CN109909055B - Rapid cleaning method for entomopathogenic nematodes - Google Patents
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Abstract
The invention provides a method for quickly cleaning entomopathogenic nematodes. The method comprises the following steps: putting the culture mixture obtained by the entomopathogenic nematode solid culture method into a washing bucket of a washing machine, sequentially performing single washing, dewatering, rinsing and dewatering or single washing, dewatering, rinsing, dewatering and dewatering by setting a washing program of the washing machine, and collecting the discharge liquid of the washing machine; standing the obtained discharge liquid for precipitation, and discarding the supernatant; then stirring and cleaning the precipitate with distilled water to obtain an entomopathogenic nematode concentrated solution; and finally adding benzisothiazolin-3-one, adsorbing by using a sponge, sealing and packaging, and storing at low temperature. The method is simple, the used devices are easy to obtain, the recovery efficiency of the nematodes can be effectively improved, the recovery time is shortened, the damage to the nematodes is small, the quality of the nematodes is improved, the survival rate of the nematodes is high, and the infection power of the nematodes is strong.
Description
Technical Field
The invention relates to a method for quickly cleaning entomopathogenic nematodes, and belongs to the technical field of biological propagation and culture.
Background
Entomopathogenic nematodes are nematodes that are parasitic in nature in insects and include the families Steinernemamidae [ Steinernemanidae carbopacapsae (Sc-2) ] and Heterodera [ Herorpha bifida bacteriophora (Hb-1) ]; the nematodes enter the body of the insect from stomata, mouths, anus or internode membranes of the insect in an infected state (IJ) of the third age, and then release symbiotic bacteria of Xenorhabdus (symbiotic with Steelenchus) or Photorhabdus (symbiotic with Heterodera heterorhabdus) carried by the nematodes, and the nematodes and the symbiotic bacteria breed and release toxic substances to cause the death of the insect. The entomopathogenic nematode can effectively control soil-inhabiting and boring pests of agricultural, forestry and horticultural plants, such as Chinese chive maggot, grub, peach fruit borer, yellow flea beetle and the like, has the characteristics of wide hosts, strong pathogenicity and no harm to people and livestock, and is a novel biological insecticide.
The culture mode of entomopathogenic nematodes is mainly divided into living culture and isolated culture; the isolated culture is divided into solid culture and liquid culture according to different states of culture matrixes. The requirements of the solid culture method on technical difficulty and culture conditions are obviously lower than that of liquid culture, and the pathogenicity and activity of the nematodes produced by the solid culture method are also better than those of the liquid culture method, so that the solid culture method is particularly suitable for companies entering the industry newly or mature companies to rapidly expand the scale when the market demand is large; furthermore, the solid culture method of the entomopathogenic nematodes has a good development prospect.
The existing solid culture method mainly comprises the processes of symbiotic bacteria separation preparation, nematode propagation, harvesting, cleaning, storage and the like, wherein the cleaning is an important step, and aims to separate nematodes from solid cultures as much as possible and completely and quickly so as to obtain entomopathogenic nematode products with high purity and good impregnation capability and survival rate. The existing cleaning method for entomopathogenic nematodes mostly adopts a standing soaking method, the extraction time of the nematodes is long, the extraction rate is only 80-90%, and the extraction rate needs to be further improved. For example, chinese patent document CN106172259A discloses a method for cleaning entomopathogenic nematodes, which comprises the steps of: (a) soaking the nematodes and the solid culture in a larger container filled with clear water, wherein a movable screen is arranged in the container, and a ventilation and oscillation device is arranged in the container; (b) standing and separating, allowing the nematodes to pass through a screen, leaving impurities such as sponge and the like, and allowing about 95% of nematodes in the infection period to climb out of the culture; (c) in the industrial production, the nematode stock solution without impurities is added with diatomite powder according to the proportion of 10:1, solid-liquid separation is carried out by a filter press, the pressure of the filter press is set to be less than 3 kg, and the obtained diatomite powder containing nematodes is counted, subpackaged and stored. Said invention is aimed at the nematode harvesting, cleaning and storing problems of entomopathogenic nematode solid culture method, and said method can obtain good harvesting effect, and has small damage to nematode, and the nematode survival rate in the nematode product made in the later stage is high. However, the entomopathogenic nematode cleaning method adopts a standing soaking method, so that the extraction time of the nematodes is long, the extraction rate is to be improved, and relevant data such as the impregnation capability and the survival rate of the entomopathogenic nematodes are not involved.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a method for quickly cleaning entomopathogenic nematodes, which aims at the problems of quickly cleaning, harvesting and storing the nematodes after the solid culture of the entomopathogenic nematodes is finished. The method is simple, the used devices are easy to obtain, the recovery efficiency of the nematodes can be effectively improved, the recovery time is shortened, the damage to the nematodes is small, the quality of the nematodes is improved, the survival rate of the nematodes is high, and the infection power of the nematodes is strong.
The technical scheme of the invention is as follows:
a method for rapidly cleaning entomopathogenic nematodes comprises the following steps:
(1) cleaning: putting the culture mixture obtained by the entomopathogenic nematode solid culture method into a washing bucket of a washing machine, sequentially performing single washing, dewatering, rinsing and dewatering or single washing, dewatering, rinsing, dewatering and dewatering by setting a washing program of the washing machine, and collecting the discharge liquid of the washing machine;
(2) concentration and separation: standing and precipitating the discharge liquid obtained in the step (1), and discarding the supernatant; then stirring and cleaning the precipitate with distilled water to obtain an entomopathogenic nematode concentrated solution;
(3) and (3) storage: and (3) adding benzisothiazolin-3-one into the entomopathogenic nematode concentrated solution obtained in the step (2), adsorbing by using sponge, sealing and packaging, and storing at low temperature.
According to the invention, in the step (1), the culture mixture obtained by the entomopathogenic nematode solid culture method is: culturing entomopathogenic nematodes by a solid culture method, and culturing substances obtained in a container after the culture, wherein the substances comprise a culture medium, broken sponge, entomopathogenic nematodes and symbiotic bacteria; the solid culture method of the entomopathogenic nematodes is carried out according to the prior art.
Preferably, in the step (1), a solid culture method is adopted to culture the entomopathogenic nematodes for 18-30 days to obtain a culture mixture; preferably, the entomopathogenic nematodes are cultured by a solid culture method for 20-26 days to obtain a culture mixture. The culture time is the optimal time for culturing the entomopathogenic nematodes, and the obtained culture mixture has the highest yield of the nematodes in the infection stage and the minimum proportion of the nematodes in other states.
According to the invention, in the step (1), the washing machine is a pulsator single-drum full-automatic washing machine.
Preferably, in the step (1), the volume ratio of the water injection amount to the culture mixture in the washing machine is 1-4: 1.
according to the invention, in the step (1), the rotation speed of single washing is 30-50 rpm, the rotation speed of rinsing is 30-50 rpm, and the rotation speed of dewatering is 300-500 rpm.
According to the invention, in the step (1), 90-98% of entomopathogenic nematodes in the culture mixture are in the effluent of the washing machine.
According to the invention, in the step (1), the single washing time is 4-12 min, preferably 8 min; the rinsing time is 3-5 min, preferably 4 min; the dewatering time is 1-3 min, preferably 1 min; the total cleaning time is 9-31 min.
According to the invention, in the step (2), the discharged liquid is settled for 20-30 min in an inclined water tank; the water tank is a cuboid water tank, the length-width ratio in the water tank is 2-3: 1, and the included angle between the water tank and the horizontal plane is 10-45 degrees; along the inclined direction, a clear liquid outlet is formed in the lower side face of the water tank, and the distance between the clear liquid outlet and the bottommost part of the water tank is 5-20 cm; the bottommost part of the water tank is provided with an entomopathogenic nematode concentrated solution outlet; and after the discharged liquid is settled, the supernatant flows out through a clear liquid discharge port.
Preferably, in the step (2), the stirring and cleaning are performed in an inclined water tank; stirring and cleaning for 1-5 times by using distilled water, wherein the stirring and cleaning time is 1-2 min; standing for 5-20 min after stirring and cleaning every time, and enabling supernatant to flow out through a clear liquid outlet.
According to the invention, in the step (3), the addition amount of the benzisothiazolin-3-one is 0.5-2% of the mass of the entomopathogenic nematode concentrated solution. The benzisothiazolin-3-one acts to inhibit fungal proliferation.
According to the invention, in the step (3), the mass ratio of the entomopathogenic nematode concentrated solution to the sponge is preferably 5-9: 1-5.
Preferably, in step (3), the sponge is made of crushed sponges, and the volume of each crushed sponge is 0.2-27 cm3Preferably 0.5 to 8cm3(ii) a The pore density of each crushed sponge is 25 to 35 ppi.
According to the present invention, in the step (3), the low-temperature storage is preferably performed at 4 to 10 ℃.
The beneficial effects are as follows:
1. the invention aims at solving the problems of nematode cleaning, harvesting and storing in the entomopathogenic nematode solid culture method. The cleaning method is simple, the used appliances are easy to obtain, the nematodes are promoted to climb out and precipitate by means of the cleaning program of the impeller type washing machine and the special cleaning principle of the impeller type washing machine, and the climbing-out of the nematodes is promoted as completely as possible by the rinsing and dewatering programs, and the time used in the cleaning process is short (9-31 min); the dehydration process in the cleaning process can further promote the complete crawling of the nematodes and improve the extraction rate of the nematodes, thereby shortening the time for cleaning; in addition, the culture mixture is directly added into a washing barrel of a washing machine for washing, the method is simple, and the complete crawling of nematodes is facilitated. Compared with the traditional standing and soaking cleaning method, the method disclosed by the invention can greatly shorten the cleaning and recovery time, improve the recovery efficiency of the nematodes and improve the yield of the nematodes; the obtained nematodes have good quality, high vitality, high survival rate and strong infection.
2. The cleaning method adopted by the invention has moderate cleaning force and time, does not generate negative influence on the physiological indexes of the nematodes and can improve the quality of the nematodes in the impregnation period; the invention preferably adopts an inclined water tank for standing, precipitating, stirring and cleaning, and the method is simple; the subsequent sponge that utilizes stores, but sponge reuse practices thrift the cost. The cleaning method of the invention has very important significance for the scale production of entomopathogenic nematodes and the biological control of soil insects.
3. The traditional cleaning method of entomopathogenic nematodes is a standing soaking method, the cleaning step time is long (2-3 hours), the extraction time of nematodes is long, and the extraction rate is only 80-90%; the cleaning step can be completed within 31min at most, the extraction rate of the nematodes can reach 90-98%, and the extraction rate is high.
Detailed Description
The present invention will be described in further detail with reference to specific examples, but it should be understood that the present invention is not limited to the examples.
Meanwhile, the experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents, materials and devices, unless otherwise specified, are all prior art and commercially available.
In the examples, the washing machine used was a Sanyo (SANYO) DB6035BXS pulsator single-tub full-automatic washing machine.
According to the prior art, a solid culture method is adopted to culture entomopathogenic nematodes Hb-1, and a culture mixture is obtained after 22 days of culture.
Example 1
A method for rapidly cleaning entomopathogenic nematodes comprises the following steps:
(1) cleaning: the culture mixture obtained above was put into a washing tub of a washing machine (water injection amount was 40L), and the volume ratio of water injection amount to culture mixture in the washing machine was 2: 1; sequentially carrying out single washing for 8 min-dewatering for 1 min-rinsing for 4 min-dewatering for 1min by setting a washing program of the washing machine, wherein the total washing time is 19min, and collecting the discharged liquid of the washing machine; the rotating speed of the single washing is 40rpm, the rotating speed of the rinsing is 40rpm, and the rotating speed of the dewatering is 300 rpm; about 96% of the nematodes were in the effluent.
(2) Concentration and separation: standing and precipitating the discharge liquid obtained in the step (1) in an inclined water tank for 20 min; the water tank is a cuboid water tank, the length-width ratio in the water tank is 2:1, and the included angle between the water tank and the horizontal plane is 30 degrees; along the inclined direction, a clear liquid outlet is arranged on the lower side surface of the water tank, and the distance between the clear liquid outlet and the bottommost part of the water tank is 10 cm; the bottommost part of the water tank is provided with an entomopathogenic nematode concentrated solution outlet; after the discharged liquid is settled, the supernatant flows out through a clear liquid discharge port; adding distilled water, stirring and cleaning for 2 times, stirring and cleaning for 2min, standing for 10min after cleaning each time, and allowing supernatant to flow out through a clear liquid outlet to obtain a clean entomopathogenic nematode concentrated solution.
(3) And (3) storage: adding benzisothiazolin-3-one into the concentration solution of the entomopathogenic nematodes obtained in the step (2), wherein the addition amount of benzisothiazolin-3-one is 0.8% of the mass of the concentration solution of the entomopathogenic nematodes, and adsorbing with 1.54kg of crushed sponge (each crushed sponge is in the shape of a cube and has a volume of 8 cm)3The pore density is 30ppi), the mass ratio of the entomopathogenic nematode concentrated solution to the sponge is 7:3, and the entomopathogenic nematode concentrated solution and the sponge are sealed and packaged and then stored in a refrigeration house at the temperature of 4 ℃.
Example 2
A method for rapidly cleaning entomopathogenic nematodes comprises the following steps:
(1) cleaning: the culture mixture obtained above was put into a washing tub of a washing machine (water injection amount was 40L), and the volume ratio of water injection amount to culture mixture in the washing machine was 3: 1; sequentially carrying out single washing for 4 min-dewatering for 2 min-rinsing for 3 min-dewatering for 2min by setting a washing program of the washing machine, wherein the total washing time is 11min, and collecting the discharged liquid of the washing machine; the rotating speed of the single washing is 40rpm, the rotating speed of the rinsing is 40rpm, and the rotating speed of the dewatering is 500 rpm; about 95% of the nematodes were in the effluent.
(2) Concentration and separation: standing and precipitating the discharge liquid obtained in the step (1) in an inclined water tank for 25 min; the water tank is a cuboid water tank, the length-to-width ratio in the water tank is 2.5:1, and the included angle between the water tank and the horizontal plane is 10 degrees; along the inclined direction, a clear liquid outlet is arranged on the lower side surface of the water tank, and the distance between the clear liquid outlet and the bottommost part of the water tank is 5 cm; the bottommost part of the water tank is provided with an entomopathogenic nematode concentrated solution outlet; after the discharged liquid is settled, the supernatant flows out through a clear liquid discharge port; adding distilled water, stirring and cleaning for 1 time, stirring and cleaning for 2min, standing for 5min after each cleaning, and allowing supernatant to flow out through a clear liquid outlet to obtain a clean entomopathogenic nematode concentrated solution.
(3) And (3) storage: adding benzisothiazolin-3-one into the concentrated solution of entomopathogenic nematodes obtained in the step (2), wherein the addition amount of benzisothiazolin-3-one is 0.5% of the mass of the concentrated solution of entomopathogenic nematodes, and adsorbing with 1.54kg of crushed sponge (each crushed sponge is in the shape of a cube and has a volume of 8 cm)3The pore density is 30ppi), the mass ratio of the entomopathogenic nematode concentrated solution to the sponge is 5:3, and the entomopathogenic nematode concentrated solution and the sponge are sealed and packaged and then stored in a refrigeration house at the temperature of 6 ℃.
Example 3
A method for rapidly cleaning entomopathogenic nematodes comprises the following steps:
(1) cleaning: the culture mixture obtained above was put into a washing tub of a washing machine (water injection amount was 40L), and the volume ratio of water injection amount to culture mixture in the washing machine was 4: 1; sequentially carrying out single washing for 12min, dewatering for 2min, rinsing for 5min and dewatering for 2min by a quick washing program of a washing machine, wherein the total washing time is 28min, and collecting the discharged liquid of the washing machine; the rotating speed of the single washing is 40rpm, the rotating speed of the rinsing is 40rpm, and the rotating speed of the dewatering is 500 rpm; about 97% of the nematodes were in the effluent.
(2) Concentration and separation: standing and precipitating the discharge liquid obtained in the step (1) in an inclined water tank for 30 min; the water tank is a cuboid water tank, the length-width ratio in the water tank is 3:1, and the included angle between the water tank and the horizontal plane is 45 degrees; along the inclined direction, a clear liquid outlet is arranged on the lower side surface of the water tank, and the distance between the clear liquid outlet and the bottommost part of the water tank is 10 cm; the bottommost part of the water tank is provided with an entomopathogenic nematode concentrated solution outlet; after the discharged liquid is settled, the supernatant flows out through a clear liquid discharge port; adding distilled water, stirring and cleaning for 4 times, wherein the stirring and cleaning time is 1min, standing for 15min after each cleaning, and allowing supernatant to flow out through a clear liquid outlet to obtain a clean entomopathogenic nematode concentrated solution.
(3) And (3) storage: adding benzisothiazolin-3-one into the concentration solution of the entomopathogenic nematodes obtained in the step (2), wherein the addition amount of benzisothiazolin-3-one is 1.5% of the mass of the concentration solution of the entomopathogenic nematodes, and adsorbing with 1.54kg of crushed sponge (each crushed sponge is in the shape of a cube and has a volume of 8 cm)3The pore density is 30ppi), the mass ratio of the entomopathogenic nematode concentrated solution to the sponge is 9:4, and the entomopathogenic nematode concentrated solution and the sponge are stored in a refrigeration house at 10 ℃ after being sealed and packaged.
Comparative example 1
A method for cleaning entomopathogenic nematodes by standing precipitation.
Soaking the obtained culture mixture in an inclined water tank (the structure of the inclined water tank is as described in example 1) filled with 40L of clear water, wherein the water tank is provided with a movable screen and an aeration device, standing and precipitating for 3h to allow nematodes to climb out of the sponge, allowing the nematodes to leave impurities such as the sponge through the screen, removing the screen together with the solid culture, allowing the nematodes to settle to the bottom of the water tank, and allowing supernatant to flow out through a clear liquid outlet; adding distilled water, stirring and cleaning for 2 times, stirring and cleaning for 2min, standing for 10min after cleaning each time, and allowing supernatant to flow out through a clear liquid outlet to obtain a clean entomopathogenic nematode concentrated solution. The preservation procedure was as described in example 1.
Comparative example 2
A method for cleaning entomopathogenic nematodes by oscillation cleaning.
The method is the same as the comparative example 1, except that the water tank is horizontally placed on an oscillation device, the oscillation amplitude is 50rpm/h, and the water tank is oscillated and cleaned for 2 h; the other steps were in accordance with comparative example 1.
Comparative example 3
A method of cleaning entomopathogenic nematodes as described in example 1, except that: the cleaning procedure comprises single washing for 20min, dewatering for 5min, rinsing for 10min, and dewatering for 5min, wherein the total cleaning time is 55 min; the other steps were in accordance with example 1.
Comparative example 4
A method of cleaning entomopathogenic nematodes as described in example 1, except that: the cleaning procedure is that single washing is carried out for 8 min-dehydration is carried out for 1min, and the total cleaning time is 9 min; the other steps were in accordance with example 1.
Comparative example 5
A method of cleaning entomopathogenic nematodes as described in example 1, except that: the cleaning procedure is as follows: after washing for 45min for one time, collecting the discharge liquid; then adding 40L of water, washing for 45min once, and collecting the discharge liquid; adding 40L of water, washing for 45min for one time, and collecting the discharge liquid; the other steps were in accordance with example 1.
Test example 1
Nematode yield calculation
And after sealing and packaging, storing the entomopathogenic nematode concentrated solution obtained in the example 1 and the comparative examples 1 to 5 in a refrigeration house at the temperature of 4 ℃ and calculating the yield by adopting a gradient dilution method.
1ml of the concentrated solution is diluted by gradient 3 times, each time the dilution times are 10 times, 2ml of the diluted concentrated solution is taken to be put into a 6-hole cell culture plate, observed and counted by a stereoscopic microscope, and the total nematode output in the entomopathogenic nematode concentrated solution is calculated, and the results are shown in table 1.
TABLE 1 Effect of different washing methods on nematode production
As can be seen from Table 1, the concentrated solutions of entomopathogenic nematodes collected by the cleaning procedures of comparative examples 5 and 3 had the highest number of nematodes and the highest extraction rates of nematodes, which were 97%, but the cleaning times were longer, 135min and 55min, respectively, whereas the cleaning time of example 1 was only 19min, which was 116min and 36min shorter than that of comparative examples 5 and 3, respectively, but the extraction rates of nematodes were only reduced by 1 percentage point, which was 96%; the cleaning time of the comparative example 1 and the comparative example 2 which respectively adopt the standing precipitation and the oscillation cleaning modes is 180min and 120min respectively, the longest time and the longest time of the 3 rd treatment in all the cleaning modes are adopted, but the nematode extraction rate is only 86 percent and 90 percent respectively; in contrast, comparative example 4, which lacks a rinsing step, had the shortest washing time of only 9min, but the number of nematodes in the resulting nematode concentrate was the smallest, and the nematode extraction rate was only 40%, which was half as low as that of the other washing methods. As can be seen from the above results, the washing method of the present invention can obtain more nematodes with less washing time.
Test example 2
Nematode survival assay
The entomopathogenic nematode products obtained in the example 1 and the comparative examples 1 to 5 are stored in a refrigeration house at 4 ℃ for 20 days, then are taken out, are placed at room temperature for 2 hours, and are observed under a dissecting mirror to kill the nematodes; if no reaction activity of the nematode is caused by mechanical stimulation of the needle, the individual is considered dead, and the survival rate of the nematode is recorded and calculated. Nematode survival (%) (number of surviving nematodes/total number of nematodes) × 100%.
TABLE 2 Effect of different washing methods on nematode survival
| Example 1 | Comparative example 1 | Comparative example 2 | Comparative example 3 | Comparative example 4 | Comparative example 5 | |
| Nematode survival (%) | 96.3 | 89.2 | 88.7 | 79.6 | 97.7 | 90 |
As can be seen from Table 2, the nematode survival rate was the highest in the products obtained in comparative example 4 and example 1, followed by comparative example 5, comparative example 1, comparative example 2 and comparative example 3 in this order; as can be seen from the above, the survival rate of the nematodes obtained by the cleaning method of the invention is high.
Test example 3
Determination of the infectivity of nematodes
The entomopathogenic nematode concentrates obtained in example 1 and comparative examples 1 to 5 were diluted to a nematode concentration of 500 nematodes/ml, 2ml of the diluted nematode concentrates were added to culture dishes on which double-layered filter paper was laid as treatment groups, a control group of undiluted nematode concentrates was set, 10 galleria mellonella were placed in each dish, cultured at 25 ℃, and the number of galleria mellonella dead per dish was investigated every 12 hours after 24 hours, and the corrected mortality was calculated. Corrected mortality (%) - (treatment-control mortality)/(1-control mortality) ]. 100
TABLE 3 correction of mortality data
As shown by the results of Hb-1 infectivity measurement of nematodes in Table 3, the nematodes collected in comparative example 1 after 24 hours of culture gave the highest corrected mortality of greater wax moth, after example 1, followed by comparative example 4, comparative example 2, comparative example 5 and comparative example 3 in this order; after 36h of culture, except that the nematode staining power collected in the comparative example 3 is poor, the corrected mortality rate of the greater wax moth by the nematodes collected in all the cleaning methods reaches about 50%, and the corrected mortality rate of the greater wax moth by the nematodes obtained by the cleaning method in the embodiment 1 of the invention is the highest; after 48h of culture, the corrected mortality of the greater wax moth by the nematodes collected by the cleaning methods reaches over 95 percent, and the corrected mortality of the greater wax moth by the nematodes collected by the cleaning method in the embodiment 1 of the invention can reach 100 percent. As can be seen from the above, the nematode infectivity obtained by the cleaning method of the invention is stronger.
As can be seen from the above test examples 1-3, the comparative examples 5 and 3 were cleaned sufficiently, and as many nematodes were collected as possible, but the time spent was long, and the nematode survival rate and the impregnation rate of the comparative example 3 were both the lowest, indicating that the cleaning method of the comparative example 3 had a large effect on nematodes, whereas the nematode survival rate and the impregnation rate of the comparative example 5 were both higher than that of the comparative example 3 but both lower than that of the example 1 of the present invention, and the corrected mortality rate of greater wax moth on the first day of nematodes extracted by the comparative example 5 was also lower than that of the example 1, indicating that the comprehensive comparison, the example 1 of the present invention was superior to the comparative example 5 in terms of nematode extraction efficiency and quality; comparative example 4 lacks a rinsing procedure, washing is incomplete, and nematode yield and extraction rate are lowest although nematode survival rate and impregnation rate are higher; the nematode yield, survival rate and impregnation power of comparative example 1 (standing precipitation) and comparative example 2 (shaking washing) were relatively high after long-term immersion washing, but the nematode yield, survival rate and impregnation power were inferior to those of the present invention, and the washing time was too long, up to 3 hours. The yield of the nematodes obtained in the example 1 of the invention is second to that of the comparative examples 5 and 3, the survival rate and the impregnation power of the nematodes are superior to those of other cleaning methods, the cleaning time is only 19 minutes, particularly less than that of the comparative examples 5, 1 and 3, and the nematodes with high yield, high survival rate and strong infection power can be obtained in a shorter cleaning time.
Claims (9)
1. A method for rapidly cleaning entomopathogenic nematodes comprises the following steps:
(1) cleaning: putting the culture mixture obtained by the entomopathogenic nematode solid culture method into a washing bucket of a washing machine, sequentially performing single washing, dewatering, rinsing and dewatering or single washing, dewatering, rinsing, dewatering and dewatering by setting a washing program of the washing machine, and collecting the discharge liquid of the washing machine;
the washing machine is a pulsator single-cylinder full-automatic washing machine; the volume ratio of the water injection amount in the washing machine to the culture mixture is 1-4: 1; the single washing time is 4-12 min; rinsing time is 3-5 min each time; the dehydration time is 1-3 min each time; the total cleaning time is 9-31 min; the rotation speed of dehydration is 300-500 rpm;
(2) concentration and separation: standing and precipitating the discharge liquid obtained in the step (1), and discarding the supernatant; then stirring and cleaning the precipitate with distilled water to obtain an entomopathogenic nematode concentrated solution;
(3) and (3) storage: and (3) adding benzisothiazolin-3-one into the entomopathogenic nematode concentrated solution obtained in the step (2), adsorbing by using sponge, sealing and packaging, and storing at low temperature.
2. The method for rapidly washing entomopathogenic nematodes according to claim 1, wherein in the step (1), the culture mixture is obtained after the entomopathogenic nematodes are cultured for 18-30 days by a solid culture method.
3. The method for rapidly washing entomopathogenic nematodes according to claim 1, wherein in the step (1), the single washing time is 8 min; the rinsing time is 4min each time; the dehydration time is 1min each time.
4. The method for rapidly cleaning entomopathogenic nematodes according to claim 1, wherein in step (1), the rotation speed of each single washing is 30-50 rpm, and the rotation speed of each rinsing is 30-50 rpm.
5. The method for rapidly washing entomopathogenic nematodes according to claim 1, wherein in the step (2), the effluent is left to stand and settle in an inclined water tank for 20-30 min; the water tank is a cuboid water tank, the length-width ratio in the water tank is 2-3: 1, and the included angle between the water tank and the horizontal plane is 10-45 degrees; along the inclined direction, a clear liquid outlet is formed in the lower side face of the water tank, and the distance between the clear liquid outlet and the bottommost part of the water tank is 5-20 cm; the bottommost part of the water tank is provided with an entomopathogenic nematode concentrated solution outlet; and after the discharged liquid is settled, the supernatant flows out through a clear liquid discharge port.
6. The method for rapidly washing entomopathogenic nematodes according to claim 5, wherein in the step (2), the agitation washing is performed in an inclined water tank; stirring and cleaning for 1-5 times by using distilled water, wherein the stirring and cleaning time is 1-2 min; standing for 5-20 min after stirring and cleaning every time, and enabling supernatant to flow out through a clear liquid outlet.
7. The method for rapidly washing entomopathogenic nematodes according to claim 1, wherein in the step (3), the benzisothiazolin-3-one is added in an amount of 0.5-2% by mass based on the mass of the concentrate of entomopathogenic nematodes.
8. The method for rapidly cleaning entomopathogenic nematodes according to claim 1, wherein in the step (3), the mass ratio of the entomopathogenic nematode concentrated solution to the sponge is 5-9: 1-5.
9. The method for rapidly washing entomopathogenic nematodes according to claim 1, wherein in the step (3), the sponge is composed of pieces of sponge, and the volume of each piece of sponge is 0.2-27 cm3(ii) a The pore density of each crushed sponge is 25 to 35 ppi.
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Citations (8)
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| CN2145211Y (en) * | 1992-12-31 | 1993-11-03 | 吴国新 | Automatic clean water tank |
| US6474259B1 (en) * | 2001-04-30 | 2002-11-05 | Rutgers, The State University Of New Jersey | Apparatus and method for mass production of insecticidal nematodes |
| CN201758682U (en) * | 2010-06-24 | 2011-03-16 | 山东省果树研究所 | Entomopathogenic nematode cleaner |
| CN203470134U (en) * | 2013-07-31 | 2014-03-12 | 浙江大学 | Soil nematode separation and deposition device |
| CN106172259A (en) * | 2016-08-18 | 2016-12-07 | 浙江绿神天敌生物技术有限公司 | A kind of entomopathogenic nematode cleaning method |
| CN207733477U (en) * | 2018-01-18 | 2018-08-17 | 项鹏 | A kind of soybean cyst nematode Heterodera glycines separator |
| CN108967360A (en) * | 2018-06-14 | 2018-12-11 | 广东省生物资源应用研究所 | A kind of method of quick collection entomopathogenic nematode |
| CN208466767U (en) * | 2018-07-05 | 2019-02-05 | 山西大学 | A kind of entomopathogenic nematode cleaning device |
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2019
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| CN2145211Y (en) * | 1992-12-31 | 1993-11-03 | 吴国新 | Automatic clean water tank |
| US6474259B1 (en) * | 2001-04-30 | 2002-11-05 | Rutgers, The State University Of New Jersey | Apparatus and method for mass production of insecticidal nematodes |
| CN201758682U (en) * | 2010-06-24 | 2011-03-16 | 山东省果树研究所 | Entomopathogenic nematode cleaner |
| CN203470134U (en) * | 2013-07-31 | 2014-03-12 | 浙江大学 | Soil nematode separation and deposition device |
| CN106172259A (en) * | 2016-08-18 | 2016-12-07 | 浙江绿神天敌生物技术有限公司 | A kind of entomopathogenic nematode cleaning method |
| CN207733477U (en) * | 2018-01-18 | 2018-08-17 | 项鹏 | A kind of soybean cyst nematode Heterodera glycines separator |
| CN108967360A (en) * | 2018-06-14 | 2018-12-11 | 广东省生物资源应用研究所 | A kind of method of quick collection entomopathogenic nematode |
| CN208466767U (en) * | 2018-07-05 | 2019-02-05 | 山西大学 | A kind of entomopathogenic nematode cleaning device |
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| CN109909055A (en) | 2019-06-21 |
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