Abnormal wickerhamomyces anomalus was1 and applications thereof

Abstract

The present application relates to the field of biotechnology, in particular to a strain of Wickerhamomyces anomalus WAS1 and application thereof.The strain WAS1 has a preservation number of CDMCC NO:61811.It is found through experiments that the strain can be applied in preparation of organic fertilizer, can promote crop growth, improve crop yield and quality, and has a remarkable effect, and the strain provides excellent strain resources for the field of crop yield increase application and the like.

Description

[Technical Field]

[0001] This invention relates to the field of biotechnology, and in particular to Wickham yeast Anomala WAS1 and its applications. [Background Technology]

[0002] In recent decades, the use of chemical fertilizers and pesticides has played a significant role in ensuring increased and stable grain production. However, the difficulty in simultaneously achieving high yield and quality in breeding, coupled with the long-term irrational use of chemical fertilizers and pesticides, has seriously threatened the environment and agricultural product safety. Therefore, finding new, economical, efficient, and environmentally compatible agricultural methods and technologies is an urgent need to address the current environmental pollution and food safety crises. Fully utilizing and leveraging the growth-promoting and pest-resistant functions of microorganisms in the ecosystem is of significant ecological and economic importance for improving crop productivity, protecting and improving the agricultural ecological environment, and promoting sustainable agricultural development.

[0003] Yeast cultures have a complex and rich nutritional composition, containing abundant proteins and nucleic acids, making them an excellent microecological preparation and a superior fertilizer raw material. Furthermore, compared to chemical fertilizers and pesticides, microecological fertilizers are non-toxic, pollution-free, residue-free, and environmentally friendly, attracting significant attention from the planting and fertilizer industries and possessing broad development and utilization prospects in agricultural environmental protection. [Summary of the Invention]

[0004] In view of the above-mentioned efforts to discover more superior Wickerhamomyces anomalus yeasts and promote the development and utilization of Wickerhamomyces anomalus yeast resources, the purpose of this invention is to provide Wickerhamomyces anomalus WAS1 and its application in promoting crop growth.

[0005] A strain of Wickerhamomyces anomalus, named Wickerhamomyces anomalus WAS1, with accession number CDMCC NO: 61811, deposited at Guangdong Provincial Center for Microbial Culture Collection, Guangdong, China, on July 16, 2021.

[0006] The present invention also includes the application of the above-mentioned Wickerhamomyces anomalus WAS1 in the preparation of organic fertilizer.

[0007] The present invention also provides an organic fertilizer prepared from Wickerhamomycesanomalus WAS1.

[0008] Furthermore, the organic fertilizer is applied in fruit cultivation.

[0009] Furthermore, the fruit is a citrus fruit, specifically a ponkan orange.

[0010] The present invention also provides a method for preparing the above-mentioned organic fertilizer, characterized by comprising the following steps:

[0011] (1) Preparation of fermentation broth: Strain WAS1 was inoculated into PDA medium and liquid fermentation was carried out to obtain a growth-promoting bacteria fermentation broth; the number of growth-promoting bacteria spores in the fermentation broth was ≥10. 8 pcs / ml;

[0012] (2) Crush the fruit peel residue, then inoculate with the growth-promoting bacteria fermentation broth obtained in step (1), adjust the water content to 65-75%, adjust the pH to 4.5-6, and then culture in shallow trays at 27-29℃ for 6-8 days until the growth-promoting bacteria produce 10 spores. 10 The fermentation product was prepared by collecting samples per gram (g) for later use.

[0013] (3) Mix the well-rotted chicken manure compost and the fermented material obtained in step (2) at a mass ratio of 2-4:1 for secondary fermentation. Turn the compost pile 1-2 times a day during the fermentation process, keep the fermentation temperature below 50℃, and ferment for 6-8 days. After fermentation, the content of growth-promoting bacteria should reach 1.0x10⁻⁶. 8 A compost mixture is obtained when the number of cells / g or more is greater than that.

[0014] (4) The organic fertilizer can be obtained by evaporating the moisture content of the compost mixture to below 30% under the condition that the temperature does not exceed 60°C.

[0015] Furthermore, in step (1), the fermentation temperature of the abnormal Wickham yeast is 28-30℃, and the yeast is stirred during the fermentation process at a speed of 180-200 rpm for 6-8 days.

[0016] Furthermore, in step (2), the inoculation amount of the growth-promoting bacteria fermentation broth accounts for 0.5-0.5% of the mass of the fruit peel and pomace.

[0017] The above-mentioned well-rotted chicken manure compost contains >35% organic matter, 1.2-2.0% organic ammonia, and 25-30% moisture.

[0018] Fruit peel residues, such as citrus peel residues, apple peel residues, and pineapple peel residues, can be used to prepare fertilizers. This rational utilization of fruit processing byproducts has broad market prospects.

[0019] The present invention has the following beneficial effects:

[0020] The strain Wickerhamomyces anomalus WAS1 described in this application effectively promotes crop growth and increases crop yield, demonstrating significant effects. This strain provides an excellent microbial resource for applications such as crop yield enhancement. A planting trial of Ponkan oranges was conducted, showing that the fresh weight of individual Ponkan orange shoots, shoot length, number of leaves per shoot, leaf width, leaf thickness, and leaf weight all increased with the application amount of organic fertilizer prepared using the strain described in this application. This organic fertilizer exhibits a growth-promoting and biomass-increasing effect on Ponkan oranges. [Attached Image Description]

[0021] Figure 1 This is a morphological diagram of the strain WAS1 of this invention; Figure 1 Figure A shows the front view of the colony morphology of the strain growing in the culture medium; Figure B shows the back view of the colony morphology.

Detailed Implementation Methods

[0022] The present invention will be further described below with reference to the accompanying drawings, embodiments, and experiments.

[0023] Example 1:

[0024] The strain Wickerhamomyces anomalus WAS1 in this embodiment has the accession number CDMCC NO: 61811. It is deposited at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Experimental Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province, on July 16, 2021.

[0025] The above-mentioned strains were selected from mango pulp.

[0026] The strains isolated above were subjected to the following tests:

[0027] The strain was cultured on PDA plates, with three samples per plate arranged in a triangular pattern, and incubated in the dark at 28°C for 5-6 days. Colonies were picked from the edges and repeatedly subcultured for 4-5 generations until a purified strain was obtained. The purified strain was then transferred to PDA slant and stored at 4°C. The purified strain was inoculated onto PDA plates and cultured at 28°C and 75% relative humidity for 5 days.

[0028] like Figure 1 As shown, identification revealed that the colonies had relatively neat edges, lacked rose-like structures, and were grayish-yellow in color, with a darker color in the center. The reverse side of the culture dish was white. The conidiophores were beaded, unbranched or dichotomously branched, with acute-angled branches and short spore-producing spores. The conidia were colorless monospores, mostly lemon-shaped, branched, and some had flat ends. The isolated strain was 530 bp. BLAST analysis showed that the isolated strain was *Saccharomyces cerevisiae*.

[0029] Example 2:

[0030] This embodiment provides a method for preparing organic fertilizer, wherein the organic fertilizer is prepared using the strain Wickerhamomyces anomalus WAS1 described in the embodiment as a raw material. The specific method is as follows:

[0031] (1) Preparation of fermentation broth: Strain WAS1 was inoculated into PDA medium and liquid fermentation was carried out at 28℃. Stirring was performed during fermentation at a speed of 180 rpm for 6 days to obtain the growth-promoting bacteria fermentation broth; the number of growth-promoting bacteria spores in the fermentation broth was ≥10. 8 pcs / ml;

[0032] (2) Crush the fruit peel residue, then inoculate it with the fermentation broth of the growth-promoting bacteria obtained in step (1), adjust the water content to 65%, adjust the pH to 4.5, and then culture it in a shallow dish at 27℃ for 6 days until the growth-promoting bacteria produce 10 spores. 10 The inoculum was prepared by adding 1 / g of each growth-promoting bacteria to obtain fermented material for later use; the inoculum amount of the growth-promoting bacteria fermentation broth accounted for 0.5% of the mass of the fruit peel and pomace.

[0033] (3) Mix the well-rotted chicken manure compost and the fermented material obtained in step (2) at a mass ratio of 2:1 for secondary fermentation. Turn the compost pile once a day during the fermentation process. The fermentation temperature should not exceed 50℃. Fermentation should last for 6 days. After fermentation, the content of growth-promoting bacteria should reach 1.0x10⁻⁶. 8 The compost mixture is obtained by adding more than 1000 spores per gram; the germinated organic matter content of the well-rotted chicken manure compost is >35%, the organic ammonia content is 1.2%, and the moisture content is 25%.

[0034] (4) The organic fertilizer can be obtained by evaporating the moisture content of the compost mixture to below 30% under the condition that the temperature does not exceed 60°C.

[0035] Example 3:

[0036] This embodiment provides a method for preparing organic fertilizer, wherein the organic fertilizer is prepared using the strain Wickerhamomyces anomalus WAS1 described in the embodiment as a raw material. The specific method is as follows:

[0037] (1) Preparation of fermentation broth: Strain WAS1 was inoculated into PDA medium and liquid fermentation was carried out at a fermentation temperature of 29℃. During the fermentation process, the mixture was stirred at a stirring speed of 190 rpm for 7 days to obtain the growth-promoting bacteria fermentation broth; the number of growth-promoting bacteria spores in the fermentation broth was ≥10. 8 pcs / ml;

[0038] (2) Crush the fruit peel residue, then inoculate it with the fermentation broth of the growth-promoting bacteria obtained in step (1), adjust the water content to 70%, adjust the pH value to 5.0, and then culture it in a shallow dish at 28℃ for 7 days until the growth-promoting bacteria produce 10 spores. 10 The amount of fermentation product obtained is 1% of the mass of fruit peel and pomace, and the inoculation amount of the growth-promoting bacteria fermentation broth is 1%.

[0039] (3) Mix the well-rotted chicken manure compost and the fermented material obtained in step (2) at a mass ratio of 3:1 for secondary fermentation. Turn the compost pile once a day during the fermentation process. The fermentation temperature should not exceed 50℃. Fermentation should last for 7 days. After fermentation, the content of growth-promoting bacteria should reach 1.0x10⁻⁶. 8 The compost mixture is obtained by adding more than 1000 spores per gram; the germinated organic matter content of the well-rotted chicken manure compost is >35%, the organic ammonia content is 1.6%, and the moisture content is 27%.

[0040] (4) The organic fertilizer can be obtained by evaporating the moisture content of the compost mixture to below 30% under the condition that the temperature does not exceed 60°C.

[0041] Example 4:

[0042] This embodiment provides a method for preparing organic fertilizer, wherein the organic fertilizer is prepared using the strain Wickerhamomyces anomalus WAS1 described in the embodiment as a raw material. The specific method is as follows:

[0043] (1) Preparation of fermentation broth: Strain WAS1 was inoculated into PDA medium and liquid fermentation was carried out at 30℃. Stirring was performed during fermentation at a speed of 200 rpm for 8 days to obtain the growth-promoting bacteria fermentation broth; the number of growth-promoting bacteria spores in the fermentation broth was ≥10. 8 pcs / ml;

[0044] (2) Crush the fruit peel residue, then inoculate it with the fermentation broth of the growth-promoting bacteria obtained in step (1), adjust the water content to 75%, adjust the pH to 6, and then culture it in a shallow dish at 29℃ for 6-8 days until the growth-promoting bacteria produce 10 spores. 10 The inoculum was prepared by adding 1 / g of growth-promoting bacteria to obtain fermented material for later use; the inoculum amount of growth-promoting bacteria fermentation broth accounted for 1.5% of the mass of fruit peel and pomace.

[0045] (3) Mix the well-rotted chicken manure compost and the fermented material obtained in step (2) at a mass ratio of 4:1 for secondary fermentation. Turn the compost twice a day during the fermentation process. The fermentation temperature should not exceed 50℃. Fermentation should last for 8 days. After fermentation, the content of growth-promoting bacteria should reach 1.0x10⁻⁶. 8The compost mixture is obtained by adding more than 1000 spores per gram; the germinated organic matter content of the well-rotted chicken manure compost is >35%, the organic ammonia content is 2.0%, and the moisture content is 30%.

[0046] (4) The organic fertilizer can be obtained by evaporating the moisture content of the compost mixture to below 30% under the condition that the temperature does not exceed 60°C.

[0047] Experimental example:

[0048] To illustrate the effectiveness of this application, the applicant established the following treatment groups, applying fertilizers from each group to the Ponkan orange orchard, with 10-15 trees per treatment. Each treatment was applied to the soil twice a year, in February and July, with each application accounting for 50% of the total annual fertilizer. During fertilization, a 100cm long, 30cm wide, and 40cm deep trench was dug along the drip line of the tree canopy. The fertilizer was mixed with the excavated soil and then backfilled into the trench. Other orchard management was carried out according to conventional methods. Except for the fertilizer, all other methods were the same. The specific groupings are as follows:

[0049] CK group: The fertilizer used was commercially available compound fertilizer (N:P2O5:K2O=15:15:15), developed and produced by Guizhou Xiyang Fertilizer Co., Ltd., applied at 4.5kg / plant;

[0050] Group 1: The organic fertilizer described in Example 3 was used, with an annual application rate of 8.6 kg / plant;

[0051] Group 2: The organic fertilizer described in Example 3 was used, with an annual application rate of 11.3 kg / plant;

[0052] Group 3: The organic fertilizer described in Example 3 was used, with an annual application rate of 14.2 kg / plant.

[0053] According to the test, the organic fertilizer described in Example 3 has a moisture content of 40.0%, an organic matter content of 18.88 g / kg, an N:P2O5:K2O ratio of 10:5:6, and a pH value of 7.4.

[0054] 1. Effects of different fertilizer treatments on the agronomic traits of Ponkan orange plants

[0055] The effects of different fertilizers on the growth of Ponkan orange shoots were investigated and statistically analyzed, as shown in Table 1:

[0056] Table 1. Effects of different fertilizers on the growth of Ponkan orange shoots.

[0057]

[0058] As shown in Table 1, the long-term fresh weight of single saplings, single sapling length, number of leaves per sapling, leaf width, single leaf thickness, and single leaf weight of Ponkan oranges all increased with the increase of the amount of organic fertilizer applied in this application. The organic fertilizer in this application contains a large amount of nitrogen, phosphorus, potassium, various amino acids, and plant hormones, which have the effect of promoting growth and increasing biomass in Ponkan oranges.

[0059] 2. The effect of fertilization on the growth of spring shoots of Ponkan oranges

[0060] The effects of different fertilizers on the growth of spring shoots of Ponkan oranges were investigated and statistically analyzed, as shown in Table 2:

[0061] Table 2. Effects of different fertilization methods on the growth of spring shoots of Ponkan oranges.

[0062] CK group 8.35b 2.86b 6.71b Group 1 12.39a 3.23a 6.91b Group 2 12.17a 3.15ab 6.70ab Group 3 12.72a 3.21a 7.49a

[0063] Table 2 shows that the four fertilization methods significantly affected spring shoot growth. In terms of spring shoot length and thickness, the control group (CK) showed significant differences between the conventional fertilization treatment and the other three treatments. There were no significant differences among the other three treatments. However, it was observed that the spring shoot length and thickness increased after all three fertilization treatments with different fertilizer amounts. The third treatment showed the largest values ​​for both spring shoot length and thickness, with an average spring shoot length of 12.72 cm, a thickness of 3.21 mm, and an average of 7 leaves, which were 4.37 cm, 0.35 mm, and 1 leaf respectively higher than the conventional fertilization treatment. While the differences between different fertilizer amounts were relatively small, there were significant differences in spring shoot length and thickness compared to the conventional fertilization treatment.

[0064] 3. The effect of fertilization on the growth of autumn shoots of Ponkan oranges

[0065] The effects of different fertilizers on the growth of autumn shoots of Ponkan oranges were investigated and statistically analyzed, as shown in Table 3:

[0066] Table 3. Effects of different fertilization methods on the growth of autumn shoots of Ponkan oranges.

[0067] CK group 15.49d 2.49b 11.39c Group 1 19.52c 3.03b 12.68bc Group 2 24.86b 3.77a 14.40b Group 3 30.42a 4.21a 18.27a

[0068] Table 3 shows that the four fertilization methods significantly affected the growth of autumn shoots. The conventional fertilization treatment differed significantly from the other three treatments in terms of shoot length and thickness. There were no significant differences among the other three treatments, but it was observed that the length and thickness of autumn shoots increased after all three organic fertilizer treatments. The third treatment showed the highest values ​​for both shoot length and thickness, with an average shoot length of 30.42 cm, a thickness of 4.21 mm, and an average of 18 leaves, which were 14.93 cm, 1.72 mm, and 7 leaves higher than the conventional fertilization (CK) treatment, respectively. While the differences among the different organic fertilizer treatments were relatively small, there were significant differences in shoot length and thickness compared to the no-fertilization treatment.

[0069] 4. The impact of fertilization on the fruit traits of Ponkan oranges in orchards

[0070] The effects of different fertilizers on the characteristics of Ponkan oranges were investigated and statistically analyzed, as shown in Table 4:

[0071] Table 4. Effects of different fertilization methods on the fruit characteristics of Ponkan oranges.

[0072]

[0073] Table 4 shows the results of the Ponkan fruit measurements. There were significant differences in single fruit weight among the treatments: Group 3 > Group 2 > Group 1 > CK group. However, the proportion of pulp weight to fruit weight increased with increasing organic fertilizer application, while peel weight decreased. Specifically, in Group 3, pulp weight accounted for 71.25%, an increase of 6.12% compared to the CK group. This is mainly determined by the thickness and weight of the Ponkan peel. Statistical analysis showed that compared to the CK group, Group 3 had significant differences in pulp weight, peel weight, pulp-to-fruit ratio, and peel-to-fruit ratio (P<0.05).

[0074] 5. The effects of different fertilization methods on the quality of Ponkan oranges

[0075] The effects of different fertilization methods on the quality of Ponkan oranges were investigated and statistically analyzed, as shown in Table 5:

[0076] Table 5. Effects of different fertilization methods on the quality of Ponkan oranges.

[0077]

[0078] Table 5 shows the fruit quality test results, indicating significant differences in vitamin C, total sugar, and total acid among the treatments. The soluble solids content increased in the organic fertilizer group, while the total acid content decreased with increasing application of the organic fertilizer. Statistical analysis showed that in the organic fertilizer group, the total sugar content in treatment group 2 was 2.37 percentage points higher than the chemical fertilizer control, and the total acid content decreased by 0.12 percentage points. The sugar-acid ratio was significantly higher than the chemical fertilizer control (P<0.05), resulting in improved taste and increased commercial value. This is related to the easily absorbed phosphorus and potassium elements in the organic fertilizer. Increased application of the organic fertilizer led to increased phosphorus and potassium absorption by the tangerines, while the total acidity decreased.

[0079] 6. The effect of fertilization on the yield of Ponkan oranges

[0080] The impact of different fertilization methods on the yield of Ponkan oranges was investigated and statistically analyzed, as shown in Table 6:

[0081] Table 6. Effects of different fertilization methods on the yield of Ponkan oranges.

[0082]

[0083] Table 6 shows that the application of the organic fertilizer and compound fertilizer of this application significantly increased the single fruit weight and single-tree yield of Ponkan oranges compared to the CK group. The yield per tree was in the order of: Group 3 > Group 2 > Group 1 > CK group. There was no significant difference in single fruit weight among the different application methods of the organic fertilizer of this application, but there was a significant difference in single-tree yield. The single-tree yield of Groups 3, 2, and 1 was significantly higher than that of the CK group, increasing by 11.66%, 8.33%, and 4.95% respectively. The average yield per acre for different fertilizer treatments was in the order of peel fruit yield, based on actual weighing at harvest. The yield of Group 3 was 28440.0 kg·hm². -2 The yield increased by 2970.00 kg compared to the CK group, representing an increase of 11.66%. The yield ranking was: Group 3 > Group 2 > Group 1 > CK group.

[0084] 7. The impact of fertilization on soil chemical properties in orchards

[0085] The effects of different fertilizer treatments on post-harvest soil nutrients were investigated and statistically analyzed, as shown in Table 7:

[0086] Table 7 Soil nutrient indices after citrus harvest under different fertilization treatments

[0087]

[0088]

[0089] As can be seen from Table 7, under the condition of equal nutrient fertilization, the soil organic matter, total nitrogen, available nitrogen, and available phosphorus in the citrus orchards that applied the organic fertilizer of this application all showed improvement, which is consistent with the conclusion of most researchers that the application of organic fertilizer can improve soil quality.

[0090] In summary, the organic fertilizer prepared by the WAS1 strain of this application, when applied to citrus cultivation, has both growth-promoting and quality-enhancing effects. It also shows broad development and utilization prospects in agricultural environmental protection.

[0091] The above description is a detailed description of the preferred embodiments of the present invention. However, the embodiments are not intended to limit the scope of the patent application of the present invention. All equivalent changes or modifications made under the technical spirit of the present invention should fall within the patent scope covered by the present invention.

Claims

1. An abnormal strain of Wickham yeast ( Wickerhamomyces anomalus The application of WAS1 in the preparation of organic fertilizer is characterized by, The abnormal Wickham yeast WAS1 is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number CDMCC NO: 61811.

2. An organic fertilizer, characterized in that, The organic fertilizer is made from the abnormal Wickham yeast described in claim 1 ( Wickerhamomyces anomalus It was prepared from WAS1.

3. The organic fertilizer according to claim 2, characterized in that, The organic fertilizer is used in fruit cultivation.

4. The organic fertilizer according to claim 3, characterized in that, The fruit in question is a citrus fruit.

5. The method for preparing organic fertilizer according to any one of claims 2-4, characterized in that, Includes the following steps: (1) Preparation of fermentation broth: The strain WAS1 was inoculated into PDA medium and liquid fermentation was carried out to obtain the fermentation broth of growth-promoting bacteria; (2) Crush the fruit peel residue, then inoculate the fermentation liquid of the growth-promoting bacteria obtained in step (1), adjust the water content to 65-75%, adjust the pH value to 4.5-6, and then culture it in a shallow dish at 27-29℃ for 6-8 days to obtain the fermented product for later use. (3) Mix the well-rotted chicken manure compost and the fermented material obtained in step (2) in a mass ratio of 2-4:1, and carry out secondary fermentation. During the fermentation process, turn the pile over 1-2 times a day, and the fermentation temperature should not exceed 50℃. Ferment for 6-8 days to obtain a compost mixture. (4) Evaporate the moisture content of the compost mixture to below 30% to obtain the organic fertilizer.

6. The method according to claim 5, characterized in that, The fermentation temperature of the abnormal Wickham yeast in step (1) is 28-30℃, and the fermentation time is 6-8 days.

7. The method according to claim 5, characterized in that, In step (2), the amount of inoculum of the growth-promoting bacteria fermentation liquid accounts for 0.5-1.5% of the mass of the fruit peel and pomace.

Images