CN114107123B

CN114107123B - Sword fungus strain and application thereof

Info

Publication number: CN114107123B
Application number: CN202111506459.5A
Authority: CN
Inventors: 高岩; 陈未; 李江叶; 李云涛; 张维国; 刘丽珠; 续绅
Original assignee: Jiangsu Yanjiang Agricultural Science Research Institute
Current assignee: Jiangsu Yanjiang Agricultural Science Research Institute
Priority date: 2021-12-10
Filing date: 2021-12-10
Publication date: 2024-01-02
Anticipated expiration: 2041-12-10
Also published as: CN114107123A

Abstract

The invention relates to a strain of sword bacteria and application thereof, wherein the classification of the sword bacteria is named as sword bacteria @, the strain of sword bacteria is named as sword bacteria @Ensifer sp.) RE15 belonging to gram-negative bacteria, deposit number: cctccc NO: m20211334. The rice endophyte is of the genus Sword, has the cadmium resistance of 150mg/L, the salt tolerance concentration of 15%, and has stable adsorption capacity to low-concentration cadmium, strong IAA and siderophore production capacity, and stronger growth promotion capacity, can be applied to cadmium-polluted farmland for bioremediation, and is more beneficial to development and application compared with the existing soil bacteria.

Description

Sword fungus strain and application thereof

Technical Field

The invention belongs to the field of environmental microorganisms, and particularly relates to a sword fungus and application thereof.

Background

Cadmium has been recognized as one of the major toxic heavy metals most threatening to humans due to its high mobility and high toxicity. Rice is an important grain crop in China, cadmium accumulates in a large amount in roots, stems, leaves and grains of rice, not only the yield and quality of the rice and the whole farmland ecological system are affected, but also the health of human beings is endangered through a food chain.

The plant-microorganism combined repair is used as a biological repair technology, fully utilizes the symbiotic relationship of plants and microorganisms, and gradually becomes a research hot spot at home and abroad due to the characteristics of environmental friendliness, no secondary pollution and the like. On one hand, the microorganisms can enable heavy metals to be precipitated or adsorbed and fixed in soil through the passivation effect of the heavy metals, so that the occurrence form of the heavy metals is changed, and the absorption and transportation of the heavy metals by the plants are reduced; on the other hand, the microorganism can promote the plant growth through various ways, improve the tolerance of the plant to heavy metals and strengthen the repair effect of the plant. Therefore, the method for repairing heavy metal contaminated soil by strengthening plant fixation by utilizing plant growth promoting bacteria has a good application prospect.

Disclosure of Invention

The invention aims to provide a strain of sword bacteria and application thereof.

The aim of the invention can be achieved by the following technical scheme:

in a first aspect, the invention provides a strain of sword bacteria, classified as sword bacteria (Ensifer sp.) RE15, belonging to gram-negative bacteria, deposited with the following numbers: cctccc NO: m20211334. The 16S rRNA sequence is shown as SEQ ID NO: 1.

In a second aspect, the invention provides a culture method of the sword bacteria, which comprises the steps of inoculating the sword bacteria into a culture medium and culturing at 28-30 ℃.

Further, the sword bacteria are cultured at 30 ℃.

Further, the sword bacteria are cultivated under the condition that the pH=4.0-8.0; preferably 7.0.

Further, the culture medium is Luria-Bertani (LB) culture medium, and the components are as follows: 10.0g/L peptone, 5.0g/L yeast powder, 10.0g/L NaCl and the balance of water.

In a third aspect, the invention provides the use of the above-described Sword in IAA production. Fermenting and culturing the sword bacteria to produce IAA.

In a fourth aspect, the invention provides application of the sword bacteria in cadmium-resistant growth promotion of crops.

The invention screens a strain RE15 of the genus Sword from rice roots, and the concentration of cadmium in the plant body is generally lower than that of other environments, so that endophytes have weak tolerance or adsorption capability to cadmium, but have certain growth promotion performance. The endophyte cadmium resistance of the invention can reach 150mg/L, the tolerance concentration of the endophyte cadmium can reach 15 percent, the endophyte cadmium has stable adsorption capacity to low-concentration cadmium, the IAA and siderophore production capacity is strong, the IAA yield can reach 75mg/L, the endophyte cadmium has stronger growth promoting capacity, and the endophyte cadmium can be applied to cadmium-polluted farmland for bioremediation, thus being more beneficial to development and application compared with other soil bacteria in the prior art.

Drawings

FIG. 1 shows colony morphology of Sword RE15 in the present invention.

FIG. 2 is a phylogenetic relationship of Sword RE15 in the present invention.

FIG. 3 is a graph showing the change in cell density of Sword RE15 in the medium supplemented with different concentrations of cadmium in the present invention.

FIG. 4 is a graph showing the pH change of Sword RE15 in the medium supplemented with cadmium at various concentrations in the present invention.

FIG. 5 is a graph showing the change in adsorption of RE15 under different cadmium concentrations.

The biological material of the invention, which is classified and named as Sword fungus (Ensifer sp.) RE15, has been preserved in China Center for Type Culture Collection (CCTCC) for 10 months and 28 days in 2021, and has the preservation number of: cctccc NO: m20211334, address: chinese armed chinese.

Detailed Description

The invention will be described in further detail with reference to the drawings and the detailed description, in order to make the objects, technical solutions and advantages of the invention more apparent.

Example 1

Isolation and screening of endophytes

The strain is separated from rice roots in a cadmium polluted farmland, and the specific separation method is as follows:

(1) Root surface disinfection: collecting rice roots, flushing dirt on the plant surfaces by running water, and sucking water; cutting roots by using sterile scissors, and carrying out surface disinfection on the rice roots according to the steps of soaking the rice roots in 75% ethanol for 30s, washing the rice roots in sterile water for 3 times, soaking the rice roots in 0.3% sodium hypochlorite for 6min and washing the rice roots in sterile water for 5 times; adding a certain amount of sterilized water into a sterile mortar, and grinding to homogenate.

(2) And (3) separating and purifying: homogenizing the above mixture at 10 ^-3 、10 ^-4 、10 ^-5 Dilution is carried out by multiple, 100 mu L of dilution liquid is respectively coated on LB plates, the culture is carried out for 3-4 days in an inverted way at 30 ℃, single bacterial colonies are picked up, and streak purification is carried out for 3 times.

(3) Cadmium resistant strain selection: the formula of the LB solid medium containing cadmium is as follows: 10.0g peptone, 5.0g yeast powder, 10.0g NaCl,20.0g agar powder, pH 7.0; cadmium chloride mother liquor is added to lead the final concentration of cadmium to be 50mg/L, 100mg/L and 150mg/L. And streaking the single colony obtained by purification on an LB plate containing cadmium, picking the single colony with the cadmium resistance higher than 150mg/L, and obtaining two target strains which are named endophyte RE 35 and endophyte RE15 respectively, wherein the application is described in detail for the endophyte RE 15.

Example 2

Classification and identification of endophyte RE15

The bacterial strain RE15 is classified and identified by adopting a 16S rRNA gene sequence analysis method, and the specific steps are as follows:

(1) Extraction of total bacterial DNA: genomic DNA of strain RE15 was extracted using Takara doctor materials technologies Co., ltd (TaKaRa) MiniBEST bacterial genomic DNA extraction kit.

(2) Amplification of the RE15 16S rRNA Gene sequence of the Strain: the general primer 27F (5 '-AGAGTTTGATCMTGGCTCAG-3')/1492R (5 '-CGGYTACCTTGTTACGACTT-3') was usedThe PCR premix was prepared by 2×Taq Master Mix, a biological technology Co-Ltd, of Nannoopran, and the system was as follows: 2 XTaq Master Mix 25. Mu.L, F, R primer 1. Mu.L each, template DNA 1. Mu.L, dd H ₂ O was made up to 50. Mu.L; reaction cycle parameters: pre-denaturation at 95℃for 5min; cycling at 95℃for 30s,55℃for 30s,72℃for 1min, 35; total extension was carried out at 72℃for 10min.

(3) Clone sequencing analysis of PCR products: the strain was sent to general biosystems (Anhui) Inc. for sequencing, with the nucleotide sequence set forth in SEQ ID NO: 1. Nucleotide sequence comparison of the 16S rRNA gene sequence of the strain on the national center for biotechnology information website (http:// www.ncbi.nlm.nih.gov) to obtain a plurality of nucleotide sequences with homology to the gene sequence of related strains shows that the 16S rRNA sequence of the strain RE15 has more than 99 percent of homology to the gene sequence of Swiss (Ensifer), and the isolated strain is identified as Swiss (Ensifer sp.).

(4) Morphological characterization of strain RE15 was identified as follows:

sword RE15 belongs to gram-negative bacteria, the colony diameter is about 0.1-0.2cm, and the colony on the LB plate is white, round, neat in edge and moist in surface. The optimal growth conditions of the strain are as follows: pH7.0, temperature 30 ℃.

Example 3

Growth curve and growth promoting characteristic research of strain RE15

(1) Determination of strain growth curves at different cadmium concentrations: cadmium chloride powder is dissolved in deionized water to prepare cadmium chloride mother solution with the concentration of 5000mg/L, and a proper amount of the mother solution is taken and added into a triangular flask which is respectively provided with LB culture medium, so that the final concentration of Cd is 0mg/L, 5mg/L and 10mg/L respectively. Followed by high-temperature autoclaving. Culturing RE15 bacterial liquid stored in a glycerol tube on an LB plate under a sterile condition at 30 ℃ until single colonies are formed, picking single colonies, inoculating the single colonies into an LB liquid culture medium, culturing for 20 hours at 150rpm at 30 ℃ to obtain seed liquid, transferring the seed liquid into the LB culture medium with different cadmium concentrations according to the inoculum size of 5%, and sucking fermentation liquor every 2 hours to measure the absorbance value at 600 nm. The result shows that the logarithmic growth phase of the strain RE15 is about 2-16 h, and the addition of cadmium has no influence on the growth potential of the strain.

(2) Strain auxin (IAA) ability assay: tryptophan is prepared into 2.5mg/mL solution, filtering and sterilizing are carried out, 1/5LB liquid culture medium is subpackaged in test tubes, and the formula of the 1/5LB culture medium is as follows: each liter contains 2.0g peptone, 1.0g yeast powder, 2.0g NaCl, pH7.0. After autoclaving, the filtered sterilized tryptophan solution was added to give a final tryptophan concentration in the medium of 0.5mg/mL. Strain RE15 was inoculated into the above test tube and cultured at 30 ℃ for 3 days at 150 r/min. Taking the fermentation liquor in a 7mL centrifuge tube, centrifuging to obtain 1mL of supernatant, adding 50 mu L of 10mM orthophosphoric acid, and adding 2mL of Sackowski's color reagent, wherein the color reagent comprises the following formula: 2mL of 0.5M FeCl ₃ ·6H ₂ O is mixed with 98mL 35% perchloric acid, after the mixture is fully mixed, the color is developed for 30min at room temperature in a dark place, and the light absorption value is measured under the condition of 530nm wavelength. The same reaction was performed by using a sterile 1/5LB liquid medium instead of the fermentation broth as a blank, and IAA standard solutions having concentrations of 0,10,20,50,100,200mg/L were used to prepare standard curves according to the above-described methods, and IAA concentrations in the fermentation broth were calculated. The result shows that the strain RE15 has stronger IAA production capacity which reaches 75mg/L and stronger growth promotion potential.

(3) Determination of the iron-producing carrier capacity of the strain:

(1) determination of siderophore yield:

preparing CAS detection solution, adding 6.0mL of 10mmol/L hexadecyl trimethyl ammonium bromide solution into a 100mL volumetric flask, and moderately diluting with double distilled water; 1.5mL of 1mmol/L FeCl ₃ Mixing the solution with 7.5mL of 2mmol/L of chrome azurin solution, and transferring into the volumetric flask; 4.307g of anhydrous dimethylamine is weighed and dissolved in about 30mL of double distilled water, 6.25mL of 12mol/L HCl is added to obtain buffer solution with pH=pKa=5.6, and the buffer solution is transferred into the volumetric flask for constant volume. All vessels of the siderophore were tested and immersed in 5% nitric acid for 24h. The test strain is inoculated into MKB liquid culture medium after being activated, and the MKB culture medium comprises the following formula: 5.0 g/liter of casein-containing amino acid, 15.0mL of glycerol, K ₂ HPO ₄ 2.5g，MgSO ₄ ·7H ₂ O0.2 g, pH7.0. Culturing at 30deg.C for 48 hr/min, centrifuging to obtain 1.5mL supernatant, adding equivalent CAS detection solution, mixing, and light shielding for 1 hr, and measuring absorbance (A) at 630nm wavelength. Removing methanesulfonic acid at a concentration of 2-20 mu moL/LThe standard curve was made with iron amine (DFOM). The result shows that the strain RE15 has 22.69 mu moL/L siderophore production and stronger growth promoting potential.

(2) Determination of siderophore Activity:

preparing CAS detection solution, adding 6.0mL of 10mmol/L hexadecyl trimethyl ammonium bromide solution into a 100mL volumetric flask, and moderately diluting with double distilled water; 1.5mL of 1mmol/L FeCl ₃ Mixing the solution with 7.5mL of 2mmol/L of chrome azurin solution, and transferring into the volumetric flask; 4.307g of anhydrous dimethylamine is weighed and dissolved in about 30mL of double distilled water, 6.25mL of 12mol/L HCl is added to obtain buffer solution with pH=pKa=5.6, and the buffer solution is transferred into the volumetric flask for constant volume. All vessels of the siderophore were tested and immersed in 5% nitric acid for 24h. The test strain is inoculated into MKB liquid culture medium after being activated, and the MKB culture medium comprises the following formula: 5.0 g/liter of casein-containing amino acid, 15.0mL of glycerol, K ₂ HPO ₄ 2.5g，MgSO ₄ ·7H ₂ O0.2 g, pH7.0. Culturing at 30deg.C for 48 hr/min, centrifuging to obtain 1.5mL supernatant, adding equivalent CAS detection solution, mixing, and light shielding for 1 hr, and measuring absorbance (A) at 630nm wavelength. And mixing 1.5mL of non-inoculated MKB liquid culture medium with the equivalent CAS detection solution for reaction, and measuring the absorbance value by the same method to obtain a reference value (Ar). Determination of the iron carrier content according to the formula: siderophore activity units (%) = (Ar-a)/ar×100% siderophore yield was calculated. The result shows that the activity of the strain RE15 siderophore reaches 63.35%, and the strain RE15 siderophore has stronger growth promoting potential.

Example 4

Tolerance of strain RE15 to salinity

LB culture media with NaCl concentration of 1%, 2%, 5%, 10%, 15% and 20% are prepared, respectively, and 3mL are subpackaged in test tubes, and each concentration is repeated three times. Culturing RE15 bacterial liquid stored in a glycerol tube on an LB plate under a sterile condition at 30 ℃ until single colonies are formed, picking single colonies, inoculating the single colonies into an LB liquid culture medium (salt concentration is 1%), culturing the single colonies at 30 ℃ under a condition of 150rpm until the end of logarithmic growth of the strain, obtaining seed liquid, transferring the seed liquid into LB culture media with different salt concentrations according to an inoculum size of 2%, and culturing the seed liquid under a condition of 150rpm at 30 ℃ for 24 hours in a shaking way. Results showThe strain showed a salt tolerance concentration of 15%, and under this condition, the bacterial liquid OD ₆₀₀ Can reach 0.973.

Example 5

pH adaptation of strain RE15

LB media with pH values of 2, 4, 6, 8, 10 and 12 were prepared, and 3mL of the LB media were dispensed into test tubes, and each pH was repeated three times. Culturing RE15 bacterial liquid stored in a glycerol tube on an LB plate under a sterile condition at 30 ℃ until single colony is formed, picking single colony, inoculating the single colony into an LB liquid culture medium (pH 7.0), culturing the single colony at 30 ℃ under a condition of 150rpm until the end of logarithmic growth of the strain, obtaining seed liquid, transferring the seed liquid into LB culture media with different pH values according to an inoculum size of 2%, and culturing the seed liquid under a condition of 150rpm at 30 ℃ for 24 hours in a shaking way. The result shows that the application range of the strain to the pH is 4-8, and the bacterial liquid OD is realized under the conditions of the pH of 4 and 8 ₆₀₀ 0.573 and 1.219, respectively.

Example 6

Adsorption capacity analysis of strain RE15 on cadmium with different concentrations

(1) Liquid adsorption test: cadmium chloride powder is dissolved in deionized water to prepare cadmium chloride mother liquor with the concentration of 5000mg/L, and a proper amount of the mother liquor is taken and added into a triangular flask which is respectively provided with the LB culture medium, so that the final concentration of Cd is 1mg/L, 5mg/L and 10mg/L respectively. Followed by high-temperature autoclaving. Culturing RE15 bacterial liquid stored in a glycerol tube on an LB plate under a sterile condition at 30 ℃ until single colonies are formed, picking single colonies, inoculating the single colonies into an LB liquid culture medium, culturing the single colonies to the end of logarithmic growth of a strain under the condition of 30 ℃ and 150rpm, obtaining seed liquid, transferring the seed liquid into LB culture media with different cadmium concentrations according to the inoculum size of 5%, and culturing the seed liquid under the condition of 150rpm for 7 days in a shaking way.

(2) Strain growth status and pH change under cadmium stress: and respectively taking 3mL of fermentation liquor from each triangular flask and adding the fermentation liquor into a 7mL centrifuge tube for testing after 0,1,2,3,5 and 7 days. Adding 300 μl of fermentation broth into 96-well plate, and measuring OD of fermentation broth by enzyme-labeled instrument ₆₀₀ A light absorption value; the remaining solutions were pH-measured using a pH meter. As can be seen from FIG. 4, the pH of the fermentation broth after inoculation of the strain was alkaline, and the pH was up to 8.98.

(3) Determination of cadmium adsorption amount of strain: 8mL of fermentation liquor is taken in a centrifuge tube in 0,1,2,3,5 and 7 days respectively, supernatant is taken by centrifugation, cadmium concentration in the solution is analyzed by ICP-OES after acidification, and the thallus adsorption rate is calculated according to the following calculation formula:

adsorption ratio = (Ci-Cf)/ci×100%

Wherein Ci is the concentration of cadmium in the supernatant of the control sample, and mg/L;

cf is the cadmium concentration in the supernatant of the inoculation strain RE15 sample, mg/L.

As can be seen from FIG. 5, after 7 days of culture, the adsorption rates of the strain RE15 on cadmium of 1mg/L, 5mg/L and 10mg/L respectively reach 62.75%, 57.17% and 30.68%, which shows that the endophyte has a relatively stable adsorption effect on cadmium.

Example 7

Effect of Strain RE15 on Rice seed germination

Chang Nong japonica is taken as a material, full seeds are selected, soaked in 5% NaClO for 10min for sterilization, washed 3-5 times with distilled water, and germinated in a dark place in a incubator at 30 ℃ for 12h. Microbial inoculum treatment group: rice seeds that have sprouted for 12h are transferred to the bacterial solution of the strain RE15 (10 ⁸ CFU/mL) in the dark for 12h; non-inoculation control group: the seeds that have sprouted for 12 hours are placed in a dark place in an incubator at 30 ℃ for 12 hours. The seeds were then transferred to 100 plates each with 10mL of CdCl of different concentration spread on a double layer filter paper 9cm diameter plate ₂ Solutions (0, 1, 5, 10mg/L, respectively) were randomly arranged and repeated three times. The culture dish is placed in an illumination incubator for culturing for 7 days in the illumination incubator with light intensity of 3000lx, temperature of 28 ℃/20 ℃ and humidity of 70% in the dark for 14 hours. Recording the germination number of seeds after the culture is finished, randomly selecting 10 seeds, recording root length, and calculating germination rate, vitality index and tolerance index, wherein the calculation formula is as follows:

germination percentage (G) = (number of germinated seeds/number of seeds tested) ×100%

Vitality Index (VI) =s× (Gt/Dt)

Tolerance coefficient (TI) =rlm/RLc

Wherein Gt is the germination number, dt is the germination day number, and S is the total root length of the seedlings;

RLm is the length of root system in each treatment, and RLc is the length of root system of control without inoculation and cadmium addition.

The experimental results are shown in table 1:

TABLE 1 Effect of Strain RE15 on Rice seed germination

Note that: the figures show significant differences between the controls with and without bacteria (P < 0.05), and the figures show significant differences between the controls with and without bacteria (P < 0.01).

As can be seen from Table 1, after 7 days of cultivation, inoculation of strain RE15 can significantly increase the tolerance index of rice seeds at a cadmium concentration of 5 mg/L; under the condition of 10mg/L, the inoculation of the strain RE15 can obviously increase the vitality index and the tolerance index of rice seeds.

Sequence listing

<110> academy of agricultural sciences in Jiangsu province

<120> a strain of Sword fungus and application thereof

<130> xb21121001

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1556

<212> DNA

<213> Sword RE15 (Ensifer sp. RE 15)

<400> 1

ggtgacagac ggcagtgcag ctttgcattg ccttgcaggt cgacgattta gagtttgatt 60

ccttggcttc agaacgaacg ctggcggcag gcttaacaca tgcaagtcga gcgccccgca 120

aggggagcgg cagacgggcg agtaacgcgt gggaatctac ccttttctac ggaataacgc 180

agggaaactt gtgctaatac cgtataagcc cttcggggga aagatttatc gggaaaggat 240

gagcccgcgt tggattagct agttggtggg gtaaaggcct accaaggcga cgatccatag 300

ctggtctgag aggatgatca gccacattgg gactgagaca cggcccaaac tcctacggga 360

ggcagcagtg gggaatattg gacaatgggc gcaagcctga tccagccatg ccgcgtgagt 420

gatgaaggcc ctagggttgt aaagctcttt caccggtgaa gataatgacg gtaaccggag 480

aagaagcccc ggctaacttc gtgccagcag ccgcggtaat acgaaggggg ctagcgttgt 540

tcggaattac tgggcgtaaa gcgcacgtag gcggacattt aagtcagggg tgaaatccca 600

gagctcaact ctggaactgc ctttgatact gggtgtctag agtatggaag aggtgagtgg 660

aattccgagt gtagaggtga aattcgtaga tattcggagg aacaccagtg gcgaaggcgg 720

ctcactggtc cattactgac gctgaggtgc gaaagcgtgg ggagcaaaca ggattagata 780

ccctggtagt ccacgccgta aacgatgaat gttagccgtc gggcagttta ctgttcggtg 840

gcgcagctaa cgcattaaac attccgcctg gggagtacgg tcgcaagatt aaaactcaaa 900

ggaattgacg ggggcccgca caagcggtgg agcatgtggt ttaattcgaa gcaacgcgca 960

gaaccttacc agcccttgac atcccgatcg cggattacgg agacgttttc cttcagttcg 1020

gctggatcgg agacaggtgc tgcatggctg tcgtcagctc gtgtcgtgag atgttgggtt 1080

aagtcccgca acgagcgcaa ccctcgccct tagttgccag catttagttg ggcactctaa 1140

ggggactgcc ggtgacaagc cgagaggaag gtggggatga cgtcaagtcc tcatggccct 1200

tacgggctgg gctacacacg tgctacaatg gtggtgacag tgggcagcga gaccgcgagg 1260

tcgagctaat ctccaaaagc catctcagtt cggattgcac tctgcaactc gagtgcatga 1320

agttggaatc gctagtaatc gcagatcagc atgctgcggt gaatacgttc ccgggccttg 1380

tacacaccgc ccgtcacacc atgggagttg gttctacccg aaggtagtgc gctaaccgca 1440

aggaggcagc taaccacggt agggtcagcg actggggtga agtcgtaaca aggtaaccaa 1500

tctctagagg atccccgggt acccgagctc gaatcgtaat catgttgtta cgtcgt 1556

Claims

1. A strain of sword bacteria is characterized in that the strain is named as sword bacteria @ in classificationEnsifer sp.) RE15, accession number: cctccc NO: m20211334.

2. The sword bacteria of claim 1, wherein the 16S rRNA sequence is set forth in SEQ ID NO: 1.

3. The method for culturing a sword according to any one of claims 1-2, wherein the sword is inoculated into a medium and cultured at 28-30 ℃.

4. A method of culturing according to claim 3, wherein the sword is cultured at 30 ℃.

5. The method according to claim 3, wherein the sword is cultured at a pH=4.0 to 8.0.

6. The method according to claim 5, wherein the sword is cultured at pH=7.0.

7. A culture method according to claim 3, wherein the medium is LB medium comprising the following components: 10.0g/L peptone, 5.0. 5.0g/L yeast powder, 10.0g/L NaCl and the balance of water.

8. Use of the sword bacteria of any one of claims 1-2 for producing auxin (IAA).

9. The use according to claim 8, wherein IAA is produced by fermentation culture of the sword bacteria.

10. The application of the sword bacteria in any one of claims 1-2 in cadmium-resistant growth promotion of rice.