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Culture of nematoda without bacteria

A technology for pine wood nematodes and a cultivation method, which is applied in the directions of botanical equipment and methods, fungal products, lichen products, etc., can solve the problems of difficult distribution, expensive materials, and declining nematode populations.

Inactive Publication Date: 2005-07-27
赵昕
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The above-mentioned cultivation method has achieved good results relative to the previous methods, but all have the following disadvantages: 1. The materials used in the formula are more expensive and difficult to complete. 2. The aseptic pine xylophilus cultivated The individual size and reproduction rate are much lower than those of wild pine wood nematodes using corresponding fungi or black pine callus. 3. During the culture, the population of nematodes began to decline rapidly after reaching the maximum
[0016] The weak point of this method is: 1, from the volume of the pine xylophilus that obtains from culture, quantity and nematode oviposition etc. growth, breeding index aspect, compared with the pine wood nematode in the pine tree of natural susceptible disease, still can't make people feel uncomfortable. Satisfaction
2. This method is still not convenient for large-scale industrial production

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0235] 1. Cultivation and processing of Pseudomonas fluorescens (Pseudomonas fluorescens) GcM5-1A bacterial strain: (1) Preparation of NA slant medium: 32g nutrient agar powder (NA) was dissolved in 1000ml distilled water, boiled, and distributed in test tubes Middle, then at 121°C, 1.05kg / cm 2 Sterilize in a sterilizing pot for 15 minutes, invert the slope, and set aside. (2) Strain activation: Pseudomonas fluorescens (Pseudomonas fluorescens) GcM5-1A strain stored in a refrigerator at 4°C was placed on the slant medium under aseptic conditions, and cultured in a constant temperature incubator at 27°C for 2 days , and then stored in a 4°C refrigerator for later use. (3) Bacterial cultivation: Take 1 loop of the Pseudomonas fluorescens GcM5-1A strain stored in a refrigerator at 4°C with an inoculation loop, insert it into 50ml of NA slant medium, and place it at 30 Cultivate on a constant temperature shaker (128r / min) for 48h. (4) Preparation of dead Pseudomonas fluorescens...

Embodiment 2

[0261] 1. Cultivation and processing of Pseudomonas fluorescens (Pseudomonas fluorescens) GcM5-1A bacterial strain: (1) Preparation of NB liquid medium: take 96g nutrient broth (NB) and dissolve in 3000ml distilled water, boil, and pack in Erlenmeyer flask; sterilize in a 1.05kg / cm2 autoclave at 121°C for 15 minutes, and set aside. (2) Activation of bacteria. Method is with embodiment 1. (3) Cultivation of bacteria: under sterile conditions, insert the activated Pseudomonas fluorescens GcM5-1A strain into NB liquid medium, insert 1ml of activated strain into every 50ml of NB liquid medium, and place at 30°C Cultured on a constant temperature shaker (128r / min) for 4 days. (4) Obtaining of dead bacteria: put the bacteria obtained in step (3) under aseptic conditions at -40°C for 12 hours to freeze-dry at a low temperature, take it out and thaw at room temperature, and freeze-dry to obtain sterile dried dead bacteria. spare

[0262] 2. Cultivate black pine callus

[0263] C...

Embodiment 3

[0268] 1. The cultivation and treatment of Pseudomonas fluorescens GcM5-1A strain, the same method as in Example 1

[0269] 2, cultivate black pine callus, method is with embodiment 1

[0270] 3. The same culture of pine wood nematode: (1) the preparation of aseptic pine wood nematode ovum, method is the same as embodiment 1. (2) Preparation of culture medium: under sterile conditions, weigh 200 mg of the above live callus and 0.1 mg of the above dead bacteria, add 2 ml of sterile water, or prepare the culture medium according to the above ratio. (3) Cultivation of pine wood nematode: add the prepared sterile pine wood nematode eggs to the above culture solution with a sterile pipette. Put the above-mentioned culture solution at 23° C. and culture in dark for 10 days to obtain the desired sterile pine xylophilus.

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Abstract

An aseptic culture method for pine nematode includes such steps as culturing Pseudomonas fluorescens GcM5-1A (CCTCC No.M204065), deactivating, culturing the calli of black pine, deactivating, preparing the aseptic ova of pine nematode, preparing the culture liquid from deactivated cells and calli and aseptic water, adding said aseptic ova, and culturing at 15-40 deg.C for 4-20 days.

Description

(1) Technical field: [0001] The invention relates to a method for cultivating sterile pine xylophilus. (two) background technology: [0002] Pine wood nematode (Bursaphelenchus xylophilus (Steiner & Buhrer) Nickle) is a devastating disease on pine trees, mainly spread by Monochamus alternatus Hope, which can damage black pine (Pinus thunbergii), red pine (P.densiflora ), slash pine (Pelliottii), masson pine (P.massoniana) and nearly 40 species of pine trees. Pine wood nematode disease has caused a large number of pine plant deaths, and it has become a disaster. It has caused a serious threat to forestry production in our country. [0003] Although there were records about pine xylophilus disease as early as the beginning of the 20th century, it was not until the 1970s that more in-depth research on the disease began and some results were obtained. However, the mechanism of pine wilt caused by B. xylophilus has not yet been accepted by most people, especially the role of ba...

Claims

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Application Information

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IPC IPC(8): A01H15/00
Inventor 赵博光
Owner 赵昕
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