Application of nano material in promoting growth of beauveria bassiana

Abstract

The invention relates to the technical field of new materials in agricultural pest control, and discloses application of a nano material in promotion of growth of beauveria bassiana, and the nano material is SPc. According to the application of the nano material in promoting the growth of the beauveria bassiana, the nano material SPc can promote spore germination and hypha growth of the beauveria bassiana, so that the biomass and activity of the beauveria bassiana are promoted, the time required by invasion of fungi into the body surfaces of pests is effectively shortened, and meanwhile, the growth of the pests is promoted. The fatality rate of the beauveria bassiana on target pests is remarkably increased, the prevention and treatment mechanism of microorganisms is deepened, and a new way is opened up for research, development and application of biopesticides.

Description

Technical Field

[0001] The invention relates to the technical field of new materials in agricultural pest control, and in particular to the application of nano materials in promoting the growth of Beauveria bassiana. Background Art

[0002] Biocontrol fungi play a key role in the field of biological control. As biological insecticides, they are highly praised for their strong targeting and environmental friendliness, and are regarded as the core force to promote green agriculture and sustainable development. In particular, Beauveria bassiana, as an effective biocontrol fungus, is widely used in pest control because of its strong pathogenicity and easy large-scale cultivation. Under natural conditions, Beauveria bassiana can directly penetrate the outer cuticle of insects to infect. Currently, Beauveria bassiana can be used to control a variety of insects, including many species of Lepidoptera, Coleoptera, Diptera, Hemiptera, etc. Despite this, Beauveria bassiana also faces some challenges in practical applications. Affected by factors such as the external environment and the host's own immune system, Beauveria bassiana has a relatively long incubation period from contact to the final killing of the host insect, which greatly affects its timeliness in field control. New technologies are urgently needed to overcome these problems.

[0003] So far, the research on the relationship between nanomaterials and biocontrol bacteria has mainly focused on two aspects: one is that nanomaterials have an inhibitory effect on biocontrol bacteria; the other is that nanomaterials promote the invasion of biocontrol bacteria into the insect body wall. However, there is still a relative lack of research on how nanomaterials combine with Beauveria bassiana, the optimal combination ratio, and the impact on the biological characteristics of Beauveria bassiana (such as pathogenicity, reproductive capacity, etc.). It is particularly noteworthy that existing studies have shown that nanomaterials usually have certain toxic effects on biocontrol bacteria, which may interfere with the normal life cycle of Beauveria bassiana, including key links such as spore germination, hyphae growth, and infection structure formation. However, to date, there is no clear research evidence that nanomaterials can promote the germination and growth of Beauveria bassiana or improve its biological control effect. Therefore, in-depth exploration of the interaction mechanism between nanomaterials and Beauveria bassiana will not only help reveal a new field of nanobiology, but also provide theoretical support for the development of new and efficient biological control strategies. Summary of the invention

[0004] The purpose of the invention is to provide application of nano materials in promoting the growth of Beauveria bassiana.

[0005] To achieve the above object, the present invention provides the use of nanomaterials in the preparation of nanoformulations for promoting the growth and reproduction of Beauveria bassiana. The nanomaterial is a star-shaped cationic polymer (SPc) SPc, and its structural formula is as follows:

[0006]

[0007] Where n = 40000 g / mol.

[0008] Furthermore, when used, the concentration of the nanomaterial is 0.004-0.5 mg / mL.

[0009] The invention also provides the use of the nano material in preparing the nano preparation for promoting the spore germination of Beauveria bassiana and the growth of sporangium, and the nano material is the above-mentioned nano material.

[0010] Furthermore, the concentration of the nanomaterial is 0.004-0.02 mg / mL.

[0011] The present invention also provides the use of the nano material in preparing the nano preparation for promoting the hypha growth of Beauveria bassiana, and the nano material is the above-mentioned nano material.

[0012] Furthermore, the concentration of the nanomaterial is 0.004-0.5 mg / mL.

[0013] Furthermore, the nanomaterials promoted the increase in the colony diameter of Beauveria bassiana.

[0014] Furthermore, at room temperature, the particle size of the nanomaterial is 43.34±0.316 nm, the particle size of Beauveria bassiana is 409.3±1.583 nm, and the particle size of the nanopreparation is 424±0.864 nm; the nanomaterial is positively charged, Beauveria bassiana is negatively charged, and the nanopreparation is positively charged.

[0015] The present invention also provides the use of the nano material in preparing the nano preparation for promoting the membrane permeability of Beauveria bassiana, and the nano material is the above-mentioned nano material.

[0016] The present invention also provides the use of the nano material in preparing the nano preparation for promoting the absorption of nutrients by Beauveria bassiana, and the nano material is the above-mentioned nano material.

[0017] The advantages and positive effects of the application of the nanomaterials of the present invention in promoting the growth of Beauveria bassiana are:

[0018] 1. The low concentration of nanomaterial SPc in the present invention can promote the spore germination and hyphae growth of Beauveria bassiana, which not only promotes the biomass and activity of Beauveria bassiana, but also effectively reduces the time required for the fungus to invade the body surface of pests. At the same time, it significantly improves the mortality rate of Beauveria bassiana to target pests.

[0019] 2. The present invention deepens the prevention and control mechanism of microorganisms, opens up a new path for the research and development and application of biological pesticides, and is of great significance for promoting the sustainable development of agricultural production.

[0020] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a diagram of the synthesis steps of nanomaterial SPc in an embodiment of the present invention;

[0022] Figure 2 The effects of different concentrations of SPc on the spore germination of Beauveria bassiana in the examples of the present invention;

[0023] Figure 3 The effect of different concentrations of SPc on the mycelial growth of Beauveria bassiana in the examples of the present invention;

[0024] Figure 4 It is a volcano plot of differentially expressed genes in the embodiment of the present invention;

[0025] Figure 5 This is a KEGG signal pathway analysis diagram of differentially expressed genes in the embodiment of the present invention;

[0026] Figure 6 This is a GO function analysis diagram of differentially expressed genes of Beauveria bassiana in the embodiment of the present invention;

[0027] Figure 7 This is a heat map of the expression of important differential genes of Beauveria bassiana in the embodiments of the present invention;

[0028] Figure 8 This is a particle size distribution diagram of the composite of SPc and Beauveria bassiana in the embodiment of the present invention;

[0029] Fig. 9 This is a graph showing the efficacy of SPc in controlling pea aphids against Beauveria bassiana in an example of the present invention. DETAILED DESCRIPTION

[0030] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0031] Unless otherwise defined, technical or scientific terms used in the present invention shall have the common meanings understood by one having ordinary skills in the field to which the present invention belongs.

[0032] Unless otherwise defined, the instruments, equipment and reagents used in the present invention are conventionally commercially available.

[0033] Example

[0034] 1. Preparation of nanomaterial SPc:

[0035] Structural characteristics of SPc: It is a dendritic macromolecule functionalized with amino groups. Its structural formula is shown below, and the synthesis steps are shown in 1.

[0036] Nanomaterial SPc structural formula:

[0037] Where n = 40000 g / mol.

[0038] 2. Preparation of Beauveria bassiana spore suspension and SPc dilution:

[0039] Beauveria bassiana was inoculated in potato dextrose agar medium and cultured under the conditions of 28°C and 70% relative humidity. When Beauveria bassiana filled the culture dish, the mycelium was gently scraped with a sterile coating stick and placed in a 50 mL sterile centrifuge tube filled with sterile water. The centrifuge tube was placed in a vortex shaker for 5 minutes, and the mycelium was filtered through a sterile funnel equipped with 6 layers of sterile lens paper. A sterile 50 mL centrifuge tube was placed under the funnel to collect the spore suspension. The collected filtrate was centrifuged in a centrifuge at 4°C and 8000 rpm for 15 minutes. The upper solution was then discarded to collect the precipitate and diluted with sterile water to 1×10 7 10 μl of spore suspension was added to a 25×16 blood cell counting plate, and the four corner squares and the central square were selected for counting. The counting was repeated three times and the average value was calculated. Counting formula: spore number = total number of 5 squares × dilution factor × 5 × 10 4 (pcs / mL).

[0040] Preparation of nanomaterial dilution solution: After placing SPc in an ultrasonic instrument at 99 Hz for 15 min, the SPc was filtered through a 0.22 μm filter membrane and diluted with sterile water for later use.

[0041] 3. Determination of the effect of SPc on the growth and reproduction of Beauveria bassiana:

[0042] Determination of spore germination: Pipette equal amounts of SPc of different concentrations and add them to the spore suspension to make the final concentration of SPc 0.004, 0.02, 0.1, 0.5, 2.5 and 12.5 mg / mL, respectively. Sterile water was used as a control. Three groups of replicates were set up and cultured in a 25°C incubator. After 12 hours, the spore germination was observed and photographed under a microscope. Five fields of view were selected in each group to measure the length of the sporangium. Spore germination rate (%) = number of spores germinated / total number of spores × 100.

[0043] The results are as follows Figure 2As shown in the results, compared with the control group, when the final concentration of SPc was 0.004 mg / mL and 0.02 mg / mL, SPc significantly promoted the spore germination of Beauveria bassiana and increased the length of the sporophore; however, when the final concentration of SPc was 2.5 mg / mL and 12.5 mg / mL, SPc had an inhibitory effect on Beauveria bassiana, which was manifested by a decrease in the spore germination rate and a significant shortening of the sporophore length. When the final concentration of SPc was 0.1 mg / mL and 0.5 mg / mL, there was no significant difference between the treatment group and the control group.

[0044] Colony diameter measurement: The effect of different concentrations of SPc on the mycelial growth of Beauveria bassiana was determined by the bacterial plate method. Under sterile operating conditions, equal amounts of SPc liquids of different concentrations were mixed and shaken with PDA culture medium, and the stipes of Beauveria bassiana were inoculated into the center of the PDA plate using a sterile bacterial cake puncher. Each group of experiments was repeated 3 times, and sterile water was used as a control. The plates were cultured in a 25°C incubator. On the 6th day, the colony diameter was measured with a ruler and the data was recorded. The growth was observed and photographed, and the concentration with the best growth-promoting effect was selected for sampling and transcriptome sequencing.

[0045] The results are as follows Figure 3 As shown, compared with the control group, low concentration of SPc (0.004 mg / mL to 0.5 mg / mL) can significantly promote the increase of colony diameter; while high concentration of SPc (2.5 mg / mL and 12.5 mg / mL) leads to a decrease in colony diameter, indicating that low concentration of SPc treatment of Beauveria bassiana has the effect of promoting mycelial growth.

[0046] 4. Transcriptome analysis of the effects of SPc on Beauveria bassiana:

[0047] The concentration of nanomaterials with significant promoting effect (SPc = 0.02 mg / mL) was selected to treat Beauveria bassiana, and the untreated Beauveria bassiana was used as the control. Mycelium was picked to extract RNA and transcriptome sequencing was performed. The transcriptome data showed that compared with the control group, the transcriptome data of Beauveria bassiana treated with SPc showed that the expression levels of a total of 439 genes changed, of which 132 were up-regulated genes, 307 were down-regulated genes, and 10131 genes had no significant changes ( Figure 4 shown).

[0048] KEGG analysis is a database that systematically analyzes gene function and genomic information. Through pathway significant enrichment, the most important biochemical metabolic pathways and signal transduction pathways involved in differentially expressed genes can be determined. KEGG was used to find out the signal pathways that were significantly enriched in differentially expressed genes compared with the entire genome background. They were mainly energy metabolism, and the main enrichments were in sugar metabolism and lipid metabolism ( Figure 5 shown).

[0049] Based on the GO database, the functional information of differentially expressed genes in Beauveria bassiana in biological processes, cell components, molecular functions and other categories was analyzed. In the biological process component category, the differentially expressed genes were mainly classified into functional categories such as membrane integral component and extracellular region; in the cell component category, the differentially expressed genes were mainly classified into functional categories such as nucleic acid biosynthesis, antioxidant reaction, redox process, polysaccharide decomposition process and cell oxidation and detoxification; in the molecular function category, the differentially expressed genes were mainly classified into functional categories such as ribonuclease activity, transaminase activity, oxidoreductase, hydrolase and pyridoxal phosphate binding ( Figure 6 shown).

[0050] Low-concentration nanomaterials promoted the growth of Beauveria bassiana, which may be related to the absorption of nutrients by Beauveria bassiana. The target genes were screened based on this, and the genes related to sugar and lipid metabolism pathways, energy metabolism pathways, and ion-binding proteins were significantly upregulated, proving that low-concentration nanomaterials promoted the membrane permeability and absorption of nutrients by Beauveria bassiana ( Figure 7 shown).

[0051] 5. Particle size distribution of SPc and Beauveria bassiana:

[0052] Prepare SPc (final concentration: 0.02 mg / mL), Beauveria bassiana (1×10 7 The particle size and potential of each sample were measured by dynamic light scattering (DLS) and Zetasizer Nano ZS in a laser diffraction particle size analyzer (Zeta Sizer Nano Series) (Malvern Instruments, USA) at room temperature.

[0053] The results are as follows Figure 8 As shown, under room temperature conditions, the particle size of SPc is 43.34±0.316nm, the particle size of Beauveria bassiana is 409.3±1.583nm, and the particle size after the two are mixed is 424±0.864nm; the potential measurement results show that SPc is positively charged (72.83±0.872), Beauveria bassiana is negatively charged (-26.4±0.534), and the particle size after the two are mixed is positively charged (43.58±0.312).

[0054] 6. SPc enhances the efficacy of Beauveria bassiana in controlling pea aphids:

[0055] 0mg / mL, 0.02mg / mL, 0.1mg / mL and 2.5mg / mL of SPc solutions alone and a mixture of SPc and Beauveria bassiana were prepared to make the final concentrations 0mg / mL, 0.02mg / mL, 0.1mg / mL and 2.5mg / mL, respectively. Beauveria bassiana and sterile water were also set up as control groups. Each treatment was repeated three times. The mixtures were sprayed evenly on the surface of three-day-old pea aphid adults by spraying. The mortality of pea aphids was then observed on the 2nd, 4th and 6th days.

[0056] The results are as follows Fig. 9 As shown in the results, when the concentration of SPc was 2.5 mg / mL, the treatment of pea aphids with SPc alone caused dehydration of pea aphids, thereby increasing the mortality rate compared with the control group. Secondly, after SPc was compounded with Beauveria bassiana, pea aphids were treated by soaking. The results showed that when the concentration of SPc was 0.02 mg / mL and 0.1 mg / mL, the amount of hyphae growth on the surface of pea aphids increased. However, when the concentration of SPc was 2.5 mg / mL, the amount of hyphae on the surface of pea aphids was less. In addition, it was observed that the combination of SPc and Beauveria bassiana applied to pea aphids could increase the mortality rate by more than 20%, especially when the final concentration of SPc was 0.02 mg / mL, the mortality rate of pea aphids increased by about 40%. These results show that SPc can significantly promote the growth and reproduction of Beauveria bassiana, thereby improving the control efficacy of Beauveria bassiana on pea aphids, and the nanoformulation compounded with SPc and Beauveria bassiana can be used to control pea aphids.

[0057] Therefore, the present invention adopts the above-mentioned nanomaterials in promoting the growth of Beauveria bassiana. The nanomaterial SPc can promote the spore germination and hyphae growth of Beauveria bassiana, which not only promotes the biomass and activity of Beauveria bassiana, but also effectively reduces the time required for the fungus to invade the body surface of the pest. At the same time, it significantly improves the mortality rate of Beauveria bassiana to the target pest, which not only deepens the prevention and control mechanism of microorganisms, but also opens up a new path for the research and development and application of biological pesticides.

[0058] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. The use of nanomaterials in the preparation of nano preparations for promoting the growth and reproduction of Beauveria bassiana is characterized by: The nanomaterial is SPc, and its structural formula is as follows: Where n = 40000 g / mol.

2. The use according to claim 1, characterized in that: When applied, the concentration of the nanomaterial is 0.004-0.5 mg / mL.

3. The use of nanomaterials in the preparation of nanoformulations for promoting spore germination and sporangium growth of Beauveria bassiana, characterized in that: The nanomaterial is the nanomaterial described in claim 1.

4. The use according to claim 3, characterized in that: The concentration of nanomaterials is 0.004-0.02 mg / mL.

5. The use of nanomaterials in the preparation of nanoformulations for promoting the growth of Beauveria bassiana hyphae, characterized in that: The nanomaterial is the nanomaterial described in claim 1.

6. The use according to claim 5, characterized in that: The concentration of nanomaterials was 0.004-0.5 mg / mL.

7. The use according to claim 5, characterized in that: Nanomaterials promote the increase of Beauveria bassiana colony diameter.

8. The use according to claim 1, 3 or 5, characterized in that: At room temperature, the particle size of the nanomaterial is 43.34±0.316nm, the particle size of Beauveria bassiana is 409.3±1.583nm, and the particle size of the nanopreparation is 424±0.864nm; the nanomaterial is positively charged, Beauveria bassiana is negatively charged, and the nanopreparation is positively charged.

9. The use of nanomaterials in the preparation of nanoformulations for promoting membrane permeability of Beauveria bassiana, characterized in that: The nanomaterial is the nanomaterial described in claim 1.

10. The use of nanomaterials in the preparation of nanoformulations for promoting the absorption of nutrients by Beauveria bassiana, characterized in that: The nanomaterial is the nanomaterial described in claim 1.

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