CN103205389A - Streptomyces flavotricini for antagonizing botryosphaeria dothidea - Google Patents
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- 241000936770 Streptomyces flavotricini Species 0.000 title claims abstract description 10
- 230000003042 antagnostic effect Effects 0.000 title abstract description 3
- 241000555706 Botryosphaeria dothidea Species 0.000 title 1
- 244000132436 Myrica rubra Species 0.000 claims abstract description 11
- 235000008375 Decussocarpus nagi Nutrition 0.000 claims abstract description 10
- 235000014631 Myrica rubra Nutrition 0.000 claims abstract description 10
- 235000009134 Myrica cerifera Nutrition 0.000 claims abstract description 9
- 244000061457 Solanum nigrum Species 0.000 claims abstract description 9
- 241000894006 Bacteria Species 0.000 claims abstract description 6
- 238000004321 preservation Methods 0.000 claims abstract description 4
- 230000001580 bacterial effect Effects 0.000 claims description 5
- 230000002265 prevention Effects 0.000 claims description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 abstract description 10
- 201000010099 disease Diseases 0.000 abstract description 8
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 8
- 241000196324 Embryophyta Species 0.000 abstract description 6
- 239000013043 chemical agent Substances 0.000 abstract description 5
- 229960005322 streptomycin Drugs 0.000 abstract description 5
- 230000000694 effects Effects 0.000 abstract description 4
- 238000003912 environmental pollution Methods 0.000 abstract description 2
- 239000000447 pesticide residue Substances 0.000 abstract description 2
- 238000011160 research Methods 0.000 abstract description 2
- 241000219433 Myrica Species 0.000 abstract 1
- 241000187747 Streptomyces Species 0.000 description 7
- 229920001817 Agar Polymers 0.000 description 5
- 239000008272 agar Substances 0.000 description 5
- 239000002609 medium Substances 0.000 description 5
- 238000000034 method Methods 0.000 description 5
- 244000005700 microbiome Species 0.000 description 5
- 108020004465 16S ribosomal RNA Proteins 0.000 description 4
- 230000000443 biocontrol Effects 0.000 description 4
- 230000002401 inhibitory effect Effects 0.000 description 4
- 230000005764 inhibitory process Effects 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 239000000575 pesticide Substances 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 239000002689 soil Substances 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 241000186361 Actinobacteria <class> Species 0.000 description 2
- 101000610620 Homo sapiens Putative serine protease 29 Proteins 0.000 description 2
- 102100040345 Putative serine protease 29 Human genes 0.000 description 2
- 101100397225 Schizosaccharomyces pombe (strain 972 / ATCC 24843) isp3 gene Proteins 0.000 description 2
- 101100397226 Schizosaccharomyces pombe (strain 972 / ATCC 24843) isp4 gene Proteins 0.000 description 2
- 238000012300 Sequence Analysis Methods 0.000 description 2
- 244000052616 bacterial pathogen Species 0.000 description 2
- 230000000853 biopesticidal effect Effects 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
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- 230000000877 morphologic effect Effects 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 241000233866 Fungi Species 0.000 description 1
- 238000003794 Gram staining Methods 0.000 description 1
- 241000624150 Pseudomonas amygdali pv. myricae Species 0.000 description 1
- 101100397227 Schizosaccharomyces pombe (strain 972 / ATCC 24843) isp5 gene Proteins 0.000 description 1
- 238000012271 agricultural production Methods 0.000 description 1
- 230000008485 antagonism Effects 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000007613 bennett's agar Substances 0.000 description 1
- 229960000074 biopharmaceutical Drugs 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 239000006343 isp 5 medium Substances 0.000 description 1
- 238000009629 microbiological culture Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
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- 238000012360 testing method Methods 0.000 description 1
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- 241001446247 uncultured actinomycete Species 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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Abstract
本发明涉及植物病害的生物防治，属于植物保护学科中的生物防治研究与应用领域。本发明公开了一株拮抗杨梅溃疡病菌黄三素链霉菌（Streptomyces flavotricini）YZF3，它的保藏编号为CGMCC No.7529。该菌株能有效抑制杨梅溃疡病菌，效果明显优于叶枯唑，与农用链霉素相当，有防治杨梅溃疡病的潜力。与化学药剂相比，使用YZF3菌株防治植物病害没有环境污染、农药残留等问题。
The invention relates to biological control of plant diseases and belongs to the field of biological control research and application in the plant protection discipline. The invention discloses a strain of Streptomyces flavotricini (Streptomyces flavotricini) YZF3 antagonizing myrica rubra canker, and its preservation number is CGMCC No.7529. The strain can effectively inhibit the myrica canker bacterium, and the effect is obviously better than that of phylloxazole, which is equivalent to that of the agricultural streptomycin, and has the potential of preventing and treating the bayberry canker. Compared with chemical agents, the use of YZF3 strains to prevent and control plant diseases has no problems such as environmental pollution and pesticide residues.
Description
技术领域technical field
本发明涉及植物病害的生物防治，属于植物保护学科中的生物防治研究与应用领域。The invention relates to biological control of plant diseases and belongs to the field of biological control research and application in the discipline of plant protection.
背景技术Background technique
杨梅溃疡病是许多杨梅种植区的重要病害，极易造成树势衰弱，影响杨梅产量和品质。目前防治该病害的方法主要是利用化学药剂叶枯唑和农用链霉素涂抹，其他药剂防效均不够理想。从病害防治角度，仅两种药剂用于病害防治不够丰富，还需开发其它有效药剂供农业生产选择使用。为了给研制新型生物农药提供基础，丰富防治杨梅溃疡病的药剂种类，本发明中从土壤中筛选了一株放线菌YZF3菌株，其能有效抑制杨梅溃疡病菌（Pseudomonassyringae pv.myricae）生长。经鉴定该生防菌为黄三素链霉菌（Streptomyces flavotricini）。该菌株对杨梅溃疡病菌的抑制效果与农用链霉素相当，明显优于化学药剂叶枯唑。因此可以用黄三素链霉菌YZF3研发新型生物农药，用于杨梅溃疡病的生物防治。Red bayberry canker is an important disease in many bayberry planting areas, which can easily cause tree vigor to weaken and affect the yield and quality of bayberry. At present, the method of preventing and controlling the disease is mainly to use the chemical agents Yebuzol and agricultural streptomycin to smear, and the control effects of other agents are not ideal. From the perspective of disease control, only two pesticides are not enough for disease control, and other effective pesticides need to be developed for agricultural production. In order to provide a basis for the development of new biopesticides and enrich the types of agents for preventing and treating bayberry canker, an actinomycete YZF3 strain was screened from soil in the present invention, which can effectively inhibit the growth of bayberry canker (Pseudomonassyringae pv.myricae). The biocontrol bacteria was identified as Streptomyces flavotricini. The inhibitory effect of the strain on Myrica rubra canker is equal to that of agricultural streptomycin, and obviously better than that of the chemical agent eubazole. Therefore, Streptomyces flavitinus YZF3 can be used to develop new biopesticides for the biological control of bayberry canker.
发明内容Contents of the invention
针对防治杨梅溃疡病的药剂种类少问题，筛选了生防微生物黄三素链霉菌YZF3。该菌株对杨梅溃疡病菌的抑制效果与农用链霉素相当，优于化学药剂叶枯唑。目前已完成对病菌的抑制效果测定和种类鉴定。Aiming at the problem that there are few types of pesticides for the prevention and treatment of bayberry canker, the biocontrol microorganism Streptomyces aflavitinus YZF3 was screened. The inhibitory effect of the strain on Myrica rubra canker is equal to that of streptomycin used in agriculture, and better than that of the chemical agent, eclopyrazole. At present, the determination of the inhibitory effect on the pathogen and the identification of the species have been completed.
自然界蕴含着许多有益微生物，从环境中大量筛选，得到生防微生物是生物药剂研制的基础工作，也是业内惯用的工作模式。放线菌是一类产生抗生素的菌类，以杨梅溃疡病菌为目标细菌，从土壤中筛选能抑制该细菌的放线菌，是一种效率较高的筛选生防微生物的方法。There are many beneficial microorganisms in nature. Screening a large number of microorganisms from the environment to obtain biocontrol microorganisms is the basic work of the development of biopharmaceuticals, and it is also the usual working mode in the industry. Actinomycetes are a type of fungi that produce antibiotics. Taking myrica rubra as the target bacteria, screening the actinomycetes that can inhibit the bacteria from the soil is a method of screening biocontrol microorganisms with high efficiency.
本发明公开了一种拮抗杨梅溃疡病菌黄三素链霉菌（Streptomyces flavotricini）YZF3，它的保藏编号为CGMCC No.7529。The invention discloses YZF3 which antagonizes bayberry canker pathogen Streptomyces flavotricini, and its preservation number is CGMCC No.7529.
黄三素链霉菌YZF3菌株能有效抑制杨梅溃疡病菌，效果明显优于叶枯唑，与农用链霉素相当，有防治杨梅溃疡病的潜力。与化学药剂相比，使用YZF3菌株防治植物病害没有环境污染、农药残留等问题。Streptomyces aflavoyces YZF3 strain can effectively inhibit Myrica rubra canker, the effect is obviously better than that of Yebuazole, equivalent to the agricultural streptomycin, and has the potential to prevent and control Myrica rubra canker. Compared with chemical agents, the use of YZF3 strains to prevent and control plant diseases has no problems such as environmental pollution and pesticide residues.
附图说明图1是黄三素链霉菌YZF3菌株对杨梅溃疡病菌的拮抗效果。BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is the antagonism effect of Streptomyces flavitinus YZF3 strain on Myrica rubra.
图2是YZF3菌株的菌体形态（×1000）。Figure 2 is the cell morphology of YZF3 strain (×1000).
本发明所述YZF3菌株于2013年4月28日保存于中国微生物菌种保藏管理委员会普通微生物中心，（北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所），分类命名为黄三素链霉菌（Streptomyces flavotricini），保藏编号为CGMCC7529。The YZF3 strain described in the present invention was preserved on April 28, 2013 in the General Microorganism Center of China Microbiological Culture Collection Management Committee (No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences), and the classification is named Streptomyces flavotricini (Streptomyces flavotricini), the preservation number is CGMCC7529.
具体实施方式Detailed ways
（1）采集土样，去除石子等大颗粒物质，碾碎风干，过筛。(1) Collect soil samples, remove large particles such as stones, crush and air-dry, and sieve.
（2）20g样品放置于200mL无菌水中，充分震荡，以此液为母液，梯度稀释。(2) Place 20g of the sample in 200mL of sterile water, shake it fully, and use this solution as the mother solution for gradient dilution.
（3）取10-6稀释液0.1mL涂布高氏一号平板。(3) Take 0.1mL of the 10 -6 dilution solution and spread it on the No. 1 Gaoshi plate.
（4）28℃条件下培养7天。(4) Culture at 28°C for 7 days.
（5）分离不同颜色、性状的单菌落，保存。(5) Separate and save single colonies of different colors and traits.
（6）将杨梅溃疡病菌（菌株由本实验室从慈溪市杨梅园病株上分离获得，经形态和分子生物学鉴定为杨梅溃疡病菌）与30℃的细菌培养基混匀，冷却后制成平板，在平板上打孔，挖去琼脂块，在孔中滴入分离菌株的菌悬液20μL，28℃培养，观察抑菌圈大小。（7）保留有抑菌圈的菌株，进行单菌落纯化。(6) Mix Myrica rubra (the strain was isolated from the diseased strains in Cixi Yangmeiyuan in our laboratory and identified as Myrica rubra by morphology and molecular biology) with the bacterial culture medium at 30°C, and make a plate after cooling. Make a hole on the plate, dig out the agar block, drop 20 μL of the bacterial suspension of the isolated strain into the hole, incubate at 28°C, and observe the size of the inhibition zone. (7) Purify the strains with the inhibition zone for single colony.
（8）连续转代多代，用（6）方法再测菌株的抑菌圈，确定拮抗稳定性。筛筛选出的菌株命名为YZF3。如图1。图中培养皿中对照孔（左下孔）周围病菌已长满，其它3个孔滴加了YZF3菌液，在其周围形成了明显的空白圈即抑菌圈，说明YZF3菌株发酵过程中能产生抑制物质使周围的病菌不能生长。(8) Continuously transfer to multiple generations, and use the method (6) to test the inhibition zone of the strain again to determine the antagonistic stability. The strain screened out was named YZF3. Figure 1. In the picture, the control well (lower left well) in the petri dish is covered with pathogenic bacteria, and the other three wells are dripped with YZF3 bacterial solution, forming an obvious blank circle around it, which is the inhibition zone, indicating that the YZF3 strain can produce bacteria during the fermentation process. Inhibiting substances prevent surrounding germs from growing.
（9）对YZF3菌株进行种类鉴定。(9) Species identification of YZF3 strain.
（10）采用生理生化（形态特征、培养特征和生理生化特征）和分子生物学方法（16S rRNA基因序列分析）进行菌株种类鉴定。(10) Physiological and biochemical (morphological characteristics, cultural characteristics, and physiological and biochemical characteristics) and molecular biology methods (16S rRNA gene sequence analysis) were used to identify strains.
形态特征：革兰氏染色阳性；在ISP2琼脂、ISP3琼脂、ISP4琼脂、ISP5琼脂及Bennett’s琼脂培养基上生长7天后，基内菌丝发育良好，无横隔，不断裂；气生菌丝生长良好、多分支；孢子丝直、柔曲，孢子圆形或卵圆形、光滑（图2）。Morphological characteristics: Gram staining is positive; after 7 days of growth on ISP2 agar, ISP3 agar, ISP4 agar, ISP5 agar and Bennett's agar medium, the hyphae in the substrate are well developed, without septa, and do not break; aerial hyphae grow Good, multi-branched; spore filaments are straight and flexible, and the spores are round or oval and smooth (Figure 2).
培养特征：菌株YZF3的培养特征见表1：Culture characteristics: The culture characteristics of strain YZF3 are shown in Table 1:
表1菌株YZF3的培养特征Table 1 Culture characteristics of bacterial strain YZF3
生理生化特征：菌株的生理生化特征见表2。Physiological and biochemical characteristics: See Table 2 for the physiological and biochemical characteristics of the strains.
表2菌株YZF3的生理生化特征Table 2 Physiological and biochemical characteristics of strain YZF3
+，阳性；-，阴性+, positive; -, negative
以上特征与链霉菌一致The above characteristics are consistent with Streptomyces
16S rRNA基因序列分析：提取YZF3全基因组，用16S rRNA通用引物扩增，扩增的基因序列测序后在GenBank中进行Blast比较，结果表明菌株YZF3属于链霉菌属；与目前发表的相关菌株相比，菌株YZF3的16S rRNA基因序列与黄三素链霉菌（Streptomyces flavotricini）的序列相似性很高，为99.8%。YZF3菌株为黄三素链霉菌。16S rRNA gene sequence analysis: the whole genome of YZF3 was extracted, amplified with 16S rRNA universal primers, and the amplified gene sequence was sequenced and compared with Blast in GenBank. The results showed that the strain YZF3 belonged to the genus Streptomyces; compared with the related strains published so far , the 16S rRNA gene sequence of strain YZF3 had a high sequence similarity of 99.8% to that of Streptomyces flavotricini. YZF3 strain is Streptomyces aflavitine.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103966133A (en) * | 2014-05-13 | 2014-08-06 | 北京林业大学 | Streptomyces and application thereof |
| CN107058178A (en) * | 2017-03-13 | 2017-08-18 | 陕西博秦生物工程有限公司 | A kind of plain streptomycete of Huang three and microbial inoculum and its application |
| CN110093295A (en) * | 2019-05-15 | 2019-08-06 | 湖南师范大学 | The Huang Sansu streptomycete of one plant of anti-fish-pathogenic bacteria and its application |
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| US4361650A (en) * | 1978-03-24 | 1982-11-30 | Takeda Chemical Industries, Ltd. | Fermentation process of preparing demethyl maytansinoids |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103966133A (en) * | 2014-05-13 | 2014-08-06 | 北京林业大学 | Streptomyces and application thereof |
| CN107058178A (en) * | 2017-03-13 | 2017-08-18 | 陕西博秦生物工程有限公司 | A kind of plain streptomycete of Huang three and microbial inoculum and its application |
| CN107058178B (en) * | 2017-03-13 | 2020-08-04 | 陕西博秦生物工程有限公司 | Streptomyces flavetris, microbial inoculum and application thereof |
| CN110093295A (en) * | 2019-05-15 | 2019-08-06 | 湖南师范大学 | The Huang Sansu streptomycete of one plant of anti-fish-pathogenic bacteria and its application |
| CN110093295B (en) * | 2019-05-15 | 2022-08-30 | 湖南师范大学 | Streptomyces flaviviridis for resisting fish pathogenic bacteria and application thereof |
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