CN109536407B

CN109536407B - Glyphosate-resistant Brevibacillus laterosporus strain, composition and application

Info

Publication number: CN109536407B
Application number: CN201811502647.9A
Authority: CN
Inventors: 杜蓉蓉; 刘海明
Original assignee: Beijing Hangtian Hengfeng Technology Co ltd
Current assignee: Beijing Hangtian Hengfeng Technology Co ltd
Priority date: 2018-12-10
Filing date: 2018-12-10
Publication date: 2020-12-01
Anticipated expiration: 2038-12-10
Also published as: CN109536407A

Abstract

The invention relates to the field of microorganisms, and particularly relates to a glyphosate-tolerant brevibacillus laterosporus strain, a composition and application. The strain is preserved in China general microbiological culture Collection center, and the preservation numbers are as follows: CGMCC No. 16749; the preservation time is as follows: 11 and 15 days 2018. The strain has strong glyphosate resistance, so the strain has wider application range in agricultural production.

Description

Glyphosate-resistant Brevibacillus laterosporus strain, composition and application

Technical Field

The invention relates to the field of microorganisms, and particularly relates to a glyphosate-tolerant brevibacillus laterosporus strain, a composition and application.

Background

Glyphosate is a herbicide with high efficiency, low toxicity, broad spectrum and internal absorption and conduction, and non-selective foliar spraying, and is widely applied in agricultural growth (old countries, etc., 2017). Although glyphosate provides greater convenience for agricultural production, it leaves a significant amount of residue in the soil. The domestic and foreign research on glyphosate shows that the glyphosate is applied at a normal rate of 1kg hm^-2The calculation shows that the residual amount of glyphosate in the soil with the surface layer of 13cm can reach 0.45-2 mg kg^-1(ii) a In areas with large glyphosate dosage, the residual quantity is up to 10-20 mg kg^-1(Zhou et al, 2013). The residual glyphosate not only causes great pollution to soil, water and the like, but also seriously hinders the growth of soil microorganisms. As proved by research of Wuxuening et al (2016), the glyphosate preparation has certain inhibition effect on soil microorganisms and is increased along with the increase of the concentration of the medicament dose. This inhibition renders many functional microorganisms applied to the soil ineffective. Therefore, the screening of functional microorganisms with certain glyphosate tolerance has important significance for the exertion of the functions of the microorganisms.

Brevibacillus laterosporus (Brevibacillus laterosporus) is a novel biocontrol bacterium, and has the effects of resisting bacteria, killing insects, killing nematodes, dissolving phosphorus and degrading organic matters (Guo Jing et al, 2011, Liyue et al, 2015). With the research on Brevibacillus laterosporus, the inhibition of Brevibacillus laterosporus on pathogenic fungi is concerned. Studies of Zhao Jing et al (2012) find that Brevibacillus laterosporus has a strong killing effect on Phytophthora capsici, and the generated antibacterial peptide has an inhibiting effect on the growth of phytophthora capsici hyphae, the generation of sporangia and the germination of resting spores. Plum, et al (2015) found that extracellular antibiotic substances produced by Brevibacillus laterosporus could inhibit the growth of Phytophthora capsici and Rhizoctonia solani. In addition, brevibacillus laterosporus has strong antagonistic action on tobacco brown spot, rice blast (Zhang 281562008, etc.), wheat scab (Zhao Qiu Min, etc. 2006), poplar canker (Jiang et al, 2015), gram-positive bacteria such as fusarium oxysporum, etc. (Saikia et al, 2011). Therefore, the brevibacillus laterosporus is continuously popularized and applied in agricultural production, especially in the research and development and application of biological bacterial manure. Therefore, screening of the glyphosate-tolerant Brevibacillus laterosporus strain is beneficial to the application of the strain in agriculture.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The present invention addresses the foregoing needs by providing novel microbial strains, cultures, compositions, and methods useful for enhancing the health, growth, and/or yield of plants.

In order to achieve the above purpose of the present invention, the following technical solutions are adopted:

the invention relates to a separated brevibacillus laterosporus strain which is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, and the preservation number is as follows: CGMCC No. 16749; the preservation time is as follows: 11 and 15 days 2018.

According to another aspect of the invention, the invention also relates to a composition comprising a culture of a strain of Brevibacillus laterosporus as described above.

According to another aspect of the present invention, the present invention also relates to a method for culturing a Brevibacillus laterosporus strain, which comprises culturing the Brevibacillus laterosporus strain in LB liquid medium or R2A medium, or the diluted medium.

According to another aspect of the present invention, the present invention also relates to a method for enhancing the health, growth or yield of a plant, said method comprising applying an effective amount of a composition as described above to said plant or to the environment surrounding said plant.

Compared with the prior art, the Brevibacillus laterosporus strain provided by the invention has strong glyphosate resistance, so that the Brevibacillus laterosporus strain has a wider application range in agricultural production.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 shows the growth of different Brevibacillus laterosporus (comprising the glyphosate provided by the present invention) in a medium containing glyphosate isopropylamine salt.

The Brevibacillus laterosporus strain provided by the application is named as CE4, is preserved in China general microbiological culture Collection center, and has the preservation number: CGMCC No. 16749; the strain was detected as a viable strain by the depository at 11/15 in 2018 and deposited.

Detailed Description

The strain name is CE 4.

Colonies of the strain on LB are all yellow brown, flat, smooth in edges and protruding in the center; the cells are rod-shaped and the spores are lateral. The strain is obtained by ultraviolet mutagenesis of a wild strain from agricultural resources and agricultural division research institute of Chinese academy of agricultural sciences.

According to one aspect of the invention, the invention also claims mutant strains of the above strains whose 16S rRNA gene sequence is identical to SEQ ID NO:1 has at least 99% homology and the strain is capable of tolerating at least 1.0mmol L^-1Glyphosate isopropylamine salt.

In some embodiments, the 16S rRNA gene sequence of the brevibacillus laterosporus strain is identical to SEQ ID NO:1 has 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% homology.

In some embodiments, the Brevibacillus laterosporus strain is capable of tolerating 1.0mmol L^-1～2.0mmol L^-1Glyphosate isopropylamine salt, or 1.1mmol L^-1、1.2mmol L^-1、1.3mmol L^-1、1.4mmol L^-1、1.5mmol L^-1、1.6mmol L^-1、1.7mmol L^-1、1.8mmol L^-1、1.9mmol L^-1Glyphosate isopropylamine salt.

The culture medium used in the tolerance experiment of the glyphosate isopropylamine salt is 1/5LB culture medium; further, the culture temperature is 28 ℃ to 32 ℃.

In addition, the brevibacillus laterosporus strain and the mutant strain thereof can tolerate the NaCl concentration of not more than 3%; furthermore, the culture environment is R2A culture medium, and the culture time is 8-16 h.

The invention claims the Brevibacillus laterosporus strain with the above-mentioned deposit number, and the mutation is in a moderate range and still can tolerate at least 1.0mmol L^-1A mutant strain of glyphosate isopropylamine salt.

By "mutant strain of Brevibacillus laterosporus strain", it is meant a Brevibacillus laterosporus strain whose genome is highly similar to that of the CE4 strain. In the present application, the expression "Brevibacillus laterosporus strain of the invention" covers the mutant strain. The mutant strain can be defined by means of 16S rRNA homology as described above, and can also be covered by high similarity in terms of genome:

the genome of a strain of brevibacillus laterosporus comprises at most 150 mutation events, preferably at most 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30 or 20 mutation events compared to the genome of the CE4 strain. Mutational events are defined as SNPs (single nucleotide polymorphisms) or INDELs (insertions, deletions, and combinations of both). The number of mutational events was determined as follows: taking the genome of the CE4 strain as a control, mutation events present in the mutant genome were identified, each mutation event (SNP or INDEL) representing one mutation event (i.e., for example, an insertion of a sequence containing several nucleotides is considered only one mutation event). In this context, the genomic sequence of a mutant of the invention is defined by the number of mutation events contained compared to the CE4 strain, and in addition to this definition can be defined by its percent identity with the genomic sequence of the CE4 strain, where percent identity herein denotes the percentage of sequences found in the genome of one strain that are present in the genome of another strain, in particular: a) the percentage of sequences found in the genome of the CE4 strain and present in the genome of the mutant strain, or b) the percentage of sequences found in the genome sequence of the mutant strain and present in the genome of the CE4 strain. Thus, mutant strains that differ from the CE4 strain by only an insertion(s) or by only a deletion(s) have a genome with a percentage of identity of 100% to the genome of the CE4 strain, since the entire genomic sequence of one strain is completely found in the genome of the other strain. In a particular embodiment, the genomic sequence of the mutant of the invention, as defined by the number of mutational events, is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.92%, at least 99.94%, at least 99.96%, at least 99.98%, or at least 99.99% identical to the genomic sequence of the CE4 strain, wherein the percentage of identity represents the percentage of sequences found in the genome of one strain and present in the genome of another strain; identity is described in terms of comparing two genomic sequences over their full length (global alignment) and can be calculated using any program based on the Needleman-Wunsch algorithm.

The genome of the CE4 strain can be determined by techniques customary in the art.

In the practical application process, the brevibacillus laterosporus strain is necessary to be cultured in an enlarged manner to be made into a composition (in particular, a microbial agent) in order to enlarge the application range thereof in consideration of the possible need for transportation and the like.

The composition of the invention (preferably, when used as a starter culture) may be a pure culture or a mixed culture. The present invention therefore defines a pure culture as a culture in which all or substantially all of the culture consists of the same strain of Brevibacillus laterosporus of the invention. In the alternative, a mixed culture is defined as a culture comprising several microorganisms, in particular several bacterial strains, including the brevibacillus laterosporus strain of the present invention.

The composition is used in agriculture, and can be made into liquid, frozen or dried powder form; or in the form of preparations customary in the industry, such as granules, suspensions, wettable powders, emulsions or liquors.

Any carriers may be used whether they are solid or liquid, as long as they are commonly used and biologically inert for agricultural and horticultural pesticides. And is not limited to any particular carrier.

In some embodiments, when the composition is in the form of a frozen or dried powder, it includes a solid carrier;

examples of solid carriers include mineral powders such as china clay, talc, bentonite, zeolite, calcium carbonate, diatomaceous earth and White carbon (White carbon); vegetable flours such as corn flour and starch; and polyalkylene glycols of high molecular compounds such as polyvinyl alcohol. On the other hand, typical liquid carriers include various organic solvents such as decane and dodecane, vegetable oil, mineral oil and water.

In some embodiments, the solid carrier comprises one or more of peat, turf, talc, lignite, pyrophyllite, montmorillonite, alginate, filter mud, sawdust, perlite, mica, silica, quartz flour, calcium bentonite, vermiculite, kaolin, precipitated calcium carbonate, diatomaceous earth, medical stone, calcite, zeolite, white carbon, fine sand, and clay.

In some embodiments, an adjuvant is included in the composition;

surfactants, binders, stabilizers and the like, which are generally used as auxiliaries in agricultural and horticultural chemicals, may be used alone or in combination as needed, and as stabilizers, for example, antioxidants and/or pH adjusters may be used. Light stabilizers may also be used in some cases.

The total content of these adjuvants may be 0 to 80% by weight, and the content of the carrier is a value obtained by subtracting the contents of the active ingredient and the adjuvants from 100% by weight.

In some embodiments, the adjuvant comprises one or more of sodium dodecylbenzene sulfonate, sodium butylnaphthalene sulfonate, trehalose, glycerol, sodium lignosulfonate, sodium alkylnaphthalene sulfonate polycondensates, niacin, alcohol, buffer salts, sodium chloride, amino acids, vitamins, proteins, polypeptides, polysaccharides or monosaccharides, yeast extract, white carbon, tea saponin, and skim milk.

In some specific embodiments, the composition further comprises an agriculturally effective amount of a compound or composition selected from the group consisting of: nutrients, fertilizers, acaricides, bactericides, fungicides, insecticides, microbicides, nematicides and insecticides.

The above components can be matched with the bacterial strain provided by the application to realize better technical effect.

The dilution ratio of the diluted culture medium can be selected from 1/2, 1/3, 1/4, 1/5, 1/6 and 1/7; the diluent may be selected from liquids, such as water, which are not detrimental to the growth of the strain.

According to another aspect of the invention, the invention also relates to a plant seed having a coating comprising a composition as described above.

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

Examples

1. Determination of UV mutagenesis dose

The activated Brevibacillus laterosporus was inoculated into a 150ml Erlenmeyer flask containing 50ml 1/5LB liquid medium (yeast extract powder: 2.5g, tryptone: 1g, NaCl: 0.25%, pH: 7.0. + -. 0.2 to volume of 1L) and cultured overnight, washed twice with an equal amount of 0.9% physiological saline and suspended in 0.9% physiological saline, and 6ml of the suspension was placed in an ultraviolet crosslinking apparatus (energy: 2J/m)²) The coated plates were irradiated for 0,6,12,18,24,30s and the colony counts were recorded, and the results were as follows:

2. determination of glyphosate-resistant isopropylamine salt concentration of brevibacillus laterosporus

The activated Brevibacillus laterosporus is inoculated to the solution containing glyphosate isopropylamine salt (0, 0.5, 0.8, 0.9, 1.0, 2.0mmol L) by the streak method^-1) 1/5LB medium, cultured in an incubator at 30 ℃ and the growth was observed and recorded. The results are shown in the following table:

note: "+" represents growth, "-" represents no growth

3. Ultraviolet screening of brevibacillus laterosporus glyphosate-resistant isopropylamine salt strain

The activation-washing-ultraviolet mutagenesis process was repeated at a time point with a lethality of 99.97%, and 200. mu.l of the mutagenized bacterial liquid was inoculated to a medium containing 1.0mmol L^-11/5LB solid culture medium, culturing in 30 deg.C incubator, observing the growth of strain, primarily screening 4 strains of Brevibacillus laterosporus with good growth, storing and performing subsequent tests. 4 strains with 1mmol L-1 resistance are inoculated into 1/5LB liquid culture medium without glyphosate isopropylamine salt, after culture for 72h, the strain liquid is inoculated into 1/5LB solid culture medium containing 1.0mmol L-1 glyphosate isopropylamine salt, culture is carried out at 30 ℃, and the growth condition is observed. The results showed that only 1 Brevibacillus laterosporus strain (strain CE4) of the 4 strains produced a single colony. The results are shown in FIG. 1.

4. Determination of spore production capability of mutant strain and wild strain

Inoculating activated Brevibacillus laterosporus (CE-CK) and screened Brevibacillus laterosporus (CE4) with glyphosate-resistant isopropylamine salt into a 150ml Erlenmeyer flask containing 50ml R2A culture medium, culturing for 12h, adjusting OD600 to be consistent, inoculating into a new R2A culture medium, culturing for 72h, and measuring the difference of germination rate of the two strains by using a microscope, wherein the experimental results are as follows:

5. comparison of growth rates of mutagenized strains and wild strains

Inoculating activated Brevibacillus laterosporus CE-CK and CE4 into 150ml Erlenmeyer flask containing 50ml 1/5LB liquid medium, culturing for 12h, and OD₆₀₀The mixture was adjusted to be uniform, inoculated into a new 150ml Erlenmeyer flask containing 50ml 1/5LB liquid medium, and the OD of the culture solution was measured every 3 hours using an ultraviolet spectrophotometer₆₀₀The experimental results are as follows:

6. plate antagonism method for measuring antagonistic action of strain on Rhizoctonia solani

After the rhizoctonia solani is activated by a PDA culture medium, a bacterium block is clamped by tweezers and placed in the center of 1/5LB solid culture medium, activated brevibacillus laterosporus CE-CK and CE4 are respectively inoculated to the periphery of the culture medium, and the culture is carried out for 72 hours at the temperature of 30 ℃. And (5) measuring the size of the inhibition zone, and recording the experimental result.

7. Method for detecting antibiotic tolerance of strain by using sensitivity test paper

In the antibiotic sensitivity experiment of the strain, the Brevibacillus laterosporus CE-CK and CE4 are cultured overnight to reach an exponential phase, 100 mul of bacterial liquid is taken and coated on 1/5LB solid culture medium, sensitivity test paper (OXOID) is attached to the surface of the culture medium, and the transparent ring size is determined after 24h of culture.

8. Brevibacillus laterosporus salt tolerance test sample

After activated Brevibacillus laterosporus CE-CK and CE4 were inoculated into R2A medium and cultured overnight, 100. mu.l of each was inoculated into R2A medium containing 1.0%, 2.0%, 3.0%, 4.0%, and 5.0% NaCl, respectively, and cultured overnight, and the results are shown in the following table.

Note: "+" represents growth, "-" represents no growth

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Reference to the literature

Chengshi, Qiangsheng, Chan Yuan, Glyphosate action mechanism and resistance research progress [ J ]. plant protection, 2017,43(2):17-24.

The efficient expression of Bacillus laterosporus alkaline protease gene BLG4 in Bacillus subtilis WB600 is described in the university of agriculture and forestry, Nature science, 2011,40(2):165-171.

Li Yue, Yu Hui Rong, Li Wei, etc. Brevibacillus laterosporus B8 is a preliminary study on the antibacterial mechanism of two plant pathogenic bacteria [ J ] Chinese plant protection guide, 2015(3):12-16.

Wuxuebe, Sun cyanine, Rogarden, etc. influence of glyphosate on energy metabolism of soil microorganism [ J ] chemical and biological engineering, 2016:18-21.

Zhang 2815630, Zhou Li Mei, Xude Yang, etc. Brevibacillus laterosporus BL-21 antibiotic protein stability analysis and separation purification [ J ] Chinese agricultural report, 2017,33(15):43-48.

ZhaoJING, Brevibacillus laterosporus A60 antibacterial peptide separation purification and functional analysis [ D ] Beijing, academy of agricultural sciences, China 2012.

Zhao Qimin, Chenyuehua, Chuaiqing, etc. one kind of chitinase producing and fungus inhibiting Bacillus laterosporus [ J ] GUO biological preventing and treating 2006,22(s):42-46.

Jiang H,Wang X,Xiao C,et al.Antifungal activity of Brevibacillus laterosporus JX-5 and characterization of its antifungal components[J].World Journal of Microbiology&Biotechnology,2015,31(10):1-14.

Saikia R,Gogoi D K,Mazumder S,et al.Brevibacillus laterosporus,strain BPM3,a potential biocontrol agent isolated from a natural hot water spring of Assam,India[J].Microbiological Research,2011,166(3):216-225.

Zhou C F,Wang Y J,Li C C,et al.Subacute toxicity of copper and glyphosate and their interaction to earthworm(Eisenia fetida)[J].Environmental pollution,2013,180:71-77.

SEQUENCE LISTING

<110> Beijing aerospace Hengfeng science and technology GmbH

<120> glyphosate-tolerant Brevibacillus laterosporus strains, compositions and uses

<160> 1

<170> PatentIn version 3.3

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<211> 1422

<212> DNA

<213> Brevibacillus laterosporus

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ctatacatgc aagtcgagcg agggtcttcg gaccctagcg gcggacgggt gagtaacacg 60

taggcaacct gcctgtgaga ctgggataac atagggaaac ttatgctaat accggatagg 120

gttttgcttc gcctgaagcg aaacggaaag atggcgcaag ctatcactta cagatgggcc 180

tgcggcgcat tagctagttg gtgaggtaac ggctcaccaa ggcgacgatg cgtagccgac 240

ctgagagggt gaccggccac actgggactg agacacggcc cagactccta cgggaggcag 300

cagtagggaa ttttccacaa tggacgaaag tctgatggag caacgccgcg tgaacgatga 360

aggctttcgg gtcgtaaagt tctgttgtta gggaagaaac agtgctattt aaataaggta 420

gcaccttgac ggtacctaac gagaaagcca cggctaacta cgtgccagca gccgcggtaa 480

tacgtaggtg gcaagcgttg tccggaatta ttgggcgtaa agcgcgcgca ggtggctatg 540

taagtctgat gttaaagccc gaggctcaac ctcggttcgc attggaaact gtgtagcttg 600

agtgcaggag aggaaagtgg tattccacgt gtagcggtga aatgcgtaga gatgtggagg 660

aacaccagtg gcgaaggcga ctttctggcc tgtaactgac actgaggcgc gaaagcgtgg 720

ggagcaaaca ggattagata ccctggtagt ccacgccgta aacgatgagt gctaggtgtt 780

aggggtttca atacccttag tgccgcagct aacgcaataa gcactccgcc tggggagtac 840

gctcgcaaga gtgaaactca aaggaattga cgggggcccg cacaagcggt ggagcatgtg 900

gtttaattcg aagcaacgcg aagaacctta ccaggtcttg acatcccact gaccgctcta 960

gagatagagc ttcccttcgg ggcagtggtg acaggtggtg catggttgtc gtcagctcgt 1020

gtcgtgagat gttgggttaa gtcccgcaac gagcgcaacc cttatcttta gttgccagca 1080

ttcagttggg cactctagag agactgccgt cgacaagacg gaggaaggcg gggatgacgt 1140

caaatcatca tgccccttat gacctgggct acacacgtgc tacaatggtt ggtacaacgg 1200

gatgctactt cgcgagaaga tgctaatctc ttaaaaccaa tctcagttcg gattgtaggc 1260

tgcaactcgc ctacatgaag tcggaatcgc tagtaatcgc ggatcagcat gccgcggtga 1320

atacgttccc gggccttgta cacaccgccc gtcacaccac gggagtttgc aacacccgaa 1380

gtcggtgagg taaccgtaag gagccagccg ccgaagttgg tt 1422

Claims

1. Isolated Brevibacillus laterosporus（ Brevibacillus laterosporus ) The strain is preserved in China general microbiological culture Collection center, and the preservation numbers are as follows: CGMCC No. 16749; the preservation time is as follows: 11 and 15 days 2018.

2. A composition comprising a culture of the strain of claim 1.

3. The composition of claim 2, wherein the composition is in the form of a liquid, frozen or dried powder.

4. The composition of claim 3, wherein the composition comprises an adjuvant;

when the composition is in the form of a frozen or dried powder, it further comprises a solid carrier.

5. The composition of claim 4, wherein the solid carrier comprises one or more of peat, turf, talc, lignite, pyrophyllite, montmorillonite, alginate, filter press mud, sawdust, perlite, mica, silica, quartz flour, calcium bentonite, vermiculite, kaolin, precipitated calcium carbonate, diatomaceous earth, medical stone, calcite, zeolite, white carbon, fine sand, and clay.

6. The composition of claim 4, wherein the adjuvant comprises one or more of sodium dodecylbenzene sulfonate, sodium butylnaphthalene sulfonate, trehalose, glycerol, sodium lignosulfonate, sodium alkylnaphthalene sulfonate polycondensates, niacin, alcohol, buffer salts, sodium chloride, amino acids, vitamins, proteins, polypeptides, polysaccharides or monosaccharides, yeast extract, white carbon, tea saponin, and skim milk.

7. The composition according to any one of claims 2 to 6, characterized in that it further comprises an agriculturally effective amount of a compound or composition selected from the group consisting of: microbicides and insecticides.

8. The method for culturing the strain of claim 1, wherein the Brevibacillus laterosporus strain is cultured in LB liquid medium, R2A medium, or diluted medium thereof.