Mass production of spores of the sweet potato root rot fungus by the agar leaf method

Abstract

To provide means for efficiently producing fungus spores causing foot rot of sweet potato in large quantities without irradiation with ultraviolet rays.SOLUTION: A method for producing fungus spores causing foot rot of sweet potato is characterized by comprising the following steps (1) to (3) of: (1) inoculating a fungus causing foot rot of sweet potato onto a base material placed on a solid medium; making the base material a sheet-like base material with a leaf or cellulose of a plant belonging to Ipomoea as a main component; (2) culturing the fungus causing foot rot of sweet potato inoculated in the step (1) to form spores; and (3) collecting the spores formed in the step (2).SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for producing spores of the pathogenic fungus, the causal agent of sweet potato root rot. This method enables the supply of spores of the pathogenic fungus in large quantities, facilitating disease development tests and control tests of the fungus. [Background technology]

[0002] Sweet potato root rot is a serious soil-borne disease caused by the filamentous fungus Diaporthe destruens. Infected sweet potatoes first develop dark brown lesions at the base of the stem. The lesions then spread above and below ground, adversely affecting growth and resulting in poor growth. In severe cases, the plant dies, rendering it unharvestable. Infected potatoes also pose a problem due to their rotting during storage and the supply of contaminated seedlings. Since the disease was first reported in Japan in 2018, damage has worsened in Kagoshima, Miyazaki, and Okinawa, significantly impacting local industries. There are also concerns about the disease spreading nationwide. However, there are currently no effective control measures, and there is a pressing need to clarify the disease's biology and develop control measures as soon as possible.

[0003] To conduct research into plant disease control, it is important to have a method for mass-cultivating pathogenic fungi and inoculating them onto plants to induce disease. However, because the physiological characteristics of the sweet potato root rot fungus have not yet been fully elucidated, no method for producing spores of the fungus has been established. Therefore, spore production is generally achieved by culturing the fungus on an agar medium, such as potato dextrose agar (PDA), under ultraviolet irradiation, following the experimental techniques used for other filamentous fungi (Non-Patent Document 1). It is also known that other plant pathogenic fungi can mass-produce pycnidia (pycnidia) and conidia (conidia, spores) using the agar leaf disc method (Non-Patent Document 2). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Toshifumi Yuzuki, "Control of plant pathogens by utilizing light quality," Tohoku Agricultural Research, vol. 21, pp. 13-20 (1978) [Non-patent document 2] Kishi Kunihira, "Mass production of pycnidia and pycnospores by the agar leaf disc method," Plant Protection, vol. 49, pp. 129-130 (1995) Summary of the Invention [Problem to be solved by the invention]

[0005] There is no known method for efficiently preparing large quantities of spores for the sweet potato root rot fungus, and preparing spores using the plate culture method, which is generally used for plant pathogens, requires time and effort, posing a challenge to the smooth execution of research.Spore formation is also promoted in the sweet potato root rot fungus when it is left under irradiation by a BLB light or germicidal lamp during cultivation, but the impact this has on the properties of the strain has not been taken into consideration.

[0006] The present invention has been made under these circumstances, and aims to provide a means for efficiently mass-producing spores of sweet potato root rot fungus without irradiation with ultraviolet light. [Means for solving the problem]

[0007] The present inventors conducted extensive research to solve the above-mentioned problems and discovered that mass production of spores of the pathogenic fungus can be achieved by inoculating sweet potato leaves, morning glory leaves, filter paper, or cellophane placed on an agar medium with the pathogenic fungus, thereby completing the present invention. The agar leaf disc method for producing fungal spores using plant leaves is a well-known method (Non-Patent Document 2). Hydrangea and cherry leaves are considered suitable for mass production of spores using this method (Non-Patent Document 2). However, the pathogenic fungus did not grow well on these leaves, and few spores were obtained. It was unexpected that mass production of spores of the pathogenic fungus could be achieved by using sweet potato or morning glory leaves instead of hydrangea or cherry leaves. It was also unexpected that mass production of spores is possible using sheet-like substrates primarily composed of cellulose, such as filter paper and cellophane. The present invention was completed based on the above findings.

[0008] That is, the present invention provides the following [1] to [5]. [1] A method for producing spores of sweet potato root rot fungus, comprising the following steps (1) to (3): (1) A step of inoculating a substrate placed on a solid medium with sweet potato root rot fungus, wherein the substrate is a leaf of a plant of the genus Ipomoea or a sheet-like substrate mainly composed of cellulose; (2) culturing the sweet potato root rot fungus inoculated in step (1) to form spores; (3) harvesting the spores formed in step (2).

[0009] [2] The method for producing spores of the sweet potato root rot fungus according to [1], wherein the substrate is a heat-treated substrate.

[0010] [3] The method for producing spores of the sweet potato root rot fungus according to [1], wherein the substrate is a sweet potato leaf or a morning glory leaf.

[0011] [4] The method for producing spores of sweet potato root rot fungus according to [1], wherein the substrate is filter paper or cellophane.

[0012] [5] The method for producing spores of the sweet potato root rot fungus according to [1], wherein the solid medium is an agar medium. [Effects of the Invention]

[0013] The present invention provides a novel method for producing spores of the pathogenic fungus, the causal agent of sweet potato root rot. This method allows for the easy mass production of spores of the pathogenic fungus, facilitating the implementation of disease and control tests. Furthermore, since ultraviolet irradiation is not required, not only is irradiation equipment unnecessary, but mutations in the pathogenic fungus' properties can also be suppressed, contributing to the elucidation of its ecology. [Brief explanation of the drawings]

[0014] [Figure 1] Photographs showing the state of sweet potato leaves after cultivation using different heat treatment methods. [Figure 2] Photographs showing the state of various leaves after cultivation. [Figure 3] Photographs showing the state of substrates other than plant leaves after cultivation. [Figure 4] A diagram showing the number of spores of the sweet potato root rot fungus when cultured on various substrates (1). [Figure 5] Graph (2) showing the number of spores of the sweet potato root rot fungus when cultured on various substrates. [Figure 6] A graph showing the number of spores of the sweet potato root rot fungus when cultured on various substrates (3). DETAILED DESCRIPTION OF THE INVENTION

[0015] The present invention will be described in detail below. The method for producing spores of sweet potato root rot fungus of the present invention is characterized by comprising the following steps (1) to (3).

[0016] In step (1), a substrate placed on a solid medium is inoculated with sweet potato root rot fungus.

[0017] The substrate can be made from leaves of plants of the Ipomoea genus. The leaves can be used as they are, but are usually cut to an appropriate size. Examples of plants of the Ipomoea genus include sweet potato (Ipomoea batatas), morning glory (Ipomoea nil), water spinach (Ipomoea aquatica), night glory (Ipomoea alba), and lycoris quinqueradiata (Ipomoea quamoclit). The leaves of the plants used in the present invention are not particularly limited as long as they are leaves of plants of the Ipomoea genus, but it is preferable to use sweet potato or morning glory leaves. Furthermore, sheet-like substrates whose main component is cellulose can also be used as the substrate. Examples of sheet-like substrates whose main component is cellulose include paper substrates such as filter paper, cotton substrates such as gauze, and cellophane.

[0018] The substrate is preferably heat-treated. The heat treatment method is not particularly limited, and examples thereof include boiling treatment and autoclave treatment (high-temperature, high-pressure steam treatment). When boiling treatment is performed, the treatment time is not particularly limited, but is preferably 5 to 120 seconds, more preferably 10 to 60 seconds, and even more preferably 20 to 40 seconds. When autoclave treatment is performed, the temperature and treatment time are not particularly limited, but the temperature is preferably 100 to 130°C, and the treatment time is preferably 3 to 30 minutes.

[0019] The solid medium may be one used in the general agar leaf plate method (Non-Patent Document 2), for example, an agar medium. The agar medium may be a medium that does not contain nutrients (plain agar medium) or a medium that contains nutrients (for example, potato-dextrose agar medium). The agar concentration in the agar medium may be the same as that used in the general agar leaf plate method, for example, 1.0 to 2.0% by weight.

[0020] Inoculation of the sweet potato base rot fungus can be carried out in the same manner as inoculation of filamentous fungi in the general agar leaf method, for example, by placing an agar piece of the sweet potato base rot fungus colony cultured on an agar medium on a substrate.

[0021] In step (2), the sweet potato root rot fungus inoculated in step (1) is cultured to form spores.

[0022] The culture temperature is not particularly limited, but is preferably 20 to 30° C., and more preferably 23 to 28° C. The culture period is also not particularly limited, but is preferably 7 to 28 days, and more preferably 14 to 21 days.

[0023] Furthermore, the culture can be carried out under natural light, and a large amount of spores can be produced without the need for ultraviolet light irradiation using a black light or germicidal lamp.

[0024] In step (3), the spores formed in step (2) are collected.

[0025] Spores can be collected in the same manner as in the general agar leaf disc method. For example, spores can be collected by immersing spore-forming sweet potato root rot fungi in water, releasing the spores into the water, and recovering the water containing the spores. [Example]

[0026] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0027] A. Experimental Method 1. Culture Medium Preparation [1.75% plain agar medium] 17.5 g of agar was added to 1,000 mL of distilled water, sterilized in an autoclave at 121°C for 20 minutes, and poured into a petri dish (either round or square, same below) to solidify.

[0028] [0.5x potato dextrose agar medium (0.5x PDA medium)] 100 g of potatoes were cut into 1-2 cm cubes and boiled in 1,000 mL of distilled water for 30 minutes. The potato decoction was strained through gauze and adjusted to 1,000 mL with distilled water. 10 g of glucose and 20 g of agar were added, and the mixture was sterilized in an autoclave at 121°C for 20 minutes. The mixture was then poured into a petri dish and allowed to solidify. When using commercially available PDA powder reagent or commercially available potato-dextrose liquid medium powder reagent, half the specified amount was added to 1,000 mL of distilled water, and additional agar powder was added to achieve the specified concentration. The mixture was sterilized in an autoclave at 121°C for 20 minutes, and the mixture was then poured into a petri dish and allowed to solidify.

[0029] 2a. Agar leaf method (sweet potato leaves and morning glory leaves) The leaves used as leaf pieces were fully expanded, dark in color, and free of withering and insect damage. After lightly rinsing with tap water, the leaves were wiped dry with a Kimtowel or similar, and then cut to fit the size of the petri dish. The collected leaves can be stored for long periods of time by freezing them.

[0030] [When autoclaving leaves] Several leaves, each adjusted to a specific size, were stacked on top of each other in a glass petri dish and sterilized in an autoclave at 121°C for 5 minutes. Because the conditions are milder than those used in general autoclave sterilization and there is a risk of bacteria growing, the leaves were refrigerated and used promptly.

[0031] [When boiling the leaves] Boil tap water (distilled water is also acceptable) in a pot, and once boiling, add the leaves, which have been adjusted to their size, and boil for 30 to 60 seconds while gently stirring, then remove from the boiling water. Boiling alone is not sufficient for sterilization, and bacteria can grow, so adjustments are necessary before use.

[0032] Autoclaved or boiled leaves were placed on 1.75% plain agar medium or 0.5x PDA medium. Agar pieces (2 mm to 3 mm square) of 0.5x PDA medium mycelium flora of sweet potato root rot fungus, which had been cultured at room temperature (25°C) for 2 to 3 weeks, were placed on the leaves. Static culture was continued at room temperature for 2 to 3 weeks, allowing mycelium to elongate and spores to form on the leaves. Fungal culture can be carried out under natural light; near-ultraviolet irradiation is not necessary.

[0033] 2b. Agar leaf method (sheet-like substrates mainly composed of cellulose: cellophane, filter paper, gauze) The substrates used in the test were cellophane (dialysis tubing, M-25, manufactured by Kenis), filter paper (qualitative filter paper, No. 2, manufactured by Advantec Toyo), and gauze (FC Family Gauze, manufactured by Hakujuji). The cellophane was prepared by cutting open the tube, cutting it to fit the size of a petri dish, placing it in a glass petri dish, lightly immersing it in distilled water, and autoclaving it at 121°C for 20 minutes. The filter paper and gauze were cut to fit the size of the petri dish and autoclaved at 121°C for 20 minutes. The subsequent procedures were the same as in 2a above.

[0034] 3. Measurement of spore concentration 10 mL of sterilized water was poured into a petri dish (9 cm diameter) and left to stand at room temperature (25°C) for 30 minutes to release spores. The bacterial solution was collected and the spore concentration was measured using a hemocytometer. The spore concentration was adjusted appropriately and used for inoculation tests and physiological and ecological studies.

[0035] B. Experimental Results 1. Mass spore production of the sweet potato root rot fungus on sweet potato leaves Heat-treated sweet potato leaves were used to culture the sweet potato base rot fungus using the agar leaf method. Beniazuma sweet potato was used, the fungus strain F3-SP11 was used, and plain agar medium was used. The heat-treated sweet potato leaves were subjected to four different heat treatment conditions: boiling for 10 seconds, boiling for 30 seconds, boiling for 60 seconds, and steam sterilization for 5 minutes. The condition of the sweet potato leaves after culture is shown in Figure 1. Spore formation was good under all heating conditions.

[0036] 2. Agar leaf disc method for detecting sweet potato root rot fungus using various leaves The sweet potato base rot fungus was cultured using the agar leaf disc method on sweet potato (untreated), cherry, and hydrangea leaves. The fungal strain used was F3-SP11, and plain agar medium was used. Figure 2 shows the condition of the leaves of each plant after culture. The sweet potato base rot fungus did not grow well on untreated sweet potato and cherry leaves, and barely grew on hydrangea leaves.

[0037] 3. Mass spore production of root rot fungi on sites other than plant leaves The sweet potato root rot fungus was cultured using filter paper and cellophane by the agar leaf method. The fungal strain used was F3-MZ, and the agar medium used was potato-dextrose agar. The state of each substrate after culture is shown in Figure 3. Spore formation was good whether filter paper or cellophane was used.

[0038] 4. Sporulation of root rot fungi on various substrates (1) The sweet potato root rot fungus was cultured using heat-treated sweet potato leaves by the agar leaf method. The fungal strain used was F3-SP11, and the agar medium used was plain agar or potato-dextrose agar. The number of spores formed after culture is shown in Figure 4. Under all culture conditions, the number of spores formed was greater than when cultured on agar medium alone.

[0039] 5. Sporulation of root rot fungi on various substrates (2) The sweet potato root rot fungus was cultured using heat-treated sweet potato leaves or cellophane by the agar leaf method. The fungal strain used was F3-MZ, and the agar medium used was plain agar or potato-dextrose agar. The number of spores after culture is shown in Figure 5. Under all culture conditions, the number of spores formed was greater than when cultured on agar medium alone.

[0040] 6. Sporulation of root rot fungi on various substrates (3) Heat-treated sweet potato, morning glory, cherry, or hydrangea leaves were used to culture the sweet potato root rot fungus by the agar leaf plate method. The fungal strain used was F3-SP11, and the agar medium used was plain agar or potato-dextrose agar. The number of spores after culture is shown in Figure 6. When sweet potato or morning glory leaves were used, many spores were formed. On the other hand, when cherry or hydrangea leaves were used, fewer spores were formed than when sweet potato or morning glory leaves were used. [Industrial Applicability]

[0041] The present invention can be used in the agricultural field and the like.

Claims

1. A method for producing spores of sweet potato root rot fungus, comprising the following steps (1) to (3): (1) A step of inoculating a substrate placed on a solid medium with sweet potato root rot fungus, wherein the substrate is a sheet-like substrate mainly composed of leaves of a plant of the genus Ipomoea or cellulose, and the substrate has been boiled or autoclaved; (2) culturing the sweet potato root rot fungus inoculated in step (1) to form spores; (3) harvesting the spores formed in step (2).

2. A method for producing spores of sweet potato root rot fungus as described in claim 1, characterized in that the boiling treatment is for 5 to 120 seconds, and the autoclave treatment is for 3 to 30 minutes at 100 to 130°C.

3. 2. The method for producing spores of the pathogenic fungus Pseudomonas oryzae according to claim 1, wherein the substrate is a sweet potato leaf or a morning glory leaf.

4. 2. The method for producing spores of the pathogenic fungus of sweet potato root rot according to claim 1, wherein the substrate is filter paper or cellophane.

5. 2. The method for producing spores of the pathogenic fungus Pseudomonas oryzae according to claim 1, wherein the solid medium is an agar medium.

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