Acid-resistant pseudomonas koreensis CLP-7, and applications thereof

Abstract

The invention discloses an acid-resistant pseudomonas koreensis strain CLP-7 which is capable of preventing diseases and promoting growth, and can be used for biocontrol. The acid-resistant pseudomonas koreensis strain CLP-7 is preserved at China General Microbiological Culture Collection Center, on 27th, October, 2016, and the preservation number is CGMCC No.13204. The antibacterial spectrum of the acid-resistant pseudomonas koreensis strain CLP-7 is relatively large; the acid-resistant pseudomonas koreensis strain CLP-7 is capable of inhibiting growth of Phytophthora parasitica var. nicotianae, Ralstonia solanacearum, and Alternaria alternata Keissler; the antagonistic activity under acidic conditions is high, the acid-resistant pseudomonas koreensis strain CLP-7 is capable of producing siderophore, possesses protease and glucanase activity, and potassium releasing capacity. The results of biocontrol pot experiment show that the acid-resistant pseudomonas koreensis strain CLP-7 is capable of preventing under acidic soil conditions, and promoting growth and chlorophyll synthesis of tobacco seedlings in acidic soil, so that the acid-resistant pseudomonas koreensis strain CLP-7 and microorganism bacterium agents of the acid-resistant pseudomonas koreensis strain CLP-7 can be used for preventing tobacco fungi and bacterial root and stem diseases under continuous cropping or acidic soil conditions effectively, and is high in application value.

Description

Technical field

[0001] The invention belongs to the technical field of microorganisms, and specifically relates to an acid-resistant Pseudomonas korean CLP-7 and its application. Background technique

[0002] Tobacco often suffers from a variety of fungal and bacterial diseases throughout its growth period, including tobacco black shank, tobacco wilt, brown spot, wildfire and angular leaf spot. Some pathogens can be harmful in various growth stages of tobacco. For example, some pathogens only harm certain organs during a certain growth period of tobacco. For example, Alternaria alternata (Fries) Keissler only damages leaves during tobacco maturity; rhizome diseases such as Tobacco black shank (Phytophthora parasiticavar.nicotianaer) and bacterial wilt (Ralstonia solanacearum) can be harmful from the tobacco seedling stage to the maturity stage, and they are prevalent with the climate of different years. Rhizome diseases seriously affect the yield and quality of tobacco leaves. O...

Examples

Embodiment 1

[0030] Example 1 The antagonistic effect of CLP-7 and its fermentation broth on tobacco bacterial wilt, black shank and brown spot

[0031] Inoculate CLP-7 on NA plates at 30°C for 48 hours, then use the plate confrontation method, that is, inoculate black shank and Alternaria alternata (diameter 5mm) on the oat and potato dextrose medium (PDA) plates. Pick AR03 and streak equidistantly on both sides of the bacterial cake, culture it at 30℃ for 5 days, and use Oxford cup plate diffusion method to inhibit Ralstonia solanacearum; that is, inoculate CLP-7 strain into beef juice peptone liquid culture medium ( NB), 30 ℃, 150 rpm shaking culture for 2 days, take appropriate amount of fermentation broth and sterile supernatant (filtered through a bacterial filter) on the Oxford cup containing Ralstonia solanacearum peptone medium plate (NA) Inside, stand for 2 days and observe the inhibition of CLP-7 on the above pathogenic bacteria. From the observation of the antibacterial effect of...

Embodiment 2

[0033] Example 2 The inhibitory effect of CLP-7 strain on the growth of black shank and Alternaria alternata hyphae

[0034] With black shank and Alternaria alternata as target bacteria, that is, inoculating CLP-7 on NA plates at 30°C for 48 hours, and then using the plate confrontation method, that is, inoculating Alternaria alternata on oat plates and Alternaria alternata on PDA plates. Pick AR03 in the ring and streak equidistantly with the bacterial cake as the center. Incubate at 30°C for 5 days. Pick out the edge hyphae of the black shank and Alternaria alternata respectively under a microscope. The results show that after CLP-7 treatment Compared with normal hyphae, the cytoplasm in the hyphae cells of the black shank fungus cake marginal hyphae aggregates into a spherical shape, which is separated from the cell membrane and cell wall, thus affecting its normal growth ( Figure 4 ); Compared with the normal conidia, the inhibitory effect of the conidia of Alternaria alterna...

Embodiment 3

[0035] Example 3 Determination of Protease Activity of CLP-7 Strain

[0036] The protease activity was determined using protein culture medium. The protein culture medium and formula were: A: 6.4g skimmed milk powder, dissolved in 240mL water, and sterilized at 121°C for 20min; B: agar, 6.4g, constant volume to 240mL, sterilized at 121°C for 20min , A and B are sterilized and mixed separately for protease detection. The measurement method is as follows: the protein medium is dissolved and made into a plate. Take a sterile toothpick to spot the CLP-7 strain on the protein medium plate, culture for 3 days after inoculation, observe the colony growth and the size of the transparent circle, and measure the diameter of the transparent circle R 2 And colony diameter R 1 , Using R 2 / R 1 The ratio qualitatively indicates its protease activity. The results show that( Figure 7 ), CLP-7 strain has protease activity, its enzyme activity size, namely R 2 / R 1 Is 4.10. The protease activit...

Technical field

[0001] The invention belongs to the technical field of microorganisms, and specifically relates to an acid-resistant Pseudomonas korean CLP-7 and its application. Background technique

[0002] Tobacco often suffers from a variety of fungal and bacterial diseases throughout its growth period, including tobacco black shank, tobacco wilt, brown spot, wildfire and angular leaf spot. Some pathogens can be harmful in various growth stages of tobacco. For example, some pathogens only harm certain organs during a certain growth period of tobacco. For example, Alternaria alternata (Fries) Keissler only damages leaves during tobacco maturity; rhizome diseases such as Tobacco black shank (Phytophthora parasiticavar.nicotianaer) and bacterial wilt (Ralstonia solanacearum) can be harmful from the tobacco seedling stage to the maturity stage, and they are prevalent with the climate of different years. Rhizome diseases seriously affect the yield and quality of tobacco leaves. O...

Examples

Embodiment 1

[0030] Example 1 The antagonistic effect of CLP-7 and its fermentation broth on tobacco bacterial wilt, black shank and brown spot

[0031] Inoculate CLP-7 on NA plates at 30°C for 48 hours, then use the plate confrontation method, that is, inoculate black shank and Alternaria alternata (diameter 5mm) on the oat and potato dextrose medium (PDA) plates. Pick AR03 and streak equidistantly on both sides of the bacterial cake, culture it at 30℃ for 5 days, and use Oxford cup plate diffusion method to inhibit Ralstonia solanacearum; that is, inoculate CLP-7 strain into beef juice peptone liquid culture medium ( NB), 30 ℃, 150 rpm shaking culture for 2 days, take appropriate amount of fermentation broth and sterile supernatant (filtered through a bacterial filter) on the Oxford cup containing Ralstonia solanacearum peptone medium plate (NA) Inside, stand for 2 days and observe the inhibition of CLP-7 on the above pathogenic bacteria. From the observation of the antibacterial effect of...

Embodiment 2

[0033] Example 2 The inhibitory effect of CLP-7 strain on the growth of black shank and Alternaria alternata hyphae

[0034] With black shank and Alternaria alternata as target bacteria, that is, inoculating CLP-7 on NA plates at 30°C for 48 hours, and then using the plate confrontation method, that is, inoculating Alternaria alternata on oat plates and Alternaria alternata on PDA plates. Pick AR03 in the ring and streak equidistantly with the bacterial cake as the center. Incubate at 30°C for 5 days. Pick out the edge hyphae of the black shank and Alternaria alternata respectively under a microscope. The results show that after CLP-7 treatment Compared with normal hyphae, the cytoplasm in the hyphae cells of the black shank fungus cake marginal hyphae aggregates into a spherical shape, which is separated from the cell membrane and cell wall, thus affecting its normal growth ( Figure 4 ); Compared with the normal conidia, the inhibitory effect of the conidia of Alternaria alterna...

Embodiment 3

[0035] Example 3 Determination of Protease Activity of CLP-7 Strain

[0036] The protease activity was determined using protein culture medium. The protein culture medium and formula were: A: 6.4g skimmed milk powder, dissolved in 240mL water, and sterilized at 121°C for 20min; B: agar, 6.4g, constant volume to 240mL, sterilized at 121°C for 20min , A and B are sterilized and mixed separately for protease detection. The measurement method is as follows: the protein medium is dissolved and made into a plate. Take a sterile toothpick to spot the CLP-7 strain on the protein medium plate, culture for 3 days after inoculation, observe the colony growth and the size of the transparent circle, and measure the diameter of the transparent circle R 2 And colony diameter R 1 , Using R 2 / R 1 The ratio qualitatively indicates its protease activity. The results show that( Figure 7 ), CLP-7 strain has protease activity, its enzyme activity size, namely R 2 / R 1 Is 4.10. The protease activit...

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