CN1311750C

CN1311750C - Active-fermented broth and use thereof

Info

Publication number: CN1311750C
Application number: CNB2006100106815A
Authority: CN
Inventors: 李国红; 张克勤; 周薇
Original assignee: Yunnan University YNU
Current assignee: Yunnan University YNU
Priority date: 2006-02-14
Filing date: 2006-02-14
Publication date: 2007-04-25
Anticipated expiration: 2026-02-14
Also published as: CN1817139A

Abstract

一种活性发酵液及其应用，属生物农药技术领域。由如下步骤得到：a.生产菌株为枯草芽孢杆菌(Bacillus subtilis)C2，该菌株已于2005年2月28日保存于中国微生物菌种保藏委员会普通微生物中心，保藏号：CGMCC No.1318；b.将枯草芽孢杆菌(B.subtilis)保存在固体牛肉膏蛋白胨培养基或葡萄糖牛肉膏蛋白胨培养基上，使用时在液体培养基上活化36小时；c.发酵时接种100μl活化菌种于装有80ml-120ml液体牛肉膏蛋白胨培养基或葡萄糖牛肉膏蛋白胨培养基的250ml三角瓶中，在温度为25℃-37℃、静置或转速120-180rpm的条件下培养48-72小时，即可得到培养液；d.将上述培养液在转速8500rpm下离心10分钟后，取上清放入4℃的冰箱中就得到活性发酵液。该活性发酵液对松材线虫具有毒杀作用，可作为制备毒杀松材线虫制剂的应用。An active fermented liquid and its application belong to the technical field of biological pesticides. Obtained by the following steps: a. The production strain is Bacillus subtilis (Bacillus subtilis) C2, which has been preserved in the General Microorganism Center of China Committee for the Collection of Microbial Cultures on February 28, 2005, with a preservation number: CGMCC No.1318; b .Preserve Bacillus subtilis (B.subtilis) on solid beef extract peptone medium or glucose beef extract peptone medium, and activate it on liquid medium for 36 hours during use; c. 80ml-120ml of liquid beef extract peptone medium or glucose beef extract peptone medium in a 250ml triangle flask, cultured for 48-72 hours at a temperature of 25°C-37°C, standing still or at a speed of 120-180rpm to obtain culture solution; d. after centrifuging the above culture solution at 8500 rpm for 10 minutes, take the supernatant and put it in a refrigerator at 4° C. to obtain an active fermentation solution. The active fermentation liquid has poisonous effect on pine wood nematode, and can be used as an application for preparing poisonous and killing pine wood nematode preparations.

Description

A kind of active-fermented broth and application thereof

Technical field:

The present invention relates to a kind of zymotic fluid and application thereof, belong to biological pesticide technical field with eelworm-killing activity.

Background technology:

Pine wood nematode (Bursaphelenchus xylophilus) belongs to Nematoda (Nematoda), Tylenchida (Tylenchida), Aphelenchidae (Aphelenchoididae), Bursaphelenchus (Bursaphelenchus).Pine wood nematode (B.xylophilus) is the pernicious eqpidemic disease of world pine forest, causes pine forest withered in flakes, is called as " the extinction person " of pine tree, " smokeless forest fire ".This disease period of disease weak point, dead fast, the easy propagation of disease tree, difficult control cause great ecocatastrophe.Pine nematode generally takes place in Japan, the U.S., in a plurality of countries such as Korea S, Canada and Mexico distribution is arranged also.China finds in Nanjing first in nineteen eighty-two, at present in Jiangsu Province, all there is distribution in area such as Guangdong Province, Zhejiang Province, Anhui Province, Yunnan Province and Shandong, onset area 7.3hm ², 18.2 hundred million yuan of direct economic losses, the indirect forest ecological that causes are lost about 21,600,000,000 yuan.

The method of preventing and treating of existing pine nematode comprises chemical control, breeding for disease resistance, control medium insect longicorn and the processing of the wood of dying of illness etc., but effect is all not obvious or its limitation arranged.Traditional breeding method is because of the oversize and very difficult operation of time-consuming, the medicament of medium insect longicorn sprayed easily caused woodland to pollute, and chemical agent is progressively disabled to pollution that environment produces because of it.Therefore, seeking the natural medicament of preventing and treating pine nematode from occurring in nature becomes the pine wood nematode control crucial.Microorganism is one of main source that produces bioactive natural product, and seeking to have from the metabolite of microorganism has the natural products of cytotoxicity to become one of focus of research to pine wood nematode.

Summary of the invention:

The objective of the invention is from bacterium bacillus subtilis (Bacillus subtilis), to prepare zymotic fluid, be used to develop the biologic product that high-efficiency low-toxicity kills pine wood nematode (B.xylophilus) with strong inhibition pine wood nematode (B.xylophilus) effect.

The present invention is achieved in that

1, preparation active-fermented broth

This active-fermented broth obtains through following steps:

A. producing bacterial strain was bacillus subtilis (Bacillus subtilis) C2, and this bacterial strain has been stored in China Microbial Culture Preservation Commission common micro-organisms center, preserving number: CGMCC No.1318 on February 28th, 2005.

B. bacillus subtilis (Bacillus subtilis) C2 adopts beef-protein medium and glucose beef-protein medium to preserve and cultivate, and culture medium prescription is:

Beef extract-peptone test tube medium is: beef extract 5.0g, peptone 10.0g, sodium chloride 5.0g, agar 18g, water 1000ml, pH7.5;

The beef extract-peptone liquid fermentation medium is: beef extract 5.0g, peptone 10.0g, sodium chloride 5.0g, water 1000ml, pH7.5;

Glucose beef extract-peptone test tube medium is: glucose 10.0g, beef extract 5.0g, peptone 10.0g, sodium chloride 5.0g, agar 18g, water 1000ml, pH7.5;

Glucose beef extract-peptone liquid fermentation medium is: glucose 10.0g, beef extract 5.0g, peptone 10.0g, sodium chloride 5.0g, water 1000ml, pH7.5;

The C2 bacterial strain is kept on beef-protein medium or the glucose beef-protein medium, activates 36 hours on elder generation's liquid medium within during use.

C. the C2 bacterial classification 100 μ l after will activating are inoculated in the 250ml triangular flask that 80ml-120ml liquid beef-protein medium or glucose beef-protein medium are housed, in temperature is 25 ℃-37 ℃, leave standstill or rotating speed is to cultivate 48-72 hour under the condition of 120-180rpm, can obtain culture fluid.

D. with above-mentioned culture fluid after under the rotating speed 8500rpm centrifugal 10 minutes, get supernatant and put into 4 ℃ refrigerator and just obtain active-fermented broth.

2, the pine wood nematode (B.xylophilus) of preparation test usefulness

(Botrytis cinerea) is inoculated into potato culture (potato: 200.0g with the fungi Botrytis cinerea, glucose: 20g, agar 18g, water: 1000ml) on the flat board, 25 ℃ are cultured to and cover with flat board, insert a medium that has pine wood nematode, be cultured to mycelia and disappear under 25 ℃, nematode covers with that to put into 4 ℃ of refrigerators behind the flat board standby.The medium that will be loaded with nematode during use is chosen, and after with sterile water nematode being washed out, centrifugal concentrate is prepared into concentration and is 10000/every milliliter nematode suspension.

3 test methods:

The soaked with liquid method: getting the suspension that 2ml specimen solution, 20 μ l contains 200 nematodes is in the culture dish of 6cm in diameter, behind the mixing culture dish is positioned over gently in 25 ℃ the incubator.Under anatomical lens, select 10 visuals field at random, observed respectively, count disease borer population (motionless as standard, be aided with mechanical stimulus, find fault as needle point etc.) and bus borer population with nematode in fixed 12 hours, 24 hours, 36 hours and 48 hours.Each specimen is established three repetitions, and serves as that contrast is proofreaied and correct with the medium of inoculating strain not, obtains the lethality rate of specimen to nematode, just evaluates the virulence of sample to nematode with lethality rate.

It is simple that active-fermented broth of the present invention has the preparation method, high-efficiency low-toxicity, and comprehensive proterties is good, is easy to advantages such as suitability for industrialized production, can be used as preparation biopesticide nematode killing agent and uses.

Embodiment:

Be described in further detail the present invention with embodiment below, but content of the present invention is not limited thereto.

Embodiment one:

Bacillus subtilis (B.subtilis) C2 100 μ l after the activation are inoculated in (the bottled 80ml medium of 250ml triangle) in the liquid beef-protein medium,, cultivated 72 hours under the 180rpm condition in 25 ℃.Culture fluid 8500rpm after centrifugal 10 minutes, is got supernatant and does active the use as specimen.The results are shown in Table 1.

The zymotic fluid of table 1 C2 bacterial strain on beef-protein medium is to the activity of pine wood nematode

	12 hours time	24 hours time	36 hours time	48 hours time
	12 hours time	24 hours time	36 hours time	48 hours time	Lethality rate (%)	42.5	78.6	92.5	94.3

The result shows: zymotic fluid of the present invention has good toxic action to pine wood nematode (B.xylophilus), its fermenation raw liquid has strong inhibitory action to nematode, lethality rate to pine wood nematode (B.xylophilus) reached 78.6% in 24 hours, reached more than 92.5% in 36 hours, reached 94.3% in 48 hours.

Embodiment two:

Substantially with embodiment one, when difference is fermentation for leaving standstill cultivation; The bottled 100ml medium of 250ml triangle, 37 ℃ of fermented supernatant fluids of cultivating 60 hours gained are as specimen.Activity the results are shown in Table 2.

Table 2 C2 bacterial strain leaves standstill the activity of the zymotic fluid of cultivation to pine wood nematode on beef-protein medium

	12 hours time	24 hours time	36 hours time	48 hours time
	12 hours time	24 hours time	36 hours time	48 hours time	Lethality rate (%)	20.4	47.8	67.3	78.2

The result shows: zymotic fluid of the present invention has certain toxic action to pine wood nematode (B.xylophilus), its fermenation raw liquid has stronger inhibitory action to nematode, 36 hours lethality rates to pine wood nematode (B.xylophilus) reach 67.3%, 48 hour and reach 78.2%.

The test of this example is basic identical with the method that precedent adopts, and mainly is the training method difference.This example mainly is to leave standstill cultivation, has produced different results after cultivating 60 hours under 37 ℃.The zymotic fluid that the test of this example obtains significantly is lower than precedent to the activity of pine wood nematode, shows this active-fermented broth when large-scale production, must adopt fermentation tank culture.

Embodiment three:

Substantially with embodiment one, difference is that used medium is the liquid glucose beef-protein medium, training

Foster temperature is 30 ℃, and 120rpm cultivated 48 hours, the bottled 120ml medium of every 250ml triangle.Active result

See Table 3.

The zymotic fluid of table 3 C2 bacterial strain on the glucose beef-protein medium is to the activity of pine wood nematode

	12 hours time	24 hours time	36 hours time	48 hours time
	12 hours time	24 hours time	36 hours time	48 hours time	Lethality rate (%)	32.6	55.4	72.4	80.5

The result shows: zymotic fluid of the present invention has good toxic action to pine wood nematode (B.xylophilus), its fermenation raw liquid has strong inhibitory action to nematode, 36 hours lethality rates to pine wood nematode (B.xylophilus) reach 72.4%, 48 hour and reach 80.5%.This result compares with the zymotic fluid of C2 bacterial strain on beef-protein medium, to the activity of pine wood nematode a little less than, but also have the effect of tangible poisoning nematode, illustrate that this bacterium easily cultivates generation to the activated material of nematode.Active-fermented broth of the present invention can be used as preparation biopesticide nematode killing agent and uses.

Claims

1, a kind of active-fermented broth is characterized in that this active-fermented broth obtains through following steps:

A. producing bacterial strain was bacillus subtilis (Bacillus subtilis) C2, and this bacterial strain has been stored in China Microbial Culture Preservation Commission common micro-organisms center, preserving number: CGMCC No.1318 on February 28th, 2005;

Beef extract-peptone test tube medium is: beef extract 5.0g, peptone 10.0g, sodium chloride 5.0g, agar 18g, water 1000ml, pH7.5; Liquid fermentation medium is: beef extract 5.0g, peptone 10.0g, sodium chloride 5.0g, water 1000ml, pH7.5;

Glucose beef extract-peptone test tube medium is: glucose 10.0g, beef extract 5.0g, peptone 10.0g, sodium chloride 5.0g, agar 18g, water 1000ml, pH7.5; Liquid fermentation medium is: glucose 10.0g, beef extract 5.0g, peptone 10.0g, sodium chloride 5.0g, water 1000ml, pH7.5;

The C2 bacterial strain is kept on beef-protein medium or the glucose beef-protein medium, activates 36 hours on elder generation's liquid medium within during use;

C. the C2 bacterial classification 100 μ l after will activating are inoculated in the 250ml triangular flask that 80ml-120ml liquid beef-protein medium or glucose beef-protein medium are housed, in temperature is 25 ℃-37 ℃, leave standstill or rotating speed is to cultivate 48-72 hour under the condition of 120-180rpm, can obtain culture fluid;

D. with above-mentioned culture fluid after under the rotating speed 8500rpm centrifugal 10 minutes, get supernatant and promptly obtain active-fermented broth.

2, the application of the described active-fermented broth of claim 1 is characterized in that the application of this active-fermented broth as preparation pine wood nematode poisoning preparation.