JP2009232721A - Plant cultivation method using enterobacter bacterium - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a plant cultivation method using Enterobacter bacteria instead of using chemical fertilizer to supply nitrogen to plants so as to prevent contamination of groundwater due to application of nitrogen fertilizer for nitrogen supply to the plants, in crop plant cultivation. <P>SOLUTION: The plant cultivation method using Enterobacter bacteria comprises passing the infection on to the roots of germinated plants to accelerate growth of the plants. The Enterobacter bacteria are provided in order to implement the method. A carrier is provided to which the bacteria are immobilized. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a method for cultivating a plant in a low nitrogen supply state by infecting the root of the plant after germination with an enterobacter bacterium, an enterobacter bacterium for carrying out the method, and the bacterium being immobilized Relates to the carrier.

  Plant growth requires nitrogen, phosphorus and potassium, which are said to be the three major nutrients. Among them, nitrogen is supplied to plants by converting nitrogen in the air into ammonium by microorganisms called nitrogen-fixing bacteria in nature. As nitrogen-fixing bacteria, rhizobia that forms nodules in the roots of legumes and inhabit them are well known, but symbiotic nitrogen-fixing bacteria that coexist with non-legumes and bacteria alone in the air There are also living nitrogen-fixing bacteria that can fix nitrogen. Nitrogen-fixing bacteria that are loosely symbiotic to the roots of gramineous plants are relatively well known.

On the other hand, in the cultivation of crop plants that are thought to be non-symbiotic with nitrogen-fixing bacteria, nitrogen supply to these plants by nitrogen-fixing bacteria is not performed, and therefore it is necessary to put nitrogen fertilizer into the soil. However, in this case, since the utilization rate of fertilized nitrogen is only about 50% (Keldar total nitrogen measurement method, stable isotope ¹⁵ N measurement method, etc., there are many reports; for example, non-patent literature 1-5) Nitrogen not used for crops remains in the soil. Therefore, contamination of groundwater by fertilizer nitrogen is a problem that cannot be ignored. This problem grows with the growing scale of crop plants.

  To date, attempts have been made to artificially mix bacteria or fungi into soil in plant cultivation (Patent Documents 1 to 9). Many of them are for obtaining plant growth-promoting action or controlling disease-causing bacteria.

JP-A-6-7037 JP-A-6-233675 JP-A-9-194315 Japanese Patent Laid-Open No. 10-7383 JP 2003-212708 A JP 2002-233246 A JP 2004-143102 A JP 2004-189545 A JP 2004-307342 A

Ibaraki Agricultural Research Center, Horticultural Research Institute Report, No. 15, p. 29-36, 2007 Hokkaido Agricultural Experiment Collection Report 51, p. 43-54, 1984 Japanese Journal of Soil Fertilizer, Vol. 72 (No. 1) p. 88-91, 2001 Japanese Journal of Soil Fertilizer, Volume 75 (No. 1) p. 99-102, 2004 Central Agricultural Research Center Research Report, Volume 3, p. 89-97, 2003

  An ideal way to reduce the nitrogen load on the environment caused by the cultivation of crop plants as described above is to impart a biological nitrogen fixation function, like legumes, to non-legume crop plants. This has been studied by many researchers, but has not yet been developed for practical use. In this regard, nitrogen-fixing bacteria have been isolated from sugarcane, sweet potato, wild rice, etc., and their use has attracted attention (Plant and Soil, Vol. 108, p. 23-31, 1988; Soil and Microorganisms, Volume 60 ( No. 1) p.3-9, 2006; Soil Science and Plant Nutrition, Vol.46, p.759-765, 2000; Microbes and Environment, Vol.21, p.122-128, 2006; Applied and EnvironmentalMicrobiol. , Vol.67, p.5285-5293, 2001).

  Therefore, by using nitrogen-fixing bacteria, supply of nitrogen from biological nitrogen fixation to leafy vegetables and fruit vegetables that do not have biological nitrogen fixation function like legumes and have high nitrogen demand. Therefore, it is required to reduce the amount of nitrogen fertilizer input. If such a technology is realized, groundwater contamination by fertilized nitrogen will be mitigated, and at the same time, it will have an economic effect.

  The inventors of the present invention have made it possible to infect the roots of germinated non-leguminous plants with Enterobacter bacteria so that the amount of nitrogen fertilizer input is reduced compared to the normal cultivation conditions of the plant. It was found that good growth can be realized.

Accordingly, the present invention has the following features.
(1) A method for cultivating a plant, comprising infecting a root of a non-leguminous plant after germination with an Enterobacter bacterium, and lowering an input amount of nitrogen fertilizer as compared with a plant of the same species.
(2) The method according to (1), wherein the Enterobacter bacterium is symbiotic with the plant as an endophytic fungus after infection.
(3) The Enterobacter bacterium is Enterobacter sp. The method according to (1) or (2) above, which is 35 (FERM P-21484).
(4) Enterobacter sp. Enterobacter sp. 35 (FERM P-21484) or a carrier on which a mutant having nitrogen fixation ability is immobilized.
(5) use of a plant cultivation method, including infecting the roots of non-legumes after germination with Enterobacter bacteria, and lowering the amount of nitrogen fertilizer compared to the same type of plant Enterobacter sp. For Enterobacter sp. 35 (FERM P-21484) or a mutant thereof having nitrogen-fixing ability.
(6) The bacterium according to (5), which is immobilized on a carrier.
(7) Enterobacter sp. 35 (FERM P-21484) and non-legumes other than sugarcane, characterized in that the demand for nitrogenous fertilizer is less than that of plants of the same species.

  In the present specification, “nitrogen fixation” refers to a process of converting nitrogen present as nitrogen molecules in the air into nitrogen compounds such as ammonium salts that can be used by plants for growth. In this specification, the ability to perform nitrogen fixation is expressed as nitrogen fixation ability.

  In the present specification, the “symbiotic nitrogen-fixing bacterium” refers to a microorganism that is symbiotic with a plant without causing disease to the plant and has a nitrogen-fixing ability. Symbiotic nitrogen-fixing bacteria include epiphytes that inhabit plant tissue surfaces and / or surrounding soil, and endophytes that invade and inhabit plant tissues.

  Enterobacter sp. Disclosed in the present invention. 35 was isolated from sugarcane. This strain was deposited on December 27, 2007 at the National Institute of Advanced Industrial Science and Technology Patent Biological Depositary Center (Tsukuba Center Ibaraki 1-1-1 Tsukuba Center Chuo No. 6). -21484.

  The genus was identified by comparing the base sequence of the 16S rRNA gene. The partial sequence on the 5 'terminal side of the 16S rRNA gene obtained from the isolated strain is as shown in SEQ ID NO: 1 (DDBJ (DNA Data Base of Japan) / EMBL / Genbank accession number: AB256515). This sequence was used to compare the sequence identity with the database. As a result, the sequence has 98% sequence identity with the 16S rRNA sequence (Genbank accession number: AY297791.1, etc.) of some strains such as Enterobacter cloaca, the major species of the genus Enterobacter. It was. In addition, the completely identical sequence was not registered in the database. From these facts, it was considered that the isolated strain was a bacterium belonging to the genus Enterobacter (Tanaka et al., Microbes Environ., Vol. 21, No. 2, p. 122-128, 2006).

  In addition, as shown in the examples below, Enterobacter sp. 35 has the ability to infect plants other than sugarcane, which is a natural host, and to coexist with the plant as an endophytic fungus after infection.

  The Enterobacter bacterium used in the method of the present invention is not particularly limited as long as it has nitrogen fixation ability and can infect a plant, but preferably a part or all of the bacterium is endophytic as a plant after infection. Enterobacter bacteria that live together. Most preferably, the Enterobacter bacterium used in the method of the present invention is Enterobacter sp. 35 or a mutant thereof having nitrogen-fixing ability. As a method for producing a mutant strain, a method using irradiation with high energy rays such as ultraviolet rays and radiation and a mutagenic compound (for example, N-nitroso-N-methylurea, ethylmethanesulfonate, ethyleneimine, sodium azide, etc.) are used. The method etc. are mentioned. The Enterobacter bacterium that can be used in the method of the present invention may be any plant as a host.

  Whether or not the Enterobacter bacterium used in the method of the present invention is symbiotic with the plant as an endophytic fungus after infection is determined by culturing the bacteria extracted from the surface sterilized plant tissue piece in a microbiological medium. can do. Specifically, the method described in the following examples is used.

  In the present specification, the term “plant” or “crop plant” refers to a plant that is artificially cultivated for supplying food, and that requires nitrogen supply in artificial cultivation. The plants in the present invention include leaf vegetables, fruit vegetables, root vegetables, cereals, spice crops, and luxury crops. Particularly, leaf vegetable crop plants are preferable as plants in the present invention because they require application of a large amount of nitrogen fertilizer. Leaf vegetable crop plants include, but are not limited to, cabbage, broccoli, cauliflower, Chinese cabbage, lettuce, shungiku, celery, parsley, spinach, komatsuna, tsuruna, kale, mizuna, udo, onion, leek, wakegi, assaki, Examples include leek, garlic, rakkyo and asparagus. Fruit and vegetable crop plants are also preferred and include, but are not limited to, for example, tomatoes, peppers, cucumbers, tougans, pumpkins, eggplants, okras, shrimp, melons, watermelons, mangoes and strawberries. It is done.

  In the method of the present invention, the cultivation method of the plant is not particularly limited as long as it is suitable for the growth of the plant to be cultivated, and examples thereof include soil cultivation and hydroponics.

  In order to infect a plant with Enterobacter bacteria in the method of the present invention, a mode suitable for the cultivation conditions of the plant can be selected. Examples of such modes include, but are not limited to, enterobacter bacteria mixed, sprayed, irrigated, or planted in the soil where the plants after germination are to be planted or are already planted. Or the procedure of immersing the root part of a seedling in bacterial suspension is mentioned. One skilled in the art can readily determine such a manner. In the case of hydroponics, there is a procedure in which plant seedlings previously infected with the bacteria are transferred to a larger tank for hydroponics by immersing the plants in a tank containing the suspension of the bacteria, and further cultivated. desirable.

The application form of Enterobacter bacteria for plant infection in the method of the present invention may be any as long as the bacteria can efficiently infect the plant, but suspensions and solids (immobilized Cell, encapsulated bacteria, etc.). Preferably the bacterium is applied to the plant in suspension. The concentration of the suspension is not limited to 10 ⁵ cells / mL, 10 ⁶ cells / mL, 10 ⁷ cells / mL, 10 ⁸ cells / mL, 10 ⁹ cells / mL or 10 ¹⁰ cells / mL. can do. The amount of bacteria to be applied can be, for example, 10 ⁵ cells, 10 ⁶ cells, 10 ⁷ cells, 10 ⁸ cells, 10 ⁹ cells, 10 ¹⁰ cells, 10 ¹¹ cells, or 10 ¹² cells per liter of culture medium. When the bacteria are applied to a relatively wide range of soil, for example, 10 ¹³ cells are applied per are.

  The treatment for the infection of Enterobacter bacteria to the plant in the method of the invention can be carried out only once after germination, preferably at the beginning of the growth of the plant, or several times, for example twice, 3 times. It can be repeated four times, five times and six times. Preferably, the infection process is performed only once. When the infection treatment is performed a plurality of times, the period between treatments can be, for example, 3 days, 5 days, 1 week, 10 days, 2 weeks, 3 weeks, 1 month, 2 months, and 3 months.

  Enterobacter bacteria for plant infection in the method of the present invention are grown by conventional microbiological methods. For example, the bacteria are grown by culturing in an LB liquid medium at 27 ° C. for 12 hours to 36 hours, preferably 24 hours.

  By the method of the present invention, the amount of nitrogen fertilizer added to the soil or medium of crop plants can be reduced. The amount of nitrogen fertilizer can be reduced by, for example, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, or 70% or more compared to the normal cultivation conditions of the crop plant. Here, the normal cultivation conditions of the crop plant vary depending on the type of the crop plant, and those skilled in the art can appropriately determine this. The amount of nitrogen fertilizer applied in the normal cultivation process of crop plants is, for example, 3 to 30 kg per 10 ares.

  The present invention also provides a non-legume plant other than sugar cane that is symbiotic with the Enterobacter bacterium of the present invention and has less demand for nitrogenous fertilizer than normal plants of the same species. Such a plant is preferably in a seedling state. This seedling can be used for soil cultivation or hydroponics. The kind of the plant is as exemplified above. The amount of nitrogen fertilizer required for the cultivation of such a plant is, for example, 80% or less, 70% or less, 60% or less, 50%, compared to the same kind of plant not symbiotic with the Enterobacter bacterium of the present invention % Or less, 40% or less, or 30% or less.

  The present invention also provides a carrier on which the Enterobacter bacterium of the present invention is immobilized. The carrier on which the bacteria are immobilized can be used, for example, by mixing an appropriate amount in the soil where the plant is cultivated. Examples of the method for immobilizing the Enterobacter bacterium of the present invention on a carrier include suspending, mixing or immersing the carrier in a culture of the bacterium, and drying. Suitable carriers are those on which bacteria can grow, including but not limited to mineral carriers such as vermiculite, perlite, bentonite, zeolite, diatomaceous earth, and activated carbon, peat moss, pulp, straw, bagasse, bone meal, shell fossils. Organic carrier such as oil grounds and fish grounds. Preferred carriers are porous. The carrier is preferably in the form of particles. The carrier may be packaged separately from the plant cultivation container, or may be placed in the distribution process in combination with the crop cultivation container.

  The present invention will be described in more detail with reference to the following examples, but the scope of the present invention is not limited to these examples.

1. Obtaining bacterial strains and preparing bacteria for inoculation Enterobacter sp. 35 (AB256515; hereinafter referred to as “Strain 35”) was isolated from sugarcane. This strain was deposited on December 27, 2007 at the National Institute of Advanced Industrial Science and Technology Patent Biological Depositary Center (Tsukuba Center Ibaraki 1-1-1 Tsukuba Center Chuo No. 6). -21484.

  The genus was identified as follows. First, the bacteria were purely isolated, the cultured bacterial pellet was washed, and the total DNA was extracted. The 16S rRNA gene sequence was amplified by PCR using the extracted DNA as a template. Amplified products were separated by agarose gel electrophoresis, and the corresponding bands were cut out and purified. The obtained DNA was subjected to sequence analysis. Of the base sequence of the 16S rRNA gene, about 490 bases were decoded from the base sequence on the 5 'end side, and the genus was identified by homology search with a known sequence in the database.

  The 16S rRNA sequence obtained from the isolated strain is shown in SEQ ID NO: 1 (DDBJ (DNA Data Base of Japan) / EMBL / Genbank accession number: AB256515). This sequence had 98% sequence identity with the 16S rRNA sequence (Genbank accession number: AY297791.1, etc.) of some strains such as Enterobacter cloaca, a major species of the genus Enterobacter. In addition, the completely identical sequence was not registered in the database. From these facts, the isolated strain was considered to be a bacterium belonging to the genus Enterobacter.

  Herbaspirillum sp., A bacterium belonging to the genus Herbaspirillum, provided by Dr. Minamizawa (Graduate School of Life Sciences, Tohoku University). Strain B501 (hereinafter sometimes referred to as HB501) was used for comparison. HB501 was isolated from the wild rice Oryza officinalis W0012.

  A GFP gene-carrying strain for GFP expression experiments was prepared by conjugating Strain 35 or HB501 with E. coli s17-λpi carrying pTn5kmmgfpmut1 (Tanaka et al., 2006, Microbes Environ., 21, 122-128). In the following, they are sometimes referred to as Strain 35-1 and HB501 gfp1, respectively.

Bacteria for inoculation were prepared from bacterial cultures cultured in LB medium at 27 ° C. for 24 hours. First, the culture was centrifuged at 6,500 rpm for 10 minutes at 25 ° C., and the supernatant was discarded. The pellet was resuspended in sterile nitrogen-free (N-free) MS medium. The bacterial suspension concentration was adjusted to 10 ⁸ cells / mL using a Petroff-Hausser and Helber count.

2. Greenhouse Experiment (1) Plant Growth Conditions and Bacterial Inoculation Method 250 mL plastic pot containing broccoli (Brassica oleracea) (variety: green cocoon) seeds in a commercially available potting mixture (containing 110 mg nitrogen, 98 mg phosphorus, 102 mg potassium) Sowing. After 1 month of cultivation, the plants were removed and the roots of the plants were washed with sterile distilled water to remove the potting mixture. Subsequently, the plants were replanted into modified Leonardo jars. The jar is a combination of two plastic pots, 8.5 cm in diameter and 14.5 cm in height. The upper pot was filled with sterile vermiculite (700 g) soaked in water and planted here. The lower pot was charged with 300 mL of MS medium (pH 5.6) containing 0.5 mM KNO ₃ (Njoroma et al., 2006, Biol. Fertil. Soils, 43, 137-143). The medium placed in the lower pot was supplied to vermiculite in the upper pot by a mechanism provided in the Leonardo jar. Plants were maintained under semi-natural conditions in a greenhouse. One week later, the nitrogen-containing MS medium was replaced with a nitrogen-free MS medium (ie, switched to a condition without nitrogen supply).

The next day, remove the gravel covering vermiculite and uniformly pour 200 mL of the above wild-type inoculum (Strain 35 and HB501) suspension (10 ⁸ cells / mL) into vermiculite in the jar, Plants were inoculated with bacteria without breaking the condition. The same amount of sterile distilled water was poured into the control plants. After inoculation, the surface of vermiculite was covered again with sterile gravel. The temperature range during the experiment was 13 to 28 ° C. Cultivation was continued under conditions without nitrogen supply.

  Thirty days after the inoculation of the fungus (30 DAI), the plant was collected and subjected to visual inspection of the growth state and measurement of fresh weight.

(2) Analysis method The infected group and the control group (each n = 3) collected 30 days after the inoculation were washed with tap water to remove the attached vermiculite. The root moisture was removed with a paper towel and observed and weighed.

(3) Results The results are shown in FIG. 1 and FIG. In visual observation, the Strain 35 inoculated group (7-9) developed more greatly in both roots and leaves than the control group (10-12) (FIG. 1B). The HB501-inoculated group (1-3) also developed slightly larger than the control group (4-6) (FIG. 1A).

  In comparison of fresh weight, the fresh weight of the Strain 35 inoculated group (Strain 35) compared with the control group (Control) was about 176%, and the fresh weight of the HB501 inoculated group was 118% (FIG. 2). In addition, the plant height was 173.8% (average 14.9 cm) in the Strain 35 inoculated group and 180.5% (average 14.3 cm) in the HB501 inoculated group compared with the control. The diameter of the stem was 130% (average 0.48 cm) in the Strain 35 inoculated group and 131% (average 0.47 cm) in the HB501 inoculated group compared with the control. The number of leaves was 168.4% (average 8) in the Strain 35 inoculated group and 181.2% (average 7.25) in the HB501 inoculated group compared with the control.

  Therefore, the Strain 35 inoculated group had a significantly increased fresh weight compared to the control group. Also, this test clearly showed that the Strain 35 inoculated group grew well even under conditions without nitrogen supply.

3. Laboratory test (1) Plant growth conditions and bacterial inoculation method In the same manner as described above, seeds of broccoli (Brassica oleracea) (variety: green pod) were sown in a 250 mL plastic pot, and the plant was modified 30 days after sowing. Replanted into a jar. The plants were maintained at 13 ° C. (night temperature) and 25 ° C. (day temperature) under 16 hours light conditions and light / dark conditions with a luminous flux density of 60 μmol / m ² / s (generated from a white fluorescent lamp). Six days later, the nitrogen-containing MS medium was replaced with nitrogen-free (N-free) MS medium.

In the same manner as described above, plants were inoculated with 200 mL of a suspension for GFP expression inoculation (Strain 35-1 and HB501 gfp1) (10 ⁸ cells / mL). Cultivation was continued under conditions without nitrogen supply. 15 days after inoculation, 300 mL of MS medium containing K ₂ SO ₄ (16.4 g / L) was poured into vermiculite in each pot.

  Plants were harvested for analysis 8 days (8 DAI) or 30 days (30 DAI) after fungal inoculation.

  In addition to the broccoli, infection tests were similarly conducted on rice and sugarcane.

(2) Analysis method (A) Measurement of microbial population The roots and above-ground parts of the collected plants (each time point, inoculation group and control group, each n = 3) are washed with distilled water and loosely attached to the plants. Bacteria were washed away. Plants were minced into small pieces, weighed, and transferred to sterile tubes. Each sample was washed 3 times with sterile distilled water and ground with a mortar and pestle. The debris was serially diluted and plated on LB plates containing 10 μg / mL kanamycin. Green colonies formed after incubation at 28 ° C. were counted using a Nikon SMZ1500 fluorescence microscope. When counting the number of bacteria in plant tissue after surface sterilization, the sample is surface sterilized with 70% ethanol for 30 seconds and 1% NaClO for 1 minute before milling the plant, and then sterilized distilled water. It was used for analysis after washing.

(B) Observation with fluorescent microscope The plants of the inoculated group and the control group were washed three times with sterilized distilled water and subjected to observation under a fluorescent microscope. Plant roots were observed directly to determine the localization of the inoculated bacteria. The stem portion was observed after preparing a cross section of the stem after surface sterilization. The surface sterilization in this case was performed by sterilizing with 70% ethanol for 1 to 2 minutes and then washing with sterilized distilled water. Fluorescence microscopy was performed using a Nikon Eclipse E600 (Nikon, Tokyo, Japan) (Njoroma et al., Supra).

(C) Complete plant acetylene reduction assay (ARA)
Using 8 DAI and 30 DAI plants, the nitrogenase activity of the inoculated and control plants was measured. The test plant was washed with sterile distilled water and placed in a 400 mL plastic jar. Add 5 mL of nitrogen-free Fahraeus liquid medium with sugar (Fahraeus, 1957, J. Gen. Microbiol., 16, 374-381) as a carbon source to moisten the roots, and the jar with a rubber cap Sealed. The upper space of the jar was filled with acetylene gas (10% v / v) and placed in the dark at 25 ° C. After 24 hours and 48 hours, the ethylene gas concentration was measured using a Shimadzu GC-8A gas chromatograph.

(3) Results (A) Microbial population density Table 1 shows the inoculum density of strain 35-1 and HB501 gfp1 in the root and above-ground parts measured by the colony counting method.

Strain 35-1 was established in roots (8.1 × 10 ⁶ CFU / gFW) and stems (2.8 × 10 ⁶ CFU / gFW) in 8 DAI plants. Since colonization was detected even in the sample after surface sterilization, it was clear that Strain 35-1 not only appeared in the plant tissue as an epiphyte, but also entered the internal tissue of the root and stem as an endophyte. Became. At 30 DAI, the bacterial population decreased and strain 35-1 was established and endogenous in the roots, but no colonization was detected in the stem. Strain 35-1 colonization was not detected in the leaves.

HB501 gfp1 was established in the roots in 1.7 DAI plants (1.7 × 10 ⁴ CFU / gFW). HB501gfp1 colonization was not observed in the stem and leaves (Table 1). In the sample after the surface sterilization, fixing of HB501gfp1 was not observed.

  From these facts, HB501gfp1 is expressed as an epiphyte in the root of the test plant, while strain 35-1 is not only expressed as an epiphyte in broccoli, but can also infect tissues as an endophyte. Became clear.

(B) Microscopic observation In 8 DAI, Strain 35-1 was established in the cell space of the main root and adventitious root and the boundary part where a new root extends from the original root (FIGS. 3A, B, C, and C-1). Even at 30 DAI, strain 35-1 colonization in the cell space of the lower root was observed (FIGS. 3D, E and F). Strain 35-1 also invaded the stem and was established in the epidermis (FIGS. 3G and G-1) and epiphytic xylem (FIG. 3H). No foliage colonization was observed.

  In 8DAI, HB501gfp1 was localized in the rhizosphere and sometimes settled on the root surface and cell space (FIGS. 4A and B). Intensive colonization in the rhizosphere was not observed at 30 DAI, and there was a slight colonization of the fibrous roots below the main and adventitious roots (FIGS. 4C and D). Fluorescence of HB501gfp1 was not observed in stems and leaves.

  The establishment of strain 35-1 in the cell root of the newly extending root and the main root close to the extending root indicates that these sites are the invasion sites of strain 35-1 for broccoli.

  Further, in rice and sugarcane other than broccoli, fluorescence from the plant was observed, indicating infection of strain 35-1 to the plant.

(C) Nitrogenase activity in complete plants The ARA activity of strain 35-1 inoculated in complete plants was 4.4 nmol / hour / plant and 8.8 nmol / hour / plant at 8 DAI and 30 DAI, respectively (FIG. 5). ). The ARA activity of HB501gfp1 was 2.5 nmol / hour / plant and 2.07 nmol / hour / plant at 8 DAI and 30 DAI, respectively (FIG. 5).

  This indicates an increase in plant growth with a significant amount of nitrogen fixation in the Strain 35-1 inoculated plant.

(D) Others The utilization rate of nitrogen supplied from nitrogen-fixing bacteria in infected plants is estimated to be 95% or more because nitrogen is supplied inside the plant.

4). Conclusion From the above, it was clarified that Enterobacter bacteria can enhance the growth of infected plants even when they are infected with broccoli, which is a non-leguminous non-host plant, and the nitrogen supply is reduced. Furthermore, while bacteria of this genus settled in plant tissues as endophytic bacteria, the Hervaspyriram bacterium used as a comparison was present on the plant surface as epiphytic bacteria. Despite all the bacteria used for the test having nitrogen-fixing ability, the growth enhancing action and ARA activity were significantly stronger in the case of Enterobacter spp. This suggests that Enterobacter bacteria that can colonize non-host plants as endophytes exhibit better nitrogen-fixing ability than other genera and better support plant growth under low nitrogen supply conditions. It was shown that it can be done.

  Therefore, it was suggested by using the enterobacter bacterium of the present invention that the necessary amount of nitrogen fertilizer to be artificially input during cultivation is reduced.

  According to the present invention, it is possible to reduce the input of nitrogen fertilizer in the cultivation of crop plants.

It is a photograph which shows the growth state of the broccoli cultivated on the greenhouse conditions 30 days after bacteria inoculation. A shows the HB501 inoculated group (1-3) and the control group (4-6), and B shows the Strain 35 inoculated group (7-9) and the control group (10-12). It is a graph which shows the fresh weight of the broccoli cultivated on the greenhouse conditions 30 days after bacteria inoculation. Values represent the average of 3 specimens. It is a microscope picture of the strain 35-1 inoculation plant grown in the laboratory. A represents the endogenous colonization of bacteria in the cell space of 8 DAI plants. B represents the colonization of bacteria at the boundary between the main and side roots of the 8DAI plant and in the cell space. C and C-1 represent bacterial colonization at the base of the root extending from the main root in 8 DAI plants. D to F represent bacterial localization at 30 DAI, with subcellular cell gaps (D and E) as well as root surface (F) localization. G and H are photomicrographs of a cross section of a 30 DAI plant, representing the presence of bacteria in the epidermis (G and G-1) as well as metaplastic xylem tissue (H). Scale bar = 10 μm. It is a microscope picture of the HB501gfp1 inoculation plant grown in the laboratory. A represents the colonization of bacteria on the surface and rhizosphere of adventitious roots of 8 DAI plants. B represents 8DAI plant root colonization in the intercellular space. C and D represent small bacterial colonies found along the cell space of 30 DAI plant roots. Scale bar = 10 μm. It is a graph showing the acetylene reduction activity in a bacteria inoculated plant and a control plant. The value represents the average of 2 specimens.

Claims (7)

  A method for cultivating a plant, comprising infecting a root of a non-leguminous plant after germination with an Enterobacter bacterium, and lowering an input amount of nitrogen fertilizer as compared with a plant of the same species.   The method of claim 1, wherein the Enterobacter bacterium is symbiotic with the plant as an endophytic fungus after infection.   The Enterobacter bacterium is Enterobacter sp. 35. The method of claim 1 or 2, wherein the method is 35 (FERM P-21484).   Enterobacter sp. Enterobacter sp. 35 (FERM P-21484) or a carrier on which a mutant having nitrogen fixation ability is immobilized.   For use in plant cultivation methods, including infecting the roots of non-legumes after germination with Enterobacter bacteria, and lowering the input of nitrogen fertilizer compared to similar plants, Enterobacter sp. Enterobacter sp. 35 (FERM P-21484) or a mutant thereof having nitrogen-fixing ability.   The bacterium according to claim 5, which is immobilized on a carrier.   Enterobacter sp. Enterobacter sp. 35 (FERM P-21484) and non-legumes other than sugarcane, characterized in that the demand for nitrogenous fertilizer is less than that of plants of the same species.

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