Chlorella pyrenoidosa microalgae fertilizer for preventing and controlling pathogenic bacteria of farmland soil scab as well as preparation method and application of chlorella pyrenoidosa microalgae fertilizer

Abstract

The invention discloses a chlorella pyrenoidosa microalgae fertilizer for preventing and controlling pathogenic bacteria of farmland soil scab as well as a preparation method and application of the chlorella pyrenoidosa microalgae fertilizer, and belongs to the technical field of biology. The invention discloses a chlorella pyrenoidosa microalgae fertilizer for preventing and controlling pathogenic bacteria of farmland soil scab. The chlorella pyrenoidosa microalgae fertilizer is an alkaline microalgae fertilizer or an acidic microalgae fertilizer. Compared with the existing traditional chemical pesticide or biocontrol inoculant, the chlorella pyrenoidosa microalgae fertilizer disclosed by the invention has the advantages that the application amount of the chemical pesticide is greatly reduced, the abundance of pathogenic bacteria of scab in farmland soil is fundamentally blocked, controlled and inhibited, the diversity and functional activity of soil microorganisms are remarkably enhanced, and the application amount of the chemical pesticide is greatly reduced. The yield of rhizome crops such as potatoes is improved, and the disease prevention and control, soil improvement and crop productivity improvement efficiency of the rhizome crops are highly considered.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, more particularly to a kind of protein Chlorella pyrenoidosa microalgae fertilizer for controlling and preventing soil scab pathogen and its preparation method and application. BACKGROUND

[0002] Soil scab caused by pathogenic Streptomyces is an important soil-borne disease affecting root crops such as potatoes, which can survive in soil for 10 years and is considered the fourth largest soil-borne disease after potato late blight, early blight, and ring rot. The disease is widely distributed in many countries, such as the United States, the Netherlands, and 23 other countries (regions) in China. In China, scab has been reported in 19 provinces (cities) and autonomous regions, including Heilongjiang, Gansu, and Yunnan. The pathogenic bacteria causing crop scab are very complex, and new pathogenic species are constantly being discovered, including Streptomyces scabiei, Streptomyces acidiscabies, and Streptomyces tumescens.

[0003] Root crops are more susceptible to scab under conditions of continuous planting for many years, poor production management of seed potatoes, and improper fertilization, with a disease incidence of over 90% in some areas. Pathogenic Streptomyces spreads scab through soil transmission and seed transmission. During the early stages of potato tuber formation, it enters the tissue through stomata, secretes toxins to cause brown spots on the tuber surface, and as the stolon enlarges, the lesions gradually expand, eventually causing necrosis of the tissue around the infection site, roughening of the tuber surface, and tissue suberization, resulting in the formation of scab-like rough patches on the surface of potatoes, which severely affects the appearance, quality, and economic value of potatoes, causing significant economic losses to growers. Scab has become a bottleneck problem restricting the high-quality development of potato and other root crop industries.

[0004] Currently, chemical fungicides are still the main method for controlling this disease, and have a certain control effect. For example, a patent entitled "A method and agent for preventing and treating potato scab (Application No. 202411008138.6)" has invented a prevention and treatment agent with kojic acid as the main effective component, with a field control effect of 50.3%. Biological control technology using biological agents as the main measure will become a hot spot and trend in the future for the prevention and treatment of potato scab. This method mainly uses biocontrol microorganisms and their metabolites to inhibit the occurrence of diseases, and is an environmentally friendly disease control method.

[0005] There are also many scholars who have screened out biological control strains with prevention and control effect on potato scab, such as the patent "Bacteria and medicine composition for preventing and treating potato scab and application thereof (application number 202510054335.X)" and "Compound microbial agent for preventing and treating potato scab and application thereof (application number 202510051819.9)", both of which use Bacillus velezensis BEV2 and Bacillus atrophaeus FM2-4 to prevent and control potato scab, with a control effect of 59.35% to 70.17%; the patent "Bacterium for preventing and treating potato scab and preparation method and application thereof (application number 202410291229.9)" uses Paenibacillus polymyxa AF01 and Trichoderma asperellum TZ312 to prevent and control potato scab, and the pot experiment results show that the control effect can reach 75%. The above patents mainly use biocontrol strains that can directly inhibit potato scab, and through direct antagonism between biocontrol strains and scab pathogens, the inhibition effect on potato scab pathogens is achieved. The control ideas of the above patents are all direct antagonism between biocontrol strains and pathogens, and then the purpose of inhibiting pathogens is achieved, but the effects of applying biocontrol microorganisms on the abundance, species diversity and functional activity of other microbial communities in the soil are not clear.

[0006] The fundamental reason for the pathogenicity and disease of potato and other root crops scab is the decline of soil microbial diversity and functional activity. Although biocontrol microbial preparations have the characteristics of high biological safety, small toxic and side effects, etc. in preventing and controlling potato and other root crops scab, the specific functional microbial strains have poor stability in the actual planting process in the field, and the improvement of soil microbial diversity and functional activity by biocontrol microbial preparations is low, which leads to unsatisfactory control effect and is easily affected by the environment.

[0007] Therefore, how to provide a technology and biofertilizer that can improve soil microbial community diversity and functional activity, and fundamentally improve soil health level and restore soil microbial diversity and functional activity, so as to realize ecological control of potato and other root crops scab pathogens, has become a problem to be solved by those skilled in the art. SUMMARY

[0008] Therefore, how to provide a technology and biofertilizer that can improve soil microbial community diversity and functional activity, and fundamentally improve soil health level and restore soil microbial diversity and functional activity, so as to realize ecological control of potato and other root crops scab pathogens, has become a problem to be solved by those skilled in the art.

[0009] The pathogenic bacteria of Streptomyces in farmland soil cause the occurrence of scab disease of crops by infecting the root system or the epidermal tissue of tuber, causing epidermal tissue necrosis. At present, the prevention and control of the pathogenic bacteria mainly relies on chemical pesticides, which has two problems: one is that the chemical pesticides kill the beneficial bacteria in the soil while killing the pathogenic bacteria, resulting in the decrease of microbial diversity and functional activity in the soil of root and stem crops, and failing to fundamentally improve the soil micro-ecological environment; the other is that the chemical pesticides cause pesticide residues, which eventually leads to further deterioration of the soil ecological environment.

[0010] The present application creates an ecological friendly Chlorella pyrenoidosa microalgae fertilizer, which makes up for the lack of green prevention and control products and technologies for scab disease in farmland soil. On the one hand, it can effectively inhibit and reduce the abundance of scab disease pathogenic bacteria in farmland soil, and on the other hand, it can improve soil microbial abundance and thus improve soil health without pesticide residues.

[0011] In order to achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0012] The present application uses Chlorella pyrenoidosa as the algal species, and greatly reduces the cost of the culture medium in the preparation process of Chlorella pyrenoidosa microalgae fertilizer by improving and simplifying the culture medium. The microalgae liquid fertilizer prepared by the improved culture medium is rich in various active substances, which can improve the soil microbial abundance and functional activity, reduce the survival space of scab disease pathogenic bacteria in farmland soil, and thus reduce the abundance and overall proportion of scab disease pathogenic bacteria in the soil.

[0013] In order to effectively control the scab disease of root and stem crops, Chlorella pyrenoidosa is used as the target algal species to construct a Chlorella pyrenoidosa microalgae fertilizer. The liquid fertilizer is rich in various biological active ingredients such as proteins, amino acids, indole acetic acid, gibberellins and polysaccharides. After being applied to the soil, it can rapidly improve the soil microbial diversity and functional activity, greatly reduce the proportion of root and stem scab disease pathogenic bacteria in the soil, and thus reduce the risk of scab disease of root and stem crops. The specific steps are as follows:

[0014] Chlorella pyrenoidosa (GY-D12 Chlorella pyrenoidosa) liquid strain is purchased from Shanghai Guangyu Biotechnology Co., Ltd. Chlorella pyrenoidosa is inoculated into a pre-sterilized and cooled special culture medium, and after 14-18 hours of culture under full-spectrum light source (light intensity 5000-8000 lux) at a temperature of 25±1℃, followed by 6-10 hours of dark culture, the first level of algal liquid is obtained after 3-4 days of culture. The first level of algal liquid is inoculated into a pre-sterilized special culture medium at a volume percentage of 8-15%, and is cultured at 25-30℃ by using aeration pump for 20-40 minutes at intervals of 6-8 hours. After 5-7 days of culture, Chlorella pyrenoidosa alkaline microalgae fertilizer is obtained, and the density of Chlorella pyrenoidosa in the microalgae fertilizer is 6.00×10 6cfu / mL ~ 1.0 x 10 7 cfu / mL, the alkaline microalgae fertilizer is alkaline and the pH value is above 8.5. The special medium ratio is: ammonium chloride 1.00 g / L, magnesium sulfate 0.06 g / L, potassium dihydrogen phosphate 0.03 g / L, sodium bicarbonate 0.02 g / L, glucose 0.02 g / L, and all the above materials are standard analytical reagents.

[0015] The acidic microalgae fertilizer of chlorella pyrenoidosa: the acidic microalgae fertilizer is added with glutamic acid and glycine in the alkaline microalgae fertilizer, and the amount of glutamic acid and glycine is 3-6 g / L; the microalgae fertilizer is acidic and the pH value is between 6.0-7.0.

[0016] Secondly, in the potato seedling stage, tuber setting stage and tuber bulking stage, the protein core small ball algae microalgae fertilizer is applied by drip irrigation, and is applied by 3 times of water flushing, and the flushing amount of each time is 20-50 liters / mu, and the total amount of the protein core small ball algae liquid applied per mu is 60-150 liters.

[0017] Finally, 15 days before the potato is harvested, the incidence of potato tuber scab is observed, the surface soil of the potato tuber is collected, the total DNA of the soil is extracted by using a soil DNA kit (Omega Bio-tek, Norcross, GA, U.S.) kit, and the copy number of the scab pathogenic gene txtAB in the soil is quantitatively detected by using real-time fluorescent quantitative q-PCR, and the amplification primers used for determining the scab pathogenic gene txtAB are StrepF (5'-GCAGGACGCTCACCAGGTAGT-3'), StrepR (5'

[0018] -ACTTCGACACCGTTGTCCTCAA-3'), the length of the amplified target fragment is 72 bp, and the amplification condition is 95 DEG C pre-denaturation for 5 min; 95 DEG C denaturation for 10 s, 55 DEG C annealing for 10 s, 72 DEG C extension for 20 s, 40 cycles. The incidence of potato tuber skin and the amount of scab pathogenic bacteria in the soil are compared to determine the control effect of the microalgae liquid fertilizer on the scab of the root and stem crops-potato.

[0019] The present application prepares a protein core small ball algae microalgae fertilizer rich in various beneficial active substances, improves the soil microbial diversity and functional activity, greatly reduces the proportion of soil scab pathogenic bacteria in the soil, and further reduces the risk of scab disease of root and stem crops, compared with the traditional chemical pesticide control method, the protein core small ball algae microalgae fertilizer prepared by the present application has no pesticide residue and toxic side effects, so as to realize the green and ecological prevention and control of the scab disease of the root and stem crops, and the application of the protein core small ball algae microalgae fertilizer can also improve the availability of soil available nitrogen and phosphorus nutrients, and improve the yield and quality of potato.

[0020] ​Compared with the prior art, the protein Chlorella pyrenoidosa microalgae fertilizer and the preparation method and application thereof provided by the present application can significantly reduce the application amount of chemical pesticides, fundamentally control and inhibit the abundance of the C. pyrenoidosa pathogen in farmland soil, significantly enhance the soil microbial diversity and functional activity, and improve the yield of root crops such as potatoes, and highly consider the disease control, soil improvement and crop yield improvement of root crops. BRIEF DESCRIPTION OF DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced as follows. Obviously, the drawings in the following description are only embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of the provided drawings.

[0022] Figure 1 The growth conditions of the protein Chlorella pyrenoidosa microalgae fertilizer are prepared for the general standard medium (BG11) and the improved medium (special medium).

[0023] Figure 2 The protein Chlorella pyrenoidosa microalgae fertilizer is prepared by using the special medium.

[0024] Figure 3 The soil total bacteria, the abundance and the proportion of the C. pyrenoidosa pathogen in different treatment groups of the test point 1 in Shenmu City are shown in the table. 8 .

[0025] Figure 4 The actual photos of the potato C. pyrenoidosa disease occurrence in different treatment groups of the test point 1 in Shenmu City are shown in the table.

[0026] Figure 5 The soil total bacteria, the abundance and the proportion of the C. pyrenoidosa pathogen in different treatment groups of the test point 2 in Shenmu City are shown in the table.

[0027] Figure 6 The soil total bacteria, the abundance and the proportion of the C. pyrenoidosa pathogen in different treatment groups of the test point 1 in Dingbian County are shown in the table.

[0028] Figure 7 The soil total bacteria, the abundance and the proportion of the C. pyrenoidosa pathogen in the treatment group and the control group of the test point 2 in Dingbian County are shown in the table. DETAILED DESCRIPTION

[0029] With reference to the accompanying drawings, the technical solutions in the embodiments of the present application will be clearly and completely described in the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all the other embodiments obtained by those skilled in the art without creative work fall within the scope of the present application.

[0030] (1) The present application provides a Chlorella pyrenoidosa microalgae fertilizer for reducing the abundance of Alternaria solani in the soil of potato farmland. The density of Chlorella pyrenoidosa in the microalgae fertilizer is 6.00×10 6 cfu / mL to 1.00×10 7 cfu / mL or more. The Chlorella pyrenoidosa (GY-D12 Chlorella pyrenoidosa) liquid strain is purchased from Shanghai Guangyu Biotechnology Co., Ltd.

[0031] (2) Implementation process:

[0032] The Chlorella pyrenoidosa microalgae fertilizer can reduce the abundance of Alternaria solani in the soil of rhizome crops and improve the yield of rhizome crops, and is a new type of prevention and control method. The Chlorella pyrenoidosa microalgae fertilizer is applied by irrigation at the seedling stage, tuber formation stage and tuber bulking stage of potatoes. The Chlorella pyrenoidosa microalgae fertilizer is applied by irrigation with water by using drip irrigation. The drip irrigation belt used should meet the national legal standards. The drip irrigation belt type can be an internal inlaid drip irrigation belt, a labyrinth drip irrigation belt or a patch type drip irrigation belt. The water output of each drip head is uniform, and the water flow rate of each drip head can be 1-3 liters / hour. The application amount of the Chlorella pyrenoidosa microalgae fertilizer in the three times of irrigation is 20-50 L / acre, which can be any one of 20 L / acre, 30 L / acre and 50 L / acre.

[0033] In order to further illustrate the present application, the implementation process and effects of the microalgae liquid fertilizer provided by the present application are described in detail below with reference to the accompanying drawings and examples.

[0034] Example 1: An alkaline microalgae fertilizer for reducing the abundance of Alternaria solani in the soil of rhizome crops

[0035] The Chlorella pyrenoidosa (GY-D12 Chlorella pyrenoidosa) liquid strain is inoculated into a special culture medium which has been sterilized and cooled. After 14 hours of light culture at a temperature of 25±1℃ and under full-spectrum light source (light intensity 5000 lux), 10 hours of dark culture is performed. After 4 days of culture, a first-level culture algal liquid (first-level strain) is obtained. The first-level culture algal liquid is inoculated into a special culture medium which has been sterilized at a volume percentage of 12%. The culture is performed at 26℃ by using an aeration pump for 40 minutes at intervals of 6 hours. After 7 days of culture, an alkaline Chlorella pyrenoidosa microalgae fertilizer is obtained. The density of Chlorella pyrenoidosa in the microalgae fertilizer is 1.00×107 cfu / mL Figure 2 The alkaline microalgae fertilizer has a pH value of 9.2.

[0036] The alkaline microalgae fertilizer is prepared by replacing the special medium with a standard medium (BG11).

[0037] The optical density, cell density and dry matter quality of the microalgae cells of the microalgae fertilizer prepared by the two kinds of media are shown in Table 1. Figure 1 .

[0038] Example 2: An acidic microalgae fertilizer for reducing the abundance of the pathogen of scab disease in rhizome crops

[0039] The acidic microalgae fertilizer is prepared by adding glutamic acid and glycine to the alkaline microalgae fertilizer prepared in Example 1. The specific steps are as follows: after the preparation of the alkaline microalgae fertilizer of Chlorella pyrenoidosa is completed, 6 g / L of glutamic acid is first added to the alkaline microalgae fertilizer, and the mixture is gently stirred for 20 minutes to facilitate the dissolution of the glutamic acid. Then, 6 g / L of glycine is added, and the mixture is gently stirred for 20 minutes. Then, the mixture is left to stand at room temperature for 24 hours or more. Before application, the mixture is stirred for 10 minutes to ensure that there is no precipitate at the bottom of the microalgae fertilizer. The glutamic acid and glycine are food-grade materials that meet the national quality standards. The density of the Chlorella pyrenoidosa in the acidic microalgae fertilizer is 1.00 x 10 7 cfu / mL, and the microalgae fertilizer is acidic with a pH value of 6.2.

[0040] Example 3: A method for reducing the abundance of the pathogen of scab disease in farmland soil

[0041] According to the acid-base characteristics of the farmland soil, if the farmland soil is acidic, the alkaline microalgae fertilizer prepared by the method in Example 1 is selected. If the farmland soil is alkaline, the acidic microalgae fertilizer prepared by the method in Example 2 is selected. The specific application method is as follows:

[0042] (1) The first application of the microalgae fertilizer is carried out at the seedling stage of the rhizome crops (such as potatoes) in the farmland. The application method is drip irrigation. The dosage of the acidic microalgae fertilizer is 20-50 L per mu. The drip irrigation speed is 1.38-2 L / h, and the irrigation time is 2-4 hours. The irrigation time is appropriately shortened according to the water flow rate of the drip irrigation and the soil water content, so that the microalgae fertilizer can penetrate to the lowest root position.

[0043] (2) The second application of microalgae fertilizer for tuber-forming crops (such as potatoes) is carried out during the tuber-forming period by drip irrigation, and the amount of acidic microalgae fertilizer is 20-50 L per mu, the drip irrigation speed is 1.38-2 L / h, and the irrigation time is 2-4 hours. The drip irrigation water flow and soil moisture content are comprehensively considered, and the irrigation time is appropriately shortened, and the microalgae fertilizer can be infiltrated to the lowermost root system position.

[0044] (3) The third application of microalgae fertilizer for tuber-forming crops (such as potatoes) is carried out during the tuber bulking period by drip irrigation, and the amount of acidic microalgae fertilizer is 20-50 L per mu, the drip irrigation speed is 1.38-2 L / h, and the irrigation time is 2-4 hours. The drip irrigation water flow and soil moisture content are comprehensively considered, and the irrigation time is appropriately shortened, and the microalgae fertilizer can be infiltrated to the lowermost root system position.

[0045] Example 4 Effect verification test of alkaline microalgae fertilizer for preventing and controlling the abundance of soil scab pathogen in farmland

[0046] The test site is located in Jinjie Town, Shenmu City, Shaanxi Province, and the potato variety used in the test is Wotu No. 5. The soil type is sandy soil (pH is 7.5), and there are two test sites. Test site 1 has been continuously planted with potatoes for 3 years, and test site 2 has corn as the previous crop and Wotu No. 5 as the current crop. The microalgae fertilizer used in this example is prepared by the method in Example 1. On the one hand, in order to evaluate the reduction effect of different microalgae fertilizer application amounts on the abundance of soil scab pathogen, three treatment groups (low amount, medium amount, and high amount) are designed. The control group is only normally irrigated. The inactivation method of microalgae fertilizer is gamma ray sterilization method, which can make the microalgae fertilizer lose activity, but does not destroy the material structure of the microalgae fertilizer. The test group design and the amount of microalgae fertilizer are shown in Table 1. Potatoes are planted by ridging and mulching, and drip irrigation tape is laid under the film. In order to facilitate experimental control and functional bacteria liquid flushing, each control area is equipped with an independent irrigation valve. During the entire test period, the potato planting time and density, chemical fertilizer application during the growth period, irrigation amount, and management measures of the control area and the test area are the same. 15 days before the potato harvest, the potato tuber scab disease incidence is first observed, and then the surface soil of the potato tuber is collected. The abundance of soil scab pathogen is detected by qPCR method, and the abundance of soil scab pathogen in the control area and the test area is compared. The soil DNAkit (Omega Bio-tek, Norcross, GA, US) was used to extract total DNA from the soil. Real-time quantitative q-PCR was employed to quantify the copy number of the scab pathogen gene txtAB in the soil. The amplification primers used for determining the scab pathogen gene txtAB were StrepF (5'-GCAGGACGCTCACCAGGTAGT-3'; SEQ ID NO.1) and StrepR (5'-ACTTCGACACCGTTGTCCTCAA-3'; SEQ ID NO.2). The target fragment length was 72 bp. The amplification conditions were: 95℃ pre-deformation for 5 min; 95℃ denaturation for 10 s; 55℃ annealing for 10 s; 72℃ extension for 20 s; 40 cycles. This determined the effect of microalgae fertilizer on reducing the abundance of the scab pathogen in the root and tuber crop, potato.

[0047] Table 1. Experimental Design and Scheme at Two Experimental Sites in Shenmu City

[0048]

[0049] Shenmu City Experimental Site 1 is a sandy potato farmland that has been continuously planted for 3 years. Figure 3 It is evident that, compared to the control group, the application of microalgae fertilizer consistently increased the total abundance of bacteria in the soil, with increases ranging from 19.50% to 61.40%. Furthermore, the application of microalgae fertilizer consistently reduced the abundance of scab pathogens in the soil, with decreases ranging from 11.71% to 61.89%, and also reduced the overall proportion of scab pathogens in the soil, with decreases ranging from 16.39% to 68.03%. In addition, the greater the application rate of microalgae fertilizer, the greater its effect on increasing the total abundance of bacteria in the soil, and the stronger its ability to control scab pathogens. As shown in Table 2, compared to the control group, the average potato yield after applying microalgae fertilizer was 1.11–1.23 kg / plant, higher than the control group's 1.02 kg / plant, representing an increase of 9.27%–20.39%.

[0050] Table 2. Potato Yield at Experimental Site 1 in Shenmu City

[0051]

[0052]

[0053] After harvesting potatoes at test site 1, the incidence rate was calculated based on the potato scab disease grading and incidence rate calculation formula. Scab disease was graded from 0 to 4, specifically: Grade 0: No scabs or lesions on the tuber surface; Grade 1: Lesions on the tuber surface, covering 1%-10% of the tuber; Grade 2: Lesions on the tuber surface, covering 11%-25% of the tuber; Grade 3: Lesions on the tuber surface, covering 26%-50% of the tuber; Grade 4: Lesions on the tuber surface, covering more than 50% of the tuber, or raised or sunken scabs covering 6-25%. Incidence rate was calculated as: the number of diseased potatoes divided by the total number of potatoes surveyed × 100%. Figure 4 As shown in Table 3, compared with the control group, the application of microalgae fertilizer can reduce the incidence of potato scab. The average incidence of potato after applying microalgae fertilizer is 11.85% to 21.90%, which is lower than the 56.06% of the control group.

[0054] Table 3. Incidence of potato scab at Shenmu Experimental Site 1

[0055]

[0056] Shenmu City Experimental Site 2 is a sandy potato farmland that has been continuously planted for two years. Figure 5 It is evident that, at experimental site 2 in Shenmu City, compared to the control group, the application of microalgae fertilizer consistently increased the total abundance of bacteria in the soil by 50.43%–89.79%; it also consistently reduced the abundance of scab pathogens in the soil by 42.59%–55.84%, and decreased the overall proportion of scab pathogens in the soil by 54.69%–80.15%. Furthermore, the greater the application rate of microalgae fertilizer, the greater its effect on increasing the total abundance of bacteria in the soil, and the stronger its ability to control scab pathogens. As shown in Table 4, compared to the control group, the average potato yield after applying microalgae fertilizer was 1.26–1.31 kg / plant, higher than the control group's 1.05 kg / plant, representing a yield increase of 20.38%–25.15%.

[0057] Table 4. Potato Yield Statistics at Experimental Site 2 in Shenmu City

[0058]

[0059] Note: No scab disease occurred in the potatoes at the experimental site, so no photos of each treatment group were included.

[0060] Example 5: Verification Experiment on the Effect of Acidic Microalgae Fertilizer on Controlling the Abundance of Pathogenic Pathogens of Soil Scab in Farmland

[0061] Experimental site 1 was located in a potato planting base in Bainiijing Town, Dingbian County, Yulin City, Shaanxi Province. The soil type was moderately saline-alkali (pH 9.0), and the potato variety used in the experiment was V7. The microalgae fertilizer used in this experiment was prepared using the method described in Example 2. To evaluate the effect of different microalgae fertilizer application rates on the reduction of scab pathogen abundance in the soil, three treatment groups were designed: treatment group 1 (low amount), treatment group 2 (medium amount), and treatment group 3 (high amount); the control group received only normal irrigation. The microalgae fertilizer was inactivated using gamma ray sterilization, which deactivates the microalgae fertilizer without destroying its structural composition. The experimental group design and microalgae fertilizer application rates are shown in Table 5. Potatoes were planted using a raised bed mulching method, with drip irrigation tape laid under the mulch. For ease of experimental control and application of the functional bacterial solution, each control area was equipped with an independent irrigation valve. Throughout the entire experimental period, the potato planting time and density, chemical fertilizer application during the growing season, irrigation volume, and management measures were identical in both the control and experimental areas. Fifteen days before potato harvest, the incidence of potato tuber scab was first observed, and then samples were taken from the surface soil of the potato tubers. The soil DNAkit (Omega Bio-tek, Norcross, GA, US) kit was used to extract total DNA from the soil. The copy number of the scab pathogen gene txtAB in the soil was quantitatively detected using a real-time fluorescence quantitative q-PCR instrument to determine the effect of microalgae fertilizer on reducing the abundance of scab pathogens in root and tuber crops such as potatoes.

[0062] Table 5. Experimental Design and Scheme for Fixed-Front Test Point 1

[0063]

[0064] Experimental site 1 in Bainiijing Town, Dingbian County, is moderately saline-alkali farmland. The previous crop was corn, and potatoes were planted in the current season. Figure 6 It is evident that, compared to the control group, the application of microalgae fertilizer consistently increased the total abundance of bacteria in the soil by 54.47%–91.29%; it also consistently reduced the abundance of scab pathogens in the soil by 28.46%–54.85%, and decreased the overall proportion of scab pathogens in the soil by 52.49%–71.62%. Furthermore, the higher the application rate of microalgae fertilizer, the greater its effect on increasing the total abundance of bacteria in the soil, and the stronger its ability to control scab pathogens. As shown in Table 6, compared to the control group, the average potato yield after applying microalgae fertilizer was 1.26–1.50 kg / plant, higher than the control group's 1.18 kg / plant, representing a yield increase of 6.72%–27.00%.

[0065] Table 6. Potato Yield Statistics at Dingbian Experimental Site 1

[0066]

[0067] Note: No scab disease occurred in the potatoes at the experimental site, so no photos of each treatment group were included.

[0068] Another experimental site, Site 2, was located at a potato planting base in Bainiijing Town, Dingbian County, Yulin City, Shaanxi Province. The potato variety used in the Dingbian County experiment was Wotu No. 5, and the soil type was moderately saline-alkali with a soil pH of 8.5. The microalgae fertilizer used in this example was prepared using the method described in Example 2. To evaluate the effect of acidic microalgae fertilizer on reducing the abundance of scab pathogens in the soil, a treatment group was designed. 40 liters of acidic microalgae fertilizer were applied per acre with irrigation water each time, for a total of three applications throughout the cycle, totaling 120 liters per acre. The control group received only the same amount of water. Fertilization was applied during the potato seedling stage, tuber formation stage, and tuber enlargement stage. Potatoes were planted using a raised bed mulching method, with drip irrigation tape laid under the mulch. Each control area was equipped with an independent irrigation valve to facilitate experimental control and application of the functional bacterial solution. Throughout the experimental period, the potato planting time and density, chemical fertilizer application during the growth period, irrigation volume, and management measures were the same in both the control and experimental areas. Ten days before potato harvest, the incidence of potato tuber scab was first observed, and then samples of the surface soil from the potato tubers were taken for analysis. The soilDNAkit (Omega Bio-tek, Norcross, GA, US) kit was used to extract total DNA from the soil. The copy number of the scab pathogen gene txtAB in the soil was quantitatively detected using a real-time fluorescence quantitative q-PCR instrument to determine the effect of microalgae fertilizer on reducing the abundance of scab pathogens in root and tuber crops such as potatoes.

[0069] Depend on Figure 7 As can be seen, compared with the control group, the application of microalgae fertilizer increased the total abundance of soil bacteria by an average of 33.83%, and reduced the abundance of scab pathogens in the soil by 51.37%; it also reduced the overall proportion of scab pathogens in the soil by 63.67%. Table 7 shows that, compared with the control group, the average potato yield after applying microalgae fertilizer was 1.49 kg / plant, higher than the control group's 1.25 kg / plant, representing a yield increase of 19.75%.

[0070] Table 7. Statistics on potato yield at Experimental Site 2 in Dingbian County

[0071]

[0072] Note: No scab disease occurred in the potatoes at the experimental site, so no photos of each treatment group were included.

[0073] This invention provides a new technology for reducing the abundance of scab pathogens in potato farmland soil:

[0074] This microalgae fertilizer uses Chlorella proteoglycans as the algae species and is prepared through a special culture medium. The main components of the fertilizer are various active amino acids, vitamins, minerals, dietary fiber, nucleic acids, and chlorophyll. It has no toxic side effects on farmland soil. Applying Chlorella proteoglycans microalgae fertilizer can improve soil health, comprehensively enhance the diversity and stability of soil bacterial communities, and thus achieve long-term stable control of scab pathogens in farmland soil.

[0075] This invention provides a microalgae fertilizer that reduces the abundance of scab pathogens in potato farmland soil:

[0076] A microalgae fertilizer suitable for both alkaline and acidic farmland soils is proposed. For acidic farmland soils, it can be directly applied with irrigation water or through flushing. For alkaline farmland soils, by adding specific amino acid materials, the algae solution is adjusted to a weakly alkaline state, thus solving the problem of limited application of microalgae fertilizer in strongly alkaline farmland in northern regions.

[0077] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing a Chlorella microalgae fertilizer containing Peptone nucleus to inhibit and control soil scab pathogens in farmland, characterized in that, The Chlorella microalgae fertilizer containing protein nucleus is either an alkaline or acidic microalgae fertilizer. The alkaline microalgae fertilizer is prepared as follows: *Chlorella proteoglycans* is inoculated into a pre-sterilized and cooled special culture medium. After 14-18 hours of cultivation under full-spectrum light at 25±1℃ and 5000-8000 lux, it is followed by 6-10 hours of dark cultivation for 3-4 days to obtain a primary culture solution. The primary culture solution is then inoculated into a pre-sterilized special culture medium at a volume percentage of 8-15%. Aeration is carried out at 25-30℃ using an aeration pump for 20-40 minutes every 6-8 hours for 5-7 days to obtain the *Chlorella proteoglycans* alkaline microalgae fertilizer. The density of *Chlorella proteoglycans* in the microalgae fertilizer is 6.00 × 10⁻⁶. 6 cfu / mL~1.0×10 7 cfu / mL, the microalgae fertilizer is alkaline and has a pH value above 8.5; The preparation method of the acidic microalgae fertilizer is as follows: glutamic acid and glycine are added to the alkaline microalgae fertilizer, and the dosage of glutamic acid and glycine is 3-6 g / L; the microalgae fertilizer is acidic and the pH value is between 6.0 and 7.

0. The specific culture medium formula is as follows: ammonium chloride 1.00 g / L, magnesium sulfate 0.06 g / L, potassium dihydrogen phosphate 0.03 g / L, sodium bicarbonate 0.02 g / L, and glucose 0.02 g / L.

2. The protein-nucleated Chlorella microalgae fertilizer prepared by the method of claim 1.

3. The application of the protein-nucleated Chlorella microalgae fertilizer according to claim 2 in controlling the pathogenic bacteria of soil scab in farmland.

4. The application of the Chlorella microalgae fertilizer according to claim 2 in enhancing soil microbial diversity.

5. The application of the Chlorella microalgae fertilizer according to claim 2 in reducing the incidence of potato scab.

6. The application of the Chlorella microalgae fertilizer according to claim 2 in increasing potato yield.

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