A chemically induced strain of Helminthosporium spp. Q06 and its application

Abstract

The invention discloses a chemically induced Exserohilum rostratum Q60 and its application. The induced Exserohilum rostratum is classified and named as Exserohilum rostratum Y9511-Q06, and its deposit registration number is GDMCC No: 63100. The strain is obtained by chemical mutagenesis from the induced Exserohilum rostratum Y9511 strain isolated from the leaves of Leptochloa chinensis in rice fields, is safe and harmless to rice, has a control effect on Leptochloa chinensis, is safe in use, and is suitable for the biological control of Leptochloa chinensis in rice fields.

Description

Technical Field

[0001] The invention relates to a chemically induced Helminthosporium orientalis Q60 and also relates to the application of the induced Helminthosporium orientalis Q60, in particular to the application in preventing and controlling Leptochloa chinensis. Background Art

[0002] Weeds are important plant resources in agricultural ecosystems. They compete with crops for water, nutrients, light, and space; reduce the quality of agricultural products; hinder crop harvesting; threaten the safety of world food production and cause economic losses. Therefore, weed control has become an enduring topic in the field of agricultural production. Leptochloa chinensis (L.) Nees is an annual grass weed of the genus Leptochloa. It is distributed almost throughout the major rice-producing areas of the world. In my country, it is mainly distributed in the Yangtze River Basin and various provinces to its south, and is also distributed in Shaanxi. With the large-scale promotion of light rice cultivation technology and the extensive use of selective herbicides, the structure of weed communities in rice fields has undergone tremendous changes. Leptochloa chinensis has spread rapidly in southern rice-growing areas and has become a malignant weed second only to barnyard grass. In some rice fields, the harm has even exceeded that of barnyard grass, seriously affecting rice growth and yield, and posing a serious threat to food security.

[0003] Weed control strategies include manual weeding, mechanical weeding, cultivation measures, tillage systems, etc., but the main method used now is chemical weeding. The long-term and widespread use of chemical herbicides has led to the development of resistant weed populations, and the large accumulation of herbicide residues in the ecosystem has led to secondary pollution of the environment, which ultimately endangers human health. Therefore, the development of safe, efficient, green and environmentally friendly biological herbicides has attracted widespread attention from weed scientists.

[0004] Microbial herbicides refer to a type of microbial preparation that can make target weeds susceptible to disease and kill them by using plant pathogenic microorganisms or their metabolites under artificial control. Compared with chemical herbicides, microbial herbicides have the characteristics of abundant resources, not easy to produce resistant weeds, safe to non-target organisms, good economic benefits, and good environmental compatibility. Fungi, bacteria, viruses, and actinomycetes are potential microbial resources that can be used for microbial herbicides. Many bioherbicides have been successfully commercialized, and these products help solve problems related to the prevention and control of certain malignant weeds. Globally, there are 13 bioherbicides derived from microorganisms or natural molecules on the market. However, based on the need to improve the efficiency of weed control, the development of organic agriculture, and the public's increasing concern about environmental safety, the demand for bioherbicides is still growing. Therefore, accelerating the discovery and development of bioherbicides has important practical value and far-reaching significance for ensuring the sustainable development of agriculture.

[0005] Excellent microbial strains are the basis and key of the fermentation industry, but wild-type microorganisms directly used as biopharmaceuticals usually have some disadvantages, such as slow growth, low spore production and low cost-effectiveness. Therefore, microbial mutation breeding is necessary for the development of fungal herbicides. Summary of the invention

[0006] The first object of the present invention is to provide a chemically mutated Exserohilum rostratum Q60, which is classified and named as Exserohilum rostratum Y9511-Q06. The strain is mutated from the Exserohilum rostratum Y951 strain isolated from the leaves of Leptochloa chinensis in rice fields, and has the ability to control Leptochloa chinensis.

[0007] The Exserohilum rostratum Y9511-Q06 provided by the present invention has been sent to the Guangdong Provincial Microbiological Culture Collection Center for preservation on December 29, 2022, and received a culture collection registration number of GDMCC No: 63100. The preservation address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province.

[0008] Another object of the present invention is to provide a spore of the chemically mutated Helicoverpa orientalis Q60. The spore is obtained by culturing the chemically mutated Helicoverpa orientalis Q60.

[0009] Another object of the present invention is to provide an application of the chemically induced Helicoverpa orientalis Q60, specifically an application in the prevention and control of Leptochloa chinensis.

[0010] Specifically, it involves the use of chemically induced Helicospora orientalis Q60 in the preparation of a herbicide for controlling Leptochloa chinensis; it involves the use of spores of chemically induced Helicospora orientalis Q60 in the preparation of a herbicide for controlling Leptochloa chinensis.

[0011] Another object of the present invention is to provide a biological herbicide.

[0012] The biological herbicide comprises a fermentation liquid or a bacterial suspension or a fermentation product or a fermentation sterilization supernatant of chemically mutated Helicoverpa orientalis Q60. Alternatively, the biological herbicide comprises spores of chemically mutated Helicoverpa orientalis Q60.

[0013] The biological herbicide can be a water dispersant or a spore suspension, wherein the concentration of the spores of chemically induced Helicoverpa orifice Q60 is 10 5 ~10 7 Spores / mL.

[0014] The beneficial effects of the present invention are:

[0015] (1) The present invention provides a chemically mutated Helminthosporium orientalis Q60, namely, the Helminthosporium orientalis strain Y9511-Q06, which is obtained by chemical mutagenesis from the Y9511 strain isolated from the leaves of Leptochloa chinensis in rice fields. It is safe and harmless to rice, has a control effect on Leptochloa chinensis, is safe in use, and is suitable for the biological control of Leptochloa chinensis in rice fields.

[0016] (2) The control effect is significant and stable: The spore suspension of the Y9511-Q06 strain of Helminthosporium orientalis has a significant control effect on Leptochloa chinensis in the two-leaf and one-heart stage, and the disease index in the potted test reached more than 99%. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The germination of Helicoverpa orientalis spores after being treated with different EMS concentrations for 3 hours;

[0018] The data were analyzed using one-way ANOVA test. Different lowercase letters indicate significant differences at the P < 0.05 level, and the error bars are standard deviations.

[0019] Figure 2 The germination of Helminthosporium spores under different EMS concentrations and at different times;

[0020] CK is the blank control of phosphate buffer without EMS added; the data were analyzed by one-way ANOVA test, different lowercase letters indicate that the data reached significant differences at the P<0.05 level, and the error bars are standard deviations.

[0021] Figure 3 The difference in disease index of Helminthosporium orientalis Y9511 and its different mutant strains infecting Leptochloa chinensis.

[0022] Figure 4 The diameter of the lesions on the leaves of the original strain Y9511 and 20 mutant strains;

[0023] The data were analyzed using one-way ANOVA test. Different lowercase letters indicate significant differences at the P < 0.05 level, and the error bars are standard deviations.

[0024] Figure 5 The colony morphology of the Helminthosporium orifice strain Y9511-Q06 and the original strain Y9511 after 7 days of cultivation;

[0025] A: Colony morphology of the original strain Y9511 after 7 days of cultivation, B: Colony morphology of the mutant strain Y9511-Q06 after 7 days of cultivation.

[0026] Figure 6 The conidia morphology of the Helicoverpa orientalis strain Y9511-Q06 and the original strain Y9511;

[0027] A: conidia morphology of the original strain Y9511, B: conidia morphology of the induced strain Y9511-Q06.

[0028] Figure 7 The hyphae morphology of Helicospora orifice strain Y9511-Q06 and the original strain Y9511;

[0029] A: Mycelial morphology of the original strain Y9511, B: Mycelial morphology of the induced strain Y9511-Q06.

[0030] Figure 8 This is a diagram showing the difference in phenotypic infection spots between the Helicoverpa orientalis strain Y9511-Q06 and the original strain Y9511-X50;

[0031] A: Lesion morphology of Leptochloa chinensis 48 hours after infection with the mutant strain Y9511-X50, B: Lesion morphology of Leptochloa chinensis 48 hours after infection with the mutant strain Y9511-Q06.

[0032] Fig. 9 This is a comparison chart of rice safety tests on the Hierophora strain Y9511-Q06 and the original strain Y9511. DETAILED DESCRIPTION

[0033] Example 1 Mutagenesis screening of Helminthosporium orifice strains

[0034] 1. Study on the concentration and time conditions of EMS action

[0035] The spore solution in the beaker is evenly divided into sterilized 10mL centrifuge tubes, placed in a precooled centrifuge for centrifugation (10000rpm, 5min), and the supernatant is gently aspirated with a pipette to retain the spores after centrifugation. 8mL phosphate buffer (concentration of 0.1mol / L, pH=7.2, 0.05% Tween-80) with EMS concentrations of 0.2, 0.3, 0.4, 0.5, and 0.6mol / L is added to the spore collection tube, and an equal volume of phosphate buffer is used as a blank control. Cover the centrifuge tube lid tightly, wrap it with tin foil to avoid light, place the spore suspension in ice, and gently shake the centrifuge tube several times every 20 to 30min to allow the spores to fully contact the EMS mutagen.

[0036] After 1, 2, 2.5, 3 and 4 h of mutagenesis, the centrifuge tube containing the spore suspension was placed in a precooled centrifuge for centrifugation (10000 rpm, 5 min). After centrifugation, the supernatant was aspirated, the supernatant was discarded, and an equal volume of 5% Na 2 S 2 O 3The reaction of the EMS solution was terminated by adding an equal volume of sterile water to rinse 3 to 4 times to ensure that the EMS solution has been detoxified. Then 6 to 8 mL of sterile water was added and the spores were fully suspended by shaking.

[0037] Mix the spore suspension thoroughly, pipette 100 μL onto the prepared water agar medium plate, and carefully spread the spore liquid evenly with a sterilized triangular glass rod until the surface of the plate is dry. Place the water agar plate in a 28°C incubator and culture it. After 4 to 6 hours, check and record the spore germination status and number under an optical microscope.

[0038] After treating the spores of Helicoverpa orifice with 0.2, 0.3, 0.4, 0.5, and 0.6 mol / L EMS for 3 h, the spore germination rate was statistically analyzed. The results showed that when the EMS concentration was 0.2 mol / L, the spore germination rate was 56.67%, when it was 0.3 mol / L, the spore germination rate was 46.00%, when it was 0.4 mol / L, the spore germination rate was 31.33%, when it was 0.5 mol / L, the spore germination rate was 25.33%, and when it was 0.6 mol / L, the spore germination rate was 17.33% ( Figure 1 ). This shows that under the same treatment time, with the increase of EMS mutagen concentration, the germination rate of Helminthosporium orifice gradually decreases. The data show that the chi-square test was performed on the germination of Helminthosporium orifice after mutagenesis. The results showed that the treatment groups with different concentrations of EMS (0.2, 0.3, 0.4, 0.5, 0.6 mol / L) were significantly different from the blank control group (phosphate buffer without EMS) (P<0.05).

[0039] According to the best judgment standard of biochemical mutagenic dose, the shorter the treatment time, the smaller the effect on the spores. Therefore, EMS phosphate buffer with concentrations of 0.4, 0.5, and 0.6 mol / L was selected to treat the spores of Helicoverpa orientalis at different times.

[0040] The spores of Helicoverpa orientalis were treated with EMS phosphate buffer with concentrations of 0.4, 0.5, and 0.6 mol / L under 5 different time conditions (1h, 2h, 2.5h, 3h, and 4h). The phosphate buffer without EMS was used as a blank control. The spore suspension after each treatment was smeared on 3 water agar media, and after incubation at 28°C for 4h, the spore germination was observed under an optical microscope and the spore germination rate after treatment was calculated.

[0041] like Figure 2As shown in the figure, under the premise of the same EMS concentration, the germination rate of the spores of Helminthosporium orifice gradually decreases with the increase of treatment time. When the conidia of Helminthosporium orifice Y9511 were treated with EMS at a concentration of 0.5 mol / L for 4 hours, the spore germination rate was 26.00%, when the conidia were treated with EMS at concentrations of 0.5 and 0.6 mol / L for 3 hours, the spore germination rates were 26.67% and 20.67% respectively, and when the conidia were treated with EMS at a concentration of 0.6 mol / L for 2.5 hours, the spore germination rate was 21.33%. In the above treatments, the spore germination rate can reach about 20% to 30%, which meets the criteria for selecting the best mutagenic dose for biochemical mutagenesis, and the mutagenesis efficiency is the best.

[0042] One-way ANOVA was performed on the spores of Helicoverpa orioides treated with 0.4, 0.5, and 0.6 mol / L EMS phosphate buffer for different time periods. The results showed that the spore germination rates after treatment with 0.4, 0.5, and 0.6 mol / L EMS under five different time conditions (1h, 2h, 2.5h, 3h, 4h) were significantly different from those of the blank control group (phosphate buffer without EMS) (P<0.05).

[0043] In summary, since too high EMS concentration and too long treatment time will affect spore germination, 0.6 mol / L EMS phosphate buffer treatment for 2.5 h was selected as the optimal mutagenesis condition with the best mutagenesis effect.

[0044] 2. Screening of highly pathogenic mutagenic Helminthosporium strains

[0045] By analyzing the four indicators of the initial strain and the mutagenic strain:

[0046] 1. Colony diameter on the plate from the 1st to the 4th day of growth;

[0047] 2. Conidia production on the 14th day;

[0048] 3. The size of lesions on detached leaves;

[0049] 4. Disease index on the 7th day.

[0050] Select strains that grow fast, produce a lot of spores, are susceptible to disease quickly, and have large lesions, and inoculate the slant. Store the slant at 4℃ for rescreening.

[0051] Colony diameter: Pick a single colony from the water agar medium and plant it on a PDA plate medium. After labeling, place it in a biochemical incubator at 28°C in the dark for 3-4 days. Use a 7mm diameter puncher to punch a bacterial cake along the edge of the test colony, inoculate it in the center of the PDA medium, and culture it in a biochemical incubator at 28°C in the dark. Use a vernier caliper to measure the colony diameter every 24 hours.

[0052] Spore production: Take a piece of hyphae of the same size and age from the edge of the bacterial plate and inoculate it on a PDA plate. Culture it in the dark at 28℃ for 14 days. Use a pipette to slowly rinse the PDA plate with 10mL of sterile water and filter it into a conical flask with two layers of gauze. Take a small amount of spore suspension from the filtrate and drop it on a hemocytometer. Cover it with a coverslip and count the number of conidia under an optical microscope. Repeat three times and take the average value.

[0053] The size of the lesion on the detached leaves was determined by the needle puncture method. The upper surface of the leaf was punctured with a high-temperature sterilized needle. 2 μL of bacterial suspension was aspirated with a pipette and added to the wound surface. The leaf was cultured in a biochemical incubator at 28°C in the dark for 48 hours, and then the size of the lesion was measured with a vernier caliper.

[0054] Disease index: After culturing 51 mutant strains on PDA for 14 days, they were washed from the culture dish with 0.5‰ (v / v) Tween 80 aqueous solution, and then the mycelium was filtered through four layers of gauze to obtain a spore suspension. The concentration of the spore suspension was prepared to be 1.0×10 6 Spores / mL (counted with a hemocytometer) were sprayed on live Leptochloa chinensis at the two- to three-leaf and one-heart stage, and the starting strain was used as a control. The treated live Leptochloa chinensis was kept moisturized for 48 hours and then moved into the greenhouse for growth. Each treatment was repeated 4 times. After 7 days, the grading and disease situation were investigated. Finally, the disease index was calculated and recorded as the result of the determination of the pathogenicity of the living strain. As shown in Table 1-5 (only the data with significantly higher pathogenicity than the starting strain are listed in the table).

[0055] Disease classification standards for Leptochloa chinensis:

[0056] Level 0: The whole plant is disease-free and there are no spots on the leaves;

[0057] Grade 1: Only small brown spots the size of a pinpoint;

[0058] Level 2: There are large brown spots on the leaves of the whole plant, and there are typical or oval necrotic spots of about 1 to 2 mm evenly distributed on the leaves, accounting for about 2% of the leaf area;

[0059] Level 3: The whole plant has typical lesions, and the infected area is less than 10%;

[0060] Level 4: The whole plant has typical lesions, and the infected area accounts for about 10% to 50%;

[0061] Level 5: The whole plant has typical lesions, and the infected area accounts for about 51% to 75%;

[0062] Level 6: The whole plant has typical spots and all leaves are dead.

[0063]

[0064] In the formula, i represents the disease level, n represents the number of plants with disease level i, N represents the total number of plants surveyed, and 6 represents the highest disease level of the plants.

[0065] Table 1 Analysis of the significant differences in colony growth between the original strain and 20 mutant strains on PDA medium on the first day

[0066]

[0067]

[0068] Table 2 Analysis of the significant differences in colony growth between the original strain and 20 mutant strains on PDA medium on the second day

[0069]

[0070] Table 3 Analysis of the significant differences in colony growth between the original strain and 20 mutant strains on PDA medium on the 3rd day

[0071]

[0072] Table 4 Significant difference analysis of colony growth between the original strain and 20 mutant strains on PDA medium on the 4th day

[0073]

[0074]

[0075] Table 5 Significant difference analysis of conidia production between the original strain and 20 mutant strains on PDA medium on day 14

[0076]

[0077] Based on the three indicators of the strain after mutagenesis: colony diameter, spore production, and detached leaf spot size, the target strain was screened, and strains Q06 and Q19 were obtained as highly pathogenic strains, and strains Q10 and Q12 were obtained as low pathogenic strains. The disease index of strain X50 obtained by early mutagenesis was measured.

[0078] A disease index is calculated for the infection situation ( Figure 3 ), the disease index of the original strain Y9511 was 0.88, and the disease index of the mutant strains X50 and Q06 was 0.98. After 48 hours of infection, the lesions of Q06 were obviously larger, and the leaves lost green and wilted in a large area, which was better than Y9511-X50 (such as Figure 8 The disease index of Leptochloa chinensis infected by the mutant strain Q10 was only 0.76, while the disease index of Leptochloa chinensis infected by the mutant strains Q12 and Q19 reached 0.87, and the difference in disease index was extremely significant.

[0079] Example 2 Identification of Helminthosporium orifice strains

[0080] 1. Morphological characteristics of Helminthosporium orifice strain Y9511-Q06

[0081] On the 7th day after inoculation on PDA medium, the colonies of the mutant strain Q06 were darker in color than those of the original strain Y9511, the hyphae grew densely, the conidia were tightly attached to the surface of the colonies, and the colonies of the strain Q06 were black.

[0082] 2. Molecular biological identification of Helminthosporium orifice strain Y9511-X050

[0083] (1) Genomic DNA was extracted using the SK8259 (fungus) kit.

[0084] (2) PCR amplification

[0085] PCR reaction system:

[0086] Reagents Volume (μl) Template (genomic DNA 20~50ng / μl) 0.5 <![CDATA[10×Buffer(with Mg 2+ )]]> 2.5 dNTP (2.5 mM each) 1 Enzymes 0.2 ITS1 or ITS1(10uM) 0.5 ITS2 or ITS2(10uM) 0.5 <![CDATA[Add double-distilled H 2 O to]]> 25

[0087] PCR Cycling Conditions:

[0088]

[0089] (3) Use of primers

[0090] ITS1: 5'-AGAAGTCGTAACAAGGTTTCCGTAGG-3';

[0091] ITS2: 5'-GCTGCGTTCTTCATCGATGC-3';

[0092] as well as

[0093] Brn1: 5'-GCCAACATCGCAAACATGG-3';

[0094] Brn2: 5'-GCAAGCAGCACCGTCAATACCAAT-3'.

[0095] The genomic DNA of strains Y9511 and Y9511-Q06 was used as a template to amplify their ITS and Brn sequences, which were amplified to lengths of 231bps, 794bps and 231bps, 795bps, respectively. The amplified sequences of the strains were uploaded to the NCBI website for BLAST comparison analysis. The results showed that the ITS and Brn gene sequences of strain Y9511-Q06 were 99.60% homologous to those of Exserohilum rostratum. The strain Y9511 was identified as Exserohilum rostratum by observing the conidia and hyphae morphology of the strain and the molecular identification results.

[0096] Example 3 Genetic stability of Helicospora orifice strain Y9511-Q06

[0097] The screened mutant strain Y9511-Q06 of Helicoverpa orientalis (abbreviated as Q06 in the table) was transferred to the slant. After the mutant bacteria grew for 3 to 4 days, they were transferred to fresh PDA medium for culture, which was used as the first generation. The above steps were repeated until the 10th generation, and the colony growth diameter (4th day), spore production (14th day) and disease index of Leptochloa chinensis on the 7th day after spraying were measured for each generation of strains (Table 6).

[0098] Table 6 Genetic stability of Helminthosporium spp. Y9511-Q06

[0099]

[0100] As shown in Table 6, after 10 generations of subculture, the colony diameter length of each generation of Helminthosporium spp. strain Y9511-Q06 was stable; the conidia yield did not degenerate and remained basically stable; it had a good inhibitory effect on Leptochloa chinensis (the method was the same as the disease index survey in Example 1), and the disease index did not degenerate. In summary, the results show that the mutagenic Helminthosporium spp. strain Y9511-Q06 has good genetic stability.

[0101] Example 4 Safety of Helminthosporium orientalis strain Y9511-Q06 to rice

[0102] 1. Preparation of spore suspension of Y9511-Q06 strain

[0103] The Y9511-Q06 strain was inoculated into a 9 cm culture dish containing PDA medium and cultured at 28°C for 14 days. The strain was washed from the culture dish with a 0.5‰ (v / v) Tween 80 aqueous solution and filtered through four layers of gauze to remove the mycelium to obtain a spore suspension. The concentration of the spore suspension was prepared to be 10 6 Spores / mL.

[0104] 2. Cultivation of experimental rice

[0105] The rice variety was Ivory Rice, which was cultured in a constant temperature incubator at 28°C until the seeds germinated, then transplanted into pots, with 4 rice plants per pot, repeated 4 times, and placed outdoors for growth.

[0106] 3. Test methods

[0107] The spore suspension prepared above was sprayed on rice, and rice treated with the starting strain and rice treated with water containing Tween (the concentration of Tween aqueous solution was 0.5‰ (v / v)) without any bacterial suspension was used as a control (the spraying rate was 80 mL / m 2 ). The treated rice was kept moist for at least 48 hours before being moved into a greenhouse for growth. Each treatment was repeated 4 times. After 7 days, the rice was graded and the disease was investigated. The disease was graded and assigned values ​​according to the degree of disease on the leaves of the treated plants.

[0108] The classification of different degrees of rice diseases refers to the classification standard of Chen Yong (2001). The specific classification standards of rice diseases are as follows:

[0109] Level 0: Rice is disease-free, indicated by NS;

[0110] Level 1: Rice leaves have lesions but they do not exceed 1 / 2 of the entire leaf, indicated by HS;

[0111] Level 2: All rice leaves are dead, indicated by S.

[0112] 4. Test results:

[0113] Through the above test, the safety of strains Y9511 and Y9511-Q06 for rice was determined. The test results are shown in Table 7 and Fig. 9 The results showed that strains Y9511-Q06 and Y9511 did not infect rice. The experiment showed that the mutant strains Y9511-Q06 and Y9511 had a specific host range when used in paddy fields.

[0114] Table 7 Safety of highly pathogenic mutant strains to rice (indoor) determination

[0115]

Claims

1. A chemically induced mutagenesis strain of Exserohilumrostratum Y9511-Q60, It is characterized in that It was deposited on December 29, 2022 at the Guangdong Provincial Microbiological Culture Collection Center on the 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province, with the deposit number GDMCCNo: 63100.

2. The spores of the chemically induced Helicoverpa orientalis Y9511-Q60 according to claim 1.

3. Use of the chemically induced Helicoverpa orientalis Y9511-Q60 described in claim 1 in preventing and treating Leptochloa chinensis.

4. Use of the spores of the chemically mutated Helicospora orientalis Y9511-Q60 according to claim 2 in preventing and controlling Leptochloa chinensis.

5. Use of the chemically mutated Helicospora orientalis Y9511-Q60 according to claim 1 in the preparation of a herbicide for controlling Leptochloa chinensis.

6. Use of the spores of the chemically mutated Helicospora orientalis Y9511-Q60 according to claim 1 in preparing a herbicide for controlling Leptochloa chinensis.

7. A biological herbicide, Its characteristics are: A fermentation broth or a bacterial suspension thereof comprising the chemically mutated Helicoverpa orientalis Y9511-Q60 according to claim 1; Or it contains spores of the chemically mutated Helicoverpa orientalis Y9511-Q60 described in claim 2.

8. The bioherbicide according to claim 7, Its characteristics are: The biological herbicide is a water dispersant or a spore suspension.

9. The bioherbicide according to claim 8, Its characteristics are: The concentration of the spores of the chemically induced Helicoverpa orientalis Y9511-Q60 was 10 5 ~10 7 Spores / mL.

Images