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] This invention relates to the technical field of new materials in the prevention and control of agricultural pests and diseases, and in particular to the application of nanomaterials in promoting the growth of Beauveria bassiana. Background Technology

[0002] Biocontrol bacteria play a crucial role in the field of biological control. As biological insecticides, they are highly regarded for their strong specificity and environmental friendliness, and are considered a core force in promoting green agriculture and sustainable development. In particular, *Beauveria bassiana* (…) Beauveria bassiana As an effective biocontrol fungus, *Beauveria bassiana* is widely used in pest control due to its strong pathogenicity and ease of large-scale cultivation. Under natural conditions, *Beauveria bassiana* can directly penetrate the exodermis of insects to infect them. Currently, it can be used to control a variety of insects, including many species of Lepidoptera, Coleoptera, Diptera, and Hemiptera. However, *Beauveria bassiana* also faces some challenges in practical application. Influenced by factors such as the external environment and the host's own immune system, there is a relatively long latency period between contact and eventual killing of the host insect, which significantly affects its timeliness in field control. New technologies are urgently needed to overcome these problems.

[0003] To date, research on the interaction between nanomaterials and biocontrol bacteria has mainly focused on two aspects: first, the inhibitory effect of nanomaterials on biocontrol bacteria; and second, the promotion of biocontrol bacteria invasion of insect body walls by nanomaterials. However, research on how nanomaterials bind to *Beauveria bassiana*, the optimal binding ratio, and their impact on the biological characteristics of *Beauveria bassiana* (such as pathogenicity and reproductive capacity) remains relatively scarce. Of particular concern is that existing research indicates that nanomaterials often exhibit certain toxic effects on biocontrol bacteria, which may interfere with the normal life cycle processes of *Beauveria bassiana*, including key stages such as spore germination, hyphal 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 biocontrol efficacy. 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 developing novel and efficient biocontrol strategies. Summary of the Invention

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

[0005] To achieve the above objectives, this invention provides the application of nanomaterials in the preparation of nano-formulations that promote the growth and reproduction of Beauveria bassiana. The nanomaterial is a star-shaped cationic polymer (SPc), and its structural formula is as follows: , Where n = 40000 g / mol.

[0006] Furthermore, when applying the nanomaterials, the concentration is 0.02-0.5 mg / mL.

[0007] The present invention also provides the application of nanomaterials in the preparation of nano-preparations that promote the germination of Beauveria bassiana spores and the growth of sporophytes, wherein the nanomaterials are those described above.

[0008] Furthermore, the concentration of the nanomaterial was 0.02 mg / mL.

[0009] The present invention also provides the application of nanomaterials in the preparation of nano-preparations that promote the mycelial growth of Beauveria bassiana, wherein the nanomaterials are those described above.

[0010] Furthermore, the concentration of the nanomaterials is 0.02-0.5 mg / mL.

[0011] Furthermore, nanomaterials promote an increase in the colony diameter of Beauveria bassiana.

[0012] 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 nano-preparation is 424±0.864 nm; the nanomaterial is positively charged, Beauveria bassiana is negatively charged, and the nano-preparation is positively charged.

[0013] The present invention also provides the application of nanomaterials in the preparation of nano-formulations that promote the permeability of Beauveria bassiana membranes, wherein the nanomaterials are those described above.

[0014] The present invention also provides the application of nanomaterials in the preparation of nano-preparations that promote the absorption of nutrients by Beauveria bassiana, wherein the nanomaterials are those described above.

[0015] The advantages and positive effects of the nanomaterials described in this invention in promoting the growth of Beauveria bassiana are as follows: 1. The low concentration of nanomaterial SPc in this invention can promote the spore germination and mycelial 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 surface of pests. At the same time, it significantly improves the mortality rate of Beauveria bassiana against target pests.

[0016] 2. This invention deepens the understanding of microbial control mechanisms, opens up new avenues for the research and application of biological pesticides, and is of great significance for promoting the sustainable development of agricultural production.

[0017] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0018] Figure 1 This is a diagram illustrating the synthesis steps of the nanomaterial SPc in an embodiment of the present invention; Figure 2 This invention illustrates the effect of different concentrations of SPc on the germination of Beauveria bassiana spores in various embodiments. Figure 3 This invention illustrates the effect of different concentrations of SPc on the mycelial growth of Beauveria bassiana in various embodiments. Figure 4 This is a volcano diagram of differentially expressed genes in an embodiment of the present invention; Figure 5 This is a diagram illustrating the KEGG signaling pathway analysis of differentially expressed genes in this embodiment of the invention. Figure 6 This is a functional analysis diagram of the differentially expressed gene GO in Beauveria bassiana in an embodiment of the present invention; Figure 7 This is a heatmap showing the expression levels of important differentially expressed genes in Beauveria bassiana in this embodiment of the invention. Figure 8 This is a particle size distribution diagram of the combination of SPc and Beauveria bassiana in an embodiment of the present invention; Figure 9 This is a diagram illustrating the synergistic effect of SPc on the control of pea aphids by Beauveria bassiana in an embodiment of the present invention. Figure 10 This invention illustrates the effect of SPc on the colony diameter of different strains of Beauveria bassiana in this embodiment. Figure 11 This invention illustrates the effects of SPc treatment on the total sugar, triglyceride, protein, and ATP content of Beauveria bassiana. Figure 12 This describes the effect of SPc treatment on the growth rate and respiration rate of Beauveria bassiana in this embodiment of the invention. Detailed Implementation

[0019] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0020] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0021] Unless otherwise defined, the instruments, equipment and reagents used in this invention are all commercially available.

[0022] In this invention, Beauveria bassiana is... B. bassianaARSEF 2860 is a glyphosate-resistant strain Bb2860 isolated and improved from insects (see article "Survey of entomopathogenic fungi naturally infecting cereal aphids in irrigated cereal crops in southwestern Idaho").

[0023] Example 1. Preparation of nanomaterial SPc: Structural characteristics of SPc: It is a dendritic macromolecule functionalized with amino groups. Its structural formula is shown below, and the synthetic steps are as follows. Figure 1 As shown.

[0024] SPc structure of nanomaterials: , where n = 40000 g / mol.

[0025] 2. Preparation of Beauveria bassiana spore suspension and SPc dilution: Beauveria bassiana was inoculated into potato dextrose agar and cultured under growth conditions of 28°C and 70% relative humidity. When the culture dish was confluent, mycelia were gently scraped off with a sterile spreader and placed into a 50 mL sterile centrifuge tube containing sterile water. The centrifuge tube was vortexed for 5 min. The mycelia were filtered through a sterile funnel lined with six layers of sterile lens paper, and the spore suspension was collected in a sterile 50 mL centrifuge tube below the funnel. The collected filtrate was centrifuged at 8000 rpm for 15 min at 4°C. The supernatant was then discarded, the precipitate was collected, and diluted with sterile water to 1×10⁻⁶. 7 The spore count was set at 10 μl / mL as a spore suspension. After mixing, 10 μl of the spore suspension was dropped onto a 25×16 hemocytometer, and the four corner squares and the center square were selected for counting. The counting was repeated three times, and the average value was calculated. Counting formula: Spore count = Total number of spores in 5 squares × Dilution factor × 5 × 10 4 (pieces / mL).

[0026] Preparation of nanomaterial diluent: SPc was placed in an ultrasonic instrument at 99 Hz for 15 min, then filtered through a 0.22 μm filter membrane and diluted with sterile water for later use.

[0027] 3. Effects of SPc on the growth and reproduction of Beauveria bassiana: Spore germination determination: Equal amounts of SPc at different concentrations were added to the spore suspension to achieve final SPc concentrations of 0.004, 0.02, 0.1, 0.5, 2.5, and 12.5 mg / mL, respectively. Sterile water was used as a control. Three replicates were set up, and the samples were incubated at 25℃. After 12 h, spore germination was observed and photographed under a microscope. The length of the sporophyte was measured in five fields of view for each group. Spore germination rate (%) = number of germinating spores / total number of spores × 100.

[0028] The results are as follows Figure 2 As shown, 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 conidiophores; however, when the final concentration of SPc was 2.5 mg / mL and 12.5 mg / mL, SPc inhibited the germination of Beauveria bassiana, as evidenced by a decrease in spore germination rate and a significant shortening of the conidiophore 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.

[0029] Colony diameter measurement: The effect of different concentrations of SPc on the mycelial growth of Beauveria bassiana was determined using the mycelial disc method. Under aseptic conditions, equal volumes of SPc liquids of different concentrations were mixed with PDA medium and shaken well. The stipes of Beauveria bassiana were then inoculated onto the center of PDA plates using a sterile mycelial disc punch. Each experiment was repeated three times, with sterile water as a control. The plates were incubated at 25°C. On day 6, the colony diameter was measured and recorded. The growth was observed and photographed. Samples of the concentration with the best growth-promoting effect were taken for transcriptome sequencing.

[0030] The results are as follows Figure 3 As shown, compared with the control group, low concentrations of SPc (0.004 mg / mL to 0.5 mg / mL) significantly promoted the increase of colony diameter; while high concentrations of SPc (2.5 mg / mL and 12.5 mg / mL) led to a decrease in colony diameter, indicating that low concentrations of SPc treatment of Beauveria bassiana promote mycelial growth.

[0031] 4. Transcriptome analysis of the effects of SPc on Beauveria bassiana: The concentration of nanomaterials with a significant promoting effect (SPc = 0.02 mg / mL) was selected to treat Beauveria bassiana, with untreated Beauveria bassiana serving as a control. RNA was extracted from hyphae and transcriptome sequencing was performed. Transcriptome data showed that, compared with the control group, the expression levels of 439 genes in the SPc-treated Beauveria bassiana transcriptome changed, including 132 upregulated genes, 307 downregulated genes, and 10131 genes with no significant change. Figure 4 (As shown).

[0032] KEGG analysis is a database for systematically analyzing gene function and genomic information. Through pathway enrichment, it can identify the most important biochemical metabolic pathways and signal transduction pathways involved in differentially expressed genes. KEGG identifies signaling pathways significantly enriched in differentially expressed genes compared to the overall genomic background, primarily related to energy metabolism, with enrichment mainly in carbohydrate and lipid metabolism. Figure 5 (As shown).

[0033] Based on the GO database, the functional information of differentially expressed genes in *Beauveria bassiana* was analyzed in categories such as biological processes, cellular components, and molecular functions. In the biological process category, differentially expressed genes were mainly classified into functional categories such as membrane integral components and extracellular regions; in the cellular component category, differentially expressed genes were mainly classified into functional categories such as nucleic acid biosynthesis, antioxidant reactions, redox processes, polysaccharide catabolism, and cellular oxidative detoxification; in the molecular function category, differentially expressed genes were mainly classified into functional categories such as ribonuclease activity, transaminase activity, oxidoreductases, hydrolases, and pyridoxal phosphate binding. Figure 6 (As shown).

[0034] Low-concentration nanomaterials promote the growth of Beauveria bassiana, which is related to its absorption of nutrients. Screening for target genes based on this information revealed significant upregulation of genes related to sugar and lipid metabolism pathways, energy metabolism pathways, and ion-binding proteins. This demonstrates that low-concentration nanomaterials promote the membrane permeability and nutrient absorption of Beauveria bassiana. Figure 7 (As shown).

[0035] 5. Particle size distribution of the SPc-Beauveria bassiana compound: Prepare SPc (final concentration: 0.02 mg / mL) and Beauveria bassiana (1×10⁻⁶). 7 2 mL each of Beauveria bassiana / SPc compound solution and 2 mL of Beauveria bassiana / SPc compound solution were used to measure the particle size and potential of each sample at room temperature using a laser diffraction particle size analyzer (Zeta Sizer Nano Series) (Malvin Instruments, USA) with dynamic light scattering (DLS) and Zetasizer Nano ZS.

[0036] The results are as follows Figure 8 As shown, at room temperature, the particle size of SPc is 43.34±0.316 nm, the particle size of Beauveria bassiana is 409.3±1.583 nm, and the particle size of the two combined is 424±0.864 nm. 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 two combined are positively charged (43.58±0.312).

[0037] 6. SPc enhances the control effect of Beauveria bassiana on pea aphids: Individual SPc solutions of 0 mg / mL, 0.02 mg / mL, 0.1 mg / mL, and 2.5 mg / mL, as well as a mixture of SPc and Beauveria bassiana, were prepared to final concentrations of 0 mg / mL, 0.02 mg / mL, 0.1 mg / mL, and 2.5 mg / mL, respectively. Beauveria bassiana and sterile water were used as control groups. Each treatment was repeated three times. These mixtures were sprayed evenly onto the surface of three-day-old adult pea aphids using a spray method. Mortality of the pea aphids was observed on days 2, 4, and 6.

[0038] The results are as follows Figure 9 As shown, compared with the control group, when the concentration of SPc was 2.5 mg / mL, SPc treatment alone led to dehydration of pea aphids, thus increasing mortality. Secondly, SPc was combined with *Beauveria bassiana* and treated with pea aphids by soaking. The results showed that when the concentrations of SPc were 0.02 mg / mL and 0.1 mg / mL, the amount of mycelium on the surface of pea aphids increased. However, at the SPc concentration of 2.5 mg / mL, the amount of mycelium on the surface of pea aphids was low. Furthermore, it was observed that the combined application of SPc and *Beauveria bassiana* increased the mortality rate of pea aphids by more than 20%, especially at a final SPc concentration of 0.02 mg / mL, where the mortality rate increased by about 40%. These results indicate that SPc can significantly promote the growth and reproduction of *Beauveria bassiana*, thereby enhancing its control efficacy against pea aphids. The nano-formulation of SPc and *Beauveria bassiana* can be used to control pea aphids.

[0039] 7. Effects of different concentrations of SPc on the mycelial growth of two strains of Beauveria bassiana (Bb2860 and BbBJ): Two strains of Beauveria bassiana were inoculated into potato dextrose agar and cultured under the following conditions: 28°C and 70% relative humidity. The experiment was prepared when the Beauveria bassiana had fully colonized the culture dish.

[0040] Colony diameter measurement: The effect of different concentrations of SPc on the mycelial growth of two strains of Beauveria bassiana (Bb2860 and BbBJ) was determined using the mycelial disc method. Under aseptic conditions, equal volumes of SPc liquids of different concentrations were mixed with PDA medium and shaken well. The stipes of Beauveria bassiana were then inoculated onto the center of PDA plates using a sterile mycelial disc punch. Each experiment was repeated three times, with sterile water as a control. The plates were incubated at 28°C. On the 6th day, the colony diameter was measured and recorded. The growth was observed and photographed.

[0041] The results are as follows Figure 10As shown, compared with the control group, low concentrations of SPc (0.02-0.1 mg / mL) significantly promoted the increase in colony diameter of Beauveria bassiana strain Bb2860; while high concentrations of SPc (0.5-2.5 mg / mL) led to a decrease in colony diameter of Beauveria bassiana strain Bb2860, indicating that low concentrations of SPc treatment promoted mycelial growth of Beauveria bassiana (P < 0.001). Compared with the control group (BbBJ), different concentrations of SPc (0.02-2.5 mg / mL) had no significant growth-promoting effect on Beauveria bassiana strain BbBJ (P = 0.3101).

[0042] 8. Detection of the bioactivity of SPc against Beauveria bassiana: After treating Beauveria bassiana Bb2860 spores with SPc (0.02 mg / mL), the changes in total sugar, triglyceride, protein, and ATP content of Beauveria bassiana were detected on days 2, 4, 6, and 8 using a kit. Simultaneously, the growth rate and respiration rate of Beauveria bassiana after SPc treatment were detected on days 3, 6, and 9 using the same method.

[0043] The results showed that after SPc treatment, the total sugar, triglyceride, protein, and ATP contents of Beauveria bassiana significantly increased, further demonstrating that low-concentration nanomaterials promote the absorption of nutrients by Beauveria bassiana. Figure 11 As shown). Simultaneously, under the influence of SPc, the growth rate and respiration rate of *Beauveria bassiana* were enhanced (as shown). Figure 12 (As shown).

[0044] Therefore, this invention utilizes the aforementioned nanomaterials in promoting the growth of Beauveria bassiana. The nanomaterial SPc can promote spore germination and mycelial growth of Beauveria bassiana, not only increasing the biomass and activity of Beauveria bassiana, but also effectively reducing the time required for the fungus to invade the surface of pests. At the same time, it significantly improves the mortality rate of Beauveria bassiana against target pests, not only deepening the microbial control mechanism, but also opening up new avenues for the research and application of biological pesticides.

[0045] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. The application of nanomaterials in the preparation of nano-formulations that promote the growth and reproduction of Beauveria bassiana, characterized in that: The nanomaterial is SPc, and its structural formula is as follows: ， Where n = 40000 g / mol.

2. The application according to claim 1, characterized in that: When applied, the concentration of nanomaterials is 0.02-0.5 mg / mL.

3. The application of nanomaterials in the preparation of nano-formulations that promote the germination of Beauveria bassiana spores and the growth of sporangiophores, characterized in that: The nanomaterial is the nanomaterial described in claim 1.

4. The application according to claim 3, characterized in that: The concentration of the nanomaterial was 0.02 mg / mL.

5. The application of nanomaterials in the preparation of nano-formulations that promote the mycelial growth of Beauveria bassiana, characterized in that: The nanomaterial is the nanomaterial described in claim 1.

6. The application according to claim 5, characterized in that: The concentration of the nanomaterial was 0.02 mg / mL.

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

8. The application according to claim 1, 3, or 5, characterized in that: 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 nano-preparation is 424±0.864 nm; the nanomaterial is positively charged, Beauveria bassiana is negatively charged, and the nano-preparation is positively charged.

9. The application of nanomaterials in the preparation of nano-formulations that promote the permeability of Beauveria bassiana membranes, characterized in that: The nanomaterial is the nanomaterial described in claim 1.

10. The application of nanomaterials in the preparation of nano-formulations that promote the absorption of nutrients by Beauveria bassiana, characterized in that: The nanomaterial is the nanomaterial described in claim 1.

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