A strain of Saroma lacustrine sporosarcina ZIP-102 and its application

Abstract

The present invention relates to a strain of Zoroma lacustrine sporosarcina ZIP‑102 and an application thereof, belonging to the technical field of microorganisms; the strain has excellent iron carrier production and nitrogen fixation characteristics, and also has the ability to produce protease, amylase, lipase and cellulase, and can effectively promote the growth of tomato plants, thereby reducing the use of chemical fertilizers and alleviating the continuous cropping obstacle in the tomato planting process, and has a very broad application prospect.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and specifically to a strain of Zoroma lacustrine sporosarcina ZIP-102 and an application thereof. Background Art

[0002] tomato( Solanum lycopersicum L.) is an annual herbaceous plant of the Solanaceae family, Solanum genus, native to South America, and later spread to the world to become one of the most important vegetables in the world. my country began to grow tomatoes in the 1950s, and has been developing rapidly ever since. Currently, the places where tomatoes are grown in the world are mainly in areas with high light intensity and mild temperatures. China is one of the world's important tomato producing areas.

[0003] At present, the main mode of tomato cultivation is facility cultivation, but years of facility cultivation have led to serious obstacles to continuous cropping of tomatoes. Therefore, a large amount of chemical fertilizers are often needed to ensure the yield of tomatoes during the growth process. Among the applied chemical fertilizers, except for a small part of the nutrients used by plant growth, the remaining nutrients are gradually converted from soluble salts to insoluble inorganic salts in the soil. The continuous application of chemical fertilizers leads to the continuous accumulation of insoluble inorganic salts in the soil, which in turn leads to serious soil salinization, restricting the development of agricultural production.

[0004] At present, functional microorganisms have become a new way to solve the problem of continuous cropping in agriculture. Some microorganisms are green, pollution-free and harmless, and can convert insoluble inorganic salt ions in the soil into soluble salt ions that are easily used by plants by producing metabolites such as organic acids, enzymes, and chelates. At the same time, they improve the soil environment through nitrogen fixation and phosphorus solubilization, thereby promoting plant growth and reducing the application of chemical fertilizers. Summary of the invention

[0005] The present invention provides a strain of Zoroma lacustrine sporosarcina ZIP-102 and an application thereof. The strain is isolated and screened from tomato rhizosphere soil, has excellent iron carrier production and nitrogen fixation properties, and also has the ability to produce protease, amylase, lipase and cellulase, and can effectively promote the growth of tomato plants.

[0006] The technical solution of the present invention is as follows:

[0007] A strain of Bacillus sporesarcinia from the lake Saroma Sporosarcina saromensis ) ZIP-102, deposited on October 9, 2024 at the General Microbiology Center of China Microorganism Culture Collection Administration, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No.32154.

[0008] Among them, the 16S rDNA gene sequence of the Saroma lacustrine sporosarcina ZIP-102 is shown as SEQ ID NO.1.

[0009] The above-mentioned method for culturing the Saroma lake spore-forming ...

[0010] (1) Inoculate the ZIP-102 strain of Bacillus sporebacterium from Saroma lake onto a solid culture medium and activate the culture at 37±2°C for 24-30 hours to obtain an activated strain;

[0011] (2) Select the activated strain from step (1) and inoculate it into the seed liquid culture medium, and culture it under shaking conditions of 180-200 rpm and 37±2°C for 18-25 hours to obtain the seed liquid;

[0012] (3) The seed solution in step (2) is inoculated into the fermentation medium at an inoculation amount of 2-10% of the volume of the fermentation medium, and cultured under shaking conditions of 180-200 rpm and 37±2° C. for 24-30 h to obtain a bacterial solution of Bacillus spore-forming Sarcosinae ZIP-102.

[0013] Preferably, the solid culture medium in step (1) is NA solid culture medium.

[0014] Further preferably, the NA solid culture medium comprises the following components: 10 g / L peptone, 3 g / L beef extract, 5 g / L sodium chloride, 15 g / L agar, pH 7.0-7.5.

[0015] Preferably, the seed liquid culture medium in step (2) or the fermentation medium in step (3) is NA liquid culture medium.

[0016] Further preferably, the NA liquid culture medium comprises the following components: 10 g / L peptone, 3 g / L beef extract, 5 g / L sodium chloride, pH 7.0-7.5.

[0017] The application of the Saroma lacustrine sporosarcina ZIP-102 in producing iron carriers.

[0018] The application of the Saroma lacustrine spore-forming bacteria ZIP-102 in nitrogen fixation.

[0019] The application of the Zaroma lacustrine sporosarcina ZIP-102 in the production of protease, amylase, lipase and cellulase.

[0020] The application of the Saroma lacustrine sporosarcina ZIP-102 in promoting the growth of tomato plants.

[0021] Preferably, the application method is to use the bacterial solution of Bacillus sporesarciniae ZIP-102 to irrigate the roots of tomato plants.

[0022] More preferably, the bacterial concentration of the bacterial solution of Bacillus sporesarcinia ZIP-102 is 0.8×10 9 ~1.0×10 9 CFU / mL.

[0023] Further preferably, after the tomato plants are irrigated with water, the bacterial cell concentration in the soil is 1.6×10 6 ~2.2×10 6 CFU / g.

[0024] A microbial agent comprises the Saroma lacustrine sporosarcina ZIP-102.

[0025] Beneficial effects of the present invention:

[0026] The present invention provides a strain of Spore Sarcinia cerevisiae ( Sporosarcina saromensis ) ZIP-102, this strain has excellent iron carrier production and nitrogen fixation characteristics, and also has the ability to produce protease, amylase, lipase, and cellulase, and can effectively promote the growth of tomato plants, thereby reducing the use of chemical fertilizers and alleviating the obstacles of continuous cropping in tomato cultivation. It has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a colony photo of strain ZIP-102;

[0028] Figure 2 This is a picture of strain ZIP-102 observed under a microscope after Gram staining;

[0029] Figure 3 This is a picture of the siderophore production effect of strain ZIP-102;

[0030] Figure 4 This is a picture of the nitrogen fixation effect of strain ZIP-102;

[0031] Figure 5 This is a picture of the protease production effect of strain ZIP-102;

[0032] Figure 6 This is a picture of the amylase production effect of strain ZIP-102;

[0033] Figure 7 This is a picture of the lipase production effect of strain ZIP-102;

[0034] Figure 8 This is a picture of the cellulase production effect of strain ZIP-102;

[0035] Fig. 9 This is a picture of the growth-promoting effect of strain ZIP-102 on tomato plants. DETAILED DESCRIPTION

[0036] The following is explained in conjunction with specific embodiments:

[0037] Example 1: Isolation, screening and identification of Bacillus sporosarcina ZIP-102

[0038] A bacterial strain was isolated and screened from the tomato rhizosphere soil in Qingzhou, Shandong, my country, and we named it "ZIP-102".

[0039] The colony photos of the strain ZIP-102 obtained above on NA solid medium are shown in Figure 1 As shown in the figure, the colonies are white, round in shape, dry on the surface, and have regular wrinkles on the edges. The picture of strain ZIP-102 observed under a microscope after Gram staining is shown in the figure. Figure 2 As shown, the bacteria are short rod-shaped; Gram stain is purple, and they are Gram-positive bacteria.

[0040] The 16S rDNA sequence of the strain ZIP-102 obtained by the above screening was sequenced, and the sequencing result is shown in SEQ ID NO.1; the obtained 16S rDNA sequence was subjected to BLAST analysis with the existing sequences in the NCBI database, and strains with similar homology were selected, and the phylogenetic tree was constructed using MEGA X software, and the construction method was Neighbor-joining. The results showed that the strain ZIP-102 obtained by the above screening had a similar structure to the sporosarcina sarcoides ( Sporosarcina saromensis ) is 82% similar to the strain, and the evolutionary distance is relatively close. Combined with the physiological and biochemical characteristics of the strain, it was identified as Spore Sarcinia sarcoides ( Sporosarcina saromensis ).

[0041] Sporosarcina sarcoides Sporosarcina saromensis ) ZIP-102, deposited on October 9, 2024 in the General Microbiology Center of China Microorganism Culture Collection Administration, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No.32154.

[0042] Example 2: Cultivation of Sporosarcina sarcoides ZIP-102

[0043] (1) The ZIP-102 strain was inoculated on NA solid culture medium and activated at 37°C for 24 h to obtain an activated strain.

[0044] (2) Pick the activated strain in step (1), inoculate it into NA liquid culture medium, and culture it at 37°C and 180 rpm for 18 h to obtain seed solution.

[0045] (3) The seed solution in step (2) was inoculated into NA liquid culture medium at a volume percentage of 2%, and cultured at 37°C and 180 rpm for 24 h to obtain a bacterial cell concentration of 0.95×10 9 CFU / mL of the bacterial solution of Bacillus sporosarcina ZIP-102 from Saroma.

[0046] The components of NA solid culture medium are as follows: peptone 10 g / L, beef extract 3 g / L, sodium chloride 5 g / L, agar 15 g / L, pH 7.0-7.5; high pressure sterilization at 121°C for 20 min.

[0047] Among them, the components of NA liquid culture medium are as follows: peptone 10g / L, beef extract 3g / L, sodium chloride 5g / L, pH7.0~7.5; high-pressure sterilization at 121℃, 20min.

[0048] Example 3: Evaluation of the growth-promoting function of Bacillus sporosarcina ZIP-102

[0049] 1. Bacteria activation:

[0050] Streak the Saroma Lake Sporosarcina ZIP-102 on NA solid culture medium and culture at 37°C for 24 hours to grow a single colony, i.e., the activated strain.

[0051] 2. Evaluation of the ability of bacterial strains to produce siderophores:

[0052] Use a sterilized toothpick to pick up the activated bacteria and inoculate them in the CAS detection medium, and culture them at 28°C for 3 days. After the culture is completed, use a vernier caliper to measure the diameter of the transparent circle (D) and the colony diameter (d) in the CAS detection medium.

[0053] The presence of chlorazuron and hexadecyltrimethylammonium bromide in CAS medium will react with Fe 3+ The iron carrier produced by the bacteria combines with the chelate to form a blue chelate, taking away the Fe in the chelate. 3+ , which causes the color of the culture medium around the bacteria to change. Therefore, the iron carrier production ability of the tested bacteria can be evaluated based on the D / d value.

[0054] Among them, the components of CAS detection culture medium are as follows: chrome azuro blue S 60.5 mg / L, hexadecyltrimethylammonium bromide 72.9 mg / L, ferric chloride hexahydrate 2.645 mg / L, sodium dihydrogen phosphate dihydrate 295.25 mg / L, disodium hydrogen phosphate dodecahydrate 1213.5 mg / L, ammonium chloride 62.5 mg / L, potassium dihydrogen phosphate 37.5 mg / L, sodium chloride 62.5 mg / L, agar 9000 mg / L, pH 6.7~6.9; high pressure sterilization at 116°C for 30 min.

[0055] Photos after the training Figure 3 After measurement and calculation, the D / d value in the CAS test medium was 3.25, which indicated that Bacillus spore-forming Saroma ZIP-102 had excellent ability to produce siderophores.

[0056] 3. Evaluation of nitrogen fixation ability of bacteria:

[0057] (1) Use a sterile inoculation loop to pick up the activated bacterial strain and inoculate it into the nitrogen-free solid culture medium of Ashubei and culture it at 28°C for 5 days. After the culture is completed, observe whether there are colonies in the culture medium. If there are colonies, it means that the strain has the ability to fix nitrogen.

[0058] Photos after the training Figure 4 As shown. Figure 4 It can be seen that colonies were formed in the nitrogen-free solid culture medium of Asubei, which indicates that the Saroma Lake Sporosarcina ZIP-102 has the ability to fix nitrogen.

[0059] (2) Determination of nitrogenase activity of ZIP-102 by acetylene reduction method: ZIP-102 in logarithmic growth phase was transferred to NA liquid culture medium and cultured overnight in a shaker at 30°C and 200 rpm. The cells were collected by centrifugation at 5000 rpm for 5 min, washed three times with Ashube nitrogen-free liquid culture medium and resuspended. The O0 600 The mixture of bacteria and culture medium was 0.2. 2 mL of the mixture of bacteria and culture medium was added to the anaerobic tube as the experimental group, and the anaerobic tube with only 2 mL of Ashubei nitrogen-free liquid culture medium was used as the negative control group. The anaerobic tube was sealed with a stopper; 2 mL of acetylene gas was injected into the anaerobic tube, and after shaking evenly, it was placed in a 30°C incubator for 16 hours; after the incubation, 1 mL of gas was extracted from the anaerobic tube by a microinjector as the sample to be tested, and injected into the gas chromatograph to determine the amount of ethylene generated in the gas sample.

[0060] The parameters of the gas chromatograph were set as follows: detector temperature 180°C, injector temperature 120°C, and column box temperature 80°C.

[0061] Among them, the components of the Axubei nitrogen-free liquid culture medium are as follows: 10g glucose, 0.2g dipotassium hydrogen phosphate, 0.2g magnesium sulfate, 0.2g sodium chloride, 0.2g calcium sulfate, 5g calcium carbonate, 1000mL distilled water, pH7.0~7.2, high-pressure sterilization at 116℃ for 30min; 20g agar is additionally added to the Axubei nitrogen-free solid culture medium.

[0062] The results of the determination of ethylene production are shown in Table 1 below:

[0063] Table 1. Ethylene production measurement results

[0064] Group Ethylene peak area Negative control group ND (negative) Experimental Group 53392±12553

[0065] A standard curve of ethylene content-peak area was drawn; the ethylene production in the gas sample was calculated based on the standard curve and the measured ethylene peak area; through the ethylene production, the nitrogenase production capacity of Bacillus sporosarcina ZIP-102 from Lake Saroma was calculated to be 2.76 nmol (ethylene) / h.

[0066] Example 4: Evaluation of the enzyme production capacity of Sporosarcina sarcoides ZIP-102

[0067] 1. Bacteria activation:

[0068] Streak the Saroma Lake Sporosarcina ZIP-102 on NA solid culture medium and culture at 37°C for 24 hours to grow a single colony, i.e., the activated strain.

[0069] 2. Evaluation of the protease production ability of bacterial strains:

[0070] Use a sterilized toothpick to pick up the activated bacteria and inoculate them in a skimmed milk powder culture medium, and culture them at 28°C for 5 days. After the culture is completed, observe the color change of the culture medium around the bacteria, and use a vernier caliper to measure the diameter of the transparent circle in the culture medium (D) and the colony diameter (d), and calculate the D / d value.

[0071] The proteins present in the skimmed milk powder culture medium can be broken down into small molecular amino acids and peptides by the proteases produced by the bacteria, causing the color of the culture medium surrounding the bacteria to change from yellow-white to transparent. Therefore, the protease production ability of the tested bacteria can be evaluated by observing the color changes of the culture medium surrounding the bacteria and the D / d value.

[0072] The components of the skimmed milk powder culture medium are as follows: 0.5 g beef extract, 1 g peptone, 0.5 g NaCl, 3 g skimmed milk powder, 2 g agar, 100 mL distilled water, pH 7.0-7.2.

[0073] Photos after the training Figure 5 shown by Figure 5 It can be seen that the color of the culture medium around the bacteria changed from yellow-white to transparent, which indicates that Sporosarcina ZIP-102 has the ability to produce protease.

[0074] 3. Evaluation of the ability of bacterial strains to produce amylase:

[0075] Use a sterilized toothpick to pick up the activated bacteria and inoculate them in starch culture medium, and culture them at 28°C for 5 days. After the culture, add Lugol's iodine solution, observe the color change of the culture medium around the bacteria, and use a vernier caliper to measure the diameter of the transparent circle in the culture medium (D) and the colony diameter (d), and calculate the D / d value.

[0076] The starch present in the starch culture medium can be decomposed into small molecule sugars such as maltose and glucose by the amylase produced by the bacteria, and the starch turns blue when it meets Lugol's iodine solution. When the starch around the bacteria is enzymatically hydrolyzed, the color of the culture medium around the bacteria will change from blue-purple to transparent. Therefore, the amylase production ability of the tested bacteria can be evaluated by observing the color changes of the culture medium around the bacteria and the D / d value.

[0077] The components of the starch culture medium are as follows: 2 g soluble starch, 10 g peptone, 5 g beef extract, 5 g sodium chloride, 20 g agar, pH 7.2, dilute to 1 L with distilled water; sterilize at 121°C for 30 min.

[0078] Among them, the components of Lugol's iodine solution are as follows: 1g iodine tablets, 2g potassium iodide, and 300mL distilled water.

[0079] Photos after the training Figure 6 shown by Figure 6 It can be seen that the color of the culture medium around the bacteria changed from blue-purple to transparent, which indicates that Sporosarcina ZIP-102 has the ability to produce amylase.

[0080] 4. Evaluation of Lipase Production Capacity of Bacteria:

[0081] Use a sterilized toothpick to pick up the activated bacteria and inoculate them in the fat culture medium, and culture them at 28°C for 5 days. After the culture, observe the color change of the culture medium around the bacteria, and use a vernier caliper to measure the diameter of the transparent circle in the culture medium (D) and the diameter of the colony (d), and calculate the D / d value.

[0082] Tributyrin present in the fat culture medium can be decomposed into fatty acids and glycerol by the lipase produced by the bacteria, causing the color of the culture medium surrounding the bacteria to change from white to transparent. Therefore, the lipase-producing ability of the tested bacteria can be evaluated by observing the color change of the culture medium surrounding the bacteria and the D / d value.

[0083] The components of the fat culture medium are as follows: peptone 10 g, yeast powder 5 g, NaCl 10 g, tributyrin 2 mL, agar 20 g, distilled water 1 L, pH 7.5.

[0084] Photos after the training Figure 7 shown by Figure 7 It can be seen that the color of the culture medium around the bacteria changed from white to transparent, which indicates that Sporosarcina ZIP-102 has the ability to produce lipase.

[0085] 5. Evaluation of the ability of strains to produce cellulase:

[0086] Use a sterilized toothpick to pick up the activated bacteria and inoculate them in the cellulose culture medium, and culture them at 28°C for 5 days. After the culture is completed, observe the color change of the culture medium around the bacteria, and use a vernier caliper to measure the diameter of the transparent circle in the culture medium (D) and the colony diameter (d), and calculate the D / d value.

[0087] The sodium carboxymethyl cellulose present in the cellulose culture medium changes from colorless to red when stained with Congo red dye, and the sodium carboxymethyl cellulose can be decomposed by the cellulase produced by the bacteria, causing the color of the culture medium around the bacteria to change from red to transparent. Therefore, the cellulase production ability of the tested bacteria can be evaluated by observing the color change of the culture medium around the bacteria and the D / d value.

[0088] The components of the cellulose culture medium are as follows: 10 g of peptone, 5 g of yeast powder, 10 g of sodium chloride, 10 g of sodium carboxymethyl cellulose, 20 g of agar, and 1 L of distilled water.

[0089] Photos after the training Figure 8 shown by Figure 8 It can be seen that the color of the culture medium around the bacteria changed from red to transparent, which indicates that Sporosarcina ZIP-102 has the ability to produce cellulase.

[0090] The measurement results of D / d values ​​in the above experiments are shown in Table 2 below:

[0091] Table 2. Evaluation of enzyme production capacity of ZIP-102

[0092]

[0093] As shown in Table 2, Bacillus sporosarcina ZIP-102 has good ability to produce protease, amylase, lipase and cellulase.

[0094] Example 5: Experiment on promoting the growth of tomato seedlings by Bacillus sporesarcinia ZIP-102

[0095] This experiment was conducted in the laboratory. First, 60 pots of healthy, pest-free, and three-true-leaf tomato seedlings of the Provence variety were selected. The mass ratio of soil (arable soil) to vermiculite (to increase the looseness and air permeability of the soil) in the pots was 1:1, and the total weight was 1.5±0.1kg.

[0096] The experimental group and the control group were set up respectively, with 30 pots in each group, wherein the experimental group was applied with the bacterial solution of ZIP-102 from Lake Saroma, and the control group was applied with the same volume of sterile PBS buffer, and the experimental group and the control group were cultured in the same environment, and the experimental period was 30 days. The bacterial solution of ZIP-102 from Lake Saroma was prepared according to the culture method described in Example 2, and the application method was: using a bacterial concentration of 0.8×10 9 ~1.0×10 9 CFU / mL of the bacterial solution of ZIP-102 from the lake of Saroma was used to irrigate the roots of tomato plants, and the bacterial concentration in the soil was 1.6×10 6 ~2.2×10 6 CFU / g.

[0097] After the experiment, the appearance of the tomato plants in the experimental group and the control group were compared. Fig. 9 As shown by Fig. 9 It can be seen that the height of tomato plants in the experimental group was significantly higher than that in the control group.

[0098] After the experiment, the growth data of tomato plants in the experimental group and the control group were measured, including plant height, stem diameter, dry weight, fresh weight and leaf chlorophyll content. The method for determining leaf chlorophyll was as follows: 1g of fresh leaves were placed in liquid nitrogen for low temperature treatment for 20s, then ground and dissolved in a mortar with 20mL of anhydrous ethanol, and the absorbance was measured at 645nm and 663nm using an ultraviolet spectrophotometer. The chlorophyll content (C T ), the calculation formula is:

[0099] C a (mg / L)=12.7OD 663 -2.69OD 645 ;

[0100] C b (mg / L)=22.9OD 645 -4.68OD 663 ;

[0101] C T (mg / L)=C a +C b .

[0102] The measurement results are shown in Table 3 below:

[0103] Table 3. Growth promotion effect of ZIP-102 on tomato plants

[0104]

[0105] As shown in Table 3, compared with the control group, ZIP-102 can significantly increase the height of tomatoes and the chlorophyll content of tomato leaves, thereby effectively promoting the growth of tomato plants. Specifically, compared with the control group, the strain ZIP-102 increased the height of tomatoes by 18.34%, increased the stem diameter of tomato plants by 6.79%, increased the dry weight of tomato plants by 4.34%, increased the fresh weight of tomato plants by about 7.62%, and increased the chlorophyll content of tomato leaves by 30.56%.

Claims

1. A strain of Bacillus sporesarciniasis from the lake Saroma Sporosarcina saromensis ) ZIP-102, deposited on October 9, 2024 at the General Microbiology Center of China Microorganism Culture Collection Administration, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No.32154.

2. The method for culturing the Saroma lacustrine sporosarcina ZIP-102 according to claim 1, characterized in that: The steps include: (1) Inoculate the ZIP-102 strain of Bacillus sporebacterium from Saroma lake onto a solid culture medium and activate the culture at 37±2°C for 24-30 hours to obtain an activated strain; (2) Select the activated strain from step (1) and inoculate it into the seed liquid culture medium, and culture it under shaking conditions of 180-200 rpm and 37±2°C for 18-25 hours to obtain the seed liquid; (3) The seed solution in step (2) is inoculated into the fermentation medium at an inoculation amount of 2-10% of the volume of the fermentation medium, and cultured under shaking conditions of 180-200 rpm and 37±2° C. for 24-30 h to obtain a bacterial solution of Bacillus spore-forming Sarcosinae ZIP-102.

3. The culture method according to claim 2, characterized in that The solid culture medium in step (1) is NA solid culture medium; the seed liquid culture medium in step (2) or the fermentation culture medium in step (3) is NA liquid culture medium.

4. The use of the Saroma lacustrine sporosarcina ZIP-102 according to claim 1, characterized in that: Used to produce siderophores.

5. The use of the Saroma lacustrine sporosarcina ZIP-102 according to claim 1, characterized in that: Used for nitrogen fixation.

6. The use of the Saroma lacustrine sporosarcina ZIP-102 according to claim 1, characterized in that: It is used to produce protease, amylase, lipase and cellulase.

7. The use of the Saroma lacustrine sporosarcina ZIP-102 according to claim 1, characterized in that: Used to promote the growth of tomato plants.

8. The use according to claim 7, characterized in that The application method is to use the bacterial solution of Bacillus spore-forming Sarcosina ZIP-102 to irrigate the roots of tomato plants.

9. The use according to claim 8, characterized in that The bacterial concentration of the ZIP-102 bacterial solution of the Saroma Lake Sporosarcina was 0.8×10 9 ~1.0×10 9 CFU / mL; after root irrigation of tomato plants, the bacterial concentration in the soil was 1.6×10 6 ~2.2×10 6 CFU / g.

10. A microbial agent, characterized in that: It includes the Saroma lacustrine sporosarcina ZIP-102 described in claim 1.

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