Bidens pilosa L. enzyme and preparation method and application thereof

Abstract

The present invention belongs to the field of biotechnology, and particularly relates to a Bidens pilosa L. enzyme and its preparation method and application. The Bidens pilosa L. enzyme provided by the present invention can effectively utilize the value of Bidens pilosa L., and the Bidens pilosa L. enzyme can simultaneously promote the growth of crops and improve the disease resistance of plants. On the basis of saving natural resources, the present invention can also control the reproduction of malignant weeds, and at the same time broaden the utilization ways of Bidens pilosa L., providing support for promoting the better development of environmental protection agriculture.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a Bidens pilosa L. enzyme and a preparation method and application thereof. Background Art

[0002] Bidens alba, also known as silver plate of calendula, blind grass, one-pack needle, yellow mother, and three-leaf Bidens alba, is a plant of the genus Bidens in the Asteraceae family. Bidens alba has a strong reproductive capacity and is widely distributed in tropical and subtropical regions. Although it has certain medicinal effects, it is still a malignant weed. Bidens alba can be seen everywhere, whether in farmland, orchards, or lawns and green gardens; if this weed cannot be dealt with before it blooms, once it sets seeds, it will quickly form a large area of continuous growth, forming a large area of single superior weed population, which seriously threatens the survival of native plants. It not only seriously threatens local biodiversity, but also affects the structure and function of the ecosystem. Therefore, how to reasonably utilize Bidens alba in controlling its impact on agricultural ecology is an important goal of ecological agriculture research in recent years. Summary of the invention

[0003] In order to solve the above problems, the present invention provides a Bidens pilosa enzyme and its preparation method and application. The Bidens pilosa enzyme provided by the present invention can effectively utilize the value of Bidens pilosa, and the Bidens pilosa enzyme can promote the growth and reproduction of crops and improve the resistance of plants to diseases. On the basis of saving natural resources, the present invention can also control the reproduction of malignant weeds, and at the same time broaden the utilization of Bidens pilosa, providing support for promoting better development of environmentally friendly agriculture.

[0004] In order to achieve the above object, the present invention provides the following technical solutions:

[0005] The invention provides a Bidens pilosa L. enzyme, which comprises the following raw materials: fermented material, water and fermentation bacteria agent.

[0006] The fermented product comprises the following components in parts by weight: 5-8 parts of molasses, 20-25 parts of Bidens pilosa and 0.5-1 part of NaCl;

[0007] The mass parts of the water are 89.25 to 136 parts;

[0008] The mass of the fermentation agent used is 0.05% of the total mass of the fermentation system;

[0009] The concentration of the fermentation agent is 1×10 6 CFU / mL;

[0010] The fermenting bacterium agent includes yeast, lactic acid bacteria and Lactobacillus plantarum, and the quantity ratio of the yeast, lactic acid bacteria and Lactobacillus plantarum is 1∶1∶1.

[0011] Preferably, the source of the molasses includes sugarcane.

[0012] Preferably, the used part of the Bidens pilosa is the aboveground part and / or the whole plant.

[0013] The present invention provides a preparation method of the above-mentioned Bidens pilosa enzyme, and the preparation method includes the following steps: mixing the fermentation materials and performing the first fermentation, and adding water and a fermenting bacterium agent after the first fermentation is completed to perform the second fermentation to obtain the Bidens pilosa enzyme.

[0014] Preferably, the time of the first fermentation is 5 to 7 days;

[0015] The time of the second fermentation ≥ 50 days;

[0016] In the present invention, the first fermentation is preferably an aerobic fermentation at normal temperature, and the second fermentation is preferably an anoxic fermentation; the second fermentation is further preferably carried out in a sealed tank.

[0017] Preferably, the temperature of the second fermentation is normal temperature.

[0018] Preferably, the pH value of the fermentation system during the first fermentation and the second fermentation is the natural pH value.

[0019] The present invention also provides an application of the above-mentioned Bidens pilosa enzyme or the Bidens pilosa enzyme prepared by the above-mentioned preparation method in promoting plant growth.

[0020] The present invention also provides an application of the above-mentioned Bidens pilosa enzyme or the Bidens pilosa enzyme prepared by the above-mentioned preparation method in improving the antiviral characteristics of plants.

[0021] Preferably, the plants include passion fruit and tobacco.

[0022] The plant enzyme occurs in the process of self-fermentation, and there are uncertainties in two aspects. First, a series of chemical reactions that are difficult to control will occur during the fermentation process, and complex changes will occur in each raw material; second, different raw materials and different fermentation times have great differences in functional effects. Therefore, selecting suitable raw materials is the basic guarantee for obtaining high-quality agricultural enzymes. As an easily available plant resource, Bidens pilosa also has good application prospects. By fully utilizing Bidens pilosa in the form of preparing enzymes, natural resources can be saved, its behavior of being a noxious weed can be controlled, and at the same time the utilization ways of this plant are broadened, providing support for the better development of environmental protection agriculture.

[0023] Molasses is a by-product of the sugarcane industry, and its main use is for alcohol fermentation. However, the problem of low fermentation level of molasses has long troubled the industry. In provinces with large sugarcane production, about 3 million tons of molasses can be produced annually. Limited by the low-level molasses alcohol fermentation process, a large amount of incompletely fermented molasses can only be discharged as wastewater. Not only are limited resources not fully utilized, but it also brings great pressure to the ecological environment. The present invention uses molasses and Bidens pilosa to ferment and produce enzyme, which can better solve the problems of rational utilization of these two resources, environmental protection and agricultural ecological safety.

[0024] As can be seen from the examples, after mixing the Bidens pilosa enzyme provided by the present invention with zinc sulfate, it can effectively reduce the diseased plant rate of tobacco and reduce the disease index; after mixing with conventional water-soluble fertilizer, it can effectively improve the growth of passion fruit seedlings and also enhance the resistance of passion fruit seedlings to viruses. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Among them, the left figure is the passion fruit cuttings transplanted into a larger cup;

[0026] The middle figure is the passion fruit seedlings after being treated with Bidens pilosa enzyme and water-soluble fertilizer for 50 days;

[0027] The right figure is the passion fruit seedlings after being treated with only water-soluble fertilizer for 50 days;

[0028] Figure 2 Tobacco plants treated with Bidens pilosa enzyme solution and organic fertilizer;

[0029] Figure 3 Tobacco plants treated with only organic fertilizer;

[0030] Figure 4 Tobacco plants treated with only compound fertilizer;

[0031] Figure 5 The outer packaging of virus A used in the examples. DETAILED DESCRIPTION OF THE INVENTION

[0032] For reagents or instruments used below without specifying specific techniques or conditions, they shall be carried out according to conventional test conditions. For those not clearly stated with the instructions of reagent companies, they shall be carried out according to the conditions recommended in the instructions. For reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0033] Example 1

[0034] Preparation of Bidens pilosa enzyme

[0035] 1. By mass, take 7 parts of cane molasses purchased from the sugarcane production base in Guangxi, 23 parts of the above-ground part of the Bidens pilosa L. plant with a stem segment length less than 2 cm (removing the lower dead leaves and impurities), and 0.5 part of NaCl. Mix the above raw materials in proportion and stir evenly, then store them in a fermentation barrel for natural fermentation for 5 days. After the natural fermentation is completed, add 3.5 times the mass of the above raw materials of clear water (the clear water is distilled water or directly tap water that has been statically stored for more than 24 hours), and add a fermentation inoculum with a concentration of 1×10 6 CFU / mL (3 kinds of yeast, lactic acid bacteria and Lactobacillus plantarum, the ratio of the 3 kinds of bacteria is 1∶1∶1, the bacterial strains are all from commercial purchases, and after the bacterial strains are enlarged and cultured, they are directly used according to the use concentration). After mixing evenly under normal temperature conditions, carry out secondary fermentation for 60 days to obtain Bidens pilosa L. enzyme.

[0036] 2. By mass, take 7 parts of cane molasses purchased from the sugarcane production base in Guangxi, 23 parts of the chopped above-ground part of the Bidens pilosa L. plant (removing the lower dead leaves and impurities), and 0.5 part of NaCl. Mix the above raw materials in proportion and stir evenly, then store them in a fermentation barrel for natural fermentation for 5 days. After the natural fermentation is completed, add 3.5 times the mass of the above raw materials of clear water (the clear water is distilled water or directly tap water that has been statically stored for more than 24 hours), and add a fermentation inoculum with a concentration of 1×10 6 CFU / mL (3 kinds of yeast, lactic acid bacteria and Lactobacillus plantarum, the ratio of the 3 kinds of bacteria is 1∶1∶1, the bacterial strains are all from commercial purchases, and after the bacterial strains are enlarged and cultured, they are directly used according to the use concentration). After mixing evenly under normal temperature conditions, carry out secondary fermentation for 60 days to obtain Bidens pilosa L. enzyme.

[0037] 3. By mass, take 7 parts of cane molasses purchased from the sugarcane production base in Guangxi, 23 parts of the chopped above-ground part of the Bidens pilosa L. plant (removing the lower dead leaves and impurities), and 0.5 part of NaCl. Mix the above raw materials in proportion and stir evenly, then store them in a fermentation barrel for natural fermentation for 5 days. After the natural fermentation is completed, add 3.5 times the mass of the above raw materials of clear water (the clear water is distilled water or directly tap water that has been statically stored for more than 24 hours), and add a fermentation inoculum with a concentration of 1×10 6 CFU / mL (3 kinds of yeast, lactic acid bacteria and Lactobacillus plantarum, the ratio of the 3 kinds of bacteria is 1∶1∶1, the bacterial strains are all from commercial purchases, and after the bacterial strains are enlarged and cultured, they are directly used according to the use concentration). After mixing evenly under normal temperature conditions, carry out secondary fermentation for 60 days to obtain Bidens pilosa L. enzyme.

[0038] During the above three fermentation processes, the pH value is the natural pH.

[0039] Example 2

[0040] Effect of Bidens pilosa L. enzyme on tobacco virus disease control

[0041] This experiment was conducted in the experimental greenhouse of Guangxi Academy of Agricultural Sciences. The experimental object was tobacco, and the variety was Yunyan 97, provided by Baise Tobacco Company. Seeds were grown in seedling trays and transplanted when the seedlings grew to 4 to 5 true leaves. Four treatment groups were set up, which were:

[0042] A: Bidens pilosa L. enzyme + zinc sulfate provided in Example 1;

[0043] Treatment method: Dilute the Bidens pilosa enzyme solution 300 times, add 0.05% zinc sulfate based on the mass of the diluted solution, mix well and spray on the leaves.

[0044] B: Use only zinc sulfate;

[0045] Treatment method: Dilute 0.05% zinc sulfate with clean water, mix well and spray on leaves.

[0046] C: Virus A (Virus A developed and produced by the Northern Chemical Research Institute, product packaging see Figure 5 ), Treatment method: dilute according to the recommended dosage and spray on leaves.

[0047] D: Clean water (blank control), treatment method: spray with an equal amount of clean water.

[0048] The spray dosage is as follows: 60 liters / mu during the tobacco seedling stage; 100-120 liters / mu after the tobacco bush stage. The specific dosage will increase or decrease according to the situation of the crop.

[0049] Each treatment group was replicated 4 times, with 5 plants in each replicate. After transplanting, virus leaves were collected from the field, and 20 tobacco seedlings were planted in each treatment group. Virus juice was extracted with 0.1M phosphate buffer (the virus juice here was directly collected from virus samples in the field, mainly tobacco mosaic virus), and the virus was inoculated using the leaf juice friction method; 2 days after inoculation, each treatment group was evenly sprayed with a sprayer, and sprayed again after 7 days. The disease situation of each treatment was investigated on the 10th and 20th days after the second spraying. The disease investigation was carried out in accordance with GB / T 23222-2008, and data was collected and the incidence rate and disease index of each treatment were calculated. The test results are shown in Table 1.

[0050] Table 1 The control effect of different treatments on tobacco virus diseases

[0051]

[0052] As can be seen from Table 1, after using the Bidens pilosa L. enzyme provided in Example 1 of the present invention, the diseased plant rate of tobacco virus disease can be effectively reduced, and the disease index can also be reduced, indicating that the Bidens pilosa L. enzyme can effectively prevent and control tobacco virus disease.

[0053] Example 3

[0054] Growth promotion effect of Bidens pilosa L. enzyme on passion fruit

[0055] This experiment was set up in the experimental greenhouse of Guangxi Academy of Agricultural Sciences. The experimental object was passion fruit cuttings, and the variety was Qinmi No. 9. The experiment was carried out 7 days after the emergence, transplantation and cup changing of passion fruit cuttings. Two treatments were set:

[0056] A: Dilute the Bidens pilosa L. enzyme 400 times with water and use ordinary water-soluble fertilizer (commercially available, used according to the recommended amount). The mass ratio of the two is 1:1.

[0057] B: Only use the above-mentioned ordinary water-soluble fertilizer.

[0058] The application method of the above two treatments is spray treatment, 2 - 3 times continuously, with an interval of 7 - 10 days.

[0059] The test results are as Figure 1 shown. Figure 1 The left picture in the middle is the cutting seedlings that have been transplanted and changed to large cups without any treatment. The growth of the seedlings is not good. At the same time, the young leaves of the newly grown cutting seedlings have disease symptoms; the middle picture is the passion fruit seedlings in treatment group A after 50 days of treatment with Bidens pilosa L. enzyme and water-soluble fertilizer. The passion fruit seedlings not only grow well but also have no virus disease symptoms; the right picture is the passion fruit seedlings in treatment group B after 50 days of treatment with only water-soluble fertilizer. The growth of the seedlings is poor, and some passion fruit seedlings still have virus disease symptoms. It can be seen from this that the Bidens pilosa L. enzyme provided by the present invention also has the effects of promoting the growth of passion fruit plants and resisting diseases.

[0060] Example 4

[0061] Improvement effect of Bidens pilosa L. enzyme on the adverse effects brought by continuous cropping of tobacco

[0062] This experiment was set up in Baise City, Guangxi. The experimental object was tobacco, and the variety was Yunyan 97. It was raised in seedlings and transplanted. This experimental field has continuously planted tobacco for 4 years, and root and stem diseases such as tobacco black root rot and bacterial wilt occur relatively seriously.

[0063] Three treatments were set in the experiment:

[0064] (1) Bidens pilosa L. enzyme solution + organic fertilizer (produced by Guangxi Sanbao Agricultural Technology Co., Ltd., trade name "Xiutulong"); the dosage of organic fertilizer is 500 kg / mu (300 kg is used as base fertilizer and 200 kg is used as top dressing), and the mass ratio of Bidens pilosa L. enzyme solution to organic fertilizer is 1:300;

[0065] (2) The above-mentioned organic fertilizer;

[0066] (3) Conventional compound fertilizer (pure potassium sulfate compound fertilizer produced by Jiangsu Huachang Chemical Co., Ltd., with a nitrogen-phosphorus-potassium ratio of 15-15-15).

[0067] Application method: It is applied as base fertilizer before tobacco transplanting and as top dressing when ridging and earthing up (the cultivation method of tobacco is the conventional cultivation method).

[0068] The test results are as Figures 2 to 4 shown. Figure 2 For the treatment of applying Bidens pilosa L. enzyme solution + organic fertilizer, as Figure 2 can be seen, the tobacco grows well under the combined action of this combination, indicating that it can overcome the continuous cropping obstacle of tobacco, control the occurrence and damage of tobacco root and stem diseases, and promote the growth of tobacco at the same time.

[0069] Figure 3 For the treatment of only applying organic fertilizer, as Figure 3 can be seen, the application of organic fertilizer has a certain alleviating effect on the continuous cropping obstacle of tobacco, and can delay the occurrence time of root and stem diseases. The yellowing leaves can be seen in the figure, indicating that only using organic fertilizer still cannot better control the occurrence and development of diseases.

[0070] Figure 4 For the treatment of only using conventional compound fertilizer, as Figure 4 can be seen, the tobacco in this treatment is more obviously affected by the continuous cropping obstacle, the occurrence and damage of root and stem diseases are serious, and the plants also show premature senility.

[0071] As can be seen from the above, the Bidens pilosa L. enzyme solution provided by the present invention in combination with organic fertilizer can effectively alleviate the harm of continuous cropping of tobacco.

[0072] The above are only the preferred embodiments of the present invention. It should be noted that for the inventors of the present invention, without departing from the principle of the present invention, several improvements and refinements can still be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Preparation method of Bidens pilosa L. enzyme, characterized in that, The preparation method comprises the following steps: mixing the fermented materials and conducting the first fermentation, and after the first fermentation is completed, adding water and a fermentation inoculant to conduct the second fermentation to obtain the Bidens pilosa L. enzyme; The time for the first fermentation is 5 - 7 days; The time for the second fermentation is ≥ 50 days; The Bidens pilosa L. enzyme comprises the following raw materials: fermented materials, water and a fermentation inoculant; The fermented materials comprise the following components in parts by mass: 5 - 8 parts of molasses, 20 - 25 parts of Bidens pilosa L., and 0.5 - 1 part of NaCl; The mass of water is 89.25 - 136 parts; The usage mass of the fermentation inoculant is 0.05% of the total mass of the fermentation system; The concentration of the fermentation inoculum is 1×10 6 CFU / mL; The fermentation inoculant includes yeast, lactic acid bacteria and Lactobacillus plantarum, and the quantity ratio of yeast, lactic acid bacteria and Lactobacillus plantarum is 1∶1∶1; The used part of Bidens pilosa L. is the above - ground part and / or the whole plant.

2. The preparation method according to claim 1, characterized in that, The source of the molasses includes sugarcane.

3. The preparation method according to claim 1, characterized in that, The temperature for the second fermentation is normal temperature.

4. The preparation method according to claim 1, characterized in that, During the first fermentation and the second fermentation, the pH value of the fermentation system is the natural pH value.

5. Application of the Bidens pilosa L. enzyme prepared by the preparation method according to any one of claims 1 - 4 in promoting plant growth; The plants include passion fruit and tobacco.

6. Application of the Bidens pilosa L. enzyme prepared by the preparation method according to any one of claims 1 - 4 in improving the antiviral characteristics of plants; The plants include passion fruit and tobacco.

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