A biological fertilizer CAN1 and its preparation method, application and use method

Abstract

The present invention relates to the technical field of biological control of plant diseases, and in particular to a biological fertilizer CAN1 and a preparation method, application and use method thereof, wherein the components of the biological fertilizer CAN1 are mycelium of Rhizopus oryzae and a fermentation medium thereof. Potted plant experiments have shown that the application of the biological fertilizer CAN1 can effectively inhibit potato scab caused by Streptomyces scab, with a control effect of 67.01%; and can increase potato yield by about 10%. The biological fertilizer CAN1 can promote seed potato germination and seedling growth, and can significantly increase the activity of defense enzymes in potato tubers. The biological fertilizer CAN1 provided by the present invention can provide rich nutrients for potato growth, so that beneficial microorganisms proliferate in large quantities, and the relative number of pathogenic bacteria Streptomyces scab is sharply reduced, which does not reach the pathogenic amount, and is harmless to humans and animals and does not cause environmental pollution, and has good application and development prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of biological control of plant diseases, and in particular to a biological fertilizer CAN1 and a preparation method, application and use method thereof. Background Art

[0002] Potato scab is a soil-borne disease caused by Streptomyces scab. The phytotoxins produced by Streptomyces scab can cause brown corky spots to form on the surface of potato tubers. Once the epidermal tissue is damaged, it is easily infected by other pathogens, causing tuber rot. The decline in tuber quality and storage resistance caused by scab reduce the commercial value of potatoes. Streptomyces scab can survive in the soil for several years and spread through soil, water and diseased seed potatoes, becoming a global disease.

[0003] The main methods for preventing and controlling potato scab include chemical control, agricultural control and biological control. At present, there is no pesticide for scab Streptomyces for chemical control; agricultural control mostly adopts crop rotation, which conflicts with the demand for increasing potato production. Biological control will become the mainstream of potato scab control in the future due to its advantages such as environmental friendliness, harmlessness to human and animal health and compliance with the requirements of sustainable agricultural development. Biological control can be divided into three categories: one is the breeding of disease-resistant seed potatoes, the second is the screening of antagonistic bacteria of scab Streptomyces, and the third is the use of natural products. Due to the complexity of genetic background and the variability of pathogenic bacteria, the breeding of resistant varieties has progressed slowly. Researchers have invested a lot of energy in the screening of antagonistic bacteria. At present, a variety of antagonistic bacteria have been screened from microorganisms such as Bacillus, Streptomyces and Pseudomonas. Due to the differences in the populations of pathogenic bacteria of scab in different regions, the selection of antagonistic bacteria needs to be adapted to local conditions. The prevention and control effect of a single strain is often poor, and the development of composite bacterial agents is the development trend of biological agents. Another research hotspot is to screen suitable natural products to inhibit Streptomyces scab. It has been found that Canada thistle, Brassica crops and chitin have good inhibitory effects on Streptomyces scab.

[0004] During the potato harvest process, potatoes with no commercial value will inevitably be produced, such as infected potatoes, tiny potatoes and broken potatoes. If they are directly discarded, it will lead to a waste of resources. How to effectively utilize such potatoes with no commercial value, prepare biological fertilizers for biological control of potato scab, and develop new organic fertilizers against scab Streptomyces is a technical problem that needs to be solved. Summary of the invention

[0005] In view of this, the purpose of the present invention is to propose a biological fertilizer CAN1 and its preparation method, application and use method, so as to use potatoes that have no commercial value during the harvest process to make a fermentation medium, inoculate Rhizopus oryzae to ferment and produce a chitin-rich biological fertilizer, providing a new idea for the biological control of potato scab.

[0006] Based on the above purpose, the present invention provides a biological fertilizer CAN1, the components of the biological fertilizer CAN1 are Rhizopus oryzae mycelium and its fermentation medium.

[0007] Preferably, the Rhizopus oryzae is ATCC9396.

[0008] Preferably, the potatoes used are contaminated potatoes, tiny potatoes and broken-skin potatoes which have no commercial value.

[0009] Preferably, the spore preparation medium is: PDA with 2 g / L calcium carbonate added, sterilized at 115° C. for 15 min.

[0010] Preferably, the seed preparation medium is: PDB with 3 g / L calcium carbonate added and sterilized at 115° C. for 15 min.

[0011] Preferably, the fermentation medium includes: 200 g / L potato, 15 g / L soluble amylase, 1.5 g / L calcium chloride, 10 g / L calcium carbonate, 5 g / L yeast extract, and sterilized at 121°C for 15 min. When preparing the fermentation medium, firstly boil and mash fresh potatoes and water in a pressure cooker at a ratio of 1:4, then add amylase and calcium chloride and place in a 55°C water bath for hydrolysis overnight, and finally mix with other ingredients for sterilization.

[0012] The preparation method comprises the following steps: firstly carrying out seed culture with Rhizopus oryzae, then inoculating the Rhizopus oryzae into a potato culture medium for fermentation, and crushing the fermentation product into powder after filtering, sterilizing, filtering again, drying, and obtaining the biological bacterial fertilizer CAN1.

[0013] The seed culture method comprises washing the spores on the slant culture medium with physiological saline, inoculating the spore suspension into the seed culture medium after adjusting the spore concentration, and culturing at 27° C. and 150-200 rpm for 20-28 hours.

[0014] Optionally, the spore concentration is adjusted to 0.9 × 10 8 -1.1×10 8 cfu / mL, and the spore suspension was inoculated into the seed culture medium at a rate of 0.8-1.2%.

[0015] Optionally, the fermentation culture method is to take the seed culture medium and inoculate the seed culture into the fermentation medium at an inoculation rate of 4-6%, and ferment at 27° C. and 150-200 rpm for 8 days.

[0016] Preferably, the sterilization is performed at 121° C. for 1 hour, the drying is performed by air blast drying, and the pulverization is performed by using an air flow pulverizer.

[0017] Preferably, the drying temperature is 80°C.

[0018] Preferably, the pulverizing temperature of the air flow pulverizer is 30-50°C.

[0019] The potatoes used in the potato culture medium are potatoes without commercial value, including damaged, tiny and bacteria-contaminated potatoes during potato harvesting.

[0020] Under the preparation conditions, 66.21 g of Rhizopus oryzae mycelium can be obtained per 1000 g of potato; and 277.45 g of biological fertilizer CAN1 can be obtained per 1000 g of potato.

[0021] The present invention also provides application of the biological fertilizer CAN1 in preventing and treating potato scab and / or promoting potato yield increase.

[0022] In the present invention, chitin is not decomposed and utilized by Streptomyces scab, but can promote the growth of a large number of Gram-positive bacteria antagonistic to Streptomyces scab, so that the soil is transformed from "fungal type" to "bacterial type", and can also stimulate the plant defense mechanism, reducing the incidence and disease index of potato scab.

[0023] The present invention also provides a method for using the biological fertilizer CAN1, which comprises spreading the biological fertilizer CAN1 powder as a base fertilizer before potato planting, and the amount of the powder is 8-12% of the weight of the seed potatoes. Spreading the biological fertilizer CAN1 can achieve a 67.01% control effect of Streptomyces scab, and promote an increase in potato yield by about 10%.

[0024] Beneficial effects of the present invention:

[0025] 1. The biological fertilizer CAN1 provided by the present invention can promote the proliferation of a large number of Gram-positive bacteria that are antagonistic to Streptomyces scab, stimulate plant defense mechanisms, provide nutrients for potato growth, promote potato growth, and improve potato quality and yield. It is a new way to improve potato stress resistance through non-transgenic means.

[0026] 2. The biofertilizer CAN1 provided by the present invention is made by fermenting food-safe and environmentally safe strain Rhizopus oryzae ATCC9396 and potatoes, and the preparation process is simple. The potatoes used are contaminated potatoes, micro potatoes and broken potatoes that have no commercial value, which can turn agricultural waste into treasure, reduce production costs, and prevent contaminated potatoes from polluting the environment again.

[0027] 3. The biological fertilizer CAN1 provided by the present invention is different from common chemical reagents and chemical fertilizers, and is also different from general biological fertilizers. The microorganisms in the biological fertilizer CAN1 of the present invention are sterilized, and no exogenous organisms are introduced when used. The chitin and nutrients in the fertilizer are improved by improving the microecological environment of the crop rhizosphere, reducing the content of Streptomyces scab, and effectively inhibiting potato scab. It provides new ideas for the biological control of potato scab and provides new resources for the development of new organic fertilizers resistant to Streptomyces scab. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings in the following description are only for the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0029] Figure 1 This is a comparison chart of the use of the biological fertilizer CAN1 in the present invention for preventing and treating potato scab and increasing potato yield;

[0030] Figure 2A The total biomass in the soil of the biological fertilizer CAN1 according to the embodiment of the present invention; Figure 2B This is a graph showing the effect of the biological fertilizer on the relative content of Streptomyces scab according to an embodiment of the present invention;

[0031] Figure 3 This is a graph showing the effect of the biological fertilizer CAN1 on the plant height of potatoes at the seedling stage according to an embodiment of the present invention;

[0032] Figure 4 This is a comparison chart of the effects of the biological fertilizer CAN1 on potato roots according to an embodiment of the present invention;

[0033] Figure 5A This is a graph showing the effect of the biological fertilizer CAN1 on the activity of the defense enzyme SOD in potato tubers according to an embodiment of the present invention;

[0034] Figure 5B This is a graph showing the effect of the biological fertilizer CAN1 on the activity of the defense enzyme POD in potato tubers according to an embodiment of the present invention;

[0035] Figure 5C This is a graph showing the effect of the biological fertilizer CAN1 on the activity of the defense enzyme PPO in potato tubers according to an embodiment of the present invention. DETAILED DESCRIPTION

[0036] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments.

[0037] It should be noted that, unless otherwise defined, the technical terms or scientific terms used in the present invention should have the common meanings understood by persons having ordinary skills in the field to which the present invention belongs.

[0038] The experimental methods in the following examples are all conventional methods unless otherwise specified.

[0039] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0040] Example 1

[0041] Preparation and composition determination of biological fertilizer CAN1

[0042] 1. Preparation of biological fertilizer CAN1

[0043] The spores on the slant medium were washed off with physiological saline and the spore concentration was adjusted to about 1×10 8 cfu / mL. Take the spore suspension and inoculate it into the seed culture medium at a 1% inoculation rate, and culture it at 27°C and 180rpm for 24h. Take the seed culture medium and inoculate the seed culture into the fermentation medium at a 5% inoculation rate, and ferment it at 27°C and 180rpm for 8d. After the fermentation is completed, the fermentation product is filtered, and the filter cake is collected and sterilized at 121°C for 1h, filtered again, and then air-dried, and finally crushed with a jet mill to obtain the biological fertilizer CAN1.

[0044] Slant medium: Add 2g / L calcium carbonate to PDA and sterilize at 115℃ for 15min. Seed medium: Add 3g / L calcium carbonate to PDB medium, 100mL of liquid per 250mL seed bottle, sterilize at 115℃ for 15min. Fermentation medium: 200g / L potato, 15g / L soluble amylase, 1.5g / L calcium chloride, 10g / L calcium carbonate, 5g / L yeast extract, 200mL of liquid per 500mL fermentation bottle, sterilize at 121℃ for 15min. When preparing the fermentation medium, first boil and mash fresh potatoes and water in a pressure cooker at a ratio of 1:4, add amylase and calcium chloride, and hydrolyze overnight in a 55℃ water bath, and finally mix with other ingredients for sterilization.

[0045] 2. Determination of the composition of biological fertilizer CAN1

[0046] After the fermentation is completed, the mycelium is rinsed with a 120-mesh sieve and running water to remove the residual starch. The mycelium is sterilized at 121°C for 1 hour, and after vacuum freeze drying, it is ground into powder to calculate the mycelium yield. Method for determining the chitin content in mycelium: Take 200 mg of freeze-dried mycelium and fully hydrolyze it with 20 mL of 60% v / v sulfuric acid, and place it at room temperature for 24 hours. Add deionized water to reduce the sulfuric acid concentration to 0.5M, sterilize at 121°C in a sterilizer for 1 hour, and then adjust the solution to neutral pH with 1M NaOH, and add deionized water to 2000mL. Take 0.5mL of the above sample, add 0.5mL w / v NaNO2 and 0.5mL w / v KHSO4, shake it intermittently for 15 minutes, and centrifuge it at 250rpm for 2 minutes. Take 0.6mL of supernatant, add 0.2mL 12.5% ​​w / v NH4SO3NH2, shake for 3min, add 0.2mL 3-methyl-2-benzothiazolinone hydrazone hydrochloride, boil for 3 minutes, cool to room temperature in ice water, add 0.2mL 0.5w / v FeCl3·7H2O, let stand for 30min, and measure absorbance at 650nm. 3-methyl-2-benzothiazolinone hydrazone hydrochloride and FeCl3·7H2O must be prepared before use. Analytical grade glucosamine is used for drawing the standard curve.

[0047] Under the preparation conditions of biological fertilizer CAN1, 277.5g of biological fertilizer CAN1 can be obtained for every 1000g of fresh potato, which contains 44.1g of Rhizopus oryzae mycelium, and the chitosan content in the mycelium is 24.3%. The mycelium content in biological fertilizer CAN1 is 16.0%, and the chitosan accounts for 3.9%.

[0048] Application of biological fertilizer CAN1 in preventing and controlling potato scab and increasing potato yield

[0049] A pot experiment was conducted to verify the effect of biological fertilizer CAN1. The experiment was designed with three groups: the first group was the control group, in which no biological fertilizer CAN1 was spread in the soil and no pathogens were irrigated, and was marked as the water blank treatment group CK; the second group was irrigated with pathogens, and was marked as the 50ml pathogen treatment group SS; the third group was spread with biological fertilizer CAN1 and irrigated with pathogens, and was marked as the biological fertilizer CAN1+50ml pathogen treatment group SC. Before potato planting, the third group was spread with biological fertilizer CAN1 as the base fertilizer, and the powder dosage was 10% of the seed potato weight. The seed potatoes used in the pot experiment were Holland 15. The healthy potato seed potatoes were rinsed with clean water, and the surface of the seed potatoes was disinfected by soaking them in 0.5% NaClO solution for 1h. The disinfected potatoes were rinsed 3 times in clean water, dried and placed in a dry place for germination. The seed potatoes with bud length of 1-2 cm were sown at a depth of 10-15 cm. Watering and fertilization were carried out according to conventional plant cultivation. After 7 days of cultivation, the SS group was inoculated with pathogenic bacteria at a concentration of 1×10 7cfu / mL, and the other two groups were irrigated with 50mL of clean water. Mature potatoes were harvested 80 days after inoculation, and the incidence, disease index, and control effect of potatoes were counted.

[0050] Experimental bacterial fertilizer: biological bacterial fertilizer CAN1 prepared by the present invention.

[0051] Potting soil: The soil was collected from the experimental field of Anhui University of Technology. It is sandy soil. The soil samples were sieved through a 10-mesh sieve to remove debris and grass roots.

[0052] Experimental location: Experimental field of Anhui University of Technology, where potato scab has never occurred.

[0053] Pathogenic bacteria: Streptomyces scabies was purchased from CGMCC, with the deposit number being NO.4.1765.

[0054] Fertilization method: spreading base fertilizer.

[0055] Fertilizer dosage: 10% of seed potato weight.

[0056] Morbidity rate = number of diseased potatoes / total number of potatoes × 100%;

[0057] Disease index = ∑ (number of diseased potatoes at each level × value at each level) / (total number of potatoes × highest disease level) × 100%;

[0058] Control efficiency = (disease index of control group - disease index of treatment group) / disease index of control group × 100%.

[0059] Table 1 Grading standards for potato scab

[0060]

[0061]

[0062] Table 2 Effect of biological fertilizer CAN1 on preventing and treating potato scab:

[0063]

[0064] Table 3 Effect of biological fertilizer CAN1 on increasing potato yield:

[0065]

[0066] The results of potted plant experiments showed that the application of biological fertilizer CAN1 can effectively inhibit potato scab caused by Streptomyces scab, with a control effect of 67.01%; the application of biological fertilizer CAN1 can promote potato yield increase by about 10% ( Figure 1 ).

[0067] Effects of biological fertilizer CAN1 on total biomass and relative content of Streptomyces scab in soil

[0068] Soil samples around the tubers were collected for analysis on days 0, 9, 21, 30, 48, 60, 75, 91, and 122 of the pot experiment. Soil total DNA was extracted using the DNEASY POWER SOIL KIT kit (QIAGEN, Germany). For specific steps and precautions, please refer to the user manual (www.qiagen.com / HB-2495-005). The total DNA concentration was determined using Nanodrop 2000 (Thermo, USA). The dilution factor was determined based on the measured value, and the total DNA concentration was normalized to 1 ng / μl before q-PCR. The q-PCR absolute quantitative method was used to quantify Streptomyces causative agents. The standard used a synthetic trpB gene with an initial concentration of 1 ng / μl; the SYBR reagent used a Bio-Rad SsoAdvanced Universal Kit premix (Bio-Rad, USA); the primers used are shown in Table 4. q-PCR reaction system (20 μL): template 5 μL, upstream and downstream primers (10 μmol / L) 1 μL each, SYBR 10 μL, ddH2O 3 μL. q-PCR reaction conditions: 98℃3min; 98℃10s, 65℃30s, plate reading, 39 cycles. The melting curve was set to increase the temperature from 65℃ to 95℃, and the fluorescence signal intensity was detected once every 0.5℃ increase, and each detection lasted for 5s. Each detection contained 3 technical parallels. The experiment was designed with 5 parallel groups, each with 3 replicates: control (C, soil sample without chitin addition); 0.2% fungal chitin (F0.2); 2% fungal chitin (F2.0); 0.2% Sigma chitin (S0.2); 2% Sigma chitin (S2.0).

[0069] Table 4 Primers for the specific gene trpB of Streptomyces scab

[0070]

[0071] The change of total DNA represents the change of the overall microbial population. The total DNA of group C was relatively stable, which was in line with the experimental expectations. Exogenous additives can cause changes in the number of microbial populations in the soil, among which F2.0 had a significant effect, with a P value of 0.0002. The total DNA amount of F2.0 continued to increase and reached a peak at 30 days, which was 4.8 times that of group C; it gradually decreased after 30 days and entered a relatively stable period within 90 to 120 days, and the total DNA amount was 2.5 times that of group C. The overall trend of S2.0 was basically consistent with that of F2.0. The peak DNA concentration was 2.9 times that of group C. After the peak, the total DNA amount continued to decline, and no relative stable period appeared. In the later stage of the experiment, F and S had different effects on the number of microorganisms in the soil. The total DNA of group F was still higher than that of group C, while the total DNA of group S was lower than that of group C ( Figure 2A ).

[0072] The relative content of Streptomyces scab refers to the percentage of the absolute content of Streptomyces scab in other components to the absolute content of Streptomyces scab in the control group. As shown in Figure 2, about 70 days after chitin was added to the soil, the relative content of Streptomyces scab decreased in both the F and S groups, especially F2.0, with a significant effect, and a P value of 0.003. During the 60th to 122nd day of the experiment, the absolute content of Streptomyces scab was lower than the lower limit of q-PCR detection. F2.0 can rapidly reduce the relative content of Streptomyces scab in the early stage of the experiment, which is beneficial to reduce the damage of Streptomyces scab to potato seedlings; F2.0 affects the relative content of Streptomyces scab for a longer period of time, which is beneficial to improve potato yield and appearance ( Figure 2B ).

[0073] Effects of biological fertilizer CAN1 on potato and its defense enzymes

[0074] Potato seedlings from different groups were taken 14 days after the pot experiment. The plant height and taproot length of the seedlings were measured with a tape measure, the number of roots was counted, and the relevant data were recorded. The experimental results showed that the plant height, number of roots and taproot length of the SC group were higher than those of the control group and the diseased group ( Figure 3 , Figure 4 , Table 5). On the one hand, the biological fertilizer CAN1 can provide rich direct nutrition for potato growth; on the other hand, it can proliferate a large number of beneficial microorganisms, and the relative number of the pathogenic bacteria Streptomyces scab has decreased sharply, and it has not reached the pathogenic dosage.

[0075] Table 5 Effects of biofertilizer CAN1 on potato plant height, root length / number of root fibers

[0076]

[0077] Preparation of crude enzyme solution from potato tubers: Weigh 2 g of potato tuber sample, chop it and place it in a pre-cooled mortar, add 1 ml of 0.1 M phosphate buffer and grind it thoroughly into a homogenate, then transfer it to a 10 ml centrifuge tube and rinse the mortar with 2 ml of 0.1 M phosphate buffer. Repeat twice, transfer the mixture into a centrifuge tube, shake well, let it stand for 30 minutes, centrifuge at 8000 rpm for 15 minutes, the supernatant is the crude enzyme solution, and place it at 4°C for later use.

[0078] Determination of superoxide dismutase (SOD) activity: NBT method.

[0079] Peroxidase (POD) activity determination: guaiacol method.

[0080] Determination of polyphenol oxidase (PPO) activity: catechol method.

[0081] After watering with pathogens, the potato was stimulated to start the defense mechanism. The defense enzymes SOD, POD and PPO increased. The defense enzyme activity of potato plants with the addition of biological fertilizer CAN1 was higher than that of the group with only the addition of pathogens. The results showed that biological fertilizer CAN1 was beneficial to the defense of potato against Streptomyces scab ( Figure 5A \B\C).

[0082] The results show that the biological fertilizer CAN1 can promote the germination of seed potatoes and the growth of seedlings, and can significantly increase the activity of defense enzymes in potato tubers. The biological fertilizer CAN1 provided by the present invention can provide rich nutrients for potato growth, so that beneficial microorganisms proliferate in large quantities, and the relative number of pathogenic bacteria Streptomyces scab is sharply reduced, and does not reach the pathogenic amount. It is harmless to humans and animals and will not cause environmental pollution, and has good application and development prospects.

[0083] Those skilled in the art should understand that the discussion of any of the above embodiments is merely illustrative and is not intended to imply that the scope of the present invention (including the claims) is limited to these examples. Under the concept of the present invention, the technical features in the above embodiments or different embodiments may be combined, the steps may be implemented in any order, and there are many other variations of the different aspects of the present invention as described above, which are not provided in detail for the sake of simplicity.

[0084] The present invention is intended to cover all such substitutions, modifications and variations that fall within the broad scope of the appended claims. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. An application of a biological fertilizer CAN1 in preventing and treating potato scab caused by Streptomyces scab and / or promoting potato yield increase, wherein the components of the biological fertilizer CAN1 are Rhizopus oryzae mycelium and its fermentation medium.

2. The application according to claim 1, characterized in that: The preparation method of the biological fertilizer CAN1 is to first use Rhizopus oryzae for seed culture, then inoculate it into a potato culture medium for fermentation, and the fermentation product is filtered, sterilized, filtered again, dried, and then crushed into powder to obtain the biological fertilizer CAN1.

3. The application according to claim 2, characterized in that: The seed culture method comprises washing the spores on the slant culture medium with physiological saline, inoculating the spore suspension into the seed culture medium after adjusting the spore concentration, and culturing at 27° C. and 150-200 rpm for 20-28 h.

4. The use according to claim 3, characterized in that: The spore concentration was adjusted to 0.9×10 8 -1.1×10 8 cfu / mL, and the spore suspension was inoculated into the seed culture medium at a rate of 0.8-1.2%.

5. The application according to claim 4, characterized in that: The fermentation culture method comprises taking a seed culture medium and inoculating the seed culture into a fermentation medium at an inoculation rate of 4-6%, and fermenting for 8 days at 27° C. and 150-200 rpm.

6. The use according to claim 2, characterized in that: The sterilization is carried out at 121° C. for 1 h, the drying is carried out by air blast drying, and the pulverization is carried out by using an air flow pulverizer.

7. The use according to claim 6, characterized in that: The pulverizing temperature of the air flow pulverizer is 30-50°C.

8. The use according to claim 2, characterized in that: The potatoes used in the potato culture medium are potatoes without commercial value, including damaged, tiny and bacteria-contaminated potatoes during potato harvesting.

9. The use according to claim 1, characterized in that: Before planting potatoes, spread biological fertilizer CAN1 powder as base fertilizer, the amount of powder is 8-12% of the weight of seed potatoes.

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