Curvularia vermiculata o1-sf-04630 and application thereof in preventing and treating plutella xylostella

Abstract

The present application relates to the field of microorganism, specifically relates to the application of Tolypocladium inflatum O1-SF-04630 and its prevention and treatment of plutella xylostella, the preservation number of the Tolypocladium inflatum O1-SF-04630 strain (Tolypocladium inflatum O1-SF-04630) is CCTCC NO:M 20241094, the research has confirmed that the strain has strong pathogenicity to plutella xylostella, has the potential to be developed as biological control agent, and can be applied to the prevention and treatment of plutella xylostella pest.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microorganisms, in particular to Tolypocladium inflatum O1-SF-04630 and its application in the prevention and treatment of Plutella xylostella. BACKGROUND

[0002] Plutella xylostella L. belongs to Lepidoptera and Plutellidae, and is a worldwide important migratory pest of cruciferous vegetables and a devastating pest of cruciferous vegetables. With the use of broad-spectrum insecticides, the number and types of natural enemies are gradually decreasing, and the resistance of Plutella xylostella is becoming stronger and stronger, making the damage of Plutella xylostella more serious and the prevention and treatment more difficult. Therefore, it is an urgent need to seek green and sustainable control products. Fungal biocontrol products are green and environmentally friendly, harmless to humans and animals, and can form an epidemic in the insect population, which is a control measure that is vigorously promoted under the current green prevention and control policy. Biocontrol fungi that are effective in controlling Plutella xylostella mainly include Beauveria.sp, Metarhizium.sp and Isaria.sp. There is no report on the toxicity of fungi of the genus Tolypocladium to Plutella xylostella.

[0003] Fungi of the genus Tolypocladium can exist as saprophytes and as insect pathogens. The fungi have been isolated from soil and insects of Coleoptera (Cerambycidae), Lepidoptera (Noctuidae), Diptera (Apidae, Andrenidae, Halictidae) and Hymenoptera (Serphitidae, Formicidae). As of 2020, the International Mycological Database recorded 43 species of fungi of the genus Tolypocladium, of which Tolypocladium inflatum is the type strain of the genus Tolypocladium and also the production strain of the immunosuppressive drug Cyclosporins. Most of the research on the fungus is related to the production and yield of secondary metabolites, and Cyclosporins is the main research object. In addition, other secondary metabolites such as Ophiocordin (strong inhibitor of protease), efrapeptin, Aphidicolin, Chlamydosporol, etc. have also been studied and discussed, but the pathogenicity of the fungus itself to insects is rarely reported. SUMMARY

[0004] The purpose of the present application is to provide a strain of Tolypocladium inflatum.

[0005] Another purpose of the present application is to provide the use of the above-mentioned strain in the prevention and treatment of Plutella xylostella.

[0006] The Tolypocladium inflatum O1-SF-04630 strain according to the application is separated and purified from soil, and is identified as Tolypocladium inflatum through morphological and molecular biological analysis, and has been preserved in the China Center for Type Culture Collection (CCTCC) of Wuhan University on May 29, 2024, with a preservation number of CCTCC NO: M 20241094.

[0007] The application also provides application of the Tolypocladium inflatum O1-SF-04630 in prevention and treatment of Plutella xylostella, and a person skilled in the art can prepare the Tolypocladium inflatum O1-SF-04630 into a microbial insecticide for preventing and treating Plutella xylostella.

[0008] Compared with the prior art, the application has the following advantages:

[0009] (1) The application separates and purifies a strain, and determines that the obtained strain O1-SF-04630 is Tolypocladium inflatum through morphological observation and molecular identification; and secondly, the applicant also determines the spore production capacity of the strain and the tolerance of the strain to high temperature of 45 DEG C and ultraviolet.

[0010] (2) The application first detects the pathogenicity of Tolypocladium inflatum to Plutella xylostella, and the result shows that the strain has high pathogenicity and can effectively kill Plutella xylostella.

[0011] (3) The Tolypocladium inflatum O1-SF-04630 of the application is a biological control strain with potential significance on Plutella xylostella, and can be developed into a microbial insecticide, thereby providing a biocontrol material for green prevention and control of Plutella xylostella. BRIEF DESCRIPTION OF DRAWINGS

[0012] Figure 1 A phylogenetic tree of Tolypocladium strains constructed based on rDNA-ITS sequences is shown;

[0013] Figure 2 Morphological characteristics of Tolypocladium are shown;

[0014] Figure 3 Germination rates of Tolypocladium inflatum under high temperature and ultraviolet stress are shown;

[0015] Figure 4 Death rates of Plutella xylostella under treatment of Tolypocladium inflatum are shown, wherein a is a virulence curve of Tolypocladium inflatum on Plutella xylostella, and b is a graph of Plutella xylostella after infection.

[0016] The Tolypocladium inflatum O1-SF-04630 strain of the present invention is classified and named Tolypocladium inflatum O1-SF-04630, and was deposited in the China Center for Type Culture Collection (CCTCC) on May 29, 2024, with a deposit number of CCTCC NO: M 20241094. DETAILED DESCRIPTION

[0017] In the following examples, all results are expressed as mean ± standard error (SE), and the differences were considered significant when p < 0.05 using one-way analysis of variance using Duncan's multiple-range test (SPSS 26.0 version, SPSS).

[0018] Example 1: Isolation, purification and identification of strains

[0019] The soil isolation method of the strain is as follows: Weigh 10g of soil sample and add it to 90mL of sterile water and shake until the large soil particles are completely broken up, then let it stand for more than 30 minutes to clarify. Maintain sterile operation during the experiment to prevent miscellaneous bacteria from contaminating the soil bacterial solution. Gradient dilution is to dilute the original solution in ten-fold steps. Under sterile conditions, use a pipette to take 1mL of supernatant and add it to a test tube filled with 9mL of sterile water and shake it to dilute it to 10-1 times. Use different pipettes to take liquid from the diluted bacterial solution in turn, and gradient dilute the bacterial solution to 10 times the original solution. -1 , 10 -2 , 10 -3 Add penicillin (50 μg / mL), streptomycin (20 μg / mL), bacitracin (20 μg / mL), and tetracycline hydrochloride (20 μg / mL) to the separation medium cooled to 50-55°C and pour it into the plate. After the plate solidifies, spread it. -3 Gradient plating was performed. Three plates were plated per gradient. Culture was performed at 28°C for 3-5 days. Single colonies were selected and transferred to purification medium. Purification was performed three times and then stored at 4°C. Four strains of Toxoplasma were obtained after purification. After initial screening to determine pathogenicity, strain O1-SF-04630 was selected for identification and potential application testing.

[0020] The strain identification method is as follows:

[0021] The strains were inoculated into PSA solid medium covered with sterilized cellophane and cultured in a constant temperature incubator at 28°C for 8 days. Morphological observations were performed and hyphae were collected. Genomic DNA of each strain was extracted using a fungal genomic DNA extraction kit.

[0022] Molecular identification of the strains: The ITS region of the rDNA of the strains was amplified by PCR using the fungal universal primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3'). The ITS sequence obtained by amplification was subjected to BLAST similarity comparison on the NCBI website, and the ITS sequence information of the registered Curvularia strains downloaded from GenBank was used to construct a phylogenetic tree. The classification status of the strains was finally determined by combining morphological analysis.

[0023] The ITS1-ITS4 rDNA sequence of the test Curvularia strain was amplified using universal primers to obtain a fragment of about 500 bp in size. The sequence length of O1-SF-04630 was 558 bp, and the similarity with Polypocladium inflatum was 98.82%. The phylogenetic tree was constructed by screening the sequences of some Curvularia species in Genebank and combining the sequence of the test strain. Figure 1 The results showed that O1-SF-04630 and Polypocladium inflatum (CBS 824.70) clustered in the same branch, and the morphological characteristics were consistent with those of Polypocladium inflatum. Therefore, O1-SF-04630 was Polypocladium inflatum.

[0024] Morphological observation of the strains:

[0025] The activated conidia of Polypocladium inflatum were mixed with 0.05% Tween-80 to prepare a suspension of 1.0 x 10 7 spores / mL. A sterilized filter paper with a diameter of 5 mm was placed in the center of a PSA plate, and 10 μL of the conidial suspension was dropped on the filter paper using a micropipette. After air-drying, the plate was placed in an incubator at 28°C and cultured upside down. Each strain was repeated 3 times. The colony diameter was measured once on the 3rd day and the 7th day using an electronic digital caliper. The average value was obtained by crosswise measurement, and the colony growth rate was calculated by correcting the growth diameter (corrected growth diameter = 7d growth diameter - 3d growth diameter). After 8 days of culture, the morphology of the colonies and spores was observed, and the spore production was determined. A puncher with a diameter of 5 mm was used to take 4 colony cakes from each colony at a distance of 1 / 2 of the colony radius from the center of the colony. The colony cakes were placed in a certain amount of 0.1% Tween-80 sterile water, and vortexed to disperse the spores. The spore concentration was determined using a hemocytometer and converted into the spore production per unit area (mm 2 ) of the colony. Each strain was repeated 3 times.

[0026] The morphology of the colonies was as follows: Figure 2As shown, it can be seen that on the PSA plate, the colony is round, the middle of the colony is prominent, the periphery of the colony is annularly concave, and the mycelium is floccose white, the colony is prominent, the mycelium is radially villous, and the spore layer is white. Under a microscope, the spores are small and transparent, the conidia are spherical, subspherical to ellipsoidal, and the diameter is less than 3 μm.

[0027] The analysis of the corrected growth diameter of O1-SF-04630 on the seventh day found that the corrected growth diameter of the strain was 11.14±0.93 mm, and the analysis of the spore production found that the spore production of O1-SF-0436 on the eighth day was (2.97±0.33)×10 5 / mm 2 .

[0028] Example 2 Environmental tolerance determination

[0029] Moisture-heat resistance determination: 100 μL of spore suspension (1.0×10 7 / mL) was taken in a 1.5 mL EP tube, and was heat shocked for 0, 10, 30, 60, 120 and 300 min in a 45°C constant temperature metal bath, 30 μL of which was uniformly coated on a germination medium (1000 mL containing 0.1% glucose, 0.05% peptone, 2% agar powder) plate, and was cultured in a 28°C constant temperature incubator, and after 18 h, an agar block with an area of about 1 cm 2 was cut from each culture dish and was placed on a glass slide, and 100 spores were randomly observed under an optical microscope. The judgment standard for germination was that the length of the protrusion on the spore was greater than half of the spore itself. Each group of experiments was independently repeated 3 times. The germination of the spores was calculated and statistically analyzed. Germination rate = number of germinated spores / total number of spores × 100%.

[0030] Ultraviolet resistance determination: 30 μL of spore suspension at 1.0×10 7 / mL was uniformly coated on a germination medium plate, then the lid of the culture dish was opened, the plate was irradiated under a UV lamp (irradiation intensity was 100 μW / cm 2 at 254 nm wavelength) for 0 s, 10 s, 30 s and 60 s, and then was placed in a 28°C constant temperature incubator for inverted culture, and after 18 h, the germination rate was measured, and the determination method was the same as above. Each group of treatments was repeated 3 times.

[0031] The germination rate of T. proliferans under different time treatment at 45℃ was analyzed (Table 1), and it was found that the O1 strain had better tolerance to high temperature at 45℃. The germination rate of the strain had no significant difference under 0-120 min treatment, and the germination rate was more than 80%. The germination rate was significantly decreased by 18% (p<0.05) under 300 min treatment, but the germination rate was still 75%, indicating that T. proliferans had strong tolerance to high temperature at 45℃.

[0032] It was found that the strain was greatly affected by UV (p<0.05) and the germination rate of the strain was less than 10% when treated by UV for more than 30s. Figure 3 The germination rate of the strain was 66.3% when treated by UV for 0s. The germination rate of the strain was significantly decreased by 64.9% (p<0.05) when treated by UV-B for 10s, and the germination rate was 23.3%. The germination rate of the strain was less than 3.3% when treated by UV for 30s, and there was no significant difference in the germination rate of the strain between 30s and 60s treatment.

[0033] Table 1 Germination rate of T. proliferans under different heat shock time at 45℃

[0034] 45 °C heat stress (s) Germination rate % 0 (85.67±2.08)a 10 (83.33±4.93)a 30 (83±1)a 60 (82.67±1.15)a 120 (83±2)a 300 (75±2)b

[0035] Example 3 Determination of virulence of strains

[0036] The diet of P. xylostella was evenly distributed in 20-hole culture plates, 1 mL per hole. The feeding method was used. The spores of the strains were prepared into spore suspension with 0.05% Tween-80 sterile water, and adjusted to 1×10 8 spores / mL concentration after counting by hemocytometer. 80 μL of spore suspension was added to each hole of the culture plate with diet, and incubated at room temperature for 2 h. The three-day-old P. xylostella was randomly distributed in the culture holes, 5 per hole, and 4 holes per plate were added as one repetition, and each treatment had 3 repetitions. The control (Control-0.05% Tween) was 80 μL of sterile 0.05% Tween-80 added to the diet plate. The number of dead P. xylostella was recorded every day, and the mortality rate was calculated every day for 7 consecutive days.

[0037] When determining the median lethal concentration, 1×10 8 spores / mL spore suspension was diluted into 10 7 , 10 6 , 10 5 , 10 4 spores / mL in turn at the ratio of 1:9, and 5 decreasing concentration gradients were obtained. The inoculation treatment and observation method were the same as above. The time-dose-mortality model was used for simulation analysis, and the fitted dose effect and time effect parameters were used to estimate the time-varying median lethal concentration LC 50 .

[0038] Bioassay of P. variotii on P. xylostella showed that P. variotii O1-SF-04630 had higher virulence on P. xylostella. Figure 4 The mortality of P. xylostella began to appear on the second day after treatment, and the mortality of P. xylostella treated with O1 strain was 28.33% on the third day, which was significantly higher than that of the control strain (p<0.05) (Table 2). From the fourth to the seventh day, the cumulative mortality of P. xylostella treated with each strain gradually increased, and the mortality was significantly higher than that of the control treatment (p<0.05). On the seventh day, the cumulative mortality of O1 strain was the highest, reaching 98.3%. The infected insect bodies were treated with moisture, and obvious mycelium grew on the surface of the insect bodies, which was identified as P. variotii O1-SF-04630 (b in FIG. 1). Figure 4 The virulence regression equation was y=16.964x-16.429, R 2 value was 0.9884, and the linear relationship was good. The calculated median lethal time LT 50 of O1 strain was 3.89±0.34 days. The virulence regression equation was y=11.667x-2.3333, R 2 value was 0.8922, and the median lethal concentration LC 50 of O1 strain was 3.06×10 4 spores / mL, indicating that P. variotii O1-SF-04630 had a good control effect on P. xylostella.

[0039] Table 2 Analysis of significant difference in cumulative mortality of each strain

[0040]

[0041] Note: Different lowercase letters in the same column indicate significant difference (P<0.05).

[0042] Example 4 Pathogenicity of the strain on P. xylostella

[0043] Feeding method was used. The P. xylostella feed was evenly distributed in 20-hole culture plates, 1 mL per hole. The strain spores were prepared into a spore suspension with 0.05% Tween-80 sterile water, counted by a hemocytometer, and adjusted to a concentration of l×10 8 spores / mL, and then diluted by 1:9 to 10 7 , 10 6 , 10 5 , and 10 4Spores / mL, 5 decreasing concentration gradients were obtained. 80 μΐ of spore suspension was added to each well of the culture plate with feed, and incubated at room temperature for 2 h. The 4th instar of P. xylostella were randomly allocated to the culture wells, 5 per well, 4 wells were added to each plate, a total of 20, 3 replicates for each treatment. The blank control (Control-0.05% Tween) was 80 μΐ of sterile 0.05% Tween-80 added to the feed plate. The number of adult emergence was observed on the 10th day, and the emergence rate was calculated, Emergence rate = (number of emerged adults / total number of adults) * 100.

[0044] The results show (Table 2) that on the 10th day after treatment, P. xylostella had emerged in succession, and the emergence rate of P. xylostella in the blank control was the highest, 58.3%, significantly higher than all the bacterial liquid treatment groups (p < 0.01); the number of adult emergence in the treatment group was inversely proportional to the concentration of bacterial liquid, and the emergence rate of P. xylostella in the 10 4 The emergence rate of P. xylostella was the highest, 41.7%, in the 10 8 The emergence rate of P. xylostella was the lowest, only 10%, in the 10 5 , 10 6 , 10 7 The emergence rate of P. xylostella was less than 32% in the 10

[0045] Table 2

[0046]

[0047] The above examples are only for understanding the technical solutions of the present application, and do not limit the protection scope of the present application.

Claims

1. Curvularia expansum O1-SF-04630 strain ( Tolypocladium inflatum O1-SF-04630), characterized in that The Toxoplasma intumescentis O1-SF-04630 strain ( Tolypocladium inflatum O1-SF-04630) is deposited in CCTCC NO:M 20241094.

2. The Toxoplasma inflata O1-SF-04630 strain according to claim 1 ( Tolypocladium inflatum O1-SF-04630) for use in controlling diamondback moths.

3. comprising the Toxoplasma inflata O1-SF-04630 strain according to claim 1 ( Tolypocladium inflatum O1-SF-04630).

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