium removal ability of Cy28 (a budding short-stalked mold)

[0052] Soil filtrate experiment:

[0053] Add 10 L of deionized water to 2.5 kg of soil, shake at 150 rpm for 48 h, centrifuge at 5000 rpm for 15 min, collect the supernatant, filter through a 0.45 μm microporous membrane, sterilize, and then mix thoroughly with PDB liquid culture medium at a volume ratio of 4:1. Add 2.5g cadmium chloride hydrate to obtain Cd. 2+ A sterile mixture with a final concentration of 50 mg / L was prepared. 100 mL of the mixture was transferred to an Erlenmeyer flask and inoculated with the Cy28 bacterial agent prepared in Example 2 at a volume percentage of 1%, serving as the Cy28 group. A mixture without Cy28 was used as the blank control group (CK group), with three replicates for each group. The mixture was incubated at 28°C and 180 rpm, with samples taken every 24 hours to determine the Cd content. The Cd content was determined by centrifuging the sample at 10,000 rpm for 5 minutes, collecting the supernatant, filtering it through a 0.45 μm filter membrane, and measuring the Cd content using inductively coupled plasma optical emission spectrometry (ICP-OES).

[0054] 100 mL of the above mixture was transferred to an Erlenmeyer flask and inoculated with the Cy28 bacterial agent prepared in Example 2 at a volume percentage of 1% as the Cd group. A Cd-free mixture was used as the negative control group (ck group), with three replicates for each group. The mixture was cultured at 28°C and 180 rpm, and samples were taken every 24 hours to determine the pullulan yield. The pullulan yield was determined by alcohol extraction followed by freeze-drying under vacuum, and weighing to confirm the pullulan yield.

[0055] The Cd content detection results are as follows: Figure 5 As shown, by Figure 5 It can be seen that, compared with the blank control group, strain Cy28 can significantly reduce the Cd content in soil filtrate. By day 11 of culture, Cd in soil filtrate was almost completely removed.

[0056] The results of pullulan polysaccharide content detection are as follows: Figure 6 As shown, by Figure 6 It was found that strain Cy28 produced more pullulan in a Cd-containing environment compared to the negative control group. Specifically, on day 2 of cultivation, the pullulan production in the negative control group began to stabilize at approximately 3.5 g / L; on day 5 of cultivation, the pullulan production in the Cy28 group began to stabilize at approximately 4.5 g / L, representing an increase of approximately 28.57% compared to the negative control group.

[0057] Example 5: Application of Cy28, a budding short-stem mold, in lettuce cultivation

[0058] The Cy28 strain was applied to a potted lettuce experiment, and the following steps were followed:

[0059] Cultivation pots were 14cm (side length) × 11cm (height), containing 1.5kg of soil. Cadmium chloride (Cd) solution (2.5 ppm) was added to the soil to final concentrations of 0mg / kg, 0.5mg / kg, and 1mg / kg, respectively. After thorough mixing and equilibration for 45 days, purchased lettuce seeds were surface-sterilized. Ten seeds were sown per pot, and after germination, seedlings were thinned to 5 plants per pot. The soil was regularly irrigated to maintain moisture. The cultivation pots were divided into four groups: CK, LF, DF, and P. The CK group received deionized water, the LF group received the Cy28 inoculant prepared in Example 2, the DF group received an inactivated Cy28 inoculant as a negative control, and the P group received a 2g / L pullulan polysaccharide solution (produced by strain Cy28). Each group had three replicates. At the third leaf stage of the lettuce, trenches 1-2cm deep were dug around the lettuce roots, and the above liquid was added to the trenches at a volume of 20mL per pot. The potted plants were then cultivated in a greenhouse (temperature 10~22℃, relative humidity 30~45%, normal light) for a total cultivation period of 45 days.

[0060] After cultivation, edible tissues and roots of lettuce were collected. These were washed sequentially with 0.1M EDTA solution and distilled water, and then divided into two equal portions. One portion was inactivated at 105℃ for 30 min and dried at 65℃ to constant weight. The weight of the dried edible tissue was recorded as the dry weight. The dried edible tissue and roots were then ground, pulverized, and digested to determine the Cd content. The other portion of fresh edible tissue was used to determine the contents of vitamin C, chlorophyll, soluble protein, and IAA. Simultaneously, soil in close contact with the lettuce roots (rhizosphere soil) was collected to determine the microbial components in the soil. The methods and results are as follows:

[0061] (1) The results of the determination of the dry weight of edible tissue of lettuce are as follows: Figure 7 As shown, by Figure 7 It was found that in environments with cadmium content of 0 mg / kg, 0.5 mg / kg, and 1 mg / kg, the dry weight of edible lettuce tissue in the LF group with added Cy28 was significantly increased compared to the CK group. This indicates that Cy28 can significantly increase the dry weight of edible lettuce tissue in both cadmium-free and cadmium-containing environments. In addition, the P group with added pullulan also significantly increased the dry weight of edible lettuce tissue, indicating that pullulan produced by Cy28 is one of the components that enables it to exert its growth-promoting function.

[0062] (2) Determination of Cd content in edible tissues and roots: Accurately weigh 0.2000 g of dried sample into a polytetrafluoroethylene digestion tube, add 10 mL of concentrated nitric acid and let stand overnight, then place it in a microwave digester for microwave digestion. After the digestion, remove the acid to about 1 mL, and make up to 25 mL with distilled water. After filtering through a 0.45 μm filter membrane, the digestion solution was obtained. The Cd content in the digestion solution was detected by ICP-AES. The results are as follows: Figure 8~Figure 9 As shown. By Figure 8~Figure 9 It was found that in environments with cadmium content of 0.5 mg / kg and 1 mg / kg, the Cd content in the edible tissues and roots of lettuce in the LF group with added Cy28 and the P group with added pullulan polysaccharide was significantly reduced compared with the CK group. This indicates that Cy28 can significantly reduce the Cd content in the edible tissues and roots of lettuce, and can make the Cd content in the edible tissues meet the edible standard (cadmium limit of 0.2 mg / kg) specified in the national standard GB 2762-2022.

[0063] (3) Determination of Vitamin C Content: Fresh edible tissues were taken and the vitamin C content was determined according to the method in national standard GB 5009.86-2016. The results are as follows: Figure 10 As shown. By Figure 10It was found that in environments with cadmium content of 0 mg / kg, 0.5 mg / kg, and 1 mg / kg, the vitamin C content of edible lettuce tissues in the LF group with added Cy28 and the P group with added pullulan polysaccharide were significantly increased compared with the CK group. This indicates that Cy28 can significantly increase the vitamin C content of edible lettuce tissues in both cadmium-free and cadmium-containing environments.

[0064] (4) Chlorophyll content determination: Accurately weigh 2.0g of fresh edible tissue, add a small amount of calcium carbonate, quartz sand and 95% ethanol solution and grind, centrifuge at 10000rpm for 2min, remove the supernatant, and measure the absorbance at 665nm and 649nm using an enzyme-linked immunosorbent assay (ELISA) reader. Calculate the total chlorophyll content (C0) according to the following formula. 总 ).

[0065] C a =13.95 A665 -6.88 A649 ;

[0066] C b =24.96 A649 -7.32 A665 ;

[0067] C 总 =C a +C b .

[0068] The calculation results are as follows Figure 11 As shown, by Figure 11 It was found that in environments with cadmium content of 0 mg / kg, 0.5 mg / kg, and 1 mg / kg, the chlorophyll content of edible lettuce tissues in the LF group with added Cy28 and the P group with added pullulan polysaccharide were significantly increased compared with the CK group. This indicates that Cy28 can significantly increase the chlorophyll content of edible lettuce tissues in both cadmium-free and cadmium-containing environments.

[0069] (5) Determination of soluble protein content: Accurately weigh 2.0g of fresh edible tissue, add 5mL of distilled water and grind into a homogenate. Centrifuge at 12000×g for 20min at 4℃, collect the supernatant, which is the soluble protein extract. Determine the soluble protein content in the extract according to the method in national standard GB5009.5-2016. The results are as follows: Figure 12 As shown. By Figure 12It was found that in environments with cadmium content of 0 mg / kg, 0.5 mg / kg, and 1 mg / kg, the soluble protein content of edible lettuce tissues in the LF group with added Cy28 and the P group with added pullulan polysaccharide were significantly increased compared with the CK group. This indicates that Cy28 can significantly increase the soluble protein content of edible lettuce tissues in both cadmium-free and cadmium-containing environments.

[0070] (6) IAA content determination: Fresh edible tissue was taken, and the content of IAA (auxin-indoleacetic acid) was determined using a plant auxin enzyme-linked immunosorbent assay kit. The results are as follows: Figure 13 As shown. By Figure 13 It was found that in environments with cadmium content of 0 mg / kg, 0.5 mg / kg, and 1 mg / kg, the IAA content of edible lettuce tissue in the LF group with added strain Cy28 was significantly increased compared with that in the CK group. This indicates that strain Cy28 can significantly increase the IAA content of edible lettuce tissue in both cadmium-free and cadmium-containing environments.

[0071] (7) Determination of microbial components in soil: Bacterial genomic DNA was extracted from rhizosphere soil samples using a rapid DNA extraction kit. The extracted DNA was amplified using universal primers 338F and 806R. The amplified fragment was the V4 region of bacterial 16S rRNA, and the nucleotide sequences of 338F and 806R are shown in SEQ ID NO. 2~3, respectively. The amplified fragment was sequenced using Illumina Hiseq 2000. Based on the sequencing results, the microorganisms in the rhizosphere soil included... Pseudarthrobacter The genus *Pseudorobacter* typically possesses various beneficial functions such as biodegradation and biomineralization.

[0072] In different treatment groups Pseudarthrobacter The relative content of microorganisms such as Figure 14 As shown. By Figure 14 It was found that in environments with cadmium content of 0.5 mg / kg and 1 mg / kg, the rhizosphere soil of lettuce in the LF group with added Cy28 and the P group with added pullulan polysaccharide showed better results. Pseudarthrobacter The content of all microorganisms was significantly increased compared to the control group, indicating that strain Cy28 can significantly increase the content of microorganisms in the soil under cadmium conditions. Pseudarthrobacter It contains microorganisms, which helps to improve the soil.

[0073] In summary, the budding short-stem mold Cy28 provided by this invention can reduce the Cd content in soil and produce more pullulan in Cd-containing environments. When applied to lettuce cultivation, it can significantly increase the dry weight of edible lettuce tissues and reduce the Cd content in both cadmium-free and cadmium-containing environments, bringing the Cd content in edible lettuce tissues to edible standards. Furthermore, it can increase the vitamin C, chlorophyll, soluble protein, and IAA content of edible lettuce tissues. Simultaneously, it can significantly increase beneficial microorganisms in the soil under cadmium conditions. Pseudarthrobacter The content of these elements helps to improve the soil.

Claims

1. A short-stalked fungus Cy28, characterized in that, The short-stalked fungus ( Aureobasidium Cy28 sp. was deposited on March 31, 2025 at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with accession number CCTCCNO: M 2025660.

2. The method for culturing Cy28 of claim 1, characterized in that, Includes the following steps: (1) Inoculate Cy28 of the short-skinned dermal strain onto PDA solid medium and activate it at 28-30℃ to obtain the activated strain; (2) Inoculate the activated strain from step (1) into PDB liquid culture medium and culture it under shaking conditions at 160~180 rpm and 28~30℃ to obtain Cy28 bacterial culture.

3. The application of Cy28, the short-stalked fungus according to claim 1, is characterized in that, It is used in the production of pullulan.

4. The application as described in claim 3, characterized in that, The short-stalked fungus Cy28 produces pullulan under cadmium conditions.

5. The application of Cy28, the short-stalked fungus according to claim 1, is characterized in that, It is used to reduce the cadmium content in soil.

6. The application of Cy28, the short-stalked fungus according to claim 1, is characterized in that, It is used in lettuce cultivation.

7. The application as described in claim 6, characterized in that, The Cy28 short-stem mold is used to promote lettuce growth, reduce cadmium content in edible tissues and roots of lettuce, and increase vitamin C, chlorophyll, soluble protein, and indoleacetic acid content in edible tissues of lettuce.

8. The application as described in claim 6, characterized in that, The Cy28 short-stalked mold was used for lettuce cultivation in a cadmium environment.

9. A live bacteria preparation, characterized in that, The active ingredient is Cy28, the short-stalked fungus described in claim 1.

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