A method for promoting the increase of amylopectin content in sorghum of jinliangnuo 1 by arbuscular mycorrhizal fungi

Abstract

The application discloses a method for promoting Jinliangnu No.1 kaffir corn to increase amylopectin content by using arbuscular mycorrhizal fungi, and the arbuscular mycorrhizal mixed fungicide is pelletized with the sterilized Jinliangnu No.1 kaffir corn seeds, and the treated kaffir corn seeds form a symbiotic body with the arbuscular mycorrhizal fungi in the growth and development process, so that the amylopectin content of the kaffir corn grains can be significantly improved. In addition, the application can improve the germination rate and the growth phenotype index of the Jinliangnu No.1 kaffir corn in the seedling stage, and solves the problem of large dosage of the arbuscular mycorrhizal fungicide in the prior art. The application is simple and convenient, has good economic benefit, social benefit and ecological benefit, and also provides technical support for sustainable planting of a Guizhou karst farmland ecological system.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microbial preparation, the technical field of seed pellet and the technical field of strain preparation, in particular to a method for promoting the increase of amylopectin content of Kengangnu No.1 kaffir corn by arbuscular mycorrhizal fungi. BACKGROUND

[0002] Brewing kaffir corn is the main raw material of Maotai-flavor and Luzhou-flavor liquor. The amylopectin content of kaffir corn is closely related to the flavoring substances of liquor, and the brewing kaffir corn with high amylopectin content is often used for making famous liquor. "Kengangnu No.1" is a medium-early-maturing conventional waxy variety, which is bred from Qinghouyang, a local variety, by the Jindong County Agricultural Technology Station in 2004. The growth period of Kengangnu No.1 is about 129 days, which is 3 days earlier than the control. The plant type is semi-compact, and the plant height is 255 cm. The bud sheath is light purple, the leaf color is green, and the total leaf number is about 16. The ear is scattered and umbrella-shaped, and the maturity is uniform and consistent. The ear length is 39 cm, the first branch number is about 46, the average ear weight is 47 g, the thousand-grain weight is 16.5 g, the kernel is red, the grain is flat and round, and the seed coat is thick and easy to thresh. The grain is waxy, and the amylopectin content accounts for 78.14% of the total starch content. Therefore, increasing the amylopectin content of Kengangnu No.1 will be beneficial to the brewing of Maotai-flavor liquor.

[0003] Arbuscular mycorrhizal (AM) fungi are ubiquitous in nature and can form mutualistic symbiosis with plants. A large number of studies have found that AM fungi can promote plant growth, enhance plant resistance, improve plant water and nutrient uptake capacity, and improve soil nutrients. As one of the ubiquitous groups of soil microorganisms, AM fungi can form mutualistic symbiosis with more than 80% of terrestrial plants. By absorbing carbohydrates and lipids produced by plants, AM fungi can accelerate the construction of carbon skeleton. The highly branched arbuscules structure in the root cortex cells is also considered to be an important functional site for nutrient exchange. In addition, the extensive extraradical mycelium system formed by the hyphae in the root epidermal cells can penetrate the soil particles and form a hyphal network with the plant roots. This network not only expands the root absorption area, but also accelerates the decomposition of soil organic matter, thus providing a mycorrhizal pathway for plants to obtain nutrients. The increase in nutrient acquisition pathways can significantly improve the quality of plant nutrients and grain products. Related studies have been reported in corn and sorghum grain. However, previous methods involved spreading AM fungal inoculum with seeds into the culture medium, which required a large amount of inoculum. Direct wet screening to select spores for inoculation into seeds and soil is a huge workload and cannot be applied to mass production. Therefore, there is an urgent need for a convenient and cost-effective method to apply this technology. Especially in the increasingly complex and severe soil environment, improving crop yield and resistance through AM fungi has important research value and application prospects for sustainable agricultural development. At present, the application of AM technology in China has opened up an important way for green and sustainable development. However, the large amount of inoculum used makes it difficult to promote.

[0004] Seed pelleting is to make the outer layer of the lighter seed with a strong shell by wrapping the pelleting material, so that the seed shape is regular and uniform. The medium for pelleting often contains different active substances, and the difference of active substances will bring different features to the seed. Seed processing (grading, priming, coating and pelleting treatment, etc.) before sowing can help to improve seed vigor and improve field emergence rate. Coating and pelleting have been used for pre-sowing treatment of sorghum seeds, which can not only prevent seed-borne diseases, but also improve emergence rate, enhance stress resistance and increase yield, etc. The powder of pelleted seed is composed of inert materials and active substances. Inert materials are the main carrier of seed pelleting, and the selected inert materials cannot affect the normal germination and growth of seeds, and cannot make the plant accumulate harmful substances during growth, which can affect other organisms. At the same time, the inert material also determines the physical and chemical properties of the pelleted seed, such as seed hardness, air permeability, storage resistance and slow-release active substances. Active substances are substances that can promote the pelleting of seeds, prevent pests and diseases during plant growth and development, improve plant stress resistance and promote plant growth, etc. Active substances can be added to pelleted seeds according to production needs, and different active substances can be added according to different actual conditions, so as to achieve the corresponding purpose. Some scholars have applied pelleting technology to sorghum, for example, adding 7.8mg / L salicylic acid and 100mg / L proline mixed coating can promote the germination and seedling growth of sorghum. In addition, using activated carbon, talc, fungicide mancozeb, carboxymethyl cellulose and gibberellin for pelleting treatment of sorghum seeds can shorten the emergence time under low temperature stress, improve the emergence rate, and reduce the incidence of smut.

[0005] The present application combines pelleting and arbuscular mycorrhizal fungi technology to improve wine sorghum, providing technical support for sustainable planting in Guizhou karst farmland ecosystem. SUMMARY

[0006] The technical problem to be solved by the present application is to provide a method for promoting the increase of amylopectin content of Jinlangu No.1 sorghum using arbuscular mycorrhizal fungi, which is a sustainable development improvement method. The method not only improves the germination rate of Jinlangu No.1 sorghum, but also solves the problem of large dosage of arbuscular mycorrhizal fungi, which can significantly improve the growth and development indicators and amylopectin content of sorghum.

[0007] To solve the above technical problems, the technical scheme of the present application is as follows: a method for promoting the increase of amylopectin content of Jinlangu No.1 sorghum using arbuscular mycorrhizal fungi, which comprises the following steps:

[0008] (1) Fungicide propagation:

[0009] ① Fill the sterilized boiling sand culture medium to 2 / 3 of the culture container, evenly spread 1020g of arbuscular mycorrhizal fungus inoculum on the sterilized boiling sand culture medium, then sow the rinsed Jinliangnu No. 1 sorghum seeds, cover with 2cm thick sterilized boiling sand culture medium, water thoroughly, and move to the light room or greenhouse for 3-5 months of culture;

[0010] ② Cut the Jinliangnu No. 1 sorghum roots after 3-5 months of culture in step ① into small pieces less than 1cm, use a microscope to select and check the spores in the inoculum using wet screening method, then mix evenly with the sterilized boiling sand culture medium used in step ① to obtain the expanded arbuscular mycorrhizal fungus inoculum, and store the expanded arbuscular mycorrhizal fungus inoculum in an ultralow temperature environment for later use;

[0011] (2) Seed treatment:

[0012] ① The Jinliangnu No. 1 sorghum seeds should meet the requirements of GB / T8231-2007 Sorghum, and the seeds to be pelleted are selected for purification, removing sand, stones or foreign crop seeds, and impurities, so that the purity of the seeds is greater than or equal to 98%, and the purity is greater than or equal to 98%;

[0013] ② Surface disinfection is performed on the seeds, especially the seeds to be sown, for conventional germination soaking;

[0014] (3) Inoculation of fungi and pelleting treatment:

[0015] ① Put the disinfected Jinliangnu No. 1 sorghum seeds into a rotating pelleting machine, add the expanded arbuscular mycorrhizal fungus inoculum in step (1) according to a weight ratio of 1:1, spray a little peach gum to form a uniform and smooth seed surface structure so that the additives can be adsorbed on the seed surface, then add the additives to form small particles centered on each seed; the arbuscular mycorrhizal fungus inoculum is formed by the inoculum of Glomus etunicatum Glomus etunicatum , Glomus mosseae Glomus mosseae and Glomus intraradices Glomus versiforme after being expanded for 4 months by brewing sorghum, and the spore density of the inoculum is ≥100 / g, containing mycelium fragments and infected root segments;

[0016] ② Increase the pellet strength: mainly used to change the shape of the seeds, increase the size, weight and gloss of the seeds, and requires low solubility, PH value between 5.5-8, conductivity below 1.0 s / m, increase the rolling machine, add additives multiple times, repeat the coating process, rapidly increase the volume or weight, and the pellets gradually become smooth and uniform, use a vibrating screen with a specific mesh size to separate the large and small pellets, and the unqualified ones can be continuously pelleted, and the qualified ones can enter the next process;

[0017] ③ Again spray into peach glue, add the expanded inoculant, when the inoculant added makes the seed reach the required particle size, spray into the peach glue idling to increase the strength and roundness, finally, the coating layer is wrapped, the pelletized seed after coloring is placed in a ventilated place, and is naturally dried.

[0018] Preferably, the sterilized boiling sand culture medium of the step (1) is obtained by sterilizing the coarse river sand by 120 DEG C water vapor for 20 minutes.

[0019] Preferably, the rinsing mode of the Jinliangnu No. 1 sorghum seed is rinsing 3-5 times by distilled water.

[0020] Preferably, the culture container in the step (1) is a circular PVC pot with a height of 55-65 cm and a diameter of 95-105 cm.

[0021] Preferably, the temperature of the ultra-low temperature in the step (1) is-80 DEG C.

[0022] Preferably, the coating layer in the step (3) is composed of the following raw materials in parts by weight: 70 parts of sodium polyacrylate, 20 parts of diamine slow-release fertilizer, 2.5 parts of naphthaleneacetic acid rooting powder, 2.5 parts of carbendazim, 2.5 parts of konjac powder and 2.5 parts of zinc sulfate powder.

[0023] Compared with the prior art, the present application has the following beneficial effects:

[0024] The present application mixes the Glomus etunicatum, Glomus mosseae and Glomus lamellosum with the sterilized Jinliangnu No. 1 seed to carry out pelletization treatment, and the root system of the treated sorghum forms a symbiotic body with the arbuscular mycorrhizal fungi, which significantly improves the growth and development indexes and amylose content of the sorghum. Glomus etunicatum Glomus mosseae Glomus versiforme The present application is simple and convenient, has good economic, social and ecological benefits, has a positive significance for realizing the carbon peak and carbon neutralization target and improving the Guizhou wine sorghum, and provides technical support for sustainable planting of the Guizhou karst farmland ecological system.

[0025] The present application is simple and convenient, has good economic, social and ecological benefits, has a positive significance for realizing the carbon peak and carbon neutralization target and improving the Guizhou wine sorghum, and provides technical support for sustainable planting of the Guizhou karst farmland ecological system.

[0026] ​​3、In addition, through actual test, the prepared pelletized Jinlangnu No. 1 highland barley seeds have increased total starch, amylopectin and protein in the mature test grain after planting for 130-135 days, and the increase amplitude is 0.4%, 2.2% and 0.036% respectively compared with the control; the fat and tannin content is reduced, and the reduction amplitude is 0.49% and 0.03% respectively; in summary, the pelletized Jinlangnu No. 1 highland barley seeds treated by the pelletization formula and method of the arbuscular mycorrhizal fungus can improve the germination rate, seedling growth traits and the total starch, amylopectin and protein content of the mature grain after normal sowing, and the fat and tannin content is slightly reduced. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 Figure 6 is the comparison of the seedling height of Jinlangnu No. 1 highland barley treated after 30 days and untreated;

[0028] Figure 2 Figure 6 is the comparison of the seedling height of Jinlangnu No. 1 highland barley treated after 30 days and untreated;

[0029] Figure 3 Figure 6 is the comparison of the seedling height of Jinlangnu No. 1 highland barley treated after 30 days and untreated;

[0030] Figure 4 Figure 6 is the comparison of the seedling height of Jinlangnu No. 1 highland barley treated after 30 days and untreated. DETAILED DESCRIPTION

[0031] The specific embodiments of the present application will be further described below in conjunction with the accompanying drawings. It should be noted that the description of the embodiments is used to help understand the present application, but does not constitute a limitation of the present application. In addition, the technical features involved in the various embodiments of the present application described below can be combined with each other as long as there is no conflict.

[0032] The present application is a method for promoting Jinlangnu No. 1 highland barley to increase amylopectin content by using arbuscular mycorrhizal fungus, which comprises three links of inoculum propagation, seed treatment and inoculation of fungus and pelletization treatment.

[0033] (1) Inoculum propagation:

[0034] ① Fill the sterilized boiling sand culture medium to 2 / 3 of the culture container, evenly spread 1020g of arbuscular mycorrhizal fungus inoculum on the sterilized boiling sand culture medium, then sow the rinsed Jinliangnu No. 1 sorghum seeds, cover with 2cm thick sterilized boiling sand culture medium, water thoroughly, and move to the light room or greenhouse for 3-5 months; the culture container in this step is a circular PVC pot with a height of 55-65cm and a diameter of 95-105cm; the sterilized boiling sand culture medium is obtained by sterilizing coarse river sand at 120℃ for 20 minutes with steam; the rinsing method of Jinliangnu No. 1 sorghum seeds is to rinse 3-5 times with distilled water; the arbuscular mycorrhizal fungus inoculum in this step is a mixed inoculum of Glomus etunicatum, Glomus mosseae and Glomus versiforme, which are dominant species in Guizhou Province and have broad-spectrum, high affinity and good effect. The public can purchase them from the Chinese Arbuscular Mycorrhizal Resource Bank.

[0035] ② Cut the roots of Jinliangnu No. 1 sorghum after 3-5 months of culture in step ① into small pieces less than 1cm, use a microscope to select spores in the inoculum by wet sieving method, then mix with the sterilized boiling sand culture medium used in step (1) to obtain the expanded arbuscular mycorrhizal fungus inoculum, and store the expanded arbuscular mycorrhizal fungus inoculum in an ultra-low temperature environment for later use; the ultra-low temperature environment in this step is a refrigerator with a temperature of-80℃;

[0036] (2) Seed treatment:

[0037] ① Jinliangnu No. 1 sorghum seeds should meet the requirements of GB / T8231-2007 Sorghum, and the seeds to be pelleted should be purified and selected to remove sand, stones or foreign crop seeds, and impurities, so that the purity of the seeds is greater than or equal to 98% and the purity is greater than or equal to 98%;

[0038] ② Surface disinfection of the seeds, especially conventional germination soaking of the seeds to be sown;

[0039] (3) Inoculation of fungi and pelleting treatment:

[0040] ① Put the disinfected Jinliangnu No. 1 sorghum seeds into a rotating pelleting machine, add the expanded arbuscular mycorrhizal fungus inoculum in step (1) according to a weight ratio of 1:1, spray a little peach gum to form a uniform and smooth seed surface structure so that the additives can be adsorbed on the surface of the seeds, then add the additives to form small particles centered on each seed;

[0041] 2) Increasing the strength of the pellets: mainly used to change the shape of the seeds, increase the size, weight and gloss of the seeds, and require low solubility, pH value between 5.5-8, conductivity below 1.0 s / m, increase the rolling device, add additives multiple times, repeat the coating process, and the volume or weight increases rapidly, the pellets gradually become smooth and uniform, and are screened by a vibrating screen with a specific mesh size to separate the size of the pellets, and the unqualified ones can continue to be pelletized, and the qualified ones enter the next process;

[0042] 3) Re-injecting peach gum and adding the expanded microbial agent, when the added microbial agent reaches the required particle size of the seeds, the peach gum is injected for idling to increase the strength and roundness, and finally a coating layer is wrapped, a dyeing agent with different dyes or color films is sprayed on the surface of the moving pellets, and the dyed pelletized seeds are placed in a ventilated place and naturally dried, the coating layer of the application is composed of the following raw materials by weight: 70 parts of sodium polyacrylate, 20 parts of diamine slow-release fertilizer, 2.5 parts of naphthalene acetic acid rooting powder, 2.5 parts of carbendazim, 2.5 parts of konjac powder, and 2.5 parts of zinc sulfate powder, the mycorrhizal fungal agent in this step is formed by expanding the microbial agent of Glomus etunicatum, Glomus mosseae and Glomus versiforme for 4 months after brewing highland barley, and the spore density of the microbial agent is ≥100 / g, containing mycelium fragments and infected root segments.

[0043] The mixture of soil, sand and vermiculite (3:1:1, v / v / v) is used as the culture medium, which is sterilized at 121℃ and 0.11 MPa for 2h, the sterilized culture medium is transferred into each PVC pot, 3 pellets of the prepared Jinlangu No.1 highland barley seeds of the application are sowed in each pot in the treatment group; the control group sows Jinlangu No.1 highland barley seeds without any treatment, and each control group has 4 repeats. Water is poured in time to keep the water holding capacity of the soil at 80%. During the culture, the pot body is rotated once a week, and the position is randomly moved every 2 weeks to eliminate the test errors caused by factors such as light and ventilation. After 7 days of culture, the germination rate is counted, after 30 days of culture, the growth index of the seedlings is counted, and after 135 days of culture, the harvested highland barley grains are obtained, and the index parameter data of each example and the blank control are measured respectively.

[0044] Example 1:

[0045] The pelletized Jinlangu No.1 seeds prepared by the application and the untreated Jinlangu No.1 seeds are alternated in temperature of 12-19℃, light and dark time of 14 / 10 hours, and the germination rate is counted after 7 days.

[0046] Germination rate index determination:

[0047] Germination rate = number of germinated seeds / number of test seeds x 100%.

[0048] Main technical index determination of pelletized seeds:

[0049] Degree of cracking = number of cracked pelletized seeds in 24h / 200 x 100%, 200 is the total number of seeds for each sample, and the pelletizing multiple = 1,000-grain weight of pelletized seeds / 1,000-grain weight of naked seeds;

[0050] Table 1: Effects of pelletizing treatment on seed germination and growth of Jinliangnu No. 1 seeds

[0051] Germination rate (%) Degree of lysis (%) Treatment 98.2±1.3 97 Untreated 79.3±0.9 ——

[0052] The experimental results show that the pelletized Jinliangnu No. 1 sorghum seeds prepared by the application have a significantly improved germination rate, uniform germination, and better growth after germination at low temperature for 7 days, and effectively improve the weight of the seeds.

[0053] Example 2:

[0054] The growth indexes of the seedlings of the pelletized Jinliangnu No. 1 seeds prepared by the application and the untreated Jinliangnu No. 1 seeds are counted after 30 days, and the results are shown in Table 2. Figures 1 to 4 As shown in Table 2, the plant height, leaf width, and leaf length are significantly improved.

[0055] Example 3:

[0056] The physicochemical indexes of the sorghum grains, including total starch, amylopectin, protein, fat, and tannin content, are detected after the sorghum is planted for 135 days.

[0057] Table 2: Grain content of Jinliangnu No. 1 sorghum seeds after treatment and without treatment

[0058]

[0059] The experimental results show that the pelletized Jinliangnu No. 1 sorghum seeds prepared by the application have increased total starch, amylopectin, and protein in the grains after the sorghum is planted for 130-135 days, and the increase amplitudes are 0.4%, 2.2%, and 0.036%, respectively; the fat and tannin content is reduced, and the reduction amplitudes are 0.49% and 0.03%, respectively.

[0060] In summary, the pelletized Jinliangnu No. 1 sorghum seeds treated by the pelletizing formula and method of the application of arbuscular mycorrhizal fungi have improved germination rate and seedling growth traits after normal sowing; the total starch, amylopectin, and protein content of the sorghum grains are improved after maturation; and the fat and tannin content is reduced.

[0061] The embodiments of the present application are described in detail above with reference to the accompanying drawings, but the present application is not limited to the described embodiments. For those skilled in the art, various changes, modifications, replacements and variations can be made to the embodiments without departing from the principles and spirits of the present application, and still fall within the protection scope of the present application. Finally, it should be noted that the contents not described in detail in the specification belong to the prior art known to those skilled in the art, and those skilled in the art can implement them without creative labor, therefore, they will not be described here again.

Claims

1. A method for promoting the increase of amylopectin content in Jinliangnuo No. 1 sorghum using arbuscular mycorrhizal fungi, characterized in that, The method includes the following steps: (1) Propagation of microbial agents: ① Fill the culture container with sterilized boiling sand culture medium to 2 / 3 full. Evenly spread 1020g of arbuscular mycorrhizal fungal agent on the sterilized boiling sand culture medium. Then plant the rinsed Jinliangnuo No. 1 sorghum seeds, cover with a 2cm layer of sterilized boiling sand culture medium, water thoroughly, and move to a light room or greenhouse for cultivation for 3-5 months. The arbuscular mycorrhizal fungal agent is *Gymnospermum juvenileum*. Glomus etunicatum Moses globosum Glomus mosseae and lamellae Glomus versiforme Mixed bacterial agents; ② Cut the roots of Jinliangnuo No. 1 sorghum, which have been cultured for 3-5 months in step ① above, into small sections less than 1 cm long. Use a microscope to examine the spores in the fungal agent using the wet sieving method. Then mix them evenly with the sterilized boiling sand medium used in step ① to obtain the propagated arbuscular mycorrhizal fungal agent. Place the propagated arbuscular mycorrhizal fungal agent in an ultra-low temperature environment for later use. (2) Seed treatment: ① Jinliangnuo No. 1 sorghum seeds should meet the requirements of GB / T8231-2007 for sorghum. Seeds to be pelleted should be purified and selected to remove sand, stones or seeds of other crops, and impurities, so that the purity of the seeds is greater than or equal to 98%. ②Disinfect the surface of the seeds and soak the seeds that are about to be sown in a conventional germination-promoting solution; (3) Inoculation with fungi and pelleting treatment: ① Place the disinfected Jinliangnuo No. 1 sorghum seeds into a rotating pelleting machine, add the arbuscular mycorrhizal fungal agent propagated in step (1) at a weight ratio of 1:1, spray in a small amount of peach gum to form a uniform and smooth seed surface structure so that the additive can be adsorbed on the seed surface, and then add the additive to form small particles centered on each seed; the arbuscular mycorrhizal fungal agent uses the arbuscular mycorrhizal fungus 'Jiayuhoujuan'. Glomus etunicatum Moses globosum Glomus mosseae and lamellae Glomus versiforme The microbial agent formed after 4 months of propagation on brewing sorghum has a spore density of ≥100 spores / g, including mycelial fragments and infected root segments; ②Increasing pellet strength: This process is used to change the shape of seeds, increase seed size, weight, and gloss. It requires low solubility, a pH value between 5.5 and 8, and a conductivity below 1.0 s / m. The pellets are enlarged, additives are added multiple times, and coating is repeated to rapidly increase volume or weight. The pellets gradually become smooth and uniform. A vibrating sieve with specific sieve holes is used to separate the pellets by size. Unqualified pellets can continue to be pelletized, while qualified pellets proceed to the next process. ③ Spray peach gum again, add the propagated inoculant. When the added inoculant makes the seeds reach the required particle size, spray peach gum and let them spin to increase strength and roundness. Finally, coat the seeds with a coating layer and spray different dyes or colored films onto the surface of the moving pellets. Place the colored pellets in a ventilated place and let them air dry naturally.

2. The method for promoting the increase of amylopectin content in Jinliangnuo No. 1 sorghum using arbuscular mycorrhizal fungi according to claim 1, characterized in that, The sterilized boiling sand culture medium in step (1) is obtained by sterilizing coarse river sand with 120°C steam for 20 minutes.

3. The method for promoting the increase of amylopectin content in Jinliangnuo No. 1 sorghum using arbuscular mycorrhizal fungi according to claim 1, characterized in that, The Jinliangnuo No. 1 sorghum seeds are washed by rinsing with distilled water 3-5 times.

4. The method for promoting the increase of amylopectin content in Jinliangnuo No. 1 sorghum using arbuscular mycorrhizal fungi according to claim 1, characterized in that, The culture container in step (1) is a round PVC basin with a height of 55-65cm and a diameter of 95-105cm.

5. The method for promoting the increase of amylopectin content in Jinliangnuo No. 1 sorghum using arbuscular mycorrhizal fungi according to claim 1, characterized in that, The ultra-low temperature in step (1) is -80℃.

6. The method for promoting the increase of amylopectin content in Jinliangnuo No. 1 sorghum using arbuscular mycorrhizal fungi according to claim 1, characterized in that, The coating layer in step (3) is composed of the following raw materials in parts by weight: 70 parts sodium polyacrylate, 20 parts diamine slow-release fertilizer, 2.5 parts naphthaleneacetic acid rooting powder, 2.5 parts carbendazim, 2.5 parts konjac powder, and 2.5 parts zinc sulfate powder.

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