Application of biochar-loaded phytophthora nicotianae toxin material in prevention and control of tobacco black shank

Abstract

The invention discloses application of a biochar-loaded phytophthora nicotianae toxin material to prevention and control of tobacco black shank, and belongs to the technical field of tobacco disease prevention and control, and the material is composed of biochar and phytophthora nicotianae toxin loaded in the biochar. The biochar and phytophthora nicotianae liquid are subjected to mixed culture, and after culture is completed, sterilization, centrifugation and drying are performed to obtain the biochar-loaded phytophthora nicotianae toxin material. The biochar prepared by the method inhibits the growth of phytophthora nicotianae under high concentration, the biochar-loaded phytophthora nicotianae toxin can enhance the antioxidant enzyme activity of tobacco seedlings and promote the growth of the tobacco seedlings, and when the biochar-loaded phytophthora nicotianae toxin is applied to soil, the tobacco growth can be promoted, and the incidence rate of tobacco black shank can be reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of tobacco disease prevention and control, and in particular relates to the application of a biochar-carrying tobacco phytotoxin material in the prevention and control of tobacco black shank disease. Background Art

[0002] Tobacco is an important economic crop in my country, with a wide planting area. However, tobacco blight (Phytophthoraparasitica var.nicotianae) is a fungal disease that infects tobacco plants under natural conditions and causes tobacco black shank disease, causing tobacco leaves to wilt and turn yellow, plants to dwarf, and roots and stems to necrotize, seriously affecting tobacco growth and economic benefits. In recent years, the gradual deterioration of the crop growth environment and improper application of farmland fertilizers have caused tobacco diseases to become increasingly serious and show a trend of continuous intensification.

[0003] In the prevention and control of tobacco diseases, the selection of disease-resistant tobacco plant varieties and the spraying of chemical pesticides have reduced the infection of pathogens to a certain extent and improved the economic benefits of tobacco. However, the selection of new disease-resistant varieties takes a long time and is difficult to breed; the long-term use of chemical pesticides will make tobacco phytophthora resistant and may damage the planting soil environment.

[0004] In recent years, biological control has become an emerging green control measure, which achieves the effect of killing and inhibiting bacteria by applying disease-resistant strains to inhibit pathogens. In addition, with the deepening of research on plant-induced disease resistance, measures to induce and stimulate plant disease resistance by using pathogen toxins have gradually received attention. Biochar is a value-added product obtained by pyrolysis of agricultural waste, with rich pore structure and specific surface area. If new plant immune inducers can be prepared by loading tobacco phytotoxins on biochar, it will provide new ideas and means for the prevention and control of tobacco black shank disease. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a novel plant immune inducer of tobacco phytotoxin carried by biochar, thereby reducing the incidence of tobacco black shank disease.

[0006] The technical solution of the present invention is: a biochar-loaded tobacco phytophthora toxin material, which consists of biochar and tobacco phytophthora toxin loaded in the biochar.

[0007] The method for preparing the above-mentioned biochar-loaded tobacco Phytophthora toxin material comprises mixing and culturing the biochar with tobacco Phytophthora liquid, sterilizing after the cultivation, centrifuging and drying to obtain the biochar-loaded tobacco Phytophthora toxin material.

[0008] Furthermore, the method for mixed culturing of the biochar and tobacco Phytophthora liquid is: first culturing tobacco Phytophthora in a liquid culture medium for 5 days, and then adding the biochar into the liquid culture medium and continuing to culture for 5 days.

[0009] Furthermore, the culture conditions are shaking at 180 r / min and a temperature of 28°C.

[0010] Furthermore, the amount of biochar added is 1 g of biochar per 25 ml of liquid culture medium.

[0011] Furthermore, the preparation method of the biochar is:

[0012] (1) washing, drying, grinding and sieving rice straw;

[0013] (2) subjecting the rice straw treated in step (1) to anaerobic pyrolysis at 700° C. to obtain straw charcoal;

[0014] (3) washing the straw charcoal treated in step (2) with distilled water until the washing liquid is neutral, drying, grinding and sieving, and sterilizing with high-pressure steam to obtain biochar.

[0015] Furthermore, in step (1), the sieving is through a 20-mesh sieve.

[0016] Furthermore, in step (2), the procedure of anaerobic pyrolysis is: heating to 700°C at a rate of 10°C / min and maintaining for 2h.

[0017] Furthermore, in step (3), the sieving refers to sieving through a 60-mesh sieve.

[0018] The application of the plant immunity inducing material described above in promoting the growth of tobacco plants or / and reducing the incidence of black shank disease of tobacco plants.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The rice straw biochar-loaded tobacco phytophthora toxin material prepared by the method of the present invention consists of rice straw and tobacco phytophthora toxin. The biochar is prepared from rice straw under anaerobic conditions. High-concentration biochar can inhibit the growth of tobacco phytophthora, and the biochar loaded with tobacco phytophthora toxin can induce and improve tobacco stress resistance and reduce the incidence of tobacco black shank disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a graph of the diameter of tobacco phytophthora colonies in Example 1.

[0022] Figure 2 This is a graph showing changes in the activity of defense enzymes in tobacco seedlings in Example 2. DETAILED DESCRIPTION

[0023] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from commercial channels unless otherwise specified.

[0024] Example 1 Effect of biochar on the growth of tobacco phytophthora

[0025] 1. Specific steps

[0026] 1.1 Biochar preparation

[0027] The rice straw to be prepared is obtained through washing, drying, grinding and passing through a 20-mesh sieve, and is placed in a muffle furnace and filled with nitrogen. The temperature is increased to 700°C at a heating rate of 10°C / min, and the pyrolysis temperature is maintained for 2 hours to obtain 700°C biochar. The biochar is washed with deionized water until the pH of the washing liquid is neutral, dried and ground and passed through a 60-mesh sieve, and then sterilized with high-pressure steam at 121°C and 30 minutes to obtain sterile biochar.

[0028] 1.2 Plate culture of tobacco phytophthora

[0029] Sterile biochar was added to potato agar solid medium at a ratio of 1.25 g / L, 2.5 g / L, 5 g / L and 10 g / L, sterilized and poured into sterile culture dishes. After cooling and solidification, a 0.5 mm tobacco phytophthora cake was placed in the center of the solid medium, and a control group without biochar addition was set up. Colony diameter was measured and toxicity effects were calculated on the 2nd, 3rd and 4th days.

[0030] 2. Experimental results

[0031] 2.1 Colony diameter of Phytophthora nicotianae

[0032] like Figure 1 As shown in the figure, the colony diameter increases with the extension of the culture time, and the colony diameter of the control group can reach 27.72cm on the 4th day. With the increase of biochar concentration, the colony diameter shows a decreasing trend. 10g / L biochar has the best inhibitory effect on the growth of tobacco phytophthora, and the colony diameter is 6.24cm, 15.92cm and 20.57cm on the 2nd, 3rd and 4th days, respectively, which is 14.99%, 22.57% and 25.79% lower than that of the control group, respectively. The results show that biochar inhibits the growth of tobacco phytophthora, and high concentration of biochar has better inhibitory effect.

[0033] 2.2 Analysis of the effect of biochar on the toxicity of Phytophthora nicotianae

[0034] As shown in Table 1, the response of tobacco phytophthora to biochar varies during different growth stages. 2The results showed that the toxicity fitting curve was good. The half effective concentration EC 50 They are 1.9533g / L, 2.1951g / L and 2.1146g / L respectively.

[0035] Table 1 Analysis of the toxicity of biochar to Phytophthora nicotianae

[0036] Cultivation time Toxicity equation <![CDATA[R 2 ]]> <![CDATA[EC 50 (g / L)]]> 2 days y = 0.0712x – 0.0021 0.9464 1.9533 3 days y=0.0414x+0.1282 0.9867 2.1951 4 days y=0.0402x+0.1669 0.9752 2.1146

[0037] Example 2 Study on the effect of biochar-loaded tobacco phytotoxin on tobacco seedlings

[0038] 1. Specific steps

[0039] 1.1 Preparation of biochar-loaded tobacco phytotoxin

[0040] The preparation of biochar is as shown in 1.1 Preparation of biochar in Example 1. A 0.5 mm diameter bacterial cake was picked from a potato glucose solid culture medium and inoculated into a potato glucose liquid culture medium, cultured at 180 r / min and 28°C for 5 days, the biochar was mixed with the cultured bacterial solution at a ratio of 1:25 (W / V), and cultured at 180 r / min and 28°C for 5 days, the charcoal-bacteria mixture was sterilized by high-pressure steam at 121°C for 30 min, and the sterilized mixture was centrifuged at 5000 r / min for 15 min to obtain biochar-loaded tobacco phytotoxin and dried, and at the same time, a bacterial solution without adding biochar was prepared to obtain a supernatant containing tobacco phytotoxin.

[0041] 1.2 Tobacco seedling cultivation

[0042] The tobacco seedling variety was Yunyan 87, and the seedlings used had 3 leaves. The supernatant containing tobacco phytophthora toxin and the biochar loaded with tobacco phytophthora toxin were diluted 10 times with distilled water, and the tobacco seedlings were placed in them and cultured in a light culture room for 15 days; the tobacco phytophthora toxin supernatant treatment group was named T1, the biochar loaded with tobacco phytophthora toxin treatment group was T2, and the distilled water culture was used as the control group (CK). The temperature and time conditions required for culture were daytime (25℃, 12h) and nighttime (20℃, 12h), and the light intensity was 5000lux.

[0043] 1.3 Seedling growth assay

[0044] At the 6th hour of cultivation, the leaves of tobacco seedlings were cut and the peroxidase activity was determined by guaiacol colorimetry, the polyphenol oxidase activity was determined by catechol colorimetry, and the phenylalanine ammonia lyase activity was determined by phenylalanine colorimetry. The agronomic traits of tobacco seedlings were determined on the 5th, 10th and 15th days of cultivation.

[0045] 2. Experimental results

[0046] 2.1 Enzyme activity of tobacco seedlings

[0047] like Figure 2 As shown in the results, the activities of peroxidase, polyphenol oxidase and phenylalanine ammonia lyase were significantly different among the groups. Compared with the control group, the treatment with tobacco phytophthora toxin supernatant significantly increased the activities of the three enzymes, with the highest values ​​of 39.73U / g / min, 19.67U / g / min and 26.59U / g / min, respectively; followed by the biochar-loaded tobacco phytophthora toxin treatment group, with the three enzyme activities of 26.04U / g / min, 11.99U / g / min and 15.58U / g / min, respectively. The results showed that the application of tobacco phytophthora toxin could induce tobacco seedlings to increase the activity of defense enzymes.

[0048] 2.2 Growth status of tobacco seedlings

[0049] As shown in Table 2, the growth of tobacco seedlings changed with the extension of the culture time. There were differences in plant height, stem diameter and leaf length between the control group, the tobacco phytotoxin supernatant treatment group and the biochar-loaded tobacco phytotoxin treatment group. Compared with the control group, the biochar-loaded tobacco phytotoxin treatment promoted the growth of tobacco seedlings, and the plant height, stem diameter and leaf length were 12.36 cm, 0.25 cm and 5.57 cm on the 15th day, respectively; while the tobacco phytotoxin supernatant treatment increased the activity of defense enzymes, but inhibited the growth of tobacco seedlings, and the plant height, stem diameter and leaf length were reduced by 1.68 cm, 0.02 cm and 1.03 cm on the 15th day compared with the control group.

[0050] Table 2 Agronomic conditions of tobacco seedling growth

[0051]

[0052]

[0053] Example 3 Study on the effect of biochar-loaded tobacco phytotoxin on tobacco growth and disease resistance

[0054] 1. Specific steps

[0055] 1.1 Experimental setup

[0056] Yunyan 87 was used as the flue-cured tobacco variety for testing, and a control group (conventional fertilization), a conventional fertilization + application of 20 g / plant biochar (prepared in Example 1) group (T1), and a conventional fertilization + application of 20 g / plant biochar loaded with tobacco phytotoxin (prepared in Example 2) group (T2) were set. During the growth period, fertilization was carried out according to local field management measures, and the fertilizers included tobacco-specific compound fertilizer (N: P2O5: K2O = 12: 12: 25), film-covered box fertilizer (N: P2O5: K2O = 5: 5: 40), and sheep manure organic fertilizer (80% sheep manure).

[0057] 1.2 Sample collection and determination

[0058] Typical flue-cured tobacco plants were selected during the clustering, vigorous growth and maturity stages of flue-cured tobacco. The agronomic traits of each stage were measured according to the flue-cured tobacco agronomic traits survey and measurement method (YC / T142-2010), and the occurrence of diseases in each stage was analyzed according to the tobacco disease and insect pest classification and survey method (GB / T 23222-2008), and the disease index was calculated.

[0059] 2. Experimental results

[0060] 2.1 Analysis of tobacco growth and diseases

[0061] As shown in Table 3, both the application of biochar and the application of biochar loaded with tobacco phytotoxin promoted the growth of flue-cured tobacco, with the maximum leaf area and leaf number reaching 1410.63 cm at maturity. 2 and 1692.22cm 2 and 19 and 21.33 pieces. In addition, the application of both can effectively inhibit the infection of tobacco by Phytophthora nicotianae, reducing the incidence rate by 18.30%-29.56% and 43.65%-47.18%, respectively. The results showed that the control effect of tobacco black shank disease on biochar-carried tobacco phytophthora toxins was better, reaching 55.82%, 44.20% and 43.48% in the three periods, respectively. Biochar-carried tobacco phytophthora toxins has the potential to improve the application of tobacco resistance to black shank disease.

[0062] Table 3 Effects of biochar-loaded tobacco phytotoxins on tobacco growth and disease resistance

[0063]

[0064]

Claims

1. A biochar-loaded tobacco phytophthora toxin material, characterized in that: The material consists of biochar and tobacco phytotoxin loaded in the biochar.

2. The method for preparing the biochar-loaded tobacco phytoncides toxin material according to claim 1, characterized in that: The biochar is mixed with tobacco phytophthora liquid and cultured. After the culture is completed, the mixture is sterilized, centrifuged and dried to obtain the biochar-loaded tobacco phytophthora toxin material.

3. The preparation method according to claim 2, characterized in that: The method for mixed culturing of the biochar and tobacco phytophthora liquid is as follows: firstly culturing tobacco phytophthora in a liquid culture medium for 5 days, and then adding the biochar into the liquid culture medium and continuing culturing for 5 days.

4. The preparation method according to claim 3, characterized in that: The culture conditions were shaking at 180 r / min and a temperature of 28°C.

5. The preparation method according to claim 3, characterized in that: The amount of biochar added was 1 g of biochar per 25 ml of liquid culture medium.

6. The preparation method according to any one of claims 2 to 5, characterized in that: The preparation method of the biochar is: (1) washing, drying, grinding and sieving rice straw; (2) subjecting the rice straw treated in step (1) to anaerobic pyrolysis at 700° C. to obtain straw charcoal; (3) washing the straw charcoal treated in step (2) with distilled water until the washing liquid is neutral, drying, grinding and sieving, and sterilizing with high-pressure steam to obtain biochar.

7. The preparation method according to claim 6, characterized in that: In step (1), the sieving is through a 20-mesh sieve.

8. The preparation method according to claim 6, characterized in that: In step (2), the procedure of anaerobic pyrolysis is: heating to 700°C at a rate of 10°C / min and maintaining for 2h.

9. The preparation method according to claim 6, characterized in that: In step (3), the sieving refers to sieving through a 60-mesh sieve.

10. Use of the biochar-loaded tobacco phytophthora toxin material according to claim 1 or the biochar-loaded tobacco phytophthora toxin material obtained by the preparation method according to any one of claims 2 to 9 in promoting the growth of tobacco plants or / and reducing the incidence of black shank disease of tobacco plants.

Images