CN1187439C - Biological toxin for preventig and controlling pine nematode and its prepn - Google Patents

Abstract

The present invention relates to a biological toxin for preventing and killing pine wood nematodes and a preparation method thereof, which belong to the technical field of biological pesticide. The strains for producing the biological toxin, namely pseudohalonectria adversaria(ZD1), is preserved in China General Microbiological Culture Collection Center on May, 26th, 2003, and the preservation number is CGMCC NO. 0931. The ZD1 is cultured in liquid and wheat grain solid, and effective water soluble and liposoluble substances are extracted from the metabolic products to prepare the nematode killing biological toxins. The present invention has excellent killing effect on pine wood nematodes and excellent application and development prospect.

Description

A kind of biotoxin and preparation method who prevents and treats pine wood nematode

Technical field:

The present invention relates to a kind of biotoxin and preparation method who prevents and treats pine wood nematode, belong to biological pesticide technical field

Background technology:

Pine wood nematode (Bursaphelenchus xylophilus) disease is the especially big crushing disaster of Pinus seeds, belongs to international important Quarantine Objects, classifies as first of the forest disease and pest, is called as " smokeless hill fire ".From nineteen eighty-two since the pine wood nematode disease is found in forest zone, the Zhongshan Tomb, Nanjing first, now in Jiangsu, many provinces and regions diffusions such as Anhui, Guangdong, Zhejiang, Shandong, Taiwan and Hong Kong spread, do great damage, and the pine forest of vast normal region constituted a serious threat for the pine forest resource of China and ecotope, natural landscape.Effectively prevent and treat medicament because of still having in the world at present, this disease be otherwise known as " pine tree cancer ".Because this disease has been approached Huang and has been gone out the landscaped area, be protection Mount Huang wonderful view, country and Anhui Province invest nearly 6,000 ten thousand yuan, to set up " Mount Huang pine wood nematode prevention system engineering ".This disease once once had been negligent of quarantine control after Guangdong Province takes place, cause once more to expand.

Both at home and abroad pine wood nematode is not still had efficacious agents and prevents and treats method at present, generally speaking, having taken place behind the pine wood nematode all is to adopt conservative control method, is about to susceptible pine tree felling and burns, and propagates to cut off.And, pine wood nematode is not then had activity at all to phytotrophy root knot nematode and the effective biotechnological formulation of Cyst nematode.Searching is to pine wood nematode effectively preventing method and medicament, and is extremely urgent.

Summary of the invention:

Purpose of the present invention is exactly the fungal bacterial strain of selecting pine wood nematode tool good insecticidal, after liquid fermenting and wheat solid fermentation of the present invention are cultivated routinely with bacterial strain, extraction is killed the pine wood nematode effective ingredient and is prepared into biotoxin from meta-bolites, in the hope of finding pine wood nematode effectively preventing medicament and method.

The fungi that the present invention adopts is Pseudohalonectria adversaria ZD1, depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center; Address: China. Beijing. the Zhong Guan-cun; Preservation date: on May 26th, 2003; The numbering CGMCC NO.0931 that preservation is registered on the books.P.adversaria (ZD1) belongs to aquatic fungi, is a strain that the inventor filters out from a large amount of aquatic fungi bacterial strains has good eelworm-killing activity to pine wood nematode bacterial strain.

The inventor has good insecticidal effect by discovering the fungi that Pseudohalonectria belongs to pine wood nematode, and screening has determined that the best bacterial strain Pseudohalonectriaadversaria ZD1 of a strain proterties carries out further research from Pseudohalonectria belongs to.The present invention with the ZD1 bacterial strain by liquid fermenting and wheat solid fermentation after, extract meta-bolites with killing activity of pine wood nematode, be prepared into biological nematicide toxin.

Fungi Pseudohalonectri.Adversaria ZD1 cultural method of the present invention (below be weight percentage):

The liquid culture based formulas is the 1-4% soybean; 1-5% glucose; 1-2% starch; The 0.1-0.5% yeast extract; 0.2-1% sodium-chlor; The 0.02-0.1% potassium primary phosphate; 0.02-0.1% sal epsom; 0.1-0.6% lime carbonate, remainder is a water.

Wheat solid culture based formulas is a 0.2-1% lime carbonate; The 1-3% soyflour; The 0.2-1% sulfate of ammoniac; 0.5-3% crosses phosphorus

Acid calcium; Wheat wheat 92%-96%.

The present invention is divided into that liquid fermenting extracts water-soluble wireworm-killing biologic toxin and the wheat solid fermentation extracts two kinds of methods of fat-soluble wireworm-killing biologic toxin.

Liquid fermenting extracts the preparation method of wireworm-killing biologic toxin:

1, the mycelium with ZD1 is inoculated on the test tube nutrient agar inclined-plane, and culture medium prescription is the PDA substratum, cultivates 6-12 days down, obtains the test tube kind for 18-26 ℃;

2, the test tube kind is inoculated in 250ml triangular flask (the every bottled 70ml) liquid nutrient medium, culture medium prescription is the aforementioned liquids culture medium prescription, cultivates in 20-30 ℃ of following shaking table, and rotating speed is 200-300rpm, incubation time 5-8 days.

3, the fermented liquid that will contain mycelia is put into whizzer, and 3600 left the heart 20 minutes, gets the water-soluble pine wood nematode biotoxin that kills after the filtration.

The wheat solid fermentation extracts the preparation method who kills the pine wood nematode biotoxin:

1, test tube kind cultural method is identical with aforementioned liquids fermentation method for producing 1;

2, adopt aforementioned wheat solid culture based formulas.After wheat soaked 24 hours with clear water, pick up and put into boiling water and boil, to boil but skin is broken for suitable.Pack into after picking up drainage and auxiliary material mixing in the triangular flask of 1000ml, every bottled 700ml, 120 ℃ of sterilizations inserted the test tube kind after 30 minutes, cultivated 15-20 days in 22-26 ℃, covered with up to mycelia.

3, the wheat that will cover with mycelia digs out, and freeze-drying with chloroform and methyl alcohol, is mixed with the solvent lixiviate in 1: 1 ratio, concentrates after the lixiviate, gets total crude extract.Crude extract is dissolved in a small amount of methyl-sulphoxide (DMSO), uses the dissolving of certain density tween 20 (Tween-20) solution again, get the fat-soluble pine wood nematode biotoxin emulsion that kills.In this emulsion, the final concentration of methyl-sulphoxide is no more than 3%, and the final concentration of Tween-20 is no more than 0.3%.

What extract with liquid fermentation and culture mainly is water-soluble wireworm-killing biologic toxin, and mainly is fat-soluble wireworm-killing biologic toxin with wheat solid fermentation production extraction.

Product of the present invention is to the test of pesticide effectiveness of pine wood nematode

The test of pesticide effectiveness 1:

1, test with medicament: press liquid cultivating method and produce the wireworm-killing biologic toxin; Leave 20 minutes after-filtration of the heart in contrast with nonvaccinated substratum 3600.

2, pine wood nematode preparation: put into 15g through 2 days corn grain of water logging bubble in the 100ml triangular flask, add water 10ml, autoclaving inserts Botrytis cinerea (Botrytis cinerea).Cultivated 4 to 7 days for 25 ℃.After treating that mycelia is paved with triangular flask, inoculation was cultivated 15 to 20 days for 28 ℃ through the pine wood nematode of 0.25% clorox surface sterilization.With sterilized water nematode is washed, making content is the nematode suspension of 15/μ l.

3, test method: cut-off footpath 6cm culture dish, the sterilization back adds 2ml test with medicament, add 20 μ l then, nematode suspension (300), put 25 ℃ of cultivations, regularly check the life or death of nematode with anatomical lens, nematode is reactionless under physiological stimulation then is defined as nematode death, calculates the drug effect of biological nematicide toxin according to mortality ratio.

Vat liquor with the substratum that do not connect bacterium compares every processing triplicate.

4, test-results

Table 1 liquid fermenting biotoxin is to the pine wood nematode insecticidal effect

Handle	12 hours			24 hours			48 hours
										Dead borer population	Mortality ratio (%)	Drug effect (%)	Dead borer population	Mortality ratio (%)	Drug effect (%)	Dead borer population	Mortality ratio (%)	Drug effect (%)
	Medicament 1	245	81.6	81.3	257	85.6	85.6	262	87.3										87
Medicament 2										244	81.3	81	254	84.6	84.3	257	85.6	85.3
	Medicament 3	209	69.6	69.3	258	86	85.6	268	89.3										89
On average										233	77.5	77.2	256	85.4	85	262	87.4	87.1
	Contrast	1			1			1

The result shows: product water-soluble biological nematicide toxin of the present invention has good insecticidal effect to pine wood nematode, and its 12 hourly average mortality ratio reaches 77.5%, is up to 81.6%; 48 hourly average mortality ratio reach 87.4%, are up to 89.3%, 48 hourly average drug effect and reach 87.1%.

The test of pesticide effectiveness 2:

1, test with medicament: wheat solid culture based formulas is 1% lime carbonate; 3% soyflour; 0.5% sulfate of ammoniac; 0.5% calcium superphosphate; Wheat wheat 95%.Extract the preparation method who kills the pine wood nematode biotoxin by aforementioned wheat solid fermentation and prepare the test with medicament.

Contrast medicament:, but do not insert the test tube kind in the wheat solid medium by aforementioned wheat solid fermenting producing method.

2, the pine wood nematode preparation is identical with the test of pesticide effectiveness 1.

3, test method is identical with the test of pesticide effectiveness 1.

4, test-results

Table 2 a wheat solid fermentation biotoxin is to the pine wood nematode insecticidal effect

Handle	12 hours			24 hours			36 hours
										Dead borer population	Mortality ratio (%)	Drug effect (%)	Dead borer population	Mortality ratio (%)	Drug effect (%)	Dead borer population	Mortality ratio (%)	Drug effect (%)
	Medicament (on average)	16	5.3	5.3	27	9	9	294	98										98
Contrast										0			0			0

The result shows: fat-soluble biological toxin of the present invention has good insecticidal effect to pine wood nematode, compare with water-soluble substances, its drug effect produces slower, not remarkable 12 hours and 24 hours drug effects, drug effect significantly also surpassed the water-soluble biological toxin in 36 hours, mortality ratio reaches 98%, shows its good application development prospect.

Embodiment:

Embodiment one:

The mycelium of Pseudohalonectria adversariaZD1 is inoculated on the test tube nutrient agar inclined-plane, and culture medium prescription is the PDA substratum, cultivates 9 days down, obtains the test tube kind for 20 ℃.

The test tube kind is inoculated in the 250ml triangular flask in (every bottled 70ml) liquid nutrient medium, culture medium prescription is 2% soybean again; 2% glucose; 0.5% starch; 0.2% yeast extract; 0.4% sodium-chlor; 0.05% potassium primary phosphate; 0.05% sal epsom; 0.2% lime carbonate, remainder is a water; Cultivate in 22 ℃ of following shaking tables, rotating speed is 250rpm, incubation time 8 days.

It is centrifugal that the fermented liquid that will contain mycelia is again put into whizzer, and rotating speed is 3600 to change, and the time is 20 minutes, after the filtration the water-soluble pine wood nematode biotoxin that kills.

Embodiment two:

Test tube kind cultural method is identical with embodiment one.

The test tube kind is inoculated on the wheat solid medium, and culture medium prescription is 1% lime carbonate; 3% soyflour; 0.5% sulfate of ammoniac; 0.5% calcium superphosphate; Wheat wheat 95%.After the wheat wheat soaked 24 hours with clear water, pick up and put into the water that boils and boil, to boil but skin is broken for suitable, pick up behind the drainage in the triangular flask with the 1000ml that packs into after auxiliary material mixes, every bottled 700ml, 120 ℃ of sterilizations are after 30 minutes, and inoculation is placed on 22 ℃ and cultivated 20 days.

Bacterial strain with solid culture digs out then, and freeze-drying with chloroform and methyl alcohol, is mixed with the solvent lixiviate in 1: 1 ratio, concentrates after the lixiviate, gets total crude extract.Crude extract is dissolved in a small amount of methyl-sulphoxide (DMSO), uses the dissolving of certain density tween 20 (Tween-20) solution again, must kill pine wood nematode biotechnological formulation emulsion.In this emulsion, the final concentration of methyl-sulphoxide is no more than 3%, and the final concentration of Tween-20 is no more than 0.3%.

Embodiment three:

Substantially with embodiment two, difference is a culture medium prescription, and its prescription is 0.5% lime carbonate; 1% soyflour; 0.5% sulfate of ammoniac; 2% calcium superphosphate; Wheat wheat 96%.

Embodiment four:

Substantially with embodiment two, difference is a culture medium prescription, and its prescription is 1% lime carbonate; 3% soyflour; 1% sulfate of ammoniac; 3% calcium superphosphate; Wheat wheat 92%.

Claims (2)

1, a kind of biotoxin of preventing and treating pine wood nematode, after pressing liquid and wheat solid culture, bacterial strain and auxiliary material extract preparation by producing, it is characterized in that by the production bacterial strain that extracts the biotoxin of preparation after the wheat solid culture be Pseudohalonectria adversaria ZD1, the ZD1 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; The process of extracting the preparation biotoxin after the wheat solid culture is:

(1) mycelium with Pseudohalonectria adversaria ZD1 is inoculated on the test tube nutrient agar inclined-plane, and culture medium prescription is the PDA substratum, cultivates 6-12 days down, obtains the test tube kind for 18-26 ℃;

(2) wheat solid culture based formulas is: 0.2-1% lime carbonate, 1-3% soyflour, 0.2-1% sulfate of ammoniac, 0.5-3% calcium superphosphate, wheat wheat 92-96%; After wheat soaked 24 hours with clear water, pick up and put into boiling water and boil, to boil but skin is broken for suitable; Pack into after picking up drainage and auxiliary material mixing in the triangular flask of 1000ml, every bottled 700ml, 120 ℃ of sterilizations inserted the test tube kind after 30 minutes, cultivated 15-20 days in 22-26 ℃, up to covering with Pseudohalonectriaadversaria ZD1 mycelia;

(3) wheat that will cover with Pseudohalonectria adversaria ZD mycelia digs out, and freeze-drying with chloroform and methyl alcohol, is mixed with the solvent lixiviate in 1: 1 ratio, concentrates after the lixiviate, gets total crude extract; Crude extract is dissolved in methyl-sulphoxide,, gets the fat-soluble pine wood nematode biotoxin emulsion that kills again with the dissolving of tween 20 solution; In this emulsion, the final concentration of methyl-sulphoxide is no more than 3%, and the final concentration of tween 20 is no more than 0.3%.

2, a kind of preparation method who prevents and treats the biotoxin of pine wood nematode, by liquid fermenting and solid fermentation preparation, solid fermentation preparation method is characterised in that:

(1) wheat solid culture based formulas is: 0.2-1% lime carbonate, 1-3% soyflour, 0.2-1% sulfate of ammoniac, 0.5-3% calcium superphosphate, wheat wheat 92-96%;

(2) to prepare the method for biological nematicide toxin be that the wheat that covers with Pseudohalonectriaadversaria ZD1 mycelia is dug out freeze-drying to the wheat solid fermentation; With chloroform and methyl alcohol, be mixed with the solvent lixiviate in 1: 1 ratio, concentrate after the lixiviate, get total crude extract, crude extract is dissolved in methyl-sulphoxide, again with the dissolving of tween 20 solution, get the fat-soluble pine wood nematode biotechnological formulation emulsion that kills; In this emulsion, the final concentration of methyl-sulphoxide is no more than 3%, and the final concentration of tween 20 is no more than 0.3%.