Glucose kit-based straw decomposition cycle rapid evaluation microplate method

Abstract

The invention discloses a glucose kit-based straw decomposition cycle rapid evaluation microplate method, and relates to the technical field of agriculture. The method comprises the following steps: taking 0.02 g of a fresh sample and 0.2 mL of water, standing at room temperature for 30 minutes, and filtering by a 25 m needle to obtain percolate; 10 L of filtrate and 1000 L of GOPOD are added, the temperature is 37 DEG C for 10 min, and A505 is read at 505 nm; and with A505 greater than or equal to 0.01 as a threshold value, corresponding to the net weight loss ratio of 4-13% (quadratic curve R = 0.90), early warning decomposition starting is performed 48 h in advance with 24 h increment greater than or equal to 0.01 or valley bottom recovery greater than or equal to 0.002. The result can be obtained within 15 min, and the method is suitable for cross-crop application, low gradient still has quadratic linearity, can be repeated in the field and is suitable for rapid decision making.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of agricultural technology, in particular to a kind of microplate method for rapid evaluation of straw decomposition period based on enzyme marker-glucozyme kit, for the rapid determination of straw decomposition start of field fresh sample, guide water supplement or add bacteria. BACKGROUND

[0002] Straw returning is an important measure to fertilize soil, but if decomposition is not timely, it will hinder the germination of the following crop, breed diseases and pests, and consume soil nitrogen. The existing determination methods (loss weight method, DNS colorimetry, microbial count, etc.) are time-consuming, complex to operate and high in cost, and cannot meet the field decision-making needs of "must know whether to start rotting within 7 days". Therefore, a rapid evaluation method of 15 min level, direct fresh sample and cross-crop is urgently needed. SUMMARY

[0003] The present application provides a fresh sample-microplate method, which uses 0.02 g fresh sample + 0.2 mL water, stands at room temperature for 30 min, filters with a 25 μm needle, takes 10 μL exudate, reacts with 1000 μL GOPOD working solution at 37 ℃ for 10 min, and reads A505 at 505 nm; A505≥0.01 is taken as the "decomposition start" threshold, corresponding to a net weight loss rate of 4-13% (R²=0.90), and a water supplement / bacteria addition signal is sent 48 h in advance. DETAILED DESCRIPTION

[0004] The present application will be described clearly and completely in combination with examples, and the described examples are only a part of the present application, not all. Other examples obtained by those skilled in the art on the premise of no creative labor based on the present application all belong to the protection scope of the present application. EXAMPLE

[0005] Sampling: rice straw crushed to ≤5 mm at the field edge, 0.02 g fresh sample was weighed, 0.2 mL deionized water was added, and it was stood at 20-30 ℃ for 30 min; filtering: filtered with a 25 μm needle filter to obtain "fresh sample exudate"; reaction: take 10 μL filtrate + 1000 μL GOPOD working solution, 37 ℃ for 10 min; determination: read A505 at 505 nm with a microplate reader; threshold: A505≥0.01 is "decomposition start"; weight loss: flush with the same bag - 65 ℃ drying to constant weight, calculate the net weight loss rate; repeat: retest every 24 h, guide water supplement / add bacteria. EXAMPLE

[0006] The operation is the same as in Example 1, except that the rice is replaced by wheat fresh sample, and the rest of the steps remain unchanged. EXAMPLE 3

[0007] Fitted with quadratic curve at 5 points (0, 24, 48, 72, 96 h):

[0008] Rice: A505 = 0.0035t² - 0.0293t + 0.065, R² = 0.90;

[0009] Wheat: A505 = 0.0009t² - 0.0176t + 0.093, R² = 0.91;

[0010] Threshold A505 ≥ 0.01 corresponds to 24 h increment ≥ 0.01 or rebound ≥ 0.002 after valley, signals 48 h in advance. Technical effects

[0011] 15 min result, much faster than 7-15 d weight loss method;

[0012] 0.02 g fresh sample directly measured, no need for drying or enzymatic hydrolysis;

[0013] Cross-crop (rice, wheat) threshold unified A505 ≥ 0.01;

[0014] Low gradient (4-13%) still quadratic R² = 0.90, 48 h early warning; room temperature + needle filter, field repeatable. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 Flowchart of the present application (fresh sample → needle filter → microplate → quadratic threshold).

[0016] Figure 2 Threshold determination logic amplification diagram.

[0017] Figure 3 A505 of rice straw in the process of decomposition 505 Quadratic fitting curve of relationship with net weight loss rate.

[0018] Figure 4 A505 of wheat straw in the process of decomposition 505 Quadratic fitting curve of relationship with net weight loss rate.

Claims

1. A microplate method for rapid evaluation of straw decomposition cycle based on a glucose reagent kit, characterized in that, The following steps are included: (1) Sampling: Take 0.02 g of fresh rice straw or corn stalks that have just been crushed from the field, add 0.2 mL of deionized water, let stand at 20–30 ℃ for 30 min, filter with a 25 µm needle filter to obtain "fresh sample exudate"; (2) Reaction: Take 10 µL of filtrate, add 1000 µL of glucose reagent kit working solution, react at 37 ℃ for 10 min; (3) Measurement: Use an enzyme-linked immunosorbent assay (ELISA) reader to measure absorbance at a wavelength of 505 nm; (4) Judgment: Take A505 ≥ 0.01 as the "decomposition initiation" threshold, corresponding to a reducing sugar release of 0.25–0.40 mg g⁻¹DW, and the quadratic curve R² = 0.90 with the net weight loss rate of 4–13% after 7 days; (5) Application: The measurement can be repeated every 24 h to guide farmers on "when to add water and when to add bacteria".

2. The method according to claim 1, characterized in that: The straw in question is freshly crushed rice straw or corn stalks from the edge of the field.

3. The method according to claim 1, characterized in that: The settling temperature is 20–30 ℃, and the settling time is 30 min.

4. The method according to claim 1, characterized in that: A 25 µm needle filter was used for filtration.

5. The method according to claim 1, characterized in that: The "decomposition initiation" threshold is A505 ≥ 0.

01.

6. The method according to claim 1, characterized in that: The net weight loss rate over 7 days was 4–13%, and the quadratic curve R² = 0.

90.

7. The method according to claim 1, characterized in that: The measurement can be repeated every 24 hours.

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