JP2024043828A - Amaranthaceae seedlings, their production methods, and fungal culture materials - Google Patents

Abstract

[Problem] To enable stable growth of seedlings of the Amaranthaceae family. [Solution] A fungus belonging to Cladophialophora chaetospira is cultivated in a culture material mainly made of a wood medium modified by adding ferrous sulfate and citric acid to wood chips of uncomposted coniferous trees. The culture material may be mainly made of a wood medium obtained by blasting uncomposted coniferous trees by crushing them with an extruder. Seedlings of plants of the Amaranthaceae family are raised using these culture materials. [Selected drawing] Figure 3

Description

The technology disclosed relates to seedlings (young plants before transplantation) of the Amaranthaceae family, a method for producing the seedlings, and a culture medium for a fungus suitable for producing the seedlings.

Examples of plants in the Amaranthaceae family include spinach, chard, amaranth, and sugar beet. Spinach is widely used as a food ingredient. Chard and amaranth are also used as ingredients, although to a lesser extent than spinach.

Sugar beets, also known as sugar beets, are used as a raw material for sugar (beet sugar). Unlike sugar cane, sugar beets are relatively resistant to the cold, so they are mainly cultivated in cold regions. Other plants in the Amaranthaceae family include globe amaranth and celosia.

By the way, soil is inhabited by a wide variety of microorganisms such as bacteria and fungi. It is known that many plants coexist with these microorganisms in a mutually complementary manner.

The use of such symbiotic microorganisms has been used to promote the growth of specific plants and to impart resistance to diseases and the environment. VA mycorrhizal fungi are widely known as a typical example.

When VA mycorrhizal fungi infect a plant, VA mycorrhizae are formed on the plant. The formation of VA mycorrhizae has been shown to have effects such as improving nutrient absorption and developing resistance to diseases and the environment. Therefore, various studies have been conducted regarding the use of VA mycorrhizal fungi for plant cultivation (Patent Document 1, Non-Patent Document 1).

Regarding the disclosed technology, a technology is disclosed in which tomato seedlings are inoculated with root endophyte (a strain belonging to Veronaeopsis simplex with the receipt number NITE AP-01933) to suppress the absorption of radioactive cesium. (Patent Document 2). Endophytes are symbiotic microorganisms that can promote the growth of certain plants or confer resistance to diseases and the environment.

The applicant has been developing woody soil suitable for agriculture, horticulture, etc., and has disclosed various technologies so far. For example, Patent Document 3 discloses that by containing iron ammonium citrate, a woody soil (a soil made from uncomposted wood) in which plants can grow well can be obtained.

Japanese Patent Publication No. 7-61257 Patent No. 6315195 Patent No. 6469142

Makoto Ogawa, “VA mycorrhizae and their functions,” Forest Situation XXX (2), 1988, P57-65

VA mycorrhizal fungi infect many plants and form VA mycorrhizae, but it is known that they rarely infect and do not form VA mycorrhizas on some limited plants. .

For example, Patent Document 1 lists plants of the Brassicaceae and Chenopodiaceae families as plants that are difficult to infect with VA mycorrhizal fungi (currently, Chenopodiaceae has been integrated into Amaranthaceae, forming the subfamily Amaranthaceae).

Furthermore, Non-Patent Document 1 lists Urticaceae, Polygonaceae, Chenopodiaceae, Amaranthaceae, Arunaceae, Portulaceae, Caryophyllaceae, and Brassicaceae as herbaceous plants that do not produce VA mycorrhizae.

In other words, it is difficult for Amaranthaceae plants such as spinach to effectively utilize VA mycorrhizal fungi. As a result, the growth of Amaranthaceae seedlings may be unstable due to the growing conditions.

For example, when spinach seedlings are grown in soil with high salinity, their growth is easily inhibited by the salt concentration, and when these seedlings are exposed to high temperatures due to abnormal weather, their growth may be inhibited by temperature damage.

Incidentally, there is a fungus that is classified into the genus Cladophialophora chaetospira (hereinafter also referred to as C. chaetospira). Fungi that belong to C. chaetospira may be useful as endophytes.

Therefore, C. Bacteria belonging to chaetospira have been isolated experimentally, but there is a problem in that hyphae are difficult to grow in a general organic medium mainly composed of peat moss, that is, it is difficult to culture. C. At present, cultivation methods and utilization methods that will lead to practical use of bacteria belonging to chaetospira have not been sufficiently studied.

Therefore, the main objective of the disclosed technology is to provide Amaranthaceae seedlings that can achieve stable growth.

One of the disclosed techniques relates to a culture material for culturing bacteria belonging to Cladophialophora chaetospira, in which uncomposted conifer wood chips are modified by containing iron sulfate and citric acid. It is characterized by being mainly made of woody medium. Alternatively, the main medium may be a woody medium that has been subjected to blast treatment by crushing non-composted coniferous wood with an extruder.

As mentioned above, C. The fungi belonging to Chaetospira have difficulty growing hyphae in a general organic medium mainly composed of peat moss, but the present inventors have found that hyphae of fungi belonging to the genus Chaetospira are difficult to grow using culture materials made from these modified or blasted products. It was found that the cells can be easily expanded, that is, can be efficiently cultured.

Moreover, these modified or blasted culture materials are mainly made from uncomposted wood, so the required amount can be obtained in a short time when needed. In other words, they are easy to handle and highly convenient.

Another disclosed technology relates to seedlings of the Amaranthaceae family, and is characterized in that the roots are inoculated with a fungus belonging to Cladophialophora chaetospira.

Although the details will be described later, C. The effect of imparting salt tolerance was observed by inoculating bacteria belonging to Chaetospira. That is, the seedlings of the Amaraceae family are C. Bacteria belonging to chaetospira can be effectively utilized.

If the roots of Amaranthaceae seedlings are infected with a fungus belonging to C. chaetospira, not only will they be salt-tolerant, but it is expected that they will also be able to confer various resistances. Therefore, it may be possible to grow Amaranthaceae seedlings stably even under conditions where conventional cultivation methods would not produce good growth.

Another disclosed technology relates to a method for producing seedlings of the Amaranthaceae family, characterized by including a step of inoculating the roots of the seedlings with a fungus belonging to C. chaetospira.

In the above step, a process of growing seedlings using a specified culture material in which the bacteria has been cultured may be performed, or a process of attaching an aqueous solution containing the bacteria to the roots of the seedlings may be performed.

Once on the roots of these seedlings, C. Once infected with a bacterium belonging to chaetospira, it becomes possible to obtain advantageous effects such as growth promotion and resistance through symbiosis with this bacterium.

It is preferable to use the above-mentioned modified or blasted product as the specified culture material. This makes it easy to handle and allows bacteria belonging to C. chaetospira to be cultured efficiently.

The above-mentioned bacterium may be the bacterium deposited with the designated institution by the present inventors with receipt number: NITE AP-03738.

This bacterium is effective against C. amaranthaceae seedlings. It has been identified as a bacterium of the genus Chaetospira, and is also available. Therefore, the disclosed technology can be easily applied.

According to the disclosed technology, seedlings of the Amaranthaceae family can be stably grown even under inappropriate growth conditions, so the growth performance of edible plants such as spinach and ornamental plants such as celosia can be improved.

FIG. 3 is a diagram for explaining a method for measuring the elongation rate of hyphae. It is a graph summarizing the measurement results of the elongation rate of hyphae. It is a graph showing the results of cultivation test 1. It is a graph showing the results of cultivation test 2.

The disclosed technology will be explained below. However, the following description is merely illustrative in nature.

<Amaryllidaceae seedling, fungus belonging to C. chaetospira>
The present inventors focused on fungi belonging to C. chaetospira and conducted a test in which they inoculated seedlings of the Amaranthaceae family with fungi belonging to C. chaetospira.

As a result, the seedlings were found to have a growth-promoting effect even under harsh conditions of high salt concentrations. The specific test results are explained below.

(Preparation of bacteria)
C. chaetospira is a fungus. There are several strains of C. chaetospira. Among them, specific strains available include a strain called "MNB12" (also called "MNB12 strain"), a strain called "H4007" (also called "H4007 strain"), and a strain called "OGR3" (also called "OGR3 strain").

The "MNB12 strain" was discovered by the present inventors and has been deposited by the present inventors at the National Institute of Technology and Evaluation's Patent Microorganisms Depositary (NPMD) (received on August 30, 2022, accession number: NITE AP-03738).

"H4007 strain" is registered with the Agricultural and Biological Resources Genebank under MAFF number 238955. "OGR3 strain" is also registered with MAFF number 238957. Therefore, these three strains are available through designated institutions.

These three strains, ie, MNB12 strain, H4007 strain, and OGR3 strain, were used in the test.

(Preparation of bacteria)
Cornmeal agar: 8.5g, malt extract: 10g, yeast extract: 1.0g, and agar: 10.5g were dissolved in 1L of water, and after high-pressure steam sterilization (121℃, 20 minutes), 20mL each was prepared. The mixture was dispensed into sterile petri dishes to prepare an agar medium. Bacteria were inoculated onto these agar media and cultured for two weeks at a temperature of 25°C in a culture chamber.

(Preparation of base material)
Mushrooms, which are representative fungi, are mass-produced through fungal bed cultivation using sawdust from coniferous trees and culture material supplemented with rice bran. However, in tests conducted by the present inventors, C. Bacteria belonging to Chaetospira (for example, MNB12 strain) could not be cultured well.

In addition, peat moss is sometimes used as a substitute for sawdust. We tried replacing sawdust with peat moss, but even with a culture medium mainly made of peat moss, bacteria belonging to C. chaetospira (e.g., MNB12 strain) could not be cultivated well.

Therefore, as a result of studying a new culture material, the present inventors added a small amount of iron sulfate and a small amount of citric acid to the wood chips formed by crushing coniferous wood, thereby modifying the wood chips. By doing so, C. It has been found that a woody medium suitable for culturing fungi belonging to Chaetospira can be obtained.

In the test, the woody medium was used as a sample as the base material for the culture material (Example 1). Specifically, cedar scraps were cut and crushed using a cutter mill. The material was sieved through a 4 mm mesh to obtain crushed wood (wood fragments) having a size of 4 mm or less that passed through the mesh. Note that the definition of mesh size here is equivalent to the nominal opening of a test sieve specified in JIS Z 8801-1.

The obtained wood chips were subjected to a modification treatment to produce the above-mentioned woody medium. In the modification treatment, 0.15 wt% of iron sulfate heptahydrate, 0.02 wt% of citric acid, 0.10 wt% of ammonium bicarbonate, and 0.10 wt% of potassium carbonate were added to the bone dry weight of the wood chips. % and 0.30 wt% of surfactant were added, respectively.

The inventors have also found that C. It has been found that a woody medium suitable for culturing fungi belonging to Chaetospira can be obtained.

In the test, the woody medium was used as the base material for the culture material (Example 2). That is, the above-mentioned woody medium was prepared by crushing cedar wood scraps with an extruder. By processing with an extruder, the cedar offcuts are crushed or ground under high pressure and then discharged to atmospheric pressure. As a result, a woody medium in a state different from that obtained by pulverization by cutting with a cutter mill or the like is obtained.

In the test, as a comparative example, wood chips that were simply crushed without any modification treatment were used as a sample (Comparative Example 1). In addition, peat moss (unmodified peat moss manufactured by Emata Co., Ltd.), which is a common substrate for culture materials, was also used as a comparative example sample (Comparative Example 2).

(Preparation of culture material)
A culture material was prepared by adding a predetermined amount of nutrients (bran) and water to each sample of Examples and Comparative Examples. Specifically, rice bran and bran (wheat bran) were mixed in a predetermined ratio to each sample, and a predetermined amount of water was added.

Specifically, in each sample for MNB12 strain and H4007 strain, sample: rice bran: bran were mixed at a ratio of 6:2:2 (volume ratio). By adding water to the mixture, the water content was adjusted to 215% based on the absolute dry weight.

In the sample for the OGR3 strain, sample: rice bran: bran were mixed at a ratio of 8:1:1 (volume ratio). The water content was adjusted by adding water to the mixture so that the water content was 195% based on the absolute dry weight.

Each sample (culture material) thus obtained was sterilized by high-pressure steam in an autoclave (121°C for 40 minutes), and then 30 mL of each was dispensed into sterile petri dishes and used for testing.

(Inoculation and cultivation of bacteria)
From the colonies formed by culturing on the agar medium, a bacterial mass and agar medium were cut out with a needle with a handle to a size of about 5 mm in diameter, and placed on the surface of each sample to inoculate the bacteria. Each sample inoculated with the bacteria was cultured in an incubation chamber at a temperature of 25°C for 15 days.

(Measurement and results of hyphal elongation speed)
As shown in FIG. 1, after culturing, two reference lines were set on the lid of the sterilized Petri dish for each sample so as to be perpendicular to each other above the approximate center of the colony. Based on these reference lines, the diameter of the colony was measured as shown by the arrow in FIG.

The average daily mycelial elongation rate (mm/day) for each sample was calculated from the initial value (diameter of the bacterial mass at the time of inoculation) and the culture period (15 days). For Comparative Example 2, only the MNB12 strain was used. The results are shown in Figure 2.

In the case of MNB12 strain, in Comparative Example 1 (woody medium without modification treatment), the average elongation rate of hyphae was 0.31 (mm/day). In Comparative Example 2 (peat moss), the rate was 0.08 (mm/day), and almost no hyphal elongation was observed.

In contrast, in Example 1 (modified wood medium), the average hyphae growth rate was 0.63 (mm/day) or more. In Example 2 (exploded wood medium), the average hyphae growth rate was 0.71 (mm/day) or more.

In the case of the H4007 strain, the average elongation rate of hyphae in Comparative Example 1 was 0.76 (mm/day). In contrast, in Example 1, the average elongation rate of hyphae was 0.86 (mm/day) or more. Furthermore, in Example 2, the average elongation rate of hyphae was 0.81 (mm/day) or more.

In the case of OGR3 strain, in Comparative Example 1, the average elongation rate of hyphae was 0.41 (mm/day). In contrast, in Example 1, the average elongation rate of hyphae was 0.46 (mm/day) or more. Moreover, in Example 2, the average elongation rate of hyphae was 0.53 (mm/day) or more.

Although there were differences in the degree, the mycelium elongation rate increased in the Examples compared to the Comparative Examples for all strains. From these test results, it was confirmed that fungi belonging to C. chaetospira can be clearly cultivated well by using a culture material based on a wood medium that has been modified or crushed.

(Mass culture of bacteria)
By increasing the amount of culture material for inoculating bacteria, a large amount of C. Bacteria belonging to Chaetospira can be cultured. In other words, C. It becomes possible to put bacteria belonging to Chaetospira and culture materials thereof into practical use.

For example, similar to the above, the culture medium is adjusted so that the ratio of wood medium:rice bran:bran is 6:2:2 (volume ratio) or 8:1:1 (volume ratio), and the culture medium is placed in a container that can be sterilized at high temperature and high pressure, such as a glass bottle. With a lid placed on the container so that it is not airtight, the container is sterilized by high pressure steam (121°C for 90 minutes). After cooling, the culture medium is inoculated with bacteria.

The container is left unsealed in a clean place where the temperature can be kept at about 25°C. If the container is then cultured for 28 days or more, a large amount of culture material containing the bacteria belonging to C. chaetospira can be obtained.

(Inoculation of Amaranthaceae seedlings with fungi)
A test was carried out in which seedlings of the Amaranthaceae family were inoculated with a fungus belonging to C. chaetospira.

C. After thoroughly pulverizing and mixing the culture material in which each of the bacteria belonging to Chaetospira grew using a sterilized miller, the culture material was added to a predetermined sterilized organic culture soil at a volume ratio of 10%. This was used as culture soil for testing (Example).

Specifically, the culture soil using MNB12 strain was designated as "Example 1", the culture soil using H4007 strain was designated as "Example 2", and the culture soil using OGR3 strain was designated as "Example 3". . A control sample to which sterile culture material was added was used as a control (comparative example).

In the test, spinach (Spinacia oleracea), sugar beet (Beta vulgaris ssp. vulgaris), and amaranth (Amaranthus cruentus) were used as plants of the Amaranthaceae family.

These various seeds were prepared and transplanted into each culture soil. In each example, the seedlings were inoculated with the fungus.

(Cultivation test 1)
A spinach cultivation test was conducted using the culture soil (Example 1) using the MNB12 strain.

Each sample (cultivating soil) in which seeds were sown was cultivated for 3 weeks under constant temperature conditions of 25°C. During the cultivation period, an LED light for cultivation was used to periodically turn on and off the light so that the daylight hours were 14 hours.

In cultivation tests, plants were subjected to high salt concentration stress. Specifically, spinach was grown and germinated under constant temperature conditions of 25°C. Then, when the true leaves opened, 50 mL of a sodium chloride aqueous solution (17.5 g/L) of a predetermined concentration was given. Furthermore, one week after the true leaves opened, 50 mL of a sodium chloride aqueous solution with the same concentration as before was given, and cultivation was continued for two weeks after the true leaves opened.

(Cultivation test results)
The results of the cultivation test are shown in Figure 3. The numerical values shown in FIG. 3 indicate the amount of growth of seedlings of each sample after cultivation. Specifically, at the end of the test, the dry weight of the above-ground portion of each of the eight individuals in each sample was measured. The average value (dry weight average value) of the measured values was defined as the growth amount.

As shown in FIG. 3, in the case of spinach, the amount of growth in the example was 459.8 mg, while the amount of growth in the comparative example was 217.5 mg.

In the spinach test, it was confirmed that the growth of seedlings was promoted more in the example than in the comparative example.

(Cultivation test 2)
Cultivation tests of plants of the Amaranthaceae family, including spinach, were carried out using the culture soils (Examples 1 to 3) in which each strain was used.

Each sample (cultivating soil) in which seeds were sown was cultivated for 4 weeks under a constant temperature condition of 25°C. During the cultivation period, an LED light for cultivation was used to periodically turn on and off the light so that the daylight hours were 14 hours.

In cultivation tests, plants were subjected to high salt concentration stress. Specifically, each plant was grown and germinated under constant temperature conditions of 25°C. Then, when the true leaves opened, 50 mL of a sodium chloride aqueous solution with a predetermined concentration (17.5 g/L) was given. Furthermore, one week after the true leaves opened, the plants were given 50 mL of aqueous sodium chloride solution at a concentration of 23.3 g/L, and cultivated for 4 weeks after sowing.

(Cultivation test results)
The results of the cultivation test are shown in Figure 4. Figure 4 shows the growth promotion rate (%) of the seedlings of each sample after cultivation relative to the comparative example (the percentage of the average dry weight of the example relative to the average dry weight of the comparative example).

As shown in FIG. 4, in the case of spinach, the growth promotion rates in Examples 1 to 3 were 192%, 119%, and 199%, respectively. In the case of sugar beet, the growth promotion rates in each of Examples 1 to 3 were 112%, 140%, and 123%, respectively. In the case of amaranth, the growth promotion rates in Examples 2 and 3 were 319% and 209%, respectively (Example 1 was not implemented).

In this way, it has been demonstrated that by inoculating the roots of Amaranthaceae seedlings with fungi belonging to C. chaetospira, the growth of the seedlings is promoted even under harsh conditions of high salt concentrations, although the degree of this promotion varies depending on the fungus strain. Infecting the roots of Amaranthaceae seedlings with fungi belonging to C. chaetospira is expected to not only promote the growth of the seedlings, but also confer various resistances to the seedlings.

Therefore, C. A method including the step of inoculating the roots of seedlings with a fungus belonging to chaetospira makes it possible to stably grow Amaranthaceae seedlings even under inappropriate growth conditions.

<Specific example of how to produce Amaranthaceae seedlings>
Based on the above test results, it is sufficient to inoculate the roots of seedlings of the Amaranthaceae family, such as spinach, with fungi belonging to C. chaetospira before or during cultivation.

Once the roots of these seedlings are infected with C. Once infected with a bacterium belonging to chaetospira, it becomes possible to obtain advantageous effects such as promoting growth and imparting resistance through symbiosis with the bacterium. C. on the roots of seedlings. Various methods can be considered for inoculating bacteria belonging to chaetospira.

For example, if the above-mentioned culture material is used, C. Bacteria belonging to Chaetospira can be cultured in large quantities at low cost. In particular, in the case of the above-mentioned culture material, since its base material is wood chips, there is an advantage that it can be used as a cultivation material in its original state.

Since seedlings are often raised in pots or seedling boxes, the pots or seedling boxes may be filled with culture material in which bacteria belonging to C. chaetospira have been cultivated, either alone or together with other culture materials, and seeds of plants of the Amaranthaceae family may be sown there or seedlings of plants of the Amaranthaceae family may be planted there and raised. In this way, the bacteria belonging to C. chaetospira can be inoculated into the roots of the Amaranthaceae seedlings simply by raising the Amaranthaceae plants in the same manner as before.

In addition, large amounts of C. Using bacteria belonging to chaetospira, C. An aqueous solution (bacterial liquid) containing a high concentration of bacteria belonging to chaetospira may be prepared, and the roots of Amaranthaceae seedlings may be immersed in the bacterial liquid to allow the bacterial liquid to adhere to the roots. In this case as well, the bacteria can be easily inoculated into a large number of seedlings.

Claims (11)

A culture material for culturing bacteria belonging to Cladophialophora chaetospira,
A culture material based on a wood medium that has been modified by adding iron sulfate and citric acid to uncomposted coniferous wood chips. A culture material for culturing a fungus belonging to Cladophialophora chaetospira,
A culture medium mainly made of woody medium made by crushing non-composted coniferous wood using an extruder. In the culture material according to claim 1 or 2,
A culture material in which the bacterium is a bacterium with receipt number: NITE AP-03738. It is a seedling of the Amaranthaceae family,
A seedling of the Amaranthaceae family whose roots are inoculated with a fungus belonging to Cladophialophora chaetospira. 5. The seedling of the Amaranthaceae family according to claim 4,
A seedling of the Amaranthaceae family, wherein the fungus is a fungus having accession number: NITE AP-03738. 1. A method for producing Amaranthaceae seedlings, comprising the steps of:
A method for producing Amaranthaceae seedlings, comprising the step of inoculating the roots of the seedlings with a fungus belonging to Cladophialophora chaetospira. 7. The method for producing Amaranthaceae seedlings according to claim 6,
The method for producing Amaranthaceae seedlings includes, in the above step, growing seedlings using a predetermined culture material in which the fungus has been cultured. The method for producing Amaranthaceae seedlings according to claim 7,
A method for producing Amaranthaceae seedlings, wherein a wood medium obtained by modifying uncomposted coniferous wood chips by containing iron sulfate and citric acid is used as the predetermined culture material. The method for producing Amaranthaceae seedlings according to claim 7,
A method for producing Amaranthaceae seedlings, wherein a woody medium obtained by crushing non-composted coniferous wood with an extruder is used as the predetermined culture material. 7. The method for producing Amaranthaceae seedlings according to claim 6,
The method for producing seedlings of the Amaranthaceae family, wherein the step comprises a treatment of attaching the aqueous solution containing the bacteria to the roots of the seedlings. The method for producing Amaranthaceae seedlings according to any one of claims 6 to 9,
A method for producing Amaranthaceae seedlings, using the fungus with receipt number: NITE AP-03738 as the fungus.