KR20120010791A - Antifungal activity of Bacillus sp. AM44 against to multiple apple mold diseases and the improvement in the quality of apples - Google Patents

Abstract

PURPOSE: A method for improving fruit product quality and preventing apple mold using Bacillus AM44 strain is provided to prevent Alternaria mali bacteria and Botryosphaeria. CONSTITUTION: A Bacillus sp. AM44(KCTC 11714BP) has an activity of preventing apple diseases. A microbial formulation for biological prevention of apple pathogen contains the strain as an active ingredient. A disinfectant for preventing plant blight contains the strain as an active ingredient. The plant blight is caused by Alternaria mali, Botryosphaeria dothidea, colletotrichum acutatum, Rhizoctonia solani AG-2-1, phytophthora cactorum, Colletotrichum gloeosporioides, or Phytophthora capsici.

Description

Improvement of apple quality and control of apple disease of Bacillus AM44 {Antifungal activity of Bacillus sp. AM44 against to multiple apple mold diseases and the improvement in the quality of apples}

The present invention isolate strain Bacillus genus AM44 ( Bacillus sp. AM44) relates to a strain and a method for controlling an apple disease using the same. More specifically, the Apple spots deciduous germs to occur throughout the growing season of the apple, causing the most severe damage. [Al Tena Leah Marley (Alternaria mali)], gyeopmunui rot fungus [Boats Rio paella Dottie Oradea (Botryosphaeria dothidea)], Bacillus genus, a novel microbial strain that controls anthrax ( Colletotrichum acutatum ) AM44 ( Bacillus sp. It relates to a biopesticide to replace chemical pesticides using AM44).

More than 30 kinds of parasitic diseases occur in apple gardens in Korea, but more than 10 diseases occur in orchards that are normally managed (Refer to low pesticide high efficiency apple pest control technology, Eom Jae-yeol, 2005). Among them, six types of diseases such as rot rot disease, anthrax disease, brown pattern disease, sooty disease and red star pattern disease need to be controlled during the growing season. (Refer to low pesticide high efficiency apple pest control technology, Um Jae-yeol, 2005, Yu et al. 2008, Uhm and Lee, 2004). In order to control apple disease, it is necessary to apply a control agent before or immediately after the pathogen is infected. Therefore, it is necessary to accurately recognize the timing of infection, and simultaneous control should be performed because the infection time of these diseases is almost identical. High Efficiency Apple Pest Control Technology, Eom Jae-yeol, 2005).

Currently, 204 of 282 patents related to biopesticides have been registered in Korea for the past 10 years, 74 of 96 microbial pesticides have been registered, and 26 have been registered for apples. In the apple garden in Korea, weed management through pest control and herbivore cultivation using natural enemies, sex pheromone traps and mating disturbances are carried out in various ways. It is true that awareness of pesticide control is low. Most apple cultivators use organic synthetic fungicides for disease control. This is because organic farming is practically difficult because no apples are easily exposed to disease and there is not enough database of organic apple farming in Korea. The degree of resistance to the disease of apple varieties varies widely, among which the disease to be controlled includes double rot and anthrax. None of the apple varieties are resistant to both of these diseases and none are susceptible to both. In addition, few fungicides have similar control effects for these two diseases (Guozheng Qin, Shiping Tian., Yong Xu / Postharvest Biology and Technology 31 (2004) 51-58).

Accordingly, the present inventors have an excellent antagonistic force against apple pathogens, isolates strains capable of stable colonization on the skin and leaves, and as a result of the identification, it was found that the genus Bacillus AM44, microbial agent using the same as well as apple disease The present invention was completed by revealing the antifungal activity of various spectra, verifying the activity through field tests, and improving the fruit quality in addition to the antifungal activity.

In the present invention, it was intended to develop a biopesticide that is effective in controlling apple diseases, and as a result of studying biological control methods using antagonists, Bacillus genus AM44 was isolated from the soil.

That is, the main object of the present invention is to provide a novel strain exhibiting a control effect on apple diseases.

Still another object of the present invention is to provide a biopesticide preparation for apple disease control using the novel strain and a method for controlling apple disease, wherein the biopesticide preparation is used.

In addition, the dilution and spray treatment of the culture of the new strain is to provide the applicability as an apple quality improver.

In addition, an object of the present invention is to use a novel strain of the above, a wide range of plant diseases causing bacteria of other crops other than apples, for example, spotted deciduous bacteria (Alternaria Mali), layered rot bacteria (Botrios paeria doti Dea), Anthrax bacterium (Coletotricum akutat), Rotting bacterium [ Rhizoctonia solani AG-2-1], Plague bacterium [ Phytophthora cactorum ], Anthrax bacterium [ Colletotrichum gloeosporioides ], Zyrocoptera [ Pythium ultimum ], Mycobacterium fungi [ Sclerotinia sclerotiorum ], Root rot bacteria [ draw carboxylic phone desk look Tansu (Cylindrocarpon destructans) cylinder], wilt fungus - Fusarium oxy sports rooms (Fusarium oxysporum)], jeommunuibyeong bacteria [Alterna Ria Panax (Alternaria panax)], jeommunuibyeong bacteria [poma in (Phoma sp.) , Gray Mold Fungus [Bortiness] Sinne LEA (Botrytis cinerea)], there is provided a sterilizing agent for the control station, such as bacteria [pi Saratov Tora to Cobb (Phytophthora capsici)].

In the present invention, in order to achieve the above object, Bacillus genus showing an antagonistic action against apple diseases Provides AM44.

The microbial agent of the present invention provides a basis for microbial pesticide preparation for apple disease control containing the culture as an active ingredient.

The present invention provides a bacterium of the genus Bacillus AM44, which is useful for controlling apple diseases, and provides a basis of a microbial pesticide preparation for controlling apple diseases. Bacillus genus according to the present invention AM44 is useful as a microbial agent for the control of apple diseases, and is expected to be used as a bioadjuvant to affect the quality of apples as well as biopesticides to replace chemical pesticides.

1 is Bacillus genus 16S rRNA gene analysis of AM44.
2 is genus Bacillus This is the identification result of AM44.
3 is a genus of Bacillus according to the culture pH and temperature The results show the antifungal activity of AM44 against apple disease [(A): pH, (B): temperature].
4 is isolated Bacillus genus After establishment of the optimal medium and culture conditions of AM44, it is the result showing the apple disease control activity according to the culture time of the strain.
5 is the result of an in vitro assay.
Figure 6 is a result of comparing the apple disease incidence of apple test packaging treated with conventional pesticides (practice), water (control) and Bacillus genus AM44 (treatment) of the present invention.
Fig. 7 shows the results of the impact evaluation (total polyphenols, sugar content, hardness, acidity) of the packaging test apples treated with conventional pesticides (practice), water (control) and Bacillus AM44 (treatment) of the present invention. .
FIG. 8 shows the quality effects (total polyphenols, sugar content, hardness, PH) results.
Figure 9 shows the decay and photo results of the storage of the apples treated with conventional pesticides (practice), water (control) and Bacillus AM44 (treatment) of the present invention.

Through the following examples will be described in more detail the present invention.

Example 1 Isolation and Selection of Antagonistic Bacteria

To select antagonistic microorganisms that inhibit the growth of apple diseases, seawater, shellfish samples, orchards and natural farmland soils throughout the country were used as mycelial samples, suspended and diluted in sterile physiological saline, and they were placed on MA, LB, and NA agar media. Inoculation was incubated at 30 ° C. for 2 days to separate microorganisms.

Among the purely isolated strains, the culture supernatant of each strain was used to select apple strains that can simultaneously control apples and apples [apple, dot, pattern, deciduous, and anthrax] (colletotricum akutatum). Finally, strains that inhibit mycelial growth of apple disease bacteria were selected by replacement culture.

Subsequently, the strain was identified through 16S rRNA sequencing for taxonomic identification of antagonistic bacteria (FIGS. 1 and 2).

Example 2 Establishment of Optimal Culture Conditions for Selected Antagonists

Bacillus genus according to medium condition As a result of examining the antagonistic activity of AM44, under the conditions shown in Table 1, the strain grew best and showed antagonistic activity.

Distilled water (1ℓ) Starch 30 g KNO ₃ 4 g Trypton 1 g Yeast extract 1 g MgSO ₄ ? 7H ₂ 0 0.1g

Bacillus genus according to culture pH and temperature The production of the antagonist of AM44 showed that it was stable at a wide range of pH, and at the temperature of 7 and 30 ℃, the representative apple disease-causing deciduous bacterium (Alternaria Mali) and the multiplying rot bacterium (Botrios paeria dotidea) ) And optimal antifungal activity against anthrax (colletotricum akutatum) (FIG. 3).

In addition, as a result of investigating the spectrum of the control and control activity of the isolates against other phytopathogens, it was found that they showed excellent inhibitory activity against various phytopathogens (Table 2). This proves that Bacillus AM44 strain can be used as biocontrol agents of other crops.

Phytopathogenic fungi
KACC Suppression zone
(mm)
Rotting bacterium [ Rhizoctonia solani ] AG2-1

Plague Bacteria [ Phytophthora cactorum ]

Anthrax bacterium [colletotricum Gloeosporioides]
( Colletotrichum gloeosporioides) ]

Drosophila bacteria [Pitium Ultimum]
( Pythium ultimum) ]

Plague Bacteria [Phytophthora Capsiki]
( Phytophthora capsici)

Wither disease [Fusarium oxysporum]
( Fusarium oxysporum)

Anthrax bacteria [Coletotricum akutatum]
( Colletotrichum acut a tum) ]

40120

40176

40695


40705


40157


41079


40689


22.51

27.54

37.23


29.64


26.92


25.82


21.34

Example 3 Bacillus genus In vitro control of AM44 cultures

In order to verify whether the antagonism of isolated bacteria has antagonism not only on the medium, but also on the apple fruit, Fuji (adverb) apple fruit grown in Y farm, Waryong-myeon, Andong-si, Gyeongsangbuk-do, Korea was used. The cultivated fruit was first washed with sterile distilled water and then washed once more with 3% sodium hypochlorite, and then irradiated with cotton soaked with 70% alcohol and irradiated with UV for about 10 minutes before being used for the experiment. To induce the apple fruit, the wound is made with a sterilized glass rod (5 mm), and then the culture medium of the isolated bacteria and the spotted deciduous bacterium (Alteraria Mali), the apple disease, and the layered rot bacterium (Botrios paeria) Dotidea) and spore suspensions of anthrax (Coletotricum akutatum) were treated together for 1 hour, followed by control (medium), infection (pathogen spore suspension), and treatment (pathogen spore suspension + Bacillus genus AM44 culture) Treatment). The dried apples were incubated for 10 days in an incubator at 25 ° C. and confirmed the pathogenesis of each bacterium and the pathogenesis of the isolated bacterium for these pests.

As a result, the control (medium) did not find any unusual incidence and other abnormalities in the wounded area even after 10 days.However, in the case of the infected (homologous spore suspension), each of the three pathogens was treated as soon as possible. The onset began to appear from day 1, and after 10 days, the symptoms showed an infection that could be judged visually. However, in the treatment group (pathogen spore suspension + Bacillus AM44 and simultaneous treatment), all three strains showed only the infection symptom similar or very weak (Fig. 5).

Example 4 Bacillus genus Control Effect of Apple Bacteria by Field Test Using AM44 Culture Solution

Prototypes made with the AM44 strain of Bacillus genus were used to process prototypes at Y Farm, Waryong-myeon, Andong-si, Gyeongsangbuk-do starting August 1, 2009, and then harvested six times every 15 days until the harvest date of November 06, 2009. Treatment over The treatment method was spraying by each treatment section by using automatic spraying machine (180 × 480 × 330, 20ℓ), which was treated by 2ℓ per tree. This is because the water pressure of the sprayer is not high and a certain amount is not sprayed uniformly. The amount was treated more than the amount to treat the medicine uniformly. Each treatment was divided into control (water treatment), practice (farm pesticide treatment) and treatment (Bacillus AM44 prototype treatment), and three trees were selected for each treatment. At harvest time, the incidence, yield, total polyphenols, acidity, sugar content and hardness were examined.

Control Value (%) = 100-(Control Incidence / Control Incidence) X 100

As a result, the weather conditions of 2009 were suitable for the development of various apple diseases, but the test packaging of spotted deciduous bacteria (Alteraria Mali), layered rot bacteria (Botrios paeria dotidea), and anthrax (colletotricum aku) Although the incidence rate of TATUM) was very low, the collected data of some cases showed that the control group (microbial agent) did not show a higher control rate than the conventional treatment (chemical pesticide treatment), but was weaker than the control group. Hana showed efficiency. However, if the change in the treatment method or other environmental factors, such as optimum will show a better control rate (Fig. 6).

Total polyphenols, sugar content, hardness, and acidity were investigated to determine whether the microbial treatments affected apple quality improvement. As a result, the total polyphenol content tended to be slightly higher in the treatment group, and in the case of sugar content, the sample treated with the microbial agent was about 1 brix than the conventional treatment (chemical pesticide) and no control treatment (water treatment). Raised. In addition, even in the case of hardness, the sample treated with the microbial agent showed better results than the other treatments, and in the case of acidity, it was not able to confirm the difference (Fig. 7).

Example 5 Bacillus genus Shelf-life experiment of fruits grown after control effect of apple pests by field test using AM44 culture

end. Hypotonic results

The total polyphenol content, sugar content, hardness, and acidity were examined in the 0 ° C. storage cell at intervals of up to 160 days for 20 days. As a result, the microbial treatment group (Bacillus AM44) remained superior to the control and the conventional treatments (FIG. 8). .

I. Incidence study during hypotonic experiment

As a result of examination of the decaying department during the 160 days storage test in the 0 ° C. reservoir, as shown in Table 3, it was about 15% in the control, 10% in the conventional and 10% in the microbial treatment. However, in the case of microbial agents, anthrax was not developed. In particular, as shown in FIG. 9, it was found that the size of the lesions was smaller in the microbial treatment group than in the size of the lesions caused by the fruit of the control or the practice. It is believed that the microbial agent buried in the worm inhibited the mycelia of pathogens in the fruit.

Control (water treatment) Practice (Pesticide Treatment) Treatment tool (microbial agent) Zhejiang Test Quantity 100 100 100 Spotted deciduous 2 3 2 Double rot 11 5 8 anthrax 2 2 0

Example 6 Bacillus genus Storage shelf life test using AM44 culture

For the commercialization of microbial products, the shelf life of the product must be stable. Therefore, the Bacillus genus AM44 culture solution was stored under these conditions for 100 days in consideration of the low temperature storage at 4 ℃ and room temperature and observed the stability of the microbial preparation. As a result, Bacillus AM44 strain culture medium was found to maintain more than 80% activity in both cases stored at 4 ℃ and room temperature (Table 4). It is judged that the antagonist produced by the Bacillus AM44 strain is a stable substance, and that not all farms have a low temperature storage facility, which may occur when the microbial agent is stored at room temperature or during the distribution of the microbial agent. It is considered to be an efficient preparation for time.

Spotted deciduous bacteria
(Alternaria Mali) Double rot
(Botrios Paeria Dotidea) Anthrax
(Coletotricum Akutatum) 4 ℃ Room temperature 4 ℃ Room temperature 4 ℃ Room temperature 0 days 35.34 35.34 34.89 34.89 33.27 33.27 20 days 35.94 35.48 34.48 34.70 33.41 33.35 40 days 35.52 32.98 34.60 32.29 33.18 32.70 60 days 33.59 31.65 34.44 30.78 33.97 30.51 80 days 32.87 30.11 32.68 29.10 31.69 28.05 100 days 31.46 29.45 30.39 27.98 30.11 27.94

Korea Research Institute of Bioscience and Biotechnology KCTC11714BP 20100618

<110> ANDONG NATIONAL UNIVERSITY INDUSTRY-ACADEMY COOPERATION <120> Antifungal activity of Bacillus sp. AM44 against to multiple          apple mold diseases and the improvement in the quality of apples <130> ADU-01 <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 1391 <212> DNA <213> Bacillus sp. AM44 <400> 1 tggcggcgtg cctaatacat gcaagtcgag cggacagatg ggagcttgct ccctgatgtt 60 agcggcggac gggtgagtaa cacgtgggta acctgcctgt aagactggga taactccggg 120 aaaccggggc taataccgga tggttgtctg aaccgcatgg ttcagacata aaaggtggct 180 tcggctacca cttacagatg gacccgcggc gcattagcta gttggtgagg taacggctca 240 ccaaggcgac gatgcgtagc cgacctgaga gggtgatcgg ccacactggg actgagacac 300 ggcccagact cctacgggag gcagcagtag ggaatcttcc gcaatggacg aaagtctgac 360 ggagcaacgc cgcgtgagtg atgaaggttt tcggatcgta aagctctgtt gttagggaag 420 aacaagtgcc gttcaaatag ggcggcacct tgacggtacc taaccagaaa gccacggcta 480 actacgtgcc agcagccgcg gtaatacgta ggtggcaagc gttgtccgga attattgggc 540 gtaaagggct cgcaggcggt ttcttaagtc tgatgtgaaa gcccccggct caaccgggga 600 gggtcattgg aaactgggga acttgagtgc agaagaggag agtggaattc cacgtgtagc 660 ggtgaaatgc gtagagatgt ggaggaacac cagtggcgaa ggcgactctc tggtctgtaa 720 ctgacgctga ggagcgaaag cgtggggagc gaacaggatt agataccctg gtagtccacg 780 ccgtaaacga tgagtgctaa gtgttagggg gtttccgccc cttagtgctg cagctaacgc 840 attaagcact ccgcctgggg agtacggtcg caagactgaa actcaaagga attgacgggg 900 gcccgcacaa gcggtggagc atgtggttta attcgaagca acgcgaagaa ccttaccagg 960 tcttgacatc ctctgacaat cctagagata ggacgtcccc ttcgggggca gagtgacagg 1020 tggtgcatgg ttgtcgtcag ctcgtgtcgt gagatgttgg gttaagtccc gcaacgagcg 1080 caacccttga tcttagttgc cagcattcag ttgggcactc taaggtgact gccggtgaca 1140 aaccggagga aggtggggat gacgtcaaat catcatgccc cttatgacct gggctacaca 1200 cgtgctacaa tggacagaac aaagggcagc gaaaccgcga ggttaagcca atcccacaaa 1260 tctgttctca gttcggatcg cagtctgcaa ctcgactgcg tgaagctgga atcgctagta 1320 atcgcggatc agcatgccgc ggtgaatacg ttcccgggcc ttgtacacac cgcccgtcac 1380 accacgagag t 1391

Claims (5)

Bacillus sp. AM44 strain (KCTC 11714BP) with apple disease control activity. Bacillus genus AM44 strain (KCTC 11714BP), characterized in that it contains as an active ingredient, microbial agent for biological control of apple pathogenic fungi. Bacillus genus AM44 strain (KCTC 11714BP) according to claim 1, incubated in distilled water having a composition of starch 3%, KNO ₃ 0.4%, tryptone 0.1%, yeast extract 0.1% and MgSO ₄ ~ 7H ₂ 0 0.01% Way. The apple spots leaves bacteria containing Bacillus AM44 strain (KCTC 11714BP) according to claim 1 as an active ingredient [Alterna Ria Mali (Alternaria mali)], gyeopmunui rot fungi [boat Rio Spa Area Dottie Oradea (Botryosphaeria dothidea)] , Anthrax [ Colletotrichum acutatum ], rot fungus [ Rhizoctonia solani AG-2-1], blight [ Phytophthora cactorum ], anthrax Fungi [ Colletotrichum gloeosporioides ], fungus [ Pythium ultimum ], fungal pathogens [ Sclerotinia sclerotiorum ], root rot disease Fungi [ Cylindrocarpon destructans ], wilt fungus [ Fusarium oxysporum ], spots germs [ Alternaria panax ], spots germs [Poma spoma sp. Gray Mold Disease [Botrytis sinne LEA (Botrytis cinerea)], reverse germs [pi Saratov Tora to Cobb (Phytophthora capsici)] fungicide for plant disease control according to the. A method of improving apple quality, characterized in that the culture of Bacillus AM44 strain (KCTC 11714BP) obtained by the culture method according to claim 3 is used.

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