A method and application of enhancing the ability of Vallisneria natans to resist heavy metal cadmium stress

Abstract

The invention relates to the field of water environment pollution control, and specifically provides a method for enhancing the ability of Vallisneria vallisneria to resist heavy metal cadmium stress and application thereof, wherein resistant plant endophytes are extracted from herbaceous plants collected from cadmium-contaminated areas, and colonized on Vallisneria vallisneria to enhance the ability of Vallisneria vallisneria to resist heavy metal cadmium stress; the method for enhancing the ability of Vallisneria vallisneria to resist heavy metal cadmium stress effectively improves the threshold of the ability of Vallisneria vallisneria to resist heavy metal cadmium stress, improves the survival rate of Vallisneria vallisneria in cadmium-contaminated sediment, increases the cadmium enrichment ability of Vallisneria vallisneria, strengthens its cadmium pollution restoration ability, and enables Vallisneria vallisneria to be better applied in the field of water ecological restoration.

Description

Technical Field

[0001] The invention relates to the field of water environment pollution control, and in particular to a method for enhancing the ability of Vallisneria vallisneria to resist heavy metal cadmium stress and application thereof. Background Art

[0002] Due to the rough development and utilization of natural resources by humans in the past and the development of industrial and agricultural production, environmental pollution caused by heavy metals has become increasingly serious. In particular, some heavy metals have strong toxic side effects on organisms. Take cadmium as an example. It is a highly toxic, difficult to degrade, and easily accumulated heavy metal element. It is one of the priority controlled pollutants in my country's water environment. Cadmium that enters the water environment migrates and deposits in the bottom mud, and is released into the water body with wind and wave disturbances, forming repeated pollution and causing great harm to aquatic plants and animals.

[0003] At present, the treatment of heavy metal pollution in sediments is mainly carried out by physical, chemical and biological restoration methods. Among them, plant restoration has become one of the most popular methods due to its low cost, low risk of secondary pollution and beautiful landscape. In particular, submerged plants have the characteristics of large biomass, can be planted in all waters, and directly act on sediments. They can be widely used in the restoration of heavy metal pollution in sediments. Among them, Vallisneria has the advantages of wide distribution, strong reproductive capacity, easy management and maintenance, and stable community. It is often used as the main community-building species in the reconstruction and restoration of submerged plant communities.

[0004] Therefore, enhancing the ability of Vallisneria sinensis to resist heavy metal cadmium stress is of great significance to the control of water environment pollution. Summary of the invention

[0005] In view of the shortcomings of the prior art described above, the purpose of the present invention is to provide a method and application for enhancing the ability of Vallisneria sinensis to resist heavy metal cadmium stress, so as to solve the problem that Vallisneria sinensis is difficult to survive or has a low survival rate in cadmium-contaminated sediments during water ecological restoration in the prior art.

[0006] To achieve the above object and other related objects, the present invention provides a method for enhancing the ability of Vallisneria vallisneriae to resist heavy metal cadmium stress, comprising the following steps:

[0007] 1) Extraction of resistant plant endophytes: Collect cadmium hyperaccumulating herbs from cadmium-contaminated areas, sterilize, grind, add buffer solution to mix evenly, and inoculate into sterile LB solid medium containing cadmium, culture at a constant temperature and aseptically, then select colonies and transfer them to new sterile LB solid medium, purify, and store the obtained resistant endophytes for future use;

[0008] 2) Preparation of resistant plant endophyte suspension: inoculate the resistant endophyte obtained in step 1) into LB liquid medium, culture to logarithmic phase, centrifuge, resuspend in sterile saline, and resuspend in sterile water to dilute to OD 600 =0.5~0.9;

[0009] 3) Vallisneria planting: plough the bottom mud surface of the first Vallisneria planting pond, mix CdCl2 with the ploughed bottom mud to prepare cadmium-containing bottom mud, select Vallisneria seedlings and plant them in the first Vallisneria planting pond, and return the water;

[0010] 4) Colonization of resistant plant endophytes: 15 to 20 days after the Vallisneria seedlings are planted, bleaching powder is applied to the water of the first Vallisneria planting pond. 5 to 10 days later, the resistant endophyte suspension obtained in step 2) is evenly added to the water of the first Vallisneria planting pond to colonize the resistant endophytes on the Vallisneria;

[0011] 5) Thinning and transplanting: Thinning is performed 50 to 70 days after the first Vallisneria planting pond is planted. The surface of the bottom mud of the second Vallisneria planting pond is plowed, and CdCl2 is mixed with the plowed bottom mud to prepare cadmium-containing bottom mud. Vallisneria plants from the first Vallisneria planting pond are selected and planted in the second Vallisneria planting pond, and the method of step 4) is repeated in the second Vallisneria planting pond.

[0012] The present invention also provides an application of the above-mentioned method for enhancing the ability of Vallisneria vallisneria to resist heavy metal cadmium stress in the field of water ecological restoration.

[0013] As described above, the method and application of the present invention for enhancing the ability of Vallisneria vallisneria to resist heavy metal cadmium stress has the following beneficial effects:

[0014] The method for enhancing the ability of Vallisneria to resist heavy metal cadmium stress of the present invention effectively improves the threshold of Vallisneria's ability to resist heavy metal cadmium stress, improves the survival rate of Vallisneria in cadmium-contaminated sediment, increases the cadmium enrichment ability of Vallisneria, strengthens its cadmium pollution repair ability, and enables Vallisneria to be better applied in the field of water ecological restoration. DETAILED DESCRIPTION

[0015] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0016] When a numerical range is disclosed herein, the above range is deemed to be continuous and includes the minimum and maximum values ​​of the range, as well as each value between such minimum and maximum values. Further, when a range refers to an integer, each integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be merged. In other words, unless otherwise indicated, all ranges disclosed herein should be understood to include any and all sub-ranges included therein. For example, a specified range from "1 to 10" should be deemed to include any and all sub-ranges between a minimum of 1 and a maximum of 10. Exemplary sub-ranges of ranges 1 to 10 include, but are not limited to, 1 to 6.1, 3.5 to 7.8, 5.5 to 10, etc.

[0017] In addition, it should be understood that one or more method steps mentioned in the present invention do not exclude the existence of other method steps before or after the combination step or the insertion of other method steps between these explicitly mentioned steps. Unless otherwise specified, the numbering of each method step is only a convenient tool for identifying each method step, and is not intended to limit the order of arrangement of each method step or to define the scope of implementation of the present invention. Changes or adjustments in their relative relationships should also be regarded as the scope of implementation of the present invention without substantially changing the technical content.

[0018] In the present invention, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features.

[0019] The first aspect of the present invention provides a method for enhancing the ability of Vallisneria vallisneria to resist heavy metal cadmium stress, comprising the following steps:

[0020] 1) Extraction of resistant plant endophytes: Collect cadmium hyperaccumulating herbs from cadmium-contaminated areas, sterilize, grind, add buffer solution to mix evenly, and inoculate into sterile LB solid medium containing cadmium, culture at a constant temperature and aseptically, then select colonies and transfer them to new sterile LB solid medium, purify, and store the obtained resistant endophytes for future use;

[0021] 2) Preparation of resistant plant endophyte suspension: inoculate the resistant endophyte obtained in step 1) into LB liquid medium, culture to logarithmic phase, centrifuge, resuspend in sterile saline, and resuspend in sterile water to dilute to OD 600 =0.5~0.9, 0.5~0.6, 0.6~0.7, 0.7~0.8 or 0.8~0.9;

[0022] 3) Vallisneria planting: plough the bottom mud surface of the first Vallisneria planting pond, mix CdCl2 with the ploughed bottom mud to prepare cadmium-containing bottom mud, select Vallisneria seedlings and plant them in the first Vallisneria planting pond, and return the water;

[0023] 4) Colonization of resistant plant endophytes: 15 to 20 days after the Vallisneria seedlings are planted, bleaching powder is applied to the water of the first Vallisneria planting pond. 5 to 10 days later, the resistant endophyte suspension obtained in step 2) is evenly added to the water of the first Vallisneria planting pond to colonize the resistant endophytes on the Vallisneria;

[0024] 5) Thinning and transplanting: Thinning is performed 50 to 70 days after the first Vallisneria planting pond is planted. The surface of the bottom mud of the second Vallisneria planting pond is plowed, and CdCl2 is mixed with the plowed bottom mud to prepare cadmium-containing bottom mud. Vallisneria plants from the first Vallisneria planting pond are selected and planted in the second Vallisneria planting pond, and the method of step 4) is repeated in the second Vallisneria planting pond.

[0025] In some embodiments of the present invention, in step 1), the cadmium hyperaccumulating herb is a herb whose above-ground part absorbs heavy metals more than 10 times that of ordinary plants. In a preferred embodiment of the present invention, the cadmium hyperaccumulating herb is Sedum alfredii;

[0026] In some embodiments of the present invention, in step 1), the herbaceous plant has good growth, tall plants, and a well-developed root system.

[0027] In some embodiments of the present invention, in step 1), the buffer is a phosphate buffer with a pH of 7.2 to 7.4.

[0028] In some embodiments of the present invention, in step 1), the disinfection sequentially comprises the steps of: sterile water cleaning, 95% alcohol surface disinfection, 20-40%, 20-25%, 25-30%, 30-35% or 35-40% hydrogen peroxide + 2-5%, 2-3%, 3-4% or 4-5% sodium hypochlorite surface disinfection, 95% alcohol surface disinfection, and sterile water cleaning. In a preferred embodiment of the present invention, the disinfection sequentially comprises the steps of: sterile water cleaning, 95% alcohol surface disinfection, 30% hydrogen peroxide + 3% sodium hypochlorite surface disinfection, 95% alcohol surface disinfection, and sterile water cleaning.

[0029] The specific steps of disinfection include: fully washing the plants with sterile water and performing surface disinfection: 95% alcohol (1 min) → 30% hydrogen peroxide + 3% sodium hypochlorite (30 min) → 95% alcohol (1 min) → rinsing with sterile water three times.

[0030] In some embodiments of the present invention, in step 1), the LB solid culture medium formula is: NaCl 10 g, peptone 10 g, yeast powder 5 g, agar 15 g, distilled water 1000 mL, pH 7.2~7.4.

[0031] In some embodiments of the present invention, in step 1), the cadmium content in the LB solid culture medium is 1.5-3.5 mmol / L, for example, 1.5-2.0 mmol / L, 2.0-2.5 mmol / L, 2.5-3.0 mmol / L or 3.0-3.5 mmol / L.

[0032] In some embodiments of the present invention, in step 1), the temperature of the constant temperature sterile culture is 20-30°C, for example, 20-23°C, 23-25°C, 25-27°C or 27-30°C.

[0033] In some embodiments of the present invention, in step 1), the constant temperature sterile culture time is 6 to 8 days, for example 6 to 7 days or 7 to 8 days.

[0034] In some embodiments of the present invention, in step 1), the storage for standby use is storage in a glycerol tube.

[0035] In some embodiments of the present invention, in step 2), the LB liquid culture medium formula is: NaCl 10 g, peptone 10 g, yeast powder 5 g, distilled water 1000 mL.

[0036] In some embodiments of the present invention, in step 2), the centrifugal force of the centrifugation is 10000-14000 g, for example, 10000-11000 g, 11000-12000 g, 12000-13000 g or 13000-14000 g.

[0037] In some embodiments of the present invention, in step 2), the culture temperature during the culture to the logarithmic phase is 20-30° C., for example, 20-23° C., 23-25° C., 25-27° C. or 27-30° C.

[0038] In some embodiments of the present invention, in step 3), the planting density of the Vallisneria in the first Vallisneria planting pond is 9 to 16 clumps / m 2 , 9~11 clusters / m 2 , 11~13 clusters / m 2 13~15 clusters / m 2 or 15~16 clusters / m 2 , 3~5 buds / cluster, 3~4 buds / cluster or 4~5 buds / cluster.

[0039] In some embodiments of the present invention, in step 3), the Vallisneria salsa seedlings are Vallisneria salsa seedlings with well-developed and strong root systems.

[0040] In some embodiments of the present invention, in step 3), the cadmium content of the cadmium-containing sludge is 5-15 mg / kg, for example, 5-7 mg / kg, 7-9 mg / kg, 9-11 mg / kg, 11-13 mg / kg or 13-15 mg / kg.

[0041] In some embodiments of the present invention, in step 3), the surface layer of the bottom mud is 5 to 100 cm. For example, 5 to 10 cm, 10 to 20 cm, 20 to 30 cm, 30 to 40 cm, 40 to 50 cm, 50 to 60 cm, 60 to 70 cm, 70 to 80 cm, 80 to 90 cm or 90 to 100 cm. In a preferred embodiment of the present invention, in step 5), the surface layer of the bottom mud is 30 cm.

[0042] In some embodiments of the present invention, in step 3), the backwater is backwater to a water depth of 40-60 cm, for example, 40-45 cm, 45-50 cm, 50-55 cm or 55-60 cm.

[0043] In some embodiments of the present invention, in step 4), the amount of bleaching powder applied is 5-10 ppm, for example, 5-6 ppm, 6-7 ppm, 7-8 ppm, 8-9 ppm or 9-10 ppm.

[0044] In some embodiments of the present invention, in step 4), the amount of the resistant endophyte suspension added to the water of the first Vallisneria lappa planting pond is 1-3% V / V, for example, 1-2% V / V or 2-3% V / V.

[0045] In some embodiments of the present invention, in step 5), the planting density of the Vallisneria in the second Vallisneria planting pond is 9 to 16 clumps / m 2 , 9~11 clusters / m 2 , 11~13 clusters / m 2 13~15 clusters / m 2 or 15~16 clusters / m 2 , 3~5 buds / cluster, 3~4 buds / cluster or 4~5 buds / cluster.

[0046] In some embodiments of the present invention, in step 5), the cadmium content of the cadmium-containing sludge is 15-35 mg / kg, for example, 5-7 mg / kg, 7-9 mg / kg, 9-11 mg / kg, 11-13 mg / kg or 13-15 mg / kg.

[0047] In some embodiments of the present invention, in step 5), the surface layer of the bottom mud is 5 to 100 cm. For example, 5 to 10 cm, 10 to 20 cm, 20 to 30 cm, 30 to 40 cm, 40 to 50 cm, 50 to 60 cm, 60 to 70 cm, 70 to 80 cm, 80 to 90 cm or 90 to 100 cm. In a preferred embodiment of the present invention, in step 5), the surface layer of the bottom mud is 30 cm.

[0048] In some embodiments of the present invention, in step 5), the backwater is backwater to a water depth of 40-60 cm, for example, 40-45 cm, 45-50 cm, 50-55 cm or 55-60 cm.

[0049] In some embodiments of the present invention, in step 5), the Vallisneria plants selected in the first Vallisneria planting pond are Vallisneria plants with well-developed and strong root systems.

[0050] The second aspect of the present invention provides an application of the above-mentioned method for enhancing the resistance of Vallisneria vallisneria to heavy metal cadmium stress in the field of water ecological restoration.

[0051] Example 1 Test on the maximum lethal concentration of Cd against resistant plant endophytes

[0052] (1) Test plants: Sedum alfredii, collected from an abandoned lead-zinc mine in Quzhou, Zhejiang Province.

[0053] (2) Test steps:

[0054] a. Wash the Sedum alfredii plants thoroughly with sterile water and disinfect their surfaces (95% alcohol (1 min) → 30% hydrogen peroxide + 3% sodium hypochlorite (30 min) → 95% alcohol (1 min) → rinse with sterile water three times).

[0055] b. Place the disinfected Sedum alfredii plants in a sterilized mortar, grind into juice, add an appropriate amount of phosphate buffer solution (pH=7.2~7.4), stir evenly, let stand for 10 minutes, and then stir quickly. Take 200μL and inoculate into sterilized LB solid culture medium containing 0, 0.5, 1.5, 2.5, 3.5, 4, and 4.5 mmol / L Cd, respectively, and culture in a constant temperature sterile box at 20~30℃ for one week, and observe the colonies. The results are shown in Table 1.

[0056] Table 1

[0057] Serial number Cd content (mmol / L) Number of colonies 11 0 Several, countless 12 0.5 32 13 1.5 7 14 2.5 4 15 3.5 1 16 4 0 17 4.5 0

[0058] Combined with the data in Table 1, it can be seen that when the Cd concentration of solid LB medium is ≤0.5mmol / L, the number of colonies is large; when the Cd concentration of solid LB medium is ≥4mmol / L, no colonies grow. Therefore, in order to screen out endophytes with strong resistance, the Cd concentration of the screening medium should be between 1.5 and 3.5mmol / L.

[0059] Example 2 Test on the ability of Vallisneria serrata to resist heavy metal Cd stress

[0060] (1) Test plants: evergreen dwarf Vallisneria ovalis, purchased from Chibi Xinhe Aquatic Plant Co., Ltd.

[0061] (2) Test steps:

[0062] a. The indoor temperature was controlled at 20℃. Under the light conditions, 9 1×1×1.2 glass cylinders were prepared, and the bottom was covered with pond mud of 50cm. The No. 21 cylinder was used as the control group (CK). The top 30cm of the bottom mud in cylinders 22 to 29 was fully plowed, and CdCl2 was added to the plowed bottom mud and mixed thoroughly to obtain 5, 10, 15, 20, 25, 30, 35, and 40 mg / kg of cadmium-containing bottom mud. The Vallisneria spp. seedlings with well-developed and strong root systems were selected at 9 to 16 clumps / m 2 , 3 to 5 buds / clump were planted on the cadmium-treated pond mud and the water was returned to a depth of 60 cm.

[0063] b. After 60 days of planting, the height and fresh weight of Vallisneria were measured. Three plants were taken from each device for sampling, rinsed with ultrapure water, and the residual water on the surface of the plants was absorbed with absorbent paper. The fresh weight was weighed on an electronic balance and the height of the plants was measured with a ruler (when measuring the height of the plants, the highest leaf vein of the whole plant was taken for measurement). The results are shown in Table 2.

[0064] Table 2

[0065] Serial number Cadmium concentration (mg / kg) Fresh weight (g) Plant height(cm) 21 CK 3.21±0.67 32.8±2.1 22 5 2.62±0.52 27.1±1.8 23 10 2.32±0.41 25.1±1.1 24 15 2.27±0.38 24.2±2.3 25 20 2.08±0.49 22.9±1.3 26 25 1.92±0.22 21.28±0.8 27 30 1.88±0.21 20.12±1.3 28 35 1.57±0.87 18.21±0.5 29 40 1.45±0.22 17.77±0.3

[0066] Combined with the data in Table 2, it can be seen that when the Cd concentration in the sediment is greater than 15 mg / kg, the growth of evergreen dwarf Vallisneria is significantly restricted.

[0067] Example 3 Effects of resistant plant endophytes on the ability of Vallisneria odorifera to resist heavy metal Cd stress

[0068] (1) Test plants:

[0069] Vallisneria was purchased from Xinhe Aquatic Plant Co., Ltd., Chibi City;

[0070] Sedum southeasternensis: mined from abandoned lead-zinc mines in Quzhou, Zhejiang Province.

[0071] (2) Test steps:

[0072] a. Wash the plants thoroughly with sterile water and disinfect their surfaces (95% alcohol (1 min) → 30% hydrogen peroxide + 3% sodium hypochlorite (30 min) → 95% alcohol (1 min) → rinse with sterile water three times).

[0073] b. Place the disinfected plant in a sterilized mortar, grind it into juice, add an appropriate amount of phosphate buffer solution, stir evenly, let it stand for 10 minutes, and then stir quickly. Take 200μL and inoculate it into a sterilized LB solid culture medium containing 2mmol / L Cd, and culture it in a constant temperature sterile box at 25℃ for one week. Randomly select 3 colonies to a new culture medium, purify it once, and store it in a glycerol tube. Inoculate several isolated resistant endophytes into LB liquid culture medium respectively, culture them at 25℃ to the logarithmic phase, centrifuge the bacteria at 12000, resuspend them with sterile saline, repeat 3 times, and resuspend and dilute them to OD with sterile water for the last time. 600 =0.7, for standby use.

[0074] c. The indoor temperature was controlled at 25℃. Under the light conditions, 4 1×1×1.2 glass tanks were prepared, and the bottom was covered with pond mud of 50cm. Tank 31 was used as the control group (CK). Tanks 32 to 34 had the top 30cm of the bottom mud fully plowed, and CdCl2 was added to the plowed bottom mud and mixed thoroughly, so that the cadmium concentration of the bottom mud in tank 32 was 15mg / kg, and the cadmium concentration of the bottom mud in tanks 33 and 34 was 25mg / kg.

[0075] d. Select Vallisneria serrata seedlings with well-developed and strong root systems and plant them at 9-16 clumps / m 2 , 3 to 5 buds / clump were planted in glass tanks No. 31 and No. 32, and the water was returned to a depth of 60 cm.

[0076] e. 20 days after planting Vallisneria, choose a sunny day and evenly apply bleaching powder to the water in the Vallisneria culture tank at 8ppm to kill suspended microorganisms in the water and microorganisms on the surface of Vallisneria. One week later, evenly sprinkle fresh resistant endophyte suspension into glass tanks No. 31 and No. 32 at a ratio of 2% to allow the above endophytes to colonize on Vallisneria.

[0077] f. After 60 days of planting, thin out the seedlings and transplant them. After transplanting, the number of 32 glass fiber reinforced Vallisneria still maintains 9-16 clumps / m 2 .

[0078] g. Transplant the Vallisneria ovalis with developed and strong root system from the No. 32 glass steel and plant it in the No. 33 and No. 34 glass cylinders. 20 days after planting the Vallisneria ovalis, choose a sunny day and evenly apply bleaching powder to the water in the Vallisneria ovalis culture cylinder at 8ppm to kill the suspended microorganisms in the water and the microorganisms on the surface of the Vallisneria ovalis. One week later, evenly sprinkle the No. 34 glass cylinder with a 2% suspension of fresh resistant endophytes to colonize the endophytes. The No. 33 glass cylinder remains unchanged.

[0079] h. In the glass jars No. 31 and No. 32, 3 plants of Vallisneria were randomly selected after 60 days of planting, and the plant height, fresh weight, and Cd content in the aboveground and underground parts of Vallisneria were measured. In the glass jars No. 33 and No. 34, 60 days after Vallisneria was planted, the plant height, fresh weight, and Cd content in the aboveground and underground parts of Vallisneria were also measured. The results are shown in Table 3.

[0080] Table 3

[0081] Serial number Fresh weight (g) Plant height(cm) Cd content in aboveground parts (mg / kg) Cd content in underground part (mg / kg) Enrichment factor (BFAS) Transfer Factor (TF) 31 3.32±0.77 32.19±0.55 / / / / 32 2.88±0.38 28.98±1.7 36.28±0.12 192.32±0.6 7.93 0.19 33 2.23±0.24 24.18±3.2 60.11±0.78 169.23±0.98 4.52 0.36 34 2.96±0.25 29.12±2.1 38.25±0.34 240.15±0.89 6.88 0.16

[0082] Combined with the data in Table 3, it can be seen that although the growth of Vallisneria in the No. 31 glass cylinder was restricted after adding the endophytic suspension once, the growth restriction of Vallisneria in the No. 31 glass cylinder was less affected than that in the No. 32 glass cylinder. By comparing the Vallisneria in the No. 33 and No. 34 glass cylinders, it was found that when the Vallisneria was transplanted to the more polluted sediment, the fresh weight and plant height of the Vallisneria with the endophytic suspension were significantly better than those without the endophytic suspension, and the plant's ability to enrich heavy metals was enhanced and the transport coefficient was reduced. This shows that the addition of the endophytic suspension can enrich more heavy metals in the roots, reduce the transport of heavy metals to the stems and leaves, and thus reduce the toxic effects of heavy metals on the stems and leaves.

[0083] Example 4 Effect of resistant plant endophytes on the ability of Vallisneria odorifera to resist heavy metal Cd stress

[0084] (1) Test plants:

[0085] Vallisneria was purchased from Xinhe Aquatic Plant Co., Ltd., Chibi City;

[0086] Sedum southeasternensis: mined from abandoned lead-zinc mines in Quzhou, Zhejiang Province.

[0087] (2) Test steps:

[0088] a. Wash the plants thoroughly with sterile water and disinfect their surfaces (95% alcohol (1 min) → 30% hydrogen peroxide + 3% sodium hypochlorite (30 min) → 95% alcohol (1 min) → rinse with sterile water three times).

[0089] b. Place the disinfected plant in a sterilized mortar, grind it into juice, add an appropriate amount of phosphate buffer solution, stir evenly, let it stand for 10 minutes, and then stir quickly. Take 200μL and inoculate it into a sterilized LB solid culture medium containing 2.5mmol / L Cd, and culture it in a constant temperature sterile box at 25℃ for one week. Randomly select 3 colonies to a new culture medium, purify it once, and store it in a glycerol tube. Inoculate several isolated resistant endophytes into LB liquid culture medium respectively, culture them at 25℃ to the logarithmic phase, centrifuge the bacteria at 13000g, resuspend them with sterile saline, repeat 3 times, and resuspend and dilute them to OD with sterile water for the last time. 600 =0.8, for standby use.

[0090] c. The indoor temperature was controlled at 28℃. Under the light conditions, 4 1×1×1.2 glass tanks were prepared, and the bottom was covered with 50cm of pond mud. No. 41 was used as the control group (CK). No. 42 and No. 44 fully plowed the top 30cm of the bottom mud, added CdCl2 and mixed it with the plowed bottom mud, respectively, to achieve a cadmium concentration of 15mg / kg in the bottom mud of No. 42, and a cadmium concentration of 35mg / kg in the bottom mud of No. 43 and No. 44.

[0091] d. Select Vallisneria serrata seedlings with well-developed and strong root systems and plant them at 9-16 clumps / m 2 , 3-5 buds / clump were planted in No. 41 and No. 42 fiberglass and water was returned to a depth of 60cm.

[0092] e. 20 days after planting Vallisneria, choose a sunny day and evenly apply bleaching powder to the water in the Vallisneria culture tank at a concentration of 10ppm to kill suspended microorganisms in the water and microorganisms on the surface of Vallisneria. One week later, evenly sprinkle fresh resistant endophyte suspension at a ratio of 3% into glass tanks No. 41 and No. 42 to allow the above endophytes to colonize on Vallisneria.

[0093] f. After 60 days of planting, thin out the seedlings and transplant them. After transplanting, the 42# glass fiber reinforced Vallisneria still maintains 9~16 clumps / m 2 .

[0094] g. Transplant the Vallisneria ovalis with developed and strong root system from No. 42 and plant it in the glass tanks No. 43 and 44. 20 days after planting the Vallisneria ovalis, choose a sunny day and evenly apply bleaching powder to the water in the Vallisneria ovalis culture tank at a rate of 10ppm to kill the suspended microorganisms in the water and the microorganisms on the surface of the Vallisneria ovalis. One week later, evenly sprinkle the fresh resistant endophyte suspension at a rate of 3% into the glass fiber reinforced plastic No. 44 to colonize the endophytes. The glass tank No. 43 remains unchanged.

[0095] h. In the glass jars No. 41 and No. 42, 3 plants of Vallisneria were randomly selected after 60 days of planting, and the plant height, fresh weight, and Cd content in the aboveground and underground parts of Vallisneria were measured. In the glass jars No. 43 and No. 44, 60 days after Vallisneria was planted, the plant height, fresh weight, and Cd content in the aboveground and underground parts of Vallisneria were also measured. The results are shown in Table 4.

[0096] Table 4

[0097] Serial number Fresh weight (g) Plant height(cm) Cd content in aboveground parts (mg / kg) Cd content in underground part (mg / kg) Enrichment factor (BFAS) Transfer Factor (TF) 41 3.47±0.21 35.8±0.9 / / / / 42 3.05±0.12 31.14±2.9 38.74±0.42 200.41±0.55 8.01 0.18 43 2.08±0.49 22.9±1.3 58.48±0.33 178.54±0.48 4.88 0.33 44 2.77±0.43 26.17±2.1 48.18±0.76 275.64±1.27 6.93 0.17

[0098] Combined with the data in Table 4, it can be seen that although the growth of Vallisneria in glass cylinder No. 41 was restricted after adding endophytic suspension once, the growth restriction of Vallisneria in cylinder No. 41 was less affected than that in glass cylinder No. 42; when Vallisneria in cylinders No. 43 and No. 44 was compared, it was found that when Vallisneria was transplanted to the more polluted sediment, the fresh weight and plant height of Vallisneria with endophytic suspension added were significantly better than those without endophytic suspension added, and the plant's ability to enrich heavy metals was enhanced and the transport coefficient was reduced. This shows that the addition of endophytic suspension can enrich more heavy metals in the roots, reduce the transport of heavy metals to stems and leaves, and thus reduce the toxic effects of heavy metals on stems and leaves.

[0099] In summary, the method of enhancing the ability of Vallisneria sinensis to resist heavy metal cadmium stress of the present invention increases the cadmium enrichment ability of Vallisneria sinensis and strengthens its cadmium pollution remediation ability, so that Vallisneria sinensis can be better applied in the field of heavy metal contaminated sediment remediation.

[0100] Therefore, the present invention effectively overcomes various shortcomings in the prior art and has high industrial utilization value.

[0101] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. A method for enhancing the ability of Vallisneria tatarica to resist heavy metal cadmium stress, characterized in that: The following steps are involved: 1) Extraction of resistant plant endophytes: collecting cadmium hyperaccumulating herbaceous plants from cadmium-contaminated areas, disinfecting, grinding, adding buffer solution to mix evenly, and inoculating them into a sterile LB solid medium containing cadmium, culturing them at a constant temperature and aseptically, then selecting colonies and transferring them to a new sterile LB solid medium, purifying them, and storing the obtained resistant endophytes for later use, wherein the cadmium content in the LB solid medium is 1.5-3.5 mmol / L; 2) Preparation of resistant plant endophyte suspension: The resistant endophyte obtained in step 1) was inoculated into LB liquid medium, cultured to the logarithmic phase, centrifuged, resuspended in sterile saline, and resuspended in sterile water to an OD of 600 =0.5~0.9, for standby use; 3) Vallisneria planting: plowing the bottom mud surface of the first Vallisneria planting pond, mixing CdCl2 with the plowed bottom mud, preparing cadmium-containing bottom mud, wherein the cadmium content of the cadmium-containing bottom mud is 5-15 mg / kg, selecting Vallisneria seedlings and planting them in the first Vallisneria planting pond, and returning the water; 4) Colonization of resistant plant endophytes: 15 to 20 days after the Vallisneria seedlings are planted, bleaching powder is applied to the water of the first Vallisneria planting pond. 5 to 10 days later, the resistant endophyte suspension obtained in step 2) is evenly added to the water of the first Vallisneria planting pond to colonize the resistant endophytes on the Vallisneria; 5) Thinning out seedlings and transplanting: Thin out seedlings 50 to 70 days after planting Vallisneria in the first Vallisneria planting pond; plow the surface of the bottom mud of the second Vallisneria planting pond, mix CdCl2 with the plowed bottom mud, and prepare cadmium-containing bottom mud, wherein the cadmium content of the cadmium-containing bottom mud is 15 to 35 mg / kg; select Vallisneria plants from the first Vallisneria planting pond and plant them in the second Vallisneria planting pond, and repeat the method of step 4) in the second Vallisneria planting pond.

2. The method for enhancing the ability of Vallisneria vallisneriae to resist heavy metal cadmium stress according to claim 1, characterized in that: In step 1), the cadmium hyperaccumulating herbaceous plant is a herbaceous plant whose above-ground part absorbs heavy metals more than 10 times that of ordinary plants; And / or, in step 1), the herbaceous plant has good growth, tall plants and well-developed root systems; And / or, in step 1), the buffer is a phosphate buffer with a pH of 7.2 to 7.

4.

3. The method for enhancing the ability of Vallisneria vallisneriae to resist heavy metal cadmium stress according to claim 1, characterized in that: In step 1), the disinfection comprises the following steps in sequence: sterile water cleaning, 95% alcohol surface disinfection, 20-40% hydrogen peroxide + 2-5% sodium hypochlorite surface disinfection, 95% alcohol surface disinfection, and sterile water cleaning.

4. The method for enhancing the ability of Vallisneria vallisneriae to resist heavy metal cadmium stress according to claim 1, characterized in that: In step 1), the LB solid medium formula: NaCl 10g, peptone 10g, yeast powder 5g, agar 15g, distilled water 1000mL, pH 7.2~7.

4.

5. The method for enhancing the ability of Vallisneria vallisneriae to resist heavy metal cadmium stress according to claim 1, characterized in that: In step 1), the temperature of the constant temperature sterile culture is 20-30°C; And / or, in step 1), the constant temperature sterile culture time is 6 to 8 days; And / or, in step 1), the storage for standby use is storage in a glycerol tube.

6. The method for enhancing the ability of Vallisneria vallisneriae to resist heavy metal cadmium stress according to claim 1, characterized in that: In step 2), the LB liquid culture medium formula: NaCl 10g, peptone 10g, yeast powder 5g, distilled water 1000mL And / or, in step 2), the centrifugal force of the centrifugation is 10000-14000g; And / or, in step 2), the culture temperature during the culture to the logarithmic phase is 20-30°C.

7. The method for enhancing the ability of Vallisneria vallisneriae to resist heavy metal cadmium stress according to claim 1, characterized in that: In step 3), the planting density of the Vallisneria in the first Vallisneria planting pond is 9 to 16 clumps / m 2 , 3-5 buds / cluster; And / or, in step 3), the Vallisneria seedlings are Vallisneria seedlings with well-developed and strong root systems; And / or, in step 3), the bottom mud surface layer is 5 to 100 cm of the surface layer; And / or, in step 3), the backwater is backwater to a water depth of 40 to 60 cm.

8. The method for enhancing the ability of Vallisneria vallisneriae to resist heavy metal cadmium stress according to claim 1, characterized in that: In step 4), the amount of bleaching powder applied is 5 to 10 ppm; And / or, in step 4), the amount of the resistant endophyte suspension added to the water of the first Vallisneria planting pond is 1-3% V / V.

9. The method for enhancing the ability of Vallisneria vallisneriae to resist heavy metal cadmium stress according to claim 1, characterized in that: In step 5), the planting density of the Vallisneria in the second Vallisneria planting pond is 9 to 16 clumps / m 2 , 3-5 buds / cluster; And / or, in step 5), the bottom mud surface layer is 5 to 100 cm of the surface layer; And / or, in step 5), the backwater is backwater to a water depth of 40 to 60 cm; And / or, in step 5), the Vallisneria plants selected in the first Vallisneria planting pond are Vallisneria plants with well-developed and strong root systems.

10. Use of the method for enhancing the resistance of Vallisneria vallisneria to heavy metal cadmium stress according to any one of claims 1 to 9 in the field of water ecological restoration.