CN112266877B - Helminthosporium rosthornii mutagenesis and application thereof in preventing and treating moleplant seed - Google Patents

Abstract

The invention discloses a mutant Helminthosporium rosthornii and application thereof in preventing and treating moleplant seed. The strain is classified and named as Exserohilustratum Y9511-X050, the preservation number is GDMCC NO:60804, and the strain is preserved in Guangdong province microbial strain preservation center of No. 59 building and No. 5 building of Michelia Tokyo No. 100 college in Guangdong province, guangzhou City in 2019, 10 and 8 days. The strain is obtained by chemical mutagenesis of a Helminthosporium rosthornii Y9511 strain separated from a paddy field moleplant seed leaf, is safe and harmless to rice, has a control effect on moleplant seed, is safe in application, and is suitable for biological control of the paddy field moleplant seed.

Description

A strain of Mutagenic Helminthrospora rostatus and its application in the control of Aurantia chinensis

technical field

The invention belongs to the field of microbes, and in particular relates to a mutagenized Helminthrospora rostralis and its application in the prevention and treatment of Aurantia chinensis.

Background technique

Weeds are important plant resources in agricultural ecosystems, which compete with crops for water, nutrients, light, and space; reduce the quality of agricultural products; hinder crop harvest; threaten the security of world food production and cause economic losses. Therefore, weed control has become an enduring subject in the field of agricultural production. Leptochloachinensis (L.) Nees is an annual grass weed belonging to the genus Leptochloachinensis. Its distribution is almost all over the world's major rice producing areas. In my country, it is mainly distributed in the Yangtze River Basin and various provinces to the south, and it is also distributed in Shaanxi. With the large-scale popularization of rice light cultivation techniques and the extensive use of selective herbicides, the weed community structure in rice fields has undergone tremendous changes. Qianjinzi has spread rapidly in southern rice areas and has become a malignant weed second only to barnyardgrass. Weeds are even more harmful than barnyard grass in some rice fields, seriously affecting the growth and yield of rice, and posing a serious threat to food security.

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

Microbial herbicide refers to a microbial preparation that can use plant pathogenic microorganisms or their metabolites under human-controlled conditions to make target weeds susceptible to disease and lethal. Compared with chemical herbicides, microbial herbicides have abundant resources and are not easy to It produces resistant weeds, is safe to non-target organisms, has good economic benefits, and has good environmental compatibility. Fungi, bacteria, viruses, and actinomycetes are potential microbial resources that can be used for microbial herbicides. A number of biological herbicides have been successfully commercialized and these products help to solve problems related to the prevention and control of some noxious weeds. Worldwide, there are 13 bioherbicides on the market that are derived from microorganisms or natural molecules. 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 biological herbicides is still growing. Therefore, accelerating the discovery and development of biological herbicides has important practical value and far-reaching significance to ensure the sustainable development of agriculture.

Excellent microbial strains are the basis and key of the fermentation industry, but wild-type microorganisms directly used as biological agents usually have some disadvantages, such as slow growth, low sporulation and low potency. Therefore, microbial mutation breeding is necessary for the development of fungal herbicides.

The present invention aims to provide a mutagenic Helminthrospora rostatus and its application in preventing and treating D. chinensis.

Contents of the invention

The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art, and provide a strain of Umbilocystis sp.

Another object of the present invention is to provide the application of the mutagenized Umbilocystis rostatus.

The object of the present invention is achieved through the following technical solutions:

A mutagenized strain of Umbilocorium rostralis, named Exserohilumrostratum Y9511-X050, with a preservation number of GDMCC NO:60804, which was preserved in Guangzhou, Guangdong Province on October 8, 2019 Guangdong Microbial Culture Collection Center (GDMCC) on the 5th Floor, Building 59, Compound, No. 100 Xianlie Middle Road.

The present invention also provides a spore of the above-mentioned mutagenized Umbilatus rostralis.

The preparation method of the spores of the mutagenized Umbilocystis rostatus comprises the following steps: inoculating the mutagenized Umbilocystis rostis on the PDA medium, culturing at 28°C to 30°C, and then The hyphae were removed by filtration to obtain the spores of the mutagenized Helminthosporium rostralis.

The temperature of the cultivation is preferably 28°C.

The time of said culturing is preferably 14 days.

The filtering is filtering with gauze; preferably filtering with four layers of gauze.

The application of the spores of the mutagenic Umbilocystis rostis or the described mutagenic Umbilocystolum in the preparation of biological herbicides.

The said grass is a weed of the genus Stephania; preferably Paddy Stephania.

A biological agent for the biological control of Stephania chinensis, the active ingredient of which is the spores of the above-mentioned mutagenized Umbilocystis rostis and/or the above-mentioned mutagenized Umbilocystis.

The concentration of the spores of the mutagenized Umbilatus rostralis in the biological preparation is 10 ⁵ -10 ⁷ spores/mL; preferably 10 ⁶ spores/mL.

The biological preparation may also contain a pesticide acceptable carrier; the carrier includes an emulsifier, water and the like.

The emulsifier is preferably Tween 80.

The concentration of the emulsifier in the biological preparation is 0.5‰ by volume.

The application of the mutagenized Heldosporium rostralis, the spores of the mutagenized Umbilicalsporium rostatus, and/or the biological agent used for the biological control of D. chinensis.

Compared with the prior art, the present invention has the following advantages and effects:

(1) In order to solve the polluted environment and unsafe problems of chemical control in the prior art, the present invention provides a kind of mutagenous Helminthrospora rostralis bacterial strain Y9511-X050, and this bacterial strain is obtained from the leaves of the daughter of a paddy field. The isolated Y9511 strain is obtained through chemical mutagenesis, is safe and harmless to rice, and has a control effect on A. chinensis, is safe in use, and is suitable for biological control of A. chinensis in paddy fields.

(2) The method of use is simple and practical: make the water emulsion of Helminthrospora rostralis Y9511-X050, and the process is simple and practical.

(3) The control effect is remarkable and stable: the spore suspension of Umbilatus rostralis Y9511-X050 strain has a significant control effect on A. chinensis in the stage of two leaves and one heart, and the disease index of the pot test reaches more than 99%.

Description of drawings

The invention will be further described by using the accompanying drawings, but the embodiments in the accompanying drawings do not constitute any limitation to the present invention. For those of ordinary skill in the art, on the premise of not paying creative work, they can also be obtained according to the following drawings: Additional drawings.

Fig. 1 is after chemical mutagenesis of the Umbilophila rostralis strain Y9511 of the present invention, by considering the same weight of four indexes, sorting by the largest principal component and mapping the ordinal numbers to the ordinal numbers one by one to screen the best mutagenized strain .

Fig. 2 is the spore morphology figure of the umbilicalspora bacterial strain Y9511-X050 and starting strain Y9511 of the present invention; wherein, figure a is the umbilicalspora bacterial strain Y9511; bacterial strain Y9511-X050.

Fig. 3 is the mycelia morphology diagram of the umbilical sp. rostral strain Y9511-X050 and the starting strain Y9511 of the present invention; wherein, figure a is the umbilical sp. rostral strain Y9511; bacterial strain Y9511-X050.

Fig. 4 is the bacterium colony morphology figure of the umbilical sp. rostral bacterial strain Y9511-X050 and starting bacterial strain Y9511 of the present invention; Wherein, figure a is the umbilical sp. bacterial strain Y9511-X050.

Fig. 5 is the rice safety figure of Umbilocorium rostralis bacterial strain Y9511-X050 and starting bacterial strain Y9511 of the present invention; Wherein, Fig. a is Umbilocordomonas rostatus bacterial strain Y9511; Helminthes sp. strain Y9511-X050.

Detailed ways

The present invention is further described in conjunction with the following examples.

The present invention will be further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field. Unless otherwise specified, reagents, culture medium, etc. used in the present invention can be obtained commercially; wherein:

The formula of the PDA medium is: 40.1 g of potato dextrose agar (PDA), 5 g of agar, dissolved in 1 L of water.

The formula of potato sucrose liquid culture medium is as follows: take 250g of potatoes, boil them in cold water and filter them with gauze to obtain the filtrate, and set the volume to 1000mL.

The mutagenesis screening of embodiment 1 Umbilatus rostralis bacterial strain

1. Screening for the best time for mutagenesis of Umbilocys rosatus

Use a sampler to pick out the mycelia of Umbilocystis roti Y9511 that have been cultivated on the plate for 5 days and grow vigorously around the colony, and insert it into the potato sucrose liquid medium with an appropriate amount of glass beads, and shake it at 28°C. Cultivate for 48 hours at a speed of 200r/min. After taking it out, suck 10mL of the bacterial solution and grind it fully in a sterile grinder to obtain the mycelium suspension. Take 2 mL of 0.2 mol/L pH4.6 acetate buffer solution in a test tube, add 1 mL of mycelia suspension and shake well, immediately add 1 mL of 0.2 mol/L sodium nitrite solution and mix evenly, so that the treatment concentration of nitrous acid is 0.05 mol/L, at room temperature for 5min, 10min, 15min, 20min, 25min, 30min, 35min, 40min respectively, after the time is up, add 2mL of 0.07mol/L Ph8.6Na ₂ HPO ₄ solution to terminate the reaction, and absorb the bacteria with different treatments Spread 0.1mL of liquid solution on PDA plate, 4 parallel samples in each group, take mycelium suspension without chemical mutagenesis as control, and calculate the lethal rate after culturing at 28°C for at least 3d (days) in the dark to avoid bacterial colony After growing up, mycelia cross-linked each other to affect counting, and the optimal mutagenesis time range was determined according to the lethality rate. After the colony within the optimal mutagenesis time range grows, use a sterile glass puncher to take an equal amount of bacterial block along the outer edge of the colony where the growth is vigorous, transfer it to the center of the PDA plate, and select 51 strains, numbered It is Y9511-X001, Y9511-X002, Y9511-X003...Y9511-X049, Y9511-X050, Y9511-X051.

Table 1 The relationship between the optimal time frame of chemical mutagenesis and the mortality rate

Note: The data in the table are mean ± standard error, and different capital letters represent significant differences at p<0.05 (the same below).

As shown in Table 1, when the chemical mutagenesis time is 20min, the mutagenesis lethality rate is 75% (the mutagenesis lethality rate is between 70%-80%, which is the best lethality rate), so 20min is selected as the best mutagenesis time .

2. Screening of highly pathogenic mutagenous strains of Umbilocystis sp.

By analyzing the four indicators of the initial strain and the mutant strain: 1. The colony diameter of the 1st-4th day of growth on the plate; 2. The amount of spore production on the 14th day; Select bacterial strains with fast growth, high spore production, fast susceptibility, and large lesions, and inoculate the slant. Store the slant at 4°C for re-screening.

Colony diameter: measured with a ruler (Table 2, 3, 4, 5);

Sporulation: hemocytometer measures the number of spores under a microscope (Table 6);

Detached leaf lesion size: measure lesion size (table 7) after inoculating spore suspension 3d on detached leaf;

Disease index: After cultivating 51 mutant strains on PDA for 14 days, they were washed from the petri dish with 0.5‰ (v/v) Tween 80 aqueous solution, and then four layers of gauze were used to filter out mycelia to obtain a spore suspension. The concentration of the prepared spore suspension is 1.0×10 ⁶ spores/mL (counted with a hemocytometer), sprayed on the living Azalea chinensis with two to three leaves and one heart stage, and using the starting strain as a control, the treated live Azalea chinensis is moisturized After culturing for 48 hours, they were moved into the greenhouse for growth, and each treatment was repeated 4 times. After 7 days, classify and investigate the incidence, and finally calculate the disease index and record it as the measurement result of the pathogenicity of the strain in vivo. As shown in Table 8 (only the data of pathogenicity significantly higher than the starting strains are listed in the table).

Grading standards for diseases of Qianjinzi:

Grade 0: The whole plant is disease-free and there are no spots on the leaves;

Grade 1: There are only small brown spots the size of pinpoints;

Grade 2: There are large brown spots on the leaves of the whole plant, and typical or oval necrotic lesions of about 1-2 mm are evenly distributed on the leaves, accounting for about 2% of the leaf area;

Grade 3: The whole plant has typical lesion, and the infected area is less than 10%;

Grade 4: There are typical lesion spots on the whole plant, and the infected area accounts for about 10% to 50%;

Grade 5: There are typical lesion spots on the whole plant, and the infected area accounts for about 51% to 75%;

Grade 6: There are typical disease spots on the whole plant, and all leaves are dead.

In the formula, i represents the disease level, n represents the number of plants with the disease level i, N represents the total number of plants under investigation, and 6 represents the highest level of plant disease. Significant analysis of the colony growth difference of the 1st day on the PDA medium between the starting strain and 51 mutagenized strains in table 2

Note: The data in the table are mean ± standard error, and different capital letters represent significant differences at p<0.05.

Table 2 Significant analysis of the difference in colony growth between the starting strain and 51 mutagenized strains on the PDA medium on the 1st day

Note: The data in the table are mean ± standard error, and different capital letters represent significant differences at p<0.05.

Table 3 Significant analysis of the colony growth differences between the starting strain and 51 mutagenized strains on the PDA medium on the 2nd day

Note: The data in the table are mean ± standard error, and different capital letters represent significant differences at p<0.05.

Significant analysis of the colony growth difference of the 3rd day on the PDA medium between the starting strain and the 51g mutagenic strain in table 4

Note: The data in the table are mean ± standard error, and different capital letters represent significant differences at p<0.05.

Significant analysis of the colony growth difference of the 4th day on the PDA medium between the starting strain and 51 mutagenized strains in table 5

Note: The data in the table are mean ± standard error, and different capital letters represent significant differences at p<0.05.

Table 6 Significant difference analysis of sporulation production between the starting strain and 51 mutagenized strains on the 14th day

Note: The data in the table are mean ± standard error, and different capital letters represent significant differences at p<0.05.

Table 7 Determination of the pathogenicity of the starting strains and 51 mutagenized strains on the detached leaves of D. japonicus

Note: The data in the table are mean ± standard error, and different capital letters represent significant differences at p<0.05.

Table 8 The disease index of the starting strains and 51 mutagenized strains to the live Phalaenopsis chinensis (indoor determination)

Note: The data in the table are mean ± standard error, and different capital letters represent significant differences at p<0.05.

According to the four indicators of the strain after the mutagenesis: the comprehensive consideration of the diameter of the colony, the amount of sporulation, the size of the lesion on the detached leaf, and the disease index, the strain with the best traits was screened out. For the above 4 indicators, we consider the same weight. Because the distribution of each index is different and not uniform (uniform) distribution, we calculate the ordinal number (ie rank) of each index for more analysis later. Due to the significant correlation between the indicators, we used principal component analysis (PCA) method to find the largest principal component, and use it to measure the pros and cons of strain traits. We sorted the maximum principal components of the 4 indicators of the strains and mapped the ordinal numbers to the (0,1) score interval one by one, and the closer the score is to 1, the better the strain's traits. In order to check the sensitivity (sensitivity) between each observation and the final score, we use the Monte Carlo method (Monte Carlo, MC, method) to perturb each observation within its biologically reasonable value range (perturbation) . The stability of the final fraction of strains was checked and a 95% confidence interval (95% CI) was generated.

The genetic stability of embodiment 2 Umbilatus rostralis bacterial strain Y9511-X050

Transfer the screened mutant strain of Umbilatus rostralis to the slant, and after the mutant grows for 3 to 4 days, transfer it to fresh PDA medium for cultivation as the first generation; repeat The above steps are until the 10th generation, and the colony growth diameter (4d), spore production (14d) and the disease index (Table 9) of the 7d after spraying are measured for each generation of strains.

Table 9 Genetic Stability of Umbilamispora rostralis Y9511-X050

Note: The data in the table are mean ± standard error, and different capital letters represent significant differences at p<0.05.

As shown in Table 9, after 10 generations of subculture, the diameter and length of the colonies of each generation of Umbilocys rostralis strain Y9511-X050 were stable; the yield of conidia did not degenerate and was basically stable; Inhibitory effect (method is the same as the investigation of the disease index in embodiment 1), and the disease index does not appear to degenerate under the situation. In summary, the results showed that the mutagenized Umbilatus rostatus strain Y9511-X050 had good genetic stability.

Embodiment 3 identification of Umbilatus umbilicale strains

1. Morphological characteristics of Umbilophila rostatus strain Y9511-X050

(1) Using the petri dish culture method, inoculate the strains Y9511 and Y9511-X050 on the PDA medium, and cultivate them at 28°C. After the tissue grows, isolate single spores, pick a single colony and purify it on the PDA medium. Used for classification identification. Pick the purified bacterial strain and place it on a glass slide with an appropriate amount of sterile water, observe the strain morphology on the microscope line, and use the starting strain Y9511 as a contrast, its spore and hyphae morphology are shown in Figure 2 and Figure 3, from Figure 2a, As can be seen in Figure 3a, the mycelium of Y9511 is grayish brown to dark brown, irregularly branched, and the conidia are 50-120×12-22μm in size, with many septa, and most of them have 6-15 septa. The middle and lower parts are slightly thicker, long beak-shaped, blunt and rounded at both ends and lighter in color, around the umbilical point and at the top. It can be seen from Figure 2b and Figure 3b that, unlike Y9511, the conidia of Y9511-X050 are 50-122×10-22μm, and the hyphae and conidia are darker than the original strain Y9511.

(2) Pick the purified strain and put it on a new PDA medium, culture it at 28°C for 14 days, observe the shape and color of the colony, as shown in Figure 4a and 4b. The colonies of Umbilatus rostralis Y9511 and Y9511-X050 are round, spreading, and have aerial hyphae. The initial hyphae are white and then turn grayish brown to dark brown. Sporulation begins at about 7 days. The spores gradually increased until they spread to the edge, and the edge of the colony was still gray-brown; the back of the colony was also gray-black. The difference is that the aerial hyphae of Y9511 are longer, while those of Y9511-X050 are shorter.

2. Molecular biological identification of Umbilocortium rostatus strain Y9511-X050

(1) Genomic DNA was extracted according to the SK8259 (fungus) kit.

(2) PCR amplification

PCR reaction system:

Reagent Volume (μl) Template (genome DNA 20～50ng/μl) 0.5 10×Buffer(with Mg²⁺) 2.5 dNTP (2.5mM each) 1 enzyme 0.2 ITS1 or ITS1(10uM) 0.5 ITS2 or ITS2(10uM) 0.5 Add double distilled H₂O to 25

PCR cycling conditions:

(3) Use primers

ITS1: 5'-AGAAGTCGTAACAAGGTTTCCGTAGG-3';

ITS2: 5'-GCTGCGTTTCTTCATCGATGC-3';

as well as

Brn1: 5'-GCCAACATCGCAAACATGG-3';

Brn2: 5'-GCAAGCAGCACCGTCAATACCAAT-3'.

Using the genomic DNA of strains Y9511 and Y9511-X050 as templates, their ITS and Brn sequences were amplified to lengths of 257bps, 860bps and 268bps, 860bps, respectively, and the amplified sequences of the strains were uploaded to the NCBI website for BLAST comparison. The results showed that the ITS and Brn gene sequences of strain Y9511-050 had 99.40% homology with Exserohilumrostratum. By observing the conidia and hyphae morphology of bacterial strain Y9511, as well as molecular identification results, the bacterial strain was identified as Umbilocystis rostralis. We uploaded the sequence data to GenBank and obtained accession number: BankIt 2272786 (Seq1MN587722).

The strain was named Umbilophila rostralis Y9511-X050, and it was preserved on October 8, 2019 at the Guangdong Provincial Microbiological Culture Collection Center (referred to as GDMCC), the deposit number is GDMCC NO:60804.

Example 4 The safety of Umbilocorcus rostralis strain Y9511-X050 to rice

1. Preparation of spore suspension of Y9511-X050 strain

Inoculate the Y9511-X050 strain into a 9 cm petri dish containing PDA medium, culture it at 28°C for 14 days, wash it off the petri dish with 0.5‰ (v/v) Tween 80 aqueous solution, and pass through four layers The mycelia were filtered out with gauze to obtain a spore suspension, and the concentration of the prepared spore suspension was 10 ⁶ spores/mL.

2. Rice for cultivation experiment

The rice variety is Ruanhuayou 6100 (purchased from Guangdong Huanongda Seed Industry Co., Ltd.), cultivated in a constant temperature incubator at 28°C until the seeds emerged, and then transplanted into pots, with 4 rice plants per pot, repeated 4 times, Grow outdoors.

3. Test method

The rice was sprayed with the spore suspension of the above configuration, and the rice treated with the starting strain and the water containing Tween (the concentration of the Tween aqueous solution was 0.5‰ (v/v)) was used as a control ( The spraying amount is 80mL/m ² ). The treated live rice was cultured in moisture for at least 48 hours, and then moved into the greenhouse for growth, and each treatment was repeated 4 times. After 7 days, the disease was graded and the disease was investigated, and the disease was graded and assigned according to the degree of disease on the leaves of the treated plants.

The grading of different rice disease degrees refers to the grading standards of Chen Yong (2001). The specific rice disease grading standards are as follows:

Grade 0: Rice has no disease, represented by NS;

Grade 1: Rice leaves have diseased spots but no more than 1/2 of the whole leaf, represented by HS;

Grade 2: The whole leaf of rice is dead, indicated by S.

4. Test results:

Through the above tests, the safety of strains Y9511 and Y9511-X050 to rice was determined. The test results are shown in Table 10 and Figure 5. The results showed that neither strain Y9511-X050 nor strain Y9511 infected rice. The experiments showed that when the mutagenized strain Y9511-X050 and strain Y9511 were used in paddy fields, their host range was specific.

Table 10 The safety (indoor) determination of highly pathogenic mutagenic strains to rice

The above-mentioned embodiment is a preferred embodiment of the present invention, but the embodiment of the present invention is not limited by the above-mentioned embodiment, and any other changes, modifications, substitutions, combinations, Simplifications should be equivalent replacement methods, and all are included in the protection scope of the present invention.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than limiting the protection scope of the present invention, although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand , the technical solution of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solution of the present invention.

Claims (10)

1. A strain of mutagenic Exserohilumrostratum (Exserohilumrostratum) Y9511-X050, characterized in that: its preservation number is GDMCCNO: 60804, and it was preserved at No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province on October 8, 2019. Guangdong Provincial Microbial Culture Collection Center on the 5th floor of Building 59 of the hospital. 2. A spore of the mutagenic Umbilocystis rostralis Y9511-X050 according to claim 1. 3. the preparation method of the spore of the mutagenic Helminthrospora rostralis Y9511-X050 described in claim 2, is characterized in that, comprises the steps: inoculating the mutagenic Helminthosporium rostatus on the PDA medium, culturing it at 28° C. to 30° C., and then filtering and removing the hyphae to obtain the spores of the mutagenic Umbilocystis rostatus; The time of described culture is 14 days; Described filtration is to filter with gauze. 4. The application of the spores of the mutagenized Umbilocystis rostralis Y9511-X050 described in claim 1 or the spores of the mutagenized Umbilocystoris Y9511-X050 described in claim 2 in the preparation of biological herbicides. 5. The application according to claim 4, characterized in that: the grass is a weed of the genus Stephica. 6 . The application according to claim 5 , characterized in that: the control organism of the biological herbicide is the daughter of paddy field. 7. A biological agent for the biological control of Stephania chinensis, characterized in that: its active ingredient is the mutagenic Helminthrospora rostralis Y9511-X050 described in claim 1 and/or the decoy described in claim 2 The spores of Helminthromyces varizoris Y9511-X050. 8. The biological agent for the biological control of Stephania chinensis according to claim 7, characterized in that: the concentration of the spores of Umbilocystis rostralis Y9511-X050 mutagenized in the biological agent is 10 ⁵ -10 ⁷ spores/mL. 9. the biological agent for the biological control of Stephania chinensis according to claim 8, characterized in that: The biological preparation may also contain a pesticide acceptable carrier; Described carrier is emulsifying agent and water; Described emulsifying agent is Tween 80; The concentration of the emulsifier in the biological preparation is 0.5‰ by volume. 10. The new strain of the mutagenic Umbilicalspora rostralis Y9511-X050 described in claim 1 or the mutagenic Umbilicalsporium rostatus Y9511-X050 spores described in claim 2, and/or claims 7- 9. The application of any one of the biological agents for the biological control of D. japonicus in the prevention and control of D. japonicus.

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