CN102293201B - New purpose of coronatine - Google Patents

Abstract

The invention discloses a new purpose of coronatine. The new purpose is an application of coronatine in promoting the cold resistance of plants. Coronatine has high biological activities when coronatine is used as a cold resistant agent of paddy rice, and the consumption amount is low to less than 1.0g/hectare, so the cost is reduced; in addition, coronatine belongs to natural products, and the environmental compatibility is good, so problems of residues and residual effects do not exist.

Description

Coronatine improves the cold resistant purposes of paddy rice

Technical field

The present invention relates to the novelty teabag of coronatine.

Background technology

Paddy rice originates from the torrid zone, is typically to like hotwork thing.Paddy rice is higher to ambient temperature conditions, turns green between tillering stage after nursery stage in early spring, rice transplanting, and particularly the paddy pollen mother cell reduction division phase of containing is easier to the harm being subject to interim low temperature, brings have a strong impact on to Rice Production.

Coronatine (coronatine) is a kind of nosotoxin that pseudomonad produces.

Coronatine has following structural formula:

Coronatine carries out industrial fermentation and produces acquisition (ZL 200510011466.2; ZL 200510085280.1; ZL200710099033.6; ZL 200810119153.2).

Summary of the invention

An object of the present invention is to provide the novelty teabag of coronatine.

One of novelty teabag of coronatine provided by the present invention is that coronatine is promoting the application in plant cold resistance.

In above-mentioned application, the application concentration of described coronatine is 0.01 ~ 10mg/L or 0.1 ~ 1mg/L, is specially 0.2 ~ 1mg/L, then is specially 0.1mg/L, 0.2mg/L, 0.5mg/L or 1.0mg/L.

Two of the novelty teabag of coronatine provided by the present invention is coronatines as the application in Plant Cold-resister.

In above-mentioned application, in described Plant Cold-resister, also comprise the upper acceptable carrier of formulations of pesticide processing.

The various formulations of Plant Cold-resister can be prepared according to the conventional production process of formulations of pesticide manufacture field.Such as make active component mix with one or more carriers, be then made into required formulation.

Carrier can be the upper acceptable carrier system of processing, specifically refers to the material meeting following condition: it and active component are convenient to be applied to pending site after preparing, such as, can be plant, seed or soil; Or be conducive to storage, transport or operation.Carrier can be the farm chemical carrier of formulations of pesticide manufacture field routine, such as: the agriculturally acceptable carrier such as dispersant, wetting agent, binding agent, emulsifier, these dispersants, wetting agent, binding agent, emulsifier respectively have characteristic, and those skilled in the art can know.

In above-mentioned arbitrary described application, described plant is crops, is specially paddy rice.

Last object of the present invention is to provide the method utilizing coronatine to promote plant cold resistance.

The method utilizing coronatine to promote plant cold resistance provided by the present invention, comprising the steps: between the shooting stage of plant, is that the coronatine aqueous solution of 0.01 ~ 10mg/L is sprayed on plant leaf surface by concentration.

In said method, described concentration is specially 0.1 ~ 1mg/L or 0.2 ~ 1mg/L, then is specially 0.1mg/L, 0.2mg/L, 0.5mg/L or 1.0mg/L.

In said method, described plant is crops, is specially paddy rice.

Experiment proves, using the cold-resistance agent of coronatine as paddy rice, can carry out inducing plant generation cold resistance by Direct spraying after dilution in the surface of plant.Coronatine process in the mass range concentration of 0.01 ~ 10mg/L paddy rice can alleviate paddy rice cold damage generation and to rice safety.Per hectare uses can be low to moderate less than 1.0 grams, reduces cost; In addition, coronatine belongs to natural products, and Environmental compatibility is good, there is not residual residual effect problem.

Accompanying drawing explanation

Fig. 1 is the liquid chromatogram collection of illustrative plates of coronatine standard items.

Fig. 2 is the liquid phase chromatic graph spectrum of obtained coronatine sample.

Embodiment

The experimental technique used in following embodiment if no special instructions, is conventional method.

Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.

The coronatine used in embodiment 2 and 3 all prepares according to method described in embodiment 1.

Rice varieties cultivates rice 12, is numbered black careful rice 2006009.

Coronatine (COR) standard items purchased from Sigma-Aldrich company, content > 95%, production code member C8115-1MG.

Embodiment 1, prepare coronatine

To be seeded to by recombinant bacterium Pseudomonas syringae pv.glycinea MW123 (preserving number CGMCC No.2533) containing final concentration be the kanamycin of 10 μ g/L and final concentration is in the MG liquid nutrient medium of the streptomycin of 30 μ g/L, it is 28 DEG C in temperature, the shaking table of rotating speed 260rpm is cultivated 48 hours, obtains primary seed solution.

MG liquid nutrient medium is composed as follows:

Solid culture medium: add 1.5% agar powder

121 DEG C of sterilizings 20 minutes

HSC medium is composed as follows:

A liquid:

B liquid:

Glucose 20g

Distilled water 10mL

A liquid was 115 DEG C of sterilizings 15 minutes, and B liquid was 121 DEG C of sterilizings 20 minutes

Be sub-packed in by 600ml MG liquid nutrient medium in three 500ml triangular flasks, namely every bottle contains 200ml medium; Being inoculated in above-mentioned three 500ml triangular flasks by cultivating the primary seed solution obtained respectively by 1%-2.5% inoculum concentration, being 28 DEG C in temperature, the shaking table of rotating speed 260rpm being cultivated 48 hours, obtains secondary seed solution.

5 liters of HSC medium through conventional high-pressure hot steam sterilization method are loaded in the fermentation tank of 7 liters, secondary seed solution is accessed wherein by 1%-2.5% inoculum concentration, air agitation, temperature be 18 DEG C, initial pH ferments under being the condition of 6.0-7.0, fermentation time reaches 7 days tanks at present.

Get obtained zymotic fluid 1000ml, centrifugal 20min under 4 DEG C of conditions, collect supernatant, and with the hydrochloric acid of 1.0M, the pH value of supernatant is transferred to 2.5; The supernatant obtained is extracted with ethyl acetate 3 times (120ml ethyl acetate: 100ml supernatant), discards aqueous phase, collect organic facies; Organic facies is concentrated into 15ml at 35 DEG C, the sodium carbonate liquor of the 50mM of concentrate pH 10.5 is extracted 3 times (15ml sodium carbonate liquor: 15ml concentrate), discard organic facies, collect aqueous phase; Aqueous phase is adjusted to pH 2.5, is then extracted with ethyl acetate 3 times (15ml: 15ml ethyl acetate), discards aqueous phase, collect organic facies; Organic facies is concentrated evaporate to dryness, uses 20ml methanol suspension, get 0.5ml and detect for high performance liquid chromatography.During high performance liquid chromatography detects the mobile phase used by 0.05% trifluoroacetic acid solution (pH 2.9) and acetonitrile by 1: 9 proportions become.Testing sample carries out under retaining the condition of 10min at the speed of determined wavelength 208nm place, 2ml/min, sample size 25 μ l, each sample.According in coronatine standard items retention time determination extract whether containing coronatine, and by comparatively determining the content of coronatine with coronatine standard items peak area ratio.

Under above testing conditions, the retention time of coronatine is 7.7 minutes, and as shown in Figure 1, the collection of illustrative plates of laboratory sample as shown in Figure 2 for the collection of illustrative plates of coronatine standard items.3 repetitions are established in experiment.Result shows that the average yield of coronatine is 200.5mg/L zymotic fluid.

According to the coronatine content recorded, extracting and adding methyl alcohol in the organic facies of evaporate to dryness, be deployed into 0.05% coronatine soluble concentrate, apply for following examples.

Embodiment 2, coronatine inducing paddy rice seedling experiment of cold-resistance

For examination rice varieties for cultivating rice 12.Diameter is that the little polypots of 20cm loads paddy soils, and full rice paddy seed is by after selected for 20% sodium chloride solution, and with saturated liquor natrii hypochloritis's sterilizing 30min, flowing water is cleaned, lower 48 hours of room temperature, with clear water contrast, and vernalization 24 hours under 30 DEG C of dark conditions after cleaning.Choosing the consistent seed that germinates is sown in polypots, every basin 100 seeds, at light intensity 4000Lux, cultivate in the artificial lighting incubator of temperature 25 ± 1 DEG C, every day, irradiation 12 hours, treated that paddy growth sprays 1.0mg/L, 0.5mg/L respectively to two leaf one heart stages, 0.2mg/L and 0.1mg/L coronatine solution, every polypots spraying liquid amount 2.0ml; Do medicament contrast with natural abscisic acid (S-ABA) 20mg/L, repeat 4 times.Process and polypots is inserted 5 ± 1 DEG C of low temperature incubators after 48 hours and carry out cold damage process, 48 hours " Invest, Then Investigate " water rice seedling blight numbers, calculate seeding rate, clean seedling, removing seed, cut Miao Yugen, blot with water suction and take fresh weight respectively afterwards, at 80 DEG C, dry 48 as a child took dry weight (the results are shown in Table 1).

The process of table 1 coronatine is to the cold-resistant effect of paddy rice

Coronatine inducing paddy rice cold resistance result shows, the coronatine of different quality concentration can improve the planting percent of rice paddy seed, and Seedling Quality in Rice increases, and effect is better than contrast medicament S-ABA.

Embodiment 3, the cold resistant field trial of coronatine inducing paddy rice

For examination rice varieties for cultivating rice 12, testing and carrying out in Heilongjiang Province eight or five O Farm Rice scientific and technological park in 2010.Between the shooting stage, 0.05% coronatine soluble liquid is diluted 2500 times (coronatine active ingredient concentration is 0.2mg/L), carry out blade face by the amount of liquid medicine of 750 kilograms/hectare and evenly spray 1 time; With the solvable pulvis of 0.1%S-ABA be contrast medicament, dilute during use 2500 times (S-ABA active ingredient concentration is 0.4mg/L), amount of liquid medicine with coronatine process, and does blank with clear water.Plot area 52.5 square metres, repeats for 4 times.Process latter 15 days by intelligence control system and community ground temperature is stabilized in 18 degrees Celsius and maintains 2 days, carry out low temperature treatment.Investigate grain number per spike, sterile grain rate, thousand kernel weight during results, and measure cell production (the results are shown in Table 2).

Paddy rice receives interim low temperature in the pollen mother cells phase of containing, and cause sterile grain rate higher, improve the grouting of seed, add rice yield after coronatine process, effect is better than S-ABA process.

The process of table 2 coronatine is to the cold-resistant effect of paddy rice

Claims (5)

1. the application of coronatine in promoting Rice Resistance cold;

Described coronatine is prepared as follows and obtains:

To be seeded to by the recombinant bacterium Pseudomonas syringae pv.glycinea MW123 of preserving number CGMCC No.2533 containing final concentration be the kanamycin of 10 μ g/L and final concentration is in the MG liquid nutrient medium of the streptomycin of 30 μ g/L, it is 28 DEG C in temperature, the shaking table of rotating speed 260rpm is cultivated 48 hours, obtains primary seed solution;

MG liquid nutrient medium is composed as follows:

Solid culture medium: add 1.5% agar powder;

121 DEG C of sterilizings 20 minutes;

HSC medium is composed as follows:

A liquid:

B liquid:

Glucose 20g

Distilled water 10mL

A liquid was 115 DEG C of sterilizings 15 minutes, and B liquid was 121 DEG C of sterilizings 20 minutes;

Be sub-packed in by 600ml MG liquid nutrient medium in three 500ml triangular flasks, namely every bottle contains 200ml medium; Being inoculated in above-mentioned three 500ml triangular flasks by cultivating the primary seed solution obtained respectively by 1%-2.5% inoculum concentration, being 28 DEG C in temperature, the shaking table of rotating speed 260rpm being cultivated 48 hours, obtains secondary seed solution;

5 liters of HSC medium through conventional high-pressure hot steam sterilization method are loaded in the fermentation tank of 7 liters, secondary seed solution is accessed wherein by 1%-2.5% inoculum concentration, air agitation, temperature be 18 DEG C, initial pH ferments under being the condition of 6.0-7.0, fermentation time reaches 7 days tanks at present;

Get obtained zymotic fluid 1000ml, centrifugal 20min under 4 DEG C of conditions, collect supernatant, and with the hydrochloric acid of 1.0M, the pH value of supernatant is transferred to 2.5; The supernatant obtained is extracted with ethyl acetate 3 times, 120ml ethyl acetate: 100ml supernatant, discards aqueous phase, collects organic facies; Organic facies is concentrated into 15ml at 35 DEG C, the sodium carbonate liquor of the 50mM of concentrate pH 10.5 is extracted 3 times, 15ml sodium carbonate liquor: 15ml concentrate, discards organic facies, collect aqueous phase; Aqueous phase is adjusted to pH 2.5, is then extracted with ethyl acetate 3 times, 15ml:15ml ethyl acetate, discards aqueous phase, collects organic facies; Organic facies is concentrated evaporate to dryness, uses 20ml methanol suspension, get 0.5ml and detect for high performance liquid chromatography; During high performance liquid chromatography detects the mobile phase used by pH 2.9,0.05% trifluoroacetic acid solution and acetonitrile by 1: 9 proportions become; Testing sample carries out under retaining the condition of 10min at the speed of determined wavelength 208nm place, 2ml/min, sample size 25 μ l, each sample; According in coronatine standard items retention time determination extract whether containing coronatine, and by comparatively determining the content of coronatine with coronatine standard items peak area ratio;

Under above testing conditions, the retention time of coronatine is 7.7 minutes;

According to the coronatine content recorded, extracting and adding methyl alcohol in the organic facies of evaporate to dryness, be deployed into 0.05% coronatine soluble concentrate, obtain final product.

2. application according to claim 1, is characterized in that: the application concentration of described coronatine is 0.01 ~ 10mg/L.

3. coronatine is as the application in Cold-resistant agent for rice;

Described coronatine is prepared as follows and obtains:

To be seeded to by the recombinant bacterium Pseudomonas syringae pv.glycinea MW123 of preserving number CGMCC No.2533 containing final concentration be the kanamycin of 10 μ g/L and final concentration is in the MG liquid nutrient medium of the streptomycin of 30 μ g/L, it is 28 DEG C in temperature, the shaking table of rotating speed 260rpm is cultivated 48 hours, obtains primary seed solution;

MG liquid nutrient medium is composed as follows:

Solid culture medium: add 1.5% agar powder;

121 DEG C of sterilizings 20 minutes.

HSC medium is composed as follows:

A liquid:

B liquid:

Glucose 20g

Distilled water 10mL

A liquid was 115 DEG C of sterilizings 15 minutes, and B liquid was 121 DEG C of sterilizings 20 minutes;

Be sub-packed in by 600ml MG liquid nutrient medium in three 500ml triangular flasks, namely every bottle contains 200ml medium; Being inoculated in above-mentioned three 500ml triangular flasks by cultivating the primary seed solution obtained respectively by 1%-2.5% inoculum concentration, being 28 DEG C in temperature, the shaking table of rotating speed 260rpm being cultivated 48 hours, obtains secondary seed solution;

5 liters of HSC medium through conventional high-pressure hot steam sterilization method are loaded in the fermentation tank of 7 liters, secondary seed solution is accessed wherein by 1%-2.5% inoculum concentration, air agitation, temperature be 18 DEG C, initial pH ferments under being the condition of 6.0-7.0, fermentation time reaches 7 days tanks at present;

Get obtained zymotic fluid 1000ml, centrifugal 20min under 4 DEG C of conditions, collect supernatant, and with the hydrochloric acid of 1.0M, the pH value of supernatant is transferred to 2.5; The supernatant obtained is extracted with ethyl acetate 3 times, 120ml ethyl acetate: 100ml supernatant, discards aqueous phase, collects organic facies; Organic facies is concentrated into 15ml at 35 DEG C, the sodium carbonate liquor of the 50mM of concentrate pH 10.5 is extracted 3 times, 15ml sodium carbonate liquor: 15ml concentrate, discards organic facies, collect aqueous phase; Aqueous phase is adjusted to pH 2.5, is then extracted with ethyl acetate 3 times, 15ml:15ml ethyl acetate, discards aqueous phase, collects organic facies; Organic facies is concentrated evaporate to dryness, uses 20ml methanol suspension, get 0.5ml and detect for high performance liquid chromatography; During high performance liquid chromatography detects the mobile phase used by pH 2.9,0.05% trifluoroacetic acid solution and acetonitrile by 1: 9 proportions become; Testing sample carries out under retaining the condition of 10min at the speed of determined wavelength 208nm place, 2ml/min, sample size 25 μ l, each sample; According in coronatine standard items retention time determination extract whether containing coronatine, and by comparatively determining the content of coronatine with coronatine standard items peak area ratio;

Under above testing conditions, the retention time of coronatine is 7.7 minutes;

According to the coronatine content recorded, extracting and adding methyl alcohol in the organic facies of evaporate to dryness, be deployed into 0.05% coronatine soluble concentrate, obtain final product.

4. application according to claim 3, is characterized in that: also comprise the upper acceptable carrier of formulations of pesticide processing in described Plant Cold-resister.

5. utilize coronatine to promote the method that paddy rice is cold-resistant, comprising the steps: between the shooting stage of paddy rice, is that the coronatine aqueous solution of 0.01 ~ 10mg/L is sprayed on rice leaf by concentration;

Described coronatine is prepared as follows and obtains:

To be seeded to by the recombinant bacterium Pseudomonas syringae pv.glycinea MW123 of preserving number CGMCC No.2533 containing final concentration be the kanamycin of 10 μ g/L and final concentration is in the MG liquid nutrient medium of the streptomycin of 30 μ g/L, it is 28 DEG C in temperature, the shaking table of rotating speed 260rpm is cultivated 48 hours, obtains primary seed solution;

MG liquid nutrient medium is composed as follows:

Solid culture medium: add 1.5% agar powder;

121 DEG C of sterilizings 20 minutes.

HSC medium is composed as follows:

A liquid:

B liquid:

Glucose 20g

Distilled water 10mL

A liquid was 115 DEG C of sterilizings 15 minutes, and B liquid was 121 DEG C of sterilizings 20 minutes;

Be sub-packed in by 600ml MG liquid nutrient medium in three 500ml triangular flasks, namely every bottle contains 200ml medium; Being inoculated in above-mentioned three 500ml triangular flasks by cultivating the primary seed solution obtained respectively by 1%-2.5% inoculum concentration, being 28 DEG C in temperature, the shaking table of rotating speed 260rpm being cultivated 48 hours, obtains secondary seed solution;

5 liters of HSC medium through conventional high-pressure hot steam sterilization method are loaded in the fermentation tank of 7 liters, secondary seed solution is accessed wherein by 1%-2.5% inoculum concentration, air agitation, temperature be 18 DEG C, initial pH ferments under being the condition of 6.0-7.0, fermentation time reaches 7 days tanks at present;

Get obtained zymotic fluid 1000ml, centrifugal 20min under 4 DEG C of conditions, collect supernatant, and with the hydrochloric acid of 1.0M, the pH value of supernatant is transferred to 2.5; The supernatant obtained is extracted with ethyl acetate 3 times, 120ml ethyl acetate: 100ml supernatant, discards aqueous phase, collects organic facies; Organic facies is concentrated into 15ml at 35 DEG C, the sodium carbonate liquor of the 50mM of concentrate pH 10.5 is extracted 3 times, 15ml sodium carbonate liquor: 15ml concentrate, discards organic facies, collect aqueous phase; Aqueous phase is adjusted to pH 2.5, is then extracted with ethyl acetate 3 times, 15ml:15ml ethyl acetate, discards aqueous phase, collects organic facies; Organic facies is concentrated evaporate to dryness, uses 20ml methanol suspension, get 0.5ml and detect for high performance liquid chromatography; During high performance liquid chromatography detects the mobile phase used by pH 2.9,0.05% trifluoroacetic acid solution and acetonitrile by 1: 9 proportions become; Testing sample carries out under retaining the condition of 10min at the speed of determined wavelength 208nm place, 2ml/min, sample size 25 μ l, each sample; According in coronatine standard items retention time determination extract whether containing coronatine, and by comparatively determining the content of coronatine with coronatine standard items peak area ratio;

Under above testing conditions, the retention time of coronatine is 7.7 minutes;

According to the coronatine content recorded, extracting and adding methyl alcohol in the organic facies of evaporate to dryness, be deployed into 0.05% coronatine soluble concentrate, obtain final product.