<i>S. roseoverticillatus </i>Sr-63 and its application
11457634 · 2022-10-04
Assignee
Inventors
- Donghua Jiang (Jinhua, CN)
- Matida Suwantammarong (Jinhua, CN)
- Yunzhang Ding (Jinhua, CN)
- Tingting Shi (Jinhua, CN)
- Mengxia Lv (Jinhua, CN)
Cpc classification
International classification
Abstract
The application belongs to the field of biotechnology and microbiology, in particular to a strain of Streptomyces roseoverticillatus (Sr-63) which antagonizes the Rice Bacterial Blight and its application in the prevention and treatment of plant diseases. The application discloses a strain of S. roseoverticillatus (Sr-63) with the accession number CCTCC No.: M 2019261. It is also disclosed the application of the S. roseoverticillatus (Sr-63): used for controlling Rice Bacterial Blight.
Claims
1. A method for controlling Rice Bacterial Blight, comprising administering an S. roseoverticillatus strain to rice in need thereof, wherein the S. roseoverticillatus strain is Streptomyces roseoverticillatus Sr-63 strain with accession number CCTCC No.: M 2019261.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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(6) Note: A is the control, with the addition of sterile water; B is the fermentation filtrate of S. roseoverticillatus (Sr-63).
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DETAILED DESCRIPTION OF THE EMBODIMENTS
(8) The invention will be further described in combination with specific embodiments, but the scope of protection of the invention is not limited thereto.
Example 1
Isolation, Purification, Screening and Identification of S. Roseoverticillatus
(9) 1. Strain
(10) (1) Actinomycetes: actinomycetes from different habitats of soil (e.g. paddy field, lawn, tomato field, potato field, Ophiopogon japonicus field, etc.) are isolated, purified, screened and identified, and preserved for identification.
(11) (2) P6 race of Xanthomonas oryzae pv. oryzae.
(12) 2. Medium
(13) (1) The solid medium of Gauze's Medium No. 1: soluble starch 20 g, KNO.sub.3 1 g, K.sub.2HPO.sub.4 0.5 g, MgSO.sub.4.7H.sub.2O 0.5 g, NaCl 0.5 g, FeSO.sub.4.7H.sub.2O 0.01 g, agar 20 g, water 1000 ml, pH 7.2˜7.4. It is used for isolation, purification and identification of actinomycetes.
(14) (2) The liquid medium of Gauze's Medium No. 1: the formula is the same as (1) without agar; it is used for liquid fermentation of actinomycete strains.
(15) (3) The solid medium of Xanthomonas oryzae pv. oryzae, Xoo (Xoo solid medium): potato 300 g, sucrose 15 g, Ca (NO.sub.3).sub.20.5 g, NaH.sub.2PO.sub.4.12H.sub.2O 2.0 g, tryptone 5.0 g, agar 20 g, water 1000 ml, pH 6.5. It is used for solid culture of P6 race of Xanthomonas oryzae pv. oryzae.
(16) (4) The liquid medium of Xanthomonas oryzae pv. oryzae, Xoo (Xoo culture medium): the formula is without agar, and the rest is the same as (3); it is used for fermentation of P6 race of Xanthomonas oryzae pv. oryzae.
(17) 3. Experimental Method
(18) 3.1 Isolation, Purification and Preservation of Actinomycetes
(19) 1 g natural air-dried soil sample (moisture content≤3%) was taken to put into 99 ml sterile water and shaken on a shaker at 120 r/min for 30 min. The supernatant was diluted by gradient (10.sup.−2˜10.sup.−5), and 100 μl of the supernatant was evenly coated on Gauze's Medium No. 1 at a suitable concentration (10.sup.−3). The supernatant was cultured in a 28° C. incubator for 48 hours until the colony appeared. A single colony with typical actinomycete colony characteristics was selected and transferred to another Gauze's Medium No. 1, and the pure strain was obtained after three times of scribing purification; the strain was numbered and stored in a refrigerator at 4° C. for standby.
(20) 3.2 Screening of Actinomycetes Against Bacterial Blight of Rice
(21) 3.2.1 Preliminary Screening of Co Culture Method
(22) The P6 race of Xanthomonas oryzae pv. oryzae was inoculated into Xoo culture medium and activated on shaking table (180 r/min, 28° C.). When the bacterial density reached OD.sub.600 of 0.6, 100 μl bacterial solution was evenly coated on Xoo solid culture medium. The activated actinomycete cake (diameter 4 mm) was inoculated in the middle of Xoo solid culture medium, cultured at 28° C. for 48 hours, and the bacteriostatic circle was observed. The antagonistic actinomycetes were screened according to the existence and size of bacteriostatic circle and preserved with 25% glycerol.
(23) 3.2.2 Oxford Cup Screen
(24) (1) Fermentation of actinomycetes: The antagonistic actinomycete strains were inoculated into Gauze's Medium No. 1 plate and cultured in 28° C. incubator for 3 days; The actinomycete cake was made by a 4 mm punch and inoculated into a 250 ml conical flask filled with 100 ml liquid medium of Gauze's Medium No. 1 and cultured on shaking table (160 r/min, 28° C.) for 5 days. The fermentation liquid was placed in a 50 ml centrifuge tube for 12000 r/min and centrifuged for 10 minutes. The supernatant was filtered with 0.22 μm organic filter membrane to remove the residual spores and obtain the fermentation filtrate. (2) Culture of the P6 race of Xanthomonas oryzae pv. oryzae: P6 race of Xanthomonas oryzae pv. oryzae was inoculated in Xoo culture medium, which was activated on shaking table (180 r/min, 28° C.). When the density of bacteria reached OD.sub.600 of 0.6, 100 μl of bacteria solution was evenly coated on Xoo solid culture medium. An Oxford cup method was applied to 200 μl of fermentation filtrate of actinomycetes to determine the inhibition of each actinomycetes on P6 race of Xanthomonas oryzae pv. oryzae. The actinomycetes with strong antagonistic effect were selected according to the size of bacteriostatic circle.
(25) 3.3 Identification of Actinomycete Target Strains Against Rice Bacterial Blight
(26) A small amount of mycelium of actinomycetes target strain was picked out with tweezers and transferred to Gauze's Medium No. 1 plate, for an activate culture for 3 days; The actinomycete cake (with a diameter of 4 mm) was inoculated on the new Gauze's Medium No. 1 plate and cultured at 28° C. for 5 days, the morphological characteristics of the substrate mycelium, aeromycelium and spore hyphae were observed with microscope, and photographed.
(27) The genomic DNA of the target strain was extracted, the 16S rRNA gene was amplified and sent to Sangon Biotech (Shanghai) Co., Ltd for sequencing. The 16S rRNA gene sequence obtained by sequencing was submitted to GenBank and analyzed by blast comparison to determine the type of actinomycete.
(28) 4. Experimental Results
(29) 4.1 Acquisition of Pure Actinomycetes
(30) 65 pure strains of actinomycetes were isolated and purified from soil samples of different habitats. The colonies of 5 representative pure strains of actinomycetes cultured in Gauze's Medium No. 1 for 5 days are shown in
(31) 4.2 Screening of Actinomycetes Against Bacterial Blight of Rice
(32) 65 strains of actinomycetes were screened by co-culture method and Oxford cup method. The results show that different strains of actinomycetes have different inhibition on bacterial blight. One actinomycete strain, Sr-63 strain, with a diameter of 50 mm, was isolated from the rhizosphere soil of Ophiopogon bodiieri.
(33) 4.3 Identification Results of Actinomycete Sr-63
(34) Morphological characteristics of strain Sr-63: cultured at 28° C. for 5 days on Gauze's Medium No. 1, the colony is small and the surface is pink velvet; under the microscope, the mycelia in the substrate and aerial mycelia are branched and slender, mature aerial mycelium forms polysporic chain, and the spore hyphae is open-loop (
(35) Sequence analysis of 16S rRNA gene: the result shows that its length is 1461 bp (
(36) According to the morphological characteristics of strain Sr-63, combined with the key list of Streptomyces, strain Sr-63 was identified as Streptomyces roseoverticillatus.
(37) The Sr-63 strain was deposited, and the depository information is as follows: deposit name: Streptomyces roseoverticillatus Sr-63, depository institution: China Center for Type Culture Collection, depository address: Wuhan University, Wuhan, China, accession number CCTCC No.: M 2019261, deposit date Apr. 15, 2019.
Example 2
Determination of the Antibacterial Spectrum of the Fermentation Broth of S. Roseoverticillatus Sr-63
(38) 1. Strain
(39) (1) S. roseovarticulatus Sr-63 strain.
(40) (2) Four representative plant pathogenic fungi: Fusarium solani, Gibberella fujikuroi, Fusarium oxysporum f sp. momordicae and Alternaria solani were used to test the antimicrobial spectrum of the fermentation broth of Sr-63 strain against plant pathogenic fungi.
(41) (3) Four representative plant pathogenic bacteria: Xanthomonas oryzae pv. oryzicola, Xanthomonas campestris pv. glycines, Xanthomonas campestris pv. vesicatoria, and Pseudomonas suringae pv. glycinea were used to test the antimicrobial spectrum of the fermentation broth of Sr-63 strain against plant pathogenic bacteria.
(42) 2. Medium
(43) (1) PDA medium: potato 200 g, glucose 20 g, agar 20 g, water 1 L, pH 6.0˜6.5. It was used to culture four kinds of plant pathogenic fungi.
(44) (2) Beef extract peptone medium: beef extract 3 g, peptone 10 g, NaCl 5 g, with water added to 1000 ml, pH adjusted to 7.5 (beef extract peptone agar medium was added with 18 g agar powder). It was used for the culture of four plant pathogenic bacteria.
(45) (3) Gauze's Medium No. 1 was used for the fermentation seed culture of Streptomyces Sr-63.
(46) (4) Gauze's Medium No. 1 was used for the fermentation of Streptomyces Sr-63.
(47) 3 Experimental Method
(48) 3.1 Activation and Culture of Plant Pathogenic Fungi
(49) Four pathogenic fungi stored at 4° C. were inoculated in PDA medium and activated at 28° C. for 2 days. Then they were transferred to a new PDA medium and cultured at 28° C. for 4 days to determine the antifungal spectrum.
(50) 3.2 Activation and Culture of Plant Pathogenic Bacteria
(51) Four plant pathogenic bacteria stored at 4° C. were inoculated in beef extract peptone medium and activated at 28° C. for 2 days. Then they were transferred to a new beef extract peptone medium and cultured at 28° C. for 2 days to determine the antibacterial spectrum.
(52) 3.3 Determination of Antimicrobial Spectrum of Streptomyces Sr-63
(53) (1) Preparation of Fermentation Filtrate of Streptomyces Sr-63
(54) The S. roseoverticillatus (Sr-63) was inoculated into the medium plate of Gauze's Medium No. 1, and cultured in a 28° C. incubator for 4 days. A cake of S. roseoverticillatus (Sr-63) was taken from a 4 mm punch and inoculated into a 250 ml conical flask filled with 40 ml Gauze's Medium No. 1, it was shaken on a shaking table for 5 days (160 r/min, 28° C.). The fermentation solution was placed in a 50 ml centrifuge tube at 12000 r/min for 10 minutes. The supernatant was filtered by 0.22 μm organic filter membrane to remove the residual spores and obtain the fermentation filtrate for standby.
(55) (2) Determination of Antifungal Spectrum of Plant Pathogens
(56) The fermentation filtrate was mixed with PDA solid medium at 50° C. with a volume ratio of 1:9, then poured into the culture dish to obtain a plate containing the fermentation filtrate. A 6 mm diameter cake with 4 plant pathogenic fungi to be tested was placed in the center of the plate. It was cultured at 28° C. for 72 hours. The diameter of four plant pathogenic fungi was measured by cross method. Three groups of parallel repeats were conducted and the average value was taken. The inhibition rate of mycelium growth was calculated according to the following formula.
Mycelium growth inhibition rate %=(diameter of the control colony—diameter of the treated colony)/(diameter of the control colony −6)×100
(57) (3) Determination of Antibacterial Spectrum of Plant Pathogens
(58) Four plant pathogenic bacteria were inoculated into beef extract peptone medium respectively, and were activated on shaking table (180 r/min, 28° C.). When the bacterial density reached OD.sub.600 of 0.6, 100 μl bacterial solution was coated on the solid medium of beef extract peptone. The inhibition of the fermentation broth of 200 μL S. roseoverticillatus (Sr-63) on the four plant pathogenic bacteria was determined by the Oxford cup method.
(59) 4. Experimental Results
(60) 4.1 Inhibition of Streptomyces Sr-63 on Four Plant Pathogenic Fungi
(61) The results show that the fermentation broth of S. roseoverticillatus (Sr-63) has certain inhibitory effect on four plant pathogenic fungi. The mycelial growth inhibition rates of Fusarium solani, Gibberella fujikuroi, Fusarium oxysporum f.sp. momordicae and Alternaria solani respectively were 12.62%, 11.02%, 9.44% and 7.43% (Table 1).
(62) TABLE-US-00001 TABLE 1 Inhibitory effect of fermentation filtrate of S. roseoverticillatus (Sr-63) on four plant pathogenic fungi Mycelium growth inhibition Pathogenic fungi rate (%) Fusarium solani 12.62 ± 1.56 Gibberella fujikuroi 11.02 ± 1.35 Fusarium oxysporum f. sp. momordicae 9.44 ± 1.16 Alternaria solani 7.43 ± 0.89 note: Mycelium growth inhibition rate (%) = (diameter of the control colony − diameter of the treated colony)/(diameter of the control colony − 6) × 100
(63) 4.2 Inhibition of Fermentation Filtrate of S. Roseoverticillatus (Sr-63) on the Four Plant Pathogenic Bacteria
(64) The results show that the fermentation filtrate of S. roseoverticillatus (Sr-63) has some inhibition on four representative plant pathogenic bacteria, among which the inhibition on Xanthomonas oryzae pv. oryzicola was relatively good (Table 2), and diameter of bacteriostatic circle was about 28 mm.
(65) TABLE-US-00002 TABLE 2 Inhibitory effect of fermentation filtrate of S. roseoverticillatus (Sr-63) on the four plant pathogenic bacteria Diameter of bacteriostatic Plant pathogenic bacteria circle (mm) Xanthomonas oryzae pv. oryzicola 28.33 ± 3.96 Xanthomonas campestris pv. glycines 21.50 ± 2.81 Xanthomonas campestris pv. vesicatoria 18.50 ± 1.73 Pseudomonas syringae pv. glycinea 15.50 ± 1.47
Example 3
Study on the Control Effect of the Fermentation Liquid of S. Roseoverticillatus (Sr-63) on Rice Bacterial Blight
(66) 1. Pathogens and Rice Strains
(67) Pathogen: P6 race of Xanthomonas oryzae pv. oryzae.
(68) Rice strain: Zhonghua 11 rice strain susceptible to P6 race of Xanthomonas oryzae pv. oryzae.
(69) 2. Experimental Method
(70) 2.1 Preparation of Fermentation Filtrate of S. Roseoverticillatus (Sr-63): the Method is the Same as Eexample 2.
(71) 2.2 The Control Effect of Fermentation Filtrate of S. Roseoverticillatus (Sr-63) on Rice Bacterial Blight
(72) According to the methods in the references.sup.[2,3], the leaves of Zhonghua 11 rice line were treated by the fermentation filtrate of S. roseoverticillatus (Sr-63); the inoculation of the P6 race of Xanthomonas oryzae pv. oryzae suspension and the statistics of disease were according to the methods of references [4,5].
(73) (1) Preparation of P6 race of Xanthomonas oryzae pv. oryzae suspension: P6 race of Xanthomonas oryzae pv. oryzae suspension was inoculated on Xoo solid medium by plate scribing method and activated for 48 h; One ring of P6 race of Xanthomonas oryzae pv. oryzae suspension was inoculated into Xoo solid medium by scraping with inoculating ring and cultured on shaking bed at 28° C. and 180 r/min for 48 h. When the OD value reached 0.6, it was ready for use.
(74) (2) Method of inoculating P6 race of Xanthomonas oryzae pv. oryzae into rice leaves: when the rice grows to tillering stage, the rice leaf tip was horizontally cut off by 1.5 cm with a pair of scissors sterilized and dipped in P6 race of Xanthomonas oryzae pv. oryzae suspension.sup.[4,5].
(75) (3) Experimental design of controlling Rice Bacterial Blight: in the experiment, the distilled water blank control group (CK1), the P6 race of Xanthomonas oryzae pv. oryzae suspension treatment control group (CK2), method 1 treated group (treatment 1), method 2 treated group (treatment 2) and method 3 treated group (treatment 3) were set.
(76) a:CK1: the sterilized scissors were dipped in the distilled water, then the leaves were cut by the scissors, and the inoculated parts were kept moist.
(77) b:CK2: the sterilized scissors were dipped in the suspension of P6 race of Xanthomonas oryzae pv. oryzae, then the leaves were cut by the scissors, and the inoculated parts were kept moist.
(78) c:T1(treatment 1): the fermented filtrate of S. roseoverticillatus (Sr-63) was first sprayed with a watering can, according to the amount of 1 ml/cm.sup.2 fermented filtrate every 1 hour, three times in total. After 12 hours, P6 race of Xanthomonas oryzae pv. oryzae suspension was inoculated according to the method of control group (CK2), and the inoculated part was kept moist.
(79) d: T2(treatment 2): the leaves were cut and inoculated with P6 race of Xanthomonas oryzae pv. oryzae suspension, and 6 hours after the fermentation filtrate of S. roseoverticillatus (Sr-63) was sprayed; the method, the amount of fermentation filtrate and moisture retention were the same as treatment 1.
(80) e:T3(treatment 3): the leaves were cut and inoculated with P6 race of Xanthomonas oryzae pv. oryzae suspension, and 12 hours after the fermentation filtrate of S. roseoverticillatus (Sr-63) was sprayed; the method, the amount of fermentation filtrate and moisture retention were the same as treatment 1.
(81) After the occurrence and development of rice leaf lesions have stabilized, record and count the incidence. The lesion inhibition rate was calculated.
Lesion inhibition rate (%)=(lesion length of the CK2—lesion length of the treated)/lesion length of the CK2×100
(82) 3. Experimental Results of Control Effect of Different Treatments on Rice Bacterial Blight
(83) The lengths of Rice Bacterial Blight lesions measured by different treatment methods are shown in
(84) The results show that the fermentation filtrate of S. roseoverticillatus (Sr-63) has a significant effect on the control of Rice Bacterial Blight in Zhonghua 11 rice strain susceptible to P6 race of Xanthomonas oryzae pv. oryzae. The earlier spraying, the better the effect of disease prevention.
(85) The following references, which are mentioned above, are incorporated by reference herein in their entirety:
(86) [1] Arshad H M I, Saleem K, Khan J A, et al. Pathogenic diversity of Xanthomonas oryzae pv. oryzae isolates collected form Punjab Province of Pakistan [J]. European Journal of Plant Pathology, 2017, 147(3): 639-651. (Arshad H M I, Saleem K, Khan J A, et al.;
(87) [2] Liu Qinying, Xie Xiangcong, QiYuping, et al. Screening & Identification of Endophytic Fungus against Rice White Leaf Blight Xanthomonas oryzae pv. oryzae [J]. Journal of Microbiology, 2013, 33(4): 4-8;
(88) [3] Tian Xiaowei, Long Jianyou, Bai Hongjin, et al. Studies on Fungicidal Activity of Metabolic Products of Actinomycetes[J]. Plant Protection, 2004, 30(2): 51-54;
(89) [4] Zhao Xianfeng, Zhai Wenxue, Li Ping, et al. Field Tests and Analyses of Different Xa21-transgenic Hybrid Rice Combinations[J]. Acta Agronomica Sinica, 2002, 28(4): 521-527; and
(90) [5] Liu Yongfeng, Lu Fan, Chen Zhiyi, et al. Analysis on the Resistance of Rice Varieties (lines) to Rice Bacterial Leaf Blight in Jiangsu Province[J]. Plant Protection, 2001, 5: 3-6.
(91) Contrast
(92) The rest of Streptomyces roseoverticillatus obtained in the process of the application is detected according to the method described in treatment 1 of example 3 above, and the results are compared with that of S. roseoverticillatus (Sr-63) of the application, as shown in Table 3 below:
(93) TABLE-US-00003 TABLE 3 Strain Source Lesion length (mm) Sr-63 Screening by the 5.4 application Sr-4 Screening by the 40.8 application Sr-38 Screening by the 55.2 application
(94) Finally, it should be noted that the above listed are only a few of specific embodiments of the present application. Obviously, the present application is not limited to the above embodiments, and there can be many variations. All variations that can be directly derived from or associated with the contents of the disclosure by those skilled in the art shall be considered as the protection scope of the present invention.