Aureobasidium pullulans Cy28 and application thereof

Abstract

The invention relates to aureobasidium pullulans Cy28 and application thereof, and belongs to the technical field of microorganisms, the aureobasidium pullulans Cy28 can reduce the content of Cd in soil and can generate more pullulan in a Cd-containing environment; when the culture medium is applied to lettuce planting, the dry weight of edible tissues of the lettuce can be remarkably increased in a cadmium-free or cadmium-containing environment, and the content of Cd in the edible tissues and roots of the lettuce is reduced, so that the content of Cd in the edible tissues of the lettuce reaches the edible standard; in addition, the vitamin C content, the chlorophyll content, the soluble protein content and the IAA content of edible tissues of the lettuce can be improved; meanwhile, the content of beneficial microorganism Pseudarthrobacter in the soil can be remarkably increased in a cadmium environment, and the effect of improving the soil is achieved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial technology, in particular to a strain of Aureobasidium pullulans Cy28 and its application. BACKGROUND

[0002] Cadmium (Cd) is one of the most toxic heavy metals and is listed as one of the core pollutants for prevention and control of heavy metal pollution in China. Overuse of chemical fertilizers and pesticides, mining, smelting, discharge of industrial wastewater containing cadmium, and direct use of untreated municipal wastewater in farmland are the main reasons for cadmium pollution in farmland. Cadmium pollution in farmland can inhibit soil microbial activity and reduce crop yield. In light or moderate cadmium-contaminated soil, food crops, vegetables, fruits, and pasture plants can accumulate cadmium in their bodies and enter the human body through the food chain, posing a serious threat to human health.

[0003] In-situ immobilization technology is an important means of repairing soil heavy metal pollution, especially suitable for large-area light and moderate cadmium-contaminated farmland. The core principle is to change the chemical form of cadmium (such as converting it into a poorly soluble compound) by adding a passivation agent to reduce the bioavailability and mobility of cadmium. However, continuous addition of organic and inorganic passivation agents can have harmful effects on soil properties, structure, and ecosystems.

[0004] Microbial in-situ immobilization technology can confine functional microorganisms (such as degrading bacteria and mineralizing bacteria) to carriers or specific areas, allowing them to exert their metabolic activity in contaminated environments. It is a cutting-edge means in the field of environmental remediation. Currently, most reports in the existing technology about simultaneously achieving heavy metal degradation and promoting plant growth are combinations of multiple microorganisms or combinations of microorganisms with certain carriers to exert multiple functions in soil. However, there are relatively few reports on single strains that combine plant growth promotion, heavy metal degradation, and soil improvement functions. Therefore, developing a multifunctional microbial strain is a pressing problem in the field of agricultural microbial technology. SUMMARY

[0005] In view of the deficiencies in the prior art, the present application provides an Aureobasidium pullulans Cy28 and its application.

[0006] The technical scheme of the present application is as follows: An Aureobasidium pullulans Cy28, wherein the Aureobasidium pullulans (Aureobasidium sp.) Cy28 was deposited with the China Center for Type Culture Collection on March 31, 2025, at the address of China. Wuhan. Wuhan University, and the deposit number is CCTCC NO: M2025660. Aureobasidium The ITS rDNA sequence of the Aureobasidium pullulans Cy28 is shown in SEQ ID NO. 1.

[0007]

[0008] ​The culture method of the Aureobasidium pullulans Cy28 comprises the following steps: (1) inoculate the Aureobasidium pullulans Cy28 to PDA solid culture medium, and activate culture under the condition of 28-30 DEG C, to obtain an activated strain; (2) inoculate the activated strain in step (1) to PDB liquid culture medium, and oscillation culture under the condition of 160-180 rpm and 28-30 DEG C, to obtain the Aureobasidium pullulans Cy28 bacterial liquid.

[0009] The application of the Aureobasidium pullulans Cy28 is applied to produce pullulan.

[0010] Preferably, the Aureobasidium pullulans Cy28 produces pullulan in a cadmium environment.

[0011] The application of the Aureobasidium pullulans Cy28 is applied to reduce the cadmium content in soil.

[0012] The application of the Aureobasidium pullulans Cy28 is applied to lettuce planting.

[0013] Preferably, the Aureobasidium pullulans Cy28 is applied to promote the growth of lettuce, reduce the cadmium content in edible tissues and roots of lettuce, increase the vitamin C content, chlorophyll content, soluble protein content and indole acetic acid content in edible tissues of lettuce.

[0014] Preferably, the Aureobasidium pullulans Cy28 is used for lettuce planting in a cadmium environment.

[0015] The application of the Aureobasidium pullulans Cy28 is applied to improve soil, and the improved soil is to increase the content of Pseudomonas in cadmium contaminated soil. Pseudarthrobacter

[0016] A live bacterial preparation with the Aureobasidium pullulans Cy28 as an effective component.

[0017] The beneficial effects of the present application are as follows: The present application provides an Aureobasidium pullulans Cy28, which can reduce the Cd content in soil, and can produce more pullulan in a Cd-containing environment; when applied to lettuce planting, it can significantly increase the dry weight of edible tissues of lettuce in a cadmium-free or cadmium-containing environment, reduce the Cd content in edible tissues and roots of lettuce, so that the Cd content in edible tissues of lettuce reaches the edible standard, and additionally, it can increase the vitamin C content, chlorophyll content, soluble protein content and IAA content in edible tissues of lettuce; at the same time, it can significantly increase the content of Pseudomonas in soil in a cadmium environment, and play a role in improving soil. Pseudarthrobacter BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 ​​A colony morphology chart of strain Cy28; Figure 2 A growth curve chart of strain Cy28; Figure 3 A polysaccharide production curve chart of strain Cy28 over time; Figure 4 FTIR detection chart of polysaccharide produced by strain Cy28 and pullulan standard; Figure 5 Strain Cy28's ability to remove Cd in soil filtrate; Figure 6 Strain Cy28's ability to produce pullulan in Cd-containing and non-Cd-containing environments; Figure 7 Dry weight of lettuce edible tissue; Figure 8 Cadmium content in lettuce edible tissue; Figure 9 Cadmium content in lettuce roots; Figure 10 Vitamin C content in lettuce edible tissue; Figure 11 Chlorophyll content in lettuce edible tissue; Figure 12 Soluble protein content in lettuce edible tissue; Figure 13 IAA content in lettuce edible tissue; Figure 14 Relative content of microorganisms in the rhizosphere soil of lettuce Pseudarthrobacter DETAILED DESCRIPTION

[0019] The following will be described in conjunction with specific examples: Example 1: Isolation, screening and identification of Aureobasidium pullulans Cy28 Fresh grapes were purchased from the market in Shandong Province, China. The grape skin was taken and ground in a mortar with an appropriate amount of physiological saline. Gradient dilution was then performed to obtain sample suspensions with gradients of 10 -3 , 10 -4 , and 10 -5 . The sample suspensions were then spread on PDA solid medium containing 200 μg / mL chloramphenicol and incubated at 28°C for 48 h until single colonies were formed. Single colonies were picked and cultured. A strain was finally isolated, which had a morphology on PDA solid medium as shown in Figure 1 The strain was black with an irregular round edge and a smooth surface, and was named "Cy28".

[0020] ​The ITS rDNA gene sequence of the strain Cy28 was entrusted to Shengong Bioengineering (Shanghai) Co., Ltd. for sequencing, and the sequencing result is shown as SEQ ID NO. 1; the obtained ITS rRNA sequence was subjected to similarity comparison with the existing gene sequences in the NCBI database, and the result showed that the similarity of the strain Cy28 with Aureobasidium pullulans was more than 99%, and in combination with the physiological characteristics of the strain, the strain was identified as Aureobasidium pullulans Aureobasidium pullulans ).

[0021] Aureobasidium pullulans Aureobasidium sp. Cy28 was preserved in the China Center for Type Culture Collection on March 31, 2025, the address is: China. Wuhan. Wuhan University, and the preservation number is CCTCC NO: M 2025660.

[0022] Example 2: Preparation of the Aureobasidium pullulans Cy28 microbial agent The following steps were performed: (1) The Aureobasidium pullulans Cy28 was inoculated on PDA solid medium, and activated culture was performed at 28°C for 48h to obtain an activated strain; (2) The activated strain was inoculated in PDB liquid medium, and shaking culture was performed at 180rpm and 28°C for 72h to obtain the Aureobasidium pullulans Cy28 bacterial liquid; (3) The Aureobasidium pullulans Cy28 bacterial liquid was transferred to a sterile bottle, centrifuged at 5000rpm for 5min, the bacterial bodies were collected, washed with deionized water and resuspended, the concentration of the bacterial liquid was adjusted to 5x10 8 cfu / mL to obtain the Cy28 microbial agent.

[0023] Example 3: Ability of Aureobasidium pullulans Cy28 to produce pullulan The following steps were performed: The activated strain of Example 2 was inoculated in PDB liquid medium, and shaking culture was performed at 180rpm and 28°C for 120h, during which sampling was performed once every 12h, the absorbance OD 600 of the sample was detected at 600nm by a spectrophotometer, and then a growth curve was drawn with time as the horizontal coordinate and absorbance as the vertical coordinate, and the result is shown in Figure 2 : The OD 600 value of the strain Cy28 was the highest at about 60h of culture, and the bacterial bodies reached the maximum biomass.

[0024] 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.

[0025] 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.

[0026] Example 4: Cadmium removal ability of Cy28 (a budding short-stalked mold) Soil filtrate experiment: 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).

[0027] 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.

[0028] The results of the Cd content test are as follows: Figure 5 As shown, by Figure 5It can be seen that compared with the blank control group, strain Cy28 can significantly reduce the Cd content in the soil filtrate. On the 11th day of culture, the Cd in the soil filtrate was almost completely removed.

[0029] The detection results of pullulan content are shown in Table 2. Figure 6 Figure 6 It can be seen that compared with the negative control group, strain Cy28 can produce more pullulan in the Cd-containing environment. Specifically, on the 2nd day of culture, the pullulan yield in the negative control group began to stabilize at about 3.5 g / L; on the 5th day of culture, the pullulan yield in the Cy28 group began to stabilize at about 4.5 g / L, which was about 28.57% higher than that in the negative control group.

[0030] Example 5: Application of Aureobasidium pullulans strain Cy28 in lettuce planting Strain Cy28 was applied to a lettuce pot experiment, which was carried out according to the following steps: The cultivation pot was 14 cm (side length) x 11 cm (height), containing 1.5 kg of soil. 2.5 g of cadmium chloride was added to the soil to obtain a final concentration of Cd of 0 mg / kg, 0.5 mg / kg and 1 mg / kg, respectively. After mixing well with the soil and balancing for 45 days, the purchased lettuce seeds were surface sterilized, and 10 seeds were sown in each pot. After germination, thinning was performed to obtain 5 plants per pot. The soil was regularly irrigated to maintain moisture. The cultivation pots were divided into CK group, LF group, DF group and P group. Deionized water was added to the CK group, Cy28 microbial agent prepared in Example 2 was added to the LF group, inactivated Cy28 microbial agent was added to the DF group as a negative control, and a solution of pullulan (produced by strain Cy28) with a concentration of 2 g / L was added to the P group. Each group had 3 parallel samples. At the third leaf stage of the lettuce, a 1-2 cm deep trench was dug around the roots of the lettuce, and the above-mentioned liquid was added to the trench at a volume of 20 mL per pot. The pots were cultivated in a greenhouse (temperature 10-22℃, relative humidity 30-45%, normal light) for a total cultivation period of 45 days.

[0031] After cultivation, the edible tissues of the lettuce and the lettuce roots were collected, washed with 0.1 M ethylenediaminetetraacetic acid (EDTA) solution and distilled water, and then divided into two equal parts. One part was inactivated at 105℃ for 30 min and dried at 65℃ to constant weight. The weight of the dried edible tissues was recorded as the dry weight. The dried edible tissues and roots were ground and digested to determine the Cd content. The other part of the fresh edible tissues was used to determine the contents of vitamin C, chlorophyll, soluble protein and IAA. At the same time, the soil closely combined with the lettuce roots (rhizosphere soil) was collected to determine the microbial components in the soil. The determination methods and results are as follows: (1) The determination results of the dry weight of the edible tissues of the lettuce are shown in Table 3.​Figure 7 As shown in Figure 7 It can be seen that in the environment with cadmium content of 0 mg / kg, 0.5 mg / kg and 1 mg / kg, the dry weight of the edible tissue of the lettuce in the LF group with strain Cy28 added was significantly higher than that in the CK group, which indicated that strain Cy28 could significantly improve the dry weight of the edible tissue of the lettuce in the environment without cadmium or with cadmium. In addition, the P group with pullulan added could also significantly improve the dry weight of the edible tissue of the lettuce, which indicated that the pullulan produced by strain Cy28 was one of the components that made it play a function of promoting growth.

[0032] (2) Determination of Cd content in edible tissue and roots: 0.2000 g of dried sample was accurately weighed in a polytetrafluoroethylene digestion tube, 10 mL of concentrated nitric acid was added and placed overnight, then placed in a microwave digestion instrument for microwave digestion. After the end, the acid was chased to about 1 mL, and distilled water was used to dilute to 25 mL. After filtration through a 0.45 μm filter membrane, the digestion solution was obtained, and the Cd content in the digestion solution was detected by ICP-AES. The determination results are shown in Figure 8~Figure 9 As shown in Figure 8~Figure 9 It can be seen that in the environment with cadmium content of 0.5 mg / kg and 1 mg / kg, the Cd content in the edible tissue and roots of the lettuce in the LF group with strain Cy28 added and the P group with pullulan added was significantly lower than that in the CK group, which indicated that strain Cy28 could significantly reduce the Cd content in the edible tissue and roots of the lettuce, and could make the Cd content in the edible tissue reach the edible standard (cadmium limit 0.2 mg / kg) stipulated in the national standard GB 2762-2022.

[0033] (3) Determination of vitamin C content: fresh edible tissue was taken, and the content of vitamin C therein was detected according to the method in the national standard GB 5009.86-2016. The results are shown in Figure 10 As shown in Figure 10 It can be seen that in the environment with cadmium content of 0 mg / kg, 0.5 mg / kg and 1 mg / kg, the vitamin C content in the edible tissue of the lettuce in the LF group with strain Cy28 added and the P group with pullulan added was significantly higher than that in the CK group, which indicated that strain Cy28 could significantly improve the vitamin C content in the edible tissue of the lettuce in the environment without cadmium or with cadmium.

[0034] (4) Determination of chlorophyll content: 2.0 g of fresh edible tissue was accurately weighed, a small amount of calcium carbonate, quartz sand and 95% ethanol solution was added for grinding, centrifuged at 10000 rpm for 2 min, the supernatant was removed, and the absorbance was measured at 665 nm and 649 nm respectively by an enzyme marker. The total chlorophyll content (C 总 ).

[0035] Ca =13.95 A665 -6.88 A649 ; C b =24.96 A649 -7.32 A665 ; C 总 =C a +C b .

[0036] 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.

[0037] (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 12 It 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.

[0038] (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.

[0039] (7) Microbial component determination in soil: bacterial genomic DNA of rhizosphere soil samples is extracted by using a rapid DNA extraction kit, the extracted DNA is amplified by using universal primers 338F and 806R, the amplified fragments are V4 regions of bacterial 16S rRNA, wherein the nucleotide sequences of 338F and 806R are shown as SEQ ID NO. 2-3; the amplified fragments are subjected to high-throughput sequencing by using an illumina Hiseq 2000, and according to the sequencing results, it is analyzed that the microorganisms in the rhizosphere soil include Pseudarthrobacter Pseudomonas, which usually has various beneficial functions such as biodegradation and biomineralization.

[0040] The relative content of Pseudomonas microorganisms in different treatment groups is shown as Pseudarthrobacter The relative content of Pseudomonas microorganisms in different treatment groups is shown as Figure 14 It can be obtained that in the environment with a cadmium content of 0.5 mg / kg or 1 mg / kg, the content of Pseudomonas microorganisms in the rhizosphere soil of the lettuce in the LF group added with the strain Cy28 and the P group added with pullulan is significantly increased compared with the CK group. Figure 14 The content of Pseudomonas microorganisms in the LF group added with the strain Cy28 and the P group added with pullulan is significantly increased compared with the CK group, which indicates that the strain Cy28 can significantly increase the content of Pseudomonas microorganisms in the soil in the cadmium environment, thereby playing a role in improving the soil. Pseudarthrobacter The content of Pseudomonas microorganisms in the LF group added with the strain Cy28 and the P group added with pullulan is significantly increased compared with the CK group, which indicates that the strain Cy28 can significantly increase the content of Pseudomonas microorganisms in the soil in the cadmium environment, thereby playing a role in improving the soil. Pseudarthrobacter The content of Pseudomonas microorganisms in the LF group added with the strain Cy28 and the P group added with pullulan is significantly increased compared with the CK group, which indicates that the strain Cy28 can significantly increase the content of Pseudomonas microorganisms in the soil in the cadmium environment, thereby playing a role in improving the soil.

[0041] In conclusion, the Aureobasidium pullulans Cy28 provided by the present application can reduce the Cd content in the soil, and can produce more pullulan in the Cd-containing environment; when applied to the cultivation of lettuce, the dry weight of the edible tissue of the lettuce can be significantly increased, the Cd content in the edible tissue and the roots of the lettuce can be reduced, the Cd content in the edible tissue of the lettuce can reach the edible standard, in addition, the vitamin C content, the chlorophyll content, the soluble protein content and the IAA content of the edible tissue of the lettuce can be increased; meanwhile, the content of Pseudomonas microorganisms in the soil can be significantly increased in the cadmium environment, thereby playing a role in improving the soil. Pseudarthrobacter the content of Pseudomonas microorganisms in the soil can be significantly increased in the cadmium environment, thereby playing a role in improving the soil.

Claims

1. A budding short-stalked mold Cy28, characterized in that, 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: M 2025660.

2. The method for culturing Cy28, a budding short-stalked fungus, as described in claim 1, is characterized in that... Includes the following steps: (1) Inoculate the budding short-stalked fungus Cy28 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 the budding short-stalked fungus Cy28 according to claim 1, characterized in that, It is used in the production of pullulan.

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

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

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

7. The application as described in claim 6, characterized in that, The aforementioned Cy28 budding 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 budding short-stem mold Cy28 was used for lettuce cultivation in a cadmium environment.

9. The application of the budding short-stalked fungus Cy28 according to claim 1, characterized in that, It is applied to improve soil, wherein the improved soil increases the content of *Pseudomonas* microorganisms in cadmium-contaminated soil.

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

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