A method for promoting radish tuber growth and nutrient metabolism using earthworms combined with nano-lanthanum oxide.

Abstract

This invention discloses a method for promoting the growth and nutrient metabolism of radish tubers using earthworms in conjunction with nano-lanthanum oxide, belonging to the field of nano-agricultural regulation technology. The method of this invention, by simultaneously adding specific amounts of La2O3NMs and a specific density of *Pheretima spp.* in the soil, significantly promotes the tuber enlargement of cherry radishes, increases the net photosynthetic rate and sucrose content of cherry radish leaves, upregulates the expression of IAA biosynthetic genes in the radish epidermis, and ultimately promotes tuber epidermal growth. Furthermore, it increases the content of functional nutrients (D-tryptophan and sinapic acid) in the cherry radish tuber pulp and antioxidants (kaempferol, trifolin, calciferol 3-O-arabinoside, phlorizin) in the tuber epidermis, and increases the abundance of IAA-producing bacteria (flavobacterium) in the rhizosphere, but decreases the abundance of nitrogen cycle-related denitrifying microorganisms (Massilia), thereby increasing the rhizosphere nitrate nitrogen content and promoting nutrient absorption, growth, and physiological metabolism of cherry radish tubers.

Description

Technical Field

[0001] This invention relates to a method for promoting the growth and nutrient metabolism of radish tubers by combining earthworms with nano-lanthanum oxide, belonging to the field of nano-agricultural regulation technology. Background Technology

[0002] Existing methods to promote the enlargement (yield) and nutritional quality of cherry radish tubers mainly involve increasing the application of chemical fertilizers or plant growth regulators and other chemical agricultural products. This method involves high input and low output, and is prone to causing nitrogen and phosphorus runoff and non-point source pollution of water bodies.

[0003] In the past, agricultural production has relied heavily on traditional chemical fertilizers to increase fruit and vegetable yields. This has exacerbated soil compaction, structural damage, and a sharp decline in biodiversity, leading to reduced soil quality and fertility. This severely restricts vegetable crop yields and lowers the nutritional quality of fruits and vegetables. Furthermore, while plant growth regulators have relatively few side effects, excessive or excessive intake can still cause adverse health effects, such as endocrine disorders. Therefore, it is crucial to address how to improve soil quality by leveraging the ecological services provided by soil organisms and how to combine nanotechnology with other methods to promote the growth and nutritional quality of fruits and vegetables. Summary of the Invention

[0004] To address the aforementioned technical problems and application objectives, this invention applies earthworms combined with La2O3 nanomaterials (La2O3 NMs) to cherry radish production, establishing an optimal planting system where earthworms combined with nano-lanthanum oxide promote radish tuber enlargement and the synthesis of functional nutrients. Furthermore, the application process of this invention is simple, easy to operate, and is green, safe, and environmentally friendly.

[0005] The technical solution of the present invention is as follows:

[0006] This invention provides a method for promoting the growth of radish tubers using earthworms in conjunction with nano-lanthanum oxide.

[0007] This invention provides a method for increasing the content of functional nutrients in radish tuber pulp by using earthworms in combination with nano-lanthanum oxide; the functional nutrients include D-tryptophan and sinapic acid.

[0008] This invention provides a method for increasing the sinapic acid content in radish root pulp by using earthworms in combination with nano-lanthanum oxide.

[0009] This invention provides a method for increasing the content of antioxidant substances in radish epidermis by using earthworms in combination with nano-lanthanum oxide; the antioxidant substances include kaempferol, trifolin, calciferol 3-O-arabinoside, and phlorizin.

[0010] In one embodiment of the present invention, the method involves mixing nano-lanthanum oxide with soil, adding earthworms, and then planting radish seeds for growth.

[0011] In one embodiment of the present invention, the amount of nano-lanthanum oxide added to the soil is 0-100 mg / kg. Specifically, 0 mg / kg, 10 mg / kg, 50 mg / kg, and 100 mg / kg are optional.

[0012] In one embodiment of the present invention, the earthworm addition density relative to the soil is 0-3 earthworms / 1.5kg soil.

[0013] In one embodiment of the present invention, the temperature conditions for planting radish seeds for growth are 20-25℃.

[0014] In one embodiment of the present invention, the relative humidity condition for planting radish seeds for growth is 60% ± 5%.

[0015] In one embodiment of the present invention, the light / dark cycle for planting radish seeds is 14 / 10h.

[0016] In one embodiment of the present invention, the earthworm used is an endophytic-deep-dwelling earthworm, specifically Metaphire guillelmi, with an individual size of 1–1.5 g / worm.

[0017] The present invention has the following beneficial technical effects:

[0018] Adding 50 mg / kg La2O3 NMs and two William's ringworms to the soil is the best combination of earthworms and nano-fertilizers. It significantly promotes the tuber enlargement of cherry radishes and can ultimately increase the tuber biomass of cherry radishes by up to 82.5%.

[0019] Adding 50 mg / kg La2O3 NMs and two William's ringworms to the soil simultaneously increased the net photosynthetic rate of cherry radish leaves and the sucrose content in the leaves by 47.6% and 56.4%, respectively. Furthermore, it significantly upregulated the expression of IAA biosynthesis genes in the radish epidermis, ultimately increasing the auxin (IAA) content in the radish epidermis by up to 53.6%, thus promoting tuber epidermal growth.

[0020] Adding 50 mg / kg La2O3 NMs and two William's ringworms to the soil significantly increased the content of functional nutrients (D-tryptophan and sinapic acid) in the pulp of cherry radish tubers and antioxidants (kaempferol, trifolin, kaempferol 3-O-arabinoside, and phloretin) in the tuber epidermis.

[0021] Adding 50 mg / kg La2O3 NMs and 2 William's ringworms to the soil simultaneously increased the abundance of IAA-producing bacteria (Flavobacterium) in the rhizosphere, but decreased the abundance of nitrogen cycle-related denitrifying microorganisms (Massilia), thereby increasing the nitrate nitrogen content in the rhizosphere and promoting nutrient absorption, growth, and physiological metabolism of cherry radish tubers. Attached Figure Description

[0022] Figure 1 This is a TEM image of La2O3 NMs.

[0023] Figure 2 A comparative graph showing the effects of different inoculation densities of earthworms and different application concentrations of La2O3 NMs on the growth of cherry radish tubers.

[0024] Figure 3 The effect of 50 mg / kg La2O3 NMs combined with two earthworms (EW) on net photosynthetic capacity (a) and sucrose content (b) of cherry radish leaves is shown in the figure.

[0025] Figure 4 Comparative figure showing the effects of 0.50 mg / kg La2O3 NMs combined with two earthworms (EW) on the expression (ac) of IAA synthesis gene and the content (d) of IAA in the epidermis of cherry radish tubers.

[0026] Figure 5 A comparative graph showing the effects of 50 mg / kg La2O3 NMs combined with two earthworms (EW) on the functional nutrients in the pulp (ab) and epidermis (cf) of cherry radish tubers.

[0027] Figure 6 The effect of 50 mg / kg La2O3 NMs combined with two earthworms (EW) on rhizosphere bacterial abundance (a) and nitrate nitrogen content (b) is shown in the figure. Detailed Implementation

[0028] The present invention will be further described below with reference to specific embodiments.

[0029] The La2O3 nanomaterials involved in this invention were purchased from Sigma-Aldrich. TEM images show that the average size distribution of the La2O3 nanomaterials is approximately 58 nm. Figure 1 The physical and chemical properties of the experimental soil were as follows: pH (water) 7.2, soil organic carbon 10.8 g / kg, and total nitrogen 1.2 g / kg.

[0030] TEM characterization: The size and morphology of nano-lanthanum oxide were characterized using a JEM-2100 transmission electron microscope.

[0031] Cherry radish tuber biomass: The mass of cherry radish tubers was determined using an electronic balance.

[0032] Net photosynthetic rate: The second leaf from the bottom of the cherry radish plant was selected, and its net photosynthetic rate was measured using a photosynthesis meter (CIRAS-3, PP-Systems, USA).

[0033] Expression level of IAA transcribed gene: The expression of IAA transcribed gene in radish epidermis was determined by sampling and quantitative real-time PCR.

[0034] Plant hormones, functional nutrients (D-tryptophan, sinapic acid), and antioxidants (kaempferol, trifolin, kaempferol 3-O-arabinoside, and phloretin) were determined by liquid chromatography-mass spectrometry (LC-MS). The results were normalized using an internal standard (2-chloro-1-phenylalanine) to calculate the relative content of each substance.

[0035] Starch and soluble sugars in grains: The content of starch and soluble sugars in grains was determined by the anthrone method.

[0036] Calculation method:

[0037] Percentage increase = (Treated plants - Untreated plants) / Control group plants × 100%.

[0038] Example 1: Application of earthworms and La2O3 nanomaterials to promote the growth of cherry radishes and increase the content of functional nutrients in the radish root pulp and antioxidants in the radish peel.

[0039] Different concentrations (0 mg / kg, 10 mg / kg, 50 mg / kg, 100 mg / kg) of La2O3 nanomaterials and 1.5 kg of soil were vigorously mixed and placed into 2 L pots. Wilhelm's ringed coelomic earthworms (EW) were added at a density of 0-3 worms per pot. Cherry radish seeds (Pioneer) were purchased from Hezhiyuan Seed Industry and sterilized by surface disinfection with a 5% sodium hypochlorite solution before germination and transplanting. Growth conditions included day / night temperatures of 25℃ / 20℃, a photoperiod of 14 / 10h (i.e., 14 hours of light followed by 10 hours of darkness), and a relative humidity of 60±5%.

[0040] The control group (CK) consisted of no La2O3 nanomaterials and no *Vibrio wieniensis*.

[0041] The results are as follows Figure 2 As shown, both earthworms and La2O3 nanomaterials alone have a certain promoting effect on the enlargement of radish tubers. Compared with the control group, the application of La2O3 nanomaterials alone increased the biomass of cherry radish tubers by 54.2%, while the simultaneous addition of 50 mg / kg La2O3 nanomaterials and 2 William's ringworms to the soil increased the radish tuber yield by as much as 82.5%.

[0042] Determining the effect of earthworms combined with La2O3 nanomaterials on the photosynthetic properties of cherry radishes:

[0043] The results are as follows Figure 3 As shown, in the control group, the net photosynthetic rate and sucrose content of cherry radish leaves were 12.68 μmol CO2 m -2 s -1 And 0.77 mg / g. Compared with the control group, the simultaneous addition of 50 mg / kg La2O3 nanomaterials and 2 William's ringworms to the soil increased the net photosynthetic rate and sucrose content of cherry radish leaves by 47.6% and 56.4%, respectively. Figure 3 These leaves, through increased net photosynthesis and sucrose content, provide a sufficient energy base for the enlargement of radish tubers.

[0044] Determining the effect of earthworms combined with La2O3 nanomaterials on IAA in cherry radish root epidermis:

[0045] The results are as follows Figure 4 As shown, the IAA content in the epidermis of cherry radishes in the control group was 26.65 ng / g. Compared to the control group, the simultaneous addition of 50 mg / kg La2O3 nanomaterials and two William's ringworms to the soil significantly upregulated the expression levels of the IAA biosynthesis transcription genes CL3618.Contig4_All, CL6699.Contig2_All, and Unigene2606_All in the radish root epidermis by 123.6%, 39.9%, and 46.8%, respectively. Figure 4 The auxin IAA content in the radish tuber epidermis was increased to 53.6% (40.95 ng / g), thereby promoting the growth of the radish epidermis and laying the cellular structural foundation for tuber enlargement.

[0046] Determining the effect of earthworms combined with La2O3 NMs on antioxidant metabolites in the root epidermis of cherry radish:

[0047] When 50 mg / kg La2O3 nanomaterials and two William's ringworms were added to the soil simultaneously, the relative contents of functional nutrients—D-tryptophan (functions: regulating nerve rhythm, etc.) and sinapic acid (functions: anti-oxidation, delaying skin aging, softening blood vessels, etc.)—in the radish tuber pulp were 2.895 × 10⁻⁶. 9 and 2.828×10 7 Compared with the control group, soil supplementation with 50 mg / kg La2O3 nanomaterials and two William's ringworms increased the levels of functional nutrients—tryptophan and sinapic acid—in radish tuber pulp by 103.4% and 799.3%, respectively. Figure 5(a, b). When earthworms were used alone, the sinapic acid content in the radish root pulp was 2.314 × 10⁻⁶. 6 When La2O3 nanomaterials are used alone, the sinapic acid content in the radish root pulp is 3.651 × 10⁻⁶. 6 Therefore, it can be seen that earthworms combined with nano-lanthanum oxide have a significant synergistic effect.

[0048] Meanwhile, the levels of antioxidants in the cherry radish peel—kaempferol, trifolin, calciferol 3-O-arabinoside, and phlorizin—increased by 231.0%, 187.7%, 243.5%, and 537.1%, respectively. Figure 5 (cf), thereby improving the nutritional quality of cherry radishes.

[0049] Determining the effects of earthworms combined with La2O3 nanomaterials on rhizosphere nitrogen content and bacterial community in cherry radish:

[0050] Compared with the control group, the simultaneous addition of 50 mg / kg La2O3 NMs and 2 William's ringworms to the soil increased the abundance of IAA-producing bacteria (Flavobacterium) in the rhizosphere, but decreased the abundance of nitrogen cycle-related denitrifying microorganisms (Massilia). Figure 6 a) It increased the rhizosphere nitrate nitrogen content (68.16 mg / g) by as much as 137.7% ( Figure 6 b) promotes nutrient absorption, growth, and physiological metabolism in cherry radish tubers.

[0051] The embodiments provided above are not intended to limit the scope of the invention, nor are the described steps intended to limit the order of execution. Any obvious modifications made to the invention by those skilled in the art based on existing common knowledge also fall within the scope of protection defined by the claims.

Claims

1. A method for promoting the growth of radish tubers by combining earthworms with nano-lanthanum oxide, wherein the method involves mixing nano-lanthanum oxide with soil, adding earthworms, and then planting radish seeds for growth; the amount of nano-lanthanum oxide added relative to the soil is 10-100 mg / kg.

2. The method according to claim 1, characterized in that, The addition amount of nano-lanthanum oxide relative to the soil is 50 mg / kg.

3. The method according to claim 1, characterized in that, The density of earthworms added to the soil is 1-3 earthworms per 1.5 kg of soil.

4. The method according to claim 1, characterized in that, The optimal temperature for growing radish seeds is 20-25℃.

5. The method according to claim 1, characterized in that, The relative humidity for growing radish seeds is 60% ± 5%.

6. The method according to claim 1, characterized in that, The light / dark cycle for planting radish seeds is 14 / 10 h.

7. The method according to claim 1, characterized in that, The earthworms used were endophytic-deep-dwelling earthworms, specifically *Metaphire guillelmi*, with an individual size of 1-1.5 g / worm.

8. A method for increasing the content of functional nutrients in radish tuber pulp by combining earthworms with nano-lanthanum oxide; the functional nutrients are selected from D-tryptophan and sinapic acid, and the method is to mix nano-lanthanum oxide with soil, add earthworms, and then plant radish seeds for growth; the amount of nano-lanthanum oxide added relative to the soil is 10-100 mg / kg.

9. The method according to claim 8, characterized in that, The addition amount of nano-lanthanum oxide relative to the soil is 50 mg / kg.

10. The method according to claim 8, characterized in that, The density of earthworms added to the soil is 1-3 earthworms per 1.5 kg of soil.

11. The method according to claim 8, characterized in that, The optimal temperature for growing radish seeds is 20-25℃.

12. The method according to claim 8, characterized in that, The relative humidity for growing radish seeds is 60% ± 5%.

13. The method according to claim 8, characterized in that, The light / dark cycle for planting radish seeds is 14 / 10 h.

14. The method according to claim 8, characterized in that, The earthworms used were endophytic-deep-dwelling earthworms, specifically *Metaphire guillelmi*, with an individual size of 1-1.5 g / worm.

15. A method for increasing the content of antioxidant substances in radish epidermis by combining earthworms with nano-lanthanum oxide; the antioxidant substances are selected from kaempferol, trifolin, kanfibroin 3-O-arabinoside, and phlorizin; the method is to mix nano-lanthanum oxide with soil, add earthworms, and then plant radish seeds for growth; the amount of nano-lanthanum oxide added relative to the soil is 10-100 mg / kg.

16. The method according to claim 15, characterized in that, The relative addition amount of nano-lanthanum oxide to the soil is 50 mg / kg.

17. The method according to claim 15, characterized in that, The density of earthworms added to the soil is 1-3 earthworms per 1.5 kg of soil.

18. The method according to claim 15, characterized in that, The optimal temperature for growing radish seeds is 20-25℃.

19. The method according to claim 15, characterized in that, The relative humidity for growing radish seeds is 60% ± 5%.

20. The method according to claim 15, characterized in that, The light / dark cycle for planting radish seeds is 14 / 10 h.

21. The method according to claim 15, characterized in that, The earthworms used were endophytic-deep-dwelling earthworms, specifically *Metaphire guillelmi*, with an individual size of 1-1.5 g / worm.

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