CN1439270A

CN1439270A - Preparing method for lilacinin against nematoda eggs

Info

Publication number: CN1439270A
Application number: CN 02103056
Authority: CN
Inventors: 汪洪
Original assignee: Individual
Current assignee: Individual
Priority date: 2002-02-20
Filing date: 2002-02-20
Publication date: 2003-09-03

Abstract

A pseudo-lilacinin as insecticide with high pathogenic rate to nemic ova is prepared from the conventional culture medium through adding the chitin as the particular inductive substrate to it and separating chitinase, which can degradate the shell of nemic ova.

Description

Preparation method to the Paecilomyces lilacinus microbial inoculum of the high pathogenicity rate of line eggs

The present invention relates to a kind of nematicide biopesticide and preparation method thereof.

Nematode is second largest class crop pest except that insect, and it is widely distributed, can be parasitic and endanger multiple kinds of crops, and crop yield is reduced, quality decline.According to estimates, therefore national agriculture forestry planting industry annual and loss that cause is about 23,300,000,000 yuan.

The control nematode removes breeding resistant variety on the agricultural, and outside crop rotation and the physical control, the most effective means are chemical control, and chloropicrin, furans pellet, go out kairine and Nemacur etc. are common drug.Yet these medicines also and then to agricultural product cause severe contamination also for simultaneously agricultural land soil.

Given this, people begin to look for new method in addition from ecological view.The P.Jatava in Peru international potato research center in 1979 is separated to a kind of through being accredited as the nematodosis fungal pathogens of Paecilomyces lilacinus (Paecilomyces lilacinus) first from the sick ovum of Meloidogyne incognita, many subsequently national science workers have made big quantity research to this, and determine that it also is root-knot nematode (Meloidogyme SPP), cyst roundworm (Heterodera SPP), the puncture disease fungus of nematode multiple nematodes such as (Tylenchulus SPP) partly.And Paecilomyces lilacinus is the bacterium of commonly seeing in the soil, and to crops and person poultry harmless, therefore the biological control microorganism that is used as the kill harmful nematode with it has very big potentiality.

The eighties in last century, introduce to the market by first nematicide biopesticide " Biocon " in the world that Philippine produces, prevent and treat the effect of potato Meloidogyne incognita and hairworm and furans is red or the kairine that goes out is similar with it, therefore potato yield also increases by 20.3～36.5%.China also had two patents (publication number 1149393,1149394) to disclose to utilize brewery or sugar refinery waste liquid to produce wireworm-killing biologic agricultural chemicals---the method for Paecilomyces lilacinus microbial inoculum in 1997.

Yet, the above-mentioned Paecilomyces lilacinus bacterial strain that obtains that from line eggs, separates, use usually that the prescription composition is bred in a large number and the microbial inoculum that obtains, its nematicide effect is unsettled, its infection parasitic rate to line eggs can increase with the algebraically of breeding and reduce, and the nematicide effect also just reduces thereupon.This is because Paecilomyces lilacinus parasitizes line eggs and at first needs mycelia to pass the nematode chorion, and thick periostracum needs the chitinase degraded to form the hole mycelia can to stretch and enter in the ovum in the nematode chorion.Paecilomyces lilacinus can produce the chitinase justacrine to the extracellular inducing under the chitinous condition of culture of substrate, induces substrate or induces the substrate deficiency if lack in its living environment, then seldom secretes this kind of enzyme.Chitinase all is an induced enzyme to a lot of fungies.

Purpose of the present invention aims to provide a kind of preparation method that can stablize maintenance to the Paecilomyces lilacinus microbial inoculum of the high infection rate of line eggs, basis of the present invention is for producing this kind fungi wireworm-killing biologic agricultural chemicals, must both utilize this kind fungi that from sick ovum, separates, in the cultivation of the bacterial strain that obtains, all must allocate the inducing substance chitin in whole one, two, three generations (be the seed inclined-plane, shake bottle and the fermentation) medium.Paecilomyces lilacinus owing to be in the induced environment all the time, just can keep the synthetic and secretion of chitinase under this condition, the microbial inoculum that obtains of producing also just remains its infection rate high to line eggs, and the result of use of biopesticide can obtain to guarantee.

Technical scheme of the present invention is as follows:

Step 1: by separating the Paecilomyces lilacinus bacterial strain in the root knot nematode disease ovum.Get the root knot that the cultivation tomato has infected nematode, therefrom collect anosis nematode pieces of an egg,, use rinsed with sterile water then with 10% clorox sterilization 2 minutes.Under sterile working, the anosis pieces of an egg of surface sterilizing are moved on the cultured Paecilomyces lilacinus culture, cultivated 10 days down at 25 ± 2 ℃.Take out and infected the ovum of fungi, and use the clorox surface sterilization, with behind the rinsed with sterile water several, move into oat agar medium (OMA) and go up cultivation again, isolate the bacterial strain of parasitic capacity.

Step 2: one-level is cultivated: will contain 2～5% chitin fine powders with 3% for the conventional autoclaving of good OMA medium packing and put into the inclined-plane, and isolated Paecilomyces lilacinus bacterial classification of step accesss in, cultivation is about 7 days under 26 ± 1 ℃.

Step 3: secondary is cultivated: medium contains 1.0 liters of potato extracts, glucose 20.0 grams, and chitin fine powder 10～40 grams (is good with 20 grams) are transferred PH to 5.5～6.5, and inoculation one-level culture behind conventional autoclaving was 26 ± 1 ℃ of vibrations 5～8 days.

Step 4: three grades of cultivations: medium is the oat meal or the slag 5% of cracking rice; Cavings about 85% (its amount is adjusted with chitin powder amount); It is about 10% to contain the chitin powder-material, and its amount of allocating into is decided on chitin content, makes part by weight＞1% of chitin in whole medium, best 〉=3%.The chitin powder-material can be that the mycelium of shrimp and crab shells powder, maggot shell powder, dried silkworm chrysalis meal, fungi such as aspergillus niger (citric acid fermentation slag), edible mushroom take waste material, the Rhizopus oryzae (lactic acid fermentation slag) after the fruit body, the thalline of the mould fungies such as (Absidia) of colter all can be used.

Above powder adds suitable quantity of water to be mixed thoroughly, becomes to pinch not drip, and loosing of touching is degree.Behind moist heat sterilization, inoculation secondary culture was cultivated about 10 days at 28 ± 1 ℃.The culture vacuum is drained, pulverize.

With the Paecilomyces lilacinus microbial inoculum that this method is produced, every gram contains spore 〉=2 * 10 ⁸Individual.This microbial inoculum is inoculated on the oat agar medium (OMA), when treating that bacteria growing is better, move into the nematode pieces of an egg through the no germ infection of surface sterilization, co-incubation is after 8 days, check to find that the nematode pieces of an egg 100% have been infected by Paecilomyces lilacinus, have all found mycelial growth in the ovum.Without chitin and line eggs repeatedly the Paecilomyces lilacinus of directional induction (control strain of test) with this understanding to infection rate＜30% of line eggs.

Paecilomyces lilacinus has stable high infection rate to line eggs after directional induction repeatedly.Therefore can be used as a kind of novel biological nematocide, be used for grain (wheat, corn), cotton, oil (peanut, soybean), sugar [yielding (beet, sugarcane), tea, potato (sweet potato, potato), (banana really, citrus, pineapple, peach, kiwi fruit, red bayberry, Momordica grosvenori, persimmon, fig, grape, strawberry), tobacco, melon (cucumber, muskmelon, watermelon, balsam pear), dish (tomato, capsicum, eggplant, carrot, Kidney bean) control of crop nematodiasis such as, the chemical nematicide of contaminated environment and agricultural product can be substituted fully, and crop yield can be improved.

Embodiment 1:

To in line eggs, isolate and the Paecilomyces lilacinus bacterial classification inoculation of purifying to through the autoclaved potato that contains the accent PH6.0 of shrimp shell meal 10%---in the dextrose culture-medium, vibrated 6 to 7 days down at 26 ± 1 ℃; Insert at last through autoclaved solid medium (solid medium contains about 100 grams of shrimp shell meal, cavings 850 gram, slag 50 grams of cracking rice, and adds water and stirs evenly agglomeratingly to what pinch, and that touches promptly looses), cultivated 10 days down at 28 ± 1 ℃.Culture after vacuum drying, pulverize microbial inoculum.Microscopy is counted every gram and is contained spore 〉=2 * 10 ⁸Individual.The all alternative shrimp and crab shells powder of fly maggot powder, dried silkworm chrysalis meal.

Embodiment 2:

All programs are all with embodiment 1, but replace shrimp shell meal with the citric acid fermentation slag, and corresponding minimizing cavings consumption.The citric acid fermentation pulp water is washed till more than the PH5.5, drying.

In the slant medium OMA that one-level is cultivated, add 15% citric acid fermentation slag;

In the shake-flask culture base PDA that secondary is cultivated, add 10% citric acid fermentation slag;

In the per kilogram solid medium of three grades of cultivations, contain citric acid fermentation slag 150 grams, cavings 800 grams, slag 50 grams of cracking rice;

As not having the citric acid fermentation slag, also available (Rhizopus oryzae) lactic acid fermentation slag replaces.

Embodiment 3:

With shrimp shell meal solid-state cultivation and in the solid medium of three grades of cultivations of the mould culture replacement of the colter of sterilizing-drying.The solid medium component is: the dry mould culture 95% of colter, oat meal or the slag 5% of cracking rice.All the other are all with embodiment 1 or embodiment 2.The cultivation waste material that edible mushroom is adopted after the fruit body also can replace the mould culture of colter.

Claims

A kind of preparation method of the Paecilomyces lilacinus microbial inoculum to the high pathogenicity rate of line eggs is characterized in that:

1, enters the Paecilomyces lilacinus bacterial strain of production link, all direct separation screening from the nematodosis ovum;

2, produce used one, two, three medium, add the chitin about 3%, 2% and 3% respectively;

3, the chitin that is adopted can shrimp, crab shell, maggot shell, silkworm chrysalis shell and some fungies (as aspergillus niger, Rhizopus oryzae, colter is mould and edible mushroom cultivate waste material etc.) thalline in select for use.