Method for promoting rice growth by using metarhizium anisopliae as rice endophyte

Abstract

The invention relates to the technical field of agricultural planting, in particular to a method for promoting rice growth by using metarhizium anisopliae as rice endophyte, which comprises the following steps: step 1, preparing metarhizium anisopliae spore suspension with the concentration of 1 * 10 * 8 / mL; step 2, soaking rice seeds in the spore suspension for 12 hours; 3, the treated seeds are subjected to soil culture or water culture, and the culture conditions are that the day and night temperature is set to be 26 DEG C, and the photoperiod is set to be 16 L: 8 D; and 4, colonizing the metarhizium anisopliae in root, stem and leaf tissues of the rice to promote the growth of the rice and enhance the resistance to brown planthopper and spodoptera frugiperda. According to the invention, various growth indexes of rice can be obviously improved, and the highest synergism of plant height, root length, fresh weight and dry weight under the soil culture condition is 37.9%, 10.2%, 22.7% and 19.9% respectively; under the water culture condition, the contents are respectively 46.0%, 14.8%, 50.6% and 13.0%.

Description

Technical Field

[0001] The present invention relates to the technical field of agricultural planting, and in particular to a method for promoting rice growth by utilizing Metarhizium muscardine as an endophyte of rice. Background Art

[0002] Rice is a vital food crop worldwide, particularly in Asia, where it accounts for over 90% of the world's cultivated area. China's rice production accounts for nearly 50% of the country's total grain output, making it crucial for food security. However, rice is often threatened by pests such as the brown planthopper and the fall armyworm, severely impacting yield and quality. While traditional chemical pesticides can control pests, they lead to environmental pollution and pesticide residues, threatening ecological balance and human health. Therefore, the development of environmentally friendly biological control technologies has become a research hotspot.

[0003] Metarhizium anisopliae, an entomopathogenic fungus, is widely used in biological pest control, exhibiting broad-spectrum insecticide activity while being harmless to humans, livestock, and crops. Recent studies have shown that Metarhizium anisopliae can colonize plant tissues as an endophyte, promoting plant growth and enhancing insect resistance. However, its endophytic nature, growth-promoting mechanisms, and insect resistance in rice have not been fully investigated.

[0004] To sum up the above problems, we proposed a method to promote rice growth by using Metarhizium muscardine as an endophyte of rice. Summary of the Invention

[0005] The object of the present invention is to provide a method for promoting rice growth by utilizing Metarhizium anisopliae as a rice endophyte, so as to solve the problems raised in the above-mentioned background technology.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] A method for promoting rice growth by using Metarhizium anisopliae as a rice endophyte comprises the following steps:

[0008] Step 1: Prepare a spore suspension of Metarhizium anisopliae with a concentration of 1×10 8 / mL;

[0009] Step 2, soaking the rice seeds in the spore suspension for 12 hours;

[0010] Step 3: The treated seeds are cultured in soil or water, and the culture conditions are set to a day and night temperature of 26°C and a photoperiod of 16L:8D;

[0011] Step 4: Metarhizium muscardine colonizes in the root, stem, and leaf tissues of rice, promoting rice growth and enhancing resistance to brown planthoppers and fall armyworms.

[0012] Preferably, the spore suspension is prepared with 0.05% Tween 80, and the spores of Metarhizium are collected after being cultured on PDA medium at 25° C. for 5 to 7 days.

[0013] Preferably, the rice growth indicators include germination rate, plant height, root length, fresh weight and dry weight. The plant height efficiency under soil culture conditions is increased by up to 37.9%, and the fresh weight efficiency under hydroponic conditions is increased by up to 50.6%.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] Endophytic colonization of Metarhizium: By soaking rice seeds in a suspension of Metarhizium spores, it colonizes in the root, stem and leaf tissues of rice.

[0016] Growth-promoting effect: Under soil and hydroponic conditions, rice seeds treated with Metarhizium anisum showed higher germination rate, plant height, root length, fresh weight and dry weight.

[0017] Insect-resistant effect: Rice colonized with Metarhizium anisopliae can significantly reduce the survival rate of brown planthoppers and fall armyworms, and improve the insect resistance of rice.

[0018] Advantages of the invention: Highly efficient growth promotion: Metarhizium anisopliae significantly improves rice growth indicators. Under soil culture conditions, the highest increases in plant height, root length, fresh weight and dry weight are 37.9%, 10.2%, 22.7% and 19.9%, respectively; under hydroponic conditions, the increases are 46.0%, 14.8%, 50.6% and 13.0%, respectively.

[0019] Significant insect resistance: reduces the survival rate of brown planthoppers and fall armyworms, and enhances rice's defense against pests.

[0020] Environmentally friendly: Reduce the use of chemical pesticides, reduce environmental pollution, and meet the needs of sustainable agricultural development.

[0021] Easy to operate: through seed soaking method, the process is simple and easy to promote and apply. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a schematic diagram of the selective culture of Metarhizium anisopliae in rice roots of the present invention.

[0023] Figure 2 Schematic diagram for comparing the morphologies of the strains of the present invention.

[0024] Figure 3 This is a schematic diagram of the selective cultivation of Metarhizium anisopliae in rice stems and leaves of the present invention.

[0025] Figure 4 Schematic diagram for comparing the morphologies of the strains of the present invention.

[0026] Figure 5This is a schematic diagram of the effect of endophytic Metarhizium on rice germination.

[0027] Figure 6 Schematic diagram of the effect of endophytic Metarhizium on rice plant height and root length.

[0028] Figure 7 Schematic diagram of the effect of endophytic Metarhizium on the fresh weight and dry weight of rice.

[0029] Figure 8 This is a schematic diagram of the effect of the endophytic treatment of Metarhizium muscardine on the mortality of brown planthoppers.

[0030] Figure 9 This is a schematic diagram of the effect of the endophytic treatment of Metarhizium muscardine of the present invention on the mortality rate of Spodoptera frugiperda. DETAILED DESCRIPTION

[0031] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0032] Example 1:

[0033] Experimental verification of the growth-promoting effect of Metarhizium anisopliae on rice

[0034] 1. Experimental Methods

[0035] 1. Operation process

[0036] (1) Preparation of spore suspension: Select a laboratory-preserved strain of Metarhizium anisopliae, inoculate it into PDA medium, and culture it in a constant temperature incubator at 25°C for 7 days. During this period, the colony gradually changes from white to dark green, and a large number of conidia are formed. Use a sterile scraper to carefully scrape the spores, add a solution containing 0.05% Tween-80, and prepare a spore suspension with a concentration of 1×10^8 cells / mL. The preparation process strictly controls the aseptic conditions, and a microscope is used to count the spores to ensure the accuracy of the concentration.

[0037] (2) Seed treatment: Select high-quality rice seeds Taichung Local No. 1 (TN1), place 100 healthy rice seeds on a culture dish (repeat three times), and sterilize the seeds in a 2% sodium hypochlorite solution after soaking in clean water for 2 minutes. Subsequently, transfer the seeds to 70% ethanol for sterilization for 1 minute. After sterilization, rinse the seeds with sterile distilled water, each rinse lasting 30 seconds, and rinse 2 to 3 times in total to remove residual sodium hypochlorite and ethanol. The seeds were divided into experimental and control groups. The experimental group was soaked in a suspension of green anisopliae spores for 12 hours, and the control group was soaked in sterile water for 12 hours. The soaking process was carried out in a sterile culture dish, and the seeds were evenly dispersed in the suspension or clean water to ensure sufficient contact.

[0038] (3) Rice cultivation: Soil culture: The treated seeds were planted in pots containing nutrient soil. The soil was sandy loam (pH 6.5), with 10 seeds per pot. The day and night temperature was set at 26°C, the photoperiod was 16 hours during the day and 8 hours at night, and the pots were irrigated with distilled water to 85% of their capacity daily to ensure that the soil maintained appropriate moisture. The cultivation was continued for 25 days, and the growth of the rice seeds was observed and recorded during this period. No fertilizer was applied and no sterilization treatment was performed during the entire process.

[0039] Hydroponics: Place treated seeds in Hoagland's nutrient solution (pH 6.5) in groups of 10 seeds each. Secure them to foam boards with the roots immersed in the nutrient solution. Set the day and night temperatures to 24°C and 20°C, with a photoperiod of 16 hours during the day and 8 hours at night. Change the nutrient solution every three days to ensure a stable nutrient supply.

[0040] (4) Colonization assay: 14 days after rice planting, roots, stems, and leaves were randomly collected from the experimental and control rice plants, cut into 1 cm segments, and surface-sterilized. Similar to seed sterilization, the samples were placed on PDA medium and cultured at 25°C for 5–7 days. The growth of Metarhizium colonies was observed to confirm the endophytic colonization of Metarhizium. Figure 1 , 3).

[0041] (5) Determination of growth indicators: After soaking for 12 hours, the number of rice seeds that germinated was counted every 24 hours, and the seed germination rate was recorded (calculation formula: germination rate = number of germinated seeds / total number of seeds × 100%). Every 5 days of cultivation, for a total of 25 days, 5 rice seedlings were randomly selected from each of the four different treatment groups, namely the hydroponic control treatment group, the hydroponic green muscardine treatment group, the soil culture control treatment group, and the soil culture green muscardine treatment group. The observation was repeated three times and the following four indicators were calculated. Use a ruler to accurately measure the rice plant height (from base to top) and root length (main root length), and use an electronic balance to weigh the fresh weight (directly weigh the whole plant) and dry weight (weighed after drying in a 60°C oven for 24 hours). Repeat 3 times for each group and take the average value.

[0042] (6) Data analysis: SPSS software was used to perform t-test to compare the differences in germination rate, plant height, root length, fresh weight and dry weight between the experimental group and the control group, with the significance level set at P < 0.05.

[0043] 2. Results and Analysis

[0044] The experimental results are presented in a number of accompanying figures to visually demonstrate the growth-promoting effect of Metarhizium on rice growth. The following points are analyzed in detail:

[0045] 1. Colonization: Figure 1 As shown in Figure 3, typical Metarhizium anisopliae colonies formed on PDA medium from the root, stem, and leaf samples of rice in the experimental group. These colonies were dark green, had regular edges, and covered the sample surface, indicating successful colonization of the fungus within the rice tissue. No colonies were observed in the control group samples, eliminating the possibility of exogenous contamination. This result confirms that Metarhizium anisopliae can effectively colonize rice tissue through seed soaking, providing a basis for its growth-promoting effect.

[0046] 2. Germination rate: Figure 5 As shown in the results, the seed germination rates of the experimental group treated with Metarhizium anisopliae on days 2, 3, and 4 were significantly higher than those of the control group (P<0.05). By day 5, rice seed germination had stabilized, with the final germination rates of 91% and 88% for the Metarhizium-treated and control groups, respectively, showing no significant difference (P>0.05). This suggests that Metarhizium anisopliae promotes the initial germination of rice seeds but has no significant effect on their final germination rate.

[0047] 3. Soil culture growth indicators: such as Figure 7 、 8 As shown in the figure, the plant height, root length, fresh weight and dry weight of the experimental group under soil culture conditions were significantly higher than those of the control group (P<0.05).

[0048] The specific data are as follows: Plant height: The plant height of rice treated with Metarhizium anisopliae reached the highest growth rate of 37.9% on the 10th day compared with the control group. The bar graph shows that the plant height of the experimental group is about 1.4 times that of the control group, indicating that Metarhizium anisopliae significantly promotes the growth of the aboveground part of rice ( Figure 6 C).

[0049] Root length: The root length of rice treated with Metarhizium anisopliae increased by up to 10.2% on the 25th day compared with the control group. The bar graph shows that the difference in root length is small, indicating that the fungus has a relatively mild effect on promoting root growth ( Figure 6 D).

[0050] Fresh weight: The fresh weight of rice treated with Metarhizium anisopliae increased by up to 22.7% on the 25th day compared with the control group. The height of the fresh weight column in the histogram was significantly higher than that of the control group, reflecting the increase in the overall biomass of the plant ( Figure 7 C).

[0051] Dry weight: The dry weight of rice treated with Metarhizium anisopliae increased by up to 19.9% ​​on the 10th day compared to the control group. The bar graph shows that the difference in dry weight is significant, indicating that the fungus promotes the accumulation of dry matter in rice ( Figure 7 D).

[0052] 4 Hydroponic growth indicators: such as Figure 7 、 8 As shown, the growth index of the experimental group under hydroponic conditions increased more significantly (P<0.05). Specific data are as follows: Plant height: The dry weight of rice treated with Metarhizium anisopliae increased by up to 46.0% on the 20th day compared to the control group. The bar graph shows that the plant height of the experimental group is 1.5 times that of the control group, indicating that the fungus has a stronger promoting effect on the aboveground part under hydroponic conditions ( Figure 6 A).

[0053] Root length: The dry weight of rice treated with Metarhizium anisopliae increased by up to 14.8% on the 20th day compared to the control group. The bar graph shows a significant difference in root length, indicating that the hydroponic environment is more conducive to the symbiotic effect between fungi and roots ( Figure 6 B).

[0054] Fresh weight: The dry weight of rice treated with Metarhizium anisopliae increased by up to 50.6% on the 20th day compared to the control group. The fresh weight column height in the histogram was more than 1.5 times that of the control group, indicating a significant increase in biomass. This may be due to the optimization of the growth-promoting effect of the fungus by the nutrient solution environment. Figure 7 A).

[0055] Dry weight: The dry weight of rice treated with Metarhizium anisopliae increased by up to 13.0% on the 25th day compared to the control group. The bar graph shows that the difference in dry weight is small but still significant, indicating that the accumulation of dry matter is subject to certain restrictions ( Figure 7 B).

[0056] The results showed that Metarhizium anisopliae significantly promoted rice growth through endophytic colonization, which may be achieved through the following mechanisms: first, it enhanced the efficiency of rice's absorption of nutrients such as nitrogen and phosphorus; second, it secreted growth-promoting metabolites (such as flavonoids or steroids) to stimulate plant development; third, it improved photosynthesis efficiency and increased biomass accumulation ( Figure 7 and Figure 8 The more pronounced growth-promoting effect under hydroponic conditions may be due to the reduced competition among soil microorganisms in the nutrient solution, which promotes the symbiotic relationship between the fungus and rice roots. The significant increase in germination rate further suggests that Metarhizium may accelerate germination by regulating seed metabolism. These results demonstrate the potential of Metarhizium as an endophyte for promoting rice growth.

[0057] Example 2: Experimental verification of the insect-resistant effect of Metarhizium endophytic rice

[0058] 1. Experimental Methods

[0059] 1. Operation process

[0060] (1) Preparation of spore suspension and seed treatment: Same as Example 1, preparation of spore suspension: Select a laboratory-preserved Metarhizium strain, inoculate it into PDA medium, and place it in a constant temperature incubator at 25°C for 7 days. During this period, the colony gradually changes from the initial white color to dark green, and a large number of conidia are formed. Use a sterile scraper to carefully scrape the spores, add a solution containing 0.05% Tween-80, and prepare a spore suspension with a concentration of 1×10^8 / mL. The preparation process strictly controls the aseptic conditions, and a microscope is used to count the spores to ensure the accuracy of the spore concentration.

[0061] Seed treatment: Select high-quality rice seeds Taichung Local No. 1 (TN1), and place 100 healthy rice seeds each on a culture dish (repeat three times). After soaking the seeds in clean water, sterilize them in a 2% sodium hypochlorite solution for 2 minutes. Subsequently, transfer the seeds to 70% ethanol for sterilization for 1 minute. After sterilization, rinse the seeds with sterile distilled water, each rinse lasting 30 seconds, and rinse 2 to 3 times in total to remove residual sodium hypochlorite and ethanol. The seeds were divided into experimental and control groups. The experimental group was soaked in a suspension of green muscardine spores for 12 hours, and the control group was soaked in sterile water for 12 hours. The soaking process was carried out in a sterile culture dish, and the seeds were evenly dispersed in the suspension or clean water to ensure sufficient contact.

[0062] (2) Rice cultivation: Same as in Example 1, soil cultivation: The treated seeds were planted in pots containing nutrient soil (sandy loam (pH 6.5), with 10 seeds per pot. The diurnal temperature was set at 26°C, the photoperiod was 16 hours during the day and 8 hours at night, and the pots were irrigated with distilled water daily to 85% of their capacity, ensuring that the soil maintained appropriate moisture. The cultivation was continued for 25 days, and the growth of the rice seeds was observed and recorded during this period. No fertilizer was applied and no sterilization treatment was performed during the entire process.

[0063] Hydroponics: Place treated seeds in Hoagland's nutrient solution (pH 6.5) in groups of 10 seeds each. Secure them to foam boards with the roots immersed in the nutrient solution. Set the day and night temperatures to 24°C and 20°C, with a photoperiod of 16 hours during the day and 8 hours at night. Change the nutrient solution every three days to ensure a stable nutrient supply.

[0064] (3) Colonization assay: 14 days after rice planting, roots, stems, and leaves were randomly collected from the experimental and control rice plants, cut into 1 cm segments, and disinfected on the surface. The same disinfection treatment was applied to the seeds, and the samples were placed on PDA medium and cultured at 25°C for 5-7 days. The growth of Metarhizium colonies was observed to confirm its endophytic colonization. Figure 1 , 3).

[0065] (4) Insect Resistance Test: Brown Planthopper Test: 14-day-old rice plants from the experimental and control groups were inoculated with 20 adult brown planthoppers per plant and placed in an insectary at 28°C and 70% relative humidity. Survival was observed daily, and the survival rate after 7 days was recorded (survival rate = number of surviving adults / initial number of adults × 100%).

[0066] Fall armyworm experiment: 20 second-instar larvae of S. frugiperda were seeded on rice leaves from the experimental and control groups (10 leaves per group). The leaves were then placed in an insectary at 28°C and 70% relative humidity. Survival was observed daily, and the survival rate after 7 days was recorded (survival rate = number of surviving larvae / initial number of larvae × 100%).

[0067] (5) Data analysis: SPSS software was used to perform t-test to compare the survival rates of brown planthoppers and fall armyworms between the experimental group and the control group, with the significance level set at P < 0.05.

[0068] 2. Results and Analysis

[0069] The experimental results clearly show the effect of Metarhizium on rice insect resistance through the attached figure. The following points are analyzed in detail:

[0070] Colonization: Figure 1 As shown in Figure 3, dense colonies of Metarhizium anisopliae formed on PDA culture medium in the root, stem, and leaf samples of rice from the experimental group. The colonies were dark green, covering the sample surface, and had clear edges, indicating that the fungus had successfully colonized the rice tissue. No colonies were observed in the control group samples, confirming the absence of exogenous fungal contamination. This result provides a biological basis for the insect-resistant effect of Metarhizium anisopliae, demonstrating that it can exert its sustained effects within rice tissues through endophytic colonization.

[0071] Brown planthopper survival rate: Figure 8 As shown, adult brown planthoppers fed on rice seedlings treated with either Metarhizium or water. The results showed that the mortality rate for brown planthoppers fed on rice seedlings treated with Metarhizium was 17.78%, while the mortality rate for those fed on rice seedlings treated with water was 11.11%. Statistical analysis showed a significant difference in adult brown planthopper mortality between the two treatments, with Metarhizium treatment significantly increasing adult brown planthopper mortality.

[0072] Fall armyworm survival rate: Figure 9 As shown in the figure, fall armyworm larvae fed on rice seedlings treated with either Metarhizium or water. The results showed that the mortality rate of larvae fed on rice seedlings treated with Metarhizium was 33.33%, while the mortality rate of larvae fed on rice seedlings treated with water was 13.33%. Statistical analysis showed a significant difference in mortality between the two treatments, with Metarhizium treatment significantly increasing the mortality rate.

[0073] The results showed that Metarhizium anisopliae releases toxic metabolites (such as lysergic acid amide) or induces the expression of rice defense genes through endophytic colonization in rice tissues, significantly reducing the survival rate of brown planthoppers and fall armyworms. Possible mechanisms include: first, the toxic compounds secreted by the fungus directly kill the pests; second, it indirectly inhibits the survival of pests by enhancing the immune response of rice (such as the accumulation of secondary metabolites); third, the symbiotic relationship between fungi and rice may trigger compensatory growth and reduce the feeding efficiency of pests. The stability of the anti-insect effect under soil and hydroponic conditions shows that this method has good environmental adaptability and is suitable for application in different cultivation scenarios. The endophytic colonization of Metarhizium anisopliae ensures its continuous effect in rice tissues, providing an efficient and long-lasting solution for biological control of pests.

[0074] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A method for promoting rice growth by using Metarhizium anisopliae as an endophyte of rice, characterized in that: The following steps are involved: Step 1: Prepare a spore suspension of Metarhizium anisopliae with a concentration of 1×10 8 / mL; Step 2, soaking the rice seeds in the spore suspension for 12 hours; Step 3: The treated seeds are cultured in soil or water, and the culture conditions are set to a day and night temperature of 26°C and a photoperiod of 16L:8D; Step 4: Metarhizium muscardine colonizes in the root, stem, and leaf tissues of rice, promoting rice growth and enhancing resistance to brown planthoppers and fall armyworms.

2. The method for promoting rice growth by using Metarhizium anisopliae as an endophyte of rice according to claim 1, characterized in that: The spore suspension is prepared with 0.05% Tween 80, and the Metarhizium anisopliae is cultured on a PDA culture medium at 25° C. for 5 to 7 days to collect the spores.

3. The method for promoting rice growth by using Metarhizium anisopliae as an endophyte of rice according to claim 1, characterized in that: The rice growth indicators include germination rate, plant height, root length, fresh weight and dry weight. The plant height efficiency under soil culture conditions is increased by up to 37.9%, and the fresh weight efficiency under hydroponic conditions is increased by up to 50.6%.

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