JP2010000078A - Process for producing plant activator, activation method, activity promoter and method for applying the promoter - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To activate a plant and promote the activity by using a product of Trichoderma harzianum SK-5-5 strain. <P>SOLUTION: The activation of a plant and the promotion of the activity can be achieved by culturing Trichoderma harzianum SK-5-5 strain in a solid medium and extracting its product, or by applying the conidiospores of the above strain. There provided are a process for producing the plant activator having such characteristics, an activation method, an activity promotor and a method for applying the promotor. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to the production of a plant activity-imparting agent for the purpose of obtaining a filamentous fungus extracted from a medium of Trichoderuma hurzianum SK-5-5 (Trichoderuma hurzianum SK-5-5) and a substance having antibacterial properties against the bacterium. Regarding the method. Another invention relates to a method for imparting plant activity for the purpose of imparting resistance to pathogenic bacteria to the roots, stems, leaves, etc. of plants by substances produced by conidia of Trichoderma harzianum SK-5-5. The present invention relates to a plant activity promoter and a method for applying the same.

  Trichoderma is a fungus that has been put to practical use as a microbial pesticide for disease control, and Trichoderuma lignourum has been registered as a pesticide for control of tobacco silkworm (Agricultural Chemical Registration No. 7023). In the UK and France, microbial pesticides (F-stop) are also registered, which are effective against vegetable wilt and seedling rot, which contain Trichoderuma hurzianum as an active ingredient.

  Recently, the need for pesticide reduction and ecosystem conservation type disease control is strongly advocated, and the biological control method is considered to have little impact on the environment, so research is being conducted energetically. Some bacteria and fungi isolated from plant roots and rhizospheres promote plant growth. Plant growth-promoting rhizobacteria (PGPR) and plant growth-promoting fungi (plant) It is called growth-promoting fungi (PGPF).

  The useful rhizosphere microorganisms PGPR and PGPF are known not only to promote plant growth but also to suppress various soil diseases. Recently, the fact that not only soil diseases but also ground diseases are also clarified, and it has been introduced that the PGPR and PGPF were found to be involved in the induction of systemic resistance of plants (1999). June issue, this month's agricultural magazine).

  In addition, it has been reported that fungi such as Phoma, Trichoderma, Fusarium, Penicillium, and Sterile isolated and selected from cucumber shiba show induction resistance against anthracnose in cucumber (June 1999 issue, this month's agricultural journal). ).

Japanese Patent Laid-Open No. 6-192028

June 1999 issue, “Agriculture of the Month”

  The present invention has been studied as an object to obtain a substance having antibacterial properties against plant pathogenic bacteria, and Trichoderuma hurzianum SK-5-5 (Trichoderma harzianum SK-5-5) is isolated from soil in Tokachi region, Hokkaido. This bacterium is widely antagonizing against soil-borne pathogenic fungi, which accounts for 80% of plant pathogens. Currently, microbial pesticides (living bacteria) are present in Rhizoctonias such as turf brown and large batches. It is under development as a formulation. The antagonism of the Trichoderma haldianum SK-5-5 bacterium promotes cytoplasmic aggregation and kills by contacting the other hyphae and coiling. It is presumed from the appearance observation process that the activity is not so high as to show a circle of inhibition on a medium such as an antibiotic produced by penicillium (Penicillium sp) and the like, and is expressed by contact between mycelia.

It is considered that the cytoplasmic aggregation action is brought about by some substance produced by Trichoderma harzianum SK-5-5, and it is a first object to investigate this.
The Trichoderma harzianum SK-5-5 bacterium used in the present invention has been deposited with the Japanese Industrial Technology Institute, Microbial Industrial Research Institute under the accession number “Seikenken No. 13327”. The Institute of Fine Arts and Technology has requested transfer from the original deposit to a deposit based on the Budapest Treaty and has been assigned a deposit number of BP-4346.

The conventional PGPR and PGPF are effective against anthracnose for specific plants (for example, cucumber). As a result of future research, improvement of the method of use, etc., against other plants and pathogens Even though it may prove effective, it is still considered as a future problem for specific plants and pathogens.
However, the fact that the conventional form of biological pesticides, which simply sterilizes pathogenic bacteria, is changing in the direction of imparting resistance to the rhizomes of plants, includes important suggestions for future plant cultivation.

  The present invention was conceived to be consistent with the potential of the biopesticides during various experiments on Trichoderma harzianum SK-5-5, and further, the method of use, the target plant, etc. were selected, and the results of the research The present invention has been completed. The present invention is very promising as having a great influence on future plant cultivation, and there is no doubt that it will be one of the important means for supporting future agriculture and the like.

  The present invention presumes that the substance produced by Trichoderma harzianum SK-5-5 bacterium causes cell agglutination (antibacterial activity). When the bacterium is cultured, purified and identified, it is antibacterial against filamentous fungi or bacteria. The substance with the property was obtained.

  In another aspect of the invention, in the cultivation of plants, conidia of Trichoderma harzianum SK-5-5 are coexisted in the overburden and proliferated, thereby imparting rhizome activity of various plants with the product, As a result, it has been found that the conventional problems have been improved and the practical problems have been established.

  That is, the invention of the activity-imparting agent is a plant activity-imparting agent characterized in that it is a substance obtained by culturing Trichoderma harzianum SK-5-5 bacteria in a solid medium and extracting them, and having antibacterial activity against filamentous fungi. Another invention is a substance obtained by culturing Trichoderma harzianum SK-5-5 bacteria in a liquid medium and extracting the plant, characterized by having antibacterial activity against a kind of bacteria (Staphylococcus aureus 209p) It is an agent.

  Next, the invention of the production method is such that Trichoderma harzianum SK-5-5 is inoculated into a solid medium, left to stand at 25 ° C. to 30 ° C. for 7 to 15 days, extracted, and extracted. And after inoculating Trichoderma harzianum SK-5-5 in a liquid medium and shaking culture at 25 ° C. to 30 ° C. for 4 to 10 days. Extracting to obtain the above-mentioned activity imparting agent, a method for producing a plant activity imparting agent.

  In this invention, a rice medium was used as the solid medium to be used. The rice medium was a fixed medium composed of 100% rice, 3% soybean meal, and 10% sterilized water, and sterilized water was added so that the surface of the medium was not dried during the cultivation.

  In this invention, the liquid medium to be used is glucose 3.0% to 5.0%, polypeptone 0.5%, Nacl 0.8%, yeast extract 0.2%, calcium carbonate 1.0%.

  As described above, it has been clarified that the fixed medium and the liquid medium generate different substances depending on the respective characteristics. Although the mechanism for such a result is not clear, it produces different substances depending on differences in stimulation and other actions on Trichoderma harzianum SK-5-5, and on the characteristics of the medium constituents. Even if it is a culture medium of components other than a liquid culture medium, producing | generating the same substance is considered enough, and future research subjects, such as improvement of production efficiency, are great.

  Next, the invention of the activity imparting method is a plant characterized in that, in plant cultivation, an application means is added to the conidia of Trichoderma harzianum SK-5-5 and coexisted in the covering soil imparting activity to the plant. In the plant cultivation, an application means is added to the conidia of Trichoderma harzianum SK-5-5, and it is allowed to coexist in the covering soil that imparts the activity to the plant. A plant activity imparting method characterized by sustaining production of a substance having antibacterial activity. In addition, during plant cultivation, an application means is added to the conidia of Trichoderma harzianum SK-5-5, and it is allowed to coexist in the cover soil that imparts activity to the plant, and conditions for promoting the growth of the conidia are provided. The method for imparting plant activity characterized by the above, wherein the conidia application means are plant seed treatment, mixing of the cover soil with the conidia, spraying of the conidia, irrigation or embedding. Further, the growth promotion condition is that the soil temperature is 15 ° C. to 30 ° C. and the water content is 30% or more, and the other growth promotion condition is to impart air permeability or aeration that can grow on the soil. .

  Next, another invention is a plant activity promoter characterized in that conidia of Trichoderma harzianum SK-5-5 are attached to porous ceramic particles and other carriers, and is an aseptically treated inorganic particle Plant activity characterized by mixing conidia of Trichoderma harzianum SK-5-5, or adhering Trichoderma harzianum SK-5-5, and mixing the inorganic particles with other particles It is an accelerator. In addition, the conidia of Trichoderma harzianum SK-5-5 were cultured, separated to an appropriate amount together with the medium, attached to a sterile porous particle together with nutrients (for example, chitosan), and then packaged for the predetermined amount. A plant activity promoter characterized by the growth of Trichoderma harzianum SK-5-5 conidia in a liquid medium, which is attached to a sterile porous particle with nutrients, and then the porous particle is placed in place. It is a plant activity promoter characterized by being packaged for fixed quantity.

Another aspect of the invention is a method for applying a plant activity promoter, characterized in that the plant activity promoter described above is mixed into seedling soil, or is sprayed on soil or on a pavement at the time of seedling. In this case, the amount of the activity promoter used is 5 to 10 g of 5 × 10 ⁴ / g to 5 × 10 ⁹ / g of Trichoderma haldianum SK-5-5 per 1 m ² or 5 to 100 g mixed per 1 m ^{3 of} cultivated soil. To do. Plants in the invention are sugar beet, melon, corn, paddy rice, cabbage and onion, etc., and it is confirmed that all of the plants from which roots, stems, leaves and fruits are collected are effective, and the effect is also increased in yield. Increased sugar content and other potentials became clear.

  The conidia of Trichoderma harzianum SK-5-5 in the above invention were cultured, and the isolated and purified product was examined. From the results of UV absorption spectrum, mass spectrometry and NMR measurement, Aib (α-Aminoisobutyric acid) was obtained. The polypeptide contained was determined to be Peptibols. Peptibols antibiotics contain α-Aminoisobutyric acid (Aib) in the amino acid sequence, and are characterized by an N-terminal Acetyl group and a C-terminal Amino alcohol bond (mostly Phenylalaniol groups). ). As for the purified product, A component, B component, and D component, the partial amino acid sequence was estimated by mass spectrometry as follows, and the partial structure was searched with the existing amino acid sequence of Peptibols. There was no new Peptibols system. E could not be determined because it was difficult to estimate the amino acid sequence.

UV absorption wavelength: terminal absorption of all four components A component = 1933
B component = 1949 or 1964
D component = 1810
E component = 1829
Estimated structure:
A component: Ac-Aib-Ala-Aib-Aib-Aib-Aib-Gln-Aib-Aib -......
B component: Ac-Aib-Ala-Aib-Aib-Val-Aib-Gln-Aib-Aib -......
D component: Ac-Aib-Ala-Aib-Aib-Aib -.....
As described above, the product is recognized as an amino acid having a novel sequence, but is absorbed from the roots of the plant, reaches the stem and leaves, and then disappears. Therefore, it is unknown whether the roots, stems, and leaves remain in the plants at the time of harvest, but some changes are made to the DNA of the plants when they are absorbed by the plants (eg, young shoots after germination). It is presumed that there is something or remains. This is because plants that have once obtained antibacterial properties are recognized as having antibacterial properties regardless of fission formation such as rhizomes. Therefore, it is similar to immunity imparting (activity imparting), and if applied to seedlings or relatively young plants (substantially during plant growth), there is no need for re-use, once it can be activated This is because the efficacy is maintained throughout the life of the plant.

  When the conidia of Trichoderma harzianum SK-5-5 of this invention are mixed in the cover soil at the time of plant seedling or sowing, the amount of product increases with the growth of the conidia, Since it is absorbed and reaches the foliage, and the activity is imparted to the whole (as is the case with immunity), it is time for the plant to grow and collect roots or fruits (for example, 1 to 6 months after sowing). However, it was confirmed that the antibacterial property was maintained. Therefore, many plants were imparted with activity by a single treatment (dispersion or other means), and their objectives could be fully achieved.

  The conidia carrier in the present invention is porous ceramic particles (for example, barley stone particles), which are immersed in chitosan liquid and water is evaporated. Although the size of the particles is not specified, a diameter of about 0.5 mm to 5 mm is preferable because of easy handling. The carrier is not limited to porous ceramic particles (natural or artificial), and those that do not adversely affect the conidia can be used.

Trichoderma harzianum SK-5-5 bacteria previously cultured on the ceramic particles are attached to 5 × 10 ⁴ / g to 5 × 10 ⁹ / g and sprayed to 5 to 100 g per 1 m ² . In this case, it may be sprayed together with water, but irrigation may be used for the purpose of watering or when it is used to uniformly infiltrate the conidia and distribute it in the soil. is there.

When the amount of Trichoderma harzianum SK-5-5 bacterium is less than 5 × 10 ⁴ / g, it is proliferated if the bacterium is not inhibited from breeding, but may not grow for some reason. , For the time being. However, since the growth of bacteria varies significantly depending on the environment, it is considered that even if a small amount (for example, less than 5 g) of bacteria grows along with the growth of the plant, it is possible to obtain the required amount of product by environmental preparation after application. It is done.

On the other hand, the reason why the upper limit of the amount of bacteria was set to 5 × 10 ⁹ / g or less was that it was considered unnecessary because of the relationship between the production substances. When the growth conditions of the fungus are poor, it is necessary to apply a relatively high concentration, but it is not considered necessary to exceed 5 × 10 ⁹ / g. The Trichoderma harzianum SK-5-5 bacterium in the present invention is usually given as conidia, but it may be preferable to give it as a thick film spore when the environment during application is severe (for example, at high temperature or cold).

In the above invention, when Trichoderma harzianum SK-5-5 bacterium is cultured in a solid medium, liquid medium or other medium, and the product is extracted and applied as a plant activity-imparting agent, for example, 0 .1 to 1 g / m ^{2 is} required.

In addition, when spraying conidia, the lower limit number of bacteria (for example, 5 × 10 ⁹ / g) of the conidia is necessary.

  The plant activity-imparting agent of the present invention is applied only once at the time of fungi, and does not need to be applied until after harvesting. This is different from general pesticides, and its mechanism is unknown, but it has some effect on the DNA of the plant, or the produced substance remains in trace amounts in the roots, stems and leaves of the plant and exhibits the above characteristics It is estimated to be.

  In the above, when conidia are applied, it is confirmed that the conidia rapidly loses vitality and eventually disappears if the conidia grows sufficiently on the pavement and reaches saturation. It was done. However, conidial spores may partially remain, and if the growth conditions are improved, they may proliferate again.

In the above invention, Trichoderma haldianum SK-5-5 was cultured in a solid medium or a liquid medium, and the components A, B, C, D, and E in the product were isolated and purified. The molecular weight is A component = 192, B component = 206, C component = 168, D ₁ = 154, D ₂ = 220, and the amino acid or its peripherals are used for plant activation. Presumed to be effective.

Therefore, by applying conidia of Trichoderma harzianum SK-5-5 at a predetermined concentration (for example, 5 × 10 ⁵ or more in 1 g) or more (mixed in soil or sprayed in root rhizosphere of root), It has been confirmed that the objective is achieved. Perhaps there must be an optimal component or mixing ratio depending on the plant.

  Accordingly, the Trichoderma harzianum SK-5-5 bacterium can be cultured in a large amount by a conventional method, and the product can be extracted and purified to obtain a plant activity-imparting agent. The product A, B component, C component, D component, and E component are known.

  The invention is effective for various plants, but according to experimental results (including during the experiment), it has been confirmed that it is effective for rice, corn, sugar beet, melon, potato, sweet potato, strawberry, onion, cabbage and the like. It was.

According to this invention, when Trichoderma harzianum SK-5-5 bacterium was cultured in a solid medium, as shown in Table 2, it was confirmed that the fungus exhibited antibacterial properties.
When Trichoderma harzianum SK-5-5 was cultured in a liquid medium, it was recognized that it was effective against bacteria as shown in Table 2.
According to the present invention, at the initial stage of plant growth, the plant is activated by mixing an appropriate amount of Trichoderma harzianum SK-5-5 bacteria into the floor soil or by applying to the rhizosphere such as spraying during growth. The roots, stems and leaves can be improved, disease prevention and resistance against pathogenic bacteria can be imparted. This not only drastically reduces the occurrence of disease at the time of growth, but also has various effects such as promoting the growth of plants, increasing the yield, and improving the sugar content.
However, the effect is presumed to be caused by a conidial spore product of Trichoderma harzianum SK-5-5 (for example, a novel amino acid). Since it is recognized that all disappear at the end of growth, there is no effect of any meaning, and there is an effect that no adverse effect is seen even in continuous use. Furthermore, since the product is recognized as a novel amino acid and its peripheral substances, it is harmless even if it remains in a small amount on the leaves and stems of plants.

  The Trichoderma harzianum SK-5-5 bacterium of the present invention grows in soil, so that if the environmental conditions are suitable for growth, even if the initial spraying concentration is insufficient, the necessary amount is increased by the growth. After replenishing the product and achieving the intended purpose, it has a characteristic of self-destruction, and there is no risk of soil, plant or other environmental damage, as well as impact on the human body. Furthermore, it was estimated that the disadvantages of continuous cropping would be resolved for crops with poor continuous cropping.

The HPLC analysis graph in A culture medium culture | cultivation of the microbe of this invention. The enlarged graph of FIG. The graph which similarly shows the UV absorption wavelength of A component. Similarly graph showing the _{^{1 H-NMR (CD 3 OD}} ) of the A component. (A), (b) The graph of the mass spectrometry measurement of A component similarly. The graph which similarly shows the UV absorption wavelength of B component. Similarly graph showing the ¹ H-NMR of the B component. (A), (b) The graph of the mass spectrometry measurement of B component similarly. The graph which similarly shows the UV absorption wavelength of D component. Similarly graph showing the ¹ H-NMR of the D component. (A), (b) The graph of the mass spectrometry measurement of D component similarly. The graph which similarly shows the UV absorption wavelength of E component. Similarly graph showing the ¹ H-NMR of E component. (A), (b) The graph of the mass spectrometry measurement of E component similarly. The graph which similarly shows the UV absorption wavelength of A component. The graph which similarly shows HPLC of A component. The graph of the mass spectrometry of the detected A peak of FIG. The graph which similarly shows the UV absorption wavelength of B component. The graph which similarly shows HPLC of B component. 20 is a graph of mass spectrometry of the detected A peak in FIG. 19. The graph which similarly shows the UV absorption wavelength of C component. The graph which similarly shows HPLC of C component. 22 is a graph of mass spectrometry of the detected A peak in FIG. Similarly, mass analysis graph of C component. Similarly graph showing the ¹ H-NMR peaks of the component C _(CD 3 OD). The structural drawing of C component similarly. The graph which similarly shows the UV absorption wavelength of D component. The graph which similarly shows HPLC of D component. (A) The graph which shows D1 component similarly detected by UV (lambda) 230nm. (B) A graph showing the D2 component detected by the difference of RT1 and the D1 component by UV absorption at the same time. (A) The graph which similarly shows the mass spectrometry of D1 component detected in FIG. (B) The graph which similarly shows the mass spectrometry of D2 component detected in FIG. The systematic diagram of the culture extract refinement | purification process of a rice culture medium. The systematic diagram of the culture extract refinement | purification process of B culture medium I similarly. The structure figure of the derivative | guide_body of the novel substance (MW: 198) 1,2,3 similarly. Similarly, when applied to an onion, an explanatory diagram of the growth survey section before wintering. Similarly, the onion valve diameter and leaf area correlation diagram.

  This invention is a plant activity-imparting agent characterized by culturing Trichoderma harzianum SK-5-5 bacteria in a solid medium or a liquid medium to produce antibiotics. In addition, the invention of the production method inoculates Trichoderma harzianum SK-5-5 in a solid medium or liquid medium such as a rice medium, and at a predetermined temperature (for example, 25 ° C. to 30 ° C.) for a predetermined time (for example, 4 days to 30 days). This is a method for producing a plant activity-imparting agent, characterized by purifying and extracting after culturing for 2 days).

  The invention of the activity imparting method is one in which conidia of Trichoderma harzianum SK-5-5 are coexisted in the soil covering near the rhizosphere. In this case, the conidia have porous ceramic particles as a carrier. In this way, it is preferable because it is easy to spray and sufficient oxygen replenishment can be ensured, and the porous ceramic particles can be supplemented with chitosan and other nutrients necessary for the growth of conidia. Even more preferred. By doing so, the growth of conidia can be made smooth and reliable, and the number of isolated products of useful substances inevitably increases.

  Next, the invention of the activity imparting agent has a soil temperature of 15 ° C. to 30 ° C. and a water content of 30% or more in order to improve the growth environment in the soil, but there is no need for other miscellaneous bacteria treatment. In some cases. Further, the invention of the application method includes adhesion to seeds, spraying, mixing with soil, and infiltration by spraying watering.

(Test bacteria name)
Trichoderuma hurzianum SK-5-5 (Trichoderma harzianum SK-5-5)
The test bacterium received a slant of agar medium from Hokkaido Green Kosan in January 1997. The test bacteria were inoculated into the following slant culture medium, cultured at 28 ° C. for 5 days, and stored in a refrigerator.

Agar slope medium composition Oatmeal 5.0%
Sucrose 5.0%
Agar 1.0%
Refrigerated after incubation at 28 ° C for 5 days

(culture)
Cultivation was performed in two types of rice medium (A medium) and liquid medium (B medium I) using a 500 ml Erlenmeyer flask. The condition of the medium A was stationary culture at 28 ° C. (10 ml of sterilized water was added during the culture), and the culture of the medium B was shake culture at 28 ° C. at 270 rpm.
Medium composition: A medium 100% rice
Soybean ground 3%
Sterile water 10%
Medium composition: B medium I
Glucose 5.0%
Polypeptone 0.5%
NaCl 0.8%
Yeast extract 0.2%
Calcium carbonate 1.0%

(Test method)
All tests were performed in vitro by the paper disk plate method, and the following bacteria were used as test bacteria.

(1) Rhizoctonia solani (AG-1IA)
A medium having a composition of PDA (Nissui) 1.3% and Chloramphenicol 0.002% was used as an assay medium. The tip of Rhizoctonia hyphae was removed with a cork borer and placed in the center of the plate medium, and a paper disk soaked with broth was placed to observe the growth of the hyphae.

(2) Botrytis cinerea
A medium comprising a composition of Potato-extract 20.0%, Sucrose 2.0%, and agar 1.5% was used as the assay medium. The test was carried out by placing the sample on a paper disc, air-drying and then placing on a petri dish. The petri dish was placed in an incubator and cultured, and it was observed whether or not a blocking circle was formed around the paper disk.

(3) Antibacterial spectrum test bacteria The antibacterial spectrum was measured using the following test bacteria.
Bacillus subtilis ATCC6633
Micrococcus luters ATCC6633
Staphylococcus aureus 209P
Escherichia coli NIHJ
Saccharomyces cerevisiae SHY3
Candida albicans M9001
Candida pseudotropicalis M9035
Cryptococcus neoformans M9010
Debaryomyces hansenii M9011
Trigonopsis variabilis M9031
Schizosaccharomyces pombe M9025
Hansenula schneggi IAM4269

(Culture method)
(1) A medium culture method
Rhizoctonia solani (AG-1IA) active product Isolation and purification of the culture extract actives of medium A against the filamentous fungus Rhizoctonia solani (AG-1IA).
100 g of rice and 3 g of soybean meal were added to a 500 ml Erlenmeyer flask of A medium (1 kg of rice medium) to prepare 10 mediums under the same conditions. After sterilization, inoculate with ase and shake at 280 ° C for 10 days (shake the flask several times in the middle so that the bacteria can grow well. Add sterile water when the surface of the medium has dried.) Extraction was performed by adding 2 liters of% acetone water. Here, 2 liters of 50% acetone extract of the culture was obtained.

(2) B medium culture method
Staphylococcus aureus 209P active product Isolation and purification of active product in B medium culture extract against bacterium Staphylococcus aureus 209P.

  For the purpose of producing more antibacterial active ingredients of B medium I, which is a liquid medium, four types I to IV having different ratios of nitrogen source and carbon source in the medium composition were compared. Two 500 ml Erlenmeyer flasks were used for each medium, and after 5 days of culture, the culture solution was concentrated to 5 times and used for the assay.

(3) B medium I culture method medium B medium (Glucose 5.0%, Polypeptone 0.5%, NaCl 0.8%, Yeast extract 0.2%) , Calcium carbonate 1.0%) In a 500 ml Erlenmeyer flask, 100 ml of medium was prepared. After sterilization, the cells were inoculated and cultured at 28 ° C. for 5 days with shaking. A total of 1.5 liters of the culture solution and an equal amount of acetone were added to obtain 3 liters of 50% acetone extract.

(Purification method)
(1) A medium culture extract purification method A medium culture 50% acetone extract 2 liters of acetone was distilled off, and then 1 liter was adsorbed through HP-20-Sephadex column (60.0 cm x 4.5 cm φ). . After washing with an equal volume of water, it was eluted with an equal volume of 50% acetone and then with an equal volume of 100% acetone. After each fraction was assayed with the assay bacterium Rhizoctonia solani (AG-1IA), it was confirmed that the active substance was eluted in the 100% acetone eluate. Subsequently, 1 liter of eluate with 100% acetone was concentrated to obtain 1098 mg of a crude product, and 100 mg of the crude product was applied to a developing solvent methanol-based LH-20 column (100.0 cm × 2.0 cmφ). Fractions were assayed to confirm active fractions. These active fractions were collected and then developed on silica gel TLC (ethyl acetate: acetic acid: water = 5: 1: 1) and subjected to a color reaction of molybdenum sulfate. As a result, a spot was detected around Rf = 0.3. At the same time, these spots were not detected in the developed part of the inactive fraction. From this, it was estimated that the active substance was a spot detected in the vicinity of Rf = 0.3, and further purification was advanced. After silica gel PTLC development (ethyl acetate: acetic acid: water = 5: 1: 1), scraping and methanol extraction, the product was isolated and purified using HPLC. The isolated and purified product was identified by performing instrumental analysis (FIG. 31).

(2) B medium I culture extract purification method B medium culture 50% acetone extract 3 liters of acetone distilled off (1.5 liters), then 1.5 liters in activated carbon adsorption column (60.0 cm x 2.0 cm φ) Passed through and adsorbed. After washing with an equal volume of water, it was eluted with an equal volume of 50% acetone followed by an equal volume of 100% acetone. As a result of assaying each fraction using the test bacterium Staphylococcus aureus 209P, it was confirmed that the active substance was eluted in the 100% acetone elution part. The eluate (1.5 liters) of 100% acetone was concentrated, partitioned and extracted with ethyl acetate, followed by LH-20 using methanol as a developing solvent, and isolated and purified by HPLC. The isolated and purified product was identified by performing instrumental analysis (FIG. 32).

(Isolated and purified biological activity method)
(1) A medium isolated and purified product evaluation method 4-component biological evaluation method A medium comprising PDA (Nissui) 1.3% and Choloramphenicol 0.002% was used as the assay medium. The hyphae of Rhizoctonia solani (AG-1IA), which had been extracted with a cork borer, was placed on the center of the plate medium, and a paper disk impregnated with the isolated substance was placed on the side to observe the state of hyphal elongation.

(2) B medium I isolated and purified product evaluation biological evaluation of C component (MW168)
The isolated and purified component C (MW: 168) is novel and retains a very simple structure. The biological evaluation was expanded and carried out because of interest as a mother nucleus.

(3) Evaluation of bacteria
Bioassay
Put the agar medium with the test bacteria in a petri dish and harden. A paper disk containing the sample is placed on it and cultured at 37 ° C. for 18 hours, and then the formation of a growth inhibition circle is confirmed.

(a) Strains used
Four strains were used: Staphylococcus aureus 209P, Pseudomonas syringal (tobacco wildfire fungus), Xanthomonoas Campestris pv.citri (citrus canker), Erwinia sp (plum canker).

(b) using the spent medium before bouillon medium for culturing (DIFCO), cultured overnight, diluted × 10 ^2, which mixed with 0.5% top layer of the medium, the bacteria measurement medium MYCIN AGAR (MIKUNI Chemistry) was used.
Upper layer-MYCIN AGAR 1.5% (Mikuni Chemical) + Bouillon 2% medium (DIFCO)
Add 0.5% of the diluted culture broth overnight at 102.
Lower layer-MYCIN AGAR 2.0% (Mikuni Chemical)

(c) Antibacterial measurement A paper disk was impregnated with an isolated and purified product prepared at 1000 pppm, 500 ppm, and 250 ppm, air-dried, placed on a petri dish, cultured at 37 ° C. for 18 hours, and observed for the presence of growth-inhibiting circles.

(4) Measurement of antimicrobial activity (MIC) against staphylococci
(a) Strains used
A total of 22 strains of S. aureusu 209P JC-1, 20 clinical isolates of Staphylococcus aureus (MSSA, MRSA each 10 strains) preserved in the 1G and the reference strain E. coli NIHJ JC-2 were used.

(b) Antibacterial drugs used
MW: 168, methicllin (DMPPC, Stafcillin for injection, Lot.No.FSB 19,900μg / mg, Ariyu Pharmaceutical), vancomycin (VCM, Lot.No.41H0457,10750SIGMA)

(c) Medium used Mueller-Hinton agar (MIA: Difco) was used for antibacterial activity measurement, and Mueller Hinton broth (MHB: Difco) was used for preculture.

(d) Antibacterial activity measurement The minimum inhibitory concentration (MIC) of each drug against the strain used was measured by the agar plate dilution method in accordance with the standard method of the Japanese Society of Chemotherapy. The strain is smeared on MHA, and the colony grown by overnight culture at 37 ° C is cultured overnight at 37 ° C in MHB, and the bacterial solution is diluted 100 times (only E. Coli is diluted 1000 times). Bacterial fluid was used.

(result)
(1) As a result of the test bacteria, the culture medium activities of A medium and B medium I are as shown in Table 2.
The antibacterial spectrum was measured using the following test bacteria.

  As a result of the assay in the culture medium A extract, it was observed that the mycelium of Rhizoctonia solani (AG-1IA) was avoiding the paper disk of the medium A culture extract. It was inferred that the active substance in the medium A culture medium produced by this bacterium produced a substance that hinders mycelial growth. For this reason, isolation and purification were attempted from the medium A culture medium using as an index a substance exhibiting inhibitory activity against Rhizoctonia solani (AG-1IA).

  Further, as a result of the assay in the culture extract of B medium I, activity was not confirmed in Botrytis cinerea and Rhizoctoniasolani, and formation of growth inhibition circles was confirmed only in bacteria. This indicates that this bacterium has antibacterial activity other than antagonism and may produce antibiotics. In particular, since the growth inhibition circle formed in Staphylococcus aureus 209P was clear, it was decided to carry out isolation and purification in B medium I using the active substance against bacteria Staphylococcus aureus 209P as an index. The antibacterial spectrum of the A medium culture and the B medium I culture were different, and it seemed that they produced different strains. Each active product was isolated and purified.

(2) Culture results
(a) Culture result of A medium Ten medium culture flasks (rice medium 1 kg) of 500 ml Erlenmeyer flasks were statically cultured at 28 ° C. for 10 days to obtain 2 liters of 50% acetone extract of the culture.

(b) B medium examination and culture results The activity of B medium I and B medium II shown in Table 1 and the strong activity of B medium I were confirmed. Production was confirmed. From this result, shaking culture was performed using B medium I at 28 ° C. for 5 days.

(3) Purification results
(a) Results of culture and purification of medium A Using the active fraction after silica gel PTLC treatment as a crude product, purity was confirmed by HPLC (0.05% TFA-containing MeOH / H ₂ O). In the analysis, a gradient from 0% to 100% methanol was applied from 0 to 30 minutes, and an analysis was performed at a flow rate of 1 ml / min from 30 minutes to 100% methanol. RT (retention time) was detected at 32.5 minutes (Fr65) ( FIG. 1). At first glance, it seemed as if a single substance peak was detected, but the presence of multiple peaks became apparent by enlargement (FIG. 2). From the fastest RT, they were called A, B, C, D, and E (FIG. 2), and four of them were isolated and purified. The components and amount of the isolated and purified product were A component 7.2 mg, B component 12.1 mg, D component 3.5 mg, and E component 4.5 mg. Peak C was a small amount and purification was abandoned.

(b) B medium I culture purification result It was confirmed that the active Fr41 to 54 had activity from the assay of each fraction and recovered. HPLC (0.05% TFA-containing MeOH / H ₂ O) was collected and analyzed, and the detected peaks were counted as A, B, C, D, E from the fastest RT, and the presence of 5 components was confirmed. Of these, 8.1 mg of component C was isolated and purified.

(4) Results of instrument analysis
(a) Based on the UV absorption spectrum, mass spectrometry and NMR measurement results (FIGS. 3 to 14) of the A medium isolated and purified product, it was determined that the polypeptide containing Aib (α-Aminoisobutyric acid) was a Peptibols system. Peptibols antibiotics contain α-Aminoisobutyric acid (Aib) in the amino acid sequence, and are characterized by an N-terminal Acetyl group and a C-terminal Amino alcohol bond (mostly Phenylalaniol groups) . As for the purified product, A component, B component, and D component, the partial amino acid sequence was estimated by mass spectrometry as follows, and the partial structure was searched with the existing amino acid sequence of Peptibols. There was no new Peptibols system. E could not be determined because it was difficult to estimate the amino acid sequence.

UV absorption wavelength: terminal absorption of all four components (FIGS. 3, 6, 9, and 12)
A component = 1933 (FIG. 5)
B component = 1949 or 1964 (FIG. 8)
D component = 1810 (FIG. 11)
E component = 1829 (Fig. 14)
Estimated structure A component: Ac-Aib-Ala-Aib-Aib-Aib-Aib-Gln-Aib-Aib -...
B component: Ac-Aib-Ala-Aib-Aib-Val-Aib-Gln-Aib-Aib -...
D component: Ac-Aib-Ala-Aib-Aib-Aib -...

(b) B medium I (liquid medium) active substance As a result of instrumental analysis, it was estimated that component C is a novel substance having a molecular weight of 168 (C ₈ H ₈ O ₄ ). Although it was judged that isolation and purification of other components were very difficult and impossible, LC / MS was performed. The UV absorption wavelength of component A was terminal (FIG. 15). The HPLC was performed using a column Capcel pac C18 UG120 (2 × 150 mm), 1% acetic acid: acetonitrile = 98: 2, flow rate 0.2 ml / min, and greenhouse conditions. A component was detected at RT 7.48 minutes (FIG. 16). As a result of mass spectrometry of the fraction in which the A component was detected, it was estimated that the molecular weight was 192 (FIG. 17). Similarly, the same analysis was performed on the B component, and the B component was detected at RT 12, 45 minutes of HPLC (FIG. 19) with the UV absorption wavelength at the end (FIG. 18). As a result of mass spectrometry, it was estimated that the molecular weight was 206 (FIG. 20). Component C is a fraction that could be isolated and purified. The UV absorption wavelength of component C was 270 nm (FIG. 21), and HPLC RT was detected at 15.92 minutes (FIG. 22). As a result of mass spectrometry, it was estimated that the molecular weight was 168 (FIGS. 23 and 24). The dissolved in a solvent CD 3 _OD, measured NMR (FIG. 25), includes a five-membered ring was analyzed, it was estimated that a novel substance having a conjugated bond. The estimated structure is shown in FIG. The D component had a UV absorption wavelength around 230 nm (FIG. 27), and HPLC RT was detected at 30.28 minutes (FIG. 28). However, HPLC was performed again, and the wavelength of the UV spectrum detector was analyzed at 230 nm from the end. As a result, it was confirmed that two components (D1, D2) were present with a difference of RT 0.6 minutes (FIG. 29). As a result of the respective mass spectrometry, those having a fast UV absorption at RT of 230 nm had a molecular weight of 154 (D1). It was estimated that the molecular weight of the component detected with a delay of RT 0.6 minutes was 220 (D2) (FIG. 30). It was confirmed that these components with the molecular weights revealed were 154 (D1), 168 (C), 192 (A), 206 (B), and 220 (D2) when arranged in descending order of molecular weight.

(5) Biological evaluation results
(a) Biological evaluation results of culture medium isolated and purified from A medium Biological evaluation results of four components A, B, C, and D components were diluted from 5000 ppm and tested at 5000 ppm, 2500 ppm, 1250 ppm, and 625 ppm. As a result, it was confirmed that the A component and the E component inhibited the mycelial elongation of Rhizoctonia solani up to 1250 ppm, and the B component and the D component inhibited the mycelial elongation of Rhizoctonia solani up to 625 ppm.

(b) B medium I culture isolated and purified product biological evaluation results
(B) Bacteria genus evaluation results The biological activity of B culture (MW168) against bacteria was considered to have no antibacterial activity or weakness because no growth inhibition circle was formed.

    (B) Table 3 shows the measurement results of antibacterial activity (MIC) against staphylococci.

  As shown in Table 3, the B culture (MW168) did not show antibacterial activity at the concentration measured this time, and was considered to have no or extremely weak antibacterial activity against staphylococci.

  Discussion From the components isolated and purified from the culture medium A, 4 components of Peptibols containing Aib (α-Aminoisobutyric acid) could be isolated. Several Peptibols strains produced by Trichoderma have already been reported, but those isolated and purified this time did not match those. Moreover, as a result of the amino acid sequence of each component, it was estimated that three components are novel sequences. (It was impossible to measure the sequence of only the E component) Further, in vitro tests showed that the mycelium of Rhizoctonia solani (AG-1IA) was inhibited. From these results, it was revealed that a mycelial elongation inhibitory substance against Rhizoctonia solani (AG-1IA) was produced in the rice culture extract of this bacterium. Although it is not yet possible to assert that the substance itself is a substance that promotes and kills cytoplasmic aggregation that is expressed by contact with the partner mycelium, which is an antagonistic action of this fungus, a mycelium growth elongation inhibitor is produced in the rice culture extract of this fungus. It is clear that

In addition, the component isolated and purified from B medium I culture is a new MW: 168 (3 (3-hydroxy-cyclopropene 5-one-2yl) 2-propenoic asid). Judged that it was extremely weak. Presumably, the active body is a peripheral compound of MW: 168, and MW: 168 is a by-product of the active substance. The molecular weights of the MW: 168 peripheral compounds are MW: 154, MW: 192, MW: 206, and MW: 220, and the difference in molecular weight is 14 respectively. This is the same amount as that of one methyl group, and a difference in activity due to increase or decrease of the methyl group is also considered. In addition, although MW: 168 has a new structure, several similar compounds have been reported by structure search. Among them, the structure closest to MW: 168 (Fig. 33) was found by PGR activity screening at Tottori University, and was reported to be isolated and purified from the filamentous fungus Penicillium valiabile SOPP. Although it is a novel substance represented by the composition formula of C ₈ H ₈ O ₅ (MW: 198), its activity is reported to be weak.

  The following results were obtained when the plant activity promoter of the present invention was applied to farmland for Ginkgo melon.

Location Yayoi Kunikofu Town, Tokoro-gun, Hokkaido
Tagawa Farm Implementation period April 23, 1999-July 29, 1999 Implementation conditions Agromic SK-10 (Tricoderma harzianum SK-5-5 trademark, hereinafter the same) is applied to melon bed soil at a rate of 50 g / m ² Scattered.
5 × 10 ⁷ / g conidia were attached to barleystone with a diameter of 1 to 2 mm. Both the test group and the control group were 200 m ² , and the difference between the two groups was only whether or not the agromic SK-10 was sprayed. There was no pesticide or topdressing.

  The agromic SK-10 was sprayed on April 23, 1999, planted on April 26, 1999, and harvested on July 29, 1999. Table 4 shows the average weight and sugar content of Ginger melon.

Characteristics of the survey process
(1) The characteristics of the initial stage are remarkable root survival, large leaf shapes, thick stems and large flowers.
(2) The characteristic of the middle staircase is that the state of the vine is sharp and there is a big difference from the control zone. Of course, there is no use of pesticides, and there are no signs of seedling dying, vine cracking disease, vine wilt disease. It was.
(3) As for the characteristics at the time of harvest, the fruits were large and shaped, the net was thick, and the tension was outstanding.
(4) Regarding the taste test, the sugar content is the same throughout the fruit, the flesh is soft up to the bare skin, the sugar content is high, and the taste is smooth and refined.
(5) Fruits were trapped and could be preserved for a considerable period (more than 1.5 times that of the control plot) after harvesting.

Place of Implementation Meiji Seika Co., Ltd. Biological Science Laboratory Implementation period April 1999-August August Implementation conditions Seeding April 6
April 8-22, germination, seedling
May 7 fixed planting, spraying of agromic SK-10
2 × 10 ⁷ / g, 30-40 g / m ²
June 4-7 mating
Harvest from July 12 to 23 Fertilization and irrigation were conducted as needed during the above period. According to the above implementation, the results shown in Table 5 were obtained.

Results The sugar content was 1.5% higher in the application area. Application of SK-10 increased the amount of sucrose and suppressed glucose and fructose. The improvement in sugar content by a single application of SK-10 is a result of the translocation of the photosynthetic product and other activities provided by the interaction in the rhizosphere.

  When the plant activity promoter of this invention was applied to sugar beet, the following results were obtained.

Implementation place Shadan, Shari-cho, Hokkaido Implementation period March 12, 1999 to October 7, 1999 Implementation conditions Amount of equivalent to Agromic SK-55 (Tricoderma harzianam SK-5-5) for about 30 kg of pot soil Blended. Conidia were 2 × 10 ⁸ / g or more. No sterilization treatment for soil covering. Water irrigation was carried out once a week from late March to mid April for colonization. On July 30, 100 liters of Agromic SK-55 was sprayed onto the strain and the leaf surface by 2 liters / m ² . The difference between the treatment group and the control group is that no sterilization treatment and no treatment of Agromic SK-55 were made, and the amount of fertilization, the method of fertilization, etc. were completely the same.

Results Regarding the above implementation, the result of harvesting on July 29, 1998, the real numbers in Table 6, and the ratios in Table 7 were obtained.

  According to the example, the root weight is increased by 32% and the sugar amount is increased by 40%. Therefore, the same area increased by 40%, which is a great achievement.

  According to the above example, a remarkable increase in yield was observed by mixing at germination and application of Agromic SK-55 on July 30. Agromic SK-55 activated sugar beet roots. This suggests that nutrient absorption and other factors are rational and powerful, and that cell division is also smooth.

  In the case of sugar beet, further benefits may emerge in the future by studying the application rate and timing, such as seed treatment, soil mixing, and intermediate application.

  When the plant activity promoter of this invention was applied to paddy rice, the following results were obtained.

Location: Kiyota (Sasaki Farm), Sapporo, Hokkaido
Tani-cho, Yubari-gun, Hokkaido (Kashiyama Farm)
Hokuryu-cho, Uryu-gun, Hokkaido (Takahashi Farm)
Implementation period May 6, 1999-September 16, 1999 Implementation conditions Variety name Hoshi no Yume, Kirara 397
Agromic SK-10 (Tricoderma harzianum SK-5-5 trademark)
A ... 50 g / m ² , conidia 2 × 10 ⁸ / g
B ... 100 g / m ² , conidia 2 × 10 ⁸ / g
The control group is the same except that the Tachigalen ace solution was sprayed and topdressed (conventional method).

  Under the above-mentioned conditions, the results of paddy rice cultivation, Table 8 (Sasaki Farm), Table 9 (Kashiyama Farm), and Table 10 (Takahashi Farm) were obtained.

  According to the above example, in the case of Kirara 397, the protein was 4 to 6 points lower than the conventional section, and the difference of 1 to 3 points was confirmed for amylose (1 point is 0.5). .

  Therefore, when the taste was measured, the average was 91.0. On the other hand, when it was measured for rice that is said to have a good commercial taste, 88.0 was the highest, so it can be said that it showed the highest taste at the present time.

  Judging from the above results, it is a low pesticide organic farming method that repels pathogens and pests by activating paddy rice, and it not only improves the taste by drawing out the original power of plants but also increases the yield (20-50%) The characteristics such as being confirmed were confirmed.

  When the plant activity promoter of this invention was applied to corn, the following results were obtained.

Place of implementation Meiji Seika Co., Ltd. Biological Sciences Laboratory Period of implementation April 1999-August 30, 2005 Bacteria used: Agromic SK-10 (2 × 10 ⁷ / g)
Application method: Soil application, irrigation 150ml / pot in the second week of germination
Application description Based on the above conditions, application was performed based on the application design of Table 11.

  The place cultivated and harvested as described above, the results shown in Table 12 were obtained.

As described above, fruit enlargement effect and improved sugar content were observed. There was almost no significant difference depending on the amount of application. That is, there is no difference between the application rate of 20 g / m ^{2 and} the application rate of 200 g / m ² , and in the case of corn, harvesting is performed by applying the activity once after germination. It is estimated that it will continue until.

Therefore, if the amount of the new amino acid produced by the agromic SK-10 is sufficient to confer activity, the application rate can be sufficiently effective even at a dose of 20 g / m ² or less.

For the above reasons, if the soil has the conditions necessary for the growth of Agromic SK-10, it is recognized that even if it is 20 g / m ² or less, it is sufficiently effective. Perhaps determined by the lower limit of conditions that allow growth. Therefore, a method of growing corn under the growth conditions of agromic SK-10 for a certain period after germination can be considered.

  Agromic SK-10 (Tricoderma harzianum SK-5-5 trademark) was applied to an onion nursery bed soil, and the growth promotion effect after the seedling and planting was investigated as follows.

(1) Test material:
(a) Test seeds: Super High Gold seeds not treated with chemicals
(b) Materials to be tested: Agromic SK-10 (Trichoderma harzianum SK-5-5 5 × 10 ⁸ CFU viable agent, provided by Hokkaido Green Kosan Co., Ltd.)
(c) Test location: Imuma, Shimo-gun, Kisarazu City, Chiba Prefecture
(2) Test method:
(a) Preparation of nursery bed: The nursery bed was mountain sand soil under Chiba Prefecture, and the previous crop was a field where sweet corn was grown. Since the pH was weakly acidic soil of around 6.0, 50 g per 1 m ^{2 of} slaked lime, 1 kg of fully-ripened compost (compost containing chives), and 20 g of CDU were applied 15 days before application of Agromic SK-10. Fertilized and plowed.

(b) Agromic SK-10 application and method:
Application Date: March 11 September 11 the prepared nursery floor, test records and material Agromic SK-10 Granules to 50g sprayed per 1m ² in sowing four days ago, and mix well to the depth of the surface layer 3-5cm, water supply Water was used for watering so that the soil moisture did not become humid (weighed by hand to form dumplings, that is, optimum humidity conditions for growing fungi).

(c) Management after sowing and application of Agromic SK-10:
Sowing date: September 15, 1999 Due to the high heat and dryness of the remaining heat, the soil temperature should be as low as 20 ° C in order to improve the growth and colonization of the Trichoderma fungus in the nursery bed soil and to improve the germination of the onion. In order to keep the temperature at about 25 ° C. and to stabilize the moisture condition, rye straw was laid in a thickness of 2 to 3 cm in both sections, and a cold koji was applied thereon.

  About 70 seeds of onion seeds were sown in 50 cm between 50 cm, and after covering the soil, moderate watering was performed, and the seeds were covered by the above method until budding started.

After emergence, the straw was removed and the cold-cold tunnel was covered for about one month. In order to prepare for the fixed planting, the tunnel covering was removed, and the CDU chemical fertilizer mixed with compost was fertilized by shallowly plowing about 20 g per m ² between the strips. The seedlings were grown for the purpose of producing mature seedlings having a diameter of 0.5 to 0.6 cm and a leaf length of about 5 to 6 cm.

(d) Planting:
Planting date: November 7, 1999 (53 days after sowing)
The soil in this farm is alluvial soil, pH is weakly acidic soil of about 6.0, and the subsequent crop of sweet potatoes. 60 kg per slaked lime, 500 kg of fully-ripened compost, 60 kg of bone meal, and 60 kg of rice bran were mixed well with compost and applied. One week later, a cocoon with a ridge width of 1 m and a three-row planting was made, and 20 kg of CDU and 10 kg of superphosphate were mixed well with 300 kg of fully-ripened compost, and fertilized with a groove. The seedlings were planted in the fertilization groove at 15 cm between the plants in order of size.

(3) Survey results:
(a) Emergence and growth status: There was almost no difference in emergence between the untreated and treated groups, but seedling wilt occurred in the untreated group slightly after one week. In the subsequent growth, there was no difference until around day 25, but after removal of the tunnel and after fertilization (35 days after sowing), the leaf color became darker in the treated area, and the growth became vigorous by visual observation. It came to be able to.

(b) Seedling growth survey:
(I) Growth of seedlings before planting: Survey date November 7, 1999 (53 days after sowing) Detailed data are shown in Table 13.

  The good seedlings (large and medium seedlings) were 78% in the treated group and 65% in the untreated group. The application of Agromic SK-10 increased the percentage of good seedlings by 13%. There were no seedlings having a seedling diameter of 0.7 mm or more. Regarding the promoting effect, as shown in Table 13, the growth index increased, the growth was vigorous, and the root mass increased in the agromic SK-10 treated section. When the growth promotion rate was compared, a 27% increase in growth was observed in the SK-10 treated group than in the untreated group.

    (B) Growth investigation after planting: The growth situation before wintering was investigated.

  Investigation method: Investigation was carried out by the method shown in FIG.

    Results: Detailed results are shown in Table 14.

  In the agromic SK-10 treated group, the number of leaves emerged 0.4 more than the untreated group, the growth promotion rate was 152.5%, and the bulb diameter was 154.7%. A growth promoting effect was clearly observed by visual observation. The agromic SK-10 treatment group had a larger amount of roots, better rooting, better soil adherence to the roots, and more root hairs than the non-treatment group. Further, in the agromic SK-10 treatment group, there was a correlation between the leaf area and the bulb diameter (FIG. 35, Table 15, Table 16).

  Agromic SK-10 was applied to cabbage nursery bed soil, and the growth promotion effect after the seedling and planting was investigated as follows.

(1) Materials used:
(a) Test seeds: seeds not treated with chemicals
(b) Test material: Agromic SK-10 (Trichoderma Halzianam SK-5-5 5 × 10 ⁸ CFU, provided by Hokkaido Green Kosan Co., Ltd.)
(2) Location: Yoshida Farm, Narita City, Chiba Prefecture
(3) Application overview:
(a) Application date: September 15, 1999
(b) Sowing date: September 19, 1999
(c) Floor soil: In order to adjust the sandy loam soil of pH around 6.0 to neutral, about 50 g of slaked lime was sprayed per 1 m ² . This mature compost and fertilizer to 1m ² per 1kg, 2 weeks ago, and tillage.

Before seeding, about 50 g of agromic SK-10 was sprayed per 1 m ² . Next, about 5 cm of the surface layer was mixed with the soil and sprinkled with about 3 liters per 1 m ² , but the soil moisture did not become so humid (to the extent that it became a dumpling when grasped by hand).

(4) Survey date: December 23, 1999 (94 days after sowing)
On the day of the survey, it was confirmed that the treated area was dark in leaf color, thick and tight, and fast in heading.

Claims (7)

Plant activity characterized by inoculating Trichoderma harzianum SK-55 in a solid medium, standing at 25 to 30 ° C. for 7 to 15 days, and then extracting the product to obtain the following substance: A method for producing an imparting agent.
A component: Ac-Aib-Ala-Aib-Aib-Aib-Aib-Gln-Aib-Aib-. . . With a molecular weight of 192
B component: Ac-Aib-Ala-Aib-Aib-Val-Aib-Gln-Aib-Aib-. . . With a molecular weight of 206
C component:

. . . With a molecular weight of 168
D component: Ac-Aib-Ala-Aib-Aib-Aib-. . . And having a molecular weight of 154 or 220 Trichoderma harzianum SK-55 fungus is inoculated in a liquid medium, shaken at 25 ° C. to 30 ° C. for 4 days to 10 days, and then the product is extracted to obtain the following substance. Manufacturing method.
A component: Ac-Aib-Ala-Aib-Aib-Aib-Aib-Gln-Aib-Aib-. . . With a molecular weight of 192
B component: Ac-Aib-Ala-Aib-Aib-Val-Aib-Gln-Aib-Aib-. . . With a molecular weight of 206
C component:

. . . With a molecular weight of 168
D component: Ac-Aib-Ala-Aib-Aib-Aib-. . . And having a molecular weight of 154 or 220 In plant cultivation, the conidia of Trichoderma harzianum SK-55 is attached to the seeds of the plant, the cover soil and the conidia are mixed, or the appropriate amount of the conidia is sprayed, irrigated or embedded, A method for imparting plant activity, characterized by producing the following substances:
A component: Ac-Aib-Ala-Aib-Aib-Aib-Aib-Gln-Aib-Aib-. . . With a molecular weight of 192
B component: Ac-Aib-Ala-Aib-Aib-Val-Aib-Gln-Aib-Aib-. . . With a molecular weight of 206
C component:

. . . With a molecular weight of 168
D component: Ac-Aib-Ala-Aib-Aib-Aib-. . . And having a molecular weight of 154 or 220 In plant cultivation, the conidia of Trichoderma harzianum SK-55 is attached to the seeds of the plant, the cover soil and the conidia are mixed, or the appropriate amount of the conidia is sprayed, irrigated or embedded, The following substances are produced, the temperature of the soil where the conidia are placed is 15 ° C. to 30 ° C., the moisture is 30% or more, and air permeability or aeration that can grow on the soil is imparted. A method for imparting plant activity.
A component: Ac-Aib-Ala-Aib-Aib-Aib-Aib-Gln-Aib-Aib-. . . With a molecular weight of 192
B component: Ac-Aib-Ala-Aib-Aib-Val-Aib-Gln-Aib-Aib-. . . With a molecular weight of 206
C component:

. . . With a molecular weight of 168
D component: Ac-Aib-Ala-Aib-Aib-Aib-. . . And having a molecular weight of 154 or 220 After cultivating conidia of Trichoderma harzianum SK-55 to produce the following activity-imparting agent, it is separated into an appropriate amount together with a medium, and mixed with a bulking material composed of sterile porous particles and nutrients A plant activity promoter characterized by being packaged for a predetermined amount.
A component: Ac-Aib-Ala-Aib-Aib-Aib-Aib-Gln-Aib-Aib-. . . With a molecular weight of 192
B component: Ac-Aib-Ala-Aib-Aib-Val-Aib-Gln-Aib-Aib-. . . With a molecular weight of 206
C component:

. . . With a molecular weight of 168
D component: Ac-Aib-Ala-Aib-Aib-Aib-. . . And having a molecular weight of 154 or 220 A plant activity promoter characterized in that conidia of Trichoderma harzianum SK-55 are grown in a liquid medium to produce the following activity-imparting agent and then packaged to a predetermined amount.
A component: Ac-Aib-Ala-Aib-Aib-Aib-Aib-Gln-Aib-Aib-. . . With a molecular weight of 192
B component: Ac-Aib-Ala-Aib-Aib-Val-Aib-Gln-Aib-Aib-. . . With a molecular weight of 206
C component:

. . . With a molecular weight of 168
D component: Ac-Aib-Ala-Aib-Aib-Aib-. . . And having a molecular weight of 154 or 220 Application of a plant activity promoter characterized in that the plant activity promoter according to any one of claims 5 and 6 is mixed in seedling soil, or is sprayed on soil at the time of seedling, or sprayed on a field. Method.

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