JPWO2014083690A1 - Tick control method using novel Trichoderma spp. And prevention method of tick generation - Google Patents

Abstract

To provide a tick control method using a novel Trichoderma genus and a method for preventing the occurrence of mites. A fungus having the ability to kill insects and belonging to Trichoderma Atroviride, which is isolated from processed green tuff and has a deposit number of NITE P-1419 A method for controlling ticks, characterized by acting on ticks and / or their habitats.

Description

  The present invention relates to a tick removal method using microorganisms and a tick generation prevention method, and more particularly, to a tick removal method and a tick generation prevention method using Trichoderma spp.

  The damage to humans and livestock by mites is becoming more serious every year with global warming.

For example, the duck spider (Dermanyssus gallinae), known as an ectoparasite of poultry and birds, is a tick that is feared to cause great damage to the poultry industry. This tick, which is blood-sucking, absorbs blood at night, affects the chicken itself, and is thought to be related to anemia, whitening of the chicken crown, and a decrease in the egg-laying rate.

  On the other hand, by spraying diatomaceous earth and other mineral fine powders on wolf spiders or other ticks, or by directly applying hydrates, these mineral particles enter the gaps between joints and moving parts of wolf spiders, etc. A method of extinguishing a spider by absorbing a bodily fluid or cutting an outer skin with a mineral slice is known and widely used.

  On the other hand, a study that Trichoderma album parasitizes and kills the spider has been published (Non-patent Document 1).

  Of the genus Trichoderma, Trichoderma atroviride, which is widely distributed around the world, is a microorganism that is widely resident in the natural environment such as the fungus bed in soil. When trying to control crop diseases using these microorganisms, Trichoderma atroviride identified from an isolation source such as soil is different from the isolation site using a large tank or industrial culture facility. A method is known in which a large amount of culture is performed in a fresh environment, and then the culture is supported on silica or other mineral fine powder. Various methods for culturing and formulating them are known.

Hussein A. Kaoud et al, "Susceptibility of Poultry Red mites to Entomopathogens", International Journal of Poultry Science 9 (3), p. 259-263

  The inventors discovered and isolated a novel Trichoderma genus from among the microorganisms resident in green tuff (hereinafter sometimes referred to as “Towada stone”) mined in Hinai-cho, Odate City, Akita Prefecture Tried.

  It is known that Trichoderma spp. Have been found on natural rocks. Therefore, the possibility that the isolation source is the green tuff can be sufficiently foreseen. However, the mining method of green tuff is usually an open pit mining vertically from the ground part. In that case, the microflora adhering to the bedrock due to changes in the surrounding environment, for example, the type of resident microorganisms Are susceptible to influences that are different from each other or that the resident presence is disturbed. For this reason, it is not easy to reproducibly isolate Trichoderma spp. From the microorganisms that normally exist in green tuff.

  However, the inventors found that there is a horizontal hole-type green tuff mining site in Hinai-cho, Odate City, Akita Prefecture, at least one place in Japan. At the Towada stone quarry 30m underground in the mine, we discovered that a new Trichoderma atroviride is permanently present in the green tuff and succeeded in the isolation. did.

  Mining has been started in this tunnel since 1972, and since it is maintained at a temperature of 10 ° C and humidity of 90% throughout the year, the ecosystem of microorganisms living in the tunnel is very limited. Compared to open pit mining, the microflora in the mine has the advantage that it is hardly affected by changes in the surrounding environment.

  The inventors put polyaluminum chloride flocculant (PAC) into a basin in which wastewater containing fine processing powder generated when processing and polishing a rock block of Towada stone cut out from the Towada stone crushing yard was accumulated. Trichoderma from fine powder (commonly known as Towada stone cake) having a particle size in the range of 1 μm to 80 μm and a peak value of 5 μm, obtained by agglomerating and precipitating fine powder, collecting the precipitate and drying it -An atroviride (Trichoderma atroviride) HNT-01 strain (hereinafter sometimes referred to as "HNT-01") could be collected.

  And this Trichoderma atroviride (Trichoderma atroviride) HNT-01 strain as a new microorganism, as of September 7, 2012, the National Institute of Technology and Evaluation Patent Microorganisms Deposit Center (Kazusa Kamashika, Kisarazu City, Chiba Prefecture, Japan) 2-5-8) (Accession number NITE P-1419).

  As mentioned above, research has been published that Trichoderma album infests and kills the spider, but other species of the genus Trichoderma parasitize the spider, There are no reports of killing. In addition, as described above, the method of extermination of scabs with a fine mineral powder such as silica is widely known. However, the microorganisms that are resident in the fine mineral powders are also used for the eradication and prevention of scabs at the same time. No invention has been made.

  Therefore, the present invention provides a method for controlling mites using a novel Trichoderma genus and a method for preventing the occurrence of mites, and a mineral fine powder produced by the presence of the Trichoderma genus in a mineral fine powder. It aims at providing two points, the tick extermination method by the synergistic effect of this, and the prevention method of tick generation | occurrence | production.

  In order to solve the above problems, the following invention is proposed.

The invention of claim 1
It is a fungus belonging to Trichoderma Atroviride that has the ability to kill mites,
The fungus is characterized by being isolated from a fine powder of green tuff obtained through the following processing steps.

(1) A step of cutting the green tuff block.

(2) A step of storing wastewater containing crushed stone particles of green tuff generated by cutting.

(3) The process which stirs the stored waste_water | drain and forms the colloid of the fine powder of a green tuff in the said waste_water | drain.

(4) A step of adding a flocculant to the waste water to precipitate the colloid, sucking the precipitate, and dehydrating the sucked precipitate.

(5) A step of drying the dehydrated precipitate.

The invention of claim 2
It is a fungus characterized by having a tick insecticidal ability and NITE P-1419 as the accession number belonging to Trichoderma Atroviride.

The invention of claim 3
A method for controlling ticks, which comprises causing the fungal spore suspension according to claim 1 or 2 to act on mites and / or their habitats.

The invention of claim 4
4. The mite control method according to claim 3, wherein 1% of a nutrient source containing nitrogen and carbon is added to the spore suspension.

The invention of claim 5
A method for controlling ticks characterized by causing the fine powder of the green tuff where the fungus according to claim 1 or 2 is resident to act on ticks and / or their habitats.

The invention of claim 6
A method for preventing the occurrence of mites, wherein the fungal spore suspension according to claim 1 or 2 is allowed to act on mites and / or their habitats, and the mites are formed with the fungi mycelium. .

The invention of claim 7
The method according to claim 6, wherein a nutrient source containing nitrogen and carbon is added to the spore suspension in an amount of 1% with respect to the spore suspension.

The invention of claim 8
The fine powder of the green tuff which is the isolation source of the fungus according to claim 1 or 2 is allowed to act on the mite and / or its habitat, and the mite is resident in the fine powder of the green tuff. A method for preventing the occurrence of mites characterized by forming fungal hyphae.

  According to the present invention, a method for controlling mites using a novel Trichoderma genus and a method for preventing the occurrence of ticks, and a mineral fine powder produced by the presence of the Trichoderma genus in a mineral fine powder, It is possible to provide a method for controlling ticks and a method for preventing the occurrence of ticks by the synergistic effect of

(A) Diagram showing Trichoderma genus forming mycelial colonies on green tuff extracted at Towada stone quarry, which is a horizontal hole method, (b) Discharging lathe and water to process green tuff The figure showing a nozzle, (c) The figure showing the water storage stirring equipment which stores the waste_water | drain produced | generated when processing a green tuff, (d) The figure showing the gel of a green tuff. Prepare 2 aliquots of HNT-01 spore suspension in a plastic bag for sealer, leave Petri mites A and B in the center of each bag, and leave the ticks after 2 days. (B) Expanded view of petri dish of petri dish A, (c) Expanded view of spider spider of petri dish B, (d) Expanded spider of petri dish B FIG. Several ticks were placed in three sample tubes, 300 μL of sterilized physiological saline, 300 μL of HNT-01 spore suspension, and HNT-01 agar fragment were added thereto, and the appearance of ticks after several days was expressed. (B) HNT-01 agar fragment, (c) HNT-01 spore suspension, (d) HNT- It is an inverted photomicrograph of a black mite killed by 01 spore suspension. (A) 10 ^1-fold dilutions (10 ⁷ cells / mL) diagram showing a state of Wakumodani in HNT-01 spore suspension, HNT-01 spores (b) 10 ^2-fold dilutions (10 ⁶ cells / mL) diagram showing a state of Wakumodani in suspension, in HNT-01 spore suspension (c) 10 ^1-fold dilutions (10 ⁷ cells / mL), Wakumodani infected with HNT-01, also of Wakumodani (d) The enlarged view of a forefoot part. 10 is a diagram showing the appearance of urticae after 4 days after adding PDB medium to HNT-01 spore suspension diluted from 10 ¹ to 10 ⁷ times, among which (a) 10 ¹ times dilution (10 ⁷ cells / mL) HNT-01 spore suspension (b) 10 ² times diluted (10 ⁶ cells / mL) HNT-01 spore suspension (c) 10 ³ times diluted (10 ⁵ for HNT-01 spore suspension of cells / mL), the case of HNT-01 spore suspension (d) 10 ^4-fold dilutions (10 ⁴ cells / mL). It is the figure which microscopically observed the vaccinia mite which died in the HNT-01 spore suspension of 10 ^3- fold dilution (10 < ⁵ > cells / mL), or (a) HNT formed around the dead mite (B) Enlarged view of the leg portion of the nymph mite, (c) State of the mycelium of HNT-01 formed in the dying nymph mite, (d) Same as the foot part of the nymph mite Enlarged view.

  Embodiments of the present invention will be described below with reference to the accompanying drawings. The isolation source of the genus Trichoderma according to the present invention was a green tuff mined at the Towada stone quarry in a tunnel that is a side hole type. As mentioned above, this tunnel has been mined since 1972, and is maintained at a temperature of 10 ° C and a humidity of 90% throughout the year. Therefore, the ecosystem of microorganisms living in the tunnel is very limited. Compared with the above-mentioned open-pit mining, the microflora in the mine is almost affected by changes in the surrounding environment, that is, the type of resident microorganisms is different or the presence of the resident itself is inhibited. There are no advantages.

  As shown in FIG. 1 (a), the genus Trichoderma adheres to the block surface of the mined Towada stone and is a nutrient source (cellulose) such as a wooden pallet to be taken to the ground. , Germinate from spores to form mycelial colonies.

  The official name of Towada stone is "Quartz andesitic pumice tuff" and is known as "Green Tough". This tuff was formed by the accumulation of volcanic ash from submarine volcanoes when the Japanese archipelago was formed about 10 million years ago. When observed with an electron microscope, a porous structure in which extremely fine particles are linked together is observed. The main constituent minerals are feldspar, chlorite, quartz, and silicate minerals with about 30% amorphous crystals.

  Towada stone has been cut and commercialized from stone quarries as a building stone and bathroom flooring for about 40 years ago because of its beautiful blue-green color and non-slip heat and moisture retention. In addition, the crushed stone particles and chips generated in the lumbering process are also used as a granulating agent as a core of chemical fertilizer and chemical pesticide.

  When the Towada stone mined from the quarry is processed into the product, the Towada stone is poured along the cutter surface (FIG. 1B). And the Towada stone fine powder generated from the cut part will be included in the drainage, but the particle size of the fine powder is a constant speed and pressure controlled by a steel blade while applying water. Therefore, it is constant between 1 μm and 80 μm, and the peak value is around 5 μm.

  Along with the fine powder of Towada stone, spores of the genus Trichoderma adhering to the block surface of Towada stone also move into the wastewater together with the fine powder. This waste water is collected in one place, stored, and stirred by a motor, so that a colloid of fine powder of green tuff is formed in the waste water (FIG. 1 (c)). In addition, by this agitation work, Trichoderma spp. Is also aerated and cultured at the same time.

  After stirring for a certain period of time, flocculant (PAC: polyaluminum chloride) is put into the drainage pool, so that the green tuff colloid is settled on the floor of the pool. A clay-like gel of tuff can be obtained (FIG. 1 (d)). This clay-like gel also contains spores of the genus Trichoderma, which were resident in the block of Towada stone.

  Thereafter, the clay-like gel is naturally dried in a greenhouse, whereby a dry product of green tuff fine powder can be obtained. This fine powder size is industrially constant as described above, and is the same as the composition and size of silica and other mineral fine powder supporting microorganisms, as in general biologics. Therefore, the naturally dried green tuff fine powder can be used as a carrier for biopesticides. In addition, in the green tuff fine powder alone, as well as known research results, the fine powder enters joints and moving parts such as scabs, weakens the movement of squirrels, absorbs water, and mineral slices cover the epidermis such as scabs. Since it has been confirmed that scabs are killed by cutting, this green tuff fine powder alone can be used for insecticides of mites.

  Trichoderma atroviride HNT-01 strain was collected from the dried green tuff fine powder having such characteristics.

  Trichoderma spp. Are known to be resistant to drying by forming spores, but HNT-01 according to the present invention is also on top of the green tuff fine powder dried from the clay-like gel. The fine powder itself was confirmed by a demonstration experiment to control plant diseases. Therefore, the green tuff fine powder obtained through this series of steps and the trichoderma atroviride that resides there are considered to be extremely efficient and cost-effective biologics.

  The Trichoderma atroviride HNT-01 strain, as a new microorganism, was established on September 7, 2012, as an independent administrative agency, Product Evaluation Technology Infrastructure Organization, Patent Microorganism Depositary Center (Kazusa Kamashika, Kisarazu City, Chiba Prefecture, Japan). 5-8) and the deposit number NITE P-1419 was assigned.

(Efficacy of HNT-01 insecticide)
In this example, the following experiment was conducted on the insecticidal effect of urticae by HNT-01 suspension and HNT-01 agar section.

<Experimental material>
・ Wakumodani ・ HNT-01 Spore suspension (10 ⁸ cells / mL), culture plate of HNT-01 cultured in PDA medium ・ Sterile saline ・ Sample tube ・ Plastic bag with sealer, plastic bag with chuck

<Experiment method>
(1) Bacteria were collected from a plate of HNT-01 cultured in PDA medium using a platinum loop and suspended in 0.85% sterile physiological saline. After suspension, the mixture was filtered with a Pasteur pipette containing absorbent cotton to obtain a HNT-01 spore suspension (10 ⁸ cells / mL).

Dilute HNT-01 spore suspension with sterile physiological saline (10 ⁶ cells / mL) and dispense 10 mL in a plastic bag for sealer. Petri dish with ticks in the center of each bag A and B were allowed to stand. The petri dish had one hole with a diameter of about 3 mm, and the spider mite was adjusted so that it could freely enter and exit from the hole. Thereafter, the course was observed after 2 to 5 days.

(2) Separately from the above, several ticks are placed in three sample tubes, 300 μL of sterile physiological saline, 300 μL of HNT-01 spore suspension (10 ⁷ cfu / mL), HNT-01 agar fragment Each was followed for several days.

<Results and discussion>
(1) In the above experimental method (1), when the progress after 2 days was observed, about half of the petri dish of Petri dish A died, but the spider mite of Petri dish B was alive. When the progress was observed after 5 days, the scab mites of either petri dish were almost dead, and the survivors did not appear to move violently (Fig. 2).

(2) In the above experimental method (2), comparison was made between the HNT-01 spore suspension and the HNT-01 agar section using a sample tube to which sterile physiological saline was added as a control. As a result, only the sample tube to which the HNT-01 spore suspension was added was able to confirm the death of the vaccinia mite (Fig. 3 (c). After one week, the dead ticks were confirmed with an inverted microscope. It was confirmed that green mold was attached to the entire surface of ().

  In the sample tube to which the agar fragment of HNT-01 and sterilized physiological saline were added, it was confirmed that the spider mite moved around even after one week (FIGS. 3 (a) and 3 (b)).

  From this, it is considered that in the HNT-01 agar fragment, it was considered that the spore could not grow on the mold because it was difficult for the spores to adhere to the spider mite, and therefore, from the above experimental results, HNT-01 kills the spider mite. It is presumed that at least a spore suspension is preferable.

(Insecticidal effect of urticae by HNT-01 spore suspension given nutrients)
In this example, a potato dextrose broth (PDB) medium was added to the HNT-01 spore suspension, and the following experiment was conducted to determine whether the insecticidal effect of black spider mites can be obtained with a small number of HNT-01.

<Experimental material>
・ Wakumodani ・ HNT-01 Spore suspension (4.59 × 10 ⁸ cells / mL)
・ Sterile saline ・ Screwed glass vial ・ PDB medium

<Experiment method>
A spore suspension of HNT-01 was prepared which was diluted to 10 ⁷ times 10 ¹ times with sterile saline. About 10 to 20 ticks were transferred to glass vials, and 1 mL of each diluted spore suspension was dispensed thereto. After dispensing, the glass vial was suspended by vortexing. Thereafter, static culture was performed at 24 ° C. for 1 week.

  After culturing for 1 week, 10 μL of 1% PDB medium was added to 1 mL of the spore suspension, and culturing was continued for another 4 days. After culturing, visual confirmation and observation with a microscope were performed.

<Results and discussion>
In the week of culture, the 10 ¹ dilution (10 ⁷ cells / mL), and entanglement of precipitation and hyphae of Wakumodani, about half of the deaths were confirmed (FIG. 4 (a), (c) , (d) ). However, survival was confirmed in the dilution factor system (10 ⁶ cells / mL to 10 ¹ cells / mL) after 10 ² , although few deaths were confirmed (FIG. 4 (b)).

Therefore, the presumed nitrogen and carbon source for mycelial growth from spores is insufficient, the HNT-01 spore suspension of each dilution series to 10 ⁷ times the 10 ¹ times, a small amount ( 10 μL) of PDB medium was added for further cultivation.

As a result, when culturing for 11 days (the above-mentioned 7 days + 4 days), many duck mortality was confirmed in a dilution series up to 10 ³ -fold dilution (10 ⁵ cells / mL) (FIG. 5). In the subsequent dilution series, many surviving spiders were observed.

  In addition, as shown in FIG. 6, HNT-01 mycelium is formed in a dead or moribund state spider. From this, not only HNT-01 has an insecticidal effect on the spider mite, but also a mycelium is formed on the dead body of the spider mite, so that HNT-01 can survive for a long period of time. It is suggested that can be suppressed.

  Normally, the growth of Trichoderma genus requires a dense nutrient source such as 1/1 PDB medium. When such a nutrient source is added to the suspension, it is attracted to the nutrient source. There is a risk of increasing other germs. Therefore, the nutrient source such as the PDB medium added to the suspension need only be added about 1% with respect to the suspension. Can be obtained.

From Examples 1 and 2, it was confirmed that HNT-01 has an insecticidal effect against black mites. Moreover, when a small amount of nutrients was added to the HNT-01 spore suspension, it was suggested that the spore suspension at around 10 days and about 10 ⁵ cells / mL would greatly reduce the survival rate of the black mite. Furthermore, it was suggested that after the insecticidal effect of urticae is obtained, mycelia are formed on the dead bodies of urticae, so that it is possible to suppress generation of new urticae after insecticidal.

  The preferred embodiments of the present invention have been described above with reference to the accompanying drawings. However, the present invention is not limited to such embodiments, and various forms are possible within the technical scope grasped from the description of the claims. Can be changed.

Claims (8)

It is a fungus belonging to Trichoderma Atroviride that has the ability to kill mites,
The fungus is characterized by being isolated from a fine powder of green tuff obtained through the following processing steps.
(1) A step of cutting the green tuff block.
(2) A step of storing wastewater containing crushed stone particles of green tuff generated by cutting.
(3) The process which stirs the stored waste_water | drain and forms the colloid of the fine powder of a green tuff in the said waste_water | drain.
(4) A step of adding a flocculant to the waste water to precipitate the colloid, sucking the precipitate, and dehydrating the sucked precipitate.
(5) A step of drying the dehydrated precipitate.   A fungus having an insecticidal ability to mite and having a deposit number of NITE P-1419 belonging to Trichoderma Atroviride.   A method for controlling ticks, which comprises causing the fungal spore suspension according to claim 1 or 2 to act on ticks and / or their habitats.   The mite control method according to claim 3, wherein a nutrient source containing nitrogen and carbon is added to the spore suspension at 1% with respect to the spore suspension.   A method for controlling ticks, comprising causing the fine powder of the green tuff where the fungus according to claim 1 or 2 is permanently present to act on ticks and / or their habitat.   A method for preventing the occurrence of mites, wherein the fungal spore suspension according to claim 1 or 2 is allowed to act on mites and / or their habitats, and the mites are formed with the fungi.   The method according to claim 6, wherein a nutrient source containing nitrogen and carbon is added to the spore suspension in an amount of 1% with respect to the spore suspension.   The fine powder of the green tuff which is the isolation source of the fungus according to claim 1 or 2 is allowed to act on the mite and / or its habitat, and the mite is resident in the fine powder of the green tuff. A method for preventing the occurrence of ticks, which comprises forming fungal hyphae.

Images