CN104862245A - Carbendazim degrading bacteria MBC-6F and application thereof - Google Patents

Abstract

The invention discloses a carbendazim degrading bacteria strain MBC-6F, which is preserved on December 31, 2014 in China Center for Type Culture Collection, and has preservation No. CCTCC NO: M 2014680. The carbendazim degrading bacteria MBC-6F is isolated and screened from soil, is identified by 16S rDNA as Microbacterium sp. MBC-6F, and can directly produce specific degradation on carbendazim; by HPLC analysis, the MBC-6F can completely degrade 40 mum carbendazim within 72 hours, the optimum degradation temperature is 37 DEG C, and the optimum degradation acidity is pH value of 7.0. By HPLC-MS, it is determined that the main degradation product is 2-amino-benzimidazole and 2-hydroxyphenyl and imidazole. The invention for the first time discloses the 16S rDNA sequence, and degradation efficiency of carbendazim and analysis of products.

Description

One strain carbendazim degrading bacterium MBC-6F and application thereof

Technical field

The invention belongs to microbial technology field, relate to a kind of new fine Pseudomonas ( microbacterium), be specifically related to can the degrade fine bacterium MBC-6F of derosal (carbendazim, MBC) and this bacterium of one and the degraded of derosal is measured.

Background technology

Derosal (carbendazim), have another name called carbendazol, Bei Fen replaces, carbendazol, English trade(brand)name MBC, relative molecular mass is 191.2, and molecular formula is: C ₉h ₉n ₃0 ₂, derosal is broad-spectrum germicide, and since 2011, annual usage quantity is about 40,000 tons, and the product number of its registration in 2012 reaches 857, and formulation comprises former medicine, pulvis, suspension agent, wettable powder, suspended emulsion agent, seed coat agent etc. nearly ten kinds.In China, derosal is widely used in the control of the diseases such as wheat, fruit tree, tealeaves, vegetables, also for the post-harvest fresh-keeping of fruit.Agriculturally, be mainly used in preventing and treating Gibberella zeae (Sch.) Petch, rice sheath blight disease, rice blast, sclerotium disease; The Powdery Mildew, anthrax, black spot, gray mold etc. of fruit tree and vegetables; Seedling process can prevent and treat cereal crop smut and melon, tomato wilt etc.Industrial, derosal can be used for the fungus-proof antisepsis of the materials such as weaving, papermaking, leather, rubber.In addition, F-1991, benzimidazoles and thiophanate series bactericidal agent are in soil and do also to be work by being converted into derosal in object.

The a large amount of systemic fungicide of use continuously derosal, makes many phytopathogens to which creating resistance for many years, as botrytis cinerea pers, and Sclerotinia sclerotiorum, tomato gray mould bacterium, Pyricularia oryzae, Colletotrichum gloeosporioides Penz in Mango etc.In order to reach prevention effect, peasant increases amount of application, causes derosal content in soil to reach very high concentration through summation, result also in the problem of its residual quantity severe overweight in agricultural-food.China GB18406.1-2001 standard specifies, in vegetable and fruit, Determination of carbendazim residue must not more than 0.5mg/kg.2007, EU Committee issues 2007/12/EC instruction, revise the residue limits of F-1991 (Benomyl), derosal (Carbendazim), Topaze (Penconazole) three kinds of sterilant, it is worth noting, derosal and the F-1991 limit index in oranges and tangerines is changed to 0.05mg/kg by original 5mg/kg, strict 100 times, the maximum residue limit in edible fungi is adjusted to 0.1 mg/kg by 1 mg/kg, strict 10 times.Due to derosal stability in the environment and chronicity, therefore, by migration and biologic chain propagation naturally, very large impact is produced on soil, plant, animal and underground water.Especially in tap water and agricultural-food, derosal pollutes and exceeds standard serious, constitutes serious threat to the health of the direct or indirect humans and animals of contact derosal.

The toxic action of derosal comprises following 4 aspects: (1) is carcinogenic, mutagenic toxicity: mouse test confirms that derosal can bring out mouse liver canceration, but in conjunction with other testing datas, the plant science council (Scientific Committee on Plants) thinks and causes mouse liver tumour to be not enough to show that derosal also has the inference of carcinogenesis to people by derosal.But, derosal has mutagenesis ability to karyomit(e) really, and the derosal that had scholar to test, on the impact of Human Lymphocytes culture, found that derosal is the very strong aneuploidogen of a kind of effect, even when low dosage, also chromosomal quantity can be affected.This is because derosal can suppress the polymerization of tubulin, and tubulin has vital role to separation chromosomal in cell fission.(2) genotoxicity: derosal is a kind of endocrine disruptors, can destroy mammalian uterus and grow, the sperm of interference adult rats and testicualr development.When research finds derosal Dosages more than 50mg/kg, the obviously weight saving of visible rat testicle, seminiferous tubule atrophy and ductulus efferens locking.If rat is contacted derosal before mating, female offspring can show the development toxicity of male sex hormone characteristic, and as not having vagina, horn of uterus not exclusively grows, and shows that the toxic action of derosal may be relevant with androgen receptor dependent mechanism with male sex hormone.(3) embryotoxicity: allow the male mice of feeding derosal feed 60d and the mating of normal female mouse, although find that the conceptual quotient of female mice and average implantation number are not subject to obvious impact, still birth rate increases to some extent.If make the pregnant mouse of 6 ~ 15d catch derosal, the dosage of 35mg/kg just can produce obvious embryotoxicity, shows as to absorb tire and increase, live that tire number reduces, fetal weight alleviates, and even fetal anomaly, if dosage is increased to 160mg/kg, is then formed without fetus.(4) other toxic actions: derosal is nontoxic to honeybee, to birds low toxicity.To fish and other hydrobionts toxic, the such as LC of fresh-water fishes, Bluegill, oppossum shrimp and aquatic invertebrate ₅₀be respectively 0.36mg/L, 5.5mg/L, 0.098mg/L and 0.087mg/L.Because the settling in water can adsorb derosal, so toxicity usually can not be too high, but the biology relying on settling existence is subject to the impact of high dosage derosal possibly.Soil organic matter has strong adsorption to derosal, causes earthworm and nematode count in soil to reduce.

Derosal stable chemical nature, lower than at least Absorbable organic halogens 2 years when 50 DEG C; Slowly decompose in basic solution, raise with pH value, decompose and accelerate, and stablize in an acidic solution.Can by ultraviolet photolysis in water surrounding, and in edatope, can with soil particle combination in various degree, the main degraded by microorganism, its transformation period is 6 ~ 12 months in exposed soil, is 3 ~ 6 months in the soil having vegetation.Microbiological deterioration is the topmost removal pathway of current Organic pesticide residues.

China is large agricultural country, and be also pesticide producing and use big country, the usage quantity of annual agricultural chemicals reaches 500,000 t, wherein about has the direct entered environment of the agricultural chemicals of 80%, and the cultivated area by pesticidal contamination has 1300 ~ 1,600 ten thousand hectares at least simultaneously.Isolation and screening highly effective pesticide degradation bacteria, solve the serious problem of Soil Contamination by Chemical Pesticides by contributing to, the agricultural chemicals being reduced by rainwash or the formation of farmland seepage, to the pollution of water surrounding, improves agricultural product quality and safety, promotes the Sustainable development of China's agricultural.

Summary of the invention

For the quantity limitation of existing derosal degrading microorganism, object of the present invention is finding a kind of new carbendazim degrading bacterium derosal to efficient degradation efficiency, it can effectively be degraded derosal, for the solution of derosal Pesticide Residue is laid a good foundation, also for the research and development of derosal efficient degradation enzyme provide material.

For achieving the above object, technical scheme of the present invention is as follows: a kind of carbendazim degrading bacterium of derosal of can effectively degrading microbacteriumsp.MBC-6F, be preserved in China typical culture collection center on December 31st, 2014, preserving number is: CCTCC NO:M 2014680, preservation address: Wuhan, China Wuhan University.

1, the screening of carbendazim degrading bacterium, isolation and purification.

Get fresh soil sample sample in the enrichment medium taking derosal as sole carbon source, periodically improve carbendazim concentration to 300 mg/L, carry out acclimating cultivation, get 10 ^-1~ 10 ^-11the domestication enrichment culture liquid of gradient dilution carries out the isolation and purification culture of bacterium, obtains the carbendazim degrading bacterium purifying bacterial classification MBC-6F in soil.

2, the qualification of carbendazim degrading bacterium MBC-6F.

Identified by the micromorphology of 5000 times of scanning electron microscope to carbendazim degrading bacterium MBC-6F.The 16S rDNA molecules qualification of MBC-6F entrusts the prosperous bio tech ltd of AudioCodes, Beijing to carry out.Qualification result application Clustal X 1.83 version and MEGA 4.0 software adopts adjacent connection method phylogenetic tree construction.

3, carbendazim degrading bacterium measures the degraded of derosal.

The carbendazim degrading bacterium MBC-6F of the inoculum size inoculation purifying with 5% is in degraded substratum, HPLC-MS mensuration is carried out after timing sampling, by the assay determination carbendazim degrading bacterium MBC-6F of derosal to the degradation efficiency of derosal, and determine the degraded product of carbendazim degrading bacterium MBC-6F to derosal further.

4, the degradation condition of carbendazim degrading bacterium is inquired into.

Carbendazim degrading bacterium MBC-6F is analyzed respectively in differing temps, the degraded situation to derosal under acidity and inoculum size graded condition with single_factor method.Inquire into carbendazim degrading bacterium MBC-6F to the optimum degradation condition of derosal.

Accompanying drawing explanation

The single colonial morphology photo of Fig. 1 carbendazim degrading bacterium MBC-6F bacterial strain;

Fig. 2 carbendazim degrading bacterium MBC-6F 5000 times of stereoscan photographs ;

Fig. 3 utilizes MEGA software building carbendazim degrading bacterium MBC-6F phyletic evolution tree structure diagram;

The degraded product mass spectrum of Fig. 4 MBC-6F bacterium degraded derosal;

Fig. 5 mBC-6F bacterium is to the degradation rate of derosal;

Fig. 6 temperature is on the impact of MBC-6F degraded derosal;

Fig. 7 pH is on the impact of MBC-6F degraded derosal;

Fig. 8 inoculum size is on the impact of MBC-6F degraded derosal.

Embodiment

The accompanying drawing provided below in conjunction with contriver and concrete test example further illustrate

1, the screening of carbendazim degrading bacterium, isolation and purification:

(1) test sample: the warmhouse booth soil of in May, 2013 sampling and College of Horticulture of Xibei Univ. of Agricultural & Forest Science & Technology continuous administration derosal.

(2) substratum

LB liquid nutrient medium: NaCl 10.0 g, peptone 10.0g, yeast leaching powder 5.0g, add distilled water 1000mL, adjust ph is 7.0.

Basal salt media: NaCl 1.0 g, K2HPO4 1.5 g, KH2PO4 0.5 g, (NH4) 2,SO4 2.0 g, MgSO47H2O 0.2 g, distilled water 1000mL, pH=7.0.

LB solid medium: with LB liquid nutrient medium, adds the agar of 1% ~ 2%.

Enrichment (degraded) test substratum: same to basal salt media, adds derosal as required.

Isolation and purification culture base: same to enrichment (degraded) test substratum, adds the agar of 1% ~ 2%.

Above substratum all needs 121 DEG C of autoclaving 30min before using.

(3) concentration and separation of carbendazim degrading bacterium strain and purifying

A) in the triangular flask of 250 mL, add 100 mL enrichment mediums, sole carbon source made by the derosal simultaneously adding 5 mg, and its concentration is 50 mg/L.Regulate pH to be after 6.5,121 DEG C of autoclaving 30 min, after cooling, get fresh soil sample sample 10 g and add (establish 6 parallel, not add fresh soil sample sample for contrast), and add appropriate sterilizing granulated glass sphere, at 30 DEG C, 5 d cultivated by 150 r/min shaking tables.Leave standstill 30 min;

B) draw upper strata bacteria suspension with the inoculum size of 5%, be inoculated in 100 mL enrichment mediums, improve carbendazim concentration to 100 mg/L, under same culture condition, 5 d cultivated by shaking table.For preventing derosal photodissociation, shaking table does lucifuge process;

C) the same step b) of method, improves carbendazim concentration by the increasing amount of each 50 mg/L, and circulation domestication cultivates about one month, strengthens carbendazim concentration until 300 mg/L.

D) the enrichment culture liquid getting last domestication carries out gradient dilution (10 ^-1~ 10 ^-11), each gradient establishes 3 repetitions, coat on the isolation and purification culture base containing derosal 200 mg/L, after 30 DEG C of cultivation 5 d, variant and the bacterial strain that bacterium colony is larger in picking form, isolation and purification culture base carries out line and is separated, 30 DEG C of cultivations, obtain purebred bacterial strain, 4 DEG C of inclined-planes are preserved.

, carbendazim degrading bacterium MBC-6F qualification

(1) morphological feature

The bacterium colony of MBC-6F bacterial strain is rounded, micro-aobvious orange-yellow, neat in edge, surface elevation, moistening smooth (Fig. 1).Thalline is closely spherical, and diameter is about 3 μm (Fig. 2).

(2) molecular biology identification

MBC-6F bacterial strain 16S rDNA strain identification entrusts AudioCodes bio tech ltd, Beijing to complete.

16S rDNA sequence in 16S rDNA sequence BLAST and the Genbank of MBC-6F bacterial strain is carried out tetraploid rice by it.Application ClustalX 1.83 version and MEGA 4.0 software adopts adjacent method (neighbor-joining, NJ) phylogenetic tree construction (Fig. 3), the genetic evolution distance of MBC-6F bacterial strain with microbacteriumbelong to recently, with microbacteriumtestaceum StLB037 DNA homology is the highest, illustrates that this bacterium is fine bacterium MBC-6F on Molecular Phylogeny taxonomy.

According to morphological feature, Phylogenetic Analysis, and in conjunction with bibliographical information, determine that this bacterium is microbacteriumsp. bacterium MBC-6F is belonged to.

, carbendazim degrading bacterium measures the degraded of derosal:

(1) preparation of seed liquor

Picking one ring thalline, is seeded in the triangular flask that LB liquid nutrient medium is housed, about shaking culture 24h at 30 DEG C; Adjust its OD600 with the LB liquid nutrient medium of sterilizing and be 1.0, pipette 5mL bacterium liquid in the centrifuge tube of 10mL, at 4 DEG C, centrifugal 5min under 4000r/min, collect thalline; After washing 2 times with the basal salt media of sterilizing, suspend with isopyknic sterilizing basal salt media, for subsequent use.

(2) Degrading experiment of derosal

In Degrading experiment substratum, add the derosal of 100 mg/L, access carbendazim degrading bacterium MBC-6F seed liquor respectively with 5% inoculum size, in 25 DEG C, 150 r/min shaking culture.Cultivate every 6h timing sampling respectively, until when being cultured to 72h, with the derosal in HPLC-MS detection nutrient solution and meta-bolites, see Fig. 4 and Fig. 5.

Derosal and meta-bolites measure: by nutrient solution in centrifugal 15 min of 12000 × g, and supernatant liquor loads in the graduated test tube of tool plug, adds equal-volume acetone, vibrate after 2 min, add solid NaCl saturated to solution after, put into hot water bath and heat, make aqueous phase and NaCl precipitation in acetone layering.Take out acetone in test tube, at room temperature dry up with nitrogen, then dissolve constant volume with Chromatographic Pure Methanol, analyze with HPLC-MS.

Analyze and adopt LC-MS mass spectrograph: LCQ DECA XP Plus mass spectrograph; C18 reversed-phase column: (5C ₁₈-MS-II, 2.0 nm × 150 mm); Moving phase is methyl alcohol: water=1:1 (volume ratio), and flow velocity is 0.8 mL min-1, determined wavelength 286 nm.Adopt electro-spray ionization source, positive ion detection mode carries out LC-MS analysis to meta-bolites.

, carbendazim degrading bacterium degradation condition inquire into.

(1) temperature is on the impact of MBC-6F degraded derosal

Culture volume with 5% is got seed liquor 5 mL and is inoculated in 100 mL Degrading experiment substratum, and this substratum carbendazim concentration is 8 mg/L.Temperature is 10,20,25,30,35,40, and cultivate respectively for 45 DEG C, after shaking table 150 r/min cultivates 72 h, degradation rate is surveyed in sampling.Every temperature 3 is parallel.

Known by Fig. 6, in 7 temperature ranges of test, bacterial strain MBC-6F is to the equal degradable of derosal, but degradation effect has notable difference.When 30 DEG C, degradation rate reaches maximum, when temperature all decreases below or above degradation rate during this temperature.The optimum temperuture of bacterial strain MBC-6F degraded derosal is 30 DEG C.

(2) pH is on the impact of L-1 degraded derosal

Same 3.2.2.1, the culture volume with 5% is got seed liquor 5 mL and is inoculated in 100 mL Degrading experiment substratum, and this substratum carbendazim concentration is 8 mg/L.Temperature gets the optimum value of 3.2.2.1 test.Initial pH gets 4.0 respectively, and 5.0,6.0,7.0,8.0, after 72 h, sampling detects.

The degradation rate of pH value derosal 6.0 time is the highest as seen from Figure 7, and pH is below or above this value, and MBC-6F is poor to the degradation effect of derosal, illustrates that the suitableeest pH value of bacterial strain MBC-6F degraded derosal is 6.0.

(3) inoculum size is on the impact of L-1 degraded derosal

Under same substratum, regulate temperature, pH to optimum value, access seed liquor with the inoculum size of 1%, 3%, 5%, 7%, 9% respectively, at shaking table 150 r/min, constant temperature culture 72 h under optimum temps, carbendazim concentration in sampling analysis nutrient solution.

Fig. 8 is known, when inoculum size lower than 5% time, along with the degradation rate increasing degree of the increase derosal of inoculum size is large, show that inoculum size is in 1% ~ 5% scope, the degraded impact of inoculum size on derosal is large.When inoculum size reaches or surpasses 5%, the degradation rate rangeability of inoculum size to derosal of this scope is very little.Therefore, 5% inoculum size is selected to be most economical efficient inoculum size.

Sequence table 1

<110> Xibei Univ. of Agricultural & Forest Science & Technology

 

<120> mono-strain carbendazim degrading bacterium MBC-6F and application thereof

 

<160> 1

 

<210> 1

<211> 1116BP

<212> RNA

<213> MBC-6F

<400> 1

 

CGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGAAAGCCTGATGCAGCAACGCCGC 60

GTGAGGGACGACGGCCTTCGGGTTGTAAACCTCTTTTAGCAGGGAAGAAGCGAAAGTGA 120

CGGTACCTGCAGAAAAAGCGCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGG 180

CGCAAGCGTTATCCGGAATTATTGGGCGTAAAGAGCTCGTAGGCGGTCTGTCGCGTCTGC 240

TGTGAAAACCCGAGGCTCAACCTCGGGCCTGCAGTGGGTACGGGCAGACTAGAGTGCGGT 300

AGGGGAGATTGGAATTCCTGGTGTAGCGGTGGAATGCGCAGATATCAGGAGGAACACCGA 360

TGGCGAAGGCAGATCTCTGGGCCGTAACTGACGCTGAGGAGCGAAAGGGTGGGGAGCAAA 420

CAGGCTTAGATACCCTGGTAGTCCACCCCGTAAACGTTGGGAACTAGTTGTGGGGACCAT 480

TCCACGGTTTCCGTGACGCAGCTAACGCATTAAGTTCCCCGCCTGGGGAGTACGGCCGCA 540

AGGCTAAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGCGGAGCATGCGGATTAAT 600

TCGATGCAACGCGAAGAACCTTACCAAGGCTTGACATATACGAGAACGGGCCAGAAATGG 660

TCAACTCTTTGGACACTCGTAAACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAG 720

ATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGTTCTATGTTGCCAGCACGTAATGG 780

TGGGAACTCATGGGATACTGCCGGGGTCAACTCGGAGGAAGGTGGGGATGACGTCAAATC 840

ATCATGCCCCTTATGTCTTGGGCTTCACGCATGCTACAATGGCCGGTACAAAGGGCTGCA 900

ATACCGCAAGGTGGAGCGAATCCCAAAAAGCCGGTCCCAGTTCGGATTGAGGTCTGCAAC 960

TCGACCTCATGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCAACGCTGCGGTGAATACG 1020

TTCCCGGGTCTTGTACACACCGCCCGTCAAGTCATGAAAGTCGGTAACACCTGAAGCCGG 1080

TGGCCCAACCCTTGTGGAGGGAGCCGTCGAAGGTGA 1116

Claims (3)

1. a strain carbendazim degrading bacterium microbacteriumsp. MBC-6F bacterium, preserving number is: CCTCC No:M 2014680.

2. according to claim 1 microbacteriumsp. the 16S rDNA sequence of MBC-6F bacterium.

3. according to claim 1 microbacteriumsp. the application of MBC-6F bacterium in degraded derosal.