CN101245319A - A kind of Beauveria bassiana HFW-05 bacterial strain and application thereof - Google Patents

Abstract

The invention discloses a Beauveria bassiana HFW-O5 strain CGMCC No. 2356. Bemisia tabaci carcasses isolated from tomato greenhouses in the suburbs of Baoding. The strain is harmless to humans and the living environment of humans, and has high toxicity to pests such as whitefly, diamondback moth, and aphids. Through field efficacy tests on the control of whitefly, diamondback moth, and aphids, it has been proved that Its control effect on the three pests of whitefly, diamondback moth and aphid reaches more than 70%.

Description

A kind of Beauveria bassiana HFW-05 bacterial strain and application thereof

technical field

The invention relates to a Beauveria bassiana strain and its application, in particular to a Beauveria bassiana HFW-05 strain and its application in preventing and controlling vegetable whitefly, diamondback moth and other pests.

Background technique

In recent years, with the development of protected vegetables, whitefly and diamondback moth have become important pests of vegetables. Whitefly has been seriously harmful since the large-scale outbreak in northern my country in 1976. In recent years, Bemisia tabaci has also broken out successively in Beijing, Hebei, Xinjiang, Tianjin, Guangzhou and other places, causing serious losses of vegetables, especially facility vegetables, and in severe cases, the yield can be reduced by more than 70%. Plutella xylostella is the most resistant vegetable pest, and its production and control are extremely difficult. Whiteflies can also transmit a variety of diseases caused by viruses. For a long time, they have been controlled by chemical pesticides. Chemical pesticides pollute the environment, increase the residual harmful pesticides in vegetables, and increase the resistance of pests such as diamondback moth and whitefly, making them more difficult to control with pesticides. With the development of my country's vegetable basket project, facility vegetables and pollution-free vegetables have developed rapidly, resulting in a large number of pests such as whitefly and diamondback moth. Serious harm to vegetables, a serious threat to the production of green and pollution-free vegetables in my country and human health, and a serious threat to the export of agricultural and sideline products in my country, so the research and development of new technologies for efficient and harmless prevention and control has become an urgent problem in production question.

Contents of the invention

The purpose of the present invention is to solve the above problems in the prior art, to provide a coccidioides that has a high control effect on vegetable pests such as whitefly, diamondback moth and aphid, and has no harm to human beings and the environment in which they live. B. bassiana HFW-05 strain and its application.

To accomplish the purpose of the present invention, the technical solution of the present invention is: a Beauveria bassiana HFW-05 bacterial strain, the preservation number is CGMCC No.2356. It is isolated and obtained by the following method: collect whitefly corpses from tomato greenhouses in the suburbs of Baoding, grind the collected whitefly corpses separately, add them to a triangular flask containing 50ml sterile normal saline and glass beads, and wait 25-27 ℃, preferably 26℃, 150-200rpm, preferably 150rpm shaker for 15 minutes, after 10 minutes of rest, take 500μl of the supernatant on the ultra-quiet workbench and add it to 4.5ml sterile saline, take the above 50 μl of the suspension was evenly spread on the SDAY medium plate made of 1% peptone, 1% yeast extract, 4% glucose and 2% agar, and each treatment was repeated 3 times. It is preferably cultured in an incubator at 26°C for 5-7 days, preferably 5 days. After cultivation, pick up Beauveria bassiana colonies with a sterile toothpick, cultivate them after purification, and collect Beauveria bassiana spore powder. Use Tween 80 as a spreading agent to dilute with water to make a bacterial suspension with a concentration of 1×10 ⁸ spores/ml, and use the nymphs or larvae of Bemisia tabaci and diamondback moth as objects for biological assays, and according to the toxicity of different strains to the above pests Finally, the Beauveria bassiana strain (Beauveria bassiana) HFW-05 with high toxicity to whitefly and diamondback moth was screened out.

This bacterial strain has been preserved in the General Microorganism Center (CGMCC for short) of the China Microbiological Culture Collection Management Committee on January 24, 2008, and the preservation number is: CGMCC No.2356.

The present invention also provides a fermentation method and an application method of Beauveria bassiana HFW-05 bacterial strain, comprising the steps of:

1. Activation of strains

Using the SDAY medium mixed with 1% peptone, 1% yeast extract, 4% glucose and 2% agar, the Beauveria bassiana strain HFW-05 was inoculated on the surface of the SDAY medium, and the temperature was maintained at 25 -27°C, cultured for 5-7 days, the activated spore powder of HFW-05 was produced.

2. Cultivation of seed solution

Seed liquid culture uses SDAY liquid medium mixed with 1% peptone, 1% yeast extract and 4% glucose. Under sterile conditions, scrape the activated HFW-05 spore powder and use sterile physiological The brine is prepared into a bacterial suspension with a concentration of 1×10 ⁸ -5×10 ⁸ spores/ml, inoculated in a 14-liter liquid fermenter with an inoculation amount of 0.2-0.5%, and the temperature is kept at 25-27°C, and the rotation speed is 150 -250rpm, cultured for 1d as seed solution.

3. Solid phase fermentation

The fermentation medium is rice, soak the rice in water for 3-4 hours, remove the excess water, then add 5ml of edible oil to 1kg of rice, and mix well.

Fermentation utensils: air-permeable plastic bags with a size of 50×40cm.

Fermentation method: put 200-300 grams of the above-mentioned rice with edible oil into a plastic bag, tie the mouth of the bag tightly with a string, put it into a sterilizing pot, and sterilize it at 121°C for 30 minutes. Put 10ml of seed liquid into each bag in the ultra-clean workbench, and place them on the culture rack in a constant temperature room at 25-27°C after inoculation, and cultivate them for 5-7 days. The above cultures were collected and dried indoors. Then collect the spore powder with a 150-200 mesh vibrating sieve.

4. Formulation processing: According to the processing method of wettable powder, the fungal spore powder is made into wettable powder with 5-10 billion spores/gram.

5. How to use Beauveria bassiana

Control period: Spray control at the early stage of pests such as Bemisia tabaci and diamondback moth, spray evenly on the habitat of pests, the back of damaged leaves, near growth points, flowers, etc. Spray once every 7 days, a total of 2-3 times;

Use concentration: Dilute the wettable powder containing 5-10 billion spores per gram of Beauveria bassiana live spores to 500-1000 times liquid, and spray it with a sprayer.

Note: Sericulture areas cannot be used, because Beauveria bassiana parasitizes silkworms and will produce stiff silkworms; spraying other fungicides should be controlled within 2-3 days before and after spraying; Beauveria bassiana is sensitive to high temperature, and it is best to use it at 20-28°C It is advisable that the bacterial solution should be used as it is prepared, and the prepared medicine should be sprayed within 2 hours; it should be sprayed in the evening or on cloudy days.

Beauveria bassiana is a kind of pathogenic fungus of parasitic insects. It adheres to the epidermis or stomach of insects, reproduces rapidly, causes the death of pests, and causes epidemic infection of pests with conidia. The control effect is very high, especially in high-humidity facilities Under the conditions of cultivation, the control effect is higher. The HFW-05 bacterial strain obtained in the present invention is a new bacterial strain of Beauveria bassiana, which has no harm to human beings and the environment in which human beings live, and has a high toxicity effect on pests such as whitefly, diamondback moth and aphids. The processed preparation has been proved to have a good control effect through the field efficacy test of controlling whitefly, diamondback moth and diamondback moth in the field, and the control effect on the three pests of whitefly, diamondback moth and aphid reaches more than 70%. The solid-phase fermentation method in the present invention has the following advantages: one is to solve the problem of high cost of purchasing a solid fermentation tank. Second, the fermentation method is not restricted by environmental conditions, and the production scale can be large or small. The third is that the operation is simple, and the contamination of miscellaneous bacteria is avoided in the fermentation process. Fourth, it has the characteristics of high spore production and high fermentation efficiency.

Detailed ways

The present invention will be further described below in conjunction with specific examples.

1. The morphology and biological characteristics of the strain

The bacterial strain was cultivated on different mediums, and the culture characteristics on Sabouraud plate medium, PDA plate medium and PCA plate medium were similar, and the plate medium was streaked and cultured, and the growth was slow within 48 hours, and the hyphae grew faster thereafter. , the thickness of the bacterial lawn of the strain in each medium is Sabouraud medium > PDA medium > PCA medium, the surface of the bacterial lawn is dry, no pigment, no pigment penetrates into the medium, and no special smell. On Sabouraud's medium, the mycelium is fluffy and white at the initial stage of growth. After 5 days of cultivation, the colony is light yellow and conidia are formed; on PDA medium and PCA medium, the mycelium is powdery, and the colony is white at the initial stage. , After 4 days of culture, all the colonies were pale yellow and conidia formed. The conidia of the strain are monospores, transparent, thin-walled, spherical or nearly spherical, and the spore size is 1.7-3.2×1.5-2.7 μm.

Inoculate the bacterial block with a diameter of 6mm on each plate culture medium, 2-3 days after inoculation, mycelia growth can be seen in each medium, and the growth rate of HFW-05 strain on each medium is slightly different, and it is Sabouraud medium> PCA medium > PDA medium. The average growth rate of colony diameter within 8 days on each medium was 3.13, 2.75, 2.25mm/d.

2. Effects of different conditions on the growth and development of Beauveria bassiana HFW-05 strain

(1), the influence of temperature on the growth and development of Beauveria bassiana HFW-05 strain: the conidia suspension was inoculated in SDAY liquid medium after being treated at a high temperature between 34-50°C for a certain period of time, the results showed that 34-42°C Within the range, heat treatment for 40min and 60min had no effect on the germination rate of Beauveria bassiana spores, and even increased the germination rate in the range of 38-40°C. In the range of 44-52°C, the germination speed of spores slows down significantly after treatment for 40 minutes, but the germination rate can still reach about 80% within 60 hours; heat treatment for 60 minutes has a great influence on the germination of spores, and the germination speed and germination rate increase with temperature The germination rate of each treatment was about 20% at 72h.

(2), the effect of nutrition on the growth and development of Beauveria bassiana HFW-05 strain: 11 kinds of nitrogen sources (peanut cake powder, cottonseed powder, bean cake powder, fish meal, yeast powder, beef extract, peptone, urea, potassium nitrate, sulfuric acid ammonium, ammonium nitrate) and 14 carbon sources (Tween-20, Tween 80, CMC (sodium carboxymethyl cellulose), glycerin, mannitol, potassium carbonate, sodium carbonate, corn starch, potato starch, sweet potato starch , glucose, sucrose, maltose, lactose) on the germination rate, growth rate, sporulation and exosporal protease activity of HFW-05 strain. The results showed that the growth indexes of HFW-05 strain in organic source were higher than those in inorganic source. Among the tested carbon sources, the utilization of sucrose and glucose was the best, and the sporulation yields were 16.13×10 ⁷ spores/cm ² and 12.51×10 ⁷ spores/cm ² . The survey results of enzyme production showed that the enzyme activity of adding sucrose was the highest at 117.38μg·min ^-1 ·ml ^-1 , followed by glucose at 108.99μg·min ^-1 ·ml ^-1 ; the utilization of polysaccharide starch was significantly worse than that of single sugars and disaccharides. The utilization of inorganic carbon potassium carbonate and sodium carbonate by HFW-05 strain was poor, and the spore production and enzyme activity were far lower than those of the basal medium. In addition to conventional carbon sources, strain aids also had certain effects on the growth of Beauveria bassiana. Tween-20, Tween-80 and glycerol (tricarbonyl alcohol) promote the germination of Beauveria bassiana spores to a large extent, which can make the germination rate of the bacterial strain almost reach 100% within 24 hours, and the spore production is also high. Definitely improve.

Among the tested nitrogen sources, the growth rate of the strains in the medium added with organic nitrogen sources was significantly higher than that with inorganic nitrogen sources, and the culture medium added with bean curd powder had the highest sporulation yield, which was 15.62×10 ⁷ spores/cm ² , Followed by fish meal, cottonseed meal. The enzyme production was the largest when adding bean curd powder and yeast extract, which were 75.17μg·min ^-1 ·ml ^-1 and 74.01μg·min ^-1 ·ml ^-1 , respectively, and there was no significant difference between them. Inorganic nitrogen sources (urea, ammonium sulfate, ammonium nitrate) inhibited spore germination and growth to a certain extent, and urea and ammonium sulfate produced enzymes that were slightly higher than those of the control, but the difference was not significant; It can promote the germination and growth of spores, and the enzyme production activity is significantly higher than that of the control, and the difference is significant.

Adding 1% peptone to the basal medium of the tested carbon source, in addition to potassium carbonate and sodium carbonate, can promote the growth of Beauveria bassiana to a certain extent, and increase the spore production by 3-8 times. After adding 1% glucose on the basis of the tested nitrogen source, the spore production of the strains increased about 7 times, but there was no obvious promotion effect on the medium added with urea and ammonium sulfate.

(3) The result of culturing after ultraviolet light irradiation on the spore suspension shows that a certain intensity of ultraviolet light has no adverse effect on the germination of spores within 1 h, and even has a certain stimulating effect on the germination of spores. 4h), the spore germination rate decreased significantly until death.

3. Rejuvenation of strains

In order to maintain the insecticidal activity of the strains, the rejuvenation of the strains is carried out regularly. Prepare the spore suspension of the HFW-05 strain, immerse the young host insects in the suspension for 15 seconds, and continue to raise them to ensure a relative humidity of more than 90% within 24 hours, and ensure a humidity of more than 70% in the future. After the infected insects die, pick out the dead insects and continue to moisturize. After the growth of the Beauveria bassiana mycelia, pick the mycelia and transfer them to the culture medium after the insects are sterilized, and measure the spore production and toxicity to insects. Repeat rejuvenation if necessary.

4. Strain fermentation

(1) Activation of strains

Using the SDAY medium mixed with 1% peptone, 1% yeast extract, 4% glucose and 2% agar, the Beauveria bassiana strain HFW-05 was inoculated on the surface of the SDAY medium, and the temperature was maintained at 26 ℃, cultured for 5 days, and the activated HFW-05 spore powder was produced.

(2), the cultivation of seed solution

Seed liquid culture uses SDAY liquid medium mixed with 1% peptone, 1% yeast extract and 4% glucose. Under sterile conditions, scrape the activated HFW-05 spore powder and use sterile physiological The brine was prepared into a bacterial suspension with a concentration of 1×10 ⁸ spores/ml, inoculated in a 14-liter liquid fermenter with a 0.2% inoculum size, kept at a temperature of 26° C., and a rotational speed of 170 rpm, and cultivated for 1 d as a seed solution.

(3), solid-phase fermentation

The fermentation medium is rice, soak the rice in water for 3-4 hours, remove the excess water, then add 5ml of edible oil to 1kg of rice, and mix well.

Fermentation utensils: air-permeable plastic bags with a size of 50×40cm.

Fermentation method: put 200 grams of the above-mentioned rice with edible oil into a plastic bag, tie the mouth of the bag tightly with a string, put it into a sterilizing pot, and sterilize it for 30 minutes at a temperature of 121°C. Inject 10ml of seed solution into each bag in the ultra-clean workbench, and place them on the culture rack in a constant temperature room at 26°C after inoculation, and cultivate for 5 days. The above cultures were collected and dried indoors. Then collect the spore powder with a 200-mesh vibrating sieve.

5. Control effect

(1), Indoor toxicity to Bemisia tabaci

Raise whiteflies in the greenhouse covered with mesh cages to the stage of adult ovulation. Place the potted tomato seedlings at the 5-6 leaf stage in the breeding cage, let them lay eggs, take them out after 1 day, and raise them at 25°C. The larvae grow and develop to 1 After the 2nd instar, after investigating the population base, immerse the tomato leaves carrying Bemisia tabaci larvae in spore suspensions of various concentrations for 30 seconds, dry naturally and place them under the specified experimental conditions, and check the number of dead insects every day after inoculation until all whiteflies die or develop into adults. Simultaneously, treat Bemisia tabaci with 0.5% (V/V) OP emulsifier water as contrast, calculate the death rate with the person who dies after infection and becomes white (all following biological assay results adopt this statistical method), and each treatment is 2 leaves , set 3 repetitions.

On the 3rd day after inoculation with HFW-05, the whitefly nymphs of each treatment had no infection symptoms; on the 4th day, the infected nymphs were mostly light yellow; on the 5th day, the infected nymphs had white hyphae; As the concentration of spores increased, the mortality rate also increased accordingly.

Determination of Insecticidal Toxicity of HFW-05 Strain to Bemisia tabaci

(2), determination of toxicity to diamondback moth

Pick the same size, healthy and lively Plutella xylostella larvae at the end of the first instar as the test insects, prepare conidia into suspensions with different concentrations, spray them on the surface of the larvae of the first instar Plutella xylostella respectively, and absorb them with filter paper. More than water, cultivated at 25°C, and treated the first instar larvae with 0.5% (V/V) OP emulsifier water at the same time as a control, and calculated the mortality rate. For each treatment of 30 larvae, 3 repetitions were set. The results are shown in the table below. On the second day after inoculation with HFW-05, the Plutella xylostella larvae in each treatment died but no obvious infection symptoms were seen; on the fourth day, white hyphae could be seen on the surface of the infected larvae; the cumulative mortality rate on the sixth day at a concentration of 1×10 ⁶ spores/ml Only 31.8%, the virulence effect is not significant; but as the spore concentration increases, the mortality rate increases accordingly. At the concentration of 1×10 ⁸ spores/ml, the cumulative mortality rate on the 6th day could reach 96.6%.

Table 3 The results of the determination of the toxicity of different conidia concentrations to Plutella xylostella

(3), field control results:

A test and demonstration base has been established in Dingzhou City, the main vegetable producing area in Hebei Province. The primary preparation of Beauveria bassiana was prepared by fermentation, and field experiments were carried out for the control of whitefly. The effect is more than 70%.

Claims (3)

1. a beauveria bassiana (Beauveria bassiana) HFW-05 bacterial strain, preserving number is CGMCC No.2356.

2. the fermentation process of the described beauveria bassiana HFW-05 of claim 1 bacterial strain, it is characterized in that: it may further comprise the steps:

(1), actication of culture

Utilization is by 1% peptone, 1% yeast extract paste, 4% glucose and 2% the composite SDAY substratum of agar, beauveria bassiana bacterial strain HFW-05 is inoculated in SDAY to be cultivated on the basal plane, keep 25-27 ℃ of temperature, cultivate 5-7d, produce the spore powder of the good HFW-05 of activation;

(2), culture of seed liquid

Seed liquor is cultivated and to be used by 1% peptone, 1% yeast extract paste and 4% the composite SDAY liquid nutrient medium of glucose, under aseptic condition, scrapes the spore powder of getting the good HFW-05 of activation, and being mixed with concentration with stroke-physiological saline solution is 1 * 10 ⁸-5 * 10 ⁸The bacteria suspension of spore/ml is inoculated in 14 liters of liquid fermentation tanks with the inoculum size of 0.2-0.5%, and keeping temperature is 25-27 ℃, and rotating speed is 150-250rpm, cultivates 1d as seed liquor;

(3), solid phase fermentation

Fermention medium is a rice, soaks rice 3-4h with clear water, pulls control out and removes excessive moisture, adds the ratio of 5ml edible oil again in the 1kg rice, mixing;

The fermentation utensil: size is the plastics bag with ventilation property of 50 * 40cm;

Fermentation process: the rice 200-300 of above-mentioned adding edible oil is restrained in the plastics bag of packing into, tighten sack, in the Autoclave of packing into, under 121 ℃, sterilized 30 minutes with cotton rope; Every bag is inserted seed liquor 10ml in Bechtop, and the single lay in inoculation back is cultivated 5-7d on the culturing rack of 25-27 ℃ thermostatic chamber; With above-mentioned culture collection, in indoor airing; Collect spore powder with 150-200 purpose Vibrationsifter again.

3. the described HFW-05 bacterial strain of claim 1 is in the application of control aleyrodid, small cabbage moth and aphid insect.