CN102161976B

CN102161976B - Endophyte for promoting growth of plants and remediation of heavy metal-contaminated soil and application

Info

Publication number: CN102161976B
Application number: CN 201010609930
Authority: CN
Inventors: 罗胜联; 徐涛英; 陈亮; 饶婵; 陈觉梁; 肖潇; 刘承斌
Original assignee: Hunan University
Current assignee: Hunan University
Priority date: 2010-12-28
Filing date: 2010-12-28
Publication date: 2013-02-06
Anticipated expiration: 2030-12-28
Also published as: CN102161976A

Abstract

The invention provides an endophyte for promoting the growth of plants and the remediation of heavy metal-contaminated soil and application thereof. Bacterial liquid of endophyte SLS18 is utilized to dip-dye Sorghum bicolor L. in a mode of seed bacteria dipping to colonize the endophyte SLS18 into the Sorghum bicolor L. to accompany each stage of the growth of the Sorghum bicolor L., so that the growth of the Sorghum bicolor L. on heavy metal-contaminated marginal land and the remediation of soil are promoted. The endophyte SLS18 is separated from the stem of a hyperaccumulator, namely pokeberry root, is obtained through screening, and is an endophyte which can be successfully colonized in the Sorghum bicolor L., has a remarkable effect of promoting the growth of the Sorghum bicolor L. and has the heavy metal resistance. A method is easy to operate, and is safe and economic, no new pollution is introduced into soil environment, heavy metal-contaminated marginal land resources can be effectively utilized so as to avoid the rival of energy crops and food crops against finite cultivated land, and heavy metal-contaminated land can also be effectively remediated.

Description

Endophyte and application that a kind of Promoting plant growth and heavy-metal contaminated soil are repaired

Technical field

The invention belongs to soil pollution by heavy metal and repair the field, be specifically related to a kind of endophyte that can promote the energy crop growth, and infect plant, and improve the application method of phytoremediation soil pollution by heavy metal efficient.

Background technology

Along with the development of modernization industry, heavy metal contamination has caused serious threat to human health and the ecosystem, becomes gradually one of global environmental problem.Wherein, manganese can and cause manganese poisoning at people's cylinder accumulation, for example parkinsonism, reproductive system and immunity system pathology.And another kind of heavy metal cadmium has carcinogenic, teratogenesis, mutagenesis, can cause bone, digestive tube, vascular lesion at people's cylinder accumulation.As everyone knows, the appliable plant recovery technique is removed the heavy metal in the soil, compares with traditional physical chemistry recovery technique to have lot of advantages, such as characteristics such as cost are low, safe and effective, environmentally friendly.Especially use super accumulation plant and carry out phytoremediation, this kind of plant not only can not be subjected to the murder by poisoning of heavy metal in process of growth, can also accumulate plurality of heavy metal in its body.Yet super accumulation plant because biomass bioavailability low and heavy metal in soil is low, has been limited the phytoremediation efficient of super accumulation plant when the growth of heavy metal contamination area.Therefore, many investigators are devoted to study the method that improves super accumulation phytomass.Wherein, plant combined Plant growth-promoting effect endophyte (PGPE) technology has effectively promoted the development of the phytoremediation application of super accumulation plant.As everyone knows, PGPE can assist its host plant opposing heavy metal to accumulate in vivo to cause toxic effect, can promote the growth of host plant simultaneously.This is because PGPE has several Plant growth-promoting effect mechanism: (1) is by plant hormone Promoting plant growths such as generation indolylacetic acids (IAA); (2) assist plant from soil, to obtain the required iron ion of growth by producing siderophore, thereby make host plant obtain optimum growth conditions; (3) pass through the concentration of acc deaminase activity decreased pressure ethene precursor ACC, thereby reduce pressure ethene to the restraining effect of plant-growth.

Yet from another angle, what be worth arousing attention is to utilize these growth promotion mechanism of PGPE to promote the growth of energy crop on the marginal soil of heavy metal contamination, thereby improves the biomass of energy crop and the CO that catches from air ₂Raw material as bioenergy is very significant behave.Described marginal soil refers to because of arid, saline and alkaline is high, be subjected to heavy metal and Organic pollutants etc. to be not suitable for the soil of general crop growth.Therefore this behave can avoid energy crop and food crop to the competition of the agricultural resources such as limited arable land.Along with the day by day increase that bioenergy uses, the problem that energy crop takies the food crop arable land has become one of socioeconomic problem that needs to be resolved hurrily.According to up-to-date Economic Analysis Report, since sweet sorghum (Sorghum bicolor L.) have photosynthetic efficiency high, produce to drop into low, the characteristics such as biomass is high, drought-resistant, salt tolerant, be acknowledged as at present in the world one of potential energy crop of tool.In addition, sweet sorghum also can extract the heavy metal composition in the soil, has the potentiality for phytoremediation.Yet as far as we know, also do not have patent and bibliographical information to cross the separating obtained growth promotion of anti-heavy metal endophyte in the super accumulation plant materials at present, be applied to promote growth and the phytoremediation of energy crop on the marginal land of heavy metal contamination.

Summary of the invention

The invention provides a kind of endophyte that can promote the energy crop growth, and infect plant, and improve the application method of phytoremediation soil pollution by heavy metal efficient.The present invention is simple to operate, safety, economy, and edatope is not introduced new pollution, can effectively avoid energy crop and food crop to the competition in limited arable land, simultaneously the soil of repairing heavy metal pollution effectively.

For addressing the above problem, technical scheme provided by the invention is:

The endophyte that a kind of Promoting plant growth and heavy-metal contaminated soil are repaired, it is characterized in that, described endophyte is bacillus sp.SLS18, on December 20th, 2010, Chinese Typical Representative culture collection center in Hubei China province Wuhan City Wuhan University, carried out preservation, deposit number is CCTCCNO:M 2010359.

Described endophyte is applied to Promoting plant growth and heavy-metal contaminated soil reparation; Specifically described endophyte SLS18 is contaminated sweet sorghum seed, then the sweet sorghum sowing is grown at heavy metal (as: Mn/Cd) contaminated land, carries out soil remediation.The bacterial concentration of endophyte SLS18 is 10 ⁹Cfu mL ^-1Soak 4h in the bacterium liquid.

Described sweet sorghum seed carries out surface sterilization before soaking bacterium process is: sweet sorghum seed is soaked in 70% ethanolic soln 1min, be soaked in again 1% chlorine bleach liquor 10min, with after the rinsed with deionized water after the sterilization at least 5 times, dry and treat that seed soaks bacterium subsequently.

Sweet sorghum seed soaks the used endophyte SLS18 thalline of bacterium process for being in logarithmic phase, the endophyte culturing process is: with the SLS18 bacterial classification inoculation of 100 μ L preservations to the sterile LB substratum of 25mL, after nutrient solution placed 30 ℃ of shaking table concussions to cultivate 24h, preparation concentration was 10 ⁹Cfu mL ^-1Bacterium liquid.

Preparation concentration is 10 ⁹Cfu mL ^-1The preparation process of bacterium liquid be: get 5mL and cultivate endophyte bacterium liquid behind the 24h at 13000r/min, centrifugal under the condition of 10min, with centrifugal again after the washed with de-ionized water, then the thalline of collecting is suspended in 0.03M MgSO ₄In the solution, and be diluted to the OD that surveys ₆₀₀Be 1, namely getting corresponding concentration is 10 ⁹Cfu mL ^-1Bacterium liquid.

The sweet sorghum seed of contaminating endophyte SLS18 is seeded in the heavy metal Mn/Cd contaminated soil, the SLS18 endophyte is survived in follow each stage of sweet sorghum growth in the sweet sorghum body, promote the growth of sweet sorghum on the heavy metal contamination marginal land and the purpose of soil remediation thereby reach.

The anti-heavy metal bacterium that above-mentioned growth promotion endophyte SLS18 obtains for separation in the super accumulation plant pokeweed body, and can successfully contaminate in the sweet sorghum body.

The screening and separating process of bacterial strain of the present invention and method are with reference to a patent " a kind of method that obtains endophyte of plant of the separating " (application number: 200810107514.1) of this scientific research group.Its colony characteristics on the LB solid medium is: size 3 is to 6mm; Circular, neat in edge; Milk yellow; Smooth surface, protuberance, opaque.Its part Physiology and biochemistry Property Identification is: Gram-negative bacteria; Produce indolylacetic acid and siderophore; Its heavy metal resistance specifically shows as with MIC (minimum inhibition concentration): Mn 2000mg/L, Cd 40mg/L, Pb 500mg/L, Cu 200mg/L, Zn 100mg/L (LB substratum).By 16S rDNA is carried out sequencing analysis, endophyte SLS18 is accredited as bacillus (bacillus sp.)

Heterosis of the present invention is bright simple to operate safe, economical, edatope is not being introduced under the prerequisite of new pollution, can effectively promote energy crop to grow in the marginal land that is subjected to heavy metal contamination, simultaneously the soil of repairing heavy metal pollution.

Description of drawings

Fig. 1 is operational flowchart of the present invention;

Fig. 2 be endophyte SLS18 bacterium on the impact of the root dry weight (g) of sweet sorghum, Phytolacca acinosa, black nightshade, heavy metal-polluted soil concentration is respectively 0mg kg ^-1, Mn 2000mg kg ^-1, Cd 50mg kg ^-1

Fig. 3 be endophyte SLS18 bacterium on the impact of the over-ground part dry weight (g) of sweet sorghum, Phytolacca acinosa, black nightshade, heavy metal-polluted soil concentration is respectively 0mg kg ^-1, Mn 2000mg kg ^-1, Cd 50mg kg ^-1

Fig. 4 is that endophyte SLS18 is to sweet sorghum, Phytolacca acinosa, (the μ g strain of black nightshade accumulation Mn ability ^-1) impact, heavy metal-polluted soil concentration is respectively Mn 2000mg kg ^-1

Fig. 5 is that endophyte SLS18 is to sweet sorghum, Phytolacca acinosa, (the μ g strain of black nightshade accumulation Cd ability ^-1) impact, heavy metal-polluted soil concentration is respectively Cd 50mg kg ^-1

Embodiment:

Be intended to further specify the present invention below in conjunction with embodiment, and unrestricted the present invention.

Endophyte SLS18 is contaminated sweet sorghum simultaneously, and behind Phytolacca acinosa (P.acinosa R.) and black nightshade (S.nigrum L.) seed, distinguish Seeded growth with the three kind of plant seeds of not contaminating endophyte, carry out the test effect contrast, wherein Phytolacca acinosa and black nightshade are the super accumulation of newfound two heavy metal species plant; The test operation process is undertaken by Fig. 1.

3 parallel tests are carried out in all tests, and endophyte SLS18 is significantly improved to the sweet sorghum biomass, see Fig. 2, Fig. 3, Fig. 4 take Phytolacca acinosa and black nightshade as the statistics of reference.

Endophyte SLS18 sees Table 1 to the growth promotion rate of sweet sorghum, Phytolacca acinosa, black nightshade, and heavy metal-polluted soil concentration is respectively 0mg kg ^-1, Mn 2000mg kg ^-1, Cd 50mg kg ^-1

Table 1

Endophyte SLS18 sees Table 2 to the enhancing rate of individual plant sweet sorghum, Phytolacca acinosa, black nightshade over-ground part Heavy Metal Accumulation total amount, and heavy metal-polluted soil concentration is respectively Mn 2000mg kg ^-1, Cd 50mg kg ^-1

Table 2

Endophyte SLS18 decides the situation of growing (cfu g in sweet sorghum, Phytolacca acinosa, black nightshade body ^-1The plant fresh weight) see Table 3, heavy metal-polluted soil concentration is respectively 0mg kg ^-1, Mn 2000mg kg ^-1, Cd 50mg kg ^-1

Table 3

Improved to significant effective of the present invention biomass and the individual plants heavy metal integral dose of sweet sorghum, Phytolacca acinosa, black nightshade, endophyte SLS18 can successfully grow in the sweet sorghum body surely, and the acting in conjunction of SLS18 associating sweet sorghum is stronger than Phytolacca acinosa and black nightshade.Therefore, the present invention can realize promoting energy crop in the growth of heavy metal contamination Marginal region and the application of heavy metal reparation, thereby effectively utilize the marginal soil of heavy metal contamination, thereby avoid energy crop and food crop to the competition in limited arable land, the while is the soil of repairing heavy metal pollution effectively.

Claims

1. the endophyte repaired of a Promoting plant growth and heavy-metal contaminated soil is characterized in that, described endophyte is bacillus sp.SLS18, and deposit number is CCTCC NO:M 2010359; Described plant is sweet sorghum, Phytolacca acinosa or black nightshade; Described heavy metal is Mn and Cd.

2. the purposes of endophyte claimed in claim 1 in Promoting plant growth and heavy-metal contaminated soil are repaired; Described plant is sweet sorghum, Phytolacca acinosa or black nightshade; Described heavy metal is Mn and Cd.

3. the purposes of endophyte according to claim 2 is characterized in that, described endophyte SLS18 is contaminated sweet sorghum seed, and then the sweet sorghum sowing is grown on the ground at heavy metal polluted soil, carries out soil remediation.

4. according to claim 2 or the purposes of 3 described endophytes, it is characterized in that, is 10 with concentration ⁹CfumL ^-1The bacterium liquid of endophyte SLS18 sweet sorghum seed carried out seed soak bacterium.

5. the purposes of endophyte according to claim 4 is characterized in that, described sweet sorghum seed places bacterium liquid to soak 4h.

6. the purposes of endophyte according to claim 4, it is characterized in that, described sweet sorghum seed carries out surface sterilization before soaking bacterium process is: sweet sorghum seed is soaked in 70% ethanolic soln 1min, be soaked in again 1% chlorine bleach liquor 10min, with after the rinsed with deionized water after the sterilization at least 5 times, dry and treat that seed soaks bacterium subsequently.

7. the purposes of endophyte according to claim 4, it is characterized in that, sweet sorghum seed soaks the used endophyte SLS18 thalline of bacterium process for being in logarithmic phase, the endophyte culturing process is: with the SLS18 bacterial classification inoculation of 100 μ L preservations to the sterile LB substratum of 25mL, after nutrient solution placed 30 ℃ of shaking table concussions to cultivate 24h, preparation concentration was 10 ⁹Cfu mL ^-1Bacterium liquid.

8. the purposes of endophyte according to claim 7 is characterized in that, preparation concentration is 10 ⁹CfumL ^-1The preparation process of bacterium liquid be: get 5mL and cultivate endophyte bacterium liquid behind the 24h at 13000r/min, centrifugal under the condition of 10min, with centrifugal again after the washed with de-ionized water, then the thalline of collecting is suspended in the 0.03M MgSO4 solution, and to be diluted to the OD600 that surveys be 1, and namely getting corresponding concentration is 10 ⁹Cfu mL ^-1Bacterium liquid.