JP3328924B2 - Inoculant for plants containing plant root symbiotic microorganisms - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a novel inoculant for plants utilizing a plant root symbiotic microorganism.

[0002]

2. Description of the Related Art In the soil, there are various microorganisms which coexist with plant roots and help the growth of the plant. For example,
One of such microorganisms, rhizobia, infects plant roots, fixes nitrogen in the air and supplies it to plants, and mycorrhizal fungi also infect plant roots, It is known that by assisting the absorption of minerals and water such as the above, they promote the growth of plants infected with them, and enhance disease resistance and drought resistance.

[0003] Although such useful plant root symbiotic microorganisms are expected to be used in agriculture, forestry, and the like, great expectations are placed on them.
Simply adding these to the soil has the problem of adverse effects from other soil microorganisms, thus inhibiting infection of the plant. Even so, mycorrhizal fungi are poor in growth and require a long period of time to infect plants.

[0004] Therefore, in actual use of these plant root symbiotic microorganisms, they are protected from other soil microorganisms, and they are provided with a suitable place for growth so that infection to plants is likely to occur. Therefore, it has been studied to use it as an inoculant mixed with a carrier material or included in a carrier material. For example, various soils, clay, sand, coal, kaolin powder, sphagnum, peat, hydrophilic polymers, oil-based materials, pulp, cellulose, etc. may be added to the soil as a carrier material for plant root symbiotic microorganisms, or seeds. Inoculation methods such as direct spraying on the vegetation have been studied.

[0005]

However, an inoculant of the type using a mixture of a carrier such as soil and mycorrhizal fungi or rhizobia is insufficient in protection of these bacteria from soil microorganisms. In addition, mycorrhizal fungi or rhizobial
Inoculants of the type that contain bacteria are almost able to protect these bacteria from soil microorganisms, but not only do they need to be prepared by culturing mycorrhizal fungi or rhizobia , At least one or more additional steps for encapsulating the carrier material under appropriate conditions.

An object of the present invention is to provide a method for preparing an inoculant for a plant root symbiotic microorganism which overcomes the drawbacks of the conventional technique. That is, mycorrhizal fungi to be inoculated to plants
Rhizobium is well protected from soil microorganisms, and, in situ growth is ensured, also mycorrhizal excellent infectivity to plants
An object of the present invention is to provide a method for preparing an inoculant of rhizobia extremely easily .

[0007]

Means for Solving the Problems The present inventors have conducted intensive studies in order to solve such problems, and as a result, selected a microorganism-producing cellulose as a carrier for the inoculant , thereby obtaining mycorrhiza.
When mycelia or rhizobia are included and inoculated into plants, mycorrhizal fungi
And rhizobia are well protected from soil microorganisms, and, given the field growth, the infection rate to the plant greatly increased, moreover, microorganisms and mycorrhizal for producing cellulose
By simultaneously liquid culture and fungi or rhizobia, mycorrhizal fungi also
Is the culture and propagation process of rhizobia ,
The inventors have found that the step of encapsulating with cellulose can be performed in only one step, and have completed the present invention. That is, the present invention relates to a cell
Loose-producing Acetobacter, Pseudomonas, Achrom
Bacteria, Alcaligenes, Aerobacter, Rhizobium
Of the genus Agrobacterium, Azobacter or Sarcina
Organism and mycorrhizal fungi or rhizobia are cultivated by Hestrin-Schramm culture.
Medium, Ohta medium, Hamada medium, Hagem medium, Melin-Norkans medium
Static culture or culture using either ground or Mastutake medium
Perform shaking or swirling culture at 10-180 rpm
While simultaneously performing liquid culture, the Acetobacter
Genera, Pseudomonas, Achromobacter, Alcaligenes
Genus, Aerobacter, Rhizobium, Agrobacterium, Az
Cellulos produced from microorganisms of the genus Bacteria or Sarcina
The mycorrhizal fungi or rhizobia are contained in the medium.
Mycorrhizal fungi or rhizobia are microbial-producing cellulose
The method of making an inoculant for the plant
It is.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.

The present inventionThe inoculant created by the method of
Includes mycorrhizal or rhizobial bacteria. Mycorrhizal fungiInside,example
IfAs endomycorrhizal fungiArbuscularBelongs to mycorrhizal fungiGiga
sporaGenus,ScutellisporaGenus,GlomusGenus,Acaulospora
Genus,SclerocystisGenus,EntrophosporaGenus, orchid mycorrhiza
Belongs to fungiRhizoctoniaGenusP
ezizellaGenera etc. are ectomycorrhizal fungiAmanitaGenus,Trich
olomaGenus,RhizopogonGenus,SclerodermaGenus,PisolitusGenus
Etc. can be suitably used in the present invention, andamongIs, Example
For example, RhizobiumGenus,BradyrhizobiumGenera etc. are preferred for the present invention.
Can be used appropriately. In the case of mycorrhizal fungi, the spores, fungi
Yarn and fruiting body are used as the inoculant of the present invention.To createCan be used
In the case of rhizobia, the whole cells can be inoculated with the inoculant of the present invention.of
To createCan be used.

Further, the cellulose microbial productivity for encapsulating the these mycorrhizal fungi or Rhizobium, more in terms of effective achievement and environmental impact, biodegradable polymeric material of the water-insoluble object of the present invention use the microbial cellulose is
I do . Since cellulose is water-insoluble and used to
Inoculants containing mycorrhizal fungi or rhizobia are not destroyed by moisture in the soil during use, and effectively protect them from soil microbes until the mycorrhizal fungi or rhizobia infect the plant In addition, a place for their growth can be secured. On the other hand, since cellulose is also biodegradable, it does not decompose in soil and remain in the environment after a certain period of time.

In the present inventionIsSuch celluloseToproductionYou
MicroorganismsAsAcetobacter aceti,Acetobacter x
ylinum(Hereinafter referred to as A. xylinum).Acet
obacterGenus,PseudomonasGenus,AchromobacterGenus,Alcalig
enesGenus,AerobacterGenus,RhizobiumGenus,Agrobacterium
Genus,AzobacterGenus,SarcinaGenusUse.

Further, in the present invention, these cellulose production
By simultaneously liquid culture sexual microorganisms and mycorrhizal or the root nodule bacteria, and culturing and proliferation process of mycorrhizal fungi or rhizobial, this fungus
The step of encapsulating the rhizobia or rhizobia with cellulose in one stage.

The cellulose-producing microorganism and mycorrhizal fungi or
In liquid culture with rhizobia, the production conditions of cellulose by cellulosic microorganisms and the growth conditions of mycorrhizal fungi or rhizobia are balanced, and the amount of cellulose produced And the amount of mycorrhizal fungi or rhizobia included in the plant in the shortest possible time, an appropriate ratio as an inoculant to the plant of the present invention, that is, the optimal rate of mycorrhizal fungi or rhizobial infection to the plant Set the culture conditions so that the amount ratio is obtained.
You . For this purpose , Hestrin-Schram
m (hereinafter abbreviated as HS) medium, Ohta medium, Hamada medium, H
agem medium, Melin-Norkans medium, Ru using any such Mastutake medium. There are no particular restrictions on other culture conditions, but general
More specifically , the inoculant of the present invention can be obtained by culturing at a temperature of 16 to 30 ° C., in a dark place and at a pH of 4 to 8. Contact Do <br/>, in the mycorrhizal and rhizobia, since the optimum growth temperature is slightly different, in order to create a inoculant of the present invention more efficiently, when used mycorrhizal fungi are Temperature 20-25
When using rhizobia, the culture is preferably performed at a temperature of 25 to 30 ° C.

In addition, mycorrhizal fungi and roots contained in cellulose
The higher the amount of bacterial bacilli, the greater the rate of their transmission to plants. In order to protect against soil microorganisms, it is necessary to adequately guard these mycorrhizal fungi and rhizobia with cellulose . Therefore, the properties of mycorrhizal fungi and rhizobia (such as their resistance to soil microorganisms) and their inoculants Depending on the nature of the soil used (type and number of soil microorganisms present, etc.), the appropriate ratio that maximizes the rate of mycorrhizal fungi or rhizobia infection to plants is determined by the mycorrhizal fungi or rhizobia. Present between fungi and cellulose.

In the present invention, the cellulose productivity
The microorganisms and mycorrhizal fungi or rhizobia are cultured in a static culture or 10-1.
Simply shaking or swirling cultured in 80 rpm, naturally in the process, mycorrhizal fungi or rhizobia are enclosed in cellulose produced by cellulose-producing microorganism, inoculum is prepared for the purpose. That is, if static culture is performed,
Cellulose containing mycorrhizal fungi or rhizobia at the gas-liquid interface of the culture solution
Base film is formed.
By culturing until the inoculation is completed, a sheet-shaped inoculant can be obtained. Also,
Shake or rotate at 10-180 rpm to get granular
Is obtained. At this time, the speed is slower than 10 rpm.
Inoculants show a sheet-like shape rather than a granular shape
It is easy to do, and if it exceeds 180rpm, it will be agitated
Due to the decrease in shear force, cellulose-producing microorganisms and mycorrhiza
Bacteria and rhizobia may all have reduced activity. This
These sheet or granular inoculants can be used in actual applications.
In various aspects, for example, a granular shape
Indirectly inoculate plants by burying or mixing in or support
Or wrap a sheet-shaped material around the plant roots
You can use this for different purposes, such as inoculating plants directly.
Wear.

[0016]

Plants to be used as hosts for mycorrhizal fungi or rhizobia are not limited in type. The origin may be any of seedlings, cuttings and clones. In the case of seedlings, sowing, germination and rooting, replanting, inoculation agents created by the method of the present invention in each process of exudation, etc. can be used, and in the case of cuttings, cuttings, rooting, In each process of replanting, planting, etc., in the case of cloned seedlings, in each process of rooting, acclimatization, seedling raising, replanting, planting, etc.
Each inoculant prepared by the method of the invention can be used.

[0018]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below based on embodiments.

Example 1 Pisolitu , an ectomycorrhizal fungus
A spore was separated and collected from the fruiting body of S. s tinctrius (hereinafter, abbreviated as PT). The cells of xylinum (ATCC10245) were inoculated into a new HS medium, and shaking culture was performed at 23 ° C. in a dark place at a speed of 90 rpm.

After 10 days of cultivation, PT cells that have grown and proliferated to some extent become A. cerevisiae. Kishirinumu are in included in the microbial cellulose producing, since granular inoculum is obtained, which after germination 10 days Eucalyptus globulus (Euca
lyptus globulus : E. Described as globras. ) The seedlings were embedded in the soil near the roots of 20 seedlings in several places. Thirty days later, these seedlings were dug out, and their roots were treated with 0.05% trypan blue according to a standard method to stain the mycelium of mycorrhizal fungi and measure the infection rate. The results shown in Table 1 were obtained. Was.

[0021]

[Table 1] Comparison of infection rates for Eucalyptus globulus seedlings

In this table, the infection rate was determined by randomly extracting 30 roots among all the roots of one seedling subjected to the measurement, and of those roots stained with trypan blue. The ratio, that is, the ratio of roots infected with mycorrhizal fungi. Specifically, the root sample after trypan blue treatment was spread evenly in a petri dish with grid lines at the bottom, and the number of grid intersections located within the root contour and the stained part of the root among those intersections Was counted under a microscope (about 20 times magnification) and calculated as a percentage of [the number of intersections located in the stained part of the root] to [the number of intersections located in the contour of the root].

Comparative Example 1 Instead of the inoculant of the present invention, P
Except for using the spores of T itself,
The transmission rate of T to these seedlings was determined. Table 1 shows the results.

[Example 2] In place of PT spores, endophytic mycorrhizal fungi Gi gaspor separated and collected from soil by a wet sieve method
A granular inoculant ( Gig aspora cells are included in microbial cellulose produced by A. xylinum) was prepared in the same manner as in Example 1 except that spores belonging to the genus a were used. The infection rate to Globras seedlings was measured. Table 1 shows the results.

Comparative Example 2 Instead of the inoculant of the present invention, Gi
The infection rate of Gigaspora to those seedlings was measured in the same manner as in Example 2, except that the spores of the genus gaspora were used. Table 1 shows the results.

Example 3 Instead of PT spores, Acacci
Example 1 except that rhizobia belonging to the genus Rh izobium were isolated and collected from the root nodules of a mearnsii root and pre-cultured in YM (yeast extract / mannitol) medium at 30 ° C. for 10 days in the dark. A granular inoculum ( R hizobium cells are contained in microbial cellulose produced by A. xylinum) was prepared in the same manner as in Example 1.

This inoculant was treated with Accaccia mea 10 days after germination.
The rnsii seedlings were embedded in the soil near the roots of 20 seedlings at several locations, and after 30 days, these seedlings were dug out and the number of nodules formed on the roots was counted. Table 2 shows the results.

[0028]

[Table 2] Table 2. Comparison of infection rates for Acaccia mearnsii seedlings

Comparative Example 3 Instead of the inoculant of the present invention,
The infection rate of Rhizobium to those seedlings was measured in the same manner as in Example 3 except that Rhizobium rhizobia after preculture was used in suspension in sterilized water. Table 2 shows the results.

[0030] As is apparent from Tables 1 and 2, those of the present invention
Mycorrhiza by using the inoculant prepared by the method
The rate of fungal or rhizobial infection of plants is determined by whether these fungi or rhizobia are ectomycorrhizal or endophytic mycorrhizal. Increased more reliably than when added directly into the soil. That is, in the inoculation agent, a cellulose-producing microbial and mycorrhizal or Rhizobium, simply despite those obtained simply by liquid culture at the same time, mycorrhizal fungi and Rhizobium cellulose Productivity It is understood that the mycorrhizal fungi and rhizobial infection of the plant progressed in a relatively short period of time, sufficiently protected by cellulose produced by the microorganism.

[0031]

According to the inoculant prepared by the method of the present invention, useful mycorrhiza which is hard to infect plants by itself.
Bacteria or rhizobia By encapsulating Cellulose microbial productivity, to achieve a significant increase in the infection rate to the plant.

Moreover, according to the method of the present invention, the cellulosic
And culture and proliferation process of the scan producing microorganism, the mycorrhizal fungi or root
The Tsubukin for performing at <br/> one step without I and a step of enclosing in microbial production cellulose, it is possible to create the inoculant very easily.

Microbial-producing cellulose is biodegradable.
Because it is, inoculant created by the method of the present invention,
Be sprayed or the like in the soil, are completely degraded After a lapse of a predetermined period, the load on the environment there is no such risk by their use.

────────────────────────────────────────────────── ─── Continuation of the front page (56) References JP-A-56-8682 (JP, A) JP-A-7-298777 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) A01N 63/00 A01N 25/00 A01N 25/26 A01G 7/06 CA (STN) JICST file (JOIS)

Claims (1)

(57) [Claims] [Claim 1] Cellulose-producing Acetobacter, Pse
genus udomonas, Achromobacter, Alcaligenes, Aeroba
genus cter, genus Rhizobium, genus Agrobacterium, genus Azobacter
Or the microorganisms of the genus Sarcina, the mycorrhizal fungi or rhizobia, Hes
trin-Schramm medium, Ohta medium, Hamada medium, Hagem medium, M
Using either elin-Norkans medium or Mastutake medium
Or static culture or shaking at 10-180 rpm or
While at the same time liquid culture by performing gyratory, wherein
Acetobacter, Pseudomonas, Achromobacter, Alc
genus aligenes, genus Aerobacter, genus Rhizobium, Agrobacteri
um genus, characterized by containing the mycorrhizal fungi or rhizobial cellulose produced from microorganisms Azobacter genus or Sarcina genus, mycorrhizal fungi or nodules in microbial production cellulose
A method for preparing an inoculant for a plant containing bacteria .