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Locust microsporidia, suspension culture method and applications thereof

A locust microsporidia, locust technology, applied in the field of agricultural biology, can solve heavy and other problems

Active Publication Date: 2016-03-09
NANJING HUADEFU BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] Microsporidia locusts are used as biological insecticides to control locusts. First, their production problems must be solved. At present, the only reliable method is live reproduction. However, the propagation of Microsporidia locusts with locust gnats faces many challenges, such as heavy labor intensity, Large-scale breeding of locust gnats, crushing, and prevention of other diseases of locust gnats are required
However, the in vitro culture method of directly using suitable culture medium or suitable insect cells to culture Microsporidia locust is still in the stage of exploration.

Method used

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  • Locust microsporidia, suspension culture method and applications thereof
  • Locust microsporidia, suspension culture method and applications thereof
  • Locust microsporidia, suspension culture method and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] Example 1 Locust Microsporidia Isolation and Preliminary Identification

[0081] 1 Materials and methods

[0082] 1.1 Isolation and purification of microsporidia

[0083] Microsporidia migratiosa in the wild was isolated from Burqin County, Altay Prefecture, Xinjiang Uygur Autonomous Region. Three-day-old locust gnats (n=50) were raised in the laboratory at a room temperature of 30±1°C, fed with corn leaves mixed with a small amount of wheat bran. Homogenate the isolated microsporidia-infected tissue to make spore suspension, count the microsporidia in suspension with a hemocytometer, and feed 10 5 or 10 7 The spores are applied on the corn leaves and fed to 4-5 instar locust gnats after drying. After 24 hours of feeding, replace fresh corn leaves. Diseases and insects were kept in an inoculation room equipped with 4-watt fluorescent lamps, day and night light, and a temperature of 30±1°C. After the locust gnats have grown to old age or adult stage, collect the cor...

Embodiment 2

[0133] Embodiment 2. In vivo reproduction of isolated locust microsporidia

[0134] 1. Materials and Methods

[0135] 1.1 Materials: Nosemasp-XJ-10, multiplied by migratory locust. The locusts were collected from locust eggs in the Altay area of ​​Xinjiang Uygur Autonomous Region, and were reared indoors as test insects.

[0136] 1.2 Test method

[0137] 1.2.1 Massive proliferation of locust micropaedia

[0138] 1) The relationship between host lethal time and pathogen developmental stage

[0139] Dilute the microsporidia spore solution to 1×10 8 Spores / mL, take 10 microliters and drop it on the leaves of wheat seedlings with a diameter of 10mm. After drying, feed them to the 4th instar locust gnats subcultured in this room, one piece per head, about 10 in total. After 24 hours, the locust gnats that had eaten up all the leaves of the wheat seedlings were used as test insects, and they were raised in a large plastic tube at 30 ± 1, humidity 70%, light day and night, and f...

Embodiment 3

[0160] Example 3. Preparation of Microsporidia locust in vitro culture medium, extracellular matrix and adaptation in vitro of No. locust locust

[0161] 3.1 Self-made locust microsporidia medium MX, volume 1L, add the following substances into pure water.

[0162] Inorganic salt mixture: NaH 2 PO 4 2H 2 O507mg / L; NaHCO 3 300mg / L; KCl1720mg / L; CaCl 2 2H 2 O750mg / L; CuCl 2 ·H 2 O0.1mg / L; CoCl 2 ·6H 2 O0.03mg / L; FeSO 4 ·7H 2 O0.28mg / L; MgCl 2 4H 2 O1140mg / L; MgSO 4 ·7H 2 O3269mg / L; MnCl 2 4H 2 O0.01mg / L; NaCl1425mg / L; NaH 2 PO 4 4H 2 O580mg / L; (NH 4 ) 6 (Mo 7 o 24 )·4H2O0.02mg / L and ZnCl20.02mg / L.

[0163] Sugar mixture: D-glucose 2917mg / L; fructose 20.9mg / L; sucrose 11865mg / L; malic acid 306mg / L; alpha-ketoglutaric acid 169mg / L; succinic acid 27.4mg / L; fumaric acid 25.2mg / L; Maltose 500mg / L.

[0164] Amino acid mixture: L-alpha-alanine 131.5mg / L; beta-alanine 234mg / L; L-arginine hydrochloride 692mg / L; L-asparagine 797mg / L; L-aspartic acid 797mg / L; L-...

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Abstract

The invention discloses a locust microsporidia, a suspension culture method and applications thereof. The locust microsporidia strain is separated from Locusta migratoria L. and is prepared through 10 generations in locust body and 10 generation in-vitro culture. The preservation number is CGMCC No.11170. Moreover, the invention also provides a culture medium for in-vitro culture of locust microsporidia and a suspension culture method. In the culture medium, insect-origin, water-soluble, and de-acetylated chitin is taken as the extracellular matrix, polyvinylpyrrolidone is taken as the viscosity enhancer, and at the same time, a protein extract is added into the culture medium. The disclosed culture medium and bioreactor are used to carry out micro-carrier suspension multiplication culture, and after culture, the number of microsporidia is increased by 60 times. The invention provides a method capable of culturing a large amount of locust microsporidia and provides technical support for the massive application of locust microsporidia as a biological insecticide.

Description

technical field [0001] The invention relates to a microsporidia locust strain, and also relates to a culture medium and a cultivation method for cultivating the microsporidia locust in vitro. The invention belongs to the field of agricultural biotechnology. Background technique [0002] Locusts are pests that affect agricultural and animal husbandry production. In pastoral areas of our country, they seriously endanger grasslands. In order to kill locusts, chemical pesticides have been used in large quantities for many years. Natural enemies of locusts, causing deterioration of community function. Therefore, damage to the natural environment can be greatly reduced by using entomopathogenic microorganisms as biopesticides. [0003] Microsporidia are one of the pathogenic microorganisms of locusts, and microsporidiosis is caused by microsporidia, which reproduce through schizonts and spores in infected host cells. Since microsporidia play an important role in regulating insec...

Claims

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Application Information

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IPC IPC(8): C12N1/10A01N63/00A01P7/04C12R1/90
Inventor 陈弘飞李锦荣其他发明人请求不公开姓名
Owner NANJING HUADEFU BIOTECH CO LTD
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