Wickerhamomyces anomalus WAS1 and application thereof

Abstract

The invention relates to the technical field of biology, in particular to a strain Wickerhamomyces anomalus WAS1 and application of the strain Wickerhamomyces anomalus WAS1. The preservation number of the strain WAS1 is CDMCC (China General Microbiological Culture Collection Center) NO Experiments show that the strain can be applied to preparation of organic fertilizers, can promote crop growth and improve crop yield and quality, has a remarkable effect, and provides an excellent strain resource for the fields of crop yield increase application and the like.

Description

[Technical field]

[0001] The invention relates to the field of biotechnology, in particular to abnormal Wickham yeast WAS1 and applications thereof. [Background technology]

[0002] In recent decades, the use of chemical fertilizers and pesticides has played a huge role in ensuring the increase and stability of grain production. However, it is difficult to balance high yield and quality in breeding, and the long-term irrational use of chemical fertilizers and pesticides has seriously threatened the environment and agricultural product safety. Therefore, it is urgent to find new agricultural methods and technologies that are economical, efficient, and environmentally compatible to solve the current environmental pollution and food safety crisis. The full use and development of the growth-promoting and pest-resistant functions of microorganisms in the ecosystem has important ecological and economic significance for improving the production capacity of crops, protecting and improving the agricultural ecological environment, and promoting the sustainable development of agriculture.

[0003] The nutrient composition of yeast culture is complex and rich in nutrients, including protein, nucleic acid and other nutrients. It is a good microecological preparation and a very good fertilizer raw material. In addition, compared with chemical fertilizers and pesticides, microecological preparation fertilizers are non-toxic, pollution-free, residue-free, green and safe, and have attracted much attention from the planting and fertilizer industries. They have broad prospects for development and utilization in the protection of the agricultural natural environment. [Summary of the invention]

[0004] In view of the above-mentioned discovery of more abnormal Wickham yeasts with excellent traits and promotion of the development and utilization of abnormal Wickham yeast resources, the present invention aims to provide abnormal Wickham yeast (Wickerhamomyces anomalus) WAS1 and its application in promoting the growth of crops.

[0005] A strain of abnormal Wickham yeast, the strain is named as abnormal Wickham yeast (Wickerhamomycesanomalus) WAS1, with a preservation number of CDMCC NO: 61811; the preservation location is: Guangdong Provincial Microbiological Culture Collection Center, Guangdong, China, and the preservation time is July 16, 2021.

[0006] The present invention also includes the use of the abnormal Wickerhamomyces anomalus WAS1 in preparing organic fertilizer.

[0007] The invention also provides an organic fertilizer, which is prepared from Wickerhamomyces anomalus WAS1.

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

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

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

[0011] (1) Preparing fermentation broth: inoculating strain WAS1 into PDA medium for liquid fermentation to obtain a growth-promoting bacteria fermentation broth; the number of growth-promoting bacteria spores in the growth-promoting bacteria fermentation broth is ≥10 8 Pieces / ml;

[0012] (2) The fruit peel residue is crushed, and then the fermentation liquid of the growth-promoting bacteria obtained in step (1) is inoculated, the water content is adjusted to 65-75%, the pH value is adjusted to 4.5-6, and then the fruit peel residue is crushed, and the fermentation liquid of the growth-promoting bacteria is inoculated to 10% by weight. 10 / g, and obtain the fermented product for use;

[0013] (3) The decomposed chicken manure compost and the fermented product obtained in step (2) are mixed in a mass ratio of 2-4:1, and a secondary fermentation is performed. During the fermentation process, the compost is turned 1-2 times a day, the fermentation temperature does not exceed 50° C., and the fermentation is performed for 6-8 days. After the fermentation is completed, the content of the growth-promoting bacteria reaches 1.0×10 8 / g or more, to obtain a compost mixture;

[0014] (4) Under the condition that the temperature does not exceed 60° C., the water content of the compost mixture is evaporated to below 30%, thereby obtaining the organic fertilizer.

[0015] Furthermore, in step (1), the fermentation temperature of the abnormal Wickham yeast is 28-30° C., stirring is performed during the fermentation process, the stirring speed is 180-200 rpm, and the fermentation time is 6-8 days.

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

[0017] As mentioned above, in the decomposed chicken manure compost, the organic matter content is >35%, the organic ammonia content is 1.2-2.0%, and the water content is 25-30%.

[0018] The fruit peel residue can specifically be citrus peel residue, apple peel residue, pineapple peel residue and other fruit peel residues. The fruit peels can be used to prepare fertilizers, which is a rational use of the waste materials after fruit processing and has broad market development prospects.

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

[0020] The strain of the present application, Wickerhamomyces anomalus WAS1, can effectively promote crop growth and increase crop yields, and the effect is remarkable. The strain provides excellent bacterial resources for applications in the fields of increasing crop yields. The present application conducted a planting experiment on ponkan, and it can be seen that the fresh weight of ponkan branches, the length of a single branch, the number of leaves on a single branch, the width of a leaf, the thickness of a single leaf, and the weight of a single leaf all increase with the increase in the application amount of the organic fertilizer prepared by the strain of the present application. The organic fertilizer has the effect of promoting growth and increasing biomass on ponkan.

Brief Description of the Drawings

[0021] Figure 1 is a morphological diagram of the strain WAS1 of the present invention; Figure 1 Figure A is the front morphology of the colony of the strain growing in the culture medium; Figure B is the back morphology of the colony. [Specific implementation method]

[0022] The present invention will be further described below in conjunction with the accompanying drawings, examples and tests.

[0023] Embodiment 1:

[0024] The strain Wickerhamomyces anomalus WAS1 of this example has a deposit number of CDMCC NO: 61811. It is deposited in Guangdong Microbiological Culture Collection Center, address: 5th Floor, Dayuan Experimental Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province, and the deposit time is July 16, 2021.

[0025] The above strains were screened from mango pulp.

[0026] The strains isolated above were tested as follows:

[0027] The strain was cultured on PDA medium plates, with 3 samples on each plate in a triangular shape, and cultured in a 28°C constant temperature incubator in the dark for 5-6 days. The edge of the colony was picked and repeatedly subcultured for 4-5 generations until a purified strain was obtained, which was transferred to a PDA slant and stored at 4°C. The purified strain was inoculated on PDA and cultured for 5 days at 28°C and 75% relative humidity.

[0028] like Figure 1 As shown, the identification found that the colony edge was neat, without rosette structure, grayish yellow in color, darker in the middle of the colony, white on the back of the culture dish, beaded conidiophores, unbranched or dichotomously branched, with acute-angled branches and short conidiophores. Conidia were colorless single spores, mostly lemon-shaped, branched, and some with flat ends at the top. The isolated strain was 530 bp, and the BLAST analysis results showed that the isolated strain was abnormal Wickham yeast.

[0029] Embodiment 2:

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

[0031] (1) Preparing fermentation broth: inoculating strain WAS1 into PDA medium for liquid fermentation at a fermentation temperature of 28° C., stirring during the fermentation process at a stirring speed of 180 rpm, and fermenting for 6 days to obtain a growth-promoting bacteria fermentation broth; the number of growth-promoting bacteria spores in the growth-promoting bacteria fermentation broth is ≥10 8 Pieces / ml;

[0032] (2) The fruit peel residue was crushed, and then the fermentation liquid of the growth-promoting bacteria obtained in step (1) was inoculated, the water content was adjusted to 65%, the pH value was adjusted to 4.5, and then the mixture was cultured in a shallow dish at 27° C. for 6 days until the spore production of the growth-promoting bacteria reached 10 10 / g, and obtain the fermented product for use; wherein the inoculation amount of the growth-promoting bacteria fermentation liquid accounts for 0.5% of the mass of the fruit peel residue;

[0033] (3) The decomposed chicken manure compost and the fermented product obtained in step (2) are mixed in a mass ratio of 2:1, and a secondary fermentation is performed. The compost is turned once a day during the fermentation process. The fermentation temperature does not exceed 50° C. and the fermentation is performed for 6 days. After the fermentation is completed, the content of the growth-promoting bacteria reaches 1.0×10 8 / g or more, to obtain a compost mixture; the mature chicken manure compost has a germinated organic matter mass ratio content of> 35%, an organic ammonia mass ratio content of 1.2%, and a water content mass ratio of 25%;

[0034] (4) Under the condition that the temperature does not exceed 60° C., the water content of the compost mixture is evaporated to below 30%, thereby obtaining the organic fertilizer.

[0035] Embodiment 3:

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

[0037] (1) Preparing fermentation broth: inoculating strain WAS1 into PDA medium for liquid fermentation at a fermentation temperature of 29° C., stirring during the fermentation process at a stirring speed of 190 rpm, and fermenting for 7 days to obtain a growth-promoting bacteria fermentation broth; the number of growth-promoting bacteria spores in the growth-promoting bacteria fermentation broth is ≥10 8 Pieces / ml;

[0038] (2) The fruit peel residue is crushed, and then the fermentation liquid of the growth-promoting bacteria obtained in step (1) is inoculated, the water content is adjusted to 70%, the pH value is adjusted to 5.0, and then cultured in a shallow dish at 28° C. for 7 days until the spore production of the growth-promoting bacteria reaches 10 10 / g, and obtain the fermented product for use; wherein the inoculation amount of the growth-promoting bacteria fermentation liquid accounts for 1% of the mass of the fruit peel residue;

[0039] (3) The decomposed chicken manure compost and the fermented product obtained in step (2) are mixed in a mass ratio of 3:1, and a secondary fermentation is performed. The compost is turned once a day during the fermentation process. The fermentation temperature does not exceed 50° C. and the fermentation is performed for 7 days. After the fermentation is completed, the content of the growth-promoting bacteria reaches 1.0×10 8 / g or more, to obtain a compost mixture; the mature chicken manure compost has a germinated organic matter mass ratio content of> 35%, an organic ammonia mass ratio content of 1.6%, and a water content mass ratio of 27%;

[0040] (4) Under the condition that the temperature does not exceed 60° C., the water content of the compost mixture is evaporated to below 30%, thereby obtaining the organic fertilizer.

[0041] Embodiment 4:

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

[0043] (1) Preparing fermentation broth: inoculating strain WAS1 into PDA medium for liquid fermentation at a fermentation temperature of 30° C., stirring during the fermentation process at a stirring speed of 200 rpm, and fermenting for 8 days to obtain a growth-promoting bacteria fermentation broth; the number of growth-promoting bacteria spores in the growth-promoting bacteria fermentation broth is ≥10 8 Pieces / ml;

[0044] (2) The fruit peel residue is crushed, and then the fermentation liquid of the growth-promoting bacteria obtained in step (1) is inoculated, the water content is adjusted to 75%, the pH value is adjusted to 6, and then cultured in a shallow dish at 29° C. for 6-8 days until the spore production of the growth-promoting bacteria reaches 10 10 / g, and obtain the fermented product for use; wherein the inoculation amount of the growth-promoting bacteria fermentation liquid accounts for 1.5% of the mass of the fruit peel residue;

[0045] (3) The decomposed chicken manure compost and the fermented product obtained in step (2) are mixed in a mass ratio of 4:1, and a secondary fermentation is performed. The compost is turned twice a day during the fermentation process. The fermentation temperature does not exceed 50° C. and the fermentation is performed for 8 days. After the fermentation is completed, the content of the growth-promoting bacteria reaches 1.0×10 8 / g or more, to obtain a compost mixture; the mature chicken manure compost has a germinated organic matter mass ratio content of> 35%, an organic ammonia mass ratio content of 2.0%, and a water content mass ratio of 30%;

[0046] (4) Under the condition that the temperature does not exceed 60° C., the water content of the compost mixture is evaporated to below 30%, thereby obtaining the organic fertilizer.

[0047] Test example:

[0048] In order to illustrate the effect of this application, the applicant set up the following treatment groups, and applied the fertilizers of the groups to the ponkan orchard, with 10-15 trees treated each time. Each treatment was applied to the soil twice in February and July each year, and the amount applied each time accounted for 50% of the annual amount. During the fertilization treatment, a strip ditch with a length of 100 cm, a width of 30 cm, and a depth of 40 cm was dug along the drip line of the crown. The fertilizer was mixed with the excavated soil and then backfilled into the ditch. Other production management of the orchard was carried out uniformly according to conventional management methods. Except for the different fertilizers, other methods are the same. The specific groups are as follows:

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

[0050] Group 1: The organic fertilizer described in Example 3 was used, and the annual application amount was 8.6 kg / plant;

[0051] The second group: the organic fertilizer described in Example 3 was used, and the annual application amount was 11.3 kg / plant;

[0052] The third group: selected the organic fertilizer described in Example 3, with an annual application rate of 14.2 kg / plant.

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

[0054] 1. Effects of different fertilizer treatments on agronomic traits of ponkan plants

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

[0056] Table 1 Effects of different fertilizers on the growth of ponkan branches

[0057]

[0058] According to the results in Table 1, the long-term fresh weight of single-plant shoots, single shoot length, single-branch leaf number, leaf width, single-leaf thickness and single-leaf weight of ponkan all increase with the increase in the application amount of the organic fertilizer of the present application. The organic fertilizer of the present application contains a large amount of nitrogen, phosphorus, potassium, multiple amino acids and plant hormone substances, etc., which have the effect of promoting growth and increasing biomass of ponkan.

[0059] 2. Effect of fertilization on spring shoot growth of ponkan

[0060] The effects of different fertilizers on the growth of spring shoots of ponkan 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

[0062] Processing Spring shoot length / cm Spring shoot thickness / mm Number of leaves per spring shoot 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] As shown in Table 2, the four fertilization methods have significant effects on spring shoot growth. From the perspective of spring shoot length and spring shoot thickness, there are significant differences between the conventional fertilization of the CK group and the other three treatment methods. There is no significant difference between the other three treatment methods, but it can be found that the spring shoot length and spring shoot thickness of the three fertilization methods with different fertilizer application amounts are all on the rise. The spring shoot length and spring shoot thickness are the largest in the third group. After the third group is treated, the average length of the spring shoot is 12.72cm, the thickness is 3.21mm, and the average number of leaves can reach 7, which are higher than the conventional fertilization treatment: 4.37cm, 0.35mm, and 1 leaf, respectively. The differences between the treatments with different fertilizer amounts are small, but there are significant differences in spring shoot length and spring shoot thickness from the conventional fertilization treatment.

[0064] 3. Effect of fertilization on the growth of autumn shoots of ponkan

[0065] The effects of different fertilizers on the growth of autumn shoots of ponkan 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

[0067] Processing Autumn shoot length / cm Autumn shoot thickness / mm Number of leaves per branch in autumn shoots 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] As can be seen from Table 3, the four fertilization methods have significant effects on the growth of autumn shoots. From the perspective of autumn shoot length and autumn shoot thickness, there are significant differences between conventional fertilization and the other three treatment methods, and there are no significant differences between the other three treatment methods. However, it can be found that the length and thickness of autumn shoots after the three fertilization methods of different organic fertilizers are all on the rise. The length and thickness of autumn shoots are the largest in the third group. After the third group is treated, the average length of autumn shoots is 30.42cm, the thickness is 4.21mm, and the average number of leaves can reach 18, which are higher than the conventional fertilization CK group treatment: 14.93cm, 1.72mm, and 7. The differences between different organic fertilizer treatments are small, but there are significant differences in autumn shoot length and autumn shoot thickness compared with the no fertilization treatment.

[0069] 4. Effects of fertilization on fruit characteristics of ponkan in orchards

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

[0071] Table 4 Effects of different fertilization methods on the characteristics of ponkan fruit

[0072]

[0073] The results of the ponkan fruit test in Table 4 show that there are significant differences in the weight of each fruit in each treatment, Group 3> Group 2> Group 1> Group CK, but the proportion of pulp weight to fruit increases with the increase in the amount of organic fertilizer applied, and the weight of peel decreases. Among them, the pulp weight of Group 3 was 71.25%, an increase of 6.12% over the CK group, which was mainly determined by the thickness and weight of the ponkan peel. Statistical analysis showed that compared with the CK group, the pulp weight, peel weight, pulp to fruit ratio, and peel to fruit ratio of Group 3 were significantly different (P<0.05).

[0074] 5. Effects of different fertilization methods on the quality of ponkan fruit

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

[0076] Table 5 Effects of different fertilization methods on the quality of ponkan fruit

[0077]

[0078] As shown in Table 5, the results of fruit quality determination show that there are significant differences in vitamin C, total sugar and total acid in each treatment. The soluble solid content of the organic fertilizer group of this application has increased. As the application amount of organic fertilizer of this application increases, the total acid content in the fruit shows a downward trend. Statistical analysis shows that the total sugar of the fruit of the second group of the organic fertilizer group of this application is 2.37 percentage points higher than that of the chemical fertilizer control, and the total acid is reduced by 0.12 percentage points. The sugar-acid ratio is significantly higher than that of the control with chemical fertilizer, and the difference is significant (P<0.05). The edible taste is improved, and the commodity value of the fruit is improved. This is related to the phosphorus and potassium elements that are easily absorbed and utilized in the organic fertilizer of this application. Due to the increase in the application amount of organic fertilizer of this application, the amount of phosphorus and potassium absorbed by ponkan also increases, while the total acidity of the fruit decreases.

[0079] 6. Effect of fertilization on ponkan fruit yield

[0080] The effects of different fertilization methods on ponkan yield were investigated and statistically analyzed, as shown in Table 6:

[0081] Table 6 Effects of different fertilization methods on ponkan yield

[0082]

[0083] As shown in Table 6, after applying the organic fertilizer and compound fertilizer of the present application, the single fruit weight and single plant yield of ponkan were significantly higher than those of the CK group, and the single plant yield was as follows: Group 3> Group 2> Group 1> Group CK. There was no significant difference in the single fruit weight of fruits treated with different application methods of the organic fertilizer of the present application, but there was a significant difference in the single plant yield; the single plant yield of the third, second and first groups was significantly higher than that of the CK group, which was 11.66%, 8.33% and 4.95% higher than that of the CK group, respectively. The order of average yield per mu of different fertilizer treatments was as follows: the actual weight of the peeled fruit was harvested on site, and the equivalent yield of the third group was 28440.0 kg·hm -2 , the yield increased by 2970.00kg compared with the CK group, an increase of 11.66%. The yield ranking is: the third group > the second group > the first group > the CK group.

[0084] 7. Effect of fertilization on soil chemistry in orchards

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

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

[0087]

[0088]

[0089] It can be seen from Table 7 that under the condition of equal nutrient fertilization, the soil organic matter, total nitrogen, rapid nitrogen, rapid phosphorus and other indicators of the citrus orchard applied with the organic fertilizer of this application were improved, 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 the present application is applied in citrus cultivation, which has the effects of promoting growth and improving quality, and has broad prospects for development and utilization in the protection of agricultural natural environment.

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

Claims

1. A strain of Wickerhamomyces anomalus WAS1, characterized in that: It is deposited in Guangdong Microbiological Culture Collection Center with the deposit number CDMCC NO: 61811.

2. Application of abnormal Wickerhamomyces anomalus WAS1 according to claim 1 in preparing organic fertilizer.

3. An organic fertilizer, characterized in that: The organic fertilizer is prepared from the Wickerhamomyces anomalus WAS1 described in claim 1.

4. The organic fertilizer according to claim 3, characterized in that The organic fertilizer is used in fruit planting.

5. The organic fertilizer according to claim 4, characterized in that The fruit is citrus.

6. according to the preparation method of any one of claims 3-5 organic fertilizer, it is characterized in that, The steps include: (1) Preparing fermentation broth: inoculating strain WAS1 into PDA medium for liquid fermentation to obtain growth-promoting bacteria fermentation broth; (2) crushing the fruit peel residue, then inoculating the growth-promoting bacteria fermentation liquid obtained in step (1), adjusting the water content to 65-75%, adjusting the pH value to 4.5-6, and then culturing in a shallow dish at 27-29° C. for 6-8 days to obtain a fermentation product for use; (3) mixing the decomposed chicken manure compost and the fermented product obtained in step (2) in a mass ratio of 2-4:1, and performing secondary fermentation, turning the compost 1-2 times a day during the fermentation process, the fermentation temperature not exceeding 50° C., and fermenting for 6-8 days to obtain a compost mixture; (4) The water content of the compost mixture is evaporated to below 30% to obtain the organic fertilizer.

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

8. The method according to claim 6, characterized in that In the step (2), the inoculation amount of the growth-promoting bacteria fermentation liquid accounts for 0.5-1.5% of the mass of the fruit peel residue.

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