Method for efficiently inducing Alternaria alternata conidia and application thereof

Abstract

The invention belongs to the technical field of alternaria alternata spore induction, and particularly relates to a method for efficiently inducing alternaria alternata conidia and application. The method for efficiently inducing the Alternaria alternata conidia comprises the following steps: (1) rejuvenating Alternaria alternata; and (2) spore induction and bacterial suspension preparation. According to the method, calcium chloride and magnesium chloride are added into the PDA culture medium at the same time, under the synergistic effect of calcium chloride and magnesium chloride, alternaria alternata can be induced to generate more spores, and the carrot leaves are obviously rotted and necrotic 3-5 days after disease inoculation by spraying the prepared spore suspension liquid to the carrot leaves. The method provided by the invention solves the problem that the Alternaria alternata does not generate spores on the PDA culture medium, is simple in operation method and low in cost, and facilitates symptom observation and physiological data acquisition after plant disease inoculation.

Description

Technical Field

[0001] The invention belongs to the technical field of spore induction of Alternaria root, and particularly relates to a method for efficiently inducing conidia of Alternaria root and application thereof. Background Art

[0002] Carrot is a biennial vegetable of the genus Daucus in the family Apiaceae. It is native to Central Asia and was introduced to my country during the Yuan Dynasty. Carotene is known as "little ginseng" and is rich in nutrients. Carrots are rich in carotene, which can nourish the liver, improve eyesight, and treat night blindness. They are deeply loved by the people. However, carrots are affected by a variety of diseases during the production process, resulting in a decrease in yield and serious loss of quality.

[0003] Carrot black rot is caused by Alternaria root Alternaria radicina Meier, Drechsler & E.D.Eddy) and is a disease that occurs in carrots. The disease can occur throughout the growth period of carrots, harming leaves, stems and fleshy roots. Leaves become diseased and present dark brown spots, and in severe cases, they die. Stems and petioles become diseased and present spindle-shaped or long strips of spots, and black mold forms when humidity is high. Fleshy roots become diseased and form sunken black spots at the root head, and in severe cases, the fleshy roots rot.

[0004] It is crucial to establish an inoculation system for disease research. Therefore, it is essential to establish an inoculation system for the prevention and control of carrot black rot, and spore induction is a crucial step in the establishment of an inoculation system. At present, the methods for inducing fungal conidia include: alternating light and dark culture plates, starvation culture, and adding amino acids. There are many methods, but their induction effect on Alternaria root is not significant.

[0005] Therefore, it is necessary to explore other induction methods to induce fungal conidia, and screen out a method and application of efficiently inducing conidia of Alternaria root. Summary of the invention

[0006] In view of the deficiencies in the prior art, the present invention provides a method for efficiently inducing conidia of Alternaria root fungus and its application.

[0007] The specific technical solutions are as follows: The first object of the present invention is to provide a method for efficiently inducing conidia of Alternaria root, comprising the following steps: (1) Rejuvenation of Alternaria root: inoculate the isolated and purified mycelium of Alternaria root onto the culture medium and culture in the dark; (2) Spore induction and preparation of bacterial suspension: In a sterile environment, the mycelium of Alternaria alternata obtained by culture in step (1) is picked onto a spore induction plate containing calcium chloride and magnesium chloride in a concentration ratio of 1:1, and placed in an incubator for dark culture. The plate is scraped and washed with sterile water to obtain a spore suspension. The spores are collected by centrifugation, the concentration is adjusted, and Tween is added to promote spore adhesion to leaves to obtain a spore suspension.

[0008] Furthermore, in the step (1), after the mycelium of Alternaria root is inoculated onto the culture medium, it is cultured at 26-30° C. in the dark for 6-8 days.

[0009] Furthermore, in step (1), the culture medium used is potato dextrose agar (PDA) culture medium.

[0010] Furthermore, in step (2), the concentrations of calcium chloride and magnesium chloride added to the spore induction plate are both 0.01-0.02 g / ml.

[0011] Furthermore, in step (2), the mixture is placed in an incubator at 26-30° C. and cultured in the dark for 6-8 days.

[0012] Furthermore, in step (2), the centrifuge speed is 3000-4000 r / min, and the centrifugation is performed for 8-10 minutes.

[0013] Furthermore, in step (2), the concentration is adjusted to 1.0×10 5 ~5.0×10 5 Pieces / ml.

[0014] Furthermore, in step (2), the Tween used is Tween-20, and the volume ratio of the Tween-20 to the spore suspension is 1:1000-2000.

[0015] Furthermore, the method for efficiently inducing conidia of Alternaria root fungus further comprises the step (3) of inoculating carrot leaves: spraying the spore suspension evenly on carrot leaves of the size of five leaves and one heart, culturing in the dark and then placing in an incubator for culturing; In step (3), the dark culture time is 12 to 15 hours, and the conditions of the incubator are a light cycle of 16 hours of light, 8 hours of darkness, and a relative humidity of 80 to 90%.

[0016] The second object of the present invention is to provide an application of the above-mentioned method for efficiently inducing conidia of Alternaria root spores in the field of Alternaria root spore induction.

[0017] Compared with the prior art, the present invention has the following beneficial effects: In the method for efficiently inducing conidia of Alternaria root, calcium chloride and magnesium chloride are simultaneously added to a PDA culture medium, and under the synergistic influence of calcium chloride and magnesium chloride, the Alternaria root can be induced to produce more conidia, and the prepared spore suspension is sprayed on carrot leaves, so that the carrot leaves show obvious rot and necrosis symptoms 3 to 5 days after infection.

[0018] The method of the invention solves the problem that Alternaria root fungus does not produce conidia on a PDA culture medium, and the operation method is simple, the cost is low, and it is convenient for observing symptoms and collecting physiological data of plants after being inoculated with the disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1 This is a flow chart of the operating steps of a method for efficiently inducing conidia of Alternaria root; Figure 2 A schematic diagram of a bacterial plate and hyphae of the Alternaria root provided in Example 1 of the present invention after 6 days of rejuvenation growth, wherein A is a schematic diagram of the bacterial plate, and B is a schematic diagram of the hyphae; Figure 3 A schematic diagram of the growth state of the bacterial plate and the statistical diagram of the number of spores under the conditions of the combination of 0.01-0.02 g / ml calcium chloride and 0.01-0.02 g / ml magnesium chloride and 0.01-0.02 g / ml sodium chloride provided by the present invention, wherein A is a schematic diagram of the corresponding days of bacterial plate growth under different ratio conditions, and B is a statistical diagram of the number of spores under the corresponding days of spore induction plate growth; Figure 4 A schematic diagram of a spore suspension provided in Example 1 of the present invention, wherein A is a schematic diagram of the spore suspension before filtration, B is a schematic diagram of the spore suspension after filtration, centrifugation, and resuspension, and C is a microscope photo of spores and a schematic diagram comparing mature spores with young spores; Figure 5 A schematic diagram of the grafting of carrot seedlings and a detailed diagram of the leaves after spraying provided in Example 1 of the present invention, wherein A is a schematic diagram of the grafting of carrot seedlings and B is a detailed diagram of the leaves after spraying; Figure 6The disease state diagrams of carrot leaves at 0 days, 1 day, 2 days, 3 days, and 5 days after being infected with the disease provided in Example 1 of the present invention, as well as scanning electron microscope diagrams of diseased leaves at 0 days and 5 days after being infected with the disease, wherein A is a disease state diagram at 0 days after being infected with the disease, B is a disease state diagram at 1 day after being infected with the disease, C is a disease state diagram at 2 days after being infected with the disease, D is a disease state diagram at 3 days after being infected with the disease, E is a disease state diagram at 5 days after being infected, F is a scanning electron microscope diagram of diseased leaves at 0 days, and G is a scanning electron microscope diagram of diseased leaves at 5 days; Figure 7 This is a schematic diagram of the comparison of the DNA sequences of diseased leaves and Alternaria root provided in Example 1 of the present invention, wherein A is an ITS-amplified gel map of diseased leaves and Alternaria root, and B is a comparison map of the DNA sequences of the two. DETAILED DESCRIPTION

[0021] The principles and features of the present invention are described below in conjunction with examples and drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0022] Example 1 Reference Figure 1 , a method for efficiently inducing conidia of Alternaria root, comprising the following steps: (1) Rejuvenation of Alternaria root fungus: ① Weigh 17.5g of PDA powder and 10g of agar powder in 500ml of ddH2O, sterilize at 121℃ for 20 minutes, and when the melted culture medium cools to about 45℃, pour it into a culture dish under sterile conditions and condense it into a solid plate; ② Use an inoculation loop to gently scratch the purified Alternaria root hyphae onto the surface of the plate, place the front side in a 30℃ incubator, and culture in the dark for 6 days. When the plate is full of hyphae, use it for spore induction and subculture; Schematic diagram of the fungus plate and mycelium of Alternaria root under 6 days of rejuvenation growth, such as Figure 2 As shown; (2) Spore induction and bacterial suspension preparation: ① Weigh 1g of anhydrous calcium chloride and 1g of magnesium chloride and melt them in 100ml of ddH2O, add 3.5g of PDA powder and 2g of agar, sterilize at 121℃ for 20 minutes, and when the melted culture medium cools to about 45℃, pour it into a culture dish under sterile conditions and condense it into a solid plate to make a spore induction plate; ② Use a toothpick to pick up a small amount of hyphae from the hyphae plate cultured in step (1) and gently scratch it onto the spore induction plate. Place the plate face up in a 30°C incubator and culture it in the dark for 6 days to induce spores. ③ Place the spore induction plate grown for 6 days on a sterile table, add an appropriate amount of sterile water to the spore induction plate, scrape off the mycelium with a sterile blade, and filter it into a 50ml centrifuge tube with a double-layer sterilized gauze to obtain a spore suspension; ④ Centrifuge the spore suspension at 4000r / min for 10 minutes to collect the spores, discard the supernatant and adjust the concentration to 5×10 5 / ml, adding Tween-20 to promote the spores to adhere to the leaves, and obtaining a spore suspension; wherein each milliliter of the spore suspension contains 0.1% Tween-20 by volume; (3) Carrot leaf disease: ① When carrots have five leaves and one heart, spray the spore suspension on the carrot leaves; ② After incubating the inoculated carrot seedlings in the dark for 12 hours, move them to an incubator for incubation. The incubator conditions are a photoperiod of 16 hours of light, 8 hours of darkness, and a relative humidity of 80%.

[0023] Example 2 Reference Figure 1 , a method for efficiently inducing conidia of Alternaria root, comprising the following steps: (1) Rejuvenation of Alternaria root fungus: ① Weigh 17.5g of PDA powder and 10g of agar powder in 500ml of ddH2O, sterilize at 121℃ for 20 minutes, and when the melted culture medium cools to about 45℃, pour it into a culture dish under sterile conditions and condense it into a solid plate; ② Use an inoculation loop to gently scratch the purified Alternaria root hyphae onto the surface of the plate, place the front side in a 26℃ incubator, and culture in the dark for 8 days. When the plate is full of hyphae, use it for spore induction and subculture; (2) Spore induction and bacterial suspension preparation: ① Weigh 2g of anhydrous calcium chloride and 2g of magnesium chloride and melt them in 100ml of ddH2O, add 3.5g of PDA powder and 2g of agar, sterilize at 121℃ for 20 minutes, and when the melted culture medium cools to about 45℃, pour it into a culture dish under sterile conditions and condense it into a solid plate to make a spore induction plate; ② Use a toothpick to pick up a small amount of mycelium from the mycelium plate cultured in step (1) and gently scratch it onto the spore induction plate. Place it face up in a 26°C incubator and culture it in the dark for 8 days to induce spores. ③ Place the 8-day-old spore induction plate on a sterile table, add an appropriate amount of sterile water to the spore induction plate, scrape off the mycelium with a sterile blade, and filter it into a 50ml centrifuge tube with a double-layer sterilized gauze to obtain a spore suspension; ④ Centrifuge the spore suspension at 3000r / min for 8 minutes to collect the spores, discard the supernatant and adjust the concentration to 1×10 5 spores / ml, Tween-20 was added to promote the spores to adhere to the leaves, and a spore suspension was obtained; wherein each milliliter of the spore suspension contained 0.05% Tween-20; (3) Carrot leaf disease: ① When carrots have five leaves and one heart, spray the spore suspension on the carrot leaves; ② After incubating the inoculated carrot seedlings in the dark for 15 hours, move them to an incubator for incubation. The incubator conditions are a photoperiod of 16 hours of light, 8 hours of darkness, and a relative humidity of 90%.

[0024] Comparative Example 1 A method for inducing conidia of Alternaria root is described in Example 1, which differs from Example 1 in that 1 g of anhydrous calcium chloride and 1 g of sodium chloride are weighed and dissolved in 100 ml of ddH2O.

[0025] Comparative Example 2 A method for inducing conidia of Alternaria root is described in Example 1, which differs from Example 1 in that 2 g of anhydrous calcium chloride and 2 g of sodium chloride are weighed and dissolved in 100 ml of ddH2O.

[0026] Comparative Example 3 A method for inducing conidia of Alternaria root is described in Example 1, which differs from Example 1 in that 1 g of magnesium chloride and 1 g of sodium chloride are weighed and dissolved in 100 ml of ddH2O.

[0027] Comparative Example 4 A method for inducing conidia of Alternaria root is described in Example 1, which differs from Example 1 in that 2 g of magnesium chloride and 2 g of sodium chloride are weighed and dissolved in 100 ml of ddH2O.

[0028] test: The target crop is carrot and the target part is the leaves.

[0029] Test example: The carrot variety used for the test is "Kuroda Gosun".

[0030] Test material culture: The test material of the present invention is the carrot cultivar "Heida Gocun". The seeds were first washed with 75% anhydrous ethanol on a clean bench for 1 minute, then soaked with 40% sodium hypochlorite, shaken on a shaker at 28°C for 18 minutes, washed 10 times with anhydrous ethanol, and sown in B5 culture medium. After culturing in the dark at 28°C for 2 days, the carrots were transferred to light for culturing. After the carrots grew two cotyledons, they were transplanted into a mixed matrix of vermiculite, perlite, and soil in a volume ratio of 1:1:2. The mixture was watered thoroughly and kept in a light cycle of 16 h (light) and 8 h (dark), a day-night temperature cycle of 25°C / 20°C, and a light intensity of 150 μmol·m -2 ·s -1 and grown under conditions of 50% relative humidity; When the carrot seedlings grow to five leaves and one heart, they are inoculated. After inoculation, they are placed in a dark place for 12 hours and then transferred to an incubator with a photoperiod of 16 hours (light) and 8 hours (dark) and a relative humidity of 80%.

[0031] Test results: The growth status of the spore induction plate in Examples 1 and 2 of the present invention and Comparative Examples 1 to 4 after the corresponding number of days of growth was photographed, and the results are as follows: Figure 3 A in the embodiment of the present invention 1, 2 and comparative examples 1 to 4 of the spore induction plate after the corresponding number of days of growth were counted, the results are as follows Figure 3 As shown in B.

[0032] The method for counting the number of spores refers to the blood cell counting method, that is, after scraping and filtering the spore suspension, mix it evenly, use a rubber-tipped dropper to draw a small amount and drop it on the blood cell counting plate, cover it with a cover glass, and count the number of spores under a microscope. Count the number of spores in five squares (X1, X2, X3, X4, X5) at a time, do two parallel experiments, and take the average of three repetitions. Calculation formula: Depend on Figure 3 The results of B in Example 1 show that the number of spores under the concentration ratio condition is 1.1×10 6 / ml; the number of spores under the concentration ratio condition in Example 2 is 1.05×10 6 / ml; the number of spores under the concentration ratio condition in Comparative Example 1 was 7.4×10 5 / ml; the number of spores under the concentration ratio condition in Comparative Example 2 was 3.9×10 5 / ml; the number of spores under the concentration ratio condition in comparative example 3 is 2×10 5 / ml; the number of spores under the concentration ratio condition in Comparative Example 4 was 1.07×10 5 This shows that in the method for efficiently inducing conidia of Alternaria root spores of the embodiment of the present invention, after adding calcium chloride and magnesium chloride simultaneously to the PDA medium, more conidia of Alternaria root spores can be induced under the synergistic effect of calcium chloride and magnesium chloride.

[0033] The comparison diagram of the spore suspension before and after filtration in Example 1 of the present invention is as follows: Figure 4 As shown in A and B in the figure; young spores and mature spores photographed under an electron microscope, as shown in Figure 4 As shown in C.

[0034] Spray the spore suspension evenly onto the surface of carrot leaves through a watering can. After spraying, the surface of the leaves will appear as follows. Figure 5 shown.

[0035] The spore induction plate of Example 1 of the present invention was placed in an incubator at 30°C and cultured in the dark for 6 days to produce a large number of spores. A spore suspension was prepared and then inoculated with diseased carrot seedlings. The results were as follows: Figure 6 As shown, one day after inoculation, brown spots began to appear on the edges of the carrot leaves. Two days after inoculation, the lower leaves of the carrots turned black and rotted. Five days later, the entire carrot leaves turned black and rotted and were covered with hyphae.

[0036] DNA was extracted from the diseased leaves and Alternaria root fungus in Example 1 of the present invention, and the results were as follows: Figure 7 As shown, the band obtained by ITS amplification was about 500 bp, and the DNA sequence of the diseased leaves obtained by sequencing was consistent with the sequence of Alternaria root, confirming that the disease was caused by Alternaria root.

[0037] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for efficiently inducing conidia of Alternaria root, characterized in that: The steps include: (1) Rejuvenation of Alternaria root: inoculate the isolated and purified mycelium of Alternaria root onto the culture medium and culture in the dark; (2) Spore induction and preparation of bacterial suspension: In a sterile environment, the mycelium of Alternaria alternata obtained by culture in step (1) is picked onto a spore induction plate containing calcium chloride and magnesium chloride in a concentration ratio of 1:1, and placed in an incubator for dark culture. The plate is scraped and washed with sterile water to obtain a spore suspension. The spores are collected by centrifugation, the concentration is adjusted, and Tween is added to promote spore adhesion to leaves to obtain a spore suspension.

2. The method for efficiently inducing conidia of Alternaria root according to claim 1, characterized in that: In the step (1), after the mycelium of Alternaria root is inoculated on the culture medium, it is cultured at 26-30° C. in the dark for 6-8 days.

3. The method for efficiently inducing conidia of Alternaria root according to claim 1, characterized in that: In the step (1), the culture medium used is potato dextrose agar culture medium.

4. The method for efficiently inducing conidia of Alternaria root according to claim 1, characterized in that: In the step (2), the concentrations of calcium chloride and magnesium chloride added to the spore induction plate are both 0.01-0.02 g / ml.

5. The method for efficiently inducing conidia of Alternaria root according to claim 1, characterized in that: In the step (2), the culture medium is placed in an incubator at 26-30° C. and cultured in the dark for 6-8 days.

6. The method for efficiently inducing conidia of Alternaria root according to claim 1, characterized in that: In the step (2), the centrifuge speed is 3000-4000 r / min, and the centrifugation is performed for 8-10 minutes.

7. The method for efficiently inducing conidia of Alternaria root according to claim 1, characterized in that: In the step (2), the concentration is adjusted to 1.0×10 5 ~5.0×10 5 Pieces / ml.

8. The method for efficiently inducing conidia of Alternaria root according to claim 1, characterized in that: In the step (2), the Tween used is Tween-20, and the volume ratio of the Tween-20 to the spore suspension is 1:1000-2000.

9. The method for efficiently inducing conidia of Alternaria root according to claim 1, characterized in that: The method also includes the step (3) of inoculating carrot leaves: spraying the spore suspension evenly on carrot leaves of the size of five leaves and one heart, culturing in the dark, and then culturing in an incubator; In step (3), the dark culture time is 12 to 15 hours, and the conditions of the incubator are a light cycle of 16 hours of light, 8 hours of darkness, and a relative humidity of 80 to 90%.

10. Use of the method for efficiently inducing conidia of Alternaria root spores according to any one of claims 1 to 9 in the field of Alternaria root spore induction.

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