CN109496988B

CN109496988B - Method and device for large-scale culture of entomopathogenic nematodes

Info

Publication number: CN109496988B
Application number: CN201811297686.XA
Authority: CN
Inventors: 李小龙; 张凤琴; 何农跃
Original assignee: Individual
Current assignee: Individual
Priority date: 2018-11-01
Filing date: 2018-11-01
Publication date: 2021-06-29
Anticipated expiration: 2038-11-01
Also published as: CN109496988A

Abstract

The invention discloses a large-scale culture method and a culture device for entomopathogenic nematodes, which comprise the following steps: (1) preparing a culture medium nutrient solution, sterilizing the culture medium nutrient solution, inoculating activated nematode symbiotic bacteria in the sterilized culture medium nutrient solution, and inoculating entomopathogenic nematodes; (2) placing sponge into culture bag, compressing, and sterilizing; (3) placing the inoculated nutrient solution into a culture bag, and standing for culture; (4) soaking the nematodes and the culture cultured in the step S3 in clear water, separating out the sponge, standing, and taking the lower layer liquid. The invention reduces the volume, improves the sterilization efficiency and greatly improves the production efficiency by compressing the sponge and the culture bag. The invention utilizes the compressed negative pressure to fill the inoculation nutrient solution, the strains and the nematodes are distributed more uniformly, the culture time is shortened, the product quality is improved, and the large-scale culture is easier to realize.

Description

Method and device for large-scale culture of entomopathogenic nematodes

Technical Field

The invention relates to the technical field of biological culture, in particular to a method for large-scale culture of entomopathogenic nematodes.

Background

Entomopathogenic nematodes are novel biopesticides, have the dual characteristics of natural enemy insects and pathogenic microorganisms, can actively search and select hosts through chemoreceptors like natural enemy insects, carry symbiotic bacteria, and randomly release the symbiotic bacteria while infecting host cavities, so that the hosts are septized and die.

The entomopathogenic nematodes serving as biological insecticides can be widely applied to pest control in the aspects of agriculture, forestry, pasture, sanitation and the like, and obvious economic and social benefits are obtained. The culture method of the industrialized entomopathogenic nematodes has been developed from living culture to in vitro culture, and the in vitro culture is from aseptic culture to single bacterium culture. In the current state of the art, entomopathogenic nematodes are scaled up by single-strain culture on solid media in iron boxes. The culture scale is limited, the culture iron box device occupies large space, the sterilization efficiency before culture is low, the inoculation in the box is uneven, and the infection rate of inoculated bacteria is high. The produced product of entomopathogenic nematodes has poor effect, small batch culture scale and long culture time, and cannot meet the requirements of the market on the entomopathogenic nematodes. The invention provides a simple and convenient method and a simple and convenient device for large-scale culture of entomopathogenic nematodes, which can culture the entomopathogenic nematodes in a larger scale and improve the production efficiency.

Disclosure of Invention

The invention aims to solve the technical problem of providing a method for large-scale culture of entomopathogenic nematodes aiming at the defect of large-scale culture in the prior art.

The purpose of the invention is realized by the following technical scheme:

a method for large-scale culture of entomopathogenic nematodes is characterized by comprising the following preparation steps:

s1, preparing a culture medium nutrient solution, sterilizing the culture medium nutrient solution, inoculating activated nematode symbiotic bacteria to the sterilized culture medium nutrient solution, culturing for 2-3 days, and inoculating entomopathogenic nematodes;

s2, placing the sponge into a culture bag, compressing and sterilizing;

s3, filling the inoculated nutrient solution into a culture bag, and standing for culture;

s4, soaking the nematodes and the culture cultured in the step S3 in clear water, separating out sponge, standing, and taking the lower-layer liquid.

Further, the culture solution of step S1 includes: 4-8 wt% of corn flour, 3-7 wt% of bean flour, 1-3 wt% of lard oil, 4-8 wt% of flour, 8-12 wt% of insect powder and the balance of purified water. Preferably, corn flour 5 wt.%, bean flour 5 wt.%, lard 1 wt.%, flour 5 wt.%, insect flour 10 wt.%, and the balance purified water. Provides nutrient substances for the culture of the nematodes and the symbiotic bacteria.

Further, the sterilization temperature is 100-120 ℃, the sterilization time is 30-60 min, bacteria in the culture solution, the culture bag and the sponge are removed, subsequent bacterial infection of nematodes is prevented, and the yield of the produced nematodes is improved.

Furthermore, the inoculated entomopathogenic nematodes are nematodes in the infection stage, the adaptability of the nematodes in the infection stage is high, and the survival rate of the cultured entomopathogenic nematodes is high. The symbiotic bacteria can stimulate the nematode to sheath in the infection period, accelerate the development speed of the nematode, facilitate the shortening of the culture time of the nematode and the rapid propagation of the nematode. Further preferably, the density of the inoculated entomopathogenic nematodes in the nutrient solution is 8-15 tail/mL.

Further, in step S3, a certain amount of sterile air or pure oxygen is introduced into the culture bag to provide the air for culturing the nematodes.

Further, in the step S3, the standing culture temperature is 24-26 ℃, the culture time is 10-15 days, and the nematode content of 30 ten thousand in one sponge fragment can be achieved on average.

Further, the standing time of the step S4 is 60-120 min, so that the nematodes are guaranteed to be fully settled, and then the lower-layer nematode liquid is absorbed by the dry sponge fragments and stored at low temperature.

According to the method, the nematode culture device is a sealable culture bag, a self-sealing strip is arranged on one side of the culture bag, and a vacuum compression air nozzle and a closable liquid inlet are arranged on the culture bag. The vacuum compression air tap is used for vacuum compression of the culture bag, the volume of the culture bag is reduced, large-batch sterilization operation is facilitated, and the efficiency of large-scale culture is improved. The culture solution enters the culture bag through the liquid inlet, so that the air bacterial infection rate is reduced. The compressed culture bag and the sponge are sucked into the inoculated nutrient solution through negative pressure, and the inoculated nematodes are uniformly distributed.

Further, the sponge in the culture bag is the sponge fragment of diameter 3~5cm, and the sponge fragment can do benefit to the circulation of gas between the sponge, prevents that the middle part nematode living condition of bold sponge is uncomfortable, and the sponge piece is too little then the circulation of gas is not smooth between the sponge.

Compared with the prior art, the beneficial effects are:

according to the invention, the sponge and the culture bag are compressed, so that the sterilization efficiency of the device can be improved, and the production efficiency is further improved. After the culture bag and the sponge are compressed, the inoculated nutrient solution is filled into the culture bag through negative pressure, the strain distribution is more uniform, the culture time is shortened, and the quality of the obtained product is improved. The culture bag adopted by the invention is infused through the liquid inlet, so that the bacterial infection rate of the prior art in which the iron box is completely opened for inoculation is reduced. Meanwhile, the culture bag adopted by the invention has low cost and small occupied space, and the culture in the same space has the culture efficiency 10-20 times higher than that of an iron box, so that the large-scale production of entomopathogenic nematodes is easier.

Drawings

FIG. 1 is a schematic view of a culture bag.

Wherein, 1 self-sealing strip, 2 liquid inlet, 3 vacuum compression air cock.

Detailed Description

The following examples are further explained and illustrated, but the present invention is not limited in any way by the specific examples. Unless otherwise indicated, the methods and equipment used in the examples are conventional in the art and all materials used are conventional commercially available materials.

Example 1

s2, placing the sponge into a culture bag, vacuumizing, compressing and sterilizing;

s3, injecting the inoculated nutrient solution into a culture bag to ensure the expansion and the wetting of the sponge fragments, introducing a certain amount of sterile air, and standing for culture;

Step S1 the culture solution includes: corn flour 5 wt.%, bean flour 5 wt.%, lard 1 wt.%, flour 5 wt.%, and insect powder 10 wt.%.

The sterilization temperature is 100-120 ℃, and the sterilization time is 30-60 min.

And step S3, the standing culture temperature is 24-26 ℃, and the culture time is 10-15 days.

And step S4, the standing time is 60-120 min.

The inoculated entomopathogenic nematodes are nematodes in an infection period, and the density of the inoculated entomopathogenic nematodes in the nutrient solution is 8-15 tail/mL.

In step S3, a certain amount of sterile air or pure oxygen is introduced into the culture bag.

And step S3, a self-sealing strip is arranged on one side of the culture bag, and a vacuum compression air nozzle and a closable liquid inlet are arranged on the culture bag. The vacuum compression air tap is used for vacuum compression of the culture bag, the volume of the culture bag is reduced, large-batch sterilization operation is facilitated, and the efficiency of large-scale culture is improved. The culture solution enters the culture bag through the liquid inlet, so that the bacterial infection rate in the air is reduced. The sponge in the culture bag is sponge fragments with the diameter of 3-5 cm.

Example 2

S1, preparing a culture medium nutrient solution from 5 wt% of corn flour, 5 wt% of bean flour, 1 wt% of lard, 5 wt% of flour, 10 wt% of insect powder and the balance of water, sterilizing the culture medium nutrient solution, inoculating cultured nematode symbiotic bacteria to the sterilized culture medium nutrient solution, culturing for 2 days at 25 ℃, and then inoculating entomopathogenic nematodes;

s2, opening a self-sealing strip of the culture bag, inserting sponge into the culture bag, closing the self-sealing strip, vacuumizing through a vacuum compression air nozzle on the culture bag, closing the vacuum compression air nozzle, and placing the culture bag into a sterilization box for sterilization;

s3, pouring the inoculation culture solution of S1 into a culture bag through a liquid inlet of the culture bag, introducing a certain amount of air, and closing the liquid inlet;

s4, culturing the culture bag at 24-26 ℃ for 15 days;

s5, opening the self-sealing strips of the culture bag, pouring the nematodes and the culture into clear water, stirring, fishing out the sponge, standing and separating for 60min, and collecting sediments at the lower layer to obtain the nematodes;

s6, injecting the nematodes into the clean sponge, and storing at 4-8 ℃.

Through tests, about 30 million nematodes are cultured in one sponge fragment, the development of the nematodes is normal, the growth stages are the same, and the nematodes have the capability of infecting insects. The invention utilizes the compressed sponge and the culture bag, can improve the sterilization efficiency of the device, and further improves the production efficiency. In the sterilization process, taking a conventional sterilization box as an example, under the same specification of an iron box and a culture bag, 5-6 iron boxes used in the prior art are processed in batches, 80-120 iron boxes are processed in batches, if 10 mu of insects in a farm land or a forest land are killed, 6 iron boxes are needed to be cultivated, namely 1 sterilization batch is obtained, and 1 sterilization batch of the invention can be used for cultivating insect-killing pathogenic nematodes for 200 mu of the farm land or the forest land, so that the production efficiency and the sterilization efficiency of the nematodes are greatly improved. The inoculated nutrient solution is filled into the culture bag through negative pressure, the cultured strains and the nematodes are distributed more uniformly, the culture time is shortened, the development stages of the nematodes are in the same stage, the quality of the nematode product is improved, and the bacterial infection rate when the nematode product is in contact with air is reduced by infusing the inoculated nutrient solution through the liquid inlet of the culture bag.

Example 3

The nematode produced by the method of the present invention was compared with the effect of the insecticide pesticide on the market and sprayed to rice (quantity: 100). The sheath blight rate results are shown in Table 1, and the sheath blight rate results are shown in Table 2:

TABLE 1

TABLE 2

	Day 1	Day 10	Day 20	Day 30
					Control zone	0.62％	3.317％	2.586％	2.893％
Application area	0.58％	0.1156％	0.0537％	0
					Nematode zone	0.60	1.925％	0.112％	0.0641％

As shown in the tables 1 and 2, the nematode biopesticide prepared by the method disclosed by the invention can effectively kill pests, protect the environment, is green in development road and is in line with social benefits.

It should be understood that the above-described embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. The method for large-scale culture of entomopathogenic nematodes is characterized in that a culture device for large-scale culture of entomopathogenic nematodes is a sealable culture bag, a self-sealing strip is arranged on one side of the culture bag, and a vacuum compression air nozzle and a closable liquid inlet are arranged on the culture bag, and the preparation steps comprise:

s2, placing the sponge into a culture bag, vacuumizing and compressing through a vacuum compression air nozzle, and sterilizing;

s3, filling the inoculated culture medium nutrient solution into a culture bag from a liquid inlet, sucking the inoculated culture medium nutrient solution into the compressed culture bag and sponge through negative pressure, introducing a certain amount of sterilized air or pure oxygen, and standing for culture;

s4, soaking the nematodes and the culture cultured in the step S3 in clear water, separating out sponge, standing, and taking the lower-layer liquid;

the nutrient solution of the culture medium in the step S1 comprises: 4-8 wt% of corn flour, 3-7 wt% of bean flour, 1-3 wt% of lard oil, 4-8 wt% of flour, 8-12 wt% of insect powder and the balance of purified water;

the sterilization temperature is 100-120 ℃, and the sterilization time is 30-60 min;

the entomopathogenic nematodes being inoculated are nematodes in the infested period;

the density of the entomopathogenic nematodes inoculated in the culture medium nutrient solution is 8-15 tail/mL;

step S3, standing and culturing at 24-26 ℃ for 10-15 days;

step S4, standing for 60-120 min;

the sponge in the culture bag is broken pieces with the diameter of 3-5 cm.