CN113956981B - Pathogenic fungus with insecticidal activity and application thereof - Google Patents

Abstract

The invention relates to a white yeast strainPseudozyma flocculosaThe strain is preserved in China general microbiological culture Collection center (CGMCC) on 9, 2 and 2021 days, and the preservation number is CGMCC No.23345. The research of the invention shows that the white yeast strainPseudozyma flocculosaThe strain has high control effect on tetranychus, tarsonchus and aphid, can effectively control powdery mildew, has good economic and ecological benefits, has the characteristics of no toxicity to people and livestock, difficult generation of resistance to pests/pest mites and the like, and has good development and application prospects in the aspects of microbial preparations, biological pesticides and the like.

Description

Pathogenic fungus with insecticidal activity and application thereof

Technical Field

The invention belongs to the technical field of agricultural microorganisms, relates to an entomopathogenic microorganism strain and a control application thereof, and particularly relates to an entomopathogenic fungus with insecticidal and acaricidal activities and an application thereof.

Background

In the production process of vegetables, a plurality of pests and mites are harmed simultaneously, and serious economic loss is brought to crops. The harmful mites mainly comprise Tetranychus urticae, tetranychus truncatus, tarsonemus laterosporus and the like, and the pests comprise aphids, whiteflies, thrips and the like. The pests are tiny in size and difficult to identify by naked eyes in the field, and plant juice is absorbed by the piercing-sucking mouthparts to capture plant nutrition, so that damage is caused. Spraying chemical agents is a common mode of control against pests, but there are varying degrees of tolerance and resistance to chemical agents. Of particular concern is that the world-wide important agricultural pest mite, tetranychus urticae, is one of the most resistant and tolerant pests, and thus chemical control faces the most serious challenge. Meanwhile, harmful mites are a wide range of harmful crops, and more than 1100 host plants (Van leewen et al, 2010) including vegetables, fruit trees, cotton, corn and the like can be harmed by the harmful mites; the tetranychus urticae has the characteristics of short life cycle, strong fecundity, parthenogenesis and the like, and the characteristics cause the pests to generate different degrees of resistance to most insecticides, so that the chemical prevention and control of the pests become more difficult, and the negative effects of killing natural enemies, destroying ecology and the like caused by applying a large amount of chemical pesticides are also accompanied. Therefore, it is important to find a novel environment-friendly sustainable prevention and treatment technology, and the biological prevention and treatment means is increasingly emphasized due to the advantages of being green, safe, harmless and the like.

Researches on biological control of harmful organisms such as tetranychus urticae and the like mostly focus on the aspects of mining application of natural enemies such as predatory mites and the like, microbial control and the like. Entomopathogenic fungi are widespread in nature and, as part of the integrated pest management IPM project program, they are important biocontrol microorganisms that balance the number of insect populations in nature. At present, about 200 kinds of insect pathogenic fungi parasitizing arthropods are found in China, and besides Beauveria bassiana (Balsamo) Vuillemin, hirsutella thompsonii Fisher and the like which are used for developing fungal preparations for biological control of Tetranychus urticae, research on fungi related to mites is mostly focused on classification and ecological research, and is limited in biological control and especially on pathogenic research of mites, so that screening and applying fungi with insecticidal and acaricidal activities has important theoretical and practical significance for research and development of resistant pests such as aphids and Tetranychus urticae and harmless green control technologies of the mites.

In the early stage of research, the population of the tetranychus urticae which is bred in a laboratory is infected by an unknown bacterium, pathogenic bacteria are separated from infected mite bodies died from diseases, firstly, the species identification is carried out, then, the pathogenic potential of the pathogenic bacteria on the tetranychus urticae and other pest mites is researched, and scientific basis is provided for further research and development of biological prevention and control technology of the tetranychus urticae.

Disclosure of Invention

The invention aims to solve the technical problem of providing a material for developing a new fungal pesticide and providing an efficient strain for preventing and controlling pest mites.

The invention also aims to provide application of the strain in controlling tetranychus urticae and other pest populations.

In order to achieve the purpose, the invention adopts the following technical measures:

a white yeast strain (Pseudozyma flucculosa) is characterized in that the white yeast strain is preserved in China general microbiological culture Collection center (CGMCC) in 2021 year, 9 months and 2 days, and the preservation number is CGMCC No.23345.

The invention further provides application of the white yeast strain in pest and mite control.

Preferably, said pest is aphid; the harmful mites are tetranychid mites and tarsal mites.

More specifically, the tetranychus urticae is tetranychus urticae.

In a particular embodiment, it is controlled by spraying a spore suspension of the strain on the leaves of the plant.

Preferably, the spore suspension of the strain has a concentration of 10 ⁸ ～10 ⁹ 。

Wherein the plant is a vegetable.

More preferably, the vegetables are eggplant, cucumber.

The invention also provides a preparation for preventing and controlling pests or mites, which contains the white saccharomycete as an active ingredient. More specifically, the white yeast exists in the form of spore suspension.

The spore suspension of the strain is sprayed on plant leaves with other pests and mites such as aphids, tarsonchus laterosomus and the like, the strain can also infect the aphids and the tarsonchus laterosomus and has high concentration of 10 ⁸ ～10 ⁹ Under the condition, the test population can be infected and killed within one week, which shows that the strain can widely infect pests and mites on vegetables which happen at the same time.

On one hand, the white yeast strain has excellent infection and pathogenic activity for testing tetranychus urticae population, and has biological control potential; on the other hand, the strain of the invention is also an entomopathogenic fungus, which not only has acaricidal activity to tetranychus urticae, but also can effectively infect other pests and mites such as aphids, tarsonchus laterosomus and the like, so that the strain shows broad-spectrum insecticidal activity and has great potential in field development and application. In addition, the strain of the invention belongs to the fungus, and the powdery mildew on the plant can be effectively prevented and controlled, so that the field application is expected to realize the effect of controlling diseases and pests, and the application prospect is huge. Meanwhile, the bacterial strain has the characteristics of environmental protection, safety, no toxicity to people and livestock, difficult resistance generation of pests and mites, and the like, meets the production requirement of green healthy agricultural products, and has potential development to become a novel microbial source insecticide and acaricide.

The white-like yeast strain Pseudomonas florcultosa has been preserved in China general microbiological culture Collection center (CGMCC) in 2021, 9 and 2 days, and the preservation number is CGMCC No.23345. The preservation address is as follows: xilu No. 1, beijing, chaoyang, beijing, and institute for microbiology, china academy of sciences.

Drawings

FIG. 1 culture characteristics of PFTu-X Strain on PDA Medium

FIG. 2 spore morphology of PFTu-X strain after 24h culture

FIG. 3 phylogenetic tree of PFTu-X strain constructed by adjacency based on ITS sequence

FIG. 4 symptoms of Tetranychus urticae infestation by the PFTu-X strain

FIG. 5 symptoms of aphid infestation by the PFTu-X strain

FIG. 6 symptoms of Tarsonemus dorsalis lateral infestation by the PFTu-X strain.

FIG. 7 shows normal eggplant and cucumber plants after spraying with the bacterial liquid.

Detailed Description

EXAMPLE 1 isolation and cultivation of PFTu-X Strain

The isolation culture of pathogenic bacteria adopts a tissue isolation method and is improved, and the pathogenic bacteria are collected from a naturally dead tetranychus urticae population which is subcultured in a laboratory. Rinsing with sterile water for 5 times indoors, inoculating to a potato agar solid medium (PDA) plate by using an inoculating needle, and culturing in a constant-temperature incubator at 26 ℃ for 2-3 days. The grown hyphae were picked up on a new PDA plate and purified 3 times. Inoculating the pure bacterial colony to the slant of PDA test tube, and storing in refrigerator at 4 deg.C after the slant is full of hypha. In the previous experiments, 2 strains with certain effect were isolated from a plurality of single colonies, and the sequence determination was consistent. The test result shows that the strain has certain infectivity and lethal activity to Tetranychus urticae Koch, the 1 st strain PFTu-1 has 10 ⁷ Concentration-treated leaf mites with mites after one week period, the corrected mortality rate for the treated spider mites was 44.45% below that of the same concentration (10) ⁷ ) The infestation lethal activity of the next 2. Sup. Nd strain PFTu-X on spider mites (Table 1).

TABLE 1 preliminary test of corrected mortality of Tetranychus urticae after bacterial liquid treatment

The strain which is screened to confirm the best effect is named PFTu-X. The strain is preserved in China general microbiological culture Collection center (CGMCC) at 2021, 9 months and 2 days, and the preservation number is CGMCC No.23345.

Example 2 morphological characterization of PFTu-X Strain

After single spore purification, the strain was inoculated into a flask containing sterilized Potato Dextrose Broth (PDB), and after shaking culture for 72h on a shaker at 28. + -. 1 ℃ C, an immersion slide was prepared from the fresh culture and observed under an inverted microscope (Olympus IX 53), and a photomicrograph of the characteristic structure was taken. The colonies were observed to be white and round, with villous protrusions and wavy peripheries (FIG. 1). The strain PFTu-X grows rapidly and without rhizoids, sporangiospores are spherical ovals or cylinders with accessory silks (FIG. 2), which are presumed to be Pseudozyma floccculose compared to the description in the previous report.

Example 3 phylogenetic Tree analysis of PFTu-X Strain

The clear thallus of 7d sporangium shell was scraped from PDA plate at 28 deg.C, and total DNA was extracted according to the instruction of plant genome DNA extraction kit (DP 305-02, tiangen Biochemical technology Co., ltd., beijing). Identifying a general primer ITS 1' TCCGTAGGTGAACCTGCGG-; ITS1 (supra)/ITS 4R 5-. Reaction system: 0.1U Taq E/mL 2 XEs Mix 10. Mu.L, ITS Primer Forward/Reverse Primer (10 mM) each 1. Mu.L, DNA 2. Mu.L, ddH ₂ O6. Mu.L. And (3) PCR reaction conditions: 4min at 94 ℃; 45s at 94 ℃, 45s at 55 ℃ and 1min at 72 ℃ for 35 cycles; extension for 10min at 72 ℃. The PCR amplification product is detected by agarose gel electrophoresis and then sent to Shanghai bioengineering GmbH for sequencing, the obtained sequence is submitted to NCBI database for BLAST analysis and comparison, the BLASTn analysis result shows that the sequence of rDNA-ITS region (sequence table 1) of the strain PFTu-X and the sequence of Pseudozyma floccuosa have 99.85 percent of similarity, and the strain is also shown to be Pseudozyma floccuosa. .

Constructing a phylogenetic tree based on rDNA-ITS zone sequence of the test strain: multiple alignments were performed using DNAMAN and the confidence of the Bootstrap nodes was checked 1000 times by the Neighbor Joining (NJ) method of MEGA7.0 software. And constructing a phylogenetic tree by taking the Ganoderma lucidum as an exogenously-clustered strain PFTu-X. It also clustered with Pseudozyma floccculose in the developing tree, indicating that its relationship was highly homologous (FIG. 3).

Therefore, the invention utilizes a tissue isolation method to separate a pathogenic bacterium PFTu-X, and the pathogenic bacterium PFTu-X is identified by morphology and molecular biology, and the strain is identified as Pseudozyma floccculosa.

Example 4 virulence assay of PFTu-X Strain against Tetranychus urticae

The test tetranychus urticae sensitive strain IPP-SS is provided by plant protection research institute of Chinese academy of agricultural sciences, and after the introduction, the population is placed in a greenhouse with the temperature of 26 +/-3 ℃ for feeding on a clean, insect-free and pesticide-pollution-free 'Bifeng' kidney bean plant.

According to the Koehz's LawThe pathogenicity of pathogenic bacteria is proved. Selecting 20 female two-spotted spider mite in room, adding into fresh leaf in culture dish, taking the pathogenic bacteria fermentation liquid after 7 days of culture, and preparing 10 respectively by using sterile water according to gradient dilution method ⁹ spore/mL, 10 ⁸ spore/mL, 10 ⁷ spore/mL, 10 ⁶ spore/mL bacterial liquid, the processing method comprises the following steps: spraying the leaves of the mites for three times by using a handheld watering can, and then feeding the mites in an artificial climate box with the temperature of 26.5 +/-1 ℃ and the relative humidity of 75%. Each treatment was set to 3 replicates. After inoculation, spider mite infestations and deaths were observed daily and the control group was treated with sterile water.

Mortality and corrected mortality were calculated according to the formula: mortality (%) = number of dead mites/number of mites before treatment × 100%; corrected mortality (%) = (treatment mortality-control mortality)/(1-control mortality) × 100%. The results show that the tested strains have higher toxicity to the female tetranychus urticae koch at 4 concentrations, and the death rate of each treatment tetranychus urticae koch is over 85 percent after 1 week of treatment, wherein the death rate is 8 multiplied by 10 ⁹ The strain has higher virulence activity to adult mites under the concentration of spores/mL. The corrected mortality rate of spider mites after 9 days of treatment was 100.00%.

The specific results are as follows:

the pathogenicity of pathogenic bacteria PFTu-X to tetranychus urticae is verified in a laboratory according to the Koehz's law, and the results are shown in Table 1: the group treated with pathogenic bacteria PFTu-X had adult mite death on day 1 after the treatment, 8X 10 of them ⁹ spore/mL treatment group, the corrected mortality was the highest after 1 day, 16.60%; the virulence activity of the pathogenic bacteria from 1 to 2 days of treatment is slow, and the corrected mortality rate is 3.57 to 19.23 percent; the number of deaths of the test spider mites increased rapidly from day 3 to day 9, with a corrected mortality rate rising from 30.61% to 100.00%. Maximum concentration of 8X 10 ⁹ The spore/mL treated group had the highest virulence activity against the two-spotted spider mite within the tested concentration range, and after 9 days of each treatment, the tested spider mites all infected the fungus and died (table 2). Meanwhile, the infestation symptoms of Tetranychus urticae in the test are consistent with the natural infection symptoms, the symptoms gradually increase from day 2, and the whole mite is surrounded by hyphae at day 5 (figure 4). Tetranychus urticae of the placebo group appeared natural on test day 2Death occurred, but no hyphae were grown from the dead mites of the control group throughout the experiment.

Example 5 virulence determination of the Strain PFTu-X Tarsonemus contralaterally

Spore suspensions obtained at different concentrations were cultured as described in example 4 and the virulence activity of the PFTu-X strain towards Tarsonemus contralateral mites was measured, showing that, over time, the Tarsonemus contralateral mites were progressively infested with the fungus (FIG. 5), with a progressively higher corrected mortality rate. 2 days after the bacterial solution treatment, the tarsonse mites died in the side of the disease, and the corrected mortality rate was 10 ⁹ In the concentration treatment, the concentration is 16.95%; on day 4, the corrected mortality rate for Tarsonemus dorsalis at the highest tested concentration was over 85%; day 6 of treatment, 10 ⁶ ～10 ⁹ In the spore/mL bacterial liquid treatment, the corrected mortality rates of Tarsonemus laterospini were 66.93%, 90.17%, 100% and 100% in this order (Table 3).

Example 6 determination of the virulence of the Strain PFTu-X on aphids

Spore suspensions of different concentrations were obtained by culturing as described in example 4 and the virulence activity of the PFTu-X strain on melon aphids was measured, which showed that, over time, the test melon aphids were progressively infected by the fungus, the infestation profile being shown in figure 6 and their corrected mortality rate increasing. 1 day after the bacterial liquid treatment, aphids begin to die in each treatment, and the highest corrected death rate is 10 ⁹ In the concentration treatment, the concentration is 11.50%; on day 4, the corrected mortality rate for each concentration treatment increased significantly, with the corrected mortality rate for the test insects at the highest tested concentration being 81.86%; treatment day 5, 10 ⁶ ～10 ⁹ In the spore/mL bacterial suspension treatment, the corrected mortality rates of the test insects were 91.55%, 96.98%, 100% and 100% in this order (Table 4).

EXAMPLE 7 crop safety experiments with strains

Subjecting PFTu-X pathogenic bacteria to treatment of 10 ⁷ And 10 ⁸ After the spore suspension liquid is respectively sprayed on the leaves of eggplant, cucumber and other plants by a hand-held watering can, the plants are continuously observed for 7-10 days, abnormal symptoms such as discoloration, wilting, whitening, growth retardation and the like or similar symptoms of phytotoxicity are not generated on the leaves of the plants,as shown in FIG. 7, the treated plants were not different from the normal growing plants, indicating that they are safe for crop plants.

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Claims (9)

1. White yeast strainPseudozyma flocculosa) It is characterized in that the strain is preserved in China general microbiological culture Collection center on the 2 nd 9 th 2021 with the preservation number of CGMCC No.23345.

2. The use of the white yeast strain of claim 1 for controlling aphids and spider mites.

3. The use as claimed in claim 2, wherein the spider mites are tetranychus urticae.

4. Use according to claim 2 or 3, for controlling by spraying a spore suspension of the strain on plant leaves.

5. The use according to claim 4, wherein the spore suspension of the strain has a concentration of 10 ⁸ ~10 ⁹ spores/mL.

6. The use of claim 4, wherein the plant is a vegetable.

7. Use according to claim 6, wherein the vegetable is eggplant or cucumber.

8. A preparation for controlling melon aphids or tetranychids, which comprises the white yeast as claimed in claim 1 as an effective ingredient.

9. The formulation of claim 8, wherein said white yeast species is in the form of a spore suspension.

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