KR100411186B1 - The biocontrol agent containing aminoglycoside antibiotics for control of plant diseases - Google Patents

Abstract

The present invention relates to a plant disease control agent using aminoglycoside antibiotics, more specifically, aminoglycoside antibiotics such as paromomycin, ribostamycin, neomycin, streptomycin, etc. ( Phytophthora sp.) And Pythium sp. Bacteria have been found to have selective antimicrobial activity, such as biopesticides for controlling plant diseases such as plant diseases and stocks. activity as a biocontrol agent) and its use.

Description

The biocontrol agent containing aminoglycoside antibiotics for control of plant diseases}

The present invention relates to a plant disease control agent using aminoglycoside antibiotics, more specifically, aminoglycoside antibiotics such as paromomycin, ribostamycin, neomycin, streptomycin, etc. ( Phytophthora sp.) And Pythium sp. Bacteria have been found to have selective antimicrobial activity, such as biopesticides for controlling plant diseases such as plant diseases and stocks. activity as a biocontrol agent) and its use.

It is well known that aminoglycoside antibiotics have antimicrobial activity against gram-negative bacteria, among which streptomycin and neomycin are widely used as pharmaceutical antibiotics. Validamycin and kasugamycin, aminoglycosides of aminocyclohexanol structure, are used as water disinfectants. However, the present invention revealed for the first time that the aminoglycoside antibiotics have a control effect in vitro against plant pathogenic fungi, particularly the phytophthora, which is a late blight belonging to Oomycetes, and pisium, which is a stock bacterium. lost.

Plant diseases caused by the late blight Phytophthora and the stock bacteria Pisium are very numerous and the damage is enormous. So far, 60 kinds of late blight and more than 1,000 late blight host plants have been reported. In Korea, 16 kinds of phytophthora are known to occur in 46 various crops. Phytophthora infestans , for example, are primarily found in potatoes and tomatoes, and Phytophthora capsici occurs in the entire plant, including peppers, watermelons, cucumbers, melons, and tomatoes. It is known and a species occurs in many hosts. In particular, the phytophthora infestans and the phytophthora capsaish are causing great damage to potatoes and peppers in our country. A late blight is a hyphae that overwinters in diseased plants in the soil, and pathogens grow on the ground to become secondary infectious agents. Plague is a common disease that occurs in cool, humid environments. When a disease develops, it rapidly develops and causes significant losses in crop production. In addition, rice seed rot and wearing diseases caused by the P. corium bacteria are well known in Korea as well as all over the world, and the damage is serious. In Korea, Psidium is known as a major pathogen that causes various root rot diseases such as rice rot diseases such as tomatoes and cucumbers, as well as ginger root rot diseases, as well as rice wearing diseases. Plague bacteria belonging to Oomycetes have been included in filamentous fungi as a typical soil pathogen, but have been classified as belonging to Protist at the recent International Fungi Society. Therefore, the fungicides for controlling these pathogens also differ in their properties, and metalaxyl, dimethomorph, focetyl-A1, and the like, which are different from other fungicides, are used. However, as interest in environmentally friendly agriculture grows, interest in controlling pesticides is increasing, and many crops are hydroponicly grown by genetic engineering techniques of agriculture. In this case, the damage of late blight and wearing disease, which prefers water, has been reported. Since most existing pesticides are insoluble in water, the control rate is very weak or ineffective.

Accordingly, the inventors of the present invention have developed a method for controlling a late blight bacterium or a reputable pathogen by a biological method. ) It was found that streptomycin of skeletal structure showed specific antifungal activity against fungi such as phytopathogens, Phytophthora and Pisium, and confirmed the possibility of development as a biopesticide to control plant diseases such as late blight and wearing disease. The present invention has been completed.

Accordingly, the present invention is an aminoglycoside antibiotic, which has a selective antimicrobial effect on late blight and the pathogens, among them paromomycin and streptamine having a 2-deoxystreptamine skeletal structure. It is an object of the present invention to provide an environmentally friendly biopesticide that is effective in controlling plant diseases such as late blight and wearing disease known as heating agent using streptomycin having a skeletal structure.

Figure 1 is a photograph showing the control effect of pepper rot diseases of paromomycin, neomycin, ribostomycin antibiotic having a 2-deoxystreptamine skeletal structure and streptomycin having a streptamine structure [process concentration is 500 ppm From left, control, ribostamycin, neomycin, streptomycin, paromomycin treatment].

The present invention shows a specific antimicrobial activity against Oomycete, such as Phytophthora sp., Pythium sp., Which are phytopathogens of aminoglycoside antibiotics, and thus 2-deoxystreptamine ( It is characterized by its availability as a plant disease control agent, such as phytopathy and respiratory disease, of paromomycin with 2-deoxystreptamine) and streptomycin with streptamine.

Referring to the present invention in more detail as follows.

The present inventors collected a large number of soil samples from all over the country to screen new biopesticides for controlling plant diseases, and screened for antimicrobial activity against late blight bacteria, and strains exhibiting specific activity against fungi such as late blight and incoming bacteria. Was separated. The isolated actinomycetes were cultured in liquid and subjected to in vivo pot experiments to select one strain having the best activity. After culturing the strain in liquid, the active substance in the culture was separated and purified, and the structure was identified. As a result, it was confirmed that it is paromomycin, an aminoglycoside antibiotic having a 2-deoxystreptamine skeleton. Therefore, ribomycin, neomycin, and streptomycin with streptamine skeletal structure were purchased as aminoglycoside antibiotics with related 2-deoxystreptamine skeletal structure [Sigma Chemical Co., Ltd.]. In vitro and in vivo antimicrobial activity was tested for each concentration.

Example 1 of paromomycin in vitro Antimicrobial activity

To determine whether paromomycin is active against various types of fungi in vitro , antibiotics were added to potato dextrose broth and cultured at 25 ° C for 7 to 10 days. Observed. The fungal strains used in this assay are as follows. Colletotrichum gloeosporioides , as well as Penicillium oxalicum , Penicillium expensum , Penicillium sp., Aspergillus fla . Aspergillus flavus , Aspergillus fumigatus , Mucor ambigous , Alternaria alternata , Phytophthora infestans ATCC26081, Fishium Pythium sp. As shown in Table 1, paromomycin was inactive against several types of filamentous fungi. However, it was very active against Phytophthora infestans , a tomato and potato late blight from the American Type Culture Collection (ATCC), which inhibited the growth of bacteria at a concentration of 5 µg / ml. . This result is consistent with reports that paromomycin is known to be ineffective or low on common fungi, but the antibiotics have been shown to inhibit phytophthora infestans, a late blight, at low concentrations.

In many cases, many types of antifungal natural products obtained through screening appear to be active in vitro but not in vivo . Therefore, the activity was tested in vivo .

Example 2 of paromomycin in vivo Antimicrobial activity

In order to test whether paromomycin is effective in vivo against tomato late blight and several other plant diseases, the activity was directly tested in the greenhouse at 250 ㎍ / ml concentrations for several kinds of pathogens. It is shown in Table 2 below.

The assay for activity on in vivo is as follows.

Rice blast is sprayed enough to flow Pyricularia oryzae spores (10 ⁶ spores / ml) into rice Nakdong rice (2 ~ 3 leaves), and the inoculated rice is left in the dark for 24 hours. Disease area was investigated for 5 days in a constant temperature and humidity room with a relative humidity of 80% or higher and 26 ℃.

Rice leaf beetle blight was inoculated into the pots grown in Nakdong rice of 3-4 leaf stages treated with medicinal mycelial mass of Rhizoctonia solani evenly and incubated for 1 day in a humid room (25 ℃). After 4 days in a constant temperature and humidity room with a relative humidity of more than 80%, the diseased area of the leaf was investigated.

Tomato and pepper late blight were spore suspensions of 10 ⁵ sporangia / ml of the spore concentrations of Phytophthora infestans and Phytophthora capsici , and then cooled at 13 ° C. for 2.5 hours. The seedlings were drained, sprayed and inoculated onto the drug-treated tomato seedlings (second to third leaf), and the tomato seedlings were wet-treated at 20 ° C. for 48 hours and then in a 20 ° C. constant temperature and humidity room (relative humidity of 95% or higher). After the onset of the disease, the lesion area was examined.

Wheat red rust is Puccinia recondita , which is a synovial parasite, so it was used for inoculation of spores formed in wheat seedlings while passing directly to plants in the laboratory. Sowing seeds (light) of 5 grains in a disposable pot (6.5 cm), chemically treating seedlings seedlings grown in a greenhouse for 8 days, air-dried for 1 day, and then spraying a spore suspension (spore 0.67 g / ℓ). Inoculated and inoculated wheat seedlings were incubated for 7 days in a constant temperature and humidity room of 20 ~ 23 ℃, relative humidity 70% and then examined the lesion area.

As shown in Table 2, paromomycin has a weak effect on other pathogens or did not inhibit the occurrence of disease at all, but the effect was strong in tomato late blight, and the result was tomato inverse as in vitro activity. Showed specificity to the germ.

Example 3 of Aminoglycoside Antibiotics with Paromomycin and 2-deoxystreptamine Skeletal Structure in vitro Antimicrobial activity

To determine whether aminoglycoside antibiotics with 2-deoxystreptamine framework, including paromomycin, are active against several phytophthora strains, potato liquid medium (potato dextrose broth) The antibiotics were added to the cells and cultured at 25 ° C. for 7 to 10 days, and the growth of each assay strain was observed [Tables 3, 4, 5]. The fungal strains used in this assay were Phytophthora infestans , Phytophthora nicotianae , Phytophthora megasperma , Phytophthora palmivora , and Phytophthora palmivora . Phytophthora porri . As shown in Table 4, paromomycin inhibited growth at a concentration of 1 μg / ml against most of the fungus of the genus Phytoptotora except Phytophthora porri .

Example 4 of Paromomycin and Various Aminoglycoside Antibiotics in vivo Antimicrobial Activity Comparison

In order to compare whether other aminoglycoside antibiotics show the same activity as paromomycin, neomycin (riomycin), ribosomycin (ribostamycin) and streptomycin antibiotics were used in the same manner as in Example 2 above. In vivo control effect was assayed. As a result, as shown in the following Table 6, paromomycin showed almost 100% control against pepper blight even at 100 ㎍ / ml concentration, and streptomycin had a high control effect of about 80% at 500 ㎍ / ml. Indicated.

Example 5 Control Effects of Paromomycin against Red Pepper Plague

The control effect of paromomycin against red pepper blight was tested. In order to cause disease in the pepper, the spore concentration of phytophthora capsici ( Phytophthora capsici ) was made into 10 ⁴ zoospores / ml, and it was treated by cold treatment for 13 to 25 hours, and then spilled. Was sprayed on the red pepper seedlings (3-4 leaf). Pepper seedlings were treated for 48 hours in a 20 ° C wet room, and then onset at 20 ° C constant temperature and humidity room (relative humidity of 95% or more) for 3 days, and then examined the lesion area. As a result, as shown in Table 7, paromomycin, unlike neomycin or ribostamycin, exhibited almost 80% of the control effect against pepper blight at a concentration of 100 ㎍ / ml, and streptomycin 500 ㎍ / ml. In general, 80% of the concentrations showed a high control effect.

The above results show that the plant disease control effect of other antibiotics compared to paromomycin is shown in Table 7 above. In addition, the plants treated with ribostamycin and neomycin showed less disease than the untreated group, but showed less effect than paromomycin. On the other hand, streptomycin has a weaker control effect than paromomycin, but shows a high control effect of 80% when treated with 500 ㎍, showing the possibility of development as a control agent for fungal diseases. May be used (see FIG. 1).

Example 6 Antimicrobial Activity of Paromomycin Against Different Types of Fungi (Phytophthora and Pisium)

It is shown in Table 8 that the paromomycin used in the present invention is effective against various strains of the late blight phytophthora and the stock bacteria. Most assays for this experiment were distributed at ATCC. As a test bacterium, Phytophthora infestans ATCC 26081, Phytophthora megaspera ATCC 28004, Phytophthora palmivora ATCC 26483, Phytophthora nicotianae ATCC 15409, Phytoptophora ( Phytophthora porri ) ATCC 52729, Pythium aphanidermatum ATCC 26081, Pythium ultimum CJK01, Pythium dissotocum ATCC 60519 and Pythium sp. Used. The mycelial growth inhibitory activity was continuously diluted 2 times with paromomycin using distilled water and put it in 10 ml of potato liquid medium, and then added to the mycelial section (0.5 cm diameter) of the test strain cultured under 25 ℃ dark conditions. After culturing for 4 to 10 days, the mycelia of each strain were observed to grow. As a result, paromomycin was found in most Phytophthora species except for phytoptophora ATCC 52729 even at very low concentrations (0.5 to 1.0 μg / ml). sp.) and Pythium sp. were completely inhibited from mycelial growth. Therefore, it was demonstrated by this experiment that the paromomycin has an excellent effect against various plant late blight and stock pathogens.

Inhibition of the growth of various late blight and incoming bacterial pathogens in liquid media by paromomycin is due to fungicidal or fungistatic effects. To find out more precisely, paromycin was added to potato medium, and two mycelial fragments of Pythium aphanidermatum were added. After being left for 4 days, the mycelia fragments were collected, washed several times with sterilized distilled water, and transferred to a new potato medium to observe the mycelia growth. As a result, the growth was good in the medium without the addition of paromomycin, but did not grow at all in the added medium. Therefore, paromomycin was judged to have a bactericidal effect.

As described in detail above, the present invention finds that the aminoglyco clock antibiotics paramomycin and streptomycin, which exhibit specific antimicrobial activity against oomycete, exhibit strong antimicrobial activity against plant diseases such as late blight and rash. It became. Therefore, the aminoglyco clock antibiotics can be used as environmentally friendly biopesticides and have very useful effects on plant diseases such as plant late blight and stock diseases.

Claims (5)

Plant disease control agent comprising paromomycin as an active ingredient. The plant disease control agent according to claim 1, wherein the plant disease is plant late blight or wearing disease. delete delete delete