Method for rapidly identifying southern blight resistance of konjak plant

Abstract

The invention provides a method for rapidly identifying southern blight resistance of konjak plants, and belongs to the technical field of plant disease resistance identification. The invention provides a method for rapidly identifying southern blight resistance of konjak plants, which comprises the following steps: culturing konjak southern blight, and preparing southern blight mycelium blocks; a healthy konjak plant is selected, the southern blight silk block is attached to the base of a near-ground petiole of the konjak plant, and inoculation is completed after tight sealing; and placing the inoculated konjak plant in an incubator, culturing for 24-72 hours, and identifying. According to the method, by simulating the high-temperature and high-humidity attack environment conditions of the konjak southern blight in the field, the environment temperature is 30-35 DEG C and the humidity is controlled to be 90-95% after the southern blight silk blocks are attached to the near-earth petiole base of the konjak plant, the infection symptom can be shown in 24 hours, the infection peak is reached in 72 hours, the southern blight nucleus is gradually formed, and the purpose of rapidly identifying the plant resistance is achieved.

Description

Technical Field

[0001] The invention relates to the technical field of plant disease resistance identification, and in particular to a method for rapidly identifying the resistance of konjac plants to southern rot. Background Art

[0002] Konjac is the only important cash crop rich in glucomannan in nature. It is one of the top ten high-quality health foods recommended by the World Health Organization of the United Nations. It has the effects of reducing fat, losing weight, and reducing "three highs". my country is the largest producer of konjac, with a relatively complete set of konjac industry systems such as cultivation, processing, and product development. The planting area accounts for more than 60% of the world. It is also the world's largest exporter of konjac flour and konjac products. As an emerging characteristic industry, the konjac industry has an important position and role in the agricultural economy, and has positive significance for promoting agricultural structural adjustment, increasing farmers' income, and promoting rural economic development. However, with the continuous increase in the planting area of ​​konjac, konjac diseases have become more and more serious year by year, becoming one of the main bottlenecks restricting the development of the konjac industry.

[0003] Konjac white rot (Athelia rolfsii) is the second largest disease except konjac soft rot. Under natural conditions, the white rot pathogen does not produce conidia, but produces sclerotia on the plant's diseased remains, can survive in the soil for several years, and accumulates year after year as the initial infection source of the next year, so the disease is difficult to cure. Its pathogen often causes konjac to break off from the petiole base by secreting cell wall degrading enzymes, and in severe years, more than 60% of the plants can be caused to fall ill or even have no harvest, which seriously restricts the healthy development of the konjac industry. At present, there are fewer studies on the pathogenic mechanism of konjac white rot, and no one studies its inoculation and identification method. For konjac disease-resistant breeding, the establishment of inoculation and identification methods of different diseases and resistance evaluation systems is of vital importance for disease-resistant breeding work. At present, the plant resistance identification and evaluation system for konjac is still imperfect, lacking the method for indoor inoculation, infection, and rapid identification of konjac white rot. Therefore, the technical problems to be solved by the present invention are to establish an indoor soilless inoculation method that can quickly and efficiently identify the resistance to southern rot of konjac, and to formulate a complete identification system for the resistance to southern rot of konjac. Summary of the invention

[0004] The purpose of the invention is to provide a method for quickly identifying the resistance of konjac plants, so as to lay a foundation for the research on disease-resistant breeding of konjac.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a method for rapidly identifying the resistance of konjac plants to white rot, comprising the following steps:

[0007] (1) culturing Sclerotium rutaecarpa to prepare Sclerotium rutaecarpa mycelium blocks;

[0008] (2) selecting healthy and complete konjac plants, digging them out together with the corms, rinsing them, and attaching the mycelium block of the southern blight pathogen to the base of the petiole of the konjac plant near the ground, and sealing them tightly to complete the inoculation;

[0009] (3) The inoculated konjac plants were placed in an incubator for 24 to 72 hours for identification.

[0010] Preferably, the Saccharomyces rolfsii fungus in step (1) is cultured on a PDA plate for 3 to 5 days before being prepared into a mycelium block.

[0011] Preferably, the mycelium block in step (1) has a length and a width of 3-5 mm*3-5 mm respectively.

[0012] Preferably, the base of the petiole of the konjac plant near the ground in step (2) refers to the base of the petiole 0 to 3 cm above the ground.

[0013] Preferably, the konjac plants in step (3) are placed in a sterilized plastic box, and the humidity in the plastic box is 90% to 95%.

[0014] Preferably, the temperature of the incubator in step (3) is 30-35°C, and the culture is alternating between 8-10 hours of light and 14-16 hours of darkness.

[0015] Preferably, the identification method in step (3) is to make a cross section at the inoculation site, measure the length and width of the pathogen infection, obtain the infection area, and calculate the percentage of the infection area to the cross-sectional area of ​​the petiole.

[0016] Since konjac leaves or petioles are easily dehydrated and wilted after being separated from the body, the resistance identification process and result are affected. The present invention takes healthy and complete konjac plants away from the field and carries out soilless healthy and complete plant inoculation, rather than using isolated konjac tissues or organs. The soilless healthy and complete konjac plants can rely on their own corms to supply nutrition, thereby ensuring the good vitality of the plants during the resistance identification, and then ensuring the accuracy and credibility of the resistance identification results.

[0017] The method for rapidly identifying the resistance of konjac plants to white rot provided by the present invention simulates the high temperature and high humidity disease environment of konjac white rot in the field, ensures that the environmental temperature after inoculation is 30-35°C, and the humidity is controlled at 90%-95%, and the infection symptoms can be shown in 24 hours, the infection peak is reached in 72 hours, and the white rot nucleus is gradually formed, so as to achieve the purpose of rapidly identifying the resistance of the plant. In addition, the resistance identification of konjac plants by predecessors is generally carried out by inoculation on the corm, but the inoculation site selected by the present invention is the base of the petiole near the ground of the konjac plant, which can more quickly and accurately identify the resistance of the konjac plant to white rot. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1This is a picture of the inoculation of the konjac near the base of the petiole in Example 1;

[0019] Figure 2 This is the infection status of the petiole cross section 24 hours after inoculation in Experimental Example 1;

[0020] Figure 3 This is the petiole performance after 24h inoculation in Experimental Example 1;

[0021] Figure 4 This is the petiole performance after 48h inoculation in Experiment 1;

[0022] Figure 5 This is the petiole performance of Experimental Example 1 72 hours after inoculation. DETAILED DESCRIPTION

[0023] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0024] Example 1

[0025] Source of the pathogen of white rot: It was isolated by indoor inoculation of diseased plants of konjac in the field test site of Ankang Academy of Agricultural Sciences and cultured multiple times.

[0026] Select a sterilized open plastic box of 60cm*40cm*25cm in size, put sterile filter paper on the bottom, and spray it with sterile water to ensure that the water soaks the filter paper and a small amount of redundant water flows on the bottom of the box when it is shaken.

[0027] On a clean bench, use a sterilized scalpel to divide the Saccharomyces rolfsii pathogen that has been activated (1 sclerotium) and cultured on PDA medium for 5 days into 5 mm*5 mm mycelium blocks for later use.

[0028] Select 10 healthy konjac plants of the same age and similar petiole diameter, dig out the whole plant together with the corm, and carefully rinse the plant with tap water to avoid mechanical damage to the plant. Use a sterilized needle to poke a 1mm hole at the base of the petiole of the konjac plant 1cm above the ground. Wait for the konjac body fluid to overflow, quickly stick the mycelium block with sterilized tweezers, and seal it tightly with sealing film ( Figure 1 After the inoculation is completed, spray the plastic box with sterile water mist quickly. When the humidity in the space reaches 95%, quickly seal the plastic box with plastic wrap and place the plastic box in a 32℃ incubator. Incubate the box with 8h light and 16h dark alternately for 24h, 48h, 72h, ( Figure 3-5 ) Make a cross section at the inoculation site, that is, use a sterilized scalpel to quickly cut from the junction of the diseased and healthy parts to make a cross section ( Figure 2 ). Measure the length and width of the pathogen infection to obtain the infection area, and calculate the percentage of the infection area to the cross-sectional area of ​​the petiole.

[0029] Comparative Example 1

[0030] The method of Example 1 was adopted, except that the base of the petiole of the Amorphophallus konjac plant at 5 cm above the ground was inoculated with a mycelium block, and a total of 10 plants were inoculated.

[0031] Comparative Example 2

[0032] The method of Example 1 was adopted, except that the base of the petiole of the Amorphophallus konjac plant at 7 cm above the ground was inoculated with a mycelium block, and a total of 10 plants were inoculated.

[0033] Comparative Example 3

[0034] The method of Example 1 was adopted, except that the inoculation was completed at the base of the petiole of the Amorphophallus konjac plant at 9 cm above the ground, and a total of 10 plants were inoculated.

[0035] Comparative Example 4

[0036] The method of Example 1 was adopted, except that the inoculation was completed at the base of the petiole of the Amorphophallus konjac plant 11 cm above the ground, and a total of 10 plants were inoculated.

[0037] Experimental Example 1

[0038] Inoculation was performed according to the method of Example 1 and Comparative Examples 1 to 4, and a control group of 10 strains inoculated only with PDA culture medium was set up. After 24 hours of inoculation, Example 1, Comparative Examples 1 to 4 and the control group were identified respectively. The identification method was to make a cross section at the inoculation site with a sterilized knife, measure the infection length and width of the pathogen with a ruler, obtain the infection area, and calculate the percentage of the infection area to the handle diameter area. The results are shown in Table 1.

[0039] Table 1 Infection results at different inoculation sites

[0040]

[0041] As shown in Table 1, the inoculated part of Example 1 can show infection symptoms within 24 hours, and the petiole base of the konjac plant selected in the present invention can quickly and accurately identify the resistance of the konjac plant to white rot.

[0042] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for rapidly identifying the resistance of konjac plants to white rot. It is characterized in that The steps include: (1) culturing Sclerotium rutaecarpa to prepare Sclerotium rutaecarpa mycelium blocks; (2) selecting healthy and complete konjac plants, digging them out together with the corms, rinsing them, and attaching the mycelium block of the southern blight pathogen to the base of the petiole of the konjac plant near the ground, and sealing them tightly to complete the inoculation; (3) The inoculated konjac plants were placed in an incubator for 24 to 72 hours for identification.

2. The method according to claim 1, It is characterized in that The konjac Sclerotium rolfsii fungus described in step (1) is cultured on a PDA plate for 3 to 5 days and then prepared into a mycelium block.

3. The method according to claim 2, It is characterized in that The mycelium block in step (1) has a length and a width of 3-5 mm*3-5 mm respectively.

4. The method according to claim 3, It is characterized in that The petiole base of the konjac plant near the ground in step (2) refers to the petiole base 0 to 3 cm above the ground.

5. The method according to claim 4, It is characterized in that In step (3), the konjac plants are placed in a sterilized plastic box, and the humidity in the plastic box is 90% to 95%.

6. The method according to claim 5, It is characterized in that The temperature of the incubator in step (3) is 30-35°C, and the cells are cultured alternately with 8-10 hours of light and 14-16 hours of darkness.

7. The method according to claim 6, It is characterized in that The identification method in step (3) is to make a cross section at the inoculation site, measure the length and width of the pathogen infection, obtain the infection area, and calculate the percentage of the infection area to the cross section area of ​​the petiole.

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