CN1837371A - Ascochitine analog and application thereof - Google Patents

Abstract

The invention relates to a pseudo ascochitine and use thereof, which is prepared from production bacterial strain through the method of liquid or solid fermentation and metabolite extraction, the production bacterial strain being Pseudohalonectria adversaria ZD1 (CGMCC NO.0931) with a molecular formula of C21H16N602 and a molecular weight of 384. The pseudo ascochitine can be used for preparing pesticide preparation for the effective eradication of Phytophthora nicotianae, Pyricularia oryzae and Rhizoctonis solani.

Description

A kind of plan ascochytin and application thereof

Technical field:

The present invention relates to a kind of plan ascochytin and application thereof, belong to biological pesticide technical field.

Background technology:

More than the kind, wherein many being widely used in treated various infected by microbes diseases surplus the microbiotic of having found so far that derives from microorganism nearly ten thousand.In recent years owing to nematode, fungi etc. cause the resistance of the various pathogens of plant infectious diseases, original agricultural antibiotic, nematode killing agent antibiotic therapy effect going down, need continually develop new microbiotic, be the key of problem and invent new antibiotic activity composition.

By literature search, do not find the open report identical with the present invention.

Summary of the invention:

The objective of the invention is to carry the plan ascochytin of a kind of its chemical structure novelty of arch, intend the application of ascochytin as the preparation agricultural antibiotic.

The present invention is achieved in that

1, intends the preparation of ascochytin

(1) intends the separation screening that ascochytin is produced bacterium

Production bacterial strain of the present invention is Pseudohalonectria adversaria ZD1, and this bacterial strain is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center on May 26th, 2003, and preserving number is CGMCC NO.0931.P.adversaria ZD1 belongs to aquatic fungi, is that the inventor separates from the rotten wood of the freshwater lake in Yunnan and obtains.

(2) preparation of plan ascochytin

With Pseudohalonectria adversaria ZD1 bacterial strain, the fermentation process of the liquid or solid by the microbiotic routine makes plan ascochytin of the present invention.

Plan ascochytin of the present invention is carbon source, nitrogenous source and other nutrition source that is usually used in microorganism culturing to the nutrition source in the substratum.Wherein carbon source can be starch, dextrin, glycerine, glucose, maltose, sucrose, wheat bran etc.Nitrogenous source can be analysis for soybean powder, soybean cake powder, groundnut meal, cottonseed meal, corn steep liquor, yeast powder, peptone, ammonium salt, nitrate, and other organic or inorganic nitrogenous compound.To other nutrition source, can add some vitamin B complexess and C in right amount, reach inorganic salts, for example, salt, the metal-salt of phosphoric acid salt and potassium, calcium, iron, magnesium, manganese, zinc and so on.Also can add move, plant, the mineral wet goods is as defoamer.

Plan ascochytin of the present invention is to the same prior art of culture condition (substratum, air flow etc.), and wherein: culture temperature is 20-30 ℃, and is very fast with 24 ℃ of following mycelial growths; The pH scope of substratum is 4-9, with near neutral for well.

Intend the culture of ascochytin, extract the method for metabolite by routine and extract by above-mentioned gained.For example can utilize plan ascochytin and impurity to extract in the difference of aspects such as solubleness, ionic bond power, absorption avidity and molecular weight.Specifically, when the plan ascochytin was present in the fermented liquid, available macropore absorption resin carried out absorption, carries out desorb with aqueous methanol then.Can obtain crude extract like this, crude extract can obtain intending the faint yellow crystallization of ascochytin again by silica gel and Sephedex-LH20 gel filtration chromatography.And exist when intending ascochytin in the mycelium, then with optional organic solvent (for example methyl alcohol, ethanol, propyl alcohol, chloroform, tetrahydrofuran (THF), acetonitrile) cold soaking extracts 4 times, spissated crude extract is absorbed in the solid absorbing material, and (natural materials of Fen Suiing for example is as kaolin, clay, talcum, quartz etc.; Or the synthetic materials of pulverizing, as microgranular silica gel, aluminum oxide etc.) on, in silicagel column, carrying out chromatographic separation, sample on the dry method is that eluent carries out gradient desorption with the chloroform/methanol.The crude extract that obtains like this is similar to and extracts the operation of intending ascochytin from fermented liquid, successively by silica gel and Sephedex-LH20 gel filtration chromatography, can obtain intending the faint yellow crystallization of ascochytin.

2, intend the physics and chemistry and the biological property of ascochytin

A. molecular formula is: C ₁₄H ₁₈O ₃, molecular weight is 234 (ESI-TOF method mensuration), structural formula is as shown below:

B. physical chemical characteristics is:

Outward appearance: faint yellow powdered material;

Solvability: be dissolved in chloroform, acetone, methyl alcohol, methyl-sulphoxide etc., be insoluble to hexane and water etc.;

High resolution positive ion ESI-TOF mass spectrum: measured value is 235.1335, and calculated value is 235.1334;

Uv-absorbing UV (pyridine) λ max (ε) is: 350.0 (11947), 202.5 (20879) nm;

Infrared absorption IR (film) ν _MaxFor: 3441,2960,2925,2855,1725,1629,1544,1461,1382,1273,1160,1117,1073,1034,561cm ^-1

The nuclear-magnetism spectrum signature sees Table 1, and its dissolution solvent is CDCl ₃, 500MHz, TMS are interior mark, δ: ppm.

Table 1 is intended the nuclear-magnetism spectrum signature (CDCl of ascochytin ₃, 500MHz)

Position	13C NMR (mult.)	1H NMR (mult.，J，Hz)
			2	144.4(d)	7.29s
3	120.0(s)

4	74.6d	4.44s
			5	50.4s
6	201.4s
			7	105.5d	5.34s
8	154.7s
			9	112.2s
10	144.7s
			11	24.5d	1.61(q，7.6)
12	9.1s	0.85(t，7.6)
			13	18.8s	1.14(s)
14	18.3s	2.19(s)
			15	13.3s	1.85(s)

C. biological activity

Prove through experiment at present:

In the active determination test of excised leaf pickling process plant poisoning, when plan ascochytin strength of solution is 200ppm, treatment time, it caused a kind of perennial perennial root ruderal when being 24 hours---and tangible water stain spot and brown necrotic plaque appear in alternanthera philoxeroides (Alternanthera philoxeroides Griseb) excised leaf.

In the nematicide active testing of soaked with liquid method, when plan ascochytin strength of solution was 100ppm, the treatment time, its lethality rate to pine wood nematode was 53.8% when being 24 hours.

In the determination test of paper disk method anti-fungal activity of plant pathogenic, when plan ascochytin strength of solution is 200ppm, treatment time is when being 120 hours, and it causes the inhibition zone of Phytophthora nicotianae (Phytophthora nicotianae), Pyricularia oryzae (Pyricularia oryzae), tuber of pinellia rhizoctonia solani (Rhizoctonis solani), Exserohilum turcicum four kind of plant pathogenic fungies such as (Exserohilum turcicum) to be respectively 1.20,1.80,1.11 and 1.53cm.

Positively effect of the present invention is: intend the growth that ascochytin can be killed alternanthera philoxeroides and pine wood nematode, inhibition plant pathogenic fungi, have important research and using value in the development and use of scientific research and related drugs.

Embodiment:

Embodiment 1:

Extraction separation obtains The compounds of this invention and intends ascochytin from the solid fermentation thing of P.adversaria ZD1, and concrete steps are as follows:

A. the solid fermentation of fungi P.adversaria ZD1 is cultivated:

The PDA substratum of improvement: potato 200g, glucose 20g, yeast extract 1.0g, glycerine 6.0g, agar 17g, each 1 of medical vitamin B complexes and C, water 1000mL makes flat board, and the picking bacterial strain inserts dull and stereotyped, cultivates 7 days for 25 ℃;

B. with the mycelium freeze-drying of above-mentioned cultured P.adversaria ZD1, again with optional organic solvent (for example methyl alcohol, ethanol, propyl alcohol, chloroform, tetrahydrofuran (THF), acetonitrile) cold soaking extracts 4 times, spissated crude extract is absorbed in the solid absorbing material, and (natural materials of Fen Suiing for example is as kaolin, clay, talcum, quartz etc.; Or the synthetic materials of pulverizing, as microgranular silica gel, aluminum oxide etc.) on, in silicagel column, carrying out chromatographic separation, sample on the dry method is the eluent gradient elution with chloroform/methanol=0-30%.

C. collect the chloroform/methanol elutriant of 1%-10%, concentrating under reduced pressure.

D. above-mentioned concentrated solution is carried out chromatographic separation once more in silicagel column, sample on the dry method is the eluent gradient elution with sherwood oil/acetone=90-50%.

F. collect on the TLC plate sulfuric acid colour developing and be the sherwood oil of xanchromatic 70-80%/acetone elutriant, concentrated yellow crystal is the mixture shown in the structural formula.Through the Sephedex-LH20 separating for several times, can obtain to intend ascochytin again.

Embodiment 2:

Adopt the excised leaf pickling process to detect and intend the murder by poisoning activity experiment of ascochytin the alternanthera philoxeroides excised leaf.

1. test with medicament

To test with sample and be dissolved in a small amount of organic reagent DMSO earlier, and add certain density Tween solution again and prepare the sample test solution that concentration is 200ppm respectively.In this solution, the final concentration of the final concentration of DMSO＜3%, tween＜5 ‰.The aqueous solution that in addition DMSO of same concentrations is dissolved in the tween 20 that contains 0.5% (V/V) in contrast.

2. the preparation of the alternanthera philoxeroides of test usefulness

To pick up from Kunming interwined dragon alternanthera philoxeroides kind along the river in flowerpot.During test health is not had the alternanthera philoxeroides blade of scab and cut with scissors, water is rinsed well, embathes 1min with 70% alcohol again, uses aseptic washing 3 times standby then.

3. the testing method of alternanthera philoxeroides weeding activity

The test with medicament for preparing is poured in the flat board of the 90mm that is placed with 6 alternanthera philoxeroides, judged the power of specimen weeding activity to have or not water stain spot and necrotic plaque and scab size thereof.

4. test-results

Be 24 hours in the treatment time, when intending the ascochytin strength of solution and being 200ppm, observe it

Cause the alternanthera philoxeroides excised leaf tangible water stain spot and brown necrotic plaque to occur.

The result shows: intend ascochytin to weeds---alternanthera philoxeroides has cytotoxicity preferably.

Embodiment 3:

Adopt the nematicide activity test method of soaked with liquid method to detect the murder by poisoning activity experiment of The compounds of this invention to nematode.

1. test with medicament

Test uses the compound method of sample with embodiment 2.

2. cultivation of pine wood nematode and preparation

Pine wood nematode (Bursaphelenchus xylophilus (Steiner ﹠amp; Buhrer) cultural method Nickle): Botrytis cinerea (Botrytis cinerea) is inserted the PDA flat board, under 25 ℃, be cultured to and cover with flat board, insert a substratum that has B.xylophilus, be cultured to mycelia and disappear under 25 ℃, nematode is covered with flat board.

The substratum that will be loaded with nematode during use is chosen, and is put in the nematode separator, and after with sterilized water nematode being washed out, centrifugal concentrating is prepared into nematode suspension (about 500 every milliliter).

3. test method

It is the culture dish of 5cm that three kinds of different concns such as 200ppm, the 100ppm of The compounds of this invention, 50ppm are placed diameter for reagent liquid 2ml, the suspension that adds living nematode 20ul (about 300) nematode again behind the mixing is positioned over culture dish in 25 ℃ the incubator gently.Respectively at the mortality ratio of checking the calculating nematode in 12,24 and 48 hours.And under binocular microscope, observe observation line polypide wall changing conditions.

The method of identifying nematode death is: add 1-5 and drip 5%NaCl solution in handling flat board, observe after 2 minutes, dead worm is stiff, and the worm that lives is then curled or twisting.

Mortality ratio %=dead wire borer population/bus borer population * 100

With the test soln that does not add sample is contrast, and whole experiment triplicate is got three mean value calculation and gone out average mortality.

4. test-results

The result

Table 2. is intended ascochytin cytotoxicity result to pine wood nematode under different concns

Sample test concentration	The different treatment time (hour) interior lethality rate (%)
				12	24	36
	200ppm 100ppm 50ppm contrast	0 0 0 0	83.2 53.8 25.6 1.1				92.4 82.1 31.3 1.4

The result shows: intending ascochytin has cytotoxicity preferably to pine wood nematode.

Embodiment 4:

1. test with medicament

Test uses the compound method of sample with embodiment 2.

2. the cultivation of plant pathogenic fungi

Culture medium prescription is conventional PDA substratum, and with pathogenic fungi bacterial classification inoculation to be tested (every bottled 50mL) in the 250mL triangular flask, 25 ℃ of following shaking tables (rotating speed is 220rpm) were cultivated 36 hours.

Preparation mixes the bacterium flat board: after the sterilization of diameter 9cm culture dish, pour the aforementioned conventional PDA substratum of 45 ℃ of 20mL and the plant pathogenic fungi zymocyte liquid of 0.3mL into, fully mixing is made and is mixed the bacterium flat board.

3. test method

Scraps of paper assay method.The scraps of paper diameter that uses is 2mm, when testing, earlier the scraps of paper is dipped in the specimen solution for preparing, and the scraps of paper that will dip in soup again are placed on and mix in the middle of the bacterium flat board, cultivate 120 hours down in 25 ℃, measure antibacterial circle diameter, repeat three samples.

4. test-results

Table 3 is intended the bacteriostatic action of ascochytin (200ppm) to plant pathogenic fungi

The test fungi	The diameter of inhibition zone (cm)
					1	2	3	On average
	Phytophthora nicotianae	1.15	1.38	1.07					1.20
Pyricularia oryzae					1.84	1.80	1.76	1.80
	Tuber of pinellia rhizoctonia solani	1.20	1.05	1.08					1.11
Exserohilum turcicum					1.50	1.54	1.55	1.53

The result shows: cultivate after 120 hours, inhibition zone does not appear in contrast, and intend ascochytin the growth of the four kind of plant pathogenic fungies tested is had the obvious suppression effect, the inhibition zone maximum be 1.80cm, minimum is 1.11cm also, has shown its better resistance to plant pathogenic fungi.

Claims (2)

1, a kind of plan ascochytin by producing bacterial strain, obtains by liquid or solid fermentation and the method for extracting metabolite, it is characterized in that:

A. producing bacterial strain is Pseudohalonectria adversaria ZD1, and this bacterial strain is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center on May 26th, 2003, and preserving number is CGMCC NO.0931;

B. the molecular formula of intending ascochytin is: C ₁₄H ₁₈O ₃, molecular weight is 234, structural formula is as shown below:

2, the application of the described plan ascochytin of claim 1 is characterized in that intending the application of ascochytin as the preparation pesticide preparation.