Bacillus atrophaeus for preventing and treating wheat gibberellic disease caused by gibberellic maize and application of bacillus atrophaeus

Abstract

The invention relates to the technical field of biological control of plant diseases, in particular to application of a bacillus atrophaeus strain and a preparation thereof to control wheat gibberellic disease caused by gibberellic maize. The preservation number of the strain is CGMCC (China General Microbiological Culture Collection Center) No.29799, and the strain is classified and named as the bacillus atrophaeus strain HHQGTS13-1 (Bacillus atrophaeus HHQGTS13-1). The bacillus atrophaeus strain HHQGTS13-1 is wide in application range to natural environment conditions such as temperature and pH, and has the capability of antagonizing fusarium maydis when the temperature is 10-45 DEG C and the pH value is 4-10. The bacillus atrophaeus strain HHQGTS13-1 can be used for obviously inhibiting the growth of fusarium maydis hyphae and enabling the hyphae to be deformed; the antagonistic bacillus HHQGTS13-1 strain has an obvious inhibition effect on spore germination of fusarium maydis, the spore germination inhibition rate is 97.10%, the prevention and treatment effect of the antagonistic bacillus HHQGTS13-1 strain fermentation liquor after wheat is inoculated with fusarium maydis pathogenic bacteria is 63.43%, and the antagonistic bacillus HHQGTS13-1 strain has good potential of preventing and treating plant diseases caused by fusarium maydis.

Description

Technical Field

[0001] The present invention relates to the technical field of biological control of plant diseases, and particularly relates to the application of a strain of Bacillus atrophaeus and its preparation to control Fusarium head blight caused by Gibberella zeae. Background Art

[0002] Fusarium head blight is a climatic disease of wheat crops, which mostly occurs in areas with rainy and humid weather during the ear stage. Fusarium head blight is almost distributed in all regions of the world, and the International Maize and Wheat Improvement Center (CIMMYT) lists this disease as the most important factor affecting wheat production. It not only causes serious yield reduction, but more importantly, it deteriorates the grain quality and reduces the value of seeds for sowing. After Fusarium head blight infects wheat, it can produce a variety of mycotoxins. Among them, deoxynivalenol (DON) has the strongest toxicity, and eating it will cause acute poisoning symptoms such as dizziness, fever, nausea, vomiting, and diarrhea. In severe cases, bleeding will occur, affecting immune function and reducing fertility, etc., directly threatening the health and life safety of humans and livestock.

[0003] Gibberella zeae (Schwein.) Petch, whose anamorph is Fusarium graminearum Schwabe, is the main pathogen of Fusarium head blight in Chinese wheat. The host range of Gibberella zeae is relatively wide, and it has been recorded on dozens of crops. Especially, its colonization ability on the diseased residues of corn straw is extremely strong. Existing research shows that the population of Gibberella zeae in nature is very complex, and there is a large variability among individuals, but no strain with host specialization has been found. Although in the previous research results, the small-scale test results showed that Bacillus subtilis has a certain control effect on Fusarium head blight, the effect of large-scale application is not clear. Currently, the screened Bacillus atrophaeus has an obvious strong inhibitory effect on Gibberella zeae.

[0004] Bacillus atrophaeus has a strong ability to inhibit pathogenic bacteria, can effectively grow and colonize in the natural environment, and secrete auxin. Auxin (Indole-3-acetic acid, IAA) plays a very important role in plant morphogenesis, organogenesis, and various physiological processes, especially crucial for plant growth and development.

[0005] Therefore, screening microorganisms with a strong ability to inhibit pathogenic bacteria, which can effectively grow and colonize in the natural environment and produce IAA, has a very important role in the development of growth-promoting and disease-preventing microbial agents. Summary of the Invention

[0006] The present invention provides a strain of Bacillus atrophaeus HHQGTS13-1 that has a preventive effect against Gibberella zeae. This strain has an antagonistic effect against Gibberella zeae, can be used to control wheat scab, and can colonize in the soil, thereby achieving the purpose of preventing and resisting diseases. It is also an environmentally friendly biological control method, with advantages such as low production cost, convenient operation, non-toxic, pollution-free, and residue-free.

[0007] The Bacillus atrophaeus strain HHQGTS13-1 described in the present invention was isolated from the cultivated soil in the wheat planting area of Gansu. It was deposited with the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on January 29, 2024, with the deposit number: CGMCC No. 29799, and the taxonomic name: Bacillus atrophaeus strain HHQGTS13-1, and the deposit address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0008] Furthermore, the culture characteristics of the antagonistic Bacillus HHQGTS13-1 are that its optimal growth temperature is 25-35 °C; the highest pH value is 10.0, the lowest pH value is 4.5, and the optimal pH value is 7; it has salt tolerance and can grow in a 12% NaCl medium. It can oxidize L-arabinose and mannitol to produce acid; it can oxidize glucose but does not produce gas, and the egg yolk hydrolysis is positive; but it cannot oxidize D-xylose to produce acid.

[0009] Furthermore, the molecular biological classification of the antagonistic Bacillus HHQGTS13-1: The 16S rDNA sequence homology between the antagonistic Bacillus HHQGTS13-1 and Bacillus atrophaeus is 98%. Combining the morphological, physiological and biochemical characteristics of the strain, it is finally determined that the strain HHQGTS13-1 is Bacillus atrophaeus.

[0010] The present invention also provides a biocontrol bacterial agent. The active ingredient of the biocontrol bacterial agent is the above-mentioned Bacillus atrophaeus strain HHQGTS13-1. The Bacillus atrophaeus HHQGTS13-1 of the present invention is subjected to liquid fermentation to prepare a microbial bacterial agent with a viable bacteria count of about 1.0×10 6 ~1.0×10 8 cfu / mL.

[0011] The present invention also provides the application of the above-mentioned Bacillus atrophaeus strain HHQGTS13-1 and the biocontrol bacterial agent in preventing and controlling Gibberella zeae, and the application includes inhibiting Gibberella zeae; inhibiting the spore germination of Gibberella zeae; the field disease prevention experiment also has a good prevention and control effect by applying the fermentation broth of antagonistic Bacillus HHQGTS13-1. Inhibiting Gibberella zeae means inhibiting the growth of Gibberella zeae. The antagonistic activity of the antagonistic Bacillus HHQGTS13-1 strain against Gibberella zeae is good, and the antibacterial zone width is 9 mm. Even after culturing for 10 days, it can still maintain a high antibacterial effect; the fermentation broth of the antagonistic Bacillus HHQGTS13-1 strain has an obvious inhibitory effect on the spore germination of Gibberella zeae, and the spore germination inhibition rate is 97.10%. The prevention and control effect of the fermentation broth of the antagonistic Bacillus HHQGTS13-1 strain after inoculating the pathogen of Gibberella zeae in wheat is 63.43%.

[0012] Furthermore, the fermentation broth of the antagonistic Bacillus HHQGTS13-1 strain described in the present invention contains active ingredients that have a highly inhibitory effect on Gibberella zeae, indicating that the antagonistic Bacillus HHQGTS13-1 strain produces metabolites during the fermentation culture process that have an inhibitory effect and a teratogenic effect on Gibberella zeae.

[0013] The present invention has the following advantages compared with the prior art: 1. The antagonistic Bacillus HHQGTS13-1 strain that antagonizes Gibberella zeae in the present invention has a very significant effect in preventing and controlling Gibberella zeae, has a strong inhibitory effect on Gibberella zeae, can significantly inhibit the growth of the hyphae of Gibberella zeae and make the hyphae deformed, and can also significantly inhibit the spore germination of Gibberella zeae, and has great potential for preventing and controlling plant diseases caused by Gibberella zeae. As a bacterium in the cultivated soil of the wheat planting area, it provides an environmentally friendly, simple and effective way to prevent and control plant diseases caused by Gibberella zeae, which is beneficial to environmental protection.

[0014] 2. The antagonistic Bacillus HHQGTS13-1 of the present invention has a wide adaptability to natural environmental conditions such as temperature and pH, and has the ability to antagonize Gibberella zeae within the range of 10-45°C and pH = 4-10. The strain of the present invention is derived from the rhizosphere soil of wheat in the wheat planting area, is easy to culture and maintain, and can antagonize Gibberella zeae.

[0015] 3. The present invention solves the problem that the disease of Gibberella zeae is becoming increasingly serious at present. As a microbial pesticide, it can reduce the pollution problem of chemical pesticides and weaken the drug resistance problem of pathogenic bacteria, and has the advantages of low production cost, convenient operation, non-toxic, pollution-free and residue-free.

[0016] 4. The antagonistic Bacillus strain HHQGTS13-1 screened by the present invention has great economic value and can bring greater economic benefits to farmers.

[0017] The technical solution of the present invention will be further described in detail below through the accompanying drawings and specific embodiments. Description of the Drawings Figure 1 Colony of Bacillus atrophaeus strain HHQGTS13-1 Figure 2 Gram staining of Bacillus atrophaeus strain HHQGTS13-1 Figure 3 Antagonistic effect of Bacillus atrophaeus strain HHQGTS13-1 on Gibberella zeae on PDA medium, Figure 3 in a: Gibberella zeae growing normally, Figure 3 in b: Gibberella zeae affected by antagonistic Bacillus strain HHQGTS13-1.

[0019] Figure 4 Identification diagram of protease production function of Bacillus atrophaeus strain HHQGTS13-1 Figure 5 Identification diagram of amylase production function of Bacillus atrophaeus strain HHQGTS13-1 Figure 6 Identification diagram of cellulase production function of Bacillus atrophaeus strain HHQGTS13-1 Figure 7 Identification diagram of phytohormone production function of Bacillus atrophaeus strain HHQGTS13-1 Detailed Description of the Invention

[0020] The present invention will be described in detail below in conjunction with specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can be made. These all belong to the protection scope of the present invention.

[0021] Unless otherwise specified, the methods in the following embodiments are all conventional methods. Unless otherwise specified, the percentage contents in the following embodiments are all mass percentage contents.

[0022] Example 1 The isolation and screening method and identification of the antagonistic Bacillus strain HHQGTS13-1 in this example are as described below. The strain in this example was collected from the rhizosphere soil of wheat in the wheat planting area of Tiaoshan Farm, Jingtai County, Baiyin City, Gansu Province.

[0023] 1. Screening of antagonistic Bacillus sp. HHQGTS13-1, and the screening process includes the following steps: (1) Weigh 10 g of the rhizosphere soil of wheat in the wheat planting area collected, add it to a 250 mL conical flask containing 90 mL of sterile water and glass beads, heat it in a water bath at 80 °C for 30 min, then shake it well for 30 min, dilute it by 10-fold serial dilution, and spread it on the LB medium plate with a sterile spreading rod, using sterile water as a control. The purified bacteria are subjected to Gram staining and spore staining. The isolates showing rod-shaped cells, spore-forming, and G+ are Bacillus. Number the Bacillus isolated by the above method and observe the colony morphology. Among them, each 1000 mL of LB medium contains 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, 15 g of agar powder, and the pH is 7.0 - 7.2.

[0024] (2) Punch holes in the pathogenic fungus Gibberella zeae that has been cultured for 3 d with a 5 mm puncher. The diameter of the Gibberella zeae fungal cake is 5 mm. Transfer the fungal cake to the center of the potato dextrose agar medium (PDA). After culturing at 28 °C for 24 h, inoculate the tested Bacillus around it at equal distances from the fungus, place it in an incubator, and culture it at 28 °C in a constant temperature in an inverted position. Each confrontation experiment is repeated in parallel 3 times. After 5 d, observe and record the presence and size of the inhibition zone, and select 1 strain with the strongest inhibitory effect on Gibberella zeae. The results show that the inhibitory effect of strain HHQGTS13-1 is the best, the width of the inhibition zone is the widest, and the width of the inhibition zone reaches 9 mm (as Figure 3 ).

[0025] (3) Screen the strain with the most obvious inhibitory effect (the strongest inhibitory activity) from the PDA plate with the most obvious inhibitory effect under aseptic operation for purification culture, and obtain a strain HHQGTS13-1 with strong antagonistic effect on Gibberella zeae.

[0026] 2. Identification of the antagonistic Bacillus sp. HHQGTS13-1 strain against Gibberella zeae. The HHQGTS13-1 strain is identified by colony morphology observation, conventional physiological and biochemical methods, and molecular biology methods, and it is identified that this strain belongs to Bacillus atrophaeus.

[0027] (1) The antagonistic Bacillus sp. HHQGTS13-1 strain is milky white, opaque, with a smooth colony, a raised center, wrinkles, and a complete edge on the LB medium (1000 mL of LB medium contains 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, 15 g of agar powder, and the pH is 7.0 - 7.2) plate, and produces black pigment in the later stage of cultivation.

[0028] (2)Observation through a microscope showed that the cells of antagonistic Bacillus sp. HHQGTS13-1 were approximately 0.5 - 0.8 um × 1.5 - 2.5 um in size, without a capsule, with spores, peritrichous flagella, motile, and Gram-positive staining (as shown in Figure 1 and Figure 2 ). The results of biochemical experiments showed that antagonistic Bacillus sp. HHQGTS13-1 was motile and aerobic, with positive V-P reaction, positive methyl red reaction, positive oxidase reaction, positive catalase reaction, able to reduce nitrate, hydrolyze starch and casein, liquefy gelatin, oxidize glucose, L-arabinose, and mannitol to produce acid, unable to oxidize D-xylose to produce acid, and oxidize glucose without gas production. It could grow in LB culture medium (containing 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, 15 g of agar powder, pH 7.0 - 7.2) with a NaCl concentration of 0.5% - 12%.

[0029] Example 2 The antagonistic effect of the antagonistic Bacillus sp. HHQGTS13-1 strain of this example on Gibberella zeae was tested in the following experiment.

[0030] Confrontation culture test: Place a fresh Gibberella zeae fungal cake with a diameter of 5 mm in the center of a PDA plate. After culturing at 28°C for 24 h, inoculate the test antagonistic Bacillus sp. HHQGTS13-1 strain around the perimeter at an equal distance from the fungus. The control group was not inoculated with the antagonistic Bacillus sp. HHQGTS13-1 strain; place the PDA plate in an incubator at 28°C for confrontation culture. After culturing for 5 d, observe the antagonistic effect of the antagonistic Bacillus sp. HHQGTS13-1 strain.

[0031] The results of the confrontation culture test showed that: the Gibberella zeae in the control group grew normally (as shown in Figure 3 a), and the treatment group, that is, the antagonistic Bacillus sp. HHQGTS13-1 strain screened in the present invention, had a strong antagonistic effect on Gibberella zeae (as shown in Figure 3 b), and the width of the inhibition zone reached 9 mm.

[0032] Example 3 Determination of the ability of antagonistic Bacillus sp. HHQGTS13-1 to produce protease, amylase, and cellulase In this example, the protease-producing medium was casein medium (1000 mL): 5 g of casein, 10 g of peptone, 5 g of NaCl, 5 g of beef extract, 18 g of agar, 1000 mL of water, pH 7.0.

[0033] In this example, the amylase-producing medium was starch medium (1000 mL): 20 g of soluble starch, 10 g of peptone, 5 g of NaCl, 5 g of beef extract, 18 g of agar, 1000 mL of water, pH 7.0.

[0034] In this example, the cellulase-producing medium is carboxymethyl cellulose medium (1000 mL): 5.0 g of CMC-Na, 10 g of peptone, 5 g of NaCl, 5 g of beef extract, 16 g of agar, and 1000 mL of water.

[0035] The antagonistic Bacillus sp. strain HHQGTS13-1 cultured for 1 day was spot-inoculated on cellulose medium, casein medium, and starch medium. 3 strains of bacteria were inoculated in each petri dish, and cultured at 30 °C for 2 - 7 days, with the blank as the control and 3 replicates set; observe whether there is a clear zone around the colonies. Among them, several drops of iodine solution should be added to the starch medium before observation. For the determination of cellulase activity, stain with 1 g / L Congo red for 10 - 15 min, then pour out the staining solution, and soak with 1 mol / L NaCl for 15 min to check for the presence of a clear zone.

[0036] During the culture process, it was found that: the antagonistic Bacillus sp. strain HHQGTS13-1 can grow normally on protease medium, amylase medium, and cellulase medium, and there are obvious hydrolysis zones around the colonies. It can be clearly observed from Figure 4 、 Figure 5 and Figure 6 which indicates that the antagonistic Bacillus sp. strain HHQGTS13-1 secretes protease, amylase, and cellulase during its growth and metabolism, thereby hydrolyzing the proteins, starches, and celluloses in the medium, resulting in the appearance of a clear degradation zone around the colonies. This indicates that a series of enzymes are produced by the antagonistic Bacillus sp. HHQGTS13-1 when inhibiting the growth of Gibberella zeae pathogens and controlling plant diseases.

[0037] Example 4 Determination of the ability of strain HHQGTS13-1 to produce phytohormones The isolated strain HHQGTS13-1 was inoculated into LB liquid medium containing L-tryptophan (100 mg / L), and cultured on a shaker at 30 °C, 180 r•min -1 for 24 h. Take 100 μL of the bacterial suspension on a white ceramic plate, and then drop 100 μL of Salkowski colorimetric solution (1 mL of 0.5 mol / L FeCl3 + 50 mL of 35% HClO4). Use the uninoculated LB liquid culture as the control and add an equal volume of Salkowski colorimetric solution. Place the white ceramic plate in the dark for 30 min, take it out and observe. A red color indicates the ability to produce indole-3-acetic acid, and the darker the color, the higher the indole-3-acetic acid content. The test results show that strain HHQGTS13-1 can produce indole-3-acetic acid.

[0038] Example 5 Field experiment on the control of wheat scab by Bacillus atrophaeus HHQGTS13-1 agent 1. Preparation of biological agent The culture and fermentation broth obtained by culturing the antagonistic Bacillus strain in the following medium are used for treating Gibberella zeae. The liquid medium is LB medium. The solid medium is added with 1.5% agar. After autoclaving, it is reserved for use. When culturing in liquid, the antagonistic Bacillus HHQGTS13-1 strain of the present invention is first activated on an LB solid medium and cultured at 35 °C for 12 h. The activated antagonistic Bacillus HHQGTS13-1 strain is inoculated into a fermentation medium and cultured at 35 °C with shaking at 200 r / min for 12 h to prepare a seed solution. The seed solution is inoculated into a 250 mL Erlenmeyer flask containing 100 mL of fermentation medium at an inoculation amount of 5% and cultured with shaking at 200 r / min at 35 °C for constant temperature fermentation culture. The fermentation broth after 1 day of culture can be used to antagonize Gibberella zeae. The antagonistic Bacillus HHQGTS13-1 bacterial agent is diluted to an active ingredient content of 1.0×10 6 ~1.0×10 8 cfu / mL before use. The components of the fermentation medium are as follows: every 1000 mL of LB liquid medium contains 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, and the pH is 7.0 - 7.2.

[0039] 2. Experimental site: Wheat field in Yulin, Shaanxi 2.1 Experimental design A total of 4 treatments were set: (1) The fermentation broth of strain HHQGTS13-1 X500 (the fermentation broth was diluted 500 times), and the fermentation broth of antagonistic strain HHQGTS13-1 X500 was sprayed on the ears + irrigated at the beginning of the heading stage, beginning of the flowering stage, and full flowering stage of wheat respectively; (2) The bacterial liquid of strain HHQGTS13-1 X1000 (the fermentation broth was diluted 1000 times), and the bacterial liquid of antagonistic strain HHQGTS13-1 X1000 was sprayed on the ears + irrigated at the beginning of the heading stage, beginning of the flowering stage, and full flowering stage of wheat respectively; (3) Positive control, 50% carbendazim water dispersible granule was sprayed on the ears at the beginning of the heading stage, beginning of the flowering stage, and full flowering stage of wheat respectively; (4) Blank control (CK), water was sprayed on the ears at the beginning of the heading stage, beginning of the flowering stage, and full flowering stage of wheat respectively. Each treatment was repeated 3 times, with a total of 12 plots, and each plot was 20 m 2 .

[0040] 3. Investigation method When the disease is stable at the yellow ripening stage of wheat, the incidence of scab is investigated by the 5-point sampling method. 40 ears are investigated at each point, 200 ears are investigated in each plot, the number of diseased ears of wheat is recorded, and the diseased ear rate and control effect are calculated.

[0041] Diseased ear rate (%) = Number of diseased ears / Total number of ears investigated × 100% Disease ear control efficiency(%) = (Control disease ear rate - Treated disease ear rate) / Control disease ear rate × 100% 4. Test results The test results showed that the fermentation broth X500 of strain HHQGTS13-1 had the highest control efficiency against wheat scab, reaching 63.43%, and the fermentation broth X1000 of strain HHQGTS13-1 reached 55.32%; the control efficiency of 50% carbendazim against wheat scab was similar to that of the fermentation broth X1000 of strain HHQGTS13-1, which was 56.70%. The test data are shown in Table 1 below:

[0042] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and scope of the present invention shall be included in the protection scope of the present invention.

Claims

1. A strain of Bacillus atrophaeus with preventive effect against Fusarium graminearum causing wheat head blight is preserved in the China General Microbiological Culture Collection Center, with the preservation number: CGMCC No. 29799, and the taxonomic name: Bacillus atrophaeus strain HHQGTS13-1.

2. The optimal growth temperature of the Bacillus atrophaeus HHQGTS13-1 according to claim 1 is 25 - 35 °C; it can survive in an environment with a pH of 4.5 - 10, and the optimal pH value is 7; it can grow in a 12% NaCl medium, showing salt tolerance; it can oxidize L-arabinose and mannitol to produce acid; it can oxidize glucose but does not produce gas, and the egg yolk hydrolysis is positive; but it cannot oxidize D-xylose to produce acid.

3. The Bacillus atrophaeus HHQGTS13-1 according to claim 1 has a wide range of adaptation to natural environmental conditions such as temperature and pH, and has the ability to antagonize Fusarium graminearum within the range of 10 - 45 °C and pH = 4 - 10.

4. A method for preparing and using the fermentation broth of Bacillus atrophaeus HHQGTS13-1 for inhibiting Gibberella zeae, characterized in that, It includes the following steps: (1) The liquid medium is LB medium, and 1.5% agar is added to the solid medium, which is reserved after autoclaving.

5. (2) When culturing in liquid, the antagonistic Bacillus spore strain HHQGTS13-1 of the present invention is first activated on an LB solid medium and cultured at 35 °C for 12 h.

6. (3) The activated antagonistic Bacillus spore strain HHQGTS13-1 is inoculated into a fermentation medium and cultured at 35 °C with shaking at 200 r / min for 12 h to prepare a seed liquid.

7. (4) The seed liquid is inoculated into a 250 mL Erlenmeyer flask containing 100 mL of fermentation medium at an inoculation amount of 5%, and cultured with shaking at 200 r / min at 35 °C for constant-temperature fermentation culture. The fermentation broth after 1 day of culture can be used to antagonize Fusarium graminearum.

8. The antagonistic Bacillus strain HHQGTS13-1 agent is diluted to an active ingredient content of 1.0×10 6 ~1.0×10 8 cfu / mL before use.

9. The fermentation broth of the Bacillus atrophaeus HHQGTS13-1 strain according to claim 5 contains active ingredients with high inhibitory effect on Fusarium graminearum, indicating that the antagonistic Bacillus spore strain HHQGTS13-1 produces metabolites with inhibitory and teratogenic effects on Fusarium graminearum during the fermentation culture process.

10. A biological agent or bacteriostatic agent for inhibiting Gibberella zeae, characterized in that, It includes the Bacillus atrophaeus strain HHQGTS13-1 described in claims 1 - 5.

11. The biological agent or bacteriostatic agent according to claim 6 has a very significant effect in preventing and controlling Fusarium graminearum, can significantly inhibit the growth of Fusarium graminearum hyphae and make the hyphae deformed; it can inhibit the spore germination of Fusarium graminearum.

12. The biological agent or bacteriostatic agent according to claim 6 has an obvious inhibitory effect on the spore germination of Fusarium graminearum, and the spore germination inhibition rate is 97.10%. The preventive and control effect of the fermentation broth of the antagonistic Bacillus spore strain HHQGTS13-1 after inoculating Fusarium graminearum pathogen on wheat is 63.43%.

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