CN1439270A - Preparing method for lilacinin against nematoda eggs - Google Patents

Abstract

The invention discloses a preparation method of a Paecilomyces lilacinus fungicide with a high pathogenicity rate to nematode eggs, which can be used to produce insecticides. In the present invention, Paecilomyces lilacinus isolated from nematode eggs is cultivated by adding specific inducing substrate chitin to common culture medium of slant strains, shake flasks and solid substrates, so that it can secrete and degrade nematode eggs in large quantities Shell chitinase, thereby ensuring its efficient penetration of nematode eggs, that is, the ability to infect parasites. The bacterial agent prepared by this method does not lose the ability to infect nematode eggs due to subculture in common medium. It can be used as a nematicide pesticide for the control of nematodes in many crops.

Description

Preparation method of Paecilomyces lilacinus fungicide with high pathogenicity rate to nematode eggs

The invention relates to a biological pesticide for killing nematodes and a preparation method thereof.

Nematodes are the second largest category of crop pests except insects. They are widely distributed and can parasitize and harm a variety of crops, reducing crop yield and quality. It is estimated that the loss caused by the national agriculture and forestry planting industry is about 23.3 billion yuan every year.

The most effective way to control nematodes in agriculture is to breed resistant varieties, crop rotation and physical control, and chemical control is the most effective way. Chloropicrin, carbofuran, methacryl and grammephos are commonly used drugs. However, these drugs also cause serious pollution to farmland soil and thus to agricultural products.

In view of this, people began to find another new method from an ecological point of view. In 1979, P. Jatava of the International Potato Research Center in Peru isolated a nematode pathogenic fungus identified as Paecilomyces lilacinus from the diseased eggs of root-knot nematode incognita for the first time. A lot of research has been done, and it is determined that it is also the pathogenic fungus of various nematodes such as Meloidogyme SPP, Heterodera SPP, and Tylenchulus SPP. Moreover, Paecilomyces lilacinus is a common bacterium in the soil and is harmless to crops, humans and animals, so it has great potential as a biological control microorganism for killing harmful nematodes.

In the 1980s, the world's first nematode-killing biopesticide "Biocon" produced by the Philippines was introduced to the market. It was used to control potato root-knot nematode and golden nematode. Therefore, the yield increases by 20.3-36.5%. my country also has two patents (publication numbers 1149393, 1149394) in 1997 to disclose the method of utilizing brewery or sugar factory waste liquid to produce nematode biological pesticide---Paecilomyces lilacinus fungicide.

However, the above-mentioned Paecilomyces lilacinus strains isolated from the nematode ovum, the inoculum obtained by using common formula ingredients to reproduce in large quantities, its nematode killing effect is unstable, and its parasitic rate to the infection of nematode ovum will decrease. As the number of generations of reproduction increases and decreases, the nematocidal effect also decreases. This is because Paecilomyces lilacinus parasitizes nematode eggs firstly requires mycelium to pass through nematode eggshells, and the thick chitin layer in nematode eggshells needs to be degraded by chitinase to form porous hyphae before they can stretch into the egg. Paecilomyces lilacinus can produce chitinase and secrete it extracellularly under the culture condition with the induction substrate chitin. If the induction substrate is lacking or the induction substrate is insufficient in its living environment, it will rarely secrete this enzyme. kind of enzyme. Chitinase is an inducible enzyme for many fungi.

The object of the present invention aims to provide a kind of preparation method that can stably maintain the Paecilomyces lilacinus fungal agent of high infection rate to nematode eggs, the basis of the present invention is to produce this kind of fungus nematicide biopesticide, must utilize both from disease For this fungus isolated from eggs, in the cultivation of the obtained bacterial strains, all the first, second and third generations (ie, seed slant, shake flask and fermentation) medium must be mixed with the inducer chitin. Under such conditions, Paecilomyces lilacinus can maintain the synthesis and secretion of chitinase because it is always in the induction environment, and the bacterial agent produced will always maintain its high infection rate for nematode eggs. The effect of pesticide use can be guaranteed.

Technical scheme of the present invention is as follows:

Step 1: Isolating Paecilomyces lilacinus strains from root-knot nematode eggs. The nematode-infected root knots of cultivated tomatoes were collected, and disease-free nematode egg masses were collected therefrom, disinfected with 10% sodium hypochlorite for 2 minutes, and then rinsed with sterile water. Under aseptic operation, transfer the surface-sterilized disease-free egg masses to the well-cultured Paecilomyces lilacinus culture, and cultivate them at 25±2°C for 10 days. The eggs that have been infected with the fungus are taken out, sterilized with sodium hypochlorite, rinsed with sterile water for several times, then transferred to oat agar medium (OMA) for culture, and the strains with parasitic force are isolated.

Step 2: First-level cultivation: subpackage the OMA medium containing 2-5% chitin fine powder, preferably 3%, and sterilize it by conventional autoclaving and place it on an inclined plane, and insert the Paecilomyces lilacinus isolated in the previous step species, cultured at 26±1°C for about 7 days.

Step 3: Secondary culture: the medium contains 1.0 liter of potato extract, 20.0 grams of glucose, 10-40 grams of fine chitin powder (20 grams is better), adjust the pH to 5.5-6.5, and after conventional autoclaving Inoculate the primary culture and shake at 26±1°C for 5-8 days.

Step 4: tertiary cultivation: culture medium is 5% of oatmeal or broken rice dregs; about 85% of rice bran (the amount is adjusted with the amount of chitin powder); about 10% of chitin powder, and its The dosage depends on the content of chitin, so that the weight ratio of chitin in the whole medium is greater than 1%, and the best is greater than or equal to 3%. Chitin powdery material can be shrimp and crab shell powder, fly maggot shell powder, silkworm chrysalis powder, the mycelium of fungus such as Aspergillus niger (citric acid fermentation residue), the waste material after edible fungus takes fruiting bodies, Rhizopus oryzae (lactic acid fermentation residue). Fermented slag), fungi such as Absidia (Absidia) can be utilized.

Add appropriate amount of water to the above powder and mix well until it is kneaded without dripping, and it can disperse when touched. After being sterilized by moist heat, the secondary culture was inoculated and cultivated at 28±1°C for about 10 days. The culture is vacuum-dried and crushed.

The Paecilomyces lilacinus fungicide produced by this method contains spores ≥ 2×10 ⁸ per gram. Inoculate this bacterial agent on the oat agar medium (OMA) again, and when the bacteria grow well, move in the surface-sterilized nematode egg masses without pathogenic bacteria infection, and after co-cultivation for 8 days, it is found that the nematode egg masses have been 100% diluted. Paecilomyces purpura infection, hyphal growth has been found in the eggs. The infection rate of Paecilomyces lilacinus (the control strain of the test) without repeated directional induction of chitin and nematode eggs to nematode eggs is less than 30%.

Paecilomyces lilacinus has a stable high infection rate to nematode eggs after repeated targeted induction. Therefore, it can be used as a new type of biological nematicide for grain (wheat, corn), cotton, oil (peanut, soybean), sugar crops (beet, sugar cane), tea, potato (sweet potato, potato), fruit ( Banana, citrus, pineapple, peach, kiwi, bayberry, mangosteen, persimmon, fig, grape, strawberry), tobacco, melon (cucumber, melon, watermelon, bitter melon), vegetables (tomato, pepper, eggplant, carrot, kidney bean), etc. The control of crop nematode diseases can completely replace chemical nematicides that pollute the environment and agricultural products, and can increase crop yield.

Example 1:

Inoculate Paecilomyces lilacinus strains isolated and purified from nematode eggs into the potato-glucose medium adjusted to pH 6.0 containing 10% shrimp shell powder after autoclaving, at 26±1°C Shake for 6 to 7 days; finally insert the high-pressure sterilized solid medium (the solid medium contains about 100 grams of shrimp shell powder, 850 grams of rice bran, and 50 grams of broken rice dregs, add water and stir until it is kneaded into a ball, touch Dissipate immediately), cultivated at 28±1°C for 10 days. After the culture is dried in vacuum, it is pulverized to become the bacterial agent. Microscopic examination counted spores ≥ 2×10 ⁸ per gram. Fly maggot powder and silkworm chrysalis powder can replace shrimp and crab shell powder.

Example 2:

All procedures are the same as in Example 1, but replace shrimp shell powder with citric acid fermented dregs, and correspondingly reduce the amount of bran. The citric acid fermentation residue was washed with water until the pH was above 5.5, and dried.

Add 15% citric acid fermentation dregs in the slant medium OMA of primary culture;

Add 10% citric acid fermentation residue in the shake flask culture medium PDA of secondary culture;

Each kilogram of solid medium in the tertiary culture contains 150 grams of citric acid fermentation residue, 800 grams of rice bran, and 50 grams of broken rice residue;

If there is no citric acid fermentation residue, it can also be replaced by (Rizopus oryzae) lactic acid fermentation residue.

Example 3:

The shrimp shell powder in the solid medium of the tertiary culture was replaced by the culture of Absidia spp. cultured in solid state and sterilized and dried. The components of the solid medium are: 95% of dried Absidia culture, 5% of oat flour or broken rice dregs. All the other are with embodiment 1 or embodiment 2. The culture waste after the fruiting body of the edible fungus is harvested can also replace the Absidia mold culture.

Claims (1)

A preparation method of Paecilomyces lilacinus fungicide with high pathogenicity to nematode eggs, characterized in that: 1. Paecilomyces lilacinus strains entering the production process are all directly isolated and screened from nematode eggs; 2. Add 3%, 2% and 3% chitin to the primary, secondary, and tertiary culture medium used in production; 3. The chitin used can be selected from shrimp, crab shells, fly maggot shells, silkworm chrysalis shells and some fungi (such as Aspergillus niger, Rhizopus oryzae, Absidia mold and general edible fungus culture waste, etc.).