JP5733738B2 - Rhizobium inoculation material, inoculation method of rhizobia inoculation material, and cultivation method - Google Patents

Description

  The present invention relates to a rhizobial inoculated material, a method of inoculating a rhizobial inoculated material, and a cultivation method, and particularly to a legume inoculated material of a legume, a method of inoculating a rhizobial inoculated material, and a cultivation method.

Legumes are cultivated around the world for food crops and green manure.
Among them, green manure means to cultivate another plant and harvest it as a fertilizer without growing it for the plant to be cultivated. That is, by planting and growing a crop that becomes green manure in a fallow field or a harvested field, it can be used as a fertilizer for a plant to be sown and cultivated later.

As this plant for green manure, hairy vetch (Vicilia villasa Roth) which is one kind of leguminous plant has been frequently used. Hairy Vetch is a perennial plant that can overwinter and belongs to the genus Broadama. The Japanese names are velvet kusafuji and nayokusafuji. Hairy vetch is strong in covering power, has a feature that it has almost no choice of soil for cultivation, and is excellent in cold resistance and snow resistance, so it is cultivated or planted in the world as a plant for green manure.
Here, the leguminous plant is characterized in that it can be cultivated without applying a large amount of nitrogen fertilizer because it forms roots that are symbiotic organs with rhizobia that are soil microorganisms and fixes symbiotic nitrogen. For this reason, it becomes a food crop with efficient fertilizer versus harvesting, and is also highly efficient as green manure.
However, in leguminous plants such as hairy vetch, if there are few rhizobia with high affinity in the soil or there are many rhizobia with low nitrogen fixation activity, inoculate the legumes with the appropriate rhizobial strain. It is preferable to do.
Known rhizobial bacteria that form nodules in such leguminous plants include bacteria belonging to the genus Rhizobium, bacteria belonging to the genus Bradyrhizobium, bacteria belonging to the genus Azorhizobium, and the like. Since symbiosis between legumes and rhizobia has high host characteristics, it is possible to select these rhizobia and select a rhizobial strain for inoculum.

As a conventional nodule strain for leguminous plants, referring to Patent Document 1, hairy vetch rhizobium Y629 strain (NITE P-323) and hairy vetch rhizobium strain S625 (NITE P-322) are described. Has been. Moreover, the inoculation material using this hairy vetch rhizobia Y629 stock | strain or S625 strain | stump | stock is described (Hereafter, it is set as the prior art 1.).
The inoculation material of Prior Art 1 describes a rhizobial inoculation preparation (rhizobium inoculation material) obtained by liquid-culturing hairy vetch rhizobia strain Y629 or S625 and mixing the liquid-cultured rhizobia, peat moss and vermiculite. . Peat moss is a peat moss that has been deposited and planted, dehydrated, crushed and selected. Vermiculite is an inorganic and expansive mineral used for agriculture and horticulture, in which vermiculite is sintered at about 800 ° C. and expanded to a volume ratio of 10 times or more.
The inoculum of prior art 1 was used with peat moss: vermiculite = 1: 1. By adopting such a configuration, in addition to excellent fixation of rhizobia and retention of nourishing water, the adsorbing power to the hairy vetch seed surface is strong when inoculated, and it is suitable as an inoculation support carrier.

JP 2008-259495 A

However, the inoculating material of prior art 1 has a problem that it cannot be stored after seeding, and it must be sown immediately.
This was because peat moss and vermiculite inoculum had a weak ability to retain rhizobia, and the rhizobial was killed by drought or the number of viable bacteria was greatly reduced.
For this reason, after inoculating seeds with seeds, seeds must be sown immediately, resulting in a problem of labor.
In addition, the inoculation material using peat moss and vermiculite as a support has poor spreadability to seeds, and even when inoculated, the infection rate of the rhizobial strain is low, and in some cases no root nodules are formed.

  This invention is made | formed in view of such a condition, and makes it a subject to eliminate the above-mentioned subject.

The rhizobial inoculum of the present invention is a mixture of the rhizobia in liquid culture and zeolite in the inoculum of legumes, wherein the zeolite contains mortenite or clinoptilolite and has an average particle size of 1 It is characterized by being an ultrafine powder zeolite of 10 μm .
The inoculum of the rhizobia of the present invention is characterized in that the legume is a hairy vetch, and the rhizobia is a hairy vetch rhizobia strain Y629 (NITE P-323) . The inoculation method of the rhizobial inoculum according to the present invention produces a rhizobial inoculum prepared by mixing rhizobia of a leguminous plant that has been cultured in liquid and zeolite, and the seed of the legume and the rhizobial inoculum A method for inoculating a rhizobial inoculum to be mixed , wherein the zeolite is morphite or clinoptilolite zeolite and is an ultrafine powder zeolite having an average particle diameter of 1 to 10 μm .
The inoculation method of the rhizobial inoculation material of the present invention is characterized in that the legume is a hairy vetch, and the rhizobia is a hairy vetch rhizobia strain Y629 (NITE P-323).
The cultivation method of the present invention is characterized in that seeds of the legumes inoculated by the inoculation method of the rhizobial inoculation material are stored for about 1 to 10 months and sown.

According to the present invention, by using zeolite as a carrier for the inoculum of rhizobia, it is possible to provide an inoculum that can be stored for several months or more after inoculating seeds and reduces the labor of farming.
In addition, the rhizobial inoculum of the present invention has excellent spreadability to seeds, and can increase the density of rhizobia on the seed surface, thus increasing the infection rate of the rhizobial strain and ensuring that the legumes An inoculum for forming can be provided.

It is a conceptual diagram which shows the preparation method of the rhizobial inoculation material which concerns on embodiment of this invention. It is a photograph of the scanning electron microscope of the zeolite in tuff for the rhizobial inoculation material which concerns on embodiment of this invention. It is a photograph which shows the result of having inoculated the rhizobia to the hairy vetch seed using the rhizobial inoculation material which concerns on embodiment of this invention, and was cultivated with the sterilized medium.

<Embodiment>
(Rhizobium inoculation material using ultra-fine powder zeolite)
The inventor of the present invention has produced the above-mentioned problems when producing a rhizobial inoculum using a nodule strain for legumes as in Conventional Technique 1.
For this reason, the inventor of this invention came up with changing the support | carrier of a rhizobial inoculation material. As a result of intensive studies and experiments, it has been found that it is preferable to produce a rhizobial inoculum using ultrafine powder zeolite as a carrier. Zeolite is a white or colorless and transparent silicate (hydrated alumina silicate) porous material, and is used in various ways as an adsorbing material.
The inoculum according to the embodiment of the present invention is an inoculum for inoculating a legume crop with an excellent root nodule strain having a high nitrogen fixing ability using ultrafine powder zeolite as a support carrier.
Further, the ultrafine particle zeolite according to the present embodiment has a characteristic of being easily mixed with rhizobia because the average particle size is about 3 μm and is almost the same as the size of rhizobia.

First, with reference to FIG. 1, the manufacturing method of the rhizobial inoculation material which concerns on embodiment of this invention using the ultrafine particle zeolite is demonstrated.
The rhizobial inoculum according to the embodiment of the present invention is a large-scale liquid culture (large prep) of an excellent rhizobial strain with a high nitrogen fixation capacity suitable for the target legume crop, and the rhizobial culture solution and the ultrafine zeolite The inoculum is manufactured by mixing with powder.
In the example of FIG. 1, 200 mL of the rhizobial culture solution and 72 g of ultrafine zeolite are mixed. However, the present invention is not limited thereto, and the ultrafine zeolite can be mixed at a predetermined ratio such that the culture solution and the ultrafine zeolite have a predetermined hardness and water content.
The rhizobial inoculum prepared in this way is inoculated with the corresponding legume seeds.
The inoculum using peat moss and vermiculite of prior art 1 needed to sow seeds immediately after inoculation. However, the rhizobial inoculation material according to the present embodiment can store the inoculated seeds for several months or more, for example, about 3 to 12 months, and can eliminate the restriction of the work time.
In addition to the ultrafine zeolite, a pH adjusting agent such as CaCO ₃ can be added. As this CaCO ₃ , natural CaCO ₃ extracted from oyster shells and the like can be used.

(Selection of ultra fine powder zeolite)
Here, the type of ultrafine zeolite used for the rhizobial inoculum according to the embodiment of the present invention will be described.
Zeolite is a collective term for hydrated silicates of aluminum containing alkali metals such as sodium, potassium and calcium, and alkaline earth metals. This zeolite belongs to the tectonic silicate in the mineral classification, and it is known that there are several tens of kinds of zeolites having different compositions.
In addition, zeolite has a remarkably larger amount of water of crystallization than other minerals, and when this water of crystallization is heated, it is not released all at once, but dehydrates almost continuously. And even after dehydration, the crystal structure of the zeolite becomes porous without being destroyed. Therefore, the voids and pores that run vertically and horizontally become a zeolite-like ore (zeolite tuff) structure that looks like a sponge. Adsorbs moisture and various gases. Therefore, the dehydrated zeolite becomes an adsorbent.
Further, the texture (eyes) of zeolite is 3 to 6 angstroms (1 angstrom is one hundred millionth of a millimeter), and is generally fixed depending on the type of zeolite. Depending on the size of the eye, the molecules can be screened. Thereby, the zeolite is used as an adsorbent or a separating agent.

The rhizobial inoculum according to the embodiment of the present invention may use synthesized zeolite, but preferably uses natural zeolite containing various soil components.
In particular, it is preferable to use an ultrafine powder zeolite containing clinoptilolite having an average particle diameter of approximately 1 μm to 10 μm, particularly a zeolite having an average particle diameter of approximately 3 μm.
As this zeolite, in particular, natural zeolite produced in Niimachi, Yamamoto-gun, Akita Prefecture can be used. As the Niimachi cut stone zeolite, it is particularly preferable to use a zeolite manufactured by Sun Zeolite Industry Co., Ltd. The zeolite is present in the pumice tuff layer intervening in the Neogene Formation, and contains mortenite or clinoptilolite. The zeolite of Niimachi cutstone tuff has a fine crystal with a length of about 0.4 μm and a width of about 0.07 μm, but is homogeneous. Further, when the zeolite was heated at 300 ° C., 450 ° C., 600 ° C., 750 ° C., and 900 ° C. for 1 hour each and analyzed by X-ray, a stable structure was maintained up to about 450 ° C. The structure gradually weakens at ˜700 ° C., and at 900 ° C., the zeolite structure is completely destroyed and changed to cristobalite. As described above, the zeolite is characterized by excellent stability because it is relatively resistant to heat as a natural zeolite.
Describing with reference to the scanning electron micrograph of FIG. 2, the ultrafine particle zeolite produced in Niimachi, Yamamoto-gun, Akita Prefecture, has almost the same voids as the size of the rhizobia. For this reason, the culture solution containing the nutrients necessary for the growth of rhizobia of legumes and the rhizobia can be suitably retained in the voids of the zeolite, which is a mineral rich in water.

(Separation method of excellent rhizobia)
Among the nodules of the target legumes growing in the field, the nodules having a large nodule particle size and a red interior are collected. The nodule internal tissue fluid is applied to a YM agar medium and cultured at 30 ° C. for 7 to 14 days under aerobic conditions. The composition of this YM agar medium is the same as that of the prior art 1.
The strain is transferred from the obtained colonies to a YM liquid medium and cultured with shaking at 30 ° C. for 7 to 14 days under aerobic conditions. The composition of this YM liquid medium is also the same as that in the prior art 1.
The cultured strain is inoculated into a target legume plant and cultivated in a nitrogen-free medium for 30 days. A strain in which nodulation has been confirmed in the target legume plant is determined as a rhizobia having an affinity for the target legume plant.
A leguminous plant with nodules formed thereon is cultivated for 30 days, and the nitrogen fixation activity is measured by the acetylene reduction method. A rhizobial strain exhibiting high nitrogen fixation activity is selected as an excellent rhizobial strain. This selection method is also the same as in the prior art 1.

(Provision method of inoculation materials)
As described above, excellent rhizobia having an affinity for the target leguminous plant is isolated, cultured in liquid, and mixed with ultrafine zeolite to make an inoculum.
This inoculum is provided to farmers by filling it in a waterproof / dustproof container such as a plastic bottle.
Farmers can inoculate legume seeds with rhizobia in advance and store them. It can also be inoculated just before sowing. For this reason, the working efficiency at the time of sowing seeds can be improved.

With the configuration described above, the following effects can be obtained.
First, in the prior art 1, a rhizobial culture solution was used as a seed material of a mixture of a support material such as peat moss and vermiculite.
In addition, after mixing and inoculating seeds, seeding must be carried out immediately, and there is a problem that the workability of farm work is remarkably inferior.

On the other hand, since the inoculum according to the embodiment of the present invention uses ultrafine zeolite, drying of the surface of the rhizobial seeds is suppressed, so that the survival rate of the rhizobia increases. As a result, the seeds of legumes can be inoculated and stored at room temperature.
For this reason, it is excellent in the workability | operativity of farm work and the effect that the effort at the time of sowing can be reduced is acquired.

Moreover, the inoculum of the prior art 1 has a problem that the infection rate of the excellent nodule strain is low.
On the other hand, the average particle size of the ultrafine zeolite of the inoculum according to the embodiment of the present invention is about 1 to 10 μm and is almost the same as the size of the rhizobia, so that it is easy to mix with the rhizobia. There is.
In addition, in the inoculum of the embodiment of the present invention, since the support carrier is an ultrafine zeolite, when the inoculum and the legume seeds are mixed, the seeds are excellent in spreadability. , It is easy to permeate between the seeds and adheres easily to the seed surface.
For this reason, when inoculating leguminous plants, excellent nodule strains can be reliably infected to form nodules.

  Moreover, since the inoculum according to the embodiment of the present invention is easier to mix the rhizobial culture solution and the support (ultrafine zeolite) than the conventional inoculum, the inoculum can be easily manufactured. Is obtained.

In addition, the rhizobial inoculum according to the embodiment of the present invention is not limited to the rhizobia of legumes, and other bacteria can be retained for the inoculum.
Further, a synthesized zeolite having an average particle diameter of about 1 μm to 10 μm can be used without using natural zeolite.

Here, the Example which manufactured the inoculation material which used the ultrafine particle zeolite for the nodule bacteria inoculation material for hairy vetch of Hairy vetch rhizobium Y629 strain, and confirmed the inoculation effect of the hairy vetch actually inoculated is demonstrated.
In this example, the ultrafine particle zeolite rhizobial inoculation material of this embodiment is used to inoculate hairy vetch seeds, which are leguminous green manure crops, and the seeds stored at room temperature for 3 months in a sterile medium. Cultivation test after storage was conducted.
The details of the post-storage cultivation test will be described in detail below.

<Example 1>
(Cultivation test after storage of hairy vetch rhizobial inoculum)
First, hairy vetch rhizobia strain Y629 is cultured with shaking in 1000 mL of YM liquid medium at 30 ° C. for 5 days, and is taken out in a state where OD (Optical Density, optical density) 620 nm = 1.4. The composition of this YM liquid medium is the same as that of the prior art 1.

  Next, 200 g of the Y629 strain cultured in this YM liquid medium was sprinkled over 72 g of ultrafine zeolite to obtain a rhizobial inoculum for hairy vetch.

This hairy vetch rhizobial inoculum was mixed well with sterilized hairy vetch seeds and stored in the dark at an average room temperature of 20 ° C. for 3 months.
The hairy vetch seeds were sterilized by immersing the surface in 70% ethanol for 10 seconds and thoroughly washing off the ethanol with water. Next, after immersing in 0.5% sodium hypochlorite for 3 minutes, it was washed thoroughly with water to completely remove sodium hypochlorite.

  Hairy vetch was cultivated under low temperature conditions after 2 weeks of sowing and further at normal outside temperature for 2 weeks. During the cultivation period, a nitrogen-free culture solution was appropriately given to supplement nutrients other than nitrogen. The composition of this nitrogen-free culture solution is the same as that of the prior art 1.

After cultivating under the above conditions, the growing condition of the cultivated hairy vetch was visually observed.
With reference to FIG. 3, the cultivation result of this hairy vetch will be described.
Fig.3 (a) is the photograph of the typical plant body after cultivation of the hairy vetch of Example 1. FIG. The hairy vetch was green, and the plant length and roots were good.
FIG. 3B shows that when the roots of the hairy vetch of Example 1 are enlarged, red-brown nodules infected with rhizobia are formed, and it is found that the hairy vetch rhizobia grows. The degree of root nodule formation in FIG. 3 (b) was almost the same as the cultivation result of seeds sowed immediately after inoculation with the hairy vetch inoculation material of Prior Art 1.
On the other hand, after inoculating the seed material stored for 3 months after inoculation with the inoculation material for Hairy Vetch of Prior Art 1 (not shown), the root nodule formation is not recognized because the rhizobia is reduced. Only a few nodules were formed.

As described above, hairy vetch seeds, which are leguminous green manure crops, are inoculated with rhizobia using the inoculum according to the embodiment of the present invention, and the seeds stored for 3 months at room temperature are sown in a sterile medium. Even then, many nodules could be formed without any problem.
It was also found that seeds stored for 3 months or longer can be used within at least one year.

  Note that the configuration and operation of the above-described embodiment are examples, and it is needless to say that the configuration and operation can be appropriately changed and executed without departing from the gist of the present invention.

  The present invention can be manufactured and sold as an inoculum for legumes, and can be used industrially because it improves work efficiency due to the feature that it can be stored after inoculation.

Claims (5)

In inoculation materials of rhizobia of legumes,
Mixing the rhizobia in liquid culture and zeolite ,
Rhizobium inoculation material , wherein the zeolite is morphite or clinoptilolite zeolite and is an ultrafine powder zeolite having an average particle size of 1 to 10 μm . The legume is a hairy vetch,
The rhizobial inoculum according to claim 1 , wherein the rhizobia is a hairy vetch rhizobia Y629 strain (NITE P-323) . Produce a rhizobial inoculum mixed with rhizobia of legumes cultured in liquid and zeolite,
It is an inoculation method of a rhizobial inoculum that mixes seeds of the legumes and the rhizobia inoculum ,
The inoculation method of a rhizobial inoculation material, wherein the zeolite is an ultrafine powder zeolite having an average particle diameter of 1 to 10 µm, including mortenite or clinoptilolite zeolite . The legume is a hairy vetch,
The said rhizobia is hairy vetch rhizobia Y629 strain (NITE P-323). The inoculation method of the rhizobial inoculation material according to claim 3 characterized by things. A cultivation method, wherein seeds of the legumes inoculated by the inoculation method of the rhizobial inoculation material according to claim 3 or 4 are stored for about 1 to 10 months and sown.

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