Lecanicillium attenuatum spore production culture medium and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to a lecanicillium attenuatum spore production culture medium and application thereof. The invention provides a formula of a lecanicillium attenuatum spore production culture medium. The lecanicillium attenuatum spore production culture medium consists of the following components: 5g / L of yeast extract and 10g / L of glucose. According to the invention, the current lecanicillium attenuatum spore production culture medium is optimized, and the purposes of rapid spore production, large conidium quantity and simple preparation can be achieved.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and specifically relates to a spore-producing culture medium for Lecanopsis attenuata and its application. Background Art

[0002] Nearly half of the known insects on Earth are herbivorous pests that feed directly on crops and indirectly cause the spread of plant diseases. Currently, the most common method of pest control is the application of chemical pesticides. However, due to concerns about pesticide residues, new strategies for pest control that are environmentally friendly and cost-effective need to be developed. Insect pathogenic fungi are a class of specific parasitic microorganisms that have the remarkable ability to infect and ultimately kill insects and other arthropods, and are one of the most effective natural options for pest control (Ma, M., Luo, J., Li, C., Eleftherianos, I., Zhang, W., Xu, L., 2024. A life-and-death struggle: interaction of insects with entomopathogenic fungi across various infection stages. Front. Immunol. 14: 1329843).

[0003] Lecanicillium belongs to the order Hypocreales and is an insect pathogenic fungus that has biological control capabilities against a variety of plant pests, including aphids, whiteflies, thrips and nematodes (Liu, S., Li, J., Feng, Q., Chu, L., Tan, Z., Ji, X., Jin, P., 2023. Insecticidal effect of the entomopathogenic fungus Lecanicillium araneicola HK-1 in Aphis craccivora (Hemiptera: Aphididae). Insects 14, 860). When conidia produced by insect pathogenic fungi come into contact with insects, they penetrate the cuticle through enzymatic hydrolysis and physical action, and then multiply in the insect tissues, causing the death of the host. Subsequently, hyphae germinate from the corpse and produce dormant or infectious spores, which promote the spread of the fungus (Boomsma, JJ, Jensen, AB, Meyling, NV, Eilenberg, J., 2014. Evolutionary interaction networks of insect pathogenic fungi. Annu. Rev. Entomol. 59, 467-485.).

[0004] Therefore, the optimization of conidia culture medium is crucial to improve the biological control effect of insect pathogenic fungi such as Lecanoptera. By adjusting the nutrient composition and other conditions in the culture medium, the production of conidia can be significantly promoted, thereby enhancing the fungus's ability to infect pests and the mortality rate, reducing the use of chemical pesticides, and promoting sustainable agricultural development. In addition, the optimization of the culture medium can also help improve production efficiency, reduce costs, make biological control technology more economical and practical, and provide an environmentally friendly and efficient solution for agricultural safety production. Summary of the invention

[0005] Aiming at the problems of the prior art, in particular, the spore production of the attenuated lecanthus with the deposit number of CGMCC No.40860 is studied and optimized, and a spore production medium optimized for the attenuated lecanthus and a method for culturing the attenuated lecanthus are obtained.

[0006] The invention provides a spore production medium optimized for Lecanopsis attenuata, which comprises the following components: 4-6 g / L of yeast extract, 8-12 g / L of glucose, and the balance being sterile water.

[0007] Preferably, the following components are included: 5 g / L yeast extract, 10 g / L glucose, and the balance is sterile water.

[0008] The present invention further provides a method for preparing the optimized spore-producing medium of Lecanopsis attenuata, which comprises weighing each component according to a formula ratio, mixing and dissolving the components, and sterilizing the mixture at 121° C. for 20 minutes to obtain the optimized spore-producing medium of Lecanopsis attenuata.

[0009] The invention provides a spore production method for culturing Lecanococcus attenuata, which comprises the following steps: cutting a mycelium block of 1-2×1-2 mm in size from the edge of a fresh colony of Lecanococcus attenuata, and inoculating the block on a spore production medium optimized based on Lecanococcus attenuata as claimed in claim 1 or 2 for culturing.

[0010] Preferably, the culture is carried out in a shaking incubator at 26-30°C, with a shaking speed of 180-250 rpm for 60-90 hours.

[0011] More preferably, the culture is carried out in a shaking incubator at 28°C, with a shaking speed of 220 rpm for 70-75 hours.

[0012] Optionally, after the culture, the method further comprises filtering to remove hyphae and hyphae blocks to obtain a conidia liquid of Lecanopsis attenuata, which is optionally stored at 4° C. for later use.

[0013] Particularly preferably, the attenuated wax scale fungus has a preservation number of CGMCC No. 40860. Wherein, the attenuated wax scale fungus is classified and named as attenuated wax scale fungus (Lecanicillium attenuatum), which is preserved in the General Microbiological Center of China Microbiological Culture Collection Administration Committee, with a preservation date of October 25, 2023, a preservation number of CGMCC No. 40860, a preserved strain name of attenuated wax scale fungus 3166, and a preservation address of No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0014] The present invention provides the use of the spore production medium optimized based on Lecanopsis attenuata in culturing Lecanopsis attenuata for spore production. Preferably, the Lecanopsis attenuata has a preservation number of CGMCC No.40860.

[0015] The present invention provides that the spore count of optimized culture medium in the initial stage (12 hours) is lower, but along with the extension of incubation time, its spore count increases gradually, shows good growth potential.During 48 hours to 72 hours, the spore count of optimized culture medium increases significantly, shows that it can support the sustained growth and spore production of fungi.By contrasting the overall performance of three kinds of culture mediums, the optimized culture medium of the present invention has obvious advantages in promoting the growth and spore production of attenuated wax scale fungus, although the CM culture medium handles the absorbance higher than the optimized culture medium, there is no significant difference in spore production in 72 hours.But because the optimized culture medium formula is simple, preparation is convenient, therefore, the optimized culture medium of the present invention is the ideal selection of optimizing spore production culture medium, contributes to improving the production efficiency and the application effect of the biological control preparation based on attenuated wax scale fungus.Meanwhile, follow-up further experiments confirm that the attenuated wax scale fungus spores produced by the three kinds of culture mediums have no significant difference in the toxicity to harmful insects. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Spores of Lecanococcus attenuata produced after 72 hours of culture

[0017] Figure 2 Statistics of absorbance of bacterial solution and spore count of Lecanococcus attenuata. DETAILED DESCRIPTION

[0018] The present invention is further illustrated below with reference to the examples, but is not intended to limit the present invention.

[0019] Example 1: Optimized culture medium for spore production of Lecanopsis attenuata and spore production method thereof

[0020] 1. Culture medium preparation

[0021] 1.1 Optimize the culture medium: 5 g yeast extract and 10 g glucose per L. Sterilize at 121℃ for 20 min for later use.

[0022] 1.2CM culture medium, per L: 50ml 20×Nitrate Salts*, 1ml Trace Elements*, 10g D-Glucose, 2g Peptone, 1g Yeast Extract, 1g Casamino Acids, 1ml Vitamin Solution*. Adjust the pH to 6.5 with NaOH, sterilize at 121℃ for 20min and set aside.

[0023] 20×Nitrate Salts: 120g NaNO3, 10.4g KCl, 10.4g MgSO4·7H2O, 30.4g KH2PO4, add ddH2O to make up to 1L, store at 4℃.

[0024] 1000×Trace Elements: 2.2g ZnSO4·7H2O, 1.1g H3BO3, 0.5g MnCl2·4H2O, 0.5gFeSO4·7H2O, 0.17g CoCl2·6H2O, 0.16g CuSO4·5H2O, 0.15g Na2MnO4·2H2O, 5g Na4EDTA, add ddH2O to make up to 100mL and store at 4℃.

[0025] Vitamin Solution: 0.01g Biotin, 0.01g Pyridoxin, 0.01g Thiamine, 0.01g Riboflavin, 0.01g PABA (P aminobenzoic acid), 0.01g Nicotinic Acid, add ddH2O to 100ml, store at 4℃ away from light.

[0026] 1.3 Czapeck medium, per L: 30 g sucrose, 2 g sodium nitrate, 1 g potassium dihydrogen phosphate, 0.5 g magnesium sulfate heptahydrate, 0.5 g potassium chloride, 0.01 g ferrous sulfate. Sterilize at 121°C for 20 min and set aside.

[0027] 2. Sporulation method of Lecanococcus attenuata

[0028] 2.1 Lecanococcus attenuata was inoculated in PDA medium (per L: 200 g potato, 20 g glucose, 15-20 g agar). After 7 days of culture, about 10 mycelium pieces of 1-2×1-2 mm in size were cut from the edge of the colony and inoculated into a 400 mL conical flask containing 150 mL of culture medium.

[0029] 2.2 Place in a shaker at 28°C, 220 rpm, and culture for 72 hours.

[0030] 2.2 Use Miracloth (Cat. No. 475855, Company: Calbiochem) to filter out hyphae and hyphae blocks to obtain the conidia solution of Lecanoptera: ...

[0031] 3 Comparison of spore production in different culture media

[0032] The test results showed that as the culture time increased, the absorbance and spore count both showed an increasing trend, which indicated that the attenuated wax scale fungus continued to grow and reproduce during the culture process.

[0033] From the data, it can be seen that the number of spores in the optimized medium was low in the early stage (12 hours), but as the culture time increased, the number of spores gradually increased, showing good growth potential. During the period from 48 hours to 72 hours, the number of spores in the optimized medium increased significantly, indicating that it can support the continuous growth and spore production of the fungus.

[0034] During the period from 12 to 72 hours, the absorbance of the attenuated Lecanococcus cultured in CM medium was significantly higher than that in the other two mediums. During the period from 12 to 60 hours, the number of spores in the CM medium treatment was significantly higher than that in the optimized medium treatment, but there was no significant difference between the two after 72 hours.

[0035] The absorbance of Czapeck medium treatment did not increase significantly during 12 to 72 hours. Although the spore production increased at 48 hours, the increase in the number of spores was not significant during the entire culture period, indicating that Czapeck medium may not be as suitable for the growth and spore production of L. attenuata in terms of nutritional components as the other two culture media.

[0036] By comparing the overall performance of the three culture media, we can conclude that the optimized medium and CM medium have obvious advantages in promoting the growth and conidia of Lecanoptera attenuata. Although the absorbance of the CM medium treatment is higher than that of the optimized medium, there is no significant difference in the conidia production in 72 hours. However, due to the simple formula and convenient preparation of the optimized medium, the optimized medium is an ideal choice for optimizing conidia production medium, which helps to improve the production efficiency and application effect of biological control agents based on Lecanoptera attenuata.

[0037] Example 2: Toxicity of spores of Lecanococcus attenuata to insects

[0038] 1 Indoor toxicity determination method

[0039] Using the method described in Example 1, three culture media were used to obtain a spore suspension of Lecanococcus attenuata. The spore suspension was diluted to 10 8 spores / mL, and culture medium was used as control.

[0040] The toxicity to brown planthoppers was determined by the rice straw immersion method. Rice stalks about 10 cm long at the booting stage (retaining roots about 1 cm long, wrapped with absorbent paper and sprayed with a little water to keep them moist) were immersed in the spore suspension for 15 seconds and placed in an insect box (13.5 cm long, 9 cm wide, and 7 cm high) with small ventilation holes on the top. Three replicates were set for the spores obtained from each culture medium, and 20 second-instar nymphs of brown planthoppers were inoculated in each replicate.

[0041] The toxicity to adults of Bemisia tabaci was determined by leaf dipping method. The pepper branches and leaves were immersed in the spore suspension for 10 seconds, a small amount of water was added to a disposable cup to keep it moist, and the immersed pepper branches and leaves and adults of Bemisia tabaci were placed in the cup, and the cup was sealed with gauze. Each culture medium was repeated 3 times.

[0042] The insects were placed in a light incubator at 27°C, 85% relative humidity and a photoperiod of 16L:8D; 3-7 days after treatment, the number of second-instar nymphs of brown planthoppers or adults of whiteflies was recorded.

[0043] 3 Results of indoor toxicity test of Lecanococcus attenuata spores obtained from different culture media

[0044] The experimental results confirmed that there was no significant difference in the toxicity of the spores of Lecanococcus attenuata produced by the three culture media to brown planthoppers and whiteflies.

[0045] Table 1

[0046]

Claims

1. A spore-forming culture medium optimized for Lecanopsis attenuata, characterized in that: The invention comprises the following components: 4-6 g / L yeast extract, 8-12 g / L glucose, and the balance being sterile water.

2. The spore-forming medium optimized by Lecanopsis attenuata according to claim 1, wherein The preparation method comprises the following components: 5 g / L yeast extract, 10 g / L glucose, and the balance is sterile water.

3. A method for preparing the spore-forming culture medium optimized by Lecanopsis attenuata as claimed in claim 1, characterized in that: The components were weighed according to the formula ratio, mixed and dissolved, and sterilized at 121°C for 20 min to obtain the optimized spore production medium for Lecanopsis attenuata.

4. A method for culturing spore production of Lecanococcus attenuata, characterized in that: The method comprises the following steps: cutting a mycelium block of 1-2×1-2 mm in size from the edge of a fresh colony of Lecanococcus attenuata, and inoculating the block on the spore-forming medium optimized for Lecanococcus attenuata as claimed in claim 1 or 2 for cultivation.

5. The spore production method according to claim 4, characterized in that The culture is carried out in a shaking incubator at 26-30°C, with a shaking speed of 180-250 rpm for 60-90 h.

6. The spore production method according to claim 5, characterized in that The culture is carried out in a shaking incubator at 28°C with a shaking speed of 220 rpm for 70-75 h.

7. The spore production method according to any one of claims 4 to 6, characterized in that: After the culture, the method further includes filtering to remove hyphae and hyphae blocks to obtain the conidia liquid of the attenuated wax scale fungus, which is optionally stored at 4°C for later use.

8. The spore production method according to any one of claims 4 to 6, characterized in that: The deposit number of the attenuata lecanthus fungus is CGMCC No. 40860.

9. Use of the spore production medium optimized based on Lecanopsis attenuata according to claim 1 or 2 in culturing spore production of Lecanopsis attenuata.

10. The use according to claim 9, characterized in that The deposit number of the attenuata lecanthus fungus is CGMCC No.40860.

Images