JP6236660B2 - Plant growth promoter - Google Patents

Description

  The present invention relates to a plant growth promoter containing plant growth promotion rhizobacterium (PGPR) and a method for cultivating a plant using the same.

  Sugar beet, a sugar raw crop, is an indispensable crop for maintaining a field rotation in Hokkaido with an area of 60,000 ha. In sugar beet cultivation, there is a strong demand for lower production costs due to lower domestic consumption of sugar and price competition with imported sugar. In addition, since 2010, the yield of sugar beet has decreased, and the amount of sugar production has greatly decreased due to a decrease in the acreage, and there is a need for improved yield and stability.

  In sugar beet cultivation in Hokkaido, 90% is transplanted cultivation in which seedlings grown for about 45 days are transplanted to the field. In order to realize a stable and high yield by this transplantation cultivation, it is important to raise healthy seedlings early and transplant them in the field.

  Many microorganisms inhabit the rhizosphere or roots of plants, and it is called PGPR, and it is known that there are useful bacteria that bring plant growth-promoting effects. Growth promotion techniques using PGPR in many crops have been reported (Patent Documents 1 to 5).

  However, Amaranthaceae including sugar beet is known as a plant that does not symbiotic with the mycorrhiza. Although there are many studies on antagonistic microorganisms that suppress sugar beet pathogens, few studies on sugar beet growth-promoting microorganisms have been reported to support this prediction (Non-Patent Documents 1 to 3).

  Therefore, development of a technique for growing healthy seedlings by separating PGPR having a growth promoting effect from sugar beet and inoculating seedlings of crops such as sugar beet being raised has been awaited.

JP-A-6-31826 JP 2012-135300 A JP 2002-233246 A JP-A-9-299076 Japanese Patent Laid-Open No. 10-7383

Suslow and Schroth, 1982. Phytopathology 72: 199-206. Cakmakci et al., 1999. J. Plant Nutr. Soil Sci. 162: 437-442 Shi et al., 2011. Symbiosis 54: 159-166

  An object of the present invention is to provide a plant growth promoter containing a plant growth-promoting rhizosphere bacterium and a plant cultivation method using the same.

  The present inventors considered that PGPR having a growth promoting effect is inhabiting in the sugar beet rhizosphere, and comprehensively isolated sugar beet symbiotic bacteria. The site of the separation source was a fine root absorbing nutrients. The sampling time of the separation source was mid-July, when sugar beet is growing vigorously. As a result, the amount of dry matter increased compared to the case of no inoculation, and 13 strains in which the growth promoting effect was recognized were found, and the present invention was completed.

Therefore, the present invention is as follows.
[1] Microorganisms belonging to the genus Polaromonas, Varioborax, Novosphingobium, Nocardioides, Devosia, Mesolyzobium, Sphingomonas, Philobacterium, Bacillus, Rhizobium or Streptomyces A plant growth promoter,
[2] The plant growth promoter according to [1], wherein the plant belongs to the Amaranthaceae family,
[3] The plant growth promoter according to [2], wherein the plant is sugar beet and / or spinach.
[4] The microorganism belonging to the genus Polaromonas is HRRK103, the microorganism belonging to the genus Barrioborax is the HRRK170 strain, the microorganisms belonging to the genus Novosphingobium are the HRRK193 strain and the HRRK010 strain, and the genus Nocardioides The microorganism belonging to HRRP110 strain and the microorganism belonging to the genus Devosia are No. 184 strains, microorganisms belonging to the genus Mesozobium are HRTP027 strain and HRRK190, microorganisms belonging to the genus Sphingomonas are strains HRRP089, microorganisms belonging to the genus Philobacterium are strains HRTP192 and belong to the genus Bacillus The plant according to any one of [1] to [3], wherein the microorganism is an HRTK156 strain, the microorganism belonging to the genus Rhizobium is the HRRK005 strain, or the microorganism belonging to the genus Streptomyces is the HRTK192 strain. Growth promoter.

  By inoculating the plant growth promoter of the present invention into seeds or seedlings of crops such as sugar beet, it is possible to grow healthy seedlings with large plant bodies. This technique improves the survival and initial growth after transplanting, and can improve and stabilize the yield.

The 13 strains obtained in the present invention were deposited on June 25, 2013 at the Patent Organism Depositary, National Institute of Technology and Evaluation under the following deposit number.
Strain name Species Accession number HRRK103 Polaromonas NITE P-01607
(Polaromonas sp.)
HRRK170 Barioborax genus NITE P-01608
(Variovorax sp.)
HRRK193 Novosphingobium genite NITE P-01610
(Novosphingobium sp.)
HRRP110 Nocardioides NITE P-01612
(Nocardioides sp.)
No. 184 Debosia NITE P-01606
(Devosia sp.)
HRTP027 Mesozobium NITE P-01615
(Mesorhizobium sp.)
HRRP089 Sphingomonas genite NITE P-01611
(Sphingomonas sp.)
HRTP192 Philobacterium NITE P-01616
(Phyllobacterium sp.)
HRTK156 Bacillus NITE P-01613
(Bacillus sp.)
HRRK010 Novosphingobium NITE P-01605
(Novosphingobium sp.)
HRRK005 Rhizobium NITE P-01604
(Rhizobium sp.)
HRRK190 Mesozobium NITE P-01609
(Mesorhizobium sp.)
HRTK192 Streptomyces NITE P-01614
(Streptomyces sp.)

  For culturing the microorganism used in the present invention, any conventionally known medium can be used. Further, in addition to the liquid medium, a solid medium such as a slope medium containing agar and a plate medium can be used. By using these media, the strain can be grown to obtain a desired amount of cells.

  Any carbon source that can be assimilated by the above strain can be used as the carbon source of the medium, and examples thereof include sugars such as glucose, galactose, lactose, arabinose, mannose, and malt extract starch hydrolysate.

  Similarly, various synthetic or natural products that can be used by the strain, such as peptone, meat extract, and yeast extract, can be used as the nitrogen source.

  In accordance with a conventional method for culturing microorganisms, inorganic salts such as sodium chloride and phosphate, metal salts such as calcium, magnesium and iron, trace nutrients such as vitamins and amino acids can be added as necessary.

  The culture can be performed under aerobic conditions such as shaking culture, stationary culture, and aeration culture. The culture temperature is 20 to 30 ° C., preferably 20 to 25 ° C., and the culture period is 1 to 5 days, preferably 3 to 4 days.

  In the present invention, “plant growth promotion” refers to promoting the germination of plant seeds and / or promoting the growth of plants. The “root” refers to a portion that absorbs moisture and nutrients in soil or hydroponic liquid when a plant is cultivated.

  The microorganism of the present invention has the property of promoting germination of the seed and / or promoting the growth of the plant by bringing the microorganism into contact with or in the vicinity of the seed or root of the plant.

  Therefore, the present invention relates to a plant cultivation promoter comprising a microorganism having a property of promoting the germination of a plant and / or promoting the growth of a plant by contacting or existing in the vicinity of the seed or root of the plant. I will provide a. Moreover, this invention provides the cultivation promotion method of the plant using this cultivation promoter.

(1) Isolation of sugar beet symbiotic bacteria The sugar beet cultivar “Amahomare” was transplanted and cultured in the three-element cultivation test field in the Hokkaido Agricultural Research Center in Memuro-cho, Kasai-gun, Hokkaido. In mid-July, when growth is thriving, each of the 3 test areas, 3 areas from normal fertilization area (hereinafter referred to as NPK area), nitrogen-free fertilization area (hereinafter referred to as PK area), nitrogen and phosphate non-fertilization area (hereinafter referred to as K area) Individuals were collected and washed thoroughly, and then divided into leaf blades, petiole, roots, and fine roots and stored frozen at −30 ° C. until use.
For isolation of sugar beet symbiotic bacteria, R2A agar medium (Becton Dickinson Co., Ltd.) or TSA agar medium (Becton Dickinson Co., Ltd.), with fine roots collected from PK and K sections as the separation source, and cycloheximide 50 ppm added to the separation medium. Two types of culture medium (tryptic soy broth in which agar was added at a concentration of 1.5%) (hereinafter referred to as culture method) were used. Fine roots were ground in a mortar, diluted to an appropriate concentration with 1/15 M phosphate buffer (pH 7.0), applied to a separation medium, and cultured at 24 ° C. for 1 week. Colonies formed after culturing were exhaustively exhausted, and 200 strains, each totaling 800 strains, were isolated. Among the 800 isolated strains, 665 strains were analyzed for 16S rRNA gene to determine the genus name. On the other hand, bacteria extracted directly from roots and fine roots collected from NPK, PK and K groups were analyzed directly for bacterial 16S rRNA genes, and bacteria symbiotic to sugar beet roots and fine roots. The flora was analyzed (hereinafter, non-culture method).

  As a result of community structure analysis based on the 16S rRNA gene sequence results obtained by the culture method and the non-culture method, the symbiotic bacterial flora of sugar beet was classified into 385 OTUs (operational taxonomic units) at the species level. Symbiotic bacteria belonging to different OTUs are systematically different, and the symbiotic mechanism to plants is also considered to be different. As for 385 OTUs, the isolation frequency is high in diversity analysis based on culture method or non-culture method, and the stably detected bacteria group, 45 OTUs are expected to be useful from past papers. Selected. About the strain which belongs to these OTU, the inoculation test to a sugar beet seedling was performed by the following method, and the growth promotion effect was evaluated.

(2) Inoculation test on sugar beet seedlings The sugar beet variety “Ricca” was used for the test. Seeds were immersed in 70% ethanol for 1 minute, then sodium hypochlorite solution (effective chlorine concentration 1%, tween 20 added 0.01%) for 15 minutes, then rinsed 5 times or more with sterile distilled water. Surface sterilized. The surface sterilized seeds were allowed to germinate by standing in a dark place at 25 ° C. for 24 hours. Cell (cell size: 41 x 41 x 43.5 (H) mm, product name "Cell box 25 hole" filled with 80 ml of seedling culture soil (trade name "Pot Ace", Katakura Chikkarin Co., Ltd.) sterilized with 3 seeds confirmed to germinate ”, Meiwa Co., Ltd.) and sterilized 20 ml of sterilized seedling culture soil on the soil. The repetition of one treatment section was 12 cells. The bacterial cells that had been statically cultured in the dark at 24 ° C. for 3 to 4 days on an agar medium (R2A agar medium or TSA agar medium) were scraped with a platinum loop and suspended in sterile distilled water. The absorbance (OD value) at 600 nm of this suspension was measured, and the suspension was diluted with sterilized distilled water so that the OD value was 0.1. Inoculation was performed immediately after sowing, and 1 ml of inoculation source was irrigated per cell. The uninoculated area was inoculated with sterilized distilled water. After sowing and inoculation, the plants were cultivated in an artificial climate room adjusted to the light period: 25 ° C. for 16 hours and the dark period: 20 ° C. for 8 hours. One month after sowing, the sugar beet was carefully removed from the cell and carefully washed with water to remove the soil attached to the roots. After cutting into the above-ground part and the root part and drying at 80 ° C. for 3 days, the dry weight of each individual was measured.
As a result of the inoculation test on sugar beet seedlings, 13 strains were selected as strains in which the dry weight increased and the growth promoting effect was recognized as compared with the non-inoculated section (Table 1).

(3) Inoculation test on spinach seedlings An inoculation test on spinach belonging to the same Amaranthaceae as the sugar beet was carried out for 10 out of 13 strains that were found to have a growth promoting effect on seedlings. For the test, spinach variety “Okame (Naked Seed)” (Takii seedling) was used. Seeds were allowed to germinate at 18 ° C. for 72 hours in the dark. Cell (cell size: 45x45x50 (H) mm, product name "plug tray" filled with 50 ml of sterilized vermiculite or seedling culture soil (product name "Genki Kimikana 200", Corp Chemical Co., Ltd.) 50 holes "(Landmark Co., Ltd.) and sterilized 10 ml of sterilized seedling culture soil on it and seeded. After cotyledon development, thinning was performed so that three healthy seedlings were obtained, and the number of repetitions in one treatment area was 12 (4 cells). The bacterial cells that had been statically cultured in the dark at 25 ° C. for 3 to 4 days on an agar medium (R2A agar medium or TSA agar medium) were collected with a sterile cotton swab and suspended in sterile distilled water. Absorbance (OD value) at 600 nm of this suspension was measured and diluted with sterilized distilled water so that the OD value was 0.02 was used as the inoculation source. Inoculation was performed immediately after sowing, and 5 ml of inoculation source per cell was irrigated. The uninoculated area was inoculated with sterilized distilled water. After sowing and inoculation, cultivate in an artificial climate room adjusted to light period: 25 ° C., 16 hours, dark period: 25 ° C., 8 hours. The soil was kept dry. Two weeks after sowing, spinach was carefully extracted from the cell, carefully washed with water, and the soil attached to the roots was removed. It cut | disconnected to the above-ground part and the root part, and measured the raw weight for every individual | organism | solid.
As a result of the inoculation test on spinach, HRRK103 strain belonging to the genus Polaromonas, HRRK170 strain belonging to the genus Barrioborax, HRRP110 strain belonging to the genus Nocardioides, No. The 184 strain and the HRTP027 strain belonging to the genus Mesozobium showed a remarkable growth promoting effect, which revealed the possibility of application to spinach (Table 2).

  By inoculating the plant growth promoter of the present invention into seeds or seedlings of crops such as sugar beet, it is possible to grow healthy seedlings with large plant bodies.

Claims (4)

HRRK103 (accession number NITE P-01607) belonging to the genus Polaromonas , HRRK170 (accession number NITE P-01608) belonging to the genus Barrioborax, HRRK193 (accession number NITE P-01610) belonging to the genus Novosphingobium, or HRRK010 ( Accession number NITE P-01605) , HRRP110 belonging to the genus Nocardioides (accession number NITE P-01612) , No. belonging to the genus Debosia . 184 (Accession No. NITE P-01606) , HRTP027 (Accession No. NITE P-01615) or HRRK190 (Accession No. NITE P-01609) belonging to the genus Mesozobium, HRRP089 (Accession No. NITE P-0161) belonging to the genus Sphingomonas HRTP192 (accession number NITE P-01616) belonging to the genus Bacteria, HRTK156 (accession number NITE P-01613) belonging to the genus Bacillus , HRRK005 (accession number NITE P-01604) belonging to the genus Rhizobium , or belonging to the genus Streptomyces A plant growth promoter comprising a microorganism belonging to HRTK192 (Accession number NITE P-01614) .   The plant growth promoter according to claim 1, wherein the plant belongs to the family Amaranthaceae.   The plant growth promoter according to claim 2, wherein the plant is sugar beet and / or spinach. A method for cultivating a plant, comprising bringing the plant growth promoter according to any one of claims 1 to 3 into contact with a seed or a root of the plant.