JP2000093167A - Chlamydospore of trichoderma harzianum, its production and microbial material - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new chlamydospore of Trichoderma harzianum, usable as a microbial material or the like capable of suppressing pathogenic bacteria by readily germinating only by the impartment of water by removing nutrients from the mycelium of Trichoderma harzianum, culturing the obtained Trichoderma harzianum in a liquid starvation medium containing no vitamins, and separating the cultured material, and. SOLUTION: This new chlamydospore of Trichoderma harzianum obtained by removing nutrients from the mycelium obtained by culturing the Trichoderma harzianum, normally culturing the obtained Trichoderma harzianum in a liquid starvation medium containing no vitamins, and separating the cultured material, is useful as a microbial material or the like having activities capable of suppressing pathogenic bacteria by performing ready and active germination only by the impartment of water. The chlamydospore of the Trichoderma harzianum is obtained by culturing the Trichoderma harzianum in a nutrient medium, separating the mycelium from the nutrient medium, culturing the mycelium after removing the nutrient components in the liquid starvation medium, and separating the mycelium.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a trichoderma haldiaum chlamydospore, which is intended to obtain a chlamydospore efficiently by culturing a mycelium obtained by ordinary culture in a liquid starvation medium, and a method for producing the same. Regarding microbial materials.

[0002]

2. Description of the Prior Art It has been known that mycelial microorganisms contain a trace amount of chlamydospores. The chlamydospores are
It has been known that it has high environmental responsiveness and high resistance at high temperatures as well as at low temperatures. There is also a proposal for an invention relating to the chlamydospores of Ninbiassylpicola K-004 (FERMBP-4448) and its induction medium (JP-A-7-303481).

Further, the present applicant has previously proposed an invention for producing chlamydospores by starvating mycelial microorganisms (Japanese Patent Application No. 10-203876).

[0004]

The naturally occurring chlamydospores described above are extremely small in amount, and it has been extremely difficult to collect and effectively use such chlamydospores in such an amount that they can be used effectively.

[0005] Further, in the medium disclosed in the above-mentioned invention, it is difficult to produce large-scale chlamydospores of generally known mycelial microorganisms with high efficiency even if the microorganisms are adaptable. .

The applicant's prior invention is epoch-making in that it can produce chlamydospores of general mycelial microorganisms in large quantities, and can be expected to have a great effect by industrialization. There was room for improvement.

[0007]

Means for Solving the Problems In view of the improvement of the above problems, the present inventors have conducted intensive studies on Trichoderma harzianum, and as a result, cultivation in a liquid starvation medium is possible. The present inventors have obtained the knowledge that they can grow sufficiently and obtained the knowledge that they can efficiently produce chlamydospores in large amounts without adding vitamins to the starvation medium, and as a result of further research, they have completed the present invention.

[0008] That is, the present invention is characterized in that a nutrient component is removed from a mycelium obtained by culturing Trichoderma harzianum, and this is separated by ordinary culturing in a liquid starvation medium containing no vitamins. It is a thick spore of Harzianam.

[0009] Next, the invention of the method is that, after culturing Trichoderma harzianum in a nutrient medium, a mycelium is separated from the nutrient medium, and a nutrient component is removed from the separated mycelium.
This is a method for producing chlamydospores of Trichoderma harzianum, which comprises culturing the nutrient-removed mycelium in a liquid starvation medium and separating chlamydospores, wherein the starvation medium does not contain vitamins. In addition, raw materials containing vitamins are not added. In addition, culture of starvation medium
It is performed in the same environment as normal culture. Still another invention is a microbial material comprising trichoderma harzianum chlamydospores as an active ingredient.

[0010] The nutrient medium used in the present invention is a known L-type medium.
An eonian medium, a potato dextrose medium, and the like can be used.

Next, the starvation medium used in the present invention includes sucrose (or a carbon source such as glucose and maltose), KNO ₃ (or a nitrogen source such as NaNo ₃ ), KH ₂
PO ₄ , MgSO ₄ .7H ₂ O, CaCl ₂ , ZnSO
_{4, CuCl 2, FeSO 4,} Na 2 MoO 4 · 2H 2
A liquid starvation medium containing no vitamins consisting of O, H ₃ BO ₃ and water is effective.

[0012] The microorganism material of the present invention comprises calcium carbonate, defatted rice bran, and bran in a weight ratio of 1: 1: 1.
And finely pulverized, and mixed with a diffusing agent (for example, commercially available "Aerosol OT" (trademark), Wako Pure Chemical Industries, Ltd.), a spreading agent (for example, commercially available "Shin Rino" (trademark), Nippon Agrochemical Co., Ltd.) Company), mixed with the same amount of perlite, and added chlamydospores.

The ratio of the mixture is not limited to a mixing ratio of 1: 1: 1 by weight. Germination of chlamydospores,
It only needs to play a role as a nutrient source during growth, so it is appropriately selected according to the germination environment. Further, if the mixture is adjusted to a water content of 6% to 12% and encapsulated in a capsule for a fixed amount, it is convenient to store, transport and use the product.

The nutrients are not limited to those described above, and those that contribute to the germination and growth of chlamydospores are appropriately used.
And the ratio can be changed. The amount of chlamydospores to be added to the nutrient component may be mixed at a rate of 0.01% (weight) to 100% (weight) or diluted 10,000 times to 10,000 times depending on the purpose of use. In some cases,
In any case, the material during storage or transportation has a moisture content of 6%
It is necessary to keep １２12% to prevent premature germination.

As described above, the reason that the amount of chlamydospores significantly differs depending on the application conditions is that it differs depending on the possibility of germination and proliferation after application. Through future experiments, the most economical and effective method of use will be determined through various experiments, such as the type of pathogenic bacteria, application environment, and the possibility of germination and propagation.

The chlamydospores proposed by the present invention are obtained by subjecting chlamydospores obtained by starvation of mycelial microorganisms to a treated material to 0.03
1 to 100% by weight is mixed, or the thick spores are used after being diluted 10,000 times with the applied material. Here, the lower limit of the above-mentioned range in which the chlamydospores are mixed into the applied material or the thick film spores are diluted with the applied material is expected to have an effect of germinating the chlamydospore and suppressing the activity of the pathogenic microorganism. Above, it is preferable that the ratio be at least this level, and even if the ratio exceeds the above-mentioned upper limit, a large difference does not occur in the effect, and the ratio is determined. In this range, from the viewpoint of cost-effectiveness, the most preferable range is that the chlamydospores are mixed in the applied material in an amount of 1 to 20% by weight, or the chlamydospores are mixed in the applied material in a quantity of 5 to 100%. In this case, it is used by diluting it twice.

[0017] It is preferable that the treated material in the above is constituted so as to contain a substance which is a nutrient source of chlamydospores. If the nutrient source is finely pulverized to, for example, a size of about 0.1 to 1.0 μm and the applied treatment is configured to include the nutrient, nutrients are easily absorbed, and germination and growth of chlamydospores It is advantageous for

The chlamydospores in the present invention are asexual spores in which a storage substance accumulates at the tip or intermediate cells of the mycelium and has a large shape and a thick cell wall, and in many cases a double wall. It was also found that these spores are intended to survive in an inappropriate environment and are not originally spores in the sense of scatter or germ cells.

The yield of chlamydospores according to the invention is 3 × 10
^The yield is ⁷ / ml or more, which is a higher yield than the prior invention. The germination rate of chlamydospores in soil is 50% to 9%.
9%.

The chlamydospores of the present invention have high heat resistance and do not have a risk of being destroyed during storage, for example, up to about -5 ° C to + 70 ° C. However, it was found that it could withstand even 100 ° C. instantaneously. Therefore, the chlamydospores of the present invention can withstand the normal living temperature of living organisms on the earth. In particular, the temperature in the zone where microorganisms are used or when they are used is about 15 ° C to 40 ° C. Even if it is mass-produced and sent to any place on the earth, there is no danger of lapse due to thermal conditions during transportation, and there is no danger of a decrease in germination rate.

However, the chlamydospores of the present invention should have a water content of 6% to 12% during storage or transportation to prevent germination during storage or transportation. Need to be handled.

Hitherto, various useful microorganisms for suppressing the activity of pathogenic microorganisms have been proposed, but all of them use mycelium and conidia, so that not only the germination rate is poor, but also Due to insufficient heat management, germination became worse and sufficient effect was not exhibited in some cases, and the stability of the effect was insufficient.

However, by using chlamydospores of a useful microorganism which suppress the activity of pathogenic microorganisms, at least the chlamydospores germinate as expected and exhibit their performance sufficiently even after transportation or storage. It was found that the stability of the effect of suppressing the activity of pathogenic microorganisms was maintained.

Generally, useful microorganisms that suppress the activity of pathogenic microorganisms have a number of advantages as compared to chemical pesticides, as described below.

1. High selectivity for pathogenic microorganisms, no risk of disrupting ecosystem. 2. There is no risk of damaging the crop. 3. No harm to livestock and seafood. 4. Does not confer resistance to pest pathogens. 5. No pollution or accumulation in water, soil or crops (no resistance to long-term use).

In the above, the point that no resistance to pathogenic microorganisms is imparted means that the efficacy is not reduced or eliminated even after long-term use, which is an important property for the use of microorganisms.

For example, antibiotics which are extracted from soil bacteria or utilize their products have excellent effects in protecting humanity from diseases. However, continuous use of antibiotics imparts resistance to pathogenic bacteria. It is often experienced that the emergence of antibiotic-resistant pathogens has forced the development of new antibiotics. This one point alone shows how superior chlamydospores are.

For the commercialization of the chlamydospores, microcapsules may be encapsulated alone or mixed with conidia,
Alternatively, any conventionally known product forms such as granulation, tableting, and pulverization can be used. The only requirement for the chlamydospores of the present invention is that they be stored in a dry state in which the water content is within the range of 6% to 12%. No decrease in germination rate was observed.

[0029]

[Example] Trichoderma harzianam SK55 (FER
MBP4346) to the following medium in the tank,
After aerobically culturing at 15 ° C. to 30 ° C. for 48 hours, mycelium (for example, 5 μ or less in size) is separated from the medium,
Washing is carried out in sterilized water or physiological saline 5 to 100 times the mycelium to remove any nutrients adhering to the surface of the mycelium.

The formulation of the nutrient medium proportion glucose 6.25 g malt extract 6.25 g KH ₂ PO ₄ 1.25 g Yeast extract _{1.0 g MgSO 4 · 7H 2 O} 0.625g peptone 0.625g Distilled water 1000ml The The mycelium, from which nutrients have been removed as described above, is inoculated into the following starvation medium, and at 15 ° C to 30 ° C for 72 hours to 170 hours.
Aerobically.

The formulation of starvation medium percentage sucrose _{20 g KNO 3 1.0 g KH 2} PO 4 1.0 g MgSO 4 · 7H 2 O 0.5 g CaCl 2 0.1 g ZnSO 4 2.0mg CuCl 2 0 0.1 mg FeSO ₄ 0.2 mg Na ₂ MoO ₄ .2H ₂ O 0.2 mg H ₃ BO ₃ 0.01 mg Distilled water 1000 ml After culturing as described above, sieve through a microfilter (for example, size 5 μ to 10 μ). To separate chlamydospores).

When the experiment was carried out several times under the same conditions as described above, the yield of chlamydospores was 3 × 10 ⁷ / ml or more. In the present invention, it was confirmed that the formation rate of chlamydospores in an extreme environment (temperature and pH) was worse than that under normal conditions.

(Experimental example) After washing nutrients from the mycelium normally cultured in the same manner as in the above example, the nutrients were inoculated into the following starvation medium and aerobically cultured at 15 ° C to 30 ° C for 72 hours to 170 hours. Cultured.

The formulation of starvation medium percentage sucrose _{20 g KNO 3 1.0 g KH 2} PO 4 1.0 g MgSO 4 · 7H 2 O 0.5 g CaCl 2 0.1 g ZnSO 4 2.0mg CuCl 2 0 0.1 mg FeSO ₄ 0.1 mg Na ₂ MoO ₄ .2H ₂ O 0.2 mg H ₃ BO ₃ 0.01 mg Biotin 4.0 mg Thiamine 2.0 mg Distilled water 1000 ml After the above culture, thick film spores were separated. 10 ⁶ pieces / m
1 or less. As described above, it has been clarified that the addition of vitamins reduces the yield of chlamydospores.

It is not known whether the addition of a small amount (for example, 以下 or less) of the above-mentioned vitamin does not affect the yield, and the presence of a small amount does not affect the yield. Further, the culture environment is a starvation environment (for example, a temperature of 35 ° C. to 45 ° C.,
Although it was confirmed that it was not necessary to adjust the pH to 5-4, the effect of a change in the culture environment will be a subject for future research.

[0036]

According to the present invention, since a liquid starvation medium is used and vitamins are not included in the starvation medium, the yield of chlamydospores is significantly increased.

Further, since the culture environment in the starvation medium is the normal environment (culture environment in the general nutrient medium), culturing of chlamydospores is facilitated, and there is an effect that a large amount of chlamydospores can be easily produced. ADVANTAGE OF THE INVENTION The microbial material of this invention has the effect that chlamydospores can easily and actively germinate and control disease-causing bacteria simply by spraying them to necessary places and imparting water.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Mikiyuki Katagiri 17-38 Nittacho, Kofu City, Yamanashi Prefecture 206 Virfor Reamanano 206 (72) Inventor Yasuharu Sasaki 18-1, Odori Nishi, Chuo-ku, Sapporo, Hokkaido 3 Olympia Odori Nishi 18th Street Mansion 702 F-term (Reference) 4B065 AA70X AC20 BA23 BB31 CA60 4H011 AA01

Claims (5)

[Claims] 1. A method for removing Trichoderma harzianum from a mycelium obtained by culturing Trichoderma harzianum, and culturing and separating the nutrients in a liquid starvation medium containing no vitamins. Chlamydospores. 2. After culturing Trichoderma harzianum in a nutrient medium, the mycelium is separated from the nutrient medium, the nutrient is removed from the separated mycelium, and the mycelium having the nutrient removed is replaced with a liquid starvation medium. A method for producing chlamydospores of Trichoderma haldianam, comprising separating chlamydospores after culturing with the above method. 3. The method for producing trichoderma harzianam chlamydospores according to claim 2, wherein the starvation medium does not contain vitamins and does not add a raw material containing vitamins. 4. The method for producing chlamydospores of Trichoderma harzianam according to claim 2, wherein the cultivation of the starvation medium is performed in the same environment as the normal culture. 5. A microbial material comprising the chlamydospore of Trichoderma harzianum according to claim 1 as an active ingredient.