A method for inoculating sugarcane red rot

Abstract

The application discloses a kind of inoculation methods of sugarcane red rot, it is related to the field of agricultural technology, it solves the existing inoculation method and the problem that the operation is complicated, the disease time is long and the disease resistance is not obvious.The inoculation steps are as follows: (1) preparation of cotton swab bacterial species: in PDA culture medium, add sugarcane residue and xanthan gum, cotton swab is vertically inserted into culture medium and sterilized together, then inoculate sugarcane red rot pathogen, constant temperature dark culture, obtain cotton swab bacterial species; (2) drilling: in the ground of the sugarcane plant without red rot, the middle of the second or third internode is drilled; (3) inoculation: a single cotton swab bacterial species is inserted into the hole, and the inoculation is completed.The inoculation method is simple in operation, can promote the rapid growth of pathogenic bacteria, maintain strong pathogenic activity, fully show the disease resistance difference of different sugarcane varieties, and can meet the demand of large-scale sugarcane disease resistance identification.

Description

Technical Field

[0001] This invention relates to the field of agricultural technology, and in particular to an inoculation method for sugarcane red rot. Background Technology

[0002] Sugarcane red rot is caused by *Discocephalus falcatus* (… Colletotrichum falcatum Red rot is a common sugarcane disease caused by fungal infection. It is a global fungal disease that mainly affects the stems and leaves of sugarcane, causing the internal tissues of the stem to turn red and rot, reducing sugarcane yield and sugar content, and seriously affecting the economic benefits of sugarcane cultivation. Research on the identification of resistance to red rot and related pathogenic mechanisms requires simple and accurate artificial inoculation methods to obtain materials with different resistance levels.

[0003] Currently, the commonly used inoculation methods for sugarcane red rot are field drilling inoculation and node swab inoculation. The field drilling method involves drilling a hole in the middle of an internode on the sugarcane plant above ground level, dripping the inoculation solution into the hole using a pipette, sealing the hole with a thick layer of moist, sterile absorbent cotton, and then wrapping it with sterile plastic wrap. Throughout the inoculation period, water is sprayed 1-2 times daily to maintain a relative humidity of 75-90%. The node swab inoculation method involves selecting the 6th-8th node from the top of the stem for inoculation. The leaf sheath is removed, and absorbent cotton is soaked in the inoculation solution and wrapped around the node. The cotton is then sealed with plastic wrap. High humidity must be maintained after inoculation to promote disease development. Both methods have the following drawbacks: both require sealing and wrapping after inoculation, necessitating additional continuous moisturization, making the process cumbersome, inefficient, labor-intensive, and time-consuming. To address this issue, the applicant attempted to improve the field drilling inoculation method. This involved placing cotton swabs in a culture medium and inoculating them with the pathogen. Once the cotton was fully covered with mycelium, the swabs were directly inserted into the drill hole, eliminating the need for sealing or wrapping, resulting in high inoculation efficiency. However, in practice, the applicant found that once the cotton swabs were fully covered with mycelium, their moisture absorption and retention capacity weakened. They could not effectively absorb and retain sap seeping from sugarcane wounds, nor could they retain the sugarcane's original moisture. The inoculation site dried out easily, pathogen colonization was slow after inoculation, disease development was unstable, and differences in disease resistance were not clearly distinguishable, making it difficult to meet the requirements for standardized disease resistance identification.

[0004] Therefore, developing a simple, stable, and effective inoculation method for sugarcane red rot that can fully demonstrate differences in disease resistance has significant practical value. Summary of the Invention

[0005] To address the above shortcomings, this invention provides an inoculation method for sugarcane red rot, which solves the problems of existing inoculation methods, such as complicated operation, slow pathogen colonization after inoculation, unstable disease development, and unclear differentiation of disease resistance. The specific technical solution is as follows: A method for inoculating sugarcane with red rot disease includes the following steps: (1) Preparation of bacterial strains for cotton swabs: A. Preparation of PDA culture medium; B. Juice fresh sugarcane stalks that are 6-8 months old and free from red rot, collect the juice, and crush the juiced stalks into sugarcane residue. Mix the sugarcane residue with the juice to obtain sugarcane residue material. C. Add sugarcane bagasse and xanthan gum to the PDA culture medium, mix and stir evenly, then insert one end of a moistened cotton swab into the culture medium, seal it, sterilize it at high temperature, and cool it to obtain the cotton swab culture medium; D. Inoculate the sugarcane red rot pathogen into the cotton swab culture medium and incubate in the dark at a constant temperature to obtain cotton swab inoculum; (2) Drilling: Drill holes in the middle of the second or third internode on the ground of sugarcane plants free of red rot; (3) Inoculation: Insert one end of a single cotton swab containing the bacterial culture into the hole to complete the inoculation.

[0006] Further, in step A, the PDA culture medium is prepared by peeling and cutting 200-250g of potatoes into pieces, adding them to 1000mL of water and boiling for 20-30 minutes, filtering, adding 20-25g of glucose and 20-25g of agar to the filtrate, adding water to make up to 1000mL, and then heating until the agar melts.

[0007] Furthermore, in step B, before crushing the juiced stems, the following pretreatment is performed: first, soak the juiced stems in hot water at 75-85℃ for 2-3 hours, then remove them and soak them in a NaOH solution with a mass concentration of 0.1-0.5% for 3-5 hours, and rinse until neutral.

[0008] Furthermore, in step C, the amount of bagasse added is 10-15% of the weight of the culture medium; the amount of xanthan gum added is 0.5-1.5% of the weight of the culture medium.

[0009] Furthermore, in step C, high-temperature sterilization is sterilization at 121°C for 25-30 minutes.

[0010] Furthermore, in step D, the dark culture temperature is 28-30℃, and the dark culture time is 3-4 days.

[0011] Furthermore, in step (2), the diameter of the hole obtained by drilling is 7-10 mm and the depth is 10-12 mm.

[0012] Furthermore, in step (3), 10 sugarcane stalks are inoculated for every 20 sugarcane plants in July-August, and the inoculated sugarcane plants are 6-8 months old.

[0013] Compared with the prior art, the beneficial effects of the present invention are: 1. This invention, by adding sugarcane bagasse and xanthan gum to the PDA culture medium, provides the pathogen with nutritional conditions more closely resembling its natural infection environment, promoting rapid growth and reproduction. It also effectively enhances the water retention capacity of the swabs after inoculation, ensuring a long-term moist environment suitable for pathogen colonization at the inoculation site, eliminating the need for subsequent moisturizing procedures. Furthermore, the longer swabs extend beyond the pores, providing a sealing effect and eliminating the need for additional sealing or coating operations, simplifying the inoculation process, saving manpower and time, and significantly improving inoculation efficiency. Experiments have shown that this invention promotes rapid pathogen growth, maintains strong pathogenicity, fully demonstrates the differences in disease resistance among different sugarcane varieties, and can meet the needs of large-scale sugarcane disease resistance identification.

[0014] 2. The sugarcane bagasse material in the PDA culture medium of this invention has enhanced hygroscopicity and improved water retention after pretreatment, which can maintain a high-humidity environment in the well for 5-7 days. At the same time, the sugarcane bagasse material can also slowly release trace amounts of sugarcane-derived substances, inducing faster mycelial growth of pathogens, ensuring that the cotton swabs are loaded with a large number of mycelia, and improving the infection ability. The xanthan gum in the PDA culture medium not only promotes mycelial growth, but also has the effects of sticking mycelia and moisturizing, so that a large number of mycelia are adhered to the cotton swabs, and they are not easy to lose water and dry out when water is added, which is more conducive to the rapid infection of pathogens.

[0015] 3. This invention sets a short dark incubation time of only 3-4 days, which can prevent the cotton swabs from being completely blocked by mycelium, thus affecting their moisture absorption and retention effects. The addition of sugarcane bagasse and xanthan gum, combined with short dark incubation, can promote the large-scale generation of mycelium and ensure sufficient inoculum. Attached Figure Description

[0016] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 The bacterial strain on the cotton swabs prepared in Example 3; Figure 2 This is an inoculation diagram for Example 3; Figure 3 This is a diagram showing the infection status of pathogens after inoculation in Example 3. Detailed Implementation

[0018] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Unless otherwise defined, all technical terms used below have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing specific embodiments only and are not intended to limit the scope of protection of the present invention. Unless otherwise specifically stated, all raw materials, reagents, instruments, and equipment used in the present invention are commercially available or can be prepared by existing methods.

[0019] Example 1 A method for inoculating sugarcane with red rot disease includes the following steps: (1) Preparation of bacterial strains for cotton swabs: A. Preparation of PDA culture medium: Peel and cut 200g of potatoes into pieces, add them to 1000mL of water and boil for 20min. Filter the mixture, add 20g of glucose and 20g of agar to the filtrate, add water to make up to 1000mL, and then heat until the agar melts. B. Juice fresh sugarcane stalks free of red rot at 6 months of age, collect the juice, and soak the juiced stalks in hot water at 75°C for 2 hours. Remove them and soak them in a 0.1% NaOH solution for 3 hours. Rinse until neutral to obtain pretreated stalks. Crush the pretreated stalks into sugarcane powder residue and mix the sugarcane powder residue with the juice to obtain sugarcane bagasse. C. Add 10% by weight of sugarcane bagasse and 0.5% xanthan gum to the PDA culture medium, mix and stir evenly, then insert one end of a moistened cotton swab into the culture medium, seal it, sterilize it at 121°C for 25 minutes, cool it, and obtain the cotton swab culture medium. D. Inoculate the sugarcane red rot pathogen into the cotton swab culture medium and incubate it in the dark at 28°C for 3 days to obtain the cotton swab strain; (2) Drilling: Drill holes in the middle of the second or third internode on the ground of sugarcane plants without red rot, to obtain holes with a diameter of 7-10 mm and a depth of 10-12 mm; (3) Inoculation: Insert one end of a single cotton swab containing the bacterial culture into the hole. Inoculate 10 sugarcane stalks for every 20 sugarcane plants. Inoculate in July. The sugarcane plants should be 6 months old.

[0020] Example 2 A method for inoculating sugarcane with red rot disease includes the following steps: (1) Preparation of bacterial strains for cotton swabs: A. Preparation of PDA culture medium: Peel and cut 250g of potatoes into pieces, add them to 1000mL of water and boil for 30min. Filter the mixture, add 25g of glucose and 25g of agar to the filtrate, add water to make up to 1000mL, and then heat until the agar melts. B. Juice fresh sugarcane stalks free of red rot at 8 months of age, collect the juice, and soak the juiced stalks in hot water at 85°C for 3 hours. Remove them and soak them in a 0.5% NaOH solution for 5 hours. Rinse until neutral to obtain pretreated stalks. Crush the pretreated stalks into sugarcane powder residue and mix the sugarcane powder residue with the juice to obtain sugarcane bagasse. C. Add 15% by weight of sugarcane bagasse and 1.5% xanthan gum to the PDA culture medium, mix and stir evenly, then insert one end of a moistened cotton swab into the culture medium, seal it, sterilize it at 121°C for 30 minutes, cool it, and obtain the cotton swab culture medium. D. Inoculate the sugarcane red rot pathogen into the cotton swab culture medium and incubate it in the dark at 30°C for 4 days to obtain the cotton swab strain; (2) Drilling: Drill holes in the middle of the second or third internode on the ground of sugarcane plants without red rot, to obtain holes with a diameter of 7-10 mm and a depth of 10-12 mm; (3) Inoculation: Insert one end of a single cotton swab containing the bacterial culture into the hole. Inoculate 10 sugarcane stalks for every 20 sugarcane plants. Inoculate in August, and the sugarcane plants should be 8 months old.

[0021] Example 3 A method for inoculating sugarcane with red rot disease includes the following steps: (1) Preparation of bacterial strains for cotton swabs: A. Preparation of PDA culture medium: Peel and cut 200g of potatoes into pieces, add them to 1000mL of water and boil for 25min. Filter the mixture, add 20g of glucose and 20g of agar to the filtrate, add water to make up to 1000mL, and then heat until the agar melts. B. Juice fresh sugarcane stalks free of red rot at 7 months of age, collect the juice, and soak the juiced stalks in hot water at 80°C for 2.5 hours. Remove them and soak them in a 0.3% NaOH solution for 4 hours. Rinse until neutral to obtain pretreated stalks. Crush the pretreated stalks into sugarcane powder residue and mix the sugarcane powder residue with the juice to obtain sugarcane bagasse. C. Add 12% by weight of sugarcane bagasse and 0.8% xanthan gum to the PDA culture medium, mix and stir evenly, then insert one end of a moistened cotton swab into the culture medium, seal it, sterilize it at 121°C for 30 minutes, cool it, and obtain the cotton swab culture medium. D. Inoculate the sugarcane red rot pathogen into the cotton swab culture medium and incubate it in the dark at 28°C for 3 days to obtain the cotton swab strain; (2) Drilling: Drill holes in the middle of the second or third internode on the ground of sugarcane plants without red rot, to obtain holes with a diameter of 7-10 mm and a depth of 10-12 mm; (3) Inoculation: Insert one end of a single cotton swab containing the bacterial culture into the hole. Inoculate 10 sugarcane stalks for every 20 sugarcane plants. Inoculate in July, and the sugarcane plants should be 7 months old.

[0022] Comparative Example 1: Sugarcane bagasse and xanthan gum were not added to the PDA medium, i.e. (1) Preparation of swab inoculum: A. Preparation of PDA culture medium: Peel and cut 200g of potatoes into pieces, add them to 1000mL of water and boil for 25min. Filter the mixture, add 20g of glucose and 20g of agar to the filtrate, add water to make up to 1000mL, and then heat until the agar melts. B. Insert one end of a moistened cotton swab into the culture medium, seal it, sterilize it at 121°C for 30 minutes, cool it, and obtain the cotton swab culture medium; C. Inoculate the sugarcane red rot pathogen into the cotton swab culture medium and incubate it at a constant temperature of 28°C in the dark for 3 days to obtain the cotton swab strain.

[0023] The other steps are the same as in Example 3.

[0024] Comparative Example 2: Sugarcane bagasse was not added to the PDA medium, and the other steps were the same as in Example 3.

[0025] Comparative Example 3: No original gum was added to the PDA culture medium, and the other steps were the same as in Example 3.

[0026] Comparative Example 4: No pretreatment is performed on the stalks after juicing. That is, step B is as follows: Juice is extracted from fresh sugarcane stalks that are 7 months old and free from red rot, the juice is collected, and the juiced stalks are crushed into sugarcane powder residue. The sugarcane powder residue is mixed with the juice to obtain sugarcane residue material. Other steps are the same as in Example 3.

[0027] To illustrate the effectiveness of the present invention, the applicant conducted experiments at the Nanning Experimental Base of the Guangxi Academy of Agricultural Sciences. The experiment was divided into 8 groups: Groups 1 to 3 were the inoculation methods of Examples 1 to 3, respectively; Groups 4 to 7 were the inoculation methods of Comparative Examples 1 to 4, respectively; and Group 8 was the control group, which was inoculated using the traditional field drilling method (the method mentioned in the background art).

[0028] Each group of experiments consisted of 4 rows, 8m long, with a row spacing of 1.2m and 20 stalks per row. Each group had 3 replicates, with protective rows between groups. Each replicate was inoculated with 5 sugarcane varieties (Guitang 11 (GT11), Guitang 31 (GT31), Guitang 43 (GT43), Guitang 46 (GT46), and Guitang 29 (GT29)). Sixty days after inoculation, a disease survey was conducted by longitudinally dissecting the sugarcane stalks to observe the occurrence and development of lesions and calculating the mean score for each group. Evaluation scores were based on Table 1, with the mean score calculated as total score / number of stalks. Resistance levels were determined according to Table 2. The scoring results and resistance levels for each group are shown in Table 3-10.

[0029] Table 1 Indicator Evaluation Scores Table 2 Classification of Resistance Grades Table 3. Score results and resistance evaluation of inoculation method in Example 1 Table 4. Inoculation method scoring results and resistance evaluation in Example 2 Table 5. Score results and resistance evaluation of inoculation method in Example 3 Table 6. Scoring results and resistance evaluation of inoculation method in Comparative Example 1 Table 7. Comparative Example 2: Inoculation Method Scoring Results and Resistance Evaluation Table 8. Scoring results and resistance evaluation of inoculation method in Comparative Example 3 Table 9. Scoring results and resistance evaluation of inoculation method in Comparative Example 4 Table 10. Score results and resistance evaluation of the control group's vaccination method. The data results show that after using the inoculation method of the present invention (Examples 1 to 3), the evaluation scores of different varieties differed significantly, which can better distinguish disease resistance. In contrast, the control groups (Comparative Examples 1 to 4) had similar evaluation scores in the short term (60 days after inoculation), and the differences in disease resistance were not obvious. The inoculation method of the present invention (Examples 1 to 3) better distinguished the differences in disease resistance than the control group. This indicates that by adding pretreated sugarcane bagasse and xanthan gum to the PDA medium, the present invention can promote the rapid growth of sugarcane red rot pathogen, enabling the pathogen to maintain strong pathogenicity in the short term and quickly infect after inoculation. This fully demonstrates the differences in disease resistance among different sugarcane varieties, effectively solving the problems of slow disease development and unclear disease resistance differentiation in traditional inoculation methods. The method is simple to operate, has good inoculation consistency, and is suitable for large-scale inoculation identification of sugarcane resistance to red rot.

[0030] The foregoing description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A method for inoculating sugarcane with red rot, characterized in that, Includes the following steps: (1) Preparation of bacterial strains for cotton swabs: A. Preparation of PDA culture medium; B. Juice fresh sugarcane stalks that are 6-8 months old and free from red rot, collect the juice, and crush the juiced stalks into sugarcane residue. Mix the sugarcane residue with the juice to obtain sugarcane residue material. C. Add sugarcane bagasse and xanthan gum to the PDA culture medium, mix and stir evenly, then insert one end of a moistened cotton swab into the culture medium, seal it, sterilize it at high temperature, and cool it to obtain the cotton swab culture medium; D. Inoculate the sugarcane red rot pathogen into the cotton swab culture medium and incubate in the dark at a constant temperature to obtain cotton swab inoculum; (2) Drilling: Drill holes in the middle of the second or third internode on the ground of sugarcane plants free of red rot; (3) Inoculation: Insert one end of a single cotton swab containing the bacterial culture into the hole to complete the inoculation.

2. The inoculation method for sugarcane red rot according to claim 1, characterized in that, In step A, the PDA culture medium is prepared by peeling and cutting 200-250g of potatoes into pieces, adding them to 1000mL of water and boiling for 20-30 minutes, filtering, adding 20-25g of glucose and 20-25g of agar to the filtrate, adding water to make up to 1000mL, and then heating until the agar melts.

3. The inoculation method for sugarcane red rot according to claim 1, characterized in that, In step B, before crushing the juiced stems, the following pretreatment is performed: first, soak the juiced stems in hot water at 75-85℃ for 2-3 hours, then remove them and soak them in a NaOH solution with a mass concentration of 0.1-0.5% for 3-5 hours, and rinse until neutral.

4. The inoculation method for sugarcane red rot according to claim 1, characterized in that, In step C, the amount of bagasse added is 10-15% of the weight of the culture medium; the amount of xanthan gum added is 0.5-1.5% of the weight of the culture medium.

5. The inoculation method for sugarcane red rot according to claim 1, characterized in that, In step C, high-temperature sterilization is performed at 121°C for 25-30 minutes.

6. The inoculation method for sugarcane red rot according to claim 1, characterized in that, In step D, the dark culture temperature is 28-30℃ and the dark culture time is 3-4 days.

7. The inoculation method for sugarcane red rot according to claim 1, characterized in that, In step (2), the diameter of the hole obtained by drilling is 7-10 mm and the depth is 10-12 mm.

8. The inoculation method for sugarcane red rot according to claim 1, characterized in that, In step (3), 10 sugarcane stalks are inoculated for every 20 sugarcane plants in July-August, and the inoculated sugarcane plants are 6-8 months old.

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