Nematode enrichment method based on compound enzyme preparation

Abstract

The invention discloses a nematode enrichment method based on a compound enzyme preparation, which is characterized in that a Viscozyme L enzyme and a Cellic cTec3 enzyme are compounded, so that the quick release of two nematodes, i.e., small-stem nematode and lethal meloidogyne, is realized. The optimal extraction conditions of the caenorhabditis caenorhabditis are as follows: the volume ratio of the Cellic cTec3 enzyme to the Viscozyme L enzyme is 1 mL / 40 [mu] L, the temperature is 25 DEG C, and the time is 4.5 h; for killing meloidogyne, the release efficiency is obviously increased along with the temperature rise, and the optimal conditions are as follows: the volume ratio of the Cellic cTec3 enzyme to the Viscozyme L enzyme is 2 mL / 80 mu L, the temperature is 35 DEG C, and the time is 4.5 h. High-efficiency enrichment operation can be realized by utilizing an optimized enzyme lysis solution system, the quantity of the extracted nematodes, namely, the causticia caerulea and the killing meloidogyne, is obviously increased compared with that of the nematodes extracted by the prior art, and microscopic morphology observation proves that the biological characteristics of the nematodes are kept complete and the state is good.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a method for enriching nematodes based on a complex enzyme preparation. Background Technology

[0002] Plant parasitic nematodes mainly exist in soil and plant tissues. They are very small, and with the exception of a very few that can be directly picked from plant tissues, the vast majority require specific tools and methods for separation. Nematode isolation primarily utilizes their hydrotaxis, size, specific gravity, and differences from other impurities, employing methods such as sieving, centrifugation, and flotation. There are many methods for isolating plant parasitic nematodes, and the appropriate method should be selected based on the nematode species and research objectives. For larger nematodes such as female root-knot nematodes, cyst nematodes, and grain nematodes, a needle is generally used to pick them from plant tissues, which is time-consuming and laborious. The Behmann funnel method or direct immersion method is suitable for separating migratory nematodes from soil and plant tissues, generally requiring immersion for more than 24 hours. This method is generally only suitable for separating live nematodes; the efficiency for separating dead nematodes is low.

[0003] To address the problems of time-consuming and inefficient existing techniques for isolating nematodes from plant tissues, specifically ryegrass, this invention provides a nematode enrichment method based on a compound enzyme preparation by optimizing the components and conditions of the enzymatic hydrolysis system. Summary of the Invention

[0004] In view of the problems existing in the prior art, the purpose of this invention is to provide a method for nematode enrichment based on compound enzyme preparations.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: A method for nematode enrichment based on a complex enzyme preparation includes the following steps: S1. Combine Viscozyme L enzyme and Cellic cTec3 enzyme to obtain a compound enzyme solution; S2. Add the compound enzyme solution to phosphate buffer to obtain the enzyme lysis buffer; S3. Take plant tissue infected by plant parasitic nematodes, add it to enzyme lysis buffer, shake well until fully moistened, and then transfer it to a water bath shaker for enzyme lysis treatment. S4. After enzyme lysis, gradient rinsing and filtration are performed using sieves of different pore sizes to obtain plant parasitic nematodes; the plant parasitic nematodes are lethal granular nematodes or small rod-like nematodes; the plant tissue is ryegrass seed tissue.

[0006] Preferably, when Viscozyme L enzyme and Cellic cTec3 enzyme are combined, the volume ratio of the two is 0.5~2mL:20-80μL.

[0007] More preferably, when the plant parasitic nematode is a small rod-shaped nematode, the volume ratio of the Viscozyme L enzyme and the Cellic cTec3 enzyme is 1 mL:40 μL; when the plant parasitic nematode is a lethal granular nematode, the volume ratio of the Viscozyme L enzyme and the Cellic cTec3 enzyme is 2 mL:80 μL.

[0008] Preferably, the conditions for the enzyme lysis treatment are: an enzyme lysis treatment temperature of 25~35℃ and a treatment time of 4~6h.

[0009] More preferably, when the plant parasitic nematode is a small rod-shaped nematode, the enzyme lysis treatment temperature is 25°C and the treatment time is 4.5 h; when the plant parasitic nematode is a lethal granular nematode, the enzyme lysis treatment temperature is 35°C and the treatment time is 4.5 h.

[0010] Preferably, step S4 specifically includes: after lysis, the mixture after enzyme lysis treatment is passed through 600 μm and 25 μm mesh screens in sequence, the seed tissue is gently broken with a rubber stopper to release the nematodes in the plant tissue, the plant tissue residue is on the 600 μm mesh screen and the plant parasitic nematodes are on the 25 μm mesh screen, and the mixture is rinsed with water to collect the plant parasitic nematodes in the 25 μm mesh screen.

[0011] Preferably, the method further includes: S5, collecting the plant parasitic nematodes separated from the sieve into centrifuge tubes using a washing bottle, centrifuging, adding sucrose solution and water to the precipitate obtained by centrifugation, centrifuging again, and the plant parasitic nematodes are concentrated at the interface between the sucrose solution and water, and gently aspirating the plant parasitic nematodes with a dropper.

[0012] Beneficial effects: This invention employs a combination of "Viscozyme L enzyme" (a complex polysaccharide enzyme, Viscozyme L, a non-starch complex glycosaccharide enzyme that degrades various components such as cellulose, hemicellulose, β-glucan, xylan, and arabinogalactan) and "CelliccTec3 enzyme" (a third-generation cellulase, CellicCTec3, a highly active cellulase and hemicellulase complex), and further prepares an enzyme lysis buffer. This enzyme lysis buffer can efficiently lyse plant tissues under relatively low temperature conditions, thereby rapidly releasing and enriching nematodes (lethal granular nematodes and small rod-shaped nematodes). Attached Figure Description

[0013] Figure 1 Results of nematode enrichment (red represents lethal granulomas, blue represents small rod-shaped nematodes). Figure 2 Experimental results of enriching *Nematodes fasciculata* at different temperatures and with mixed enzyme solutions; Figure 3Experimental results of enriching lethal nematodes at different temperatures and with compound enzyme solutions; Figure 4 Microscopic images of nematodes of the order Rhizoctonia (A) and lethal nematodes (B). Detailed Implementation

[0014] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0015] Experimental materials and reagents: Samples: Ryegrass seed samples infected by lethal nematodes and small rod-shaped nematodes.

[0016] Experimental reagents: Novozymes Viscozyme L enzyme solution, Novozymes Cellic cTec3 enzyme solution, control group enzyme lysis solution (the preparation steps are as follows: first, prepare a buffer solution, each 100 mL buffer system contains 49.3 mL 0.2 M Na2HPO4 and 50.7 mL 0.1 M citric acid, and adjust the pH to 4.5 using HCl and NaOH; then add 100 μL of a specific cellulase (Cellulase from Trichoderma reesei ATCC 26921 aqueous solution) and 3 mL of a specific pectinase (Pectinase from Aspergillus aculeatus aqueous solution) to 100 mL of buffer, and mix thoroughly to obtain the enzyme lysis solution).

[0017] Sucrose, disodium hydrogen phosphate, citric acid, hydrochloric acid, sodium hydroxide, etc.

[0018] Prepare phosphate buffer: Dissolve 28.4 g of disodium hydrogen phosphate in 1 L of 0.2 M solution; prepare 19.2 g of citric acid in 1 L of 0.1 M solution; each 100 mL buffer system contains 49.3 mL of 0.2 M disodium hydrogen phosphate solution and 50.7 mL of 0.1 M citric acid solution. Adjust the pH to 4.5 using HCl and NaOH.

[0019] Preparation of compound enzyme solutions of different concentrations: Cellic cTec3 enzyme and Viscozyme L enzyme were compounded at volume ratios of 0.5 mL:20 μL, 1 mL:40 μL, and 2 mL:80 μL, respectively, to obtain compound enzyme solutions (the obtained compound enzyme solutions are respectively denoted as 0.5-20 compound enzyme solution, 1-40 compound enzyme solution, and 2-80 compound enzyme solution).

[0020] To prepare a 70% sucrose solution (100 mL): Weigh 70 g of sucrose and dissolve it in a certain amount of aqueous solution. Then transfer the solution to a 100 mL volumetric flask and bring the volume up to 100 mL.

[0021] Example 1 1.1 Enrichment and Isolation Experiment of Nematodes of the Order Rhizoctonia (1) Add the prepared compound enzyme solution to the prepared phosphate buffer to obtain ryegrass seed enzyme lysis solution; (2) Weigh 20g of ryegrass seed sample infected with small rod-shaped nematodes, add it to the ryegrass seed enzyme lysis solution, and shake well until fully moistened; (3) Transfer to a water bath shaker (180 rpm) for enzyme lysis treatment; (4) After the lysis is completed, the mixture after enzyme lysis treatment is passed through 600 μm and 25 μm sieves in sequence. The 600 μm sieve mainly contains plant tissue residue, while the 25 μm sieve contains small rod-shaped nematodes. The mixture is rinsed with water 3–5 times and the small rod-shaped nematodes in the 25 μm sieve are collected.

[0022] (5) Collect the small rod-shaped nematodes separated from the above sieve into centrifuge tubes using a wash bottle, and centrifuge at 2000 rpm for 2 minutes to remove the supernatant water; (6) Add the precipitate obtained by centrifugation to 9 mL of prepared 70% sucrose solution, stir thoroughly, and gently add about 0.5 mL of water to the upper layer. Centrifuge at 2000 rpm for 2 minutes. The nematodes will accumulate at the interface between the sucrose solution and the water. Gently aspirate the nematodes with a dropper. Wash the collected nematodes several times with water and centrifuge to remove the adhering sucrose, thereby preventing the nematodes from losing water in the sucrose solution.

[0023] 1.2 Optimization of enrichment conditions for *Nematoda* species Based on the main components of ryegrass, Cellic cTec3 enzyme solution and Viscozyme L enzyme solution were mixed at volume ratios of 0.5 mL:20 μL, 1 mL:40 μL, and 2 mL:80 μL, respectively, to obtain mixed enzyme solutions (the resulting mixed enzyme solutions were designated as 0.5-20 mixed enzyme solution, 1-40 mixed enzyme solution, and 2-80 mixed enzyme solution, respectively). These mixed enzyme solutions were added to 100 mL of phosphate buffer and stirred thoroughly to prepare ryegrass seed enzyme lysis buffer. A control group was also established: 100 mL of the pre-prepared enzyme lysis buffer was used as the control system.

[0024] Twenty g of ryegrass seed samples infected with *Bacillus spp.* were collected and added to seed enzyme lysis buffers of different concentrations, and shaken thoroughly to completely submerge the seeds. Erlenmeyer flasks were placed in shakers at 180 rpm and lysis was performed at three temperatures: 25°C, 30°C, and 35°C. The experimental groups (0.5-20 g of mixed enzyme solution, 1-40 g of mixed enzyme solution, and 2-80 g of mixed enzyme solution) were treated for 4.5 h, while the control group was treated for 6 h. After lysis, the collected nematode samples were washed. Results are shown below. Figure 1-2 Microscopic morphological observation was performed on the obtained nematodes of the order Rhizoctonia. The results are shown in [Figure 1]. Figure 4 .

[0025] Depend on Figure 1 The results show that using the enzyme lysis buffer provided by this invention as the nematode extraction and enrichment solution significantly increases the number of small rod-shaped nematodes extracted compared to the original lysis buffer.

[0026] Depend on Figure 2 The results showed that although both the experimental and control groups effectively released nematodes, the lysis efficiency of the experimental group containing the compound enzyme solution was significantly better than that of the control group. Specifically, in the experimental group using the compound enzyme solution prepared by mixing Celic cTec3 enzyme and Viscozyme L enzyme at a volume ratio of 1 mL:40 μL, the number of lethal granulosa nematodes recovered after only 4.5 h of enzymatic hydrolysis was significantly higher than that recovered in the control group under 6 h of treatment, indicating that this compound enzyme concentration can effectively shorten the treatment time and improve the nematode release efficiency.

[0027] The effect of temperature on nematode enrichment was investigated, revealing that the optimal extraction temperature for *Bacillus subtilis* nematodes was 25°C. As the temperature increased from 25°C to 35°C, the recovered quantity decreased. Therefore, the suitable conditions for enriching and extracting *Bacillus subtilis* nematodes from ryegrass seeds are: 1 mL of Celic cTec3 / Viscozyme L / 40 μL for *Bacillus subtilis* nematodes, an enrichment temperature of 25°C, and a treatment time of 4.5 h.

[0028] Figure 4Microscopic morphological observation confirmed that the biological characteristics of the nematodes of the order Rhizoctonia were intact and they were in good condition.

[0029] Example 2 2.1 Enrichment and Isolation Experiment of Lethal Nematodes (1) Add the prepared compound enzyme solution to the prepared phosphate buffer to obtain ryegrass seed enzyme lysis solution; (2) Weigh 20g of ryegrass seed sample infected with lethal nematodes, add it to the mixture, and shake well until fully moistened; (3) Transfer to a water bath shaker (180 rpm) for enzyme lysis treatment at different temperatures; (4) After the lysis is completed, the mixture after the enzyme lysis overnight is passed through 600 μm and 25 μm mesh screens in sequence. The seed tissue is gently broken with a rubber stopper to release the nematodes in the seed tissue. The 600 μm mesh screen mainly contains plant residues, while the 25 μm mesh screen contains small rod-shaped nematodes. The mixture is rinsed with water 3-5 times and the small rod-shaped nematodes in the 25 μm mesh screen are collected.

[0030] (5) Collect the small rod-shaped nematodes separated from the above sieve into centrifuge tubes using a wash bottle, and centrifuge at 2000 rpm for 2 minutes to remove the supernatant water; (6) Add the precipitate obtained by centrifugation to 9 mL of prepared 70% sucrose solution, stir thoroughly, and gently add about 0.5 mL of water to the upper layer. Centrifuge at 2000 rpm for 2 minutes. The lethal nematodes will accumulate at the interface between the sucrose solution and the water. Gently aspirate the lethal nematodes with a dropper. Wash the collected lethal nematodes several times with water and centrifuge to remove the adhering sucrose, thereby preventing the lethal nematodes from losing water in the sucrose solution.

[0031] 2.2 Optimization of enrichment conditions for lethal nematodes (Anguina funesta) Based on the main components of ryegrass, Cellic cTec3 enzyme solution and Viscozyme L enzyme solution were mixed at volume ratios of 0.5 mL:20 μL, 1 mL:40 μL, and 2 mL:80 μL to obtain compound enzyme solutions (denoted as 0.5-20 compound enzyme solution, 1-40 compound enzyme solution, and 2-80 compound enzyme solution, respectively). These compound enzyme solutions were added to 100 mL of phosphate buffer and stirred thoroughly to prepare ryegrass seed enzyme lysis buffer. A control group was also established: 100 mL of the pre-prepared enzyme lysis buffer was used as the control system.

[0032] Take 20g of ryegrass seed samples infected with lethal nematodes and add them to the above-mentioned compound enzyme lysis buffer. Shake thoroughly to completely submerge the seeds. Place the conical flasks in a shaker at 180 rpm and perform lysis treatments at three temperatures: 25℃, 30℃, and 35℃. The experimental groups (0.5-20g compound enzyme solution, 1-40g compound enzyme solution, and 2-80g compound enzyme solution) were treated for 4.5h, and the control group was treated for 6h. After lysis, wash the collected lethal nematode samples. Results are shown below. Figure 1 and Figure 3 Microscopic morphological observation was performed on the obtained lethal nematodes, and the results are shown in [Figure number missing]. Figure 4 .

[0033] Depend on Figure 1 The results show that using the enzyme lysis buffer provided by this invention as the nematode extraction and enrichment solution significantly increases the number of lethal nematodes extracted compared to the original lysis buffer.

[0034] Depend on Figure 3 The results showed that all experimental and control groups released nematodes, but the lysis efficiency of the compound enzyme solution in the experimental groups was significantly better than that in the control groups. In the experimental groups using a compound enzyme solution prepared by combining Celic cTec3 enzyme and Viscozyme L enzyme at a volume ratio of 2 mL:80 μL, the number of lethal granulocytic nematodes and small rod-shaped nematodes recovered after only 4.5 h of enzymatic hydrolysis was significantly higher than that recovered in the control group under 6 h of treatment. This result indicates that the compound enzyme concentration provided by this invention can effectively shorten the treatment time and improve the nematode release efficiency. Furthermore, by investigating the effect of temperature on the nematode enrichment and separation effect, it was found that lethal granulocytic nematodes showed the highest release efficiency at 35°C, and the number of recovered nematodes increased significantly with increasing temperature from 25°C to 35°C. Therefore, the suitable conditions for the enrichment and extraction of lethal nematodes from ryegrass seeds are: a Celic cTec3 enzyme / Viscozyme L enzyme volume ratio of 2 mL / 80 μL suitable for lethal nematodes, an extraction temperature of 35℃, and an extraction time of 4.5 h.

[0035] Figure 4 Microscopic morphological observation confirmed that the lethal nematode retained its biological characteristics and was in good condition.

[0036] In summary, this invention addresses the problems of low enrichment efficiency and long processing time for nematodes in ryegrass seed samples by researching a rapid release and enrichment technology for nematodes based on a compound enzyme preparation. Specifically, a combination of Viscozyme L enzyme and Cellic cTec3 enzyme was used to achieve rapid nematode release. For small rod-shaped nematodes, the optimal extraction conditions were: 1 mL / 40 μL Cellic cTec3 / Viscozyme L addition, 25℃ temperature, and 4.5 h extraction time. For lethal granular nematodes, the release efficiency increased significantly with increasing temperature, and the optimal conditions were: 2 mL / 80 μL Cellic cTec3 / Viscozyme L addition, 35℃ temperature, and 4.5 h extraction time. The optimized lysis buffer system enabled highly efficient enrichment, and the number of both types of nematodes extracted was significantly increased compared to the original process. Furthermore, microscopic morphological observation confirmed that the nematodes retained their biological characteristics and were in good condition.

[0037] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and its improved concept, should be covered within the scope of protection of the present invention.

Claims

1. A method for nematode enrichment based on a complex enzyme preparation, characterized in that: Includes the following steps: S1. Combine Viscozyme L enzyme and Cellic cTec3 enzyme to obtain a compound enzyme solution; S2. Add the compound enzyme solution to phosphate buffer to obtain the enzyme lysis buffer; S3. Take plant tissue infected by plant parasitic nematodes, add it to enzyme lysis buffer, shake well until fully moistened, and then transfer it to a water bath shaker for enzyme lysis treatment. S4. After enzyme lysis, gradient rinsing and filtration are performed using sieves of different pore sizes to obtain plant parasitic nematodes; the plant parasitic nematodes are lethal granular nematodes or small rod-shaped nematodes.

2. The method for nematode enrichment based on compound enzyme preparations according to claim 1, characterized in that: The plant tissue is ryegrass seed tissue; the volume ratio of Viscozyme L enzyme and Cellic cTec3 enzyme when combined is 0.5~2mL:20-80μL.

3. The method for nematode enrichment based on compound enzyme preparations according to claim 1, characterized in that: The conditions for the enzyme lysis treatment are: enzyme lysis treatment temperature of 25~35℃ and treatment time of 4~6h.

4. The method for nematode enrichment based on compound enzyme preparations according to claim 1, characterized in that: Step S4 specifically includes: after lysis, the mixture after enzyme lysis treatment is passed through 600 μm and 25 μm sieves in sequence. The 600 μm sieve contains plant tissue residue, and the 25 μm sieve contains plant parasitic nematodes. The mixture is then rinsed with water, and the plant parasitic nematodes in the 25 μm sieve are collected.

5. The method for nematode enrichment based on compound enzyme preparations according to claim 1, characterized in that: Also includes: S5. Use a wash bottle to collect the plant parasitic nematodes separated from the sieve into centrifuge tubes, centrifuge, add the precipitate obtained by centrifugation to sucrose solution and water, centrifuge again, and the plant parasitic nematodes will be concentrated at the interface between sucrose solution and water. Gently aspirate the plant parasitic nematodes with a dropper.

Images