CN117305114B - 一株联合芽孢杆菌防控番茄土传青枯病的丝足虫及其应用 - Google Patents
一株联合芽孢杆菌防控番茄土传青枯病的丝足虫及其应用 Download PDFInfo
- Publication number
- CN117305114B CN117305114B CN202311595366.3A CN202311595366A CN117305114B CN 117305114 B CN117305114 B CN 117305114B CN 202311595366 A CN202311595366 A CN 202311595366A CN 117305114 B CN117305114 B CN 117305114B
- Authority
- CN
- China
- Prior art keywords
- njau
- bacterial wilt
- heteromita
- globosa
- culture
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 230000001580 bacterial effect Effects 0.000 title claims abstract description 50
- 235000007688 Lycopersicon esculentum Nutrition 0.000 title claims abstract description 30
- 241000193830 Bacillus <bacterium> Species 0.000 title claims abstract description 13
- 240000003768 Solanum lycopersicum Species 0.000 title claims description 27
- 241000255789 Bombyx mori Species 0.000 claims abstract description 15
- 238000004321 preservation Methods 0.000 claims abstract description 12
- 241000589771 Ralstonia solanacearum Species 0.000 claims description 12
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 4
- 238000012258 culturing Methods 0.000 claims description 4
- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 4
- QHFQAJHNDKBRBO-UHFFFAOYSA-L calcium chloride hexahydrate Chemical compound O.O.O.O.O.O.[Cl-].[Cl-].[Ca+2] QHFQAJHNDKBRBO-UHFFFAOYSA-L 0.000 claims description 2
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 claims description 2
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 claims description 2
- 229910000160 potassium phosphate Inorganic materials 0.000 claims description 2
- 235000011009 potassium phosphates Nutrition 0.000 claims description 2
- 239000011780 sodium chloride Substances 0.000 claims description 2
- 239000001488 sodium phosphate Substances 0.000 claims description 2
- 229910000162 sodium phosphate Inorganic materials 0.000 claims description 2
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 claims description 2
- 241000305071 Enterobacterales Species 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 claims 1
- 238000002474 experimental method Methods 0.000 abstract description 11
- 230000000694 effects Effects 0.000 abstract description 9
- 230000000443 biocontrol Effects 0.000 abstract description 7
- 230000012010 growth Effects 0.000 abstract description 7
- 244000052616 bacterial pathogen Species 0.000 abstract description 6
- 230000005764 inhibitory process Effects 0.000 abstract description 2
- 241000227653 Lycopersicon Species 0.000 abstract 5
- 239000007788 liquid Substances 0.000 description 22
- 241000196324 Embryophyta Species 0.000 description 15
- 239000002689 soil Substances 0.000 description 13
- 238000003501 co-culture Methods 0.000 description 11
- 201000010099 disease Diseases 0.000 description 10
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 10
- 230000002265 prevention Effects 0.000 description 9
- 239000000872 buffer Substances 0.000 description 7
- 239000000126 substance Substances 0.000 description 7
- 238000002360 preparation method Methods 0.000 description 6
- 108090000623 proteins and genes Proteins 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 238000010586 diagram Methods 0.000 description 5
- 238000000034 method Methods 0.000 description 5
- 244000005700 microbiome Species 0.000 description 5
- 235000016709 nutrition Nutrition 0.000 description 5
- 230000001717 pathogenic effect Effects 0.000 description 5
- 108020004463 18S ribosomal RNA Proteins 0.000 description 4
- 241001067900 Bacillus velezensis SQR9 Species 0.000 description 4
- 241000813090 Rhizoctonia solani Species 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 3
- 241000588724 Escherichia coli Species 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- 230000003042 antagnostic effect Effects 0.000 description 3
- 229960005091 chloramphenicol Drugs 0.000 description 3
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 description 3
- 238000000576 coating method Methods 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 230000000877 morphologic effect Effects 0.000 description 3
- 230000035764 nutrition Effects 0.000 description 3
- 244000052769 pathogen Species 0.000 description 3
- 239000000575 pesticide Substances 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 241000563903 Bacillus velezensis Species 0.000 description 2
- 241000233866 Fungi Species 0.000 description 2
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 description 2
- 229930182566 Gentamicin Natural products 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 230000001651 autotrophic effect Effects 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 239000013043 chemical agent Substances 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 238000005538 encapsulation Methods 0.000 description 2
- 238000003912 environmental pollution Methods 0.000 description 2
- 230000005284 excitation Effects 0.000 description 2
- 210000003495 flagella Anatomy 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 229960002518 gentamicin Drugs 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000000242 pagocytic effect Effects 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 235000013311 vegetables Nutrition 0.000 description 2
- 241000224489 Amoeba Species 0.000 description 1
- 229930192334 Auxin Natural products 0.000 description 1
- 241000223782 Ciliophora Species 0.000 description 1
- 241000199914 Dinophyceae Species 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 201000006353 Filariasis Diseases 0.000 description 1
- 108050002220 Green fluorescent protein, GFP Proteins 0.000 description 1
- 241000190182 Heteromita globosa Species 0.000 description 1
- 241001671311 Laurus Species 0.000 description 1
- 240000008790 Musa x paradisiaca Species 0.000 description 1
- 208000031888 Mycoses Diseases 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 244000061456 Solanum tuberosum Species 0.000 description 1
- 235000002595 Solanum tuberosum Nutrition 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 230000008485 antagonism Effects 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 239000002363 auxin Substances 0.000 description 1
- 244000000005 bacterial plant pathogen Species 0.000 description 1
- 235000021015 bananas Nutrition 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 230000037237 body shape Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000011217 control strategy Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000003967 crop rotation Methods 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 230000001066 destructive effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 239000003337 fertilizer Substances 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 238000001215 fluorescent labelling Methods 0.000 description 1
- 239000005090 green fluorescent protein Substances 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- SEOVTRFCIGRIMH-UHFFFAOYSA-N indole-3-acetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CNC2=C1 SEOVTRFCIGRIMH-UHFFFAOYSA-N 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000009335 monocropping Methods 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 239000003895 organic fertilizer Substances 0.000 description 1
- 230000000243 photosynthetic effect Effects 0.000 description 1
- 238000013081 phylogenetic analysis Methods 0.000 description 1
- 230000008635 plant growth Effects 0.000 description 1
- 235000012015 potatoes Nutrition 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000000644 propagated effect Effects 0.000 description 1
- 108010054624 red fluorescent protein Proteins 0.000 description 1
- 210000003705 ribosome Anatomy 0.000 description 1
- 238000007480 sanger sequencing Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000003068 static effect Effects 0.000 description 1
- 238000005728 strengthening Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/10—Protozoa; Culture media therefor
- C12N1/105—Protozoal isolates
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
- A01N63/22—Bacillus
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P3/00—Fungicides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/10—Protozoa; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/90—Protozoa ; Processes using protozoa
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Virology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Plant Pathology (AREA)
- Environmental Sciences (AREA)
- Pest Control & Pesticides (AREA)
- Tropical Medicine & Parasitology (AREA)
- General Engineering & Computer Science (AREA)
- Agronomy & Crop Science (AREA)
- Biochemistry (AREA)
- Biomedical Technology (AREA)
- Dentistry (AREA)
- Medicinal Chemistry (AREA)
- Mycology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
本发明提供一株联合芽孢杆菌防控番茄土传青枯病的丝足虫及其应用。丝足虫NJAU‑CY1，保藏编号为CCTCC NO:C2023270。通过试验发现原生动物丝足虫NJAU‑CY1展现出对番茄青枯病较好的防治效果，其不仅能直接抑制番茄青枯病原菌的生长，还能够与生防菌芽孢杆菌联合使用，增强后者对番茄青枯病病原菌的抑制效果，表明其在番茄青枯病生物防治方面具有巨大的应用潜力。
Description
技术领域
本发明属于微生物学和植物病害生防技术领域，具体涉及一株联合芽孢杆菌防控青枯病的丝足虫Heteromita globosa及其应用。
背景技术
原生生物是指环境中除植物、真菌和动物外的单细胞真核微生物。根据其营养类型，原生生物主要可以分为四类，分别是吞噬型原生生物、自养型原生生物、共生型原生动物和腐生型原生生物。原生生物中具有吞噬功能的类群为原生动物，这一类群主要就是通过捕食细菌、真菌等微生物获得营养进行生长、繁殖。原生动物广泛地存在于地球的各种生态环境中，特别是在土壤生态系统中，原生生物具有极高的数量和丰富度，是土壤微食物网的重要组成成员。根据其结构形态，土壤中的原生动物可被分为三大类，分别是变形虫、纤毛虫和鞭毛虫。这其中鞭毛虫是使用鞭毛进行运动、捕食的原生动物。从营养方式来区分，鞭毛虫包括营光合作用的植物性鞭毛藻（包括自养与混合营养类型）和无光合作用能力的异养鞭毛虫。本发明分离、培养的丝足虫Heteromita globosa属于异养鞭毛虫。
番茄是我国种植面积排名第四的蔬菜品种，番茄产业已成为我国蔬菜产业的重要组成部分。番茄青枯病是由茄科劳尔氏菌（Ralstonia solanacearum）引起的毁灭性土传病害。R. solanacearum通过植株伤口或根尖裂缝进入植株后定殖于皮层组织，随后入侵维管束、阻碍植物水分的运输，最终导致植株枯萎、死亡。这类植物病原菌宿主范围广，能够侵染包括番茄、马铃薯和香蕉等主要农作物在内的超过200种植物，此外其在土壤中可存活多年，难以根除。番茄青枯病可造成田间番茄植株的大面积萎蔫死亡，一般田块发病率在10%~15%，重病田发病率高达80%~100%，导致番茄产量严重下降乃至绝产，进而造成重大经济损失。目前在我国番茄主要种植区均有番茄青枯病爆发的报道，严重威胁番茄产业。
目前针对青枯病的防控策略主要包括选用抗逆品种、轮作嫁接、加强田间管理、化学防治、生物防治等措施，其中化学防治为主要措施。但是针对番茄青枯病缺少有效的化学药剂，且长期使用化学农药容易使病原菌产生耐药性，导致药剂效果大大降低。此外，长期施用化学农药也会对生态环境造成污染，引发食品安全等一系列问题。针对番茄青枯病的抗病育种工作，则由于缺乏理想的抗源材料以及R. solanacearum致病菌株的分化和变异的广泛存在，进展缓慢。轮作防控青枯病需要较长的年限，且需要附近无其他R. solanacearum宿主植物的存在才可起到良好效果。鉴于以上问题，利用有益微生物来防治番茄青枯病的生物防治措施逐渐受到农业工作者的重视。
生物防治与其他方法相比，具有安全、有效、环保的特点。生防菌可以分泌抗菌物质来或通过营养竞争等方式抑制病原菌的生长，也可以产生物质如生长素等来促进植物生长，或者诱导植物的免疫应答，间接减轻病害的发生。由生防菌制备成的生防制剂对病害防治效果好，无残留不污染环境且对人畜安全。因此，为了降低番茄青枯病的发生，有必要针对该病害进行生防微生物的筛选，充分挖掘和利用根际有益微生物，将有助于减少化肥和农药的投入，减轻环境污染，实现可持续发展。
发明内容
为了解决现有技术中存在的问题，本发明提供了一株防控番茄青枯病的丝足虫。本发明所涉及的丝足虫NJAU-CY11是从江苏省南京市六合区青枯病自然发病的试验田块健康番茄植株根际土壤中分离、筛选得到的一株对青枯病原菌具有极强捕食作用的丝足虫。根据其包囊和活体形态特征、18S rDNA基因序列分析，鉴定为Heteromita globosa，并将其于2023年保藏至中国典型培养物保藏中心（地址：湖北省武汉市武昌区武汉大学保藏中心，保藏编号为CCTCC NO:C2023270）。本发明研究了该丝足虫对青枯病的防治效果，为青枯病的生物防治及生防菌剂的开发利用提供科学依据。
一株防控番茄青枯病的丝足虫Heteromita globosa NJAU-CY1，所述丝足虫保藏于中国典型培养物保藏中心，保藏地址为:中国武汉武汉大学，保藏日期为2023年8月28日，其保藏编号为CCTCC NO:C2023270。
含有本发明所述的的丝足虫Heteromita globosa NJAU-CY1的培养物。
本发明所述的丝足虫Heteromita globosa NJAU-CY1培养物的制备方法，将所述的丝足虫Heteromita globosa NJAU-CY1接种于NMAS液体培养液中，添加灭活的大肠杆菌，置于20-22℃的培养箱中，静置45-50h。
作为本发明的一种优选，所述NMAS培养液的组成为，氯化钠0.12 g/L，七水硫酸镁0.0004 g/L,六水氯化钙0.0006 g/L,磷酸钠0.142 g/L，磷酸钾0.136 g/L。
本发明所述的丝足虫丝足虫Heteromita globosa NJAU-CY1或所述的培养物在制备防治植物真菌性病害中的应用。
作为本发明的一种优选，所述植物病害的病原菌青枯菌。
作为本发明的一种优选，所述植物为番茄。
有益效果
本发明从番茄青枯病发生地块的健康植株根际分离筛选出对青枯病有极强抗性的丝足虫Heteromita globosa，细菌共培养试验表明，NJAU-CY1对青枯病具有显著的防治作用。本发明的丝足虫Heteromita globosa对连作土壤的修复及土传病害的防治具有重要意义。
附图说明
图1为本发明丝足虫Heteromita globosa NJAU-CY1休眠包囊（左）和营养体（右）形态学特征照片图。
图2为本发明原生动物NJAU-CY1的18S rDNA基因序列系统发育树。
图3为本发明实施例3中丝足虫Heteromita globosa NJAU-CY1和R. solanacearum（图中简写为RS，下同）二者液体共培养，原生动物随时间的动态变化图。
图4为本发明实施例3中丝足虫Heteromita globosa NJAU-CY1和R. solanacearum二者液体共培养，R. solanacearum随时间的动态变化图。
图5为本发明实施例3中丝足虫Heteromita globosaNJAU-CY1和R. solanacearum二者液体共培养，4d后，稀释涂布计算R. solanacearum数量的结果。
图6为本发明实施例3中丝足虫Heteromita globosa NJAU-CY1、R. solanacearum、 Bacillus velezensis SQR9 三者液体共培养实验，原生动物随时间的动态变化图。
图7为本发明实施例3中丝足虫Heteromita globosa NJAU-CY1、R. solanacearum、Bacillus velezensis SQR9三者液体共培养实验，R. solanacearum随时间的动态变化图。
图8本发明实施例中丝足虫Heteromita globosa NJAU-CY1、 R. solanacearum、Bacillus velezensis SQR9三者液体共培养实验， SQR9随时间的动态变化图。
生物材料保藏信息
丝足虫 NJAU-CY1，分类命名为Heteromita globosa NJAU-CY1，保藏于中国典型培养物保藏中心，保藏地址为:武汉大学，保藏日期为2023年8月28日，其保藏编号为CCTCCNO:C2023270。
具体实施方式
以下的实施例便于更好地理解本发明，但并不限定本发明。下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的试验材料，如无特殊说明，均购自常规生化试剂公司。以下实施例中的试验，均设置三次重复实验。
实施例1防控番茄青枯病丝足虫Heteromita globosa的分离和纯化:
本申请发明人从江苏省南京市六合区的番茄青枯病发病田块中采集健康植株的根际土壤，采集的深度范围为10-20cm。供试靶标病原菌为青枯菌劳尔氏菌（R.solanacearum），由南京农业大学土壤有机肥团队提供。细菌培养基为NB （NutrientBroth）。
采用土壤稀释法分离原生动物，随后采用液体共培养法测定原生动物对番茄青枯菌的拮抗作用。具体方法如下：将健康番茄根际土壤样品混匀，称取1g土壤加入50 mL的离心管中并添加30 mL的无菌去离子水，随后将离心管放入250 rpm、20 ℃的摇床中振荡15min，使得根际土充分混匀并释放土壤中的原生动物。将离心管从摇床中取出并静置10min 后，取上层液体加入96孔板中并添加大肠杆菌（OD=0.04）作为食物，随后在20℃避光培养条件下培养2d。 在倒置显微镜100×、200×和400×下镜检原生动物的生长情况并进行梯度稀释并在20℃下继续培养2d。 最后用毛细血管挑取单个原生动物细胞到新的96孔板中得到原生动物的纯培养。
实施例2 青枯病拮抗原生动物NJAU-CY1的鉴定:
采用形态学观察与分子生物学相结合的方法，对筛选到的原生动物进行鉴定。PCR反应体系（50 μL体系）：引物各2 μL，2× Mix 25 μL，加ddH2O补充到50 μL。引物为最常用的通用引物为25F/1256R。（25F：5’CATATGCTTGTCTCAAAGATTAAGCCA--3’）和（1256R：5’GCACCACCACCCAYAGAATCAAGAAAGAWC--3’）。反应程序：94℃预变性3min，94℃变性55s，50℃退火50s，72℃延伸1min，72℃延伸10min，35个循环后16℃保存。PCR产物于1.5%琼脂糖凝胶电泳上检测回收后，送至北京擎科生物科技有限公司进行Sanger测序，测序结果如SEQ IDNO.1所示。
在NMAS培养液中，原生动物丝足虫呈现活跃状态，上下游动， 其包囊状态是圆形，呈静止状态。图1为本发明原生动物NJAU-CY1形态学特征照片图。
使用BLAST软件，将所获得18s rDNA基因序列比对PR2（Protist RibosomalReference）数据库和NCBI网站的NT(Nucleotide Sequence Database) 数据库，利用MEGA软件对分离的原生动物进行系统发育分析。丝足虫NJAU-CY1测序结果与模式原生动物丝足虫Heteromita globosa相似性最高，达98%，因此将该分离得到的原生动物NJAU-CY1鉴定为Heteromita globosa。图2为基于18S rDNA基因序列构建的系统发育树。
将该丝足虫于2023年8月28日保藏至中国典型培养物保藏中心，保藏编号为：CCTCC NO:C2023270。
实施例3防控番茄青枯病的原生动物NJAU-CY1、庆大霉素荧光标记青枯菌，防治青枯菌的液体共培养实验。
供试青枯菌（R. solanacearum），供试原生动物为实施例2的防控青枯病丝足虫NJAU-CY1，保藏编号：CCTCC NO:C2023270。
丝足虫NJAU-CY1培养物制备：将丝足虫NJAU-CY1接种于液体NMAS培养基中，添加灭活的大肠杆菌（OD=0.04），置于恒温培养箱20℃，静置培养。48h后，吸取100 μL培养液放置在倒置显微镜下计数。
青枯病病原菌菌悬液制备：将保存好的青枯病病原菌接种到液体NB培养基中，置于恒温摇床30℃，220 rpm振荡培养1d。
设置两个对照组：10 μL青枯菌106 CFU/mL+ 90 μL NMAS缓冲液、2000个原生动物NJAU-CY1。
实验处理：2000个原生动物、10 μL青枯菌106 CFU/mL, 用NMAS培养液补足剩余的液体体积。每个处理液体体系均为100。
在0d、1d、2d、3d、4d、5d使用酶标仪测定病原菌红色荧光值RFP（Red fluorescentprotein, RFP，红色荧光激发波长：587nm，吸收波长：610nm）。同时，用显微镜视野法计算原生动物的数量。此外，在4d用平板稀释涂布法计算青枯菌的数量。
原生动物NJAU-CY1在一定程度上可以抑制青枯菌的生长，并且在和青枯菌共培养时，能迅速繁殖。研究发现Heteromita globosa NJAU-CY1显现了对R. solanacearum的捕食效应。
图3为本发明实施例3中原生动物Heteromita globosa NJAU-CY1和R. solanacearum二者液体共培养，原生动物NJAU-CY1随时间的动态变化图，其结果表明R. solanacearum能够显著促进原生动物NJAU-CY1的生长。
图4为本发明实施例3中原生动物Heteromita globosa NJAU-CY1和R. solanacearum二者液体共培养，R. solanacearum随时间的动态变化图，其结果表明原生动物NJAU-CY1抑制了R. solanacearum的生长。
图5为本发明实施例3中原生动物Heteromita globosa和R. solanacearum二者液体共培养，4d后，稀释涂布计算R. solanacearum数量的结果。
实施例4防控番茄青枯病的原生动物NJAU-CY1、庆大霉素抗性基因和红色荧光标记青枯菌、氯霉素抗性基因和绿色荧光(Green fluorescent protein , GFP)标记SQR9，防治青枯菌的液体三者共培养实验。
供试青枯菌（R. solanacearum），供试原生动物为实施例2的青枯病拮抗原生动物丝足虫NJAU-CY1，保藏编号：CCTCC NO:C2023270。
丝足虫NJAU-CY1和荧光标记的青枯病病原菌制备同上。
绿色荧光标记的贝莱斯芽胞杆菌悬液制备：将保存好的氯霉素标记的贝莱斯芽孢杆菌接种到液体LB培养基中，加工作浓度5 ug/mL氯霉素，置于恒温摇床30 ℃，220 rpm振荡培养1d。
设置6个对照组：
CK-1:10 μL青枯菌106CFU/mL + 90 μL NMAS缓冲液;
CK-2：10 μL贝莱斯芽孢杆菌SQR9 106 CFU/m + 90 μL NMAS缓冲液、
CK-3：2000个原生动物NJAU-CY1，剩余的液体用缓冲液补足;
CK-4:10 μL 青枯菌106 CFU/mL、2000个原生动物NJAU-CY1，剩余的体积用NMAS缓冲液补足;
CK-5：10 μL贝莱斯芽孢杆菌SQR9（保藏编号为CGMCC No.5808） 106 CFU/mL、2000个原生动物NJAU-CY1，剩余的体积用NMAS缓冲液补足;
CK-6：10 μL青枯菌106 CFU/mL、10 μL贝莱斯芽孢杆菌SQR9 106 CFU/mL，剩余的体积用NMAS缓冲液补足。
实验处理：2000个原生动物、10 μL青枯菌0.5×106 CFU/mL, 10 μL贝莱斯芽孢杆菌SQR9 0.5×106 CFU/mL，剩余的液体体积用NMAS缓冲液补足。以上所有处理体体系均为100 μL。
0d、2d、4d、6d使用酶标仪检测红色荧光RFP和绿色荧光GFP（激发波长：488nm，吸收波长：530nm)的荧光值。同时，用显微镜视野法计算原生动物的数量。研究发现Heteromita globosa同贝莱斯芽孢杆菌SQR9联合展现了更强的对R. solanacearum的拮抗效应。
图6为本发明实施例3中原生动物Heteromita globosa、R. solanacearum、B. velezensis SQR9三者液体共培养实验，原生动物随时间的动态变化图。研究结论:三者共培养时，明显促进了原生动物NJAU-CY1的生长。
图7为本发明实施例3中原生动物Heteromita globosa、R. solanacearum、B. velezensis SQR9三者液体共培养实验，R. solanacearum随时间的动态变化图。研究结论：三者共培养时，抑制了R. solanacearum的生长。
图8为本发明实施例中原生动物Heteromita globosa、R. solanacearum、 B. velezensis SQR9三者液体共培养实验，SQR9随时间的动态变化图。研究结论：原生动物NJAU-CY1能促进SQR9的生长。
Claims (4)
1.一株联合芽孢杆菌防控番茄青枯病的丝足虫Heteromita globosa NJAU-CY1，所述丝足虫保藏于中国典型培养物保藏中心，保藏地址为:中国武汉武汉大学，保藏日期为2023年8月28日，其保藏编号为CCTCC NO:C2023270。
2.含有权利要求1所述的丝足虫Heteromita globosa NJAU-CY1的培养物。
3. 权利要求2所述的丝足虫Heteromita globosa NJAU-CY1培养物的制备方法，其特征在于，将权利要求1所述的丝足虫Heteromita globosa NJAU-CY1接种于NMAS培养液中，添加灭活的大肠杆菌，置于20-22℃的培养箱中，静置45-50h，所述NMAS培养液的组成为，氯化钠0.12 g/L，七水硫酸镁0.0004 g/L,六水氯化钙0.0006 g/L,磷酸钠0.142 g/L，磷酸钾0.136 g/L。
4.权利要求1所述的丝足虫Heteromita globosa NJAU-CY1或权利要求2所述的培养物在制备防治番茄青枯菌中的应用。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311595366.3A CN117305114B (zh) | 2023-11-28 | 2023-11-28 | 一株联合芽孢杆菌防控番茄土传青枯病的丝足虫及其应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311595366.3A CN117305114B (zh) | 2023-11-28 | 2023-11-28 | 一株联合芽孢杆菌防控番茄土传青枯病的丝足虫及其应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN117305114A CN117305114A (zh) | 2023-12-29 |
| CN117305114B true CN117305114B (zh) | 2024-02-27 |
Family
ID=89273976
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311595366.3A Active CN117305114B (zh) | 2023-11-28 | 2023-11-28 | 一株联合芽孢杆菌防控番茄土传青枯病的丝足虫及其应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117305114B (zh) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117721022B (zh) * | 2024-02-07 | 2024-04-12 | 南京农业大学三亚研究院 | 一株促进合成菌群生物膜产量及贝莱斯芽孢杆菌丰度的原生动物变形虫及其应用 |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1081099A1 (en) * | 1999-09-06 | 2001-03-07 | Marine Biotechnology Institute Co., Ltd. | Bioremediation process using bacterivorous protozoa |
| CN116715559A (zh) * | 2023-06-28 | 2023-09-08 | 清华大学深圳国际研究生院 | 一种生物菌肥及其在调整含微塑料的土壤中微生物群落结构中的应用 |
-
2023
- 2023-11-28 CN CN202311595366.3A patent/CN117305114B/zh active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1081099A1 (en) * | 1999-09-06 | 2001-03-07 | Marine Biotechnology Institute Co., Ltd. | Bioremediation process using bacterivorous protozoa |
| CN116715559A (zh) * | 2023-06-28 | 2023-09-08 | 清华大学深圳国际研究生院 | 一种生物菌肥及其在调整含微塑料的土壤中微生物群落结构中的应用 |
Non-Patent Citations (3)
| Title |
|---|
| R.G. Mattison 等.The Soil Flagellate Heteromita globosa Accelerates Bacterial Degradation of Alkylbenzenes through Grazing and Acetate Excretion in Batch Culture.《Microbial Ecology》.2005,第49卷摘要，第142-150页. * |
| Stefan Geisen 等.Contrasting effects of soil microbial interactions on growth–defence relationships between early- and mid-successional plant communities.《New Phytologist》.2021,第233卷(第3期),摘要，第 1345-1357页. * |
| 韦中 等.土壤原生动物——研究方法及其在土传病害防控中的作用.《土壤学报》.2020,第58卷(第1期),摘要，第14-22页. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN117305114A (zh) | 2023-12-29 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111100806B (zh) | 一种耐硼赖氨酸芽孢杆菌作为底盘细胞制备的槟榔根腐病杀菌剂 | |
| CN117305114B (zh) | 一株联合芽孢杆菌防控番茄土传青枯病的丝足虫及其应用 | |
| Molina et al. | Biological contamination and its chemical control in microalgal mass cultures | |
| CN111040976B (zh) | 一株解淀粉芽孢杆菌及其应用 | |
| CN105176894B (zh) | 一种防治番茄灰霉病的解淀粉芽孢杆菌及其微生物菌剂 | |
| CN105441362A (zh) | 可杀鳞翅目昆虫、植物线虫和鞘锈菌的苏云金芽孢杆菌及应用 | |
| CN101928683B (zh) | 一种假单胞菌菌株及其应用 | |
| JP2024504510A (ja) | ミニセルの拡張可能な大量生産のためのバイオプロセスシステムおよび方法 | |
| Pal et al. | Screening, identification, and colonization of fungal root endophytes against Dematophora necatrix: a ubiquitous pathogen of fruit trees | |
| CN105567600A (zh) | 黄萎病菌拮抗菌及其应用 | |
| Moloinyane et al. | Association between chemical properties of vineyard soils and occurrence of entomopathogenic fungi causing different levels of mortality in Planococcus ficus | |
| CN105462882B (zh) | 一种用于防治作物黄萎病的铜绿假单胞菌及其应用 | |
| CN104530204B (zh) | 一种油菜抗菌肽BnPRP1及其应用 | |
| CN105969707B (zh) | 用于防治青枯病的大肠埃希氏菌及其应用 | |
| Sytnikov | How to increase the productivity of the soybean-rhizobial symbiosis | |
| CN117887580B (zh) | 一株促进香蕉生长和防控枯萎病的原生动物尾滴虫及应用 | |
| CN109804060B (zh) | 从辣椒植物体分离的Pseudozyma churashimaensis RGJ1菌株及其用途 | |
| Sagala et al. | Mass Propagation of Antagonistic Bacteria Pseudomonas fluorescens as an Environmental-Friendly Biocontrol Agent | |
| CN106565832B (zh) | 防治根结线虫的伴胞晶体蛋白组合物及其应用 | |
| Burhamzah et al. | Characterization of antibacterial-producing endophytic fungi of syzygiumpolyanthum leaves | |
| KR20030015010A (ko) | 신규한 가지검은마름병원균 ｗｔ＃３（ｋｃｃｍ１０２８３）유래 ｗｔ＃３－１유전자를 이용한 새로운생물농약 | |
| Wang et al. | Diversity and nematocidal activity of culturable bacteria from suppressive soils in Shandong Province, China | |
| AFIFI et al. | THE ABILITY OF SELECTED Trichoderma spp. TO IMPROVE WHEAT (Triticum asetivum L.) TOLERANCE UNDER HEAT, SALAINTY AND DROUGHT STRESSES THROUGH SEED TREATMENTS | |
| CN111876337B (zh) | 一种尖孢镰刀菌及其发酵方法与应用 | |
| CN114015578B (zh) | 康宁木霉菌及其发酵产物、含有其的组合物及应用 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |