JP2007077126A - Endogenous bacterium coating lettuce seed, method for producing the same, and method for controlling lettuce big-vein virus disease - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an antagonistic endogenous bacterium coating lettuce seed which has a high lettuce big-vein virus disease-controlling effect and high storage stability. <P>SOLUTION: A method for producing an antagonistic endogenous bacterium coating lettuce seed is characterized by vacuum-inoculating an endogenous bacterium exhibiting an antagonistic property against Olpidium brassicae holding lettuce big-vein virus into a lettuce seed, drying the antagonistic endogenous bacterium-inoculated lettuce seed under low temperature low moisture conditions without adopting a conventional heating ventilation drying method, or combining both the methods. The survival rate of the antagonistic endogenous bacterium on the lettuce seed coated with the antagonistic endogenous bacterium can rapidly be enhanced. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a lettuce seed coated with an endogenous bacterium that is antagonistic to the genus Orpidium that retains a lettuce big vein virus, a method for producing the seed, and a method for controlling a lettuce big bain disease.

  Lettuce Big-Bain disease is a viral disease mediated by the soil-inhabiting fungus Olpidium brassicae, and when it develops, the leaves become reticulated and the spheres become smaller, reducing the commercial value. Also, if the symptom is severe, it will not form a ball and will not become a product. It has occurred in Hyogo Prefecture, Kagawa Prefecture, Shizuoka Prefecture, Chiba Prefecture, etc., and in the case of Hyogo Prefecture with the largest occurrence scale, damage spread to 26% of the cultivation area, and further damage will be expanded in the future Is feared. At present, there are no effective registered pesticides. Therefore, cultivation using resistant varieties or control methods targeting vector bacteria such as soil disinfection are being carried out. Not. In addition, these effects are temporary and difficult to control over a long period of time.

  Therefore, a comprehensive control method for lettuce big vein disease that combines several techniques has been studied. One of them is research on biocontrol technology using antagonistic microorganisms, and Aino et al. Have already isolated endophytic bacteria effective against soil pathogenic microorganisms and confirmed their control effects ( Patent Documents 1, 2, and 3, Non-Patent Document 1). However, the method of applying endophytic bacteria to a vast field or the method of applying it to lettuce seedlings requires the production of a large amount of endophytic bacteria, which is expensive to produce and is a new material for producers. Applying is a work burden. In addition, the establishment of endogenous bacteria on lettuce roots was affected by various soil environments, cultivation conditions, and seedling cultivation soils, so it was difficult to produce stable effects.

  For producers, the simplest and cheapest method for controlling lettuce big-vein disease is to sow seeds coated with endogenous bacteria in which lettuce seeds and endogenous bacteria are integrated. If the amount of endogenous bacteria to be used is small and the endogenous bacteria are alive, it is considered that the endogenous bacteria can establish in the roots of lettuce the earliest compared to other methods. At the laboratory level, it has been confirmed that a high control effect can be obtained by attaching endogenous bacteria to lettuce seeds and immediately seeding them without drying (Non-patent Document 2). However, it has been difficult to put the method using endophytic bacteria-coated seeds into practical use. Because practical seed production of lettuce pellets consists of a granulation process, a pellet particle size and shape selection process, and a drying process. In the drying process, since heated air is blown, it is heated and dried. This is because the endogenous bacteria are easily killed, and the endobacterium is easily killed during the storage period from the production of the pellet seed to the sowing.

JP-A-9-308372 JP-A-8-268826 JP-A-7-163334 Microbial materialization: The forefront of research, (2000), edited by Takahito Suzui et al., Soft Science Journal of the Japanese Society for Plant Pathology Vol.68, No.2, p240

  An object of the present invention is to provide an antagonistic endogenous bacterial-coated lettuce seed having a high lettuce big vein disease control effect and a high storage stability.

  In order to solve the above-mentioned problems, the present inventors have proposed an endophytic bacterium that exhibits antagonistic properties against the genus Orpidium that retains the lettuce big vein virus (sometimes referred to herein as “antagonizing endophytic bacteria”). The method of stable introduction to lettuce seeds, storage stability, and its control effect were investigated.

  As a result, the present inventors surprisingly found that inoculating the antagonistic endogenous bacteria under reduced pressure before the pellet granulation step of the lettuce seed, Later, by drying under low-temperature and low-humidity conditions instead of the conventional warming-air drying, or a combination of both, the survival rate of antagonistic endogenous bacteria in lettuce seeds coated with antagonistic endogenous bacteria has been dramatically improved. It has been found that it is possible to increase it. Moreover, it discovered that the antagonistic endophytic-coated lettuce seed produced in this way had no problem with sowing and germination, and showed high control value with respect to the soil disease with respect to a crop. The phenomenon for the first time confirmed by the present inventors can be explained as follows, for example. Antagonistic endophytic bacteria can be introduced to the inside of the seed epidermis by vacuum inoculation. Unlike the dried seed surface, the inside of the seed epidermis retains enough water for the seeds to survive, so the antagonistic endogenous bacteria introduced to the inside of the seed epidermis make use of that moisture. It is estimated that the survival rate of antagonistic endogenous bacteria can be increased dramatically. In addition, by drying seeds under low-temperature and low-humidity conditions after inoculation with antagonistic endogenous bacteria, the antagonistic endogenous bacteria are less likely to be damaged by temperature or humidity. Increase.

  Furthermore, the present inventors have found that the antagonistic endobacterium-coated lettuce seeds thus produced can be stably stored for a long period of time by storing under low temperature conditions.

That is, the present invention more specifically includes the following inventions.
(1) A method for producing a lettuce seed coated with an endogenous bacterium, comprising inoculating the lettuce seed with an endophytic bacterium that is antagonistic to the genus Olpidium that retains the lettuce bigbein virus.
(2) The method according to (1), further characterized in that lettuce seeds are inoculated with endophytic bacteria under reduced pressure and then dried under low-temperature and low-humidity conditions.

(3) characterized in that an endophytic bacterium that is antagonistic to the genus Olpidium that retains the lettuce big bain virus is inoculated into lettuce seeds, and the seeds are dried under low-temperature and low-humidity conditions after inoculation. A method for producing lettuce seeds coated with live bacteria.
(4) Lettuce seeds produced by the method according to any one of (1) to (3) and coated with an endogenous bacterium that is antagonistic to the genus Orpidium that retains the lettuce big bein virus .

(5) A method for controlling a lettuce big bain disease, characterized by inoculating lettuce seeds under reduced pressure with endophytic bacteria having antagonistic properties against the genus Olpidium that retains the lettuce big bain virus, and sowing the seeds.
(6) The method according to (5), further comprising drying the seeds under low-temperature and low-humidity conditions after inoculating the lettuce seeds with endophytic bacteria under reduced pressure.

(7) Inoculating lettuce seeds with endophytic bacteria having antagonistic properties against the genus Olpidium that retains the lettuce big bain virus, drying the seeds under low-temperature and low-humidity conditions after inoculation, and sowing the seeds A method for controlling lettuce big vein disease.
(8) According to any one of (5) to (7), the lettuce seeds inoculated with endophytic bacteria are stored under low temperature and low humidity conditions between the end of drying and sowing. The method described.
In (8), “a crop seed inoculated with an antagonistic microorganism” means “a seed of a crop inoculated with an antagonistic microorganism under reduced pressure” when (8) is subordinate to (5) or (6). To do.

  The present invention relates to an antagonistic endogenous bacteria-coated lettuce seed having a high effect of controlling lettuce bigbein disease and high storage stability, a method for producing the same, and control of lettuce bigbain disease using the antagonistic endobacterium-coated lettuce seed. Provide a method.

Hereinafter, the present invention will be described in detail.
In the present invention, “lettuce seed coated with an endogenous bacterium that exhibits antagonistic properties against the genus Olpidium that retains the lettuce big vein virus” or “antagonistic endophytic-coated lettuce seed” refers to an antagonistic endogenous bacterium. Refers to the one coated with lettuce seeds. That is, as long as the antagonistic endogenous bacteria are coated on the lettuce seeds, the seeds may remain as they are, various kinds such as film-coated seeds, pellet seeds, gel-coated seeds, cedar tape, seed graphs, priming-treated seeds, etc. It may be a seed subjected to various processing. Usually, lettuce seeds are processed into pellet seeds by pellet granulation. The amount of the antagonistic endogenous bacteria coated is not particularly limited, but may be contained within a range of 10 ¹ to 10 ¹⁰ cells / grain.

  The granulation process in the case of pelletizing lettuce seeds is not particularly limited, and may be performed using a normal pellet granulator. The granulation size may be slightly larger than the lettuce seed, and the thickness of the granulation layer may be in the range of 1 nm to 50 mm. The shape after granulation is preferably spherical or rugby ball, but is not particularly limited. After granulation, it is preferable to perform selection based on the particle size and the shape of the particles in order to increase the efficiency of the sowing operation.

The seeds used in the present invention are lettuce seeds, and the variety is not particularly limited.
The endobacterium used in the present invention is not particularly limited as long as it is an endobacterium that exhibits an antagonistic property to the genus Orpidium that retains the lettuce bigbein virus. For example, Pseudomonas putida FP-16 strain (a strain isolated from the root surface of tomato, which produces an antibacterial active substance against bacterial wilt and has a high bacterial wilt control effect in the field), Pseudomonas fluo Lessence FPH9601 strain (FERM BP-5479), Pseudomonas sp. HAI00377 strain (isolated from roots of Chinese cabbage using P-1 medium (fluorescent pseudomonads selective medium) by plate dilution method) (Non-patent document 1), Pseudomonas sp. FPH-2003 strain (FERM AP-20654), FPH-2005-1 strain (FERM AP-20653), and the like.

  These antagonistic endogenous bacteria can be screened from seeds, plants, soils, etc., and isolated for use. Furthermore, seeds coated with candidate bacteria were sown in soil contaminated with Orpidium genus that mediates lettuce big bain virus, and cultured for several weeks at a suitable temperature for the growth of Orpidium genus. Can be selected as an endophytic bacterium having antagonistic properties and used in the present invention (Japanese Society for Plant Pathology, Vol. 68, No. 2, p240). Regarding the culture conditions for the antagonistic endogenous bacteria of the present invention, the conditions described in the experiment document (new edition of soil microorganism experiment method (1997) edited by Soil Microorganism Research Group, Yokendo) and the like can be used. The medium is, for example, meat extract medium, LB medium, potato dextrose (PD) medium, 1/10 PD medium, King B agar medium, etc. The culture method is, for example, petri dish, test tube, flask, jar fermenter, etc. What is necessary is just to carry out on conditions, such as standing still, shaking, and stirring, in a container, and it is not necessary to carry out on special culture conditions.

In the present invention, “vacuum inoculation” refers to creating a sealed container connected to a suction machine, putting lettuce seeds mixed and contacted with antagonistic microorganisms therein, and sucking air inside the container to reduce the negative pressure condition. After removing the air on the seed surface that has been created, the method returns the pressure to normal pressure (about 760 mmHg) to introduce antagonistic microorganisms inside the seed epidermis. The suction machine may be a widely used one. For example, an aspirator, a sucker, an oil rotary vacuum pump, or a dry vacuum pump can be used. The ultimate pressure at the time of negative pressure may be in a range where lettuce seeds and antagonistic endogenous bacteria are killed or cells are not substantially damaged, for example, 1 mmHg to 755 mmHg, preferably 100 mmHg to It is a range of 700 mmHg (expressed as a degree of vacuum when the atmospheric pressure is 0 mmHg). The time from the normal pressure to the maximum negative pressure is not particularly limited, but may be, for example, in the range of 1 second to 120 minutes. In addition, the time under the maximum negative pressure condition may be in the range of 1 minute to 100 minutes. Thereafter, the pressure is slowly returned to normal pressure, but the time for returning to normal pressure from the negative pressure condition may be in the range of 1 second to 120 minutes. It may not be preferable to leave for 120 minutes or longer under the maximum negative pressure because the viability of endophytic bacteria decreases or the germination rate significantly decreases. The number of times of vacuum inoculation treatment may be in the range of 1 to 20 times. By repeating the vacuum inoculation, the rate of introduction of endophytic bacteria into the seed may increase, but if excessively performed, the seed may be damaged, resulting in a decrease in germination rate. The sealed container can be used by creating a closed system by attaching a rubber stopper to the suction bottle or pressure bottle or sealing with a sealing tape. If the sealed system can be maintained, There are no special restrictions on materials. The size of the container can be appropriately selected according to the amounts of lettuce seeds and antagonistic endogenous bacteria, and can be selected, for example, from the range of 1 ml to 1000 m ³ . The connecting part that connects the suction device and the sealed container may be a pressure-resistant pipe that can maintain a closed system and can withstand negative pressure conditions, and should be of a material that does not harm lettuce seeds or antagonistic endogenous bacteria. There are no particular restrictions. If it is difficult to assemble the apparatus, an existing vacuum drying apparatus, a low-temperature vacuum drying apparatus, a rotary evaporator, a freeze dryer, or the like can be used.

  As a method of mixing and contacting lettuce seeds and antagonistic endogenous bacteria for vacuum inoculation, any method that is generally used may be used, and there is no particular limitation. For example, lettuce seeds are immersed in a suspension containing antagonistic endogenous bacteria, sprayed with a suspension containing antagonistic endogenous bacteria on lettuce seeds, and put into lettuce seeds containing antagonistic endogenous bacteria And then dress up. Stirring and mixing are preferable in order to increase the contact efficiency between the lettuce seeds and the antagonistic endogenous bacteria, but care should be taken because lettuce seeds may be damaged if performed excessively.

The amount of antagonistic endogenous bacteria inoculated into the lettuce seed is not particularly limited, and may be in the range of, for example, 10 ¹ to 10 ¹⁰ cells / grain.

  The method for producing an antagonistic endogenous bacterial-coated lettuce seed according to the present invention comprises the step of drying the seed under low-temperature and low-humidity conditions after inoculating the lettuce seed under reduced pressure with the antagonistic endogenous bacteria according to the method described above. It is more preferable. Alternatively, in the method for producing an antagonistic endogenous bacterial-coated lettuce seed of the present invention, the seed is dried under a low temperature and low humidity condition after inoculating the antagonistic endogenous bacteria into the lettuce seed by a method other than the above-described reduced pressure inoculation. It may be a method of performing a process. Here, “the seed is dried under low-temperature and low-humidity conditions after inoculating the antagonistic endogenous bacteria into the lettuce seeds (under reduced pressure)” means that the drying step under the low-temperature and low-humidity conditions means that the antagonistic endogenous bacteria are Any form is included as long as it is performed after the inoculation of lettuce seeds. That is, in the present invention, a step of drying lettuce seeds under low-temperature and low-humidity conditions may be performed subsequent to inoculation of lettuce seeds with antagonistic endogenous bacteria. After the inoculation, an additional treatment (for example, pellet granulation) may be performed, followed by a step of drying the seeds under low temperature and low humidity conditions. As a method of inoculating lettuce seeds with antagonistic endogenous bacteria by a method other than vacuum inoculation, lettuce is prepared by immersing lettuce seeds in a suspension containing antagonistic endogenous bacteria. Examples include, but are not limited to, methods of spraying seeds and putting lettuce seeds in a powder containing antagonistic endogenous bacteria.

In the present invention, the “low temperature and low humidity condition” in the coating seed treatment refers to a condition that is a temperature (low temperature) equal to or lower than normal temperature (about 25 ° C.) and a humidity equal to or lower than indoor humidity (low humidity).
More specifically, the “low temperature” is a temperature in the range of −80 ° C. to normal temperature, and among them, a temperature in the range of −10 ° C. to 20 ° C. is particularly desirable. “Low humidity” means indoor humidity or lower, which varies depending on indoor humidity, but is usually in the range of 0% to 80%. Of these, a range of 0% to 40% is particularly desirable. Examples of the method for reducing the temperature include a room having a cooling device or a method using a container containing a coolant, a cooler box, a refrigerator, a freezer and the like. Examples of the method for decreasing the humidity include a method using a chemical desiccant such as quick lime, a physical desiccant such as silica gel, zeolite, and clay mineral, a dehumidifier.

  The moisture content of the lettuce seeds after drying is desirably in the range of 0.01% to 20%. A more preferable moisture content is 0.1% or more and 10% or less. If the moisture content is higher than that, problems such as reduction of germination rate of lettuce seeds during storage, germination of lettuce seeds during storage, and growth of bacteria and other germs attached to lettuce seeds. appear. On the contrary, when the water content is low, the survival rate of the antagonistic endogenous bacteria is lowered. In addition, the germination rate of lettuce seeds may be reduced.

  It is desirable to store the antagonistic endobacterium-coated lettuce seed produced according to the method of the present invention under conditions that have as little influence as possible on the viable count of the antagonistic endobacterium and germination of the seed. Such conditions include low temperature and low humidity conditions. Regarding the storage conditions, “low temperature” is preferably −80 ° C. or higher and 30 ° C. or lower, and more preferably 0 ° C. or higher and 20 ° C. or lower. In terms of storage conditions, “low humidity” is preferably 0% or more and 80% or less, and more preferably 0% or more and 50% or less.

  By sowing the antagonistic endobacterium-coated lettuce seeds produced by the method of the present invention, lettuce big bain disease can be reduced or suppressed. That is, the present invention relates to a method for controlling lettuce big vein disease using an antagonistic endogenous bacterial-coated lettuce seed. For example, after sowing and raising seedlings of lettuce seeds coated with antagonistic endophytic bacteria, lettuce big The incidence of Bain's disease is reduced or suppressed.

  The method for controlling a lettuce big vein disease of the present invention can be used in combination with other disease control methods. Other disease control methods include, for example, soil disinfection treatment, chemical treatment, soil amendment treatment, and culm treatment for reducing the soil fungus density of the Orpidium spp. In addition, it is preferable to use lettuce seeds of a lettuce big bain disease resistant variety or lettuce big bain disease resistant variety as a lettuce seed coated with an antagonistic endobacterium.

  Examples of the present invention will be described below in more detail, but the present invention is not limited to these examples.

Reference Example 1: Effects of inoculation and drying of lettuce film-coated seeds on the survival rate of antagonistic endogenous bacteria As an antagonistic endogenous bacteria, Pseudomonas sp. FPH-2003 strain (FERM AP-20654) was used.

  Pseudomonas FPH-2003 strain was inoculated into a 9 cm petri dish containing King B agar medium, and statically cultured at 25 ° C. for 2 days. Bacteria were collected using a corn large stick and used as an inoculum.

  To the collected FPH-2003 strain, add 10 ml of a binder solution made of polyvinyl alcohol (PVA) or polyvinyl acetate (PVAc), and sufficiently disperse the FPH-2003 strain using a rotor (stirrer), and lettuce seeds While adding a small amount to 100 g (variety: logic), it was thoroughly stirred. The seeds were placed in a hot air circulating dryer at 25 ° C. or 35 ° C. and dried for 24 hours.

  The bacterial density of the FPH-2003 strain in seeds was determined by the following method. 100 seeds were suspended in 10 ml of sterilized water, and 10-fold, 100-fold, 1000-fold and 10000-fold dilutions were made. These bacterial suspensions were spread on a King B agar medium supplemented with streptomycin by a dilution plate method. The cells were cultured at 25 ° C. for 96 hours and judged by the appearance of colonies.

The results of Reference Example 1 are shown in Table 1. The lettuce seeds inoculated with the FPH-2003 strain using the PVA film coating agent had a bacterial density of 6.2 × 10 ⁴ cfu / grain before drying in any drying temperature range, but it was completely destroyed after 6 hours of drying. It was. In addition, lettuce seeds inoculated with the FPH-2003 strain using PVAc film coating agent had a bacterial density of 1.2 × 10 ⁵ cfu / grain before drying in any drying temperature range, but was completely destroyed after 24 hours of drying. Was. From this, it became clear that it is very difficult to survive the antagonistic endogenous bacteria after inoculating the lettuce seeds with the antagonistic endogenous bacteria and drying.

Reference Example 2: Effect of water content of lettuce pellet seeds inoculated with antagonistic endogenous bacteria on survival of antagonistic endogenous bacteria Pseudomonas (Pseudomonas sp.) FPH-2003 strain (FERM AP- 20654) was used. Pseudomonas strain FPH-2003 was inoculated into a 9 cm petri dish containing King B agar medium, and statically cultured at 25 ° C. for 2 days. Bacteria were collected using a corn large stick and used as an inoculum.

  20 ml of sterilized water was added to the collected FPH-2003 strain, and the FPH-2003 strain was sufficiently dispersed using a rotor (stirrer). This antagonistic endogenous bacterial dispersion was used for pellet granulation of lettuce seeds. Next, the pellet granulation process will be described. While rotating the granulating device Pelletizing unit (SEED PROCESSING), 100g of lettuce seeds (variety: logic) is added, and the seeds are thoroughly moistened by spraying the above-mentioned antagonistic endogenous bacterial dispersion while stirring the seeds. Let A small amount of granulating powder (a mixture of diatomaceous earth and calcium carbonate) was added to the seeds and stirred. Further, the operation of spraying the antagonistic endogenous bacterial dispersion solution and adding a small amount of granulating powder was repeated until the antagonistic endogenous bacterial dispersion solution disappeared. Thereafter, the granulating binder was switched to 3.0% polyvinyl alcohol, and pellets were granulated while alternately adding the granulating binder and the granulating powder. After granulation, only a pellet having a particle size of 3.0 to 3.5 mm was selected using a sieve. The seeds were put in a hot air circulating dryer at 30 ° C. and dried for 24 hours. The humidity of the room at this time was about 45%.

  The method similar to Reference Example 1 was used to measure the bacterial density of the FPH-2003 strain in the pelleted seeds. Further, the survival rate of the FPH-2003 strain in the pelleted granulated seed was determined by the following method. 50 pellet seeds (10 seeds × 5 repeats) were placed on a streptomycin-added King B agar medium and cultured at 25 ° C. for 96 hours. After the culture, colonies of the FPH-2003 strain were counted, and the percentage was determined from the number to determine the survival rate.

  The results of Reference Example 2 are shown in Table 2. Seeds sprayed with lettuce seeds with FPH-2003 strain have higher viability and fungal density of FPH-2003 strains as the moisture content of pellet seeds increases, that is, FPH-2003 strains decrease as pellet seeds are dried. It became clear to go. The moisture content of pellets that can be put to practical use is 0.5 to 3.0%, but it was revealed that the FPH-2003 strain was completely killed when the moisture content was lowered to 0.8%. In the case of lettuce seeds, if the moisture content exceeds 10%, it cannot be used practically because mold occurs during storage, rot starts or germinates. Therefore, it is extremely difficult to establish the antagonistic endogenous bacteria on the lettuce seeds by the conventional method in which the antagonistic endogenous bacteria are spray-inoculated on the lettuce seeds, pelletized, and then dried with warm air. There was found.

Reference Example 3: Effect of lettuce varieties on root colonization of antagonistic endogenous bacteria (1)
Antagonistic endophytic bacterium Pseudomonas sp. FPH-2003 (FERM AP-20654) was cultured on King B agar at 28 ° C for 48 hours and suspended in sterile water at 10 ⁷ cfu / ml each. did. Eight kinds of lettuce varieties were prepared and seeds were coated with an antagonistic endogenous bacterial suspension. The obtained seeds were sown on vermiculite. After seeding, the cells were cultured for 25 days under illumination at 28 ° C. for 24 hours. The root of lettuce was sterilized with 95% ethanol for 10 seconds, ground in a mortar, and antagonistic endogenous bacteria were detected by the dilution plate method using King B agar medium.
The results are shown in Table 3.

Reference Example 4: Effect of different lettuce varieties on root colonization of antagonistic endogenous bacteria (2)
A test similar to that of Reference Example 3 was carried out using the antagonistic endogenous bacterium Pseudomonas sp. FPH-2005-1 strain (FERM AP-20653), and using lettuce cultivars and commercial cultivars that were being cultivated.
The results are shown in Table 4.

  From the results of Tables 3 and 4, it was revealed that there is a considerable difference in the colonization rate of antagonistic endogenous bacteria due to the difference in the variety of lettuce. Therefore, it has been found that it is important in the production of seeds coated with an antagonistic endobacterium to confirm the affinity between the varieties of lettuce seeds and the antagonistic endogenous bacteria in carrying out the present invention. Using the combination of lettuce cultivar thus selected and antagonistic endogenous bacteria, a method for producing pellet seeds was examined.

Example 1: Method for inoculating lettuce seeds with antagonistic endogenous bacteria and the effect of heated draft drying on the survival of antagonistic endogenous bacteria As an antagonistic endogenous bacteria, Pseudomonas sp. FPH-2003 strain ( FERM AP-20654) was used. Pseudomonas (Pseudomonas sp.) Strain FPH-2003 was inoculated into a 9 cm petri dish containing King B agar medium, and statically cultured at 25 ° C. for 2 days. Bacteria were collected using a corn large stick and suspended in sterilized water to which 1/5000 Tween 80 was added. When the viable cell count was measured by the dilution plate method, it was about 1 × 10 ¹⁰ cfu / ml. 100g of lettuce seeds (variety: logic) was wrapped in a mesh and placed in a strawberry pack, a weight was placed so that it did not float, and 300ml of the bacterial suspension was poured so that the seeds would sink. The vacuum inoculation method was a negative pressure condition using a compact air pump NUP-2 (manufactured by ASONE). The pumping capacity was 12 l / min, the ultimate pressure was 300 mmHg, and the time to reach the maximum negative pressure condition was about 2 minutes. After 5 minutes of maximum negative pressure, the cock was slowly opened to return to normal pressure. The time to return to normal pressure was about 20 seconds. In order to remove surplus moisture, it was air-dried at 30 ° C. for 1 hour in a constant temperature dryer (MOV-212F) (manufactured by SANYO).

  The antagonistic endobacterium-coated lettuce seeds thus obtained were further pelletized. The pellet granulation process will be described below. The whole amount of coated lettuce seeds obtained above (about 100 g) is put into a rotating granulator Pelletizing unit (SEED PROCESSING), and a 3.0% polyvinyl alcohol for granulation is added while stirring the seeds. The seeds were sprayed and moistened. After the seeds were sufficiently moistened, a predetermined amount of granulating powder (a mixture of diatomaceous earth and calcium carbonate) was added. Furthermore, pellets were granulated while alternately adding a granulating binder and a granulating powder. After granulation, using a sieve, only pellets having a diameter of 3.0 to 3.5 mm were selected. The seeds were put in a hot air circulating dryer at 30 ° C. and dried for 24 hours. The humidity of the room at this time was about 45%.

Comparative Example 1
The FPH-2003 strain was cultured and collected in the same manner as in Example 1. The immersion treatment of the FPH-2003 strain was performed under normal pressure conditions using 100 g of lettuce seeds (variety: logic) and an antagonistic endogenous bacterial suspension. Excess water was removed and pellet granulation was performed in the same manner as in Example 1. The seeds were placed in a hot air circulating dryer at 30 ° C. and dried for 24 hours. The humidity of the room at this time was about 45%.
The survival rate of the FPH-2003 strain in pelleted granulated seeds was the same as in Reference Example 2.

  The results of Example 1 and Comparative Example 1 are shown in Table 5. In the section (Example 1) where the FPH-2003 strain was pelleted after vacuum inoculation (Example 1), the survival rate of the FPH-2003 strain hardly decreased even when dried, and the moisture content of ordinary pellets for commercial use was 1.1%. Even so, it was as high as 93%. On the other hand, in the section (Comparative Example 1) in which the FPH-2003 strain was pelletized after immersion inoculation, the survival rate of the FPH-2003 strain gradually decreased with drying, which is 1.1%, which is the moisture content of normal commercial pellets. %, The survival rate of the FPH-2003 strain was 3%, indicating that most of the FPH-2003 strains were killed. From this, it was revealed that in the production of lettuce pellet seeds, the method of inoculating the antagonistic endogenous bacteria on the lettuce seeds used under reduced pressure is effective for fixing the antagonistic endogenous bacteria on the lettuce seeds.

Example 2: Effect of Inoculation Method and Drying Method of Antagonistic Endogenous Bacteria on Lettuce Seed on Survival and Seed Germination of Antagonistic Endogenous Pseudomonas (Pseudomonas sp.) FPH-2003 A strain (FERM AP-20654) was used. Under reduced pressure inoculation and pellet granulation were performed under the same conditions as in Example 1. This seed was dried at low temperature and low humidity by the following method. A desiccator was placed in a low-temperature room at 15 ° C., and silica gel was placed therein as a desiccant. The pelleted seeds were placed in a desiccator and allowed to dry for 24 hours. At this time, the humidity in the desiccator was about 20%.

Comparative Example 2
The FPH-2003 strain was dipped in the same manner as in Comparative Example 1, and pellet granulation was performed. The seeds were placed in a hot air circulating dryer at 30 ° C. and dried for 24 hours. The humidity of the room at this time was about 45%.

  The survival rate of the FPH-2003 strain in pelleted granulated seeds was the same as in Reference Example 2. Moreover, the germination rate of seeds was confirmed by the following method. On the filter paper in which deionized water was absorbed, 150 pellet seeds (50 grains × 3 repetitions) were placed and cultivated under dark conditions at 20 ° C. for 14 days. Those that confirmed the presence of hypocotyls and radicles were counted, and the percentage was determined from the number to determine the germination rate.

  The results of Example 2 and Comparative Example 2 are shown in Table 6. In the group (Example 2) in which the FPH-2003 strain was vacuum-inoculated and dried at low temperature and low humidity at 15 ° C. (Example 2), the survival rate of the FPH-2003 strain was 100%. In Comparative Example 2), the survival rate of the FPH-2003 strain was 11%, and Example 2 had a higher survival rate than Comparative Example 2. The seed germination rate also showed a high value of 95% or more, indicating that there was no problem. From these facts, it has been clarified that the method of low-temperature, low-humidity drying after inoculating antagonistic endogenous bacteria under reduced pressure in lettuce seeds is effective for colonizing the antagonistic endogenous bacteria.

Example 3: Storage stability test of antagonistic endogenous bacteria in lettuce pellet seeds dried under low temperature and low humidity after inoculation with antagonistic endogenous bacteria (transition of survival rate of antagonistic endogenous bacteria and germination rate of seeds)
After inoculating the FPH-2003 strain under reduced pressure in Example 2, the pelleted seeds that were granulated and dried at 15 ° C at low temperature and low humidity were sealed. (1) Temperature 5 ° C, humidity 20%, (2) Temperature 15 ° C, humidity 40% (3) It was stored for 3 months under three different conditions of temperature 20/30 ° C temperature change and humidity 80%. The survival rate and seed germination rate of the FPH-2003 strain were investigated at the storage period of 0 month, 1 month, and 3 months.
The results of Example 3 are shown in Table 7.

  In the section where the lettuce pellet seeds were stored at a temperature of 5 ° C. and a humidity of 20% (Example 3-1), the survival rate of the FPH-2003 strain was 96% after 1 month of storage and 36% after 3 months of storage. there were. On the other hand, in the group (Example 3-2) where this seed was stored at a temperature of 15 ° C. and a humidity of 40%, the survival rate of the FPH-2003 strain was 30% after 1 month of storage and 0% after 3 months of storage. It was. In the section where the temperature was changed at 20/30 ° C and the humidity was 80% (Example 3-3), the survival rate of the FPH-2003 strain was 4% after 1 month of storage and 0% after 3 months of storage. It was. Regarding the germination rate of seeds, it showed a high value of 93% or more in any group, and it was found that there was no problem. From these facts, lettuce seeds were inoculated with antagonistic endogenous bacteria under reduced pressure, and after granulation, pellet seeds dried under low-temperature and low-humidity conditions were stored under low-temperature and low-humidity conditions. The survival rate was increased, and it was proved that it was effective in establishing antagonistic endogenous bacteria on seeds for a long time.

Example 4: Inhibitory effect of infection of Olpidium brassicae mediating lettuce big vein disease of seeds coated with antagonistic endogenous bacteria As antagonistic endogenous bacteria, Pseudomonas sp. FPH-2005-1 strain (FERM AP-20653) Was used. Under reduced pressure inoculation and pellet granulation were performed under the same conditions as in Example 2, followed by low temperature and low humidity drying. Seeded seeds coated with antagonistic endophytic bacteria and normal seeds (seed not coated with antagonistic endophytic bacteria) in a planter (25cm x 60cm) filled with Big-Bain disease contaminated soil and grown in a glass greenhouse . Twenty-six days after sowing, lettuce seedlings were extracted and the number of Olpidium brassicae zoosporangium formed at the root was examined under a biological microscope. In normal seeds, 86.3 infections per strain, but in the case of seeds coated with antagonistic endogenous bacteria, the number of infections with 5.5 and Olpidium brassicae could be reduced to about 1/15.
The results are shown in Table 8.

Example 5: Control effect of seed coated with antagonistic endogenous bacteria against lettuce big bene disease Pseudomonas (Pseudomonas sp.) FPH-2005-1 strain (FERM AP-20653) was used as an antagonistic endogenous bacteria. Under reduced pressure inoculation and pellet granulation were performed under the same conditions as in Example 2, followed by low temperature and low humidity drying. Seeded seeds coated with antagonistic endophytic bacteria and normal seeds (seed not coated with antagonistic endophytic bacteria) in a planter (25cm x 60cm) filled with Big-Bain disease contaminated soil and grown in a glass greenhouse . The disease was investigated 80 and 110 days after sowing. With normal seeds, the disease rate was 30.0% after 80 days of sowing and 100% after 110 days of sowing. In the seed group with antagonistic endophytic bacteria, the rate was 5.9% after 80 days of sowing and 67.7% after 110 days of sowing, which was a lower disease rate compared to normal seeds. From these results, the seeds coated with antagonistic endogenous bacteria showed a high disease-inhibiting effect against lettuce big-vein disease.
The results are shown in Table 9.

Example 6: Examination of reduced-pressure inoculation conditions Pseudomonas (Pseudomonas sp.) FPH-2005-1 strain (FERM AP-20653) was used as an antagonistic endogenous bacterium. Pseudomonas (Pseudomonas sp.) Strain FPH-2005-1 was inoculated into a 9 cm petri dish containing King B agar medium, and statically cultured at 25 ° C. for 2 days. Bacteria were collected using a corn large stick and suspended in sterile water to which 1/5000 Tween 80 was added. When the viable cell count was measured by the dilution plate method, it was about 1 × 10 ⁹ cfu / ml. Lettuce seeds 20g (variety: logic) were wrapped in a mesh and placed in a strawberry pack, a weight was placed so that it did not float, and 150ml of the bacterial suspension was poured so that the seeds would sink. In the vacuum inoculation method, the conditions of vacuum inoculation were examined using an MDA-015 pump. After inoculation under reduced pressure, it was dehydrated and dried in a constant temperature dryer at 30 ° C. for 15 hours. Detection of bacteria from seeds was performed by the following method. 100 seeds were placed on a streptomycin-added King B agar medium, cultured at 25 ° C. for 2 days, and then seeds with fluorescent colonies appeared were counted. The seed germination rate was determined by placing 150 pellet seeds (50 grains x 3 repetitions) on filter paper that had absorbed deionized water, and cultivated under dark conditions at 20 ° C for 14 days. Those that confirmed the presence of hypocotyls and radicles were counted, and the percentage was calculated from the number to determine the germination rate.

(Example 6-1) Examination of maximum negative pressure condition The pressure control valve of the pump MDA-15 was adjusted to change the maximum negative pressure condition.
(Example 6-2) Examination of return time from maximum negative pressure to normal pressure The return time to normal pressure was adjusted by adjusting the opening and closing of the air holes.
(Example 6-3) Examination of retention time of maximum negative pressure The retention time of the maximum negative pressure was changed.
As a comparative example, the detection rate and germination rate of the fungus were examined using the seeds subjected to the immersion treatment.

  The results are shown in Table 10. From the result of Example 6-1, it was found that there was no problem in both the bacteria detection rate and germination rate in the range of maximum negative pressure of 150 mmHg to 680 mmHg. From the results of Example 6-2, it was found that both the detection rate of germs and the germination rate were satisfactory when the return time from the maximum negative pressure to normal pressure was in the range of 25 to 50 seconds. From the results of Example 6-3, it was found that the retention rate of the maximum negative pressure was slightly decreased in the range of 1200 to 3600 seconds compared to 300 seconds.

  It was found that there was no problem in inoculating the lettuce seeds with endogenous bacteria within the range of the vacuum inoculation conditions of Example 6.

Example 7: Effect of inoculation method and drying method of lettuce seeds on survival of antagonistic endogenous bacteria FPH-2005-1 strain (FERM AP-20653) in the same manner as in Example 6 The test was conducted by combining inoculation under reduced pressure and low-temperature and low-humidity drying similar to Example 1, and inoculation and warm-air drying under normal temperature and normal pressure conditions similar to Comparative Example 1.

Endogenous bacteria were detected by placing pelleted seeds into 96-well microwells, injecting 100 μl of King B liquid medium supplemented with streptomycin 200 ppm and thiofamate methyl 1,000 ppm, culturing at 25 ° C. for 48 hours, and 340 nm The wells that were irradiated with ultraviolet light and showed fluorescence were counted. The fluorescence intensity was classified into three levels (+: faint fluorescence is seen. ++: fluorescence is seen. +++: strong fluorescence is confirmed). Then, the percentage of the wells showing each fluorescence intensity with respect to all the wells was determined.
The results are shown in Table 11.

  From the results in Table 11, it was confirmed that endophytic bacteria FPH-2005-1 strains inoculated by single treatment and combined treatment of reduced-pressure inoculation and low-temperature and low-humidity drying were frequently detected from lettuce pellet seeds.

  From the above results, lettuce seeds can be obtained by inoculating lettuce seeds under reduced pressure, inoculating lettuce seeds with antagonistic endogenous bacteria, then drying under low temperature and low humidity conditions, and a combination thereof. It is clear that the survival rate of the inoculated antagonistic bacteria is significantly increased. Furthermore, the antagonistic endophytic-coated lettuce seeds prepared according to the present invention showed a high control value against lettuce big bain disease. By using the present invention, it becomes possible to provide lettuce seeds that are highly effective in controlling lettuce big vein disease and have high storage stability at low cost and in a simple manner.

Claims (8)

  A method for producing a lettuce seed coated with an endogenous bacterium, comprising inoculating the lettuce seed with an endophytic bacterium having an antagonistic property to an genus Orpidium having a lettuce big bean virus.   The method according to claim 1, further comprising drying the lettuce seeds under low-temperature and low-humidity conditions after inoculating endophytic bacteria under reduced pressure.   Characterized in that an endophytic bacterium that is antagonistic to the genus Orpidium that retains the lettuce big bain virus is inoculated on a lettuce seed, and the seed is dried under low-temperature and low-humidity conditions after inoculation. A method for producing coated lettuce seeds.   A lettuce seed produced by the method according to any one of claims 1 to 3, coated with an endogenous bacterium that exhibits an antagonistic property to an genus Orpidium that retains a lettuce big bain virus.   A method for controlling a lettuce big bain disease, characterized by inoculating lettuce seeds under reduced pressure with endophytic bacteria having antagonistic properties against the genus Orpidium that retains the lettuce big bain virus, and sowing the seeds.   The method according to claim 5, further comprising drying the seeds under low-temperature and low-humidity conditions after inoculating the lettuce seeds with endogenous bacteria under reduced pressure.   Inoculating lettuce seeds with endophytic bacteria having an antagonistic property against the genus Orpidium that retains the lettuce big bain virus, drying the seeds under low-temperature and low-humidity conditions after inoculation, and sowing the seeds , A method of controlling lettuce big bain disease.   The method according to any one of claims 5 to 7, further comprising storing the lettuce seeds inoculated with endophytic bacteria under low temperature and low humidity conditions between the end of drying and sowing.