JPH0383522A - Mass-production of mycorrhiza bacterium vesicular arbuscula - Google Patents

Abstract

PURPOSE:To produce mycorrhiza bacterium Vesicular arbuscula (abbreviated as VA mycorrhiza bacterium) in high efficiency by culturing hair root in a tank, inoculating VA mycorrhiza bacterium to the hair root in a tank and culturing the bacterium in symbiotic state. CONSTITUTION:Hair root cultured in a tank is inoculated with VA mycorrhiza bacterium (an endotrophic mycorrhiza having sacciform part and arborescent part) in a tank and the bacterium is cultured in symbiotic state. The hair root is a root grown from an infected site of a plant obtained by infecting Agrobacterium rhizogenes to the stalk, leaf, root, etc., of the plant and is generated by the integration of a part of the gene of a macroplasmid (Ri plasmid) existing in Agrobacterium rhizogenes into the gene of the plant. The VA mycorrhiza bacterium is e.g. the strain of genus Gigaspora, Glomus, Sclerocystis, Accaueospora or Entrophospora.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing VA mycorrhizal fungi in large quantities in a tank.

By coexisting with plants, VA mycorrhizal fungi promote the absorption of nutrients (particularly phosphorus) by the symbiotic plants, and protect the symbiotic plants from soil diseases! To date, there have been many reports that it has favorable effects, such as protecting against bacteria. Therefore, if VA mycorrhizal fungi could be industrially produced and coexisted, it would reduce the amount of fertilizer applied to crops, improve resistance to bad cultivation environments,
It is expected that the quality of agricultural products will improve.

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.

There are two main methods for culturing VA mycorrhizal fungi that have been proposed to date:

One is a method in which plant roots and VA mycorrhizal fungi coexist and are cultured under non-sterilized conditions such as in fields, pots, and hydroponics.For this purpose, a suitable culture substrate, such as a material with a porous structure, is used. (Japanese Unexamined Patent Publication No. 60-237987), or to make the culture solution into a thin film during hydroponic cultivation (Japanese Unexamined Patent Publication No. 55-11)
8390) and the like have been developed.

However, with this method, the growth of symbiotic plants was slow, making it difficult to mass-produce VA mycorrhizal fungi, and there was a risk of contamination with microorganisms other than VA mycorrhizal fungi. This is a method in which plants are cultured in containers and coexist with VA mycorrhizal fungi.

In this case, sufficient nutrients can be provided, so in addition to normal plants, organ cultures of well-growing roots (Japanese Patent Publication No. 62-19028), hairy roots (Japanese Patent Publication No. 62-49)
037) can be used as a symbiotic partner of VA mycorrhizal fungi.

Hairy roots are caused by the hairy root disease fungus Agrobacterium rhizogenes.
) is inoculated into the stems, leaves, roots, etc. of a plant, and a part of the gene of the giant plasmid (Ri plasmid) present in Agrobacterium rhizogenes is integrated into the plant's genes in the roots that develop from the infected site. Occur. Hairy roots grow much faster than normal roots, and growth can be achieved without the use of plant hormones, which are required in organ culture of normal roots, so costs can be reduced.

In addition, hairy roots are known to stimulate spore germination and mycelium growth of VA mycorrhizal fungi, and to have many young parts near the root tips, which are easily infected by VA mycorrhizal fungi. Ori,
It has been shown that they are suitable as symbiotic partners. Furthermore, it is known that mass culture of hairy roots can be carried out in an air-lift type fermenter containing a liquid medium (
(Special Publication No. 62-49037).

However, a method using hairy roots, a method for inoculating VA mycorrhizal fungal spores in industrially advantageous mass culture, and a method for culturing in a symbiotic state have not yet been developed.

The purpose of the present invention is to develop a method for coexisting in large quantities with hairy roots and VA mycorrhizal fungi, and to provide an industrial method for producing VA mycorrhizal fungi.

The method for mass-producing VA mycorrhizal fungi of the present invention is characterized by inoculating hairy roots obtained under tank culture with VA mycorrhizal fungi in a tank and allowing them to coexist.

The present invention will be explained in more detail below.

Examples of Acrobacterium rhizogenes that can be used to produce hairy roots used in the present invention include the following.

Agrobacterium rhizogenes ATCC″11N
n25818 Agrobacterium rhizogenes AT
CC” 415834 Agrobacterium rhizogenes ATCC111Nn43057 Agrobacterium rhizogenes NCPPB” NQ8196 Agrobacterium rhizogenes NIAES″13 Hα
1724 Bacteria depository institution name *l) ATCC: American Type C
u1tureCollection, U, S, A.

*2) NCPPB: National Co11
Ection of Plant Pathogenic
Bacteria.

U, K.

*3) NIAES: Ministry of Agriculture, Forestry and Fisheries, National Institute of Agro-Environmental Science and Technology In addition, other bacteria such as Escherichia coli into which the Ri plasmid or a portion thereof has been introduced can also be used.

When plants are treated with Agrobacterium rhizogenes, part of the Ri plasmid (T-DN
A) is introduced (transformed) into the nuclear DNA of a plant cell.

Examples of methods for obtaining transformed hairy roots by introducing Ri plasmid T-DNA into plant stems, roots, leaves, etc. include:
The following methods may be mentioned.

1. Direct inoculation method to individual plants 2. Leaf disc method using leaf discs (R, B, Horsch et al., 5 CIENC
E 227゜1229 (1985)) 3. Co-culture method using plant protoplasts (Z, M, Wel et al., Plant Ce1
1 Rep, +5:93-96 (1986)) 4. Plant protoplasts and 7 Globacterium rhizogenes sphnyloplast method (R, Hain 4It, al., Plant Ce1
l Rap,.

3.60 (1984)) 5. A method in which the Ri plasmid of Agrobacterium rhizogenes or a part of the T-DNA thereof is directly injected into cells by a method such as microinjection. If you install it with
After that, it is necessary to sterilize Agrobacterium rhizogenes, and the following methods are available for this.

1) High temperature treatment (40°C) 2) Antibiotic treatment 3) Rapid cycle transplantation of hairy root tips The following methods are effective for culturing hairy roots obtained by the above methods.

In the present invention, for example, a medium conventionally used for plant tissue culture, that is, an inorganic component and a carbon source as essential components, and at least one or more selected from plant hormones, vitamins, and amino acids. It is possible to use a medium to which the above-mentioned components have been added and other components have also been added as necessary.

Inorganic components in the above medium include nitrogen, zinc, iron, steel,
Molybdenum, boron, phosphorus, cobalt, potassium, calcium, magnesium, sulfur, manganese, chlorine, sodium, iodine, etc., specifically ammonium nitrate, ammonium dihydrogen phosphate, ammonium sulfate, ammonium chloride, sodium nitrate, calcium nitrate. , potassium nitrate, zinc sulfate, ferrous sulfate, ferric sulfate, ethylenediaminetetraacetic acid iron, 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, manganese sulfate, potassium iodide, and the like.

Examples of carbon sources include sucrose and other carbohydrates, derivatives thereof, organic acids such as fatty acids, and primary alcohols such as ethanol.

Plant hormones include auxins such as indoleacetic acid (IAA), naphthaleneacetic acid (NAA), p-chlorophenoxyisobutyric acid, and 2,4-dichlorophenoxyacetic acid (2,4-D), kinetin, and benzyladenine. Examples include cytokinins such as , zeatin, and dihydrozeatin.

Vitamins include biotin and thiamin (vitamin B)
, pyridoxine (vitamin B), pantothenic acid, ascorbic acid (vitamin C), inositol, nicotinic acid, and the like.

Examples of amino acids include glycine, alanine, glutamine, cysteine, and the like.

In addition, natural products such as coconut milk, yeast extract, etc. that are said to contain vitamins, hormones, etc. can also be used.

Although various media containing the above-mentioned components can be used in the present invention, it is preferable to use a liquid medium. It should be noted that the concentrations of the components in the liquid medium can vary within a wide range. Usually, the inorganic component is about 0.1μN to about 100mM, the carbon source is about 1g/Q" 120g/Q, and the plant hormones are about 0.01μA to about 10μH.
degree, vitamins and amino acids, each about 0.
It can be about 1 mg/Q to about 100 mg/Q.

In culturing the hairy roots of the present invention, light is not necessarily necessary, and culturing in the dark is preferable for growth. The culture temperature is preferably about 0°C to about 35°C, particularly about 3°C to about 28°C. This is because the growth rate of hairy roots is low at temperatures below about 10°C, and the growth rate of hairy roots is similarly low at temperatures above about 35°C.

In the present invention, the initial planting of hairy roots in a liquid medium can vary in amount within a wide range. Usually liquid medium 50m
About 10 mg to about 1 g (fresh weight) of hairy roots for Q
It is desirable to plant it in a certain amount.

Various culture devices (tanks) can be used in the present invention. There are various types of culture vessels (fermenters) for culturing microorganisms and animal and plant cells, and in particular, the method of supplying oxygen to the culture, the method of contact with the medium (nutrients), and the method of controlling the medium components. A number of improved culture tanks have been developed. Hairy roots have a morphological characteristic of being much larger than microorganisms, so they cannot be used in culture systems that are subject to large physical shocks, such as culture equipment equipped with stirring blades that rotate at high speed. However, very good growth can be obtained if an appropriate medium and oxygen are supplied and a device that does not impose a large physical impact on the culture is used.

However, at the stage after inoculation of VA mycorrhizal fungi, VA
Since it is difficult for mycorrhizal fungi to proliferate and produce spores, this must be taken into consideration when deciding on the device when culturing is carried out in one device.

The VA mycorrhizal fungi used in the present invention include Gigaspora spp., Glomas spp.
us), Sclerocystis sp.
s), 7 Caulospora (Accaulospora)
, Entrophospora
Examples include.

Among these methods, the method for separating Gigaspora gregaria spores from soil is shown below.

First, soil from the field or potted plants is collected and suspended in water. This is passed through a sieve with a mesh size of 1 to 2 mm to remove large particles, stones, etc., and the passed liquid is passed through a sieve with a mesh size of 0.1 m and 11 mm. 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.

The spores isolated as described above may be cultured on a suitable solid medium before being inoculated into hairy roots for confirmation of sterilization and preliminary germination.

Hairy roots obtained under tank culture as described above and V
In order to have A mycorrhizal fungi coexist in the tank, VA mycorrhizal fungi are inoculated. The VA mycorrhizal fungi to be inoculated are usually spores obtained by the above method or pre-germinated spores, but spores that are mixed with other substances or that are symbiotic with roots or hairy roots can be isolated as is. It is also possible to use it as an inoculum without using it.

Inoculation of VA mycorrhizal fungi in a tank should be carried out 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 material, and injecting it into the tank. I can do it. Furthermore, VA mycorrhizal fungi can also be injected into a tank by dispersing them in a gas such as air and blowing them together.

Furthermore, infection can be facilitated by attaching and fixing a VA mycorrhizal fungi inoculum to hairy roots. This method involves suspending a VA mycorrhizal fungus inoculum in a liquid that gels under certain conditions and forms a form that is easy to adhere to hairy roots.
This is a method in which the product is applied to hairy roots and then gelled by changing the conditions.

For example, a VA mycorrhizal inoculum is suspended in a solution containing sodium alginate, the suspension is applied to the hairy roots, and then a water-soluble calcium aqueous solution such as calcium chloride is applied to the hairy roots for the purpose of gelatinizing the suspension. In this way, the sodium alginate gels, and the VA mycorrhizal fungi adhere to the hairy roots through the gel.

In addition, a VA mycorrhizal fungus inoculum was suspended in an aqueous solution of gellan gum (usually sold as a powder, which is suspended in water and then heated to obtain an aqueous solution), which similarly gels with divalent metal ions. After spraying on the roots, an aqueous ionic solution may be applied. In addition to these, natural polymeric substances such as carrageenan, agar, and chitosan, and synthetic polymeric substances such as polyvinyl alcohol and polyacrylamide can also be used.

Culture devices (tanks) used in the stages after VA mycorrhizal fungus inoculation include the following. What must be noted here is that, as mentioned above, in liquids, 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 medium so that the hairy roots do not remain in the medium at all times. By regularly raising and lowering the water level,
It is possible to provide nutrients to the hairy roots and also to supply the gas phase necessary for the growth of VA mycorrhizal fungi. 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. To adjust the water level,
This is done by installing backup tanks and pipes as appropriate.

A second example is a device using a support that supports hairy roots. In contrast to the first device, the hairy roots are pulled up from the liquid. Using a support capable of supporting the hairy roots and through which the liquid passes, such as a net, the hairy roots 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.

A third example is a device that has a porous structure and is 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 hairy roots grown therein.

A fourth example is a device that provides a liquid medium in the form of water droplets or mist. A nozzle or the like is installed as necessary, and nutrition can be given to the hairy roots even if the hairy roots are not in the liquid.

In addition, any tank can be used as long as it provides both a liquid phase that provides nutrients necessary for hairy roots and a gas phase atmosphere that allows VA mycorrhizal fungi to grow at a high rate. It is preferable to use a tank to culture hairy roots, inoculate VA mycorrhizal fungi, and culture the hairy roots and VA mycorrhizal fungi in symbiosis.

For culturing hairy roots and VA mycorrhizal fungi in a symbiotic state, the medium, culture temperature, and other conditions used for the hairy root culture described above may be maintained as they are.
Or it can be modified and used. Regarding the pH of the medium, a slightly acidic pH of 4 to 7 is preferable for hairy root culture, but a neutral pH of 6 to 8 is preferable for coexistence.

According to the present invention, V.
A mycorrhizal fungi can be efficiently produced. Therefore, the method of the present invention is extremely suitable as an industrial method for producing VA mycorrhizal fungi.

After sterilizing the seeds of Trifolium repens with a disinfectant of sodium hypochlorite solution, they were treated with Murashige Skoog (MS-) containing 3% sucrose.
Agrobacterium rhizogenes (NIAES N[L1724) carrying the Ri plasmid was inoculated onto the stems, leaves, etc. of germinated sterile plants.

After 5 weeks, the hairy roots that had developed from the inoculation site were cut out and transplanted onto a solid medium of MS-3 containing 0.5 g/Q of Kraphoran, and after 2 weeks, they were transplanted onto a new medium with the same composition. 3
This operation was repeated several times to obtain sterilized hairy roots.

MS-3 liquid medium 1Q (for hairy root culture) was placed in the tank of a 2Q airlift type plant culture device (Shibata Vario Glass Co., Ltd. fA). In addition, we assembled a device using a perister pump, a glass tube, and a silicone tube so that the water level of the medium could be changed arbitrarily during culture, and a glass tube containing a modified medium (for symbiotic culture of hairy roots and VA mycorrhizal fungi). I set up the bottle. That is, put 3Q of fresh modified MS liquid medium (altered salt concentration to half, sucrose concentration to 2%, pH to 6) in a glass bottle, connect it to the bottom of the tank with a glass tube and a silicone tube, and insert a pellister in the middle. A pump was installed to move the liquid medium in either direction. A glass filter was installed at the opening at the bottom of the tank to prevent hairy roots from entering the tube. The device was sterilized at 120°C for 60 minutes.

White clover (Trifolium repens)
Approximately 1 g (fresh weight) of hairy roots were planted in the above medium, and 2
The cells were cultured in the dark at 5°C for 20 days.

After sieving the pot-grown alfalfa soil,
Gigaspora gregaria spores were collected under a stereomicroscope and washed 30 times with sterile water to obtain sterile spores. The isolated spores were cultured for 2 weeks on a modified MS solid medium for confirmation of sterilization and preliminary germination.

After hairy root culture, the MS-3 liquid medium for hairy root culture was discarded outside the tank.

The modified MS medium was introduced from the glass bottle into the tank by operating the perister pump, the hairy roots and the inside of the tank were washed, and the medium was discarded.

After repeating this operation three times, 300 mQ of the modified MS medium was introduced into the tank.

1000 Gigaspora gregaria spores, which were confirmed to be sterile and germinated, were suspended in a modified MS medium and sprayed on one hairy root.

After culturing for 5 weeks while replacing the medium every 5 days, the hairy roots inside the tank were taken out and their fresh weight was examined. To investigate the infection rate, 50 divided roots of 1cm1 or more were randomly collected, stained with trypan blue, and examined under a microscope.
This was done by observing the number of infected individuals.

The following experiment was conducted as a comparative example.

The tank used in the example was a glass petri dish with an inner diameter of 15 cm containing 12011Q solid medium, and the other conditions were the same as in the example.

The fresh weight of hairy roots per medium IQ and the VA mycorrhizal infection rate in Examples and Comparative Examples are shown below.

Table 1 In addition, when culturing was continued for 6 months using the method described in the example without measuring weight or investigating the infection rate, a significantly higher number of spores were produced compared to conventional methods using fields, flower pots, etc. It was done.

Claims (1)

[Claims] 1. The hairy roots obtained under tank culture were treated with Vesiquier arbuscular mycorrhizal fungi (endophytic mycorrhizal fungi having a sac-like body and an arbuscular body: hereinafter referred to as VA mycorrhizal fungi). ) A method for mass-producing VA mycorrhizal fungi, characterized by inoculating and allowing them to coexist.