KR101527428B1 - Rhizopus oryzae CS108 and composition comprising thereof for preventing plant diseases - Google Patents

Abstract

The present invention relates to a composition for controlling plant diseases, which contains CS108 (Accession No. KCCM 11231P) to Rizofus Oragejie, and is more effective than the wild type in the conventional Rifofus oryzae in controlling plant diseases and promoting plant growth, It is a mutant strain of Rizofus oryzae that is effective for red pepper anthracnose, apple brown pattern bottle, apple anthracnose, bean anthracnose, pepper blight, broad-leaf rot, The present invention provides a plant disease control method and a plant growth promotion method using the strain.

Description

[0002] Rhizopus oryzae CS108 and a composition for the prevention of plant diseases,

(KCCM 11231P) having an antifungal effect against anthrax, anthrax, anthrax, pepper pest, broad-leaf rot, white-faced wing disease, The present invention relates to a microorganism preparation, which shows more resistance to pepper anthracnose, anthrax anthracnose, apple anthracnose, anthracnose, anthracnose, pepper rot, broad-leaf rot, It is about the mutant strain that shows its effect.

Recently, many researchers have made efforts to change microorganisms in the soil in order to utilize them advantageously for plant growth and human health.

Bacterization refers to the inoculation of microbial cultures to the crop field or to the growing area. This inoculation often leads to a significant increase in crop yield, but it is not yet clear what role the inoculated microorganism plays in the rhizosphere. It is not clear. Some microorganisms can survive in the rhizosphere within a period of growing crops, so that the microorganisms can maintain their density. However, some microorganisms can not settle in the rhizosphere by being rejected by the original existing rhizosphere microorganisms. At this time, the microorganisms that settle and propagate stably on the root surface are called 'rhizosphere microorganisms'.

Microbial roots settlement is a very active process in which microorganisms survive on seed surface or soil, propagate using carbohydrate and amino acid-rich seed secretions, adhere at the roots surface and continue to propagate along the growing roots . In addition, these rhizosphere microbes are passively moved to the bottom of the roots by irrigation.

As a representative microorganism of rhizosphere, Pseudomonas aeruginosa, Azotobacter, Bacillus and the like, rhizospheric microorganisms tend to have a fast growth rate, a mobility and a preference for root secretion. The kind that has a beneficial effect on the plant is called 'plant growth promoting microorganism '. In the past, the beneficial effects of plant growth promoting microorganisms were recognized only for root crops such as radish, potatoes and sugar cane. Recently, however, positive effects have been recognized in various crops such as oats, soybean, cotton, peanut, bark, rice and vegetables .

At present, we have to increase the absolute production of crops in order to provide enough food for the population increase, so the use of chemical fertilizers and pesticides is inevitable. In addition, these chemicals are also facing environmental pollution threatening the ecosystem as a whole. In this dilemma, the direction to protect our agriculture and preserve the environment can be said to be the use of environmentally friendly biological pesticides and fertilizers.

Therefore, the development of environmentally friendly biological pesticides and fertilizers is needed.

The effects of microbial inoculation on growth promotion and pest control should be recognized on both sides of the coin. That is, microorganisms having a growth promoting effect have a disease-suppressing effect, and many researchers have also found that a disease-suppressing microorganism promotes plant growth. For example, some microorganisms (Pseudomonas bacterium) that have been recognized as promoting plant growth have been reported to reduce the density of harmful bacteria and fungi in the rhizosphere, thereby reducing the disease and promoting crop growth relatively.

In other words, most of the plant growth-promoting rhizosphere microorganisms whose mechanism is known so far are known to indirectly promote crop growth by controlling harmful microorganisms.

On the other hand, rhizosphere microorganisms directly promote plant growth by producing substances (physiologically active substances) that promote plant growth. That is, these physiologically active substances are known to exert effects such as promotion of root hair growth, promotion of root and stem growth by promoting nutrient absorption by roots. As a result, plant growth-promoting microorganisms have the effect of preventing growth and promoting growth by multiplying the plant self-defense ability against invasion of the germs by changing the physiological properties of the crops by producing special physiologically active substances besides the effect of promoting growth by disease control .

Conventionally, the effect of Rizofus oligosporus on plant disease control and plant growth promotion is known (Korean Patent No. 0713059, Korean Patent Registration No. 0616408, and Korean Patent Application No. 2011-0123628) Spruce is used. Therefore, the present inventor has sought to find a Rifofus sp. Having increased activity through mutation, and developed CS108 in Rifofus oryzae with increased activity and completed the present invention.

It is an object of the present invention to provide a mutant strain excellent in the resistance to plant pathogenic bacteria and superior in plant growth promoting ability than the conventional Rizofocus oligosporus.

It is an object of the present invention to provide a microbial pesticide composition for controlling plant diseases, which contains mutant strains having excellent resistance to plant pathogenic bacteria and excellent plant growth promoting ability.

It is an object of the present invention to provide a microorganism composition for promoting plant growth, which comprises a mutant strain having excellent resistance to plant pathogenic bacteria and excellent plant growth promoting ability.

The present invention provides CS108 (Accession No. KCCM 11231P) to Rifofus oryzae having antagonistic ability against plant diseases, while having the ability to promote plant growth by mutating the Rifapus oligosporus wild type strain.

The present invention provides a pesticidal composition for controlling plant diseases, which comprises CS108 (Deposit No. KCCM 11231P) in the mutated Rizophos oryzae. Preferably, but not exclusively, the botanical bottles include pepper anthracnose, anthrax anthracnose, bean anthracnose, red pepper pest, broad-leaf rot, white-faced wing bottles, or sea bream.

Another aspect of the present invention relates to a pesticidal composition comprising a culture filtrate of CS108 (Accession No. KCCM 11231P) in a mutagenized Rizofocusoria. Such botanical bottles include pepper anthracnose, anthrax anthracnose, apple blotch, bean anthracnose, red pepper pest, broad-leaf rot, white-faced wing bottles or sea bream. The CS108 culture filtrate composition according to the present invention is effective for the treatment of plant diseases such as apple red pepper and is particularly effective for the treatment of anthrax of apple and pepper.

Another aspect of the present invention is a method for promoting plant growth characterized by treating a mutant lysophospholipid CS108 or culture filtrate thereof. Such plants include, but are not limited to, red pepper.

CS108 in the mutant Rizofus aurajijie by the present inventor was improved by about 187% in potency against plant disease and by 217% in the plant growth promoting ability in comparison with the wild type of Rizofusu oligosporus. Particularly, it has been found that CS108 is effective for the treatment of red pepper anthracnose, anthrax anthracnose, bean anthracnose, red pepper pest, broad-leaf rot, It is a fact revealed. Accordingly, CS108 in Rizophus oryzae according to the present invention is highly effective in producing peppers and controlling plant diseases in an environmentally friendly manner, and is expected to play a significant role in generating profits of farmers.

Figure 1 shows the mycelial shape of CS108 in the wild-type Rizofus oligosporus and mutated Rizofus aurajie. (A) wild-type Rizofus oligosporus at 3 days after culture; And (B) CS108 at 3 days after culture.
FIG. 2 shows the antifungal activity of CS108 against Rizophos oryzae against anthrax anthracis.
3 is an electron micrograph of CS108 at Rizofus oriji.
Fig. 4 shows antifungal activity against diplocarpon mali caused by CS108 in Rizofus oriji .
FIG. 5 is a photograph of the optical microscopic observation of the interface when treated with a culture solution of CS108 in Ripofusora oryzae ( Diplocarpon mali ). (A) Typical form of the hyphae hyphae, the outer membrane of the mycelium is clear, and the inside of the hyphae is filled with matter. (B) Rifofus oryzae was in the form of a hyphae hyphae when CS108 culture filtrate was treated. The shape of the hyphae hyphae is narrowed, the material inside the hyphae disappears, and hyphae are broken. (C) It is a form of a hyphae hyphae when the CS108 cultured filtrate was treated with Rizofus Oragei. The inside of the hyphae hyphae is empty. (D) It is a form of a hyphae hyphae when the CS108 cultured filtrate was treated with Rizofus oriji. The hyphal hyphae can be seen to be broken.
FIG. 6 shows the effect of controlling CS108 cultured filtrate on Rizofus oriji against apple anthracnose. The control was a treatment of cultured filtrate of Rifofus oryzae without culturing filtrate, a wild type treated with cultured filtrate of Rifofus oligosporus CS107 (wild type), and CS108 was treated with CS108 cultured filtrate at Rifofus orage.
FIG. 7 shows the pepper growth promoting effect of Rizofocus oligosporus wild type and mutant strain CS108 in red pepper seedlings. (A) control, (B) wild-type, (C) mutagenized Rizofus oryzae CS108.
Fig. 8 shows the ability of CS108 to degrade organic matter in Rizofusu oriji.
FIG. 9 shows the results of a phylogenetic analysis using the ITS sequence of Rizofus oryzae.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are provided only for illustrating the present invention in more detail, and the scope of the present invention is not limited to these examples

Example  One: Rizofus  Orajie Strain strain ( CS108 ) Induction and selection

1. γ-ray resistance test of Rizofus oragije

Γ-ray ( ⁶⁰ Co) irradiation was conducted by the Institute of Radiation Science to induce mutation of Rizofocus oligosporus CS107. γ-ray was irradiated with 5 ml of shake cultured sample for 2 days in PDB medium for 20 hours at a total dose of 5 ~ 17.5KGy / hr and 2.5Kgy / hr interval.

2. Isolation of the mutant strain CS108

The irradiated samples were spread on a PDA medium and cultured at 30 ° C for 3 days. After examining the morphological characteristics of the cultured colonies, colonies showing morphological and cultural characteristics different from those of the wild type of Rizofocus oligosporus were selected and cultured separately.

Antifungal activity of plant pathogens was analyzed by selecting strains which did not exhibit general property characteristics in γ-ray irradiated samples at each dose. The selected strains and plant pathogens cultured in PDA medium were made into 6 mm agar block and incubated in PDA medium. After culturing for one week with 3 replicates, the cultured diameters of the plant pathogens were measured ( Table 3 ).

3. Characteristics of the strain

(1) Cultural characteristics

The strain can be cultured in PDA (Difco) and cultured well at 25 ° C to 35 ° C.

(2) Morphological features

When cultured in a solid medium, the color turns white and gradually changes to dark gray ( Fig. 1 (B) ).

(3) Characteristics of hyphae and spores

The spores are very small, and the shape of the spores seems to pop up, and they have a cocoon.

Morphological characteristic Rizofocus oligosporus 121 (Cho.1997) Rizofus oligosporus (Domsch. 1980) Rizofocus oligosporus CS107
(Wild type) Rizofus orijeide CS108 Sporangiospore marking type  Strial Irregular ellipsoidal (elliptical) Very irregular shape A linear irregular ellipsoidal (ellipsoidal) A linear irregular ellipsoidal (ellipsoidal) Size (um) 7-10 (4-) 9-10 (-15) x (4-) 7-10 (-11) 4.16-3.12 × 13.53-11.16 4.16-3.47 x 7.57-6.13 Sporangium size (um) 100-135 80-120 109.54-136.24 113.24-203.34 Colony color Gray light brown - Dark gray brown Dark gray Rhizoid existence existence existence existence

(4) Difference from wild-type Rizofus oligosporus

 When the CS108 was compared with Rizofus Oligosporus CS107 (wild type) in Rizofus oriagen, morphologically, the sporangia spore marking form, size, and presence of carcass showed similar morphology to wild type, The size of the sporophyll is larger than that of the wild type, the color of the colony is slightly different, the formation rate of the spore is about 1 day faster than that of the wild type, and CS108 forms 170 spores per 10ul of Rizofus oolage while 125 Spores were formed, and CS108 had a large amount of spores in the modified Rizofus auraji. Also, it was identified as CS108 in Rizofus oriji which was transformed in wild type by showing difference in the optimum culture temperature and acidity.

The strain was deposited on December 13, 2011 at the Korean Society for Microbiological Conservation (Accession No .: KCCM 11231P).

Name of depository: Korea Microorganism Conservation Center (overseas)

Accession number: KCCM11231P

Funding Date: 20111213

(5) gene sequence analysis

 1. ITS Sequence Analysis

In order to confirm the genetic characteristics of CS108 in Rizofus oriji, the ITS sequence was submitted to KCCM.

ITS-5.8S rDNA sequencing of the isolates was performed in the following manner. The chromosomal DNA of this strain was suspended in physiological saline solution (0.8% NaCl, 0.0125% tween 20) and centrifuged to obtain mycelium. Lysing buffer solution (0.05M EDTA (pH 8.0), 0.3% SDS) was added to the mycelium obtained and allowed to stand for 10 minutes. The supernatant was taken and the mycelium was disrupted using sterilized glass beads. After centrifugation (14,000 rpm, 3 minutes), the supernatant was taken and the same volume of isopropanol was added to precipitate genomic DNA. The genomic DNA obtained by centrifugation was washed with 70% ethanol. The obtained genomic DNA was treated with suspending at 60 ° C for 1 hour after adding a small amount of sterilized water, and then used for PCR. PCR was performed using ITS1 (5'-TCCGTAGGTGAA CCTGCGG-3 '; SEQ ID NO: 1) and ITS4 (5'-TCCTCCGCTTATTGATATGC-3'; SEQ ID NO: 2) primers used in the ITS-5.8S rDNA sequencing analysis Respectively. Amplified purified PCR products were sequenced using ABI PRISM BigDye ™ Terminator Cycle Sequencing kits (Applied Biosystems Co.) using ABI PRISM 3730XL Analyzer (96 capillary type). The ITS sequence of CS108 in Rifofus oryzae was identical to SEQ ID NO: 3. The BLASTN program was used to compare GENEBANK ribosomal DNA sequences, and the homology of sequences was compared using Clustal X and Mega 2 programs. As a result, CS108 in Rhizopus oryzae was transformed into Rhizopus and 99% similarity with oryzae (see Fig. 9 ).

Example  2: On plant pathogens  Antifungal effect

One. Apple anthrax  Antifungal effect

On the PDA medium, CS108 and anthrax globulespores ( Coletotricum gloesporioides ) were cultured in 6mm agar block in PDA medium. After incubation for one week with 3 replicates, the cultured diameters of the plant pathogens were measured. As a result, CS108 showed higher antifungal activity against anthrax anthracnose than CS108 (Rizofus oligosporus CS107 (wild type)) ( Table 2 , Fig. 2 ) and CS108 and apple anthracnose Observation of the confluent culture interface with an electron microscope revealed that the mycelial shape of the apple anthracnose bacterium was not a normal shape but abnormally curved and distorted mycelium such as crushed ( FIG. 3 ).
The units in Table 2 are%, and the calculation of the antibacterial activity is as follows.
{(Diameter of untreated pathogen - diameter of treated pathogen) / diameter of untreated pathogen} x100

Therefore, if the% is high, it can be judged that the antimicrobial power is good. In Table 2, it was found that the effect of the antifungal against the anthrax anthracnose was higher than that of the wild type against the cultivated rizofus oriji.

Antifungal agent C. gloesporioides (%) Wild type 47.9 Rizofus orijeide CS108 51.4

delete

2. Other On plant pathogens  Antifungal effect

Antifungal activity of plant pathogens was analyzed by selecting strains which did not exhibit general property characteristics in γ-ray irradiated samples at each dose. The selected strains and plant pathogens cultured in PDA medium were made into 6 mm agar block and incubated in PDA medium. The cultured diameters of the plant pathogens were measured after one week of incubation for 3 repetitions, and the results are shown in Table 3 below.
The units in Table 3 are%, and the calculation of the antibacterial activity is as follows.
{(Diameter of untreated pathogen - diameter of treated pathogen) / diameter of untreated pathogen} x100

Therefore, if the% is high, it can be judged that the antimicrobial power is good. Table 3 shows that the effect of the antifungals on the white wing pattern germs, porcine germs, broad-leaf rot fungi, red pepper pluck, red pepper anthracnose and soybean anthracnose was higher than that of the wild-type cultivars I could.

Rizofus oligo spores wild type
(%) The mutated < RTI ID = 0.0 > CS108 < / RTI > (KCCM11231P)
(%) White wing pattern germ 26.9 50.4 Buran's disease 67.6 75.2 Fungus 68.6 70.9 Pepper blight 75.0 85.7 Pepper anthracite 62.5 69.6 Bean anthracnose 49.1 51.8

3. To apple-brown pattern germs  Antifungal effect

In the PDA medium, Rizofus oryzae CS108 and apple brown pattern germ ( Diplocarpon mali ) were made into 6 mm agar blocks and replaced by PDA medium. After three weeks of incubation for one week, the cultivation of plant pathogens was confirmed. As a result, CS108 in the Rifofus oryzae was confirmed to have antifungal activity against Diplocarpon mali ( Fig. 4 ), and the confluent culture interface between CS108 and apple brown foliage was observed with an optical microscope As a result, the mycelial shape of the apple-brown bottle was not normal but tapered, the substance in the mycelium disappeared, and hyphae such as hyphae were broken ( FIG. 5 ).

Example  3: Rizofus  Orajie CS108 Culture filtrate  Antifungal effect

In order to confirm the preventive and therapeutic effect of CS108 on Rizofus oeraji in apple anthrax, CS108 was cultivated in Rizofusu oriji for 2 days in PDB medium, and the culture was centrifuged at 3000 rpm for 30 minutes to obtain mycelium and culture The filtrate was separated.

To investigate the antifungal effect of apple anthracnose, the surface of the apple was wounded, and apple anthrax was inoculated on the spot. After the lesion of anthracnose of apple was confirmed, the cultivation filtrate of sterilized water, Rizofus oligosporus CS107 (wild type) and the cultured filtrate of CS108 on Rizofus oolage were sprayed on the surface of the apple, respectively, and the progress of apple anthrax was confirmed. As a result, the development of apple anthracnose was significantly lower than that of Rizofus oligosporus CS107 (wild type) in the cultivation solution in which cultured filtrate of Rizofus aurajiji CS108 was sprayed on the surface of red pepper inoculated with anthrax bacteria, Showed only scars at the time of inoculation and no signs of anthrax anemia occurred ( FIG. 6 ).

Example  4: Promoting growth and Rhizosphere settlement force  effect

The mutant strains CS108 were cultivated in PDB medium for 2 days with shaking, and the horticultural soil was sterilized at 121 ° C for 20 minutes under high pressure. The cultured filtrate and mycelia were diluted to 1000 ppm, and the seeds were sown with 40 lips each of the pepper seeds. Respectively. As a result, the growth of red pepper seedlings was faster than the control. However, the growth promoting effect was faster than that of Rizofocus oligosporus CS107 (wild type) (see Table 4 (unit: mm) and Fig. 7 ).

Control Wild type  CS108 198.2 (100%) 340.9 (171.5%) 430.9 (217.4%)

Example  5: Rizofus  Orajie CS108 Ability to decompose organic matter

1. Measurement of starch resolution ( amylase activity )

Culture of CS108 in Rizofusu oriji in medium containing water-soluble starch (starch 5 g, pepton 10 g, CaCl ₂ 0.5 g, MgCl ₂ 5 g, MgSO ₄ 2 g, KCl 1 g, FeSO ₄ 0.001 g, Bacto-agar 20 g / After 2 days of inoculation of the filtrate, Gram's Iodine solution was added to the medium to observe whether a clear zone was formed around the cells.

2. Measurement of fibrinolysis ( Cellulolytic activity )

Culture filtrate of CS108 in Rifofus oriji was inoculated in a Carboxymethylcelluose (CMC) medium (5 g of CMC, ₂ g of MgSO ₄ , 0.5 g of CaCl ₂ , 1 g of KCl, 0.001 g of FeSO ₄ and 20 g / L of Bacto-agar) After that, the cells were treated with 0.5% Congo-red solution to form a clear zone.

3. Protein resolution ( Proteolytic activity )

    The culture filtrate of CS108 was added to a culture medium containing gelatin (5 g, beef extract 3 g, peptone 5 g, Bacto-agar 15 g / L), and 2 days later, 1% tannic acid aqueous solution was added thereto. And whether crystals appeared.

As a result, CS108 was found to have higher antimicrobial activity and organic matter-resolving ability than wild-type strains ( Table 5 , Fig. 8 ).

division Rizofus wild type Rizofus orijeide CS108 Determination of starch resolution +++ +++++ Measurement of fiber resolution ++ +++++ Protein resolution +++ +++++

<110> B & L Agro <120> Rhizopus oligosporus CS108 and composition formulated for          preventing plant diseases <130> PN120002 <160> 3 <170> Kopatentin 2.0 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> ITS1 primer <400> 1 tccgtaggtg aacctgcgg 19 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ITS4 primer <400> 2 tcctccgctt attgatatgc 20 <210> 3 <211> 560 <212> DNA <213> Rhizopus oryzae <400> 3 ttcctctggg gtaagagaat gcttctagac tgtgaaaatt tggctgagag actcagacag 60 gtcatgggta gacctatctg gggtttgatc gatgccactc ctggtttcag gagtaccctt 120 cataataaac ctagaaattc agtattataa agtttaataa aaaacaactt ttaacaatgg 180 atctcttggt tctcgcatcg atgaagaacg tagcaaagtg cgataactag tgtgaattgc 240 atattcagtg aatcatcgag tctttgaacg cagcttgcac tctatggttt ttctatagag 300 tacgcctgct tcagtatcat cacaaaccca cacataacat ttgtttatgt ggtgatgggt 360 cgcatcgctg ttttattaca gtgagcacct aaaatgtgtg tgattttctg tctggcttgc 420 taggcaggaa tattacgctg gtctcaggat cttttttttt ggttcgccca ggaagtaaag 480 tacaagagta taatccagta actttcaaac tatgatctga agtcaggtgg gattacccgc 540 tgaacttaag catatcataa 560

Claims (14)

Raizofus Oraije CS108 (Accession No. KCCM 11231P). An agricultural chemical composition for controlling anthrax anemia comprising the microorganism of claim 1. delete A pesticide composition for controlling an apple-brown fungus disease comprising the microorganism of claim 1. A method for controlling anthrax anthracnose, which comprises treating a plant with the microorganism of claim 1 or a culture filtrate thereof. delete A method for controlling an apple-brown fungus disease, which comprises treating a plant with the microorganism of claim 1 or a culture filtrate thereof. A method for promoting plant growth, which comprises treating a plant with the microorganism of claim 1 or a culture filtrate thereof. A pesticidal composition for controlling anthrax anemia comprising the culture filtrate of the microorganism of claim 1. delete A pesticide composition for controlling an apple-brown fungus disease comprising the culture filtrate of the microorganism of claim 1. A method for preparing an agricultural chemical composition for controlling anthrax, comprising culturing the microorganism of claim 1 and extracting a culture filtrate of the microorganism. delete A method for producing an agricultural chemical composition for controlling an apple-brown fungus disease, comprising culturing the microorganism of claim 1 and extracting a culture filtrate of the microorganism.

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