CN107686823B

CN107686823B - Plant growth promoting bacterium for reducing heavy metal content in leaf vegetables and application thereof

Info

Publication number: CN107686823B
Application number: CN201710938596.3A
Authority: CN
Inventors: 何琳燕; 盛下放; 王琪; 夏丽
Original assignee: Nanjing Agricultural University
Current assignee: Nanjing Agricultural University
Priority date: 2017-09-30
Filing date: 2017-09-30
Publication date: 2020-09-29
Anticipated expiration: 2037-09-30
Also published as: CN107686823A

Abstract

The invention belongs to the field of agriculture and environmental pollution treatment application, and relates to a plant growth promoting bacterium for reducing the content of heavy metal in leafy vegetables. A bacterium XL23 for reducing heavy metal content in leafy vegetables is classified and named as Dyella jianginingsis XL23, is preserved in China center for type culture collection with the preservation date of 2016, 10 months and 10 days, and the preservation number of strains is CCTCC NO: m2016552. Dyella jiangiensis XL23 strain is used as a raw material and is activated by a culture medium to prepare a bacterial suspension. Can remove heavy metals in liquid culture medium. The inoculation in the heavy metal contaminated soil can promote the growth of vegetables, improve the quality of crops and reduce the heavy metal content of leaf vegetables.

Description

Plant growth promoting bacterium for reducing heavy metal content in leaf vegetables and application thereof

Technical Field

The invention belongs to the field of agricultural technology and environmental pollution treatment application, and relates to a plant growth promoting bacterium for reducing the content of heavy metal in leafy vegetables and application thereof.

Background

The problem of overproof heavy metals in vegetable field soil gradually becomes a prominent environmental problem which threatens the development of Chinese vegetables and needs to be solved urgently. The development of an in-situ passivation method is a better choice for repairing the existing farmland with moderately and slightly polluted soil and overproof heavy metals. The common stabilizing/passivating materials include inorganic and organic modifiers, and are various in variety, and the stabilizing/passivating materials are easy to implement and commonly utilized due to low cost. The remediation agent or the remediation agent per se has a certain content of heavy metals (such as phosphate fertilizer, compost and the like) or can significantly influence the soil property and structure (such as lime and clay passivation remediation agents), if a large amount of the passivation remediation agent is used, adverse effects such as water eutrophication, secondary heavy metal pollution, interference of trace nutrient element absorption, influence on the soil physical and chemical properties and the like can be caused, and the continuous application of the passivation remediation agent is directly influenced (Wangliang and the like, 2009; Rotao and the like, 2007).Therefore, on the premise of ensuring the safe production of vegetables and improving the ecological environment of vegetable fields, it is necessary to screen and research safe and efficient heavy metal passivators and application thereof. Giloteaux et al found H produced by bacteria₂S can react with heavy metal ions such as Cd, Cu, Fe, Pb, Hg, Ni, Zn and the like to generate metal sulfide precipitate with low solubility and be removed.

Wang et al (2017) found that strain Q2-8 reduced the As content of the edible parts of vegetables (22% -50%), and strains Q2-13 and Q3-11 reduced the Cd content of the edible parts of vegetables (21% -53%), mainly because the strains reduced the Cd content in soil in DTPA-extracted state (Wang et al, 2017).

Thus, utilizing product H₂The plant growth promoting bacteria of the S are used for fixing heavy metals in soil, reducing the content of the heavy metals in vegetables, strengthening the passivation effect of other repairing agents, improving the nutrients and the ecological environment of vegetable field soil, and greatly reducing the transportation of the heavy metals to plants, thereby ensuring the safety of agricultural products.

Disclosure of Invention

The invention aims to overcome the problems in the prior art and provide a plant growth promoting bacterium for reducing the content of heavy metal in leafy vegetables.

Another object of the present invention is to provide the use of the strain.

The purpose of the invention can be realized by the following technical scheme:

a bacterium XL23 for reducing heavy metal content in leafy vegetables is classified and named as Dyella jianginingsisXL 23, is preserved in China center for type culture collection with the preservation date of 2016, 10 months and 10 days, and the preservation number of strains is CCTCC NO: m2016552. The colonies were round, yellowish and opaque. The thallus is rod-shaped, has a width of 0.4-0.6 μm and a length of 1.2-2.4 μm, and has no spore. Citrate is utilized. Produce H₂S, nitrite reduction, urease and arginine decarboxylase reaction are positive, and methyl red and VP reaction are negative. And arabinose, galactose, fructose, maltose and glucose are utilized to produce acid.

The bacterium XL23 is applied to promotion of vegetable growth and/or reduction of heavy metal absorption of vegetables.

The heavy metal is preferably any one or more of lead, cadmium, copper and zinc.

The bacterium XL23 is applied to improving the quality of vegetables.

The bacterium XL23 is preferably applied to increase the chlorophyll content of vegetables.

The bacterium XL23 is applied to reducing the content of lead, cadmium, copper and zinc in vegetables.

A bioremediation agent comprising the bacterium XL23 according to claim 1.

The bioremediation agent of claim 1, wherein the bioremediation agent is effective for the activity of bacteria XL23

The number of bacteria is more than 5 hundred million/ml.

The XL23 strain bioremediation preparation is applied to the production of vegetables in heavy metal polluted farmlands.

Advantageous effects

The Dyella jiangiensis XL23 strain can adsorb heavy metals in a solution.

The Dyella jiangiensis XL23 strain can promote the growth of vegetables in heavy metal contaminated soil and reduce the heavy metal content of the vegetables.

The application of the Dyella jiangiensis XL23 strain in promoting plant growth and reducing the heavy metal content of leaf vegetables. Compared with the prior art, the method has the following advantages:

(1) the XL23 strain according to the invention is capable of producing hydrogen sulfide.

(2) The Dyella jiangiensis XL23 can produce indoleacetic acid, and the maximum yield of IAA can reach 24 mg.L^-1(ii) a High yielding siderophores.

(3) The Dyella jiangiensis XL23 strain can adsorb heavy metals in a solution, the Pb removal rate range of the solution is 23% -58%, the Cd removal rate range is 5% -34%, the Cu removal rate range is 9% -72%, and the Zn removal rate can reach 24% -83%.

(4) The chlorophyll content of the pakchoi inoculated with Dylla jiangiensis XL23 is obviously increased by 32%, and the overground biomass of the pakchoi is obviously increased by 22% (p < 0.01).

(5) The Pb, Cd and Zn contents of overground parts of the pakchoi inoculated with Dylla jianginingsis XL23 are respectively and obviously reduced by 78%, 20% and 47%, and Cu is not detected.

(6) The Pb, Cd and Cu contents of the lettuce leaves which are inoculated with Dyella jiangiensis XL23 are respectively and remarkably reduced by 40 percent, 36 percent and 71 percent.

Biological sample preservation information

The plant growth promoting bacteria XL23 are classified and named as Dyella jiangiensis XL23 and are preserved in China center for type culture collection (CCTCC NO): m2016552.

Detailed Description

The following claims are hereby incorporated into the detailed description of the invention, with the understanding that the invention is not to be limited in any way, as any number of modifications may be made by one within the scope of the claims and the invention is still within the scope of the claims.

The following examples are, unless otherwise indicated, all of the routine experimentation and procedures known in the art.

EXAMPLE 1 isolation and characterization of Strain XL23

The heavy metal-resistant plant growth promoting bacteria XL23 strain (CCTCC NO: M2016552) is obtained by separating and purifying rhizosphere soil of Elsholtzia splendens (Elsholtzia spleens) growing in Cu-polluted soil, and the separation and identification method comprises the following steps: gently pulling out Elsholtzia splendens from soil, collecting rhizosphere soil tightly adhered to the surface of root system with about 2mm of soil with sterile forceps, weighing 10g, placing in a triangular flask containing 90ml of sterile water, fully oscillating for 30min, standing for 10min, collecting 0.1ml of suspension, and coating on broth solid culture medium (beef extract 3.0g, peptone 5.0g, and H) by 10-fold dilution method₂O1000 ml, agar 20g, pH 7.0-7.2), culturing at 28 deg.C for 3d, selecting single colony, streaking and purifying on broth solid culture medium, and storing. Inoculating the strain into broth liquid culture medium (beef extract 3.0g, peptone 5.0g, H)₂O1000 mL, pH 7.0-7.2), 30 ℃ and 150rpm for 16h, 1.5mL of the strain is takenCentrifuging the solution in an Eppendorf centrifuge tube at 5000rpm for 5min to collect thallus, extracting total bacterial DNA by a conventional method, amplifying the bacterial 16S rDNA by PCR, comparing and analyzing an amplified product with a known 16S rDNA sequence in GenBank after sequencing, reaching 99 percent of similarity with the 16S rDNA sequence of Dyella jianginingensis SBZ3-12(T), and identifying the strain as the strain.

Example 2 ability of Strain XL23 to produce Hydrogen sulfide

Referring to the method of the test for hydrogen sulfide production by leersic (2003), hydrogen sulfide (H) is produced by decomposing sulfur-containing organic substances according to certain bacteria₂S)。H₂And S forms black precipitates when meeting heavy metal salts, so that whether hydrogen sulfide is generated or not is judged. Sterilizing the modified broth peptone medium (yeast extract 7.5g, peptone 10.0g, NaCl 5.0g, agar 20.0g, distilled water 1000mL, pH7.0) at 121 deg.C for 25min, adding FeCl, filtering and sterilizing₂Subpackaging with sterile test tubes to make the height of the culture medium be 4-5cm, cooling, solidifying, inoculating by puncture method, culturing at 30 deg.C for 1, 3, 7 days, and observing to obtain a positive culture medium when the culture medium turns black and a negative culture medium when the culture medium does not turn black. Strain XL23 is capable of producing hydrogen sulfide.

Example 3 ability of Strain XL23 to produce IAA

According to the method of Gordon and Weber (1951), YMA liquid medium was dispensed from a test tube, 4mL of each tube was sterilized at 121 ℃ and 1mL of filter-sterilized 2.5mg/mL tryptophan solution was added to the tube, so that the final concentration of tryptophan in the medium was 0.5 mg/mL. XL23(CCTCC NO: M2016552) was inoculated in the above medium and shake-cultured at 30 ℃ for 3 days. The fermentation broth was centrifuged at 12000r/min for 5min, 1mL of the supernatant was taken, 50. mu.L of 10mM orthophosphoric acid was added, 2mL of Sackowski's color developer was added, mixed well, developed at 25 ℃ in the dark for 30min, and the absorbance was measured at 530 nm. Sterile medium was treated as above for the same treatment as control zero. And (3) making a standard curve by using IAA standard solutions with the concentrations of 0, 5, 10, 20, 40 and 60mg/L according to the same method, and calculating the concentration of the IAA in the fermentation liquor. The result shows that XL23(CCTCC NO: M2016552) can produce indole acetic acid, and the maximum yield of IAA can reach 24 mg/L.

EXAMPLE 4 Strain XL23 siderophore production ability

Siderophores were determined according to wangping et al. XL23(CCTCC NO: M2016552) was inoculated in YMA liquid medium and cultured with shaking at 30 ℃ and 150rpm for 48 hours. Centrifuging the fermentation liquid at 12000rpm for 5min, collecting supernatant, mixing with equal volume CAS detection liquid, developing for 1 hr, measuring light absorption value (A) at 630nm wavelength, and zeroing with deionized water as control. And (3) fully and uniformly mixing the same volume of the sterile YMA liquid culture medium and the CAS detection solution, and determining the light absorption value by the same method to obtain a reference value (Ar). A/Ar values <1, can be considered high yielding siderophores. The result shows that the A/Ar of XL23(CCTCC NO: M2016552) fermentation liquor is 0.1, and the iron carrier is high in yield.

EXAMPLE 5 activation of Strain XL23 and preparation of bacterial suspension

XL23(CCTCC NO: M2016552) slant strain was inoculated in LB solid medium (beef extract 3.0g, peptone 5.0g, H)₂O1000 ml, agar 20g, pH 7.0-7.2), and cultured at 30 ℃ for 3 d. Then, a full and viscous XL23 colony is selected and inoculated in a liquid medium (beef extract 3.0g, peptone 5.0g, H)₂O1000 ml, pH 7.0-7.2), 30 ℃ and 150rpm, and the cell number reaches more than 5 hundred million CFU/ml by shaking culture for 20-24 h.

Example 6 adsorption of heavy metals by Strain XL23

Based on LB liquid culture medium, mother liquor (1000mg/L, national standard) of heavy metals Pb, Zn, Cu and Cd is added after sterilization, so that the final concentration of Pb and Zn is 20mg/L and the final concentration of Cu and Cd is 5 mg/L. Preparing test strains cultured for about 16h into bacterial suspension, and adjusting OD_600nmThe value is 1.0, and the culture medium containing heavy metals is inoculated into the culture medium according to the proportion of 1 percent, and the culture is carried out at 30 ℃ and the shaking speed of 180 rpm. The dynamic sampling time points are set to 0, 4, 8, 12, 24, 28, 32, 40, 50, 56, 72 h. Simultaneous setting of the adjusting OD_600nmThe same treatment was performed with the inactivated bacteria having a value of 1.0 as a control. Sampling, centrifuging, acidifying, and measuring the concentration of each heavy metal element in the supernatant by adopting ICP-OES. The removal rate of each heavy metal ion in the culture medium by the test strain is obtained by calculation, and the calculation formula is as follows:

in the formula, C₀-initial concentration of heavy metals in solution, mg/L;

C_s-concentration of heavy metals in the supernatant of the test strain, mg/L;

C_cheavy metal concentration in the supernatant of the control sample, mg/L.

The strain XL23 can normally grow in a nitrogen liquid culture medium with heavy metal ions added, wherein the slow growth period is 0-12h, the exponential growth period is 12-50h, the growth reaches the stationary phase after 50h, and the OD at the stationary phase is OD_600nmWas 1.9 (Table 1). The overall removal effect of the strain XL23 on four heavy metal ions is represented by Zn & gt Pb & gt Cu & gt Cd (Table 2). At 28h, the removal rate of the strain XL23 to Zn ions and Cu ions reaches the maximum, and the removal rates are 82.8% and 71.6%, respectively; at 32h, the removal rate of Cd ions by the strain XL23 reaches maximum 33.6%; at 40h, the strain XL23 achieves the maximum Pb ion removal rate of 58.1%.

TABLE 1 growth of the strain Dyella dianingsisXL 23 in heavy metal-containing medium

Table 2 removal ratio of heavy metals (%) -by the Strain Dyella jianginsis XL23

Example 7 effects of Strain XL23 on promoting growth of Brassica campestris and reducing heavy metal content in leaves

A potting experiment was used. The soil to be tested is obtained from surface soil (0-20cm) of farmland around Nanjing Cyanea-Gangensis lead-zinc-silver ore, the soil type is yellow brown soil, and the total lead content is 1136.52mg kg^-1The total zinc content is 1354.02mg kg^-1The total copper content was 103.77mg kg^-1The total cadmium content is 5.95mg kg^-1. And removing plant residues after soil sample collection, air-drying, and sieving by a 5mm sieve for later use. The test plant is Brassica rapa L. Filling the prepared sieved soil into hole trays, filling 80g of soil into each hole tray, watering 3d before planting and keeping in the fieldThe water holding capacity is about 60%. Selecting uniformly sized seeds of pakchoi and lettuce, adding ethanol and 30% H₂O₂(V: V1: 1) surface sterilization for 3min, and rinsing with sterile deionized water. The sterilized plant seeds were divided into 2 portions, one portion was soaked with the bacterial suspension XL23 obtained in example 5 for 4 hours to prepare an inoculated group, and the other portion was soaked with the sterilized suspension for the same time as a control group (CK), and each treatment was repeated 4 times. And (3) broadcasting the soaked plant seeds in a pot, thinning seedlings 1 week after seedling emergence, and finally reserving 3 seedlings in each pot. After the thinning-out, the inoculation treatment was performed again, and the XL23 bacterial suspension and the sterilized suspension obtained in example 5 were inoculated to the roots of the plants in pots of the inoculation group and the control group, respectively, at an inoculum size of 5mL per pot. The plants were harvested after 40 days of growth. During the growth of the plants, the plants are watered at a proper amount every day to ensure the moisture required by the growth of the plants. The relative chlorophyll content was determined using a SPAD-502 chlorophyll apparatus. The plants were removed from the pot and washed with deionized water. When harvesting, the plant is separated from the root and the overground part along the root-stem combination part. Deactivating enzymes at 105 deg.C, oven-drying at 70 deg.C, and weighing the dry weight of the roots and aerial parts. And grinding the plant sample, digesting by a nitric acid-perchloric acid method, and measuring the contents of heavy metals Pb, Cd, Zn and Cu in the plant by an atomic absorption spectrophotometer.

As can be seen from Table 3, compared with the control, the chlorophyll content of the pakchoi treated by the inoculated strain XL23 is remarkably increased by 32%, the overground part biomass and the root biomass of the pakchoi are respectively increased by 21.8% and 11.3%, wherein the growth promoting effect of XL23 on the overground part biomass reaches a very remarkable level (p < 0.01).

TABLE 3 growth promoting effect of the strain Dyella jiangiensis XL23 on seedling-stage pakchoi

As can be seen from Table 4, compared with the control, the inoculated strain XL23 remarkably reduces the Pb content of the overground part of the pakchoi by 66% (p <0.05), and remarkably reduces the Cd and Zn contents of the overground part of the pakchoi by 23% and 47% (p <0.01), respectively. The edible part of the inoculated pakchoi has no Cu detected.

TABLE 4 Effect of the Strain Dyella jiangiensis XL23 on reducing heavy metals in seedling-stage pakchoi

Example 8 Effect of Strain XL23 on reduction of lead and cadmium content in lettuce leaves

A potting experiment was used. The soil to be tested is obtained from surface soil (0-20cm) of farmland around Nanjing Cyanea-Gangensis lead-zinc-silver ore, the soil type is yellow brown soil, and the total lead content is 1136.52mg kg^-1The total cadmium content is 5.95mg kg^-1. And removing plant residues after soil sample collection, air-drying, and sieving by a 5mm sieve for later use. The test plant is lettuce Lactuca sativa. And (3) filling the prepared screened soil into a hole tray, filling 80g of soil into each hole tray, and watering 3 days before planting to keep the field water holding capacity to be about 60%. Selecting lettuce seeds with uniform size, and adding ethanol and 30% H₂O₂(V: V1: 1) surface sterilization for 3min, and rinsing with sterile deionized water. The sterilized plant seeds were divided into 2 portions, one portion was soaked with the bacterial suspension XL23 obtained in example 5 for 4 hours to prepare an inoculated group, and the other portion was soaked with the sterilized suspension for the same time as a control group (CK), and each treatment was repeated 4 times. And (3) broadcasting the soaked plant seeds in a pot, thinning seedlings 1 week after seedling emergence, and finally reserving 3 seedlings in each pot. After the thinning-out, the inoculation treatment was performed again, and the XL23 bacterial suspension and the sterilized suspension obtained in example 5 were inoculated to the roots of the plants in pots of the inoculation group and the control group, respectively, at an inoculum size of 5mL per pot. The plants were harvested after 40 days of growth. During the growth of the plants, the plants are watered at a proper amount every day to ensure the moisture required by the growth of the plants. The plants were removed from the pot and washed with deionized water. When harvesting, the plant is separated from the root and the overground part along the root-stem combination part. Deactivating enzymes at 105 deg.C, oven-drying at 70 deg.C, and weighing the dry weight of the roots and aerial parts. And grinding the plant sample, digesting by a nitric acid-perchloric acid method, and measuring the contents of heavy metals Pb, Cd, Zn and Cu in the plant by an atomic absorption spectrophotometer.

As can be seen from Table 5, compared with the control, the inoculation strain XL23 can reduce the Pb and Cd contents in lettuce leaves by 40% and 36%, respectively, and can reduce the Cu content of the edible part of the lettuce by 71%.

TABLE 5 Effect of the Strain Dyella jiangiensis XL23 on reducing heavy metals in lettuce at seedling stage

Claims

1. A bacterium XL23 for reducing heavy metal content in leafy vegetables is classified and named as Dyella jianginingsis XL23, is preserved in China center for type culture collection with the preservation date of 2016, 10 months and 10 days, and the preservation number of strains is CCTCC NO: m2016552.

2. Use of bacterium XL23 according to claim 1 for promoting the growth of vegetables and/or for reducing the absorption of heavy metals by vegetables; the heavy metal is any one or more of lead, cadmium, copper and zinc.

3. Use of bacterium XL23 according to claim 1 for improving vegetable quality.

4. Use according to claim 3, characterized in that the bacterium XL23 according to claim 1 is used for increasing the chlorophyll content of vegetables.

5. Use of bacterium XL23 according to claim 1 for reducing the lead, cadmium, copper and zinc content of vegetables.

6. A bioremediation agent comprising the bacterium XL23 according to claim 1.

7. The bioremediation agent of claim 6, wherein the bacteria XL23 is present in an effective number of 5 hundred million/ml or more.

8. Use of the bioremediation formulation of claim 6 or 7 in the production of vegetables for heavy metal contaminated farmlands; the heavy metal is any one or more of lead, cadmium, copper and zinc.