Fungal biofertilizer for promoting rice growth and its application

Abstract

The present invention discloses a fungal biofertilizer and application for promoting rice growth, and belongs to the technical field of agricultural fertilizers. The present invention provides a fungal biofertilizer and application for promoting rice growth. The fermentation of Curvularia spp. and the preparation of Curvularia spp. lysates can be performed to inoculate rice without using the fertilizers needed for planting, thereby improving the soil aggregate structure and making the soil nutrients better absorbed by rice. The Curvularia spp. biofertilizer enhances the strength of the rice base stem and improves the rice's lodging resistance. On the other hand, the roots are made strong, the taproot develops strongly in the seedling stage, and the root hairs are less, so that the nutrients are concentrated on the longitudinal growth of rice. The inoculated rice can be used without using the fertilizers needed for planting, and the growth cycle of rice is advanced. The aboveground and underground biomass of rice inoculated with the Curvularia spp. biofertilizer increases, the leaves become wider, and the leaf color deepens, which also has a positive effect on the growth of rice seedling leaves and roots.

Description

Technical Field

[0001] The invention belongs to the technical field of agricultural fertilizers, and in particular relates to a fungal biofertilizer for promoting rice growth and an application thereof. Background Art

[0002] In the past few decades, many agricultural producers have relied too much on chemical fertilizers to increase the yield of agricultural products, which has had adverse effects on the soil and the environment. [1] The systematic application of biofertilizers can establish a virtuous cycle system for the soil, and play a certain role in improving the soil ecological environment damaged by the excessive use of chemical fertilizers.

[0003] Biofertilizer is a microbial product made from the life activities of microorganisms, which contains a large number of beneficial microorganisms and has a specific fertilizer effect on crops. It is also an environmentally friendly fertilizer. Biofertilizer can be used as an auxiliary fertilizer. It can increase crop yield and quality by improving soil water and fertilizer conditions, solving soil compaction caused by chemical fertilizers, providing nutrients required for plant growth, and inducing plants to produce active substances with stress resistance. At the same time, the emergence of biofertilizers can also reduce the frequency of people's use of pesticides and chemical fertilizers, and meet the requirements of agricultural production in environmental protection. [2] The application of biofertilizers can reduce the use of chemical fertilizers, thereby reducing costs. In the short term, it can improve the quality of crops and increase their added value. Long-term application can also improve the productivity of the land. Therefore, biofertilizers can supplement and replace chemical fertilizers. [3] It is a new type of biological agent in the field of agricultural production that is highly efficient, non-toxic, harmless and pollution-free. It can simultaneously meet the requirements of agricultural product production safety, maintaining ecological balance and improving the natural environment, and has a very broad development prospect in the future fertilizer market.

[0004] At present, there are problems and shortcomings in the existing technology of biological fertilizers:

[0005] There is still a lack of some efficient bacterial resources: lack of special phosphate-dissolving bacteria that can efficiently dissolve various insoluble phosphorus in the soil, tolerate phosphorus fertilizer stress, and improve the utilization rate of phosphorus fertilizer; lack of combined nitrogen-fixing bacteria that are resistant to nitrogen repression and have efficient nitrogen fixation; lack of potassium-dissolving bacteria that can efficiently activate insoluble potassium in the soil and improve the utilization efficiency of potassium fertilizer; lack of efficient growth-promoting bacteria that can efficiently produce auxin, gibberellin, and cytokinin, etc. [4] ;

[0006] Biofertilizers have a short shelf life and poor survival effects. There are deficiencies in microbial activity and sterilization technology. The bacterial activity retention time and shelf life of some biofertilizers are short, resulting in instability in cost and quality. [4] ;

[0007] Current biological fertilizers have a single function, with more consideration given to increasing fertilizer efficiency and providing nutrition for plant growth. There is less research on their ability to inhibit specific pathogens, kill bacteria, prevent soil-borne diseases, and improve soil.

[0008] References:

[0009] [1] Bian Jianwen, Cui Yan, Yang Songqi, et al. Research progress on agricultural application of microalgae biofertilizers[J]. Chinese Journal of Soil and Fertilizer, 2020, (05): 1-9.

[0010] [2] Jin Min. Research on countermeasures for high-quality development of microbial fertilizer industry in Shandong Province[D]. Shandong Normal University, 2021

[0011] [3] Ye Jingjing, Cao Ningning, Wu Jianmei, et al. Research progress of biofertilizer and its application on mulberry trees[J]. China Sericulture, 2018, 39(02): 42-46.

[0012] [4] Fan Bingquan. Research and application progress of biofertilizer in my country[J]. Journal of Plant Nutrition and Fertilizer, 2017, 23(06): 1602-1613. Summary of the invention

[0013] The present invention provides a fungal biofertilizer for promoting rice growth and its application, wherein the fungal biofertilizer can promote above-ground and underground biomass of rice and make the root system more developed. The present invention increases fertilizer efficiency and improves soil aggregate structure through fermentation by fungi, so that soil nutrients are better absorbed by rice.

[0014] The main purpose of the present invention is to provide a method for producing a Curvularia biofertilizer that promotes rice growth, and specifically relates to a method in which the present invention ferments Curvularia and prepares Curvularia lysate, and inoculates rice without using chemical fertilizers required for planting, thereby improving the soil aggregate structure and allowing soil nutrients to be better absorbed by rice. The Curvularia biofertilizer enhances the strength of the rice base stem and improves the rice's ability to resist lodging. On the other hand, the roots are made strong, the taproot develops strongly in the seedling stage, and the root hairs are less, so that nutrients are concentrated on the longitudinal growth of rice. The inoculated rice does not need to use chemical fertilizers required for planting, and the growth cycle of rice is advanced. The aboveground and underground biomass of rice inoculated with the Curvularia biofertilizer increases, the leaves become wider, and the leaf color deepens, which also has a positive effect on the growth of rice seedling leaves and roots.

[0015] The Curvularia used in the present invention includes Curvularia CLD001 (Curvularia CLD001), which was deposited in the China General Microbiological Culture Collection Center on April 8, 2022, with the deposit number CGMCC No. 40127, and was disclosed in Chinese patent CN114907987A.

[0016] The Curvularia spp. described in the present invention mainly plays an indirect nutritional role, which is mainly reflected in:

[0017] (1) Improve soil fertility: It can decompose the insoluble phosphorus and potassium in the soil and convert them into phosphorus and potassium compounds that can be absorbed and utilized by crops, thereby increasing the supply of nutrients in the crop growth environment, increasing the organic matter in the soil, and improving the soil fertility.

[0018] (2) Improve soil structure: Curvularia can also secrete a large amount of extracellular polysaccharides, such as capsular polysaccharides and peptidoglycan. Studies have shown that the sugars produced by these beneficial microorganisms account for 0.1% of soil organic matter. They can work together with plant root secretions, soil colloids, etc. to form soil aggregate structure. In addition, they also participate in the formation of humus and improve the physical and chemical properties of the soil.

[0019] (3) Stimulate crop growth and development: The microorganisms in Curvularia can also secrete plant hormones and vitamins to stimulate and regulate crop growth and development. They can produce a variety of active substances (auxin, pantothenic acid, pyridoxine, deoxymethamphenicol, indoleacetic acid and other compounds).

[0020] The method for preparing the Curvularia spore lysate of the present invention comprises the following specific steps:

[0021] (1) CLD001 was inoculated into PDA and cultured at 28°C and 180 rpm for 15 days. The culture was centrifuged at 9000 rpm for 15 minutes and the supernatant was collected, which contained antibacterial substances.

[0022] (2) Add an equal volume of acetone to the CLD001 fermentation filtrate, shake and mix, and let stand at room temperature for several hours. Shake and mix three times during the standing process.

[0023] (3) Filter the fermentation liquid with acetone added through gauze to remove flocculent matter.

[0024] (4) Acetone was evaporated using a rotary evaporator, and an equal volume of ethyl acetate was added to the fermentation broth, the mixture was shaken and mixed, and the mixture was allowed to stand at room temperature for several hours, with the mixture shaken and mixed three times during the standing period.

[0025] (5) Pour the above liquid into a separatory funnel and let it stand for 20 minutes until it is clearly separated. Open the separatory funnel switch and collect the two liquids separately, of which the clear liquid is the organic phase and the turbid liquid is the fermentation filtrate.

[0026] (6) Concentration and evaporation: Pour the organic phase obtained by separation into a distillation flask and evaporate the organic solvent to dryness under negative pressure.

[0027] (7) After evaporation to dryness, the concentrated solution was obtained, and appropriate amounts of methanol and distilled water were added to dissolve it, and ultrasonicated for 5 minutes and placed at -20°C overnight. The frozen material was lyophilized by a freeze dryer to obtain a powdery crude extract.

[0028] Process steps: Preparation of liquid culture medium, preparation of strain seed solution, preparation of Curvularia cytolysate

[0029] 1. Preparation of Liquid Culture Medium

[0030] Measure 200g of potato, 20g of sucrose, and 1000mL of water. Wash the potato, peel it, cut it into small pieces, put it into water and boil it until it breaks when touched by a glass rod. Filter it with 6 layers of gauze, add sugar, and add water to make 1000mL of solution. The pH value is natural. After packaging, place it in a high-pressure steam sterilizer and heat it at 1.05kg / cm 2 , sterilize with high pressure steam at 121℃ for 20min, take out and refrigerate for later use.

[0031] 2. Preparation of strain seed solution

[0032] Inoculate the bacteria stored on the plate into the sterilized LB medium, place it on a shaker at 180rpm and 28℃ for 24h to activate the bacteria, draw 3% of the activated bacterial solution and inoculate it into fresh sterilized LB medium, and culture it for another 24h under the above conditions to make a seed solution with vigorous activity. [4] .

[0033] 3. Preparation of Curvularia spp. Lysate

[0034] Extract fungal substances from the PDA culture medium of the strain. The specific steps are as follows:

[0035] (1) CLD001 was inoculated into PDA and cultured at 28°C and 180 rpm for 15 days. The culture was centrifuged at 9000 rpm for 15 minutes and the supernatant was collected, which contained antibacterial substances.

[0036] (2) Add an equal volume of acetone to the CLD001 fermentation filtrate, shake and mix, and let stand at room temperature for several hours. Shake and mix three times during the standing process.

[0037] (3) Filter the fermentation liquid with acetone added through gauze to remove flocculent matter.

[0038] (4) Acetone was evaporated using a rotary evaporator, and an equal volume of ethyl acetate was added to the fermentation broth, the mixture was shaken and mixed, and the mixture was allowed to stand at room temperature for several hours, with the mixture shaken and mixed three times during the standing period.

[0039] (5) Pour the above liquid into a separatory funnel and let it stand for 20 minutes until it is clearly separated. Open the separatory funnel switch and collect the two liquids separately, of which the clear liquid is the organic phase and the turbid liquid is the fermentation filtrate.

[0040] (6) Concentration and evaporation: Pour the organic phase obtained by separation into a distillation flask and evaporate the organic solvent to dryness under negative pressure.

[0041] (7) After evaporation, a concentrated solution is obtained, and appropriate amounts of methanol and distilled water are added to dissolve it, and ultrasonicated for 5 minutes and placed at -20°C overnight. The frozen material is freeze-dried by a freeze dryer to obtain a powdered crude extract, which is the fungal biofertilizer.

[0042] Beneficial effects: The present invention provides a method for producing a Curvularia biofertilizer for promoting rice growth, and specifically relates to a method in which the present invention ferments Curvularia, prepares Curvularia lysate, and inoculates rice to effectively reduce the fertilizer required for planting, thereby improving the soil aggregate structure so that soil nutrients are better absorbed by rice, and increasing the above-ground and underground biomass of rice, making the root system more developed and enhancing the lodging resistance.

[0043] The Curvularia biofertilizer used in the present invention has various functions. The Curvularia biofertilizer enhances the strength of the rice base stem and improves the lodging resistance of the rice. On the other hand, it makes the roots strong, the taproot develops strongly in the seedling stage, and the root hairs are less, so that the nutrients are concentrated on the longitudinal growth of the rice, and the growth cycle of the rice is advanced. The aboveground and underground biomass of the rice inoculated with the Curvularia biofertilizer increases, the leaves become wider, and the leaf color deepens, which also has a positive effect on the growth of the rice seedling leaves and root system, and is conducive to stable and high yields.

[0044] The processing technology of Curvularia biofertilizer is made into active freeze-dried powder, which solves the problem that biofertilizer is easy to lose effectiveness and difficult to preserve. It is odorless, easy to apply and uniform. The use of biofertilizer does not require mixing with chemical fertilizers. Only Curvularia can be inoculated to achieve the same growth-promoting effect of using chemical fertilizers. It contains functional bacteria and organic matter, can improve the soil and promote the release of nutrients fixed by the soil, is ecological and environmentally friendly, and greatly saves the impact of chemical fertilizers on the environment. The fermentation of Curvularia is economical and environmentally friendly, has a low production cost, and is environmentally friendly. Therefore, the application of Curvularia in biofertilizers described in the present invention belongs to the exploration of efficient bacterial species resources. Curvularia fungi are mostly found in Gramineae plants and are widely distributed. They have important economic value in agriculture, industry, medicine, and environmental protection. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 The root comparison diagram of different groups, from left to right in the figure respectively represents the group with 60% bacteria and 40% compound fertilizer, the group with 100% compound fertilizer, and the CK group;

[0046] Figure 2 This is a growth status diagram of different groups. From left to right in the figure are 100% compound fertilizer group, 60% bacteria group, and CK group. DETAILED DESCRIPTION

[0047] In order to further illustrate the present invention, the technical solutions provided by the present invention are described in detail below in conjunction with embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0048] Example 1

[0049] Extract fungal substances from the PDA culture medium of the strain. The specific steps are as follows:

[0050] (1) CLD001 was inoculated into PDA and cultured at 28°C and 180 rpm for 15 days. The culture was centrifuged at 9000 rpm for 15 minutes and the supernatant was collected, which contained antibacterial substances.

[0051] (2) Add an equal volume of acetone to the CLD001 fermentation filtrate, shake and mix, and let stand at room temperature for several hours. Shake and mix three times during the standing process.

[0052] (3) Filter the fermentation liquid with acetone added through gauze to remove flocculent matter.

[0053] (4) Acetone was evaporated using a rotary evaporator, and an equal volume of ethyl acetate was added to the fermentation broth, the mixture was shaken and mixed, and the mixture was allowed to stand at room temperature for several hours, with the mixture shaken and mixed three times during the standing period.

[0054] (5) Pour the above liquid into a separatory funnel and let it stand for 20 minutes until it is clearly separated. Open the separatory funnel switch and collect the two liquids separately, of which the clear liquid is the organic phase and the turbid liquid is the fermentation filtrate.

[0055] (6) Concentration and evaporation: Pour the organic phase obtained by separation into a distillation flask and evaporate the organic solvent to dryness under negative pressure.

[0056] (7) After evaporation, a concentrated solution is obtained, and appropriate amounts of methanol and distilled water are added to dissolve it, and ultrasonicated for 5 minutes and placed at -20°C overnight. The frozen material is freeze-dried by a freeze dryer to obtain a powdered crude extract, which is the fungal biofertilizer.

[0057] Example 2

[0058] Potted experiment using the fungal biofertilizer shown in Example 1

[0059] On December 28, 2023, a potted experiment was conducted in the rice cultivation greenhouse (28°C, 16h day / 8h night) on the fifth floor of the School of Life Sciences of Jiangxi Normal University. The rice seeds used in the experiment were ZH11, provided by Xie Jiankun Laboratory of Jiangxi Normal University, Jiangxi Province.

[0060] Sandy soil was collected from the shade of the trees behind the School of Life Sciences, and compound fertilizer was purchased from Jiangsu Huachang Chemical Co., Ltd. The mixture was evenly mixed, dried, and sieved, and 7.5 kg was weighed into each pot for use.

[0061] Select healthy and plump seeds and soak them at room temperature for 48 hours, plant them in the previously treated soil, and cultivate them in a greenhouse at 28℃, 16h day / 8h night. After germination, replenish water regularly and quantitatively to ensure normal growth of the plants.

[0062] The following treatment groups were set up in this pot experiment: (1) blank control group (CK): no compound fertilizer and Curvularia spp., 3 treatments; (2) 100% compound fertilizer treatment group (compound fertilizer of type 15 nitrogen, 15 phosphorus and 15 potassium) (6.8 g), 3 treatments; (3) Curvularia spp. and compound fertilizer treatment group (60% bacteria and 40% compound fertilizer) (15.03 g bacteria + 2.72 g compound fertilizer), 3 treatments.

[0063] The 33-day-old rice seedlings were uprooted, washed and dried, and the stem width, root length, stem length, root fresh weight, stem fresh weight, root dry weight and stem dry weight were measured.

[0064] Table 1 Trait statistics

[0065] deal with Stem width (cm) Root length (cm) Stem length (cm) Root fresh weight (cm) Stem fresh weight (g) Root dry weight (g) Stem weight (g) 1 0.41 12.26 33.05 0.090 0.242 0.0371 0.0693 2 0.86 19.22 43.687 0.255 0.769 0.09647 0.2376 3 0.753 21.1967 44.4367 0.302 0.91 0.1147 0.2394

[0066] In the above table, treatment 1 is CK, which is a blank control without fertilizer, treatment 2 is 100% compound fertilizer (nitrogen 15 phosphorus 15 potassium 15 type), 100% compound fertilizer (6.8g) was applied as basal fertilizer, and treatment 3 was 60% bacteria + 40% compound fertilizer (nitrogen 15 phosphorus 15 potassium 15 type), 60% bacteria (15.03g) + 40% compound fertilizer (2.72g) was applied as basal fertilizer.

[0067] The results are as follows Figures 1-2 As shown, the root length and fresh root weight of treatment 3 are greater than those of treatments 1 and 2, and the detailed diagram of the roots of rice seedlings shows that the root growth promotion points of the group treated with Curvularia spp. increase, the number of white roots increases and is strong, and the nutrient absorption efficiency is improved. In view of the above phenomenon, the microbial fertilizer utilizes the unique effect of Curvularia spp. in enhancing rooting ability and improving nutrient absorption efficiency, and solves the problems of low quality and low yield and lodging resistance of rice due to underdeveloped root system and weak rooting ability during growth. The present invention provides a preparation method of microbial fertilizer for promoting nutrient absorption of rice.

[0068] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for improving crop resistance and growth, characterized in that: These include the application of fungal biofertilizers; During the application, 60% by weight of fungal biofertilizer and 40% by weight of compound fertilizer are applied in the form of base fertilizer; The preparation method of the fungal biofertilizer comprises the following steps: (1) inoculating seed liquid of Curvularia CLD001 into PDA for shaking culture, and centrifuging to obtain supernatant after the culture is completed; the deposit number of Curvularia CLD001 is CGMCC No.40127; (2) mixing the supernatant of step (1) and acetone in equal volumes, letting the mixture stand and filtering to remove flocculent matter and acetone in the filtrate to obtain a filtrate; (3) The filtrate of step (2) is mixed evenly with an equal volume of ethyl acetate, allowed to stand and then separated, and the organic phase is collected; (4) removing the organic solvent in the organic phase of step (3), dissolving the concentrated solution with methanol and distilled water, and freeze-drying after ultrasonic treatment to obtain the fungal biofertilizer.

2. The method according to claim 1, characterized in that: The shaking culture in step (1) includes shaking culture at 28°C and 180r / min for 15 days; centrifuging the culture at 9000r / min for 15 minutes and taking the supernatant.

3. The method according to claim 1, characterized in that: The standing process in step (2) further includes oscillating and mixing 3 times; The filtering includes filtering with gauze to remove flocculent substances.

4. The method according to claim 1, characterized in that: The ultrasonication in step (4) includes ultrasonication for 5 minutes, placing at -20°C overnight, and freeze-drying the frozen material through a freeze dryer to obtain the fungal biofertilizer in powder form.

Images