JPH04320676A - Production of fungus of mycorhiza of vesicular-arbuscular - Google Patents

Abstract

PURPOSE:To multiply the title fungus efficiently by adding an oligosaccharide to a medium in proliferation of a fungus of mycorrhiza of vesicular-arbuscular in association with a plant cultured under a condition of in vitro. CONSTITUTION:A fungus of mycorrhiza of vesicular-arbuscular (fungus of mycorrhiza of VA), a fungus of endotropic mycorrhiza having a cystidium and a dendrophysis is multiplied in an oligosaccharide-containing medium in association with a plant cultured under a condition of in vitro. Root tissue, hair root or adventitious root is preferable as the plant cultured under a condition of in vitro. Multiplication of the fungus of mycorrhiza of VA is preferably carried out at pH4-7 at 20-30 deg.C.

Description

Description: [0001] The present invention relates to a method for producing a large amount of VA mycorrhizal fungi by coexisting with plants and VA mycorrhizal fungi. [0002] By coexisting with plants, VA mycorrhizal fungi have favorable effects such as promoting the absorption of nutrients (particularly phosphorus) by the symbiotic plants and protecting the symbiotic plants from pathogenic bacteria in the soil. Many things have been reported so far. Therefore, if VA mycorrhizal fungi can be produced industrially and allowed to coexist, it can be expected to reduce the amount of fertilizer applied to crops, improve resistance to poor cultivation environments, and improve the quality of agricultural products. [0003] However, VA mycorrhizal fungi are obligate symbiotic bacteria that do not proliferate unless they coexist with plants, and cannot be cultured in pure form, making it extremely difficult to develop techniques for efficient mass production. [0004] Methods for culturing VA mycorrhizal fungi that have been proposed to date can be broadly divided into the following two methods. One is
This is a method in which VA mycorrhizal fungi are allowed to coexist with plants and cultured under conditions that are not sterilized, such as in fields, in pots, or in hydroponic cultivation, as described below. (1) In pot culture, plants are cultivated using a material having a porous structure as a culture substrate, and V.
A method for coexisting and multiplying mycorrhizal fungi (Unexamined Japanese Patent Publication No. 60-23
Publication No. 7987). (2) In hydroponic cultivation, a method of growing VA mycorrhizal fungi by making the culture solution into a thin film and constantly supplying the culture medium (Japanese Patent Laid-Open Publication No. 118390/1983)
. (3) A method for promoting the proliferation of VA mycorrhizal fungi by cultivating short-day plants, infecting them with VA mycorrhizal fungi, and further performing short-day treatment in small-capacity pot culture (Japanese Patent Application Laid-open No. 2002-100102-1).
227068). [0006] The second method is to allow VA mycorrhizal fungi and plants to coexist in a sterile container, that is, under in vitro conditions.
This is a method for propagating VA mycorrhizal fungi, and the following reports have been made. (1) A method for coexisting and propagating a root organ culture and VA mycorrhizal fungi (Japanese Patent Application Laid-open No. 19028/1983). (2) A method of coexisting and propagating hairy roots and VA mycorrhizal fungi (Japanese Patent Publication No. 62-49037). (3) A method for coexisting and propagating hairy roots and VA mycorrhizal fungi (G. Becard et al., New Phytol.
108, 211 (1988)). [0008] The term hairy root refers to the hairy root disease fungus Agrobacterium rhizogenes (Agrobacterium rhizogenes).
When Agrobacterium rhizogenes) is inoculated into the stems, leaves, roots, etc. of plants, the roots that grow from the infected area are infected with Agrobacterium.
It is generated when part of the gene of a giant plasmid (Ri plasmid) present in Rhizogenes is integrated into the plant's genes. However, the VA mycorrhizal fungi produced by the first method have the risk of being contaminated by other microorganisms (particularly plant pathogens), and are not suitable for use as a source of infection for crop seedlings. Furthermore, the production amount of VA mycorrhizal fungi was still insufficient. [0010] In addition, in the second method, although there is no risk of contamination by other microorganisms, the production amount of VA mycorrhizal fungi is still insufficient. On the other hand, regarding the effect of carbohydrates on VA mycorrhizal fungi, starch (polysaccharide) has no effect, and starch hydrolyzates such as glucose (monosaccharide) and maltose (
It has been reported that sucrose (disaccharide), and cellulose (polysaccharide) suppressed germ tube growth of VA mycorrhizal fungi (B. Mosse, Trans. Brit
．． mycol. Soc. , 42(3), 27
3 (1959). [0012] However, the effects of these carbohydrates in the symbiotic culture of plants and VA mycorrhizal fungi have not been confirmed;
It has not been possible to find a substance among carbohydrates that has an effect on the growth of mycorrhizal fungi. [0013] Furthermore, there have been several reports on the application of oligosaccharides to the cultivation of plants, as described below. (1) Cultivation method of useful plants using alginate oligosaccharide (Unexamined Japanese Patent Publication No. 6
3-101302). (2) Fruit quality improvement method using alginic acid oligosaccharide (Japanese Patent Application Laid-Open No. 1983-226220)
. (3) A method for cultivating plants using oligosaccharides that promote plant growth (Japanese Patent Application Laid-Open No. 63-215606). (4) A method for cultivating plants using a decomposed product of a polysaccharide produced by a microorganism or an oligosaccharide which is its main component (Japanese Patent Laid-Open Publication No. 79101/1982). (5) Plant growth promoter containing oligosaccharide (JP-A-1-319406). However, it is still difficult to use oligosaccharides.
There are no known examples of its application to methods for producing A mycorrhizal fungi. [0016] Problems to be Solved by the Invention The present invention relates to plants and VA.
The purpose of the present invention is to provide an industrial method for producing VA mycorrhizal fungi that can coexist with mycorrhizal fungi and multiply efficiently and can be supplied in large quantities. [Means for Solving the Problems] The method for producing VA mycorrhizal fungi of the present invention provides a method for coexisting and propagating VA mycorrhizal fungi with plants cultured under in vitro conditions, in which the medium contains oligosaccharides. Characterized by urging. Embodiments of the invention The plants used in the present invention are
Cultured under in vitro conditions, including tissue and organ cultures. Examples include tissues such as plants and calluses grown by aseptic seeding, and organ cultures such as hairy roots, adventitious roots, and adventitious buds. Since VA mycorrhizal fungi usually infect and proliferate in the roots of plants, among the above, sterile plants having root tissues and organs,
Hairy roots and adventitious roots are preferred. In the present invention, for culturing plants under in vitro conditions, for example, a medium conventionally used for plant tissue culture, that is, an inorganic component and a carbon source are essential components,
A medium can be used in which at least one component selected from plant hormones, vitamins, and amino acids is added, and other components are also added as necessary. [0021] Inorganic components in the above medium include nitrogen, zinc, iron, copper, molybdenum, boron, phosphorus, cobalt, potassium, calcium, magnesium, sulfur, manganese, chlorine, sodium, iodine, etc. Ammonium nitrate, ammonium dihydrogen phosphate, ammonium sulfate, ammonium chloride, sodium nitrate, calcium nitrate, potassium nitrate, zinc sulfate, ferrous sulfate, ferric sulfate, ethylenediaminetetraacetate, copper sulfate, molybdic acid, sodium molybdate , boric acid, phosphoric acid, monosodium phosphate, potassium phosphate, disodium phosphate, trisodium phosphate, cobalt chloride, potassium chloride,
Calcium chloride, magnesium sulfate, sodium sulfate,
Examples include manganese sulfate and potassium iodide. Carbon sources include sucrose and other carbohydrates, their derivatives, organic acids such as fatty acids, and monomers such as ethanol.
Examples include alcohols such as alcohol. Plant hormones include indole acetic acid (I
AA), naphthaleneacetic acid (NAA), p-chlorophenoxyisobutyric acid, 2,4-dichlorophenoxyacetic acid (2,
Examples include auxins such as 4-D), and cytokinins such as kinetin, benzyladenine, zeatin, and dihydrozeatin. Examples of vitamins include biotin, thiamine (vitamin B1), pyridoxine (vitamin B6), pantothenic acid, ascorbic acid (vitamin C), inositol, and nicotinic acid. Examples of amino acids include glycine, alanine, glutamine, and cysteine. In addition, natural products said to contain vitamins, hormones, etc., such as coconut milk and yeast extract, can also be used. [0026] In the present invention, the concentrations of the components in the medium can be varied within a wide range. Usually, the inorganic component is about 0.1μ
M ~ about 100mM, carbon source about 1g/l ~ 120g
The amount of plant hormones can be about 0.01 μM to about 10 μM, and the amount of vitamins and amino acids can be about 0.1 mg/l to about 100 mg/l. [0027] The pH of the medium is preferably weakly acidic, with a pH of 4 to 7. In the present invention, the medium may be solid or liquid, and when a solid medium is used, examples of medium solidifying agents include agar, gellan gum, and the like. It is also possible to culture using a culture carrier (porous material, artificial soil, etc.) impregnated with a liquid medium. [0028] In the present invention, the culture temperature is preferably about 10°C to about 40°C, particularly about 20°C to about 30°C. about 10
This is because the growth rate is low at temperatures below 40°C, and the growth rate is similarly low at temperatures above about 40°C. [0029] VA mycorrhizal fungi used in the present invention include Gigaspora, Glomus, and Sclerocystis.
ocystis), Acaulospora (Accaulo
spora), Entrophospora sp.
spora), Scutellis
pora), etc. [0030] Spores of VA mycorrhizal fungi can be collected from wild plants, soil near the roots of cultivated plants, roots of hydroponic plants, and container cultures such as hairy roots. Of these,
The method for isolating Gigaspora margarita spores from soil is shown below. First, soil from a field or a potted plant is collected and suspended in water. This is passed through a 1-2 mm mesh sieve to remove large debris, stones, etc., and the passed liquid is passed through a 0.1 mm mesh sieve. What remains on the 0.1 mm mesh sieve is collected after washing with running water, suspended in a small amount of water, and transferred to a petri dish. This is separated into dirt and spores under a stereomicroscope, and only the spores are sucked up with a pipette and transferred to another petri dish. After repeating this operation three times and transferring it to a centrifuge tube, add water and apply ultrasonic waves for several seconds to disperse the debris, and then discard the water to wash the spores. Repeat this several times. The washed spores are sterilized by washing them 20 to 30 times with sterile water, or by using antibiotics such as streptomycin, various disinfectants, and the like. Of course, this is not necessary for spores obtained under sterile culture. [0032] The spores isolated as described above may be cultured on a suitable solid medium before being inoculated into plants for confirmation of sterilization and preliminary germination. [0033] This VA mycorrhizal fungus and the in vitro plant obtained as described above are allowed to coexist and propagate. VA mycorrhizal fungi as a symbiotic inoculum are usually spores obtained by the above method or pregerminated spores, but they can also be used as a mixture with other substances or in symbiosis with roots or hairy roots. It is also possible to use the product as it is as an inoculum without separating it. [0034] VA mycorrhizal fungi can be inoculated by suspending the VA mycorrhizal fungi inoculum in a liquid such as water or a buffer solution, or in a powder or granular material such as diatomaceous earth, and bringing it into contact with the plant. This can be done by placing it near the [0035] In the present invention, oligosaccharides are contained in the medium when VA mycorrhizal fungi and plants (including tissue and organ cultures) cultured under in vitro conditions are cultured in symbiosis. Oligosaccharides are composed of about 3 to 10 monosaccharide molecules linked together, and can be obtained by decomposing polysaccharides with enzymes or acids. Oligosaccharides are a type of monosaccharide,
Classified by type of raw polysaccharide, number of bonds, method of bonding, etc.
It is named. The oligosaccharides used in the present invention include fructooligosaccharides, isomalto-oligosaccharides, gentio-oligosaccharides, galactooligosaccharides, xylooligosaccharides, alginate oligosaccharides, soybean oligosaccharides, polygalacturonic acid oligosaccharides, pectin oligosaccharides, mannan oligosaccharides, Examples include glucomannan oligosaccharides, agarooligosaccharides, cellooligosaccharides, fucoidin oligosaccharides, gum arabic oligosaccharides, and carrageenan oligosaccharides. Among these, fructooligosaccharides, isomalto-oligosaccharides, soybean oligosaccharides, and gentio-oligosaccharides, which are particularly suitable for the growth of VA mycorrhizal fungi, are preferred. Note that the oligosaccharide may be added to either or both of the plant or the culture medium for VA mycorrhizal fungi alone before coexistence with VA mycorrhizal fungi. The concentration of oligos used in the present invention can vary within a wide range;
It is preferable that the medium contains at least one of these types at a concentration of 0.01 to 20% by weight, more preferably 0.1 to 10% by weight. Other carbon sources, such as sucrose, may or may not be included. [0039] Medium components other than oligosaccharides can be used in the same manner as the above-mentioned plant culture medium, or may be modified. Furthermore, the pH of the medium, culture temperature, and other culture conditions can be the same as those used for culturing the plants, or can be used after modification. [0040] The oligosaccharide of the present invention does not realize an improvement in the production of symbiotic VA mycorrhizal fungi only by its hitherto known effect of promoting plant growth. In fact, an experiment was conducted in which the amount of plant growth was made almost the same by adjusting the amount of carbon source when using a medium containing oligosaccharides and when using a medium containing no oligosaccharides. The results showed that the growth rate of VA mycorrhizal fungi was significantly higher when using a medium containing oligosaccharides. [0041] Therefore, the fact that oligosaccharides promote the proliferation of VA mycorrhizal fungi cannot be explained by the previously known plant growth promoting effect. Furthermore, the effects of oligosaccharides at stages other than symbiotic culture with plants, such as spore germination and early mycelial growth of VA mycorrhizal fungi, cannot be found. [0042] This time, the usefulness of oligosaccharides was discovered for the first time by using them for symbiotic culture of plants and VA mycorrhizal fungi under in vitro conditions. In the present invention,
The symbiotic culture of plants and VA mycorrhizal fungi can be carried out using various culture methods. For example, the symbiotic culture is carried out as follows. [0043] As a method of co-cultivating plants obtained by aseptic seeding on a solid medium, in a sterilized container (bottle, flask, column, etc.),
Aseptically inoculate a gel medium such as agar medium or a culture carrier (porous material, artificial soil, etc.) impregnated with a liquid medium, and inoculate mycorrhizal fungi at the same time or before or after, allowing them to coexist and grow mycorrhizal fungi. There is a method for propagating. At this time, the oligosaccharide-containing medium is renewed or transplanted to a fresh medium as appropriate. [0044] Even if a root organ culture (adventitious root, hairy root) is used, VA mycorrhizal fungi can be grown by culturing it on a solid medium under sterile conditions and renewing the medium containing oligosaccharides as appropriate. I can do that. [0045] The following types of culture containers and devices can be considered when culturing is carried out using a liquid medium. What must be noted here is that in liquid, VA
Mycorrhizal fungi have difficulty multiplying and producing spores, and require a gas phase. The first example is an apparatus for culturing while controlling the water level of a liquid medium. In other words, it is a device that can lower the water level of the culture medium so that the entire plant does not remain in the culture medium at all times. In this case, the height of the device is reduced and the bottom area is increased. However, it is possible to create a device with multiple culture tanks by stacking the devices in several stages or by partitioning the inside of the device horizontally. Adjustment of water level is done by installing backup tanks and pipes as appropriate. A second example is a device using a support for supporting plants. In contrast to the first device, the plant is lifted out of the liquid. Using a support such as a net capable of supporting the plants and through which the liquid passes, the plants are lifted out of the liquid and brought into contact with the gas phase. The support may be moved up and down or rotated in a direction perpendicular to the water surface. In this case, a device capable of controlling the water level of the medium as described above may be used. The third example is a device having a porous structure and filled with a substance such as vermiculite that can hold a gas phase and a liquid phase inside. These porous materials can supply a gas phase and a liquid phase (liquid medium) to the plants grown therein. The fourth example is a device that provides a liquid medium in the form of water droplets or mist. Nozzles etc. can be installed as needed. Moreover, it can also be combined with each of the above-mentioned devices and methods. Other culture devices that are capable of introducing a gas phase can be used. Effects of the Invention According to the method of the present invention, symbiosis and proliferation of VA mycorrhizal fungi with plants can be effectively carried out under in vitro conditions, making it possible to produce VA mycorrhizal fungi on an industrial scale. Furthermore, the present invention is extremely suitable as a method for producing VA mycorrhizal fungi since it is possible to obtain VA mycorrhizal fungi that are free from contamination with other microorganisms (particularly plant pathogenic bacteria), which has been a drawback up to now. [Example] Example 1: Mass propagation of VA mycorrhizal fungi using sterile plants [0051] Seeds of white clover, eggplant, onion, and asparagus were surface sterilized and treated with MS containing 3% sucrose (Murashige). Skoog) agar medium. Among the germinated seeds, those free from bacterial contamination were selected and transplanted into a rod bottle (diameter 40 mm, height 130 mm) containing 80 ml of artificial soil (Perlite No. 5) impregnated with a medium, and at the same time, surface sterilized Gigaspora Five margarita spores were inoculated per bottle. The culture conditions at this time were a modified RC medium containing 1% fructooligosaccharide at 25° C. and a 16-hour light period and an 8-hour dark period for 3 months. During the culture period, the medium was renewed as appropriate. As a comparative example, culturing was carried out in the same manner as above except that the culture medium was an RC modified medium containing 1% sucrose. In both Examples and Comparative Examples, three rod bottles were used and cultured repeatedly. [0053] As a result, a large amount of new spores were produced in all of the plants in the examples. In the comparative example, new spores were also produced, but the number was not large. The number of newly produced spores is shown in Table 1 (average value for both Examples and Comparative Examples). [Table 1] Table 1: Number of spores produced Number of newly produced spores (pcs/bottle)
White clover Eggplant Onion Asparagus Example 1
923 816
738 711 Comparative example
261 285
227 236
Example 2: Mass production of VA mycorrhizal fungi using hairy roots (1) A medium (RC modified Medium: 5
Tomato or Hyostiamus albus hairy roots were cultured in liquid swirl for 2-3 weeks in 100 ml flasks. The grown hairy roots were treated with the previously sterilized Gigaspora margarita spores per flask.
Individually inoculated. Furthermore, the culture was carried out for 3 months while updating the medium as needed, and an attempt was made to produce VA mycorrhizal fungi. [0056] In a comparative example, culture was carried out using an RC modified medium to which only sucrose (3%) was added. In both Examples and Comparative Examples, five flasks were used and culture was repeated. [0057] [Table 2] [0058] As a result, new spores were produced in both tomatoes and Hyostiamus albus in Examples, regardless of the type of oligosaccharide. The numbers of newly produced spores in the examples are shown in Tables 3 and 4 (each flask 5
average number of books). On the other hand, in the comparative example, spore production was confirmed only in the hairy roots of tomatoes, and the number of spores was as low as 19 per flask. [Table 3] Table 3: Number of spores produced


Experiment 1
Experiment 2

Tomato Hiyos Tomato Hiyos Fructooligosaccharide
102 pieces 76 pieces
160 pieces 74 pieces Isomaltooligosaccharide 115 pieces 52 pieces 89 pieces 71 pieces
Soybean oligosaccharide 113 pieces
67 pieces 138 pieces
98 gentiooligosaccharides 12
8 pieces 39 pieces 121
[Table 4] Table 4: Number of spores produced


Experiment 3
Experiment 4

Tomato Hiyos Tomato Hiyos Fructooligosaccharide
144 pieces 69 pieces
129 70 Isomaltooligosaccharides 132 40 154 56
Soybean oligosaccharide 118 pieces
41 pieces 134 pieces
43 gentiooligosaccharides 10
9 pieces 56 pieces 117
Example 3: Mass production of VA mycorrhizal fungi using hairy roots (2) When producing VA mycorrhizal fungi using tomato hairy roots, four types of oligo The effects of using two types of sugar together were investigated. The combinations and concentrations of oligosaccharides used were as shown in Table 5, and the culture method was carried out according to Example 2. Furthermore, in a comparative example, culture was performed using an RC modified medium to which only sucrose (2%) was added (repetition: 3 bottles each). [Table 5] Table 5: Combinations and concentrations of oligosaccharides in the medium

Fructooligo Isomaltooligo Soybean oligo Genthiooligo Experiment 1 1% 1
% −
- Experiment 2 1%
-1%
- Experiment 3 1%
−
- 1% Experiment 4
-1%
1%
- Experiment 5 -
1% -
1% Experiment 6 -
−
1% 1% As a result, as shown in Table 6 below, in all combinations, it was confirmed that spore production was about 4 to 5 times that of the comparative example (20 spores/flask) (average of 3 flasks each). [Table 6] Example 4: V using hairy roots
Mass production of A mycorrhizal fungi (3) Tomato hairy roots were cultured in an RC modified medium supplemented with oligosaccharides in a 3 liter tank to attempt mass production of VA mycorrhizal fungi. Tomato hairy roots, which had been precultured in a 100 ml flask containing RC modified medium (1% fructooligosaccharide) and allowed to coexist with VA mycorrhizal fungi (Scuterispora gregaria), were placed in an air lift containing perlite (1500 ml). A 4 liter container containing RC modified medium (flask oligosaccharide) was transferred to a 3 liter tank of the type and connected to the tank.
The medium was supplied from the medium bottle using a perister pump, and cultured for 3 months. As a result, 36
18 spores were produced. Example 5: Mass production of VA mycorrhizal fungi using adventitious roots Tomato seeds were surface sterilized and sown on an MS agar medium containing 3% sucrose. Among the germinated seeds, those free from bacterial contamination were selected, the roots were cut out, and the seeds were statically cultured for 3 weeks on a 1/10 concentration MS agar medium containing 1% fructooligosaccharide using a petri dish. The grown adventitious roots were inoculated with the previously sterilized Gigaspora margarita spores, 10 per charm. In a comparative example, the culture was carried out in the same manner as described above, except that the culture medium was a 1/10 concentration MS agar medium containing 1% sucrose. In both Examples and Comparative Examples, 5 Petri dishes were used and culture was repeated repeatedly. As a result, 24 new spores were newly produced per petri dish in Example 5 weeks after spore inoculation (average number of each 5 petri dishes). On the other hand, in the comparative example, the number was as small as 5.

Claims (1)

[Claims] Claim 1: Vesicular and arbuscular mycorrhizal fungi (endophytic mycorrhizal fungi having a sac-like body and an arbuscular body: hereinafter referred to as VA mycorrhizal fungi) coexist with plants cultured under in vitro conditions. 1. A method for producing VA mycorrhizal fungi, which comprises containing an oligosaccharide in a medium during the propagation.