WO2021175201A1 - 寡养单胞菌属新物种及其应用 - Google Patents

寡养单胞菌属新物种及其应用 Download PDF

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WO2021175201A1
WO2021175201A1 PCT/CN2021/078612 CN2021078612W WO2021175201A1 WO 2021175201 A1 WO2021175201 A1 WO 2021175201A1 CN 2021078612 W CN2021078612 W CN 2021078612W WO 2021175201 A1 WO2021175201 A1 WO 2021175201A1
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strain
nematodes
stenotrophomonas
nematode
nematodicola
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PCT/CN2021/078612
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English (en)
French (fr)
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张玉琴
邓阳
刘红宇
余利岩
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中国医学科学院医药生物技术研究所
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Publication of WO2021175201A1 publication Critical patent/WO2021175201A1/zh
Priority to ZA2022/00168A priority Critical patent/ZA202200168B/en

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    • 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
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • 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
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • 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
    • C12N1/20—Bacteria; Culture media therefor

Definitions

  • the present invention relates to the field of microorganisms, in particular to a new species of Stenotrophomonas and its application.
  • Stenotrophomonas belong to the Bacteria-Proteobacteria- ⁇ -Gammaproteobacteria-Lysobacterales-Lysobacteraceae.
  • Stenotrophomonas a new bacterial genus for Xanthomonas maltophilia was established as a model species. Hugh 1980) Swings et al. 1983. Int. J. Syst. Bacteriol 43, 606-609.).
  • the genus contains 17 effective description species, most of which are isolated from the environment of sludge and sludge processors.
  • Stenotrophomonas acidaminiphila was originally discovered from the anaerobic sludge blanket of sludge processors (Assih, E., Ouattara, AS,Thierry,S.,Cayol,JL,Labat,M.,and Macarie,H.(2002).Stenotrophomonas acidaminiphila sp.nov.,a strictly aerobic bacterium isolated from an upflow anaerobic sludge blanket(UASB)reactor.”Int .J.Syst.Evol.Microbiol52,559-568.), Stenotrophomonas chelatiphaga and Stenotrophomonas daejeonensis were found from municipal sludge (Kaparullina, EN, Doronina, NV, Chistyakova, TI and
  • Stenotrophomonas chelatiphaga sp .nov. a new aerobic EDTA-degrading bacterium.Syst.Appl.Microbiol 32,157-162.)(Lee,M.,Woo,S.-G.,Chae,M.,Shin,M.-C.,Jung ,H.-M.,and Ten,LN(2011).Stenotrophomonas daejeonensis sp.nov.,isolated from sewage.Int.J.Syst.Evol.Microbiol61,598-604.), and compost (Yang,H.- C.,Im,W.-T.,Kang,MS,Shin,D.-Y.,and Lee,S.-T.(2006).Stenotrophomonas koreensis sp.nov.,isolated from compost in South Korea.Int .J.Syst.Evol.Microbiol
  • Stenotrophomonas rhizophila sp. nov. a novel plant-associated bacterium with antifungal properties.Int.J.Syst.Evol.Microbiol.52,1937-1944.), cemetery (Handa, Y., Tazato, N., Nagatsuka, Y., Koide, T., Kigawa, R., Sano ,C.,and Sugiyama,J.(2016).Stenotrophomonas tumulicola sp.nov.,a major contaminant of the stone chamber interior in the Takamatsuzuka Tumulus.Int.J.Syst.Evol.Microbiol.66,1119-1124.) , And food surfaces (Weber, M., Schunemann, W., Fuss, J., Kampfer, P., and Lipski, A.
  • Nematodes parasitic in plants are important pathogens that seriously endanger the survival and growth of plants, and have attracted widespread attention from the society.
  • the control of plant nematode diseases mainly relies on traditional methods such as chemical control methods and crop rotation, and the effects are not very satisfactory, and there are many disadvantages. Therefore, people pay more attention to the development of control preparations as substitutes for chemical pesticides, and the research on the biocontrol of nematodes has naturally become the key and focus of research.
  • nematodes are an important part of the agricultural soil environment biosphere, and their beneficial aspects are that the appropriate amount of nematodes plays an important role in maintaining the health of the soil ecological environment, such as organic matter decomposition, plant nutrient mineralization and nutrient cycling. It is an environmentally friendly organism, and humans can make full use of the beneficial functions of nematodes to loosen the soil; in vegetable, gardening and lawn pest control, some nematodes have a unique mode of action and prey on agricultural pests (such as slugs, commonly known as slugs).
  • the specific reference URL is as follows:
  • Stenotrophomonas maltophilia of the genus Stenotrophomonas has been reported to be used as a model for the development of Caenorhabditis elegans (Thomas R, Hamat RA, Neela V. (2013) Stenotrophomonas maltophilia: pathogenesis model using Caenorhabditis elegans.J Med Microbiol 62, 1777- 1779.).
  • the purpose of the present invention is to provide new species of Stenotrophomonas and its application.
  • the new species of the genus Stenotrophomonas claimed by the present invention is specifically Stenotrophomonas nematodicola W5, and its deposit number in the General Microbiology Center of the China Microbial Species Collection Management Committee is CGMCC No.19401.
  • the present invention claims a bacterial agent.
  • the active ingredient of the bacterial agent claimed in the present invention is the aforementioned nematode endophytic oligotrophomonas (Stenotrophomonas nematodicola) W5.
  • the bacterial agent may also include a carrier.
  • the carrier can be a solid carrier or a liquid carrier.
  • the solid carrier may be a mineral material, a plant material, or a polymer compound;
  • the mineral material may be at least one of clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica, and diatomaceous earth.
  • the plant material may be at least one of corn flour, soybean flour and starch;
  • the polymer compound may be polyvinyl alcohol and/or polyglycol.
  • the liquid carrier may be an organic solvent, vegetable oil, mineral oil or water; the organic solvent may be decane and/or dodecane.
  • the active ingredient may be in the form of cultured living cells, fermentation broth of living cells, filtrate of cell culture, or a mixture of cells and filtrate.
  • the bacterial agent dosage form can be a variety of dosage forms, such as liquids, emulsions, suspensions, powders, granules, wettable powders or water dispersible granules.
  • surfactants such as Tween 20, Tween 80, etc.
  • adhesives such as Tween 20, Tween 80, etc.
  • stabilizers such as antioxidants
  • pH regulators etc.
  • the present invention claims the application of the aforementioned Stenotrophomonas nematodicola W5 as a target in the preparation of preparations for trapping and killing nematodes.
  • the present invention claims the application of the aforementioned Stenotrophomonas nematodicola W5 or the aforementioned bacterial agent in the interaction with nematicidal Bacillus B16.
  • the present invention claims the application of the aforementioned endophytic Stenotrophomonas nematodicola W5 or the aforementioned bacterial agent in preventing the colonization of nematode Bacillus B16 in nematodes.
  • the present invention claims to protect the aforementioned nematode endophytic oligotrophomonas (Stenotrophomonas nematodicola) W5 or the aforementioned bacterial agent in improving the survival rate of nematodes and/or prolonging the life of nematodes (used to maintain the ecological environment). Balance).
  • the present invention claims any of the following substances:
  • the preparation achieves the trapping and killing of nematodes by inhibiting the endophytic stenotrophomonas nematodicola W5 of nematodes.
  • the nematode is a nematode with the Stenotrophomonas nematodicola W5 endogenous.
  • the present invention claims a method for preparing a preparation for attracting nematodes.
  • the method for preparing a preparation for attracting and killing nematodes as claimed in the present invention may include the following steps: preparing a preparation for attracting and killing nematodes by using the aforementioned nematode endophytic oligotrophomonas (Stenotrophomonas nematodicola) W5 as a target.
  • the preparation achieves the trapping and killing of nematodes by inhibiting the endophytic stenotrophomonas nematodicola W5 of nematodes.
  • the nematode is a nematode with the Stenotrophomonas nematodicola W5 endogenous.
  • the present invention claims a method for preventing the colonization of nematode Bacillus B16 in nematodes.
  • the method for obstructing the colonization of nematode Bacillus B16 in nematodes as claimed in the present invention may include the following steps: using the aforementioned nematode endophytic oligotrophomonas (Stenotrophomonas nematodicola) W5 or the aforementioned bacterial agent to hinder Nematicidal Bacillus B16 is colonized in nematodes.
  • the nematode can be prevented from colonizing the nematode Bacillus nematode B16 by feeding the nematode endophytic oligotrophomonas (Stenotrophomonas nematodicola) W5 or the inoculum described above.
  • the present invention claims a method for improving the survival rate of nematodes and/or extending the lifespan of nematodes.
  • the method for improving the survival rate of nematodes and/or prolonging the lifespan of nematodes claimed in the present invention may include the following steps: using the aforementioned nematode endophytic oligotrophomonas (Stenotrophomonas nematodicola) W5 or the aforementioned bacterial agent to increase Nematode survival rate and/or extension of nematode lifespan.
  • the nematode can be fed with the aforementioned Stenotrophomonas nematodicola W5 or the aforementioned bacterial agent to increase the survival rate of the nematode and/or extend the lifespan of the nematode.
  • the present invention claims a strain.
  • the strain claimed in the present invention is a strain in the strain of Stenotrophomonas nematodicola; the 16S rRNA gene sequence and SEQ of the strain in the strain of Stenotrophomonas nematodicola ID No. 1 has at least 98.7% similarity, and the average nucleotide similarity (ANI value) of the whole genome sequence is higher than 96%.
  • strain of the nematode endophytic Stenotrophomonas nematodicola strain is a gram-negative bacteria and is aerobic.
  • strain of the nematode endophytic Stenotrophomonas nematodicola strain can form moist, smooth and light yellow colonies after being cultured on NA medium at 28°C for 48 hours.
  • the growth tolerance range of the strain in the endophytic Stenotrophomonas nematodicola strain is 22-37°C, 0-5% NaCl and pH 6.0-8.0, and the optimal growth condition is 28°C , 0-1% NaCl, pH 7.0.
  • the oxidase and catalase tests of the strains of the endophytic Stenotrophomonas nematodicola strain are positive, and the nitrate reduction is negative, which can make gelatin liquefy; it can utilize dextran and D -Maltose, D-mannose, D-fructose, D-trehalose, gentiobiose, sucrose, and stachyose cannot be used.
  • the strain in the nematode endophytic oligotrophomonas (Stenotrophomonas nematodicola) strain is sensitive to polymyxin B (300IU) and rifampicin (5 ⁇ g), and to tobramycin (10 ⁇ g), streptomyces (10 ⁇ g), Netilmicin (30 ⁇ g), Tetracycline (30 ⁇ g), Clindamycin (2 ⁇ g), Novobiocin (5 ⁇ g), Erythromycin (15 ⁇ g), Vancomycin (30 ⁇ g), Gentamicin It is tolerable to vitamins (10 ⁇ g), penicillin G (10IU), chloramphenicol (30 ⁇ g), ampicillin (10 ⁇ g), cefaclor (30 ⁇ g) and kanamycin (30 ⁇ g).
  • the main fatty acids of the strain in the nematode endophytic oligotrophomonas (Stenotrophomonas nematodicola) strain are C 16:0 , Antesio-C 15:0 , Iso-C 11:0 , Iso-C 15:0 .
  • the polar lipid component of the strain in the strain of the nematode endophytic oligotrophomonas includes diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), and phosphatidylethanolamine (PE), unknown Composition of Phospholipids (PL)
  • the present invention claims the application of the strain described in the eleventh aspect above in any one of the following:
  • (B4) Improve the survival rate of nematodes and/or extend the lifespan of nematodes (used to maintain the balance of the ecological environment).
  • the present invention claims a method for preparing a preparation for attracting nematodes.
  • the method for preparing a preparation for attracting nematodes as claimed in the present invention may include the following steps: preparing a preparation for attracting nematodes by using the strain described in the eleventh aspect as a target.
  • the preparation achieves the trapping and killing of nematodes by inhibiting the strain described in the eleventh aspect of nematode endogrowth.
  • the nematodes are nematodes endogenous with the strain described in the eleventh aspect above.
  • the present invention claims a method for preventing the colonization of nematode Bacillus nematode B16 in nematodes.
  • the method for obstructing the colonization of Bacillus nematode B16 in nematodes as claimed in the present invention may include the following steps: using the strain described in the eleventh aspect above to prevent the colonization of Bacillus nematode B16 in nematodes.
  • the nematode can be prevented from colonizing the nematode Bacillus nematode B16 by feeding the strain described in the eleventh aspect above.
  • the present invention claims a method for improving the survival rate of nematodes and/or extending the lifespan of nematodes.
  • the method for improving the survival rate of nematodes and/or extending the lifespan of nematodes as claimed in the present invention may include the following steps: using the strain described in the eleventh aspect above to increase the survival rate of nematodes and/or extend the lifespan of nematodes.
  • the present invention claims the nematode endophytic oligotrophomonas (Stenotrophomonas nematodicola) W5 described in the first aspect above or the strain described in the eleventh aspect above as a target in the preparation of a preparation for trapping and killing agricultural and forestry pests. In the application.
  • the collection center registration number CGMCC No.19401
  • Figure 1 shows the test results of polar lipid components of strain W5.
  • Figure 2 shows the 16S rRNA gene sequence based on strain W5, effective species of Stenotrophomonas, and some species of Stenotrophomonas neighboring genera in the Lysobacteriaceae , taking Escherichia coli JCM 1649 T (GenBank accession no.X80725) as The outer group constructs a phylogenetic tree.
  • Figure 3 is a graph showing the inhibitory effect of Bacillus nematode B16 on strain W5.
  • the following examples facilitate a better understanding of the present invention, but do not limit the present invention.
  • the experimental methods in the following examples, unless otherwise specified, are all conventional methods.
  • the test materials used in the following examples, unless otherwise specified, are all purchased from conventional biochemical reagent stores.
  • the quantitative experiments in the following examples are all set to repeat the experiment three times, and the results are averaged.
  • the strain W5 of the present invention is isolated from the body of Caenorhabditis elegans obtained in Nanyang City, Henan province.
  • the specific separation operation is as follows: disinfect the surface of Caenorhabditis elegans with 75% alcohol for 5 minutes, and then rinse the surface of Caenorhabditis elegans with flowing sterile water for 3 minutes; in a sterile environment, use a sterile dissecting needle to dissect the Caenorhabditis elegans Caenorhabditis elegans, and then inoculate the dissected Caenorhabditis elegans into NA medium (peptone 10g, beef extract 3g, sodium chloride 5g, agar 15g, distilled water 1000mL, pH7.4), cultured at 28°C for a week, from the plate Pick a single colony and transfer it to a newly prepared NA plate, repeat purification to obtain pure bacteria, and transfer it to NA slant medium for use.
  • the obtained pure strain was preserved in liquid nitrogen and frozen at -80°C with 20% (v/v) glycerol as a protective agent.
  • the strain number is W5.
  • Strain W5 was grown on NA medium at 28°C. Morphology, physiology and biochemistry, cytochemistry, and genetic studies were conducted on this strain. Other special circumstances will be explained.
  • the growth temperature detection range of strain W5 is 4, 10, 15, 28, 30, 32, 35, 37, 40 and 45°C; the growth salt concentration (NaCl) detection range is 0-11% (0-11g/100ml) 12 (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11g/100ml) concentration gradients; the growth pH detection range is 8 (pH4, 5, 6, 7, 8, 9, 10, 11) Gradient.
  • the physiological and biochemical functions of the strains are tested using test kits API 50CH, API ZYM, and BiOLOG GEN III plates and corresponding operating methods.
  • the identification results showed that the strain W5 was a Gram-negative bacteria and was aerobic. After the strain was cultured on NA medium at 28°C for 48 hours, it could form moist, smooth and light yellow colonies.
  • the growth tolerance range of the strain is 22-37°C, 0-5% NaCl and pH 6.0-8.0, and the optimal growth conditions are 28°C, 0-1% NaCl, pH 7.0.
  • Strain W5 tested positive for oxidase and catalase, and negative for nitrate reduction, which can liquefy gelatin; it can use dextran, D-maltose, D-mannose, and D-fructose, but cannot use D-trehalose, Gentiobiose, sucrose, stachyose.
  • Table 1 shows the differences in physiological and biochemical characteristics between strain W5 and the representative strains of related strains S. rhizophila JCM 13333 T , S. tumulicola JCM 30961 T and S. bentonitica DSM 103927 T.
  • the polar lipid components include diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), and phosphatidylethanolamine (PE).
  • DPG diphosphatidylglycerol
  • PG phosphatidylglycerol
  • PE phosphatidylethanolamine
  • Phospholipids (PL) with unknown components are shown in Figure 1.
  • strain W5 The genomic DNA of strain W5 was extracted and sequenced, and the 16S rRNA gene sequence (SEQ ID No. 1) was compared online in the international authoritative bacterial taxonomy analysis database (http://www.ezbiocloud.net/) ( Kim OS, Cho YJ, Lee K, et al. 2012, Introducing EzTaxon-e: a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species. Int J Syst Evol Microbiol, 62:716-721.). The results show that the strain W5 of the present invention has the highest similarity with the species of Stenotrophomonas.
  • strain W5 is closely related to the strain of Stenotrophomonas, suggesting that the strain is a member of Stenotrophomonas.
  • Stenotrophomonas rhizophila JCM 13333 T has a similarity of 99.7% and a similarity of Stenotrophomonas bentonitica DSM103927 T that has a similarity of more than 98.65% to the 16S rRNA gene of strain W5 among the strains that have been effectively described.
  • the similarity of Stenotrophomonas tumulicola JCM 30961 T is 99.2% and 98.7%.
  • a phylogenetic tree was constructed by selecting the 16S rRNA gene sequences of all effective strains of W5, Stenotrophomonas and representative strains of neighboring genera of Stenotrophomonas in the family Lysobacteriaceae ( Figure 2).
  • strain W5 falls within the evolutionary branch of Stenotrophomonas rhizophila JCM 13333 T and Stenotrophomonas bentonitica DSM 103927 T to form a stable sub-branch. This result further supports that the strain W5 is a member of the genus Stenotrophomonas.
  • the present invention carried out the whole genome sequence determination of the strain W5.
  • the length of the obtained genome sequence is 4.4 Mbp, and the G+C content of the genome is 67.3 mol%.
  • ANI value average nucleotide similarity
  • the strain W5 of the present invention has many significant differences from existing Stenotrophomonas strains, including physiological and biochemical, cytochemical, genotype and other aspects.
  • the above data fully proves that the strain W5 of the present invention represents a new species of Stenotrophomonas nematodicola and is named Stenotrophomonas nematodicola.
  • Stenotrophomonas nematodicola W5 has been deposited at the China Common Microbial Species Collection and Management Center on January 16, 2020, and its deposit number is CGMCC No.19401.
  • Nematode endophytic Stenotrophomonas nematodicola W5 and nematocidal Bacillus B16 were respectively inoculated into NA liquid medium and cultured overnight on a shaker at 37° C. and 200 rpm.
  • Nematicidal Bacillus B16 can inhibit the nematode endophytic Stenotrophomonas nematodicola W5 ( Figure 3).
  • the nematode endophytic Stenotrophomonas nematodicola W5 of the present invention was fed with nematodes, and the nematode survival rate and lifespan were significantly increased. Comparing the nematodes fed by the strain W5 of the present invention with the nematodes fed by E.
  • the survival rate of nematodes was increased from 85% to 100%; at 70h, the survival rate of nematodes was increased from 50% to 80%; at 80h, the survival rate of nematodes was increased from 50% to 80%; Nematode survival rate was increased from 30% to 60%; at 90h, the nematode survival rate was increased from 15% to 50%; at 100h, the nematode survival rate was increased from 0 to 40%; at 140h, the nematodes fed by the strain W5 of the present invention were still There is a 10% survival rate.
  • the strain W5 of the present invention is a new species of the genus Stenotrophomonas and is named Stenotrophomonas nematodicola.
  • Stenotrophomonas nematodicola the nematode endophytic oligotrophomonas (Stenotrophomonas nematodicola) W5 provided by the present invention has the probiotic function of nematodes.
  • an appropriate amount of nematodes is essential for maintaining the stability of the soil ecosystem (nematodes are soil ecological
  • the important functional components of the system play an important role in the process of organic matter decomposition, plant nutrient mineralization and nutrient cycling). It plays an important role in the environment-friendly organisms.
  • strain W5 can be used as a target to prepare a preparation for attracting and killing nematodes.
  • the invention will have broad application prospects in agriculture and forestry biological control.

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Abstract

一种寡养单胞菌属新物种及其应用，该寡养单胞菌属新物种的代表菌株为线虫内生寡养单胞菌(Stenotrophomonas nematodicola)W5，在中国微生物菌种保藏管理委员会普通微生物中心的保藏号为CGMCC No.19401。该线虫内生寡养单胞菌(Stenotrophomonas nematodicola)W5具有线虫益生功能，适量线虫利于维持土壤生态系统稳定性。

Description

寡养单胞菌属新物种及其应用 技术领域
本发明涉及微生物领域，具体涉及寡养单胞菌属新物种及其应用。
背景技术
寡养单胞菌属属于细菌域(Bacteria)-变形菌门(Proteobacteria)-γ-变形杆菌(Gammaproteobacteria)-溶杆菌目(Lysobacterales)-溶杆菌科(Lysobacteraceae)。
寡养单胞菌属的研究工作起始于1961年，Hugh R和Ryschenkow E.首先将一株产碱菌株命名为嗜麦芽假单胞菌(Pseudomonas maltophilia)(HUGH R,RYSCHENKOW E.(1961).Pseudomonas maltophilia,an alcaligenes-like species.J Gen Microbiol26:123-32.)。这株菌被发现与黄单胞菌属(Xanthomonas)具有一定的亲缘关系，1983年，通过对该菌株的基因型、表型特征以及生理生化特性、细胞化学特性进行研究分析，将其归为黄单胞菌属(Xanthomonas)，并命名为嗜麦芽黄单胞菌(Xanthomonas maltophilia)。(Swings,J.,De Vos,P.,Van den Mooter,M.,and De Ley,J.(1983).Transfer of Pseudomonas maltophilia Hugh 1981 to the genus Xanthomonas as Xanthomonas maltophilia(Hugh 1981)comb.nov.Int.J.Syst.Bacteriol 33,409-413.)。1993年，Norberto J.Palleroni和John F.Bradbury通过对比嗜麦芽黄单胞菌(Xanthomonas maltophilia)以及其他黄单胞菌属菌株(Xanthomonasspp.)的表型特征及生理生化性质，最终将其命名为嗜麦芽寡养单胞菌(Stenotrophomonas maltophilia)，并作为模式种建立了寡养单胞菌属(Stenotrophomonas)(Palleroni,N.J.,and Bradbury,J.F.(1993).Stenotrophomonas,a new bacterial genus for Xanthomonas maltophilia(Hugh 1980)Swings et al.1983.Int.J.Syst.Bacteriol43,606-609.)。目前，该属包含17个有效描述种，多分离自污泥及污泥处理器等环境，比如，Stenotrophomonas acidaminiphila最初从污泥处理器的厌氧污泥包层发现(Assih,E.,Ouattara,A.S.,Thierry,S.,Cayol,J.L.,Labat,M.,and Macarie,H.(2002).Stenotrophomonas acidaminiphila sp.nov.,a strictly aerobic bacterium isolated from an upflow anaerobic sludge blanket(UASB)reactor."Int.J.Syst.Evol.Microbiol52,559-568.)，Stenotrophomonas chelatiphaga和Stenotrophomonas daejeonensis是从市政污泥中发现的(Kaparullina,E.N.,Doronina,N.V.,Chistyakova,T.I.and Trotsenko,Y.A.(2009).Stenotrophomonas chelatiphaga sp.nov.,a new aerobic EDTA-degrading bacterium.Syst.Appl.Microbiol 32,157-162.)(Lee,M.,Woo,S.-G.,Chae,M.,Shin,M.-C.,Jung,H.-M.,and Ten,L.N.(2011).Stenotrophomonas daejeonensis sp.nov.,isolated from sewage. Int.J.Syst.Evol.Microbiol61,598-604.)、以及堆肥(Yang,H.-C.,Im,W.-T.,Kang,M.S.,Shin,D.-Y.,and Lee,S.-T.(2006).Stenotrophomonas koreensis sp.nov.,isolated from compost in South Korea.Int.J.Syst.Evol.Microbiol 56,81-84.)，废气处理生物滤池(Finkmann,W.,Altendorf,K.,Stackebrandt,E.,and Lipski,A.(2000).Characterization of N 2O-producing Xanthomonas-like isolates from biofilters as Stenotrophomonas nitritireducens sp.nov.,Luteimonas mephitis gen.nov.,sp.nov.and Pseudoxanthomonas broegbernensis gen.nov.,sp.nov.Int.J.Syst.Evol.Microbiol50,273-282.)、膨润土(Sanchez-Castro,I.,Ruiz-Fresneda,M.A.,Bakkali,M.,Kampfer,P.,Glaeser,S.P.,Busse,H.-J.,Lopez-Fernandez,M.,Martinez-Rodriguez,P.,and Merroun,M.L.(2017).Stenotrophomonas bentonitica sp.nov.,isolated from bentonite formations.Int.J.Syst.Evol.Microbiol.67,2779-2786.)、土壤(Yoon,J.-H.,Kang,S.-J.,Oh,H.W.,and Oh,T.-K.(2006).Stenotrophomonas dokdonensis sp.nov.,isolated from soil.Int.J.Syst.Evol.Microbiol 56,1363-1367.)(Heylen,K.,Vanparys,B.,Peirsegaele,F.,Lebbe,L.,and De Vos,P.(2007).Stenotrophomonas terrae sp.nov.and Stenotrophomonas humi sp.nov.,two nitrate-reducing bacteria isolated from soil.Int.J.Syst.Evol.Microbiol57,2056-2061.)、人参田(Kim,H.-B.,Srinivasan,S.,Sathiyaraj,G.,Quan,L.-H.,Kim,S.-H.,Bui,T.P.N.,Liang,Z.-Q.,Kim,Y.-J.,and Yang,D.-C.(2010).Stenotrophomonas ginsengisoli sp.nov.,isolated from a ginseng field.Int.J.Syst.Evol.Microbiol 60,1522-1526.)、有机农业用巴西甘蔗(Ramos,P.L.,Van Trappen,S.,Thompson,F.L.,Rocha,R.C.S.,Barbosa,H.R.,De Vos,P.,and Moreira-Filho,C.A.(2011).Screening for endophytic nitrogen-fixing bacteria in Brazilian sugar cane varieties used in organic farming and description of Stenotrophomonas pavanii sp.nov.Int.J.Syst.Evol.Microbiol 61,926-931.)、植物根际土(Wolf,A.,Fritze,A.,Hagemann,M.,and Berg,G.(2002).Stenotrophomonas rhizophila sp.nov.,a novel plant-associated bacterium with antifungal properties.Int.J.Syst.Evol.Microbiol.52,1937-1944.)，墓地(Handa,Y.,Tazato,N.,Nagatsuka,Y.,Koide,T.,Kigawa,R.,Sano,C.,and Sugiyama,J.(2016).Stenotrophomonas tumulicola sp.nov.,a major contaminant of the stone chamber interior in the Takamatsuzuka Tumulus.Int.J.Syst.Evol.Microbiol.66,1119-1124.)，还有食物表面(Weber,M.,Schunemann,W.,Fuss,J.,Kampfer,P.,and Lipski,A.(2018).Stenotrophomonas lactitubi sp.nov.,and Stenotrophomonas indicatrix sp.nov.,isolated from surfaces  with food contact."Int.J.Syst.Evol.Microbiol(in press))、口腔癌患者胸膜液(Palleroni,N.J.,and Bradbury,J.F.(1993).Stenotrophomonas,a new bacterial genus for Xanthomonas maltophilia(Hugh 1980)Swings et al.1983.Int.J.Syst.Bacteriol43,606-609.)等。
对该属菌株在生态功能及其他应用方面的研究有一系列报道，如在不产生氮的情况下还原亚硝酸盐，但不能还原硝酸盐，唯一产物为N 2O(Finkmann,W.,Altendorf,K.,Stackebrandt,E.,and Lipski,A.(2000).Characterization of N 2O-producing Xanthomonas-like isolates from biofilters as Stenotrophomonas nitritireducens sp.nov.,Luteimonas mephitis gen.nov.,sp.nov.and Pseudoxanthomonas broegbernensis gen.nov.,sp.nov.Int.J.Syst.Evol.Microbiol50,273-282.)，固氮作用(Ramos,P.L.,Van Trappen,S.,Thompson,F.L.,Rocha,R.C.S.,Barbosa,H.R.,De Vos,P.,and Moreira-Filho,C.A.(2011).Screening for endophytic nitrogen-fixing bacteria in Brazilian sugar cane varieties used in organic farming and description of Stenotrophomonas pavanii sp.nov.Int.J.Syst.Evol.Microbiol 61,926-931.)，还原硝酸盐(Heylen,K.,Vanparys,B.,Peirsegaele,F.,Lebbe,L.,and De Vos,P.(2007).Stenotrophomonas terrae sp.nov.and Stenotrophomonas humi sp.nov.,two nitrate-reducing bacteria isolated from soil.Int.J.Syst.Evol.Microbiol57,2056-2061.)，利用并降解EDTA(Kaparullina,E.N.,Doronina,N.V.,Chistyakova,T.I.and Trotsenko,Y.A.(2009).Stenotrophomonas chelatiphaga sp.nov.,a new aerobic EDTA-degrading bacterium.Syst.Appl.Microbiol 32,157-162.)，抗真菌特性(Wolf,A.,Fritze,A.,Hagemann,M.,and Berg,G.(2002).Stenotrophomonas rhizophila sp.nov.,a novel plant-associated bacterium with antifungal properties.Int.J.Syst.Evol.Microbiol.52,1937-1944.)。
寄生于植物体内的线虫是严重危害植物生存和生长的重要病原，已经引起社会的广泛关注。现阶段，植物线虫病害的防治主要依靠化学防治方法和轮作等传统方法，而且效果都不是很理想，并存在不少弊端。因此人们更关注开发生防制剂以作为化学农药的替代品，对线虫的生防研究自然成为研究的重点问题和焦点问题。研究发现，线虫肠道内生菌会对杀线虫芽孢杆菌B16(Bacillus nematocida B16)产生抑制作用，阻止杀线虫芽孢杆菌B16(Bacillus nematocidaB16)在线虫体内的生长和定植。比如，杀线虫芽孢杆菌B16对线虫侵染机制的研究为线虫生防菌剂的研发提供了新思路和理论基础(Niu Qiuhong,Huang Xiaowei,Zhang Lin,et al.A Trojan horse mechanism of bacterial pathogenesis against nematodes[J].Proceedings of the National Academy of Sciences.2010,107(38):16631-16636.)。关于防治林业害虫的线虫方面 的研究在近些年逐渐成为绿色生防的热点(昆虫病原线虫共生菌杀虫活性及异小杆线虫培养条件优化[D].张奎花.甘肃农业大学2013；我国昆虫病原线虫的研究与应用现状[J].吴文丹,尹姣,曹雅忠,肖春,李克斌.中国生物防治学报.2014(06)；新型生防因子——昆虫病原线虫的研究进展[J].李星月；李其勇；符慧娟；向运佳；杨雯婷.四川农业科技.2019(1).昆虫病原线虫共生细菌对南方根结线虫卵孵化的影响[J].王鑫鹏；王从丽；李春杰.土壤与作物.2017(3).)。
另一方面，线虫作为农业土壤环境生物圈的重要组成部分，其有益方面表现为适量的线虫对于维持土壤生态环境的健康方面，如在有机质分解、植物营养矿化及养分循环过程中起重要作用，表现为环境友好类生物，人类可以充分利用其线虫有益功能疏松土壤；在蔬菜、园艺和草坪虫害防治上，一些线虫具有独特的作用模式，捕食农业害虫(如蛞蝓，俗称鼻涕虫)。具体的参考网址如下：
http://www.agrichem.cn/news/2015/10/12/m201510121165871981.shtml。
寡养单胞菌属的Stenotrophomonas maltophilia曾被报道可作为秀丽隐杆线虫发病模型研究(Thomas R,Hamat RA,Neela V.(2013)Stenotrophomonas maltophilia:pathogenesis model using Caenorhabditis elegans.J Med Microbiol 62,1777-1779.)。
发明公开
本发明的目的是提供寡养单胞菌属新物种及其应用。
第一方面，本发明要求保护的寡养单胞菌属新物种具体为线虫内生寡养单胞菌(Stenotrophomonas nematodicola)W5，其在中国微生物菌种保藏管理委员会普通微生物中心的保藏号为CGMCC No.19401。
第二方面，本发明要求保护一种菌剂。
本发明要求保护的菌剂的活性成分为前文所述的线虫内生寡养单胞菌(Stenotrophomonas nematodicola)W5。
进一步地,上述菌剂中，所述菌剂还可以包括载体。
更进一步地,所述载体可为固体载体或液体载体。
更加具体地,所述固体载体可为矿物材料、植物材料或高分子化合物；所述矿物材料可为粘土、滑石、高岭土、蒙脱石、白碳、沸石、硅石和硅藻土中的至少一种；所述植物材料可为玉米粉、豆粉和淀粉中的至少一种；所述高分子化合物可为聚乙烯醇和/或聚二醇。
更加具体地,所述液体载体可为有机溶剂、植物油、矿物油或水；所述有机溶剂可为癸烷和/或十二烷。
进一步地,所述菌剂中，所述活性成分可以以被培养的活细胞、活细胞的发酵液、细胞培养物的滤液或细胞与滤液的混合物的形式存在。
进一步地,所述菌剂剂型可为多种剂型，如液剂、乳剂、悬浮剂、粉剂、颗 粒剂、可湿性粉剂或水分散粒剂。
根据需要，所述菌剂中还可添加表面活性剂(如吐温20、吐温80等)、粘合剂、稳定剂(如抗氧化剂)、pH调节剂等。
第三方面，本发明要求保护前文所述的线虫内生寡养单胞菌(Stenotrophomonas nematodicola)W5作为靶标在制备用于诱杀线虫的制剂中的应用。
第四方面，本发明要求保护前文所述的线虫内生寡养单胞菌(Stenotrophomonas nematodicola)W5或前文所述的菌剂在与杀线虫芽孢杆菌B16互作中的应用。
第五方面，本发明要求保护前文所述的线虫内生寡养单胞菌(Stenotrophomonas nematodicola)W5或前文所述的菌剂在阻碍杀线虫芽孢杆菌B16在线虫体内定植中的应用。
第六方面，本发明要求保护前文所述的线虫内生寡养单胞菌(Stenotrophomonas nematodicola)W5或前文所述的菌剂在提高线虫存活率和/或延长线虫寿命(用于维持生态环境的平衡)中的应用。
第七方面，本发明要求保护如下任一物质：
(A1)利用前文所述的线虫内生寡养单胞菌(Stenotrophomonas nematodicola)W5制备的制备用于诱杀线虫的制剂；
所述制剂通过抑制线虫内生寡养单胞菌(Stenotrophomonas nematodicola)W5实现对线虫的诱杀。所述线虫为内生有所述寡养单胞菌(Stenotrophomonas nematodicola)W5的线虫。
(A2)利用前文所述的线虫内生寡养单胞菌(Stenotrophomonas nematodicola)W5或前文所述的菌剂制备的用于阻碍杀线虫芽孢杆菌B16在线虫体内定植的产品；
(A3)利用前文所述的线虫内生寡养单胞菌(Stenotrophomonas nematodicola)W5或前文所述的菌剂制备的用于提高线虫存活率和/或延长线虫寿命(用于维持生态环境的平衡)的产品。
第八方面，本发明要求保护一种制备用于诱杀线虫的制剂的方法。
本发明要求保护的制备用于诱杀线虫的制剂的方法，可包括如下步骤：将前文所述的线虫内生寡养单胞菌(Stenotrophomonas nematodicola)W5作为靶标制备用于诱杀线虫的制剂。
所述制剂通过抑制线虫内生寡养单胞菌(Stenotrophomonas nematodicola)W5实现对线虫的诱杀。所述线虫为内生有所述寡养单胞菌(Stenotrophomonas nematodicola)W5的线虫。
第九方面，本发明要求保护一种阻碍杀线虫芽孢杆菌B16在线虫体内定植的方法。
本发明要求保护的阻碍杀线虫芽孢杆菌B16在线虫体内定植的方法，可包 括如下步骤：利用前文所述的线虫内生寡养单胞菌(Stenotrophomonas nematodicola)W5或前文所述的菌剂来阻碍杀线虫芽孢杆菌B16在线虫体内定植。
进一步地，可通过给线虫饲喂前文所述的线虫内生寡养单胞菌(Stenotrophomonas nematodicola)W5或前文所述的菌剂来实现阻碍杀线虫芽孢杆菌B16在线虫体内定植。
第十方面，本发明要求保护一种提高线虫存活率和/或延长线虫寿命的方法。
本发明要求保护的提高线虫存活率和/或延长线虫寿命的方法，可包括如下步骤：利用前文所述的线虫内生寡养单胞菌(Stenotrophomonas nematodicola)W5或前文所述的菌剂来提高线虫存活率和/或延长线虫寿命。
进一步地，可通过给线虫饲喂前文所述的线虫内生寡养单胞菌(Stenotrophomonas nematodicola)W5或前文所述的菌剂来实现提高线虫存活率和/或延长线虫寿命。
第十一方面，本发明要求保护一种菌株。
本发明所要求保护的菌株为线虫内生寡养单胞菌(Stenotrophomonas nematodicola)菌种中菌株；所述线虫内生寡养单胞菌(Stenotrophomonas nematodicola)菌种中菌株的16S rRNA基因序列与SEQ ID No.1相比至少具有98.7％以上的相似性，且全基因组序列的平均核苷酸相似性(ANI值)高于96％。
进一步地,所述线虫内生寡养单胞菌(Stenotrophomonas nematodicola)菌种中菌株为革兰氏染色阴性细菌，好氧。
进一步地,所述线虫内生寡养单胞菌(Stenotrophomonas nematodicola)菌种中菌株在28℃下，NA培养基上培养48小时后，可形成湿润，光滑和淡黄色菌落。
进一步地,所述线虫内生寡养单胞菌(Stenotrophomonas nematodicola)菌种中菌株的生长耐受范围是22-37℃、0-5％NaCl和pH 6.0-8.0，最适生长条件是28℃、0-1％NaCl、pH 7.0。
进一步地,所述线虫内生寡养单胞菌(Stenotrophomonas nematodicola)菌种中菌株的氧化酶和过氧化氢酶测试为阳性，硝酸盐还原阴性，可使明胶液化；能利用葡聚糖、D-麦芽糖、D-甘露糖、D-果糖，不能利用D-海藻糖、龙胆二糖、蔗糖、水苏糖。
进一步地,所述线虫内生寡养单胞菌(Stenotrophomonas nematodicola)菌种中菌株对多粘菌素B(300IU)与利福平(5μg)敏感，对妥布霉素(10μg)、链霉素(10μg)、奈替米星(30μg)、四环素(30μg)、克林霉素(2μg)、新生霉素(5μg)、红霉素(15μg)、万古霉素(30μg)、庆大霉素(10μg)、青霉素G(10IU)、氯霉素(30μg)、氨苄西林(10μg)、头孢克罗(30μg)、卡那霉素(30μg)具有耐受性。
进一步地,所述线虫内生寡养单胞菌(Stenotrophomonas nematodicola)菌种中菌株的主要脂肪酸是C 16:0、Antesio-C 15:0、Iso-C 11:0、Iso-C 15:0。
进一步地,所述线虫内生寡养单胞菌(Stenotrophomonas nematodicola)菌种中菌株的极性脂成分包括二磷脂酰甘油(DPG)、磷脂酰甘油(PG)、磷脂酰乙醇胺(PE)，未知成分的磷脂(PL)
第十二方面，本发明要求保护前文第十一方面中所述菌株在如下任一中的应用：
(B1)作为靶标制备用于诱杀线虫的制剂；
(B2)与杀线虫芽孢杆菌B16互作；
(B3)阻碍杀线虫芽孢杆菌B16在线虫体内定植；
(B4)提高线虫存活率和/或延长线虫寿命(用于维持生态环境的平衡)。
第十三方面，本发明要求保护一种制备用于诱杀线虫的制剂的方法。
本发明要求保护的制备用于诱杀线虫的制剂的方法，可包括如下步骤：将前文第十一方面中所述菌株作为靶标制备用于诱杀线虫的制剂。
所述制剂通过抑制线虫内生第十一方面中所述菌株实现对线虫的诱杀。所述线虫为内生有前文第十一方面中所述菌株的线虫。
第十四方面，本发明要求保护一种阻碍杀线虫芽孢杆菌B16在线虫体内定植的方法。
本发明要求保护的阻碍杀线虫芽孢杆菌B16在线虫体内定植的方法，可包括如下步骤：利用前文第十一方面中所述菌株来阻碍杀线虫芽孢杆菌B16在线虫体内定植。
进一步地，可通过给线虫饲喂前文第十一方面中所述菌株来实现阻碍杀线虫芽孢杆菌B16在线虫体内定植。
第十五方面，本发明要求保护一种提高线虫存活率和/或延长线虫寿命的方法。
本发明要求保护的提高线虫存活率和/或延长线虫寿命的方法，可包括如下步骤：利用前文第十一方面中所述菌株来提高线虫存活率和/或延长线虫寿命。
进一步地，可通过给线虫饲喂前文第十一方面中所述菌株来实现提高线虫存活率和/或延长线虫寿命。
第十六方面，本发明要求保护前文第一方面所述的线虫内生寡养单胞菌(Stenotrophomonas nematodicola)W5或前文第十一方面所述的菌株作为靶标在制备用于诱杀农林害虫的制剂中的应用。
保藏说明
建议的分类命名：线虫内生寡养单胞菌(Stenotrophomonas nematodicola)
参椐的生物材料(株)：W5
保藏机构：中国微生物菌种保藏管理委员会普通微生物中心
保藏机构简称：CGMCC
地址：北京市朝阳区北辰西路1号院3号
保藏日期：2020年1月16日
保藏中心登记入册编号：CGMCC No.19401
附图说明
图1为菌株W5的极性脂成分检测结果。
图2为基于菌株W5、寡养单胞菌属有效种和溶杆菌科中寡养单胞菌属邻近属部分种的16S rRNA基因序列，以Escherichia coli JCM 1649 T(GenBank accession no.X80725)为外群构建系统发育树。
图3为杀线虫芽孢杆菌B16对菌株W5的抑制作用效果图。
实施发明的最佳方式
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，均为自常规生化试剂商店购买得到的。以下实施例中的定量试验，均设置三次重复实验，结果取平均值。
实施例1、菌株W5的分离与鉴定
一、菌株W5的分离筛选
本发明菌株W5分离自河南省南阳市所得的秀丽隐杆线虫体内。具体分离操作如下：用75％的酒精对秀丽隐杆线虫进行表面消毒5min，再用流动的无菌水冲洗秀丽隐杆线虫表面3min；在无菌环境下，用无菌解剖针剖开秀丽隐杆线虫，然后把剖开的秀丽隐杆线虫接种到NA培养基(蛋白胨10g，牛肉膏3g，氯化钠5g，琼脂15g，蒸馏水1000mL，pH7.4)上，28℃培养一周，从平板上挑取单菌落转接于新配制的NA平板上，反复纯化，得到纯菌，转接于NA斜面培养基上备用。
将所获得的纯菌株以20％(v/v)甘油作为保护剂，进行液氮保藏和-80℃冷冻保藏。该菌株编号为W5。
二、菌株W5的鉴定
菌株W5在28℃条件下，生长于NA培养基上，对这株菌进行形态学、生理生化、细胞化学及基因水平的研究，其他特殊情况将予以说明。
1、菌株W5的细胞形态观察和生理生化特征检测
菌株W5的生长温度检测范围为4、10、15、28、30、32、35、37、40和45℃；生长盐浓度(NaCl)检测范围为0-11％(0-11g/100ml)的12个(0、1、2、3、4、5、6、7、8、9、10、11g/100ml)浓度梯度；生长pH检测范围为4-11之间的8个(pH4、5、6、7、8、9、10、11)梯度。菌株的生理生化功能使用检测试剂盒API 50CH、API ZYM，以及BiOLOG GEN III板及相应的操作方法进行检测。菌株的其他生理生化特征，包括革兰氏染色属性、氧需求、接触酶活性、氧化酶活性、明胶水解活性，淀粉水解活性和纤维素水解活性，主要参照《常 见细菌系统鉴定手册》进行(东秀珠,蔡妙英.2001.常见细菌系统鉴定手册.北京：科学出版社)。
鉴定结果显示，菌株W5为革兰氏染色阴性细菌，好氧。菌株在28℃下，NA培养基上培养48小时后，可形成湿润，光滑和淡黄色菌落。菌株的生长耐受范围是22-37℃、0-5％NaCl和pH 6.0-8.0，最适生长条件是28℃、0-1％NaCl、pH 7.0。
菌株W5的氧化酶和过氧化氢酶测试为阳性，硝酸盐还原阴性，可使明胶液化；能利用葡聚糖、D-麦芽糖、D-甘露糖、D-果糖，不能利用D-海藻糖、龙胆二糖、蔗糖、水苏糖。对多粘菌素B(300IU)与利福平(5μg)敏感，对妥布霉素(10μg)、链霉素(10μg)、奈替米星(30μg)、四环素(30μg)、克林霉素(2μg)、新生霉素(5μg)、红霉素(15μg)、万古霉素(30μg)、庆大霉素(10μg)、青霉素G(10IU)、氯霉素(30μg)、氨苄西林(10μg)、头孢克罗(30μg)、卡那霉素(30μg)具有耐受性。
菌株W5与近缘菌种代表菌株S.rhizophila JCM 13333 T、S.tumulicola JCM 30961 T、S.bentonitica DSM 103927 T的生理生化特征差异见表1。
表1菌株W5与寡养单胞菌属近缘对照菌株生理生化特征差异表
Figure PCTCN2021078612-appb-000001
Figure PCTCN2021078612-appb-000002
注：表中，+表示为阳性，-表示为阴性。
由表1所示结果可见，本发明菌株W5与已发表的寡养单胞菌属的近缘菌株在部分生理生化特征上存在明显差异。
2、菌株W5的细胞化学特征检测
通过气相色谱(GC)、高压液相色谱(HPLC)和薄层色谱(TLC)检测菌株W5的脂肪酸、醌类型、极性脂等细胞化学组分(Sasser M.Identification of bacteria by gas ghromatography of cellular fatty acids,MIDI Technical Note 101.Newark,DE:MIDI inc；1990.Minnikin DE,O’Donnell AG,Goodfellow M,Alderson G,Athalye M et al.An integrated procedure for the extraction of bacterial isoprenoid quinones and polar lipids.J Microbiol Methods 1984；2:233–241.)。比较本发明菌株W5与近源菌种代表菌株S.rhizophila JCM 13333 T、S.tumulicola JCM 30961 T、S.bentonitica DSM 103927 T的脂肪酸成分，见表2。结果显示菌株W5的主要脂肪酸是C 16:0、antesio-C 15:0、iso-C 11:0、iso-C 15:0，同时如表2所示，菌株W5脂肪酸谱与其他近源种存在着明显差异。在菌株W5中，极性脂成分包括二磷脂酰甘油(DPG)、磷脂酰甘油(PG)、磷脂酰乙醇胺(PE)，未知成分的磷脂(PL)如图1所示。
表2菌株W5与寡养单胞菌属近源菌种代表菌株脂肪酸谱差异表
Figure PCTCN2021078612-appb-000003
Figure PCTCN2021078612-appb-000004
3、菌株W5系统进化地位的确定
提取菌株W5的基因组DNA进行测序，并将其中的16S rRNA基因序列(SEQ ID No.1)在国际权威细菌分类学分析数据库(http://www.ezbiocloud.net/)中进行在线比对(Kim OS,Cho YJ,Lee K,et al.2012,Introducing EzTaxon-e:a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species.Int J Syst Evol Microbiol,62:716-721.)。结果显示本发明菌株W5与寡养单胞菌属的菌种相似性最高。菌株W5的16S rRNA基因在数据库中比对结果显示，菌株W5与寡养单胞菌属的菌株近缘，提示该菌株为寡养单胞菌属的成员。寡养单胞菌属已经有效描述的菌种中，与菌株W5的16S rRNA基因相似性高于98.65％的最近缘菌株分别是Stenotrophomonas rhizophila JCM 13333 T相似性为99.7％，Stenotrophomonas bentonitica DSM103927 T相似性为99.2％，Stenotrophomonas tumulicola JCM 30961 T相似性为98.7％。选取W5、寡养单胞菌属所有有效种菌株以及溶杆菌科寡养单胞菌属邻近属代表菌株的16S rRNA基因序列构建系统进化树(图2)。在系统进化树上，菌株W5落在寡养单胞菌属的进化分支内，且与Stenotrophomonas rhizophila JCM 13333 T及Stenotrophomonas bentonitica DSM 103927 T聚类形成一个稳定的亚分支。该结果进一步支持菌株W5是寡养单胞菌属的一个成员。
为进一步明确菌株的系统进化地位，本发明对菌株W5进行了全基因组序列测定。获得的基因组序列长度为4.4Mbp，基因组G+C含量为67.3mol％。在EZbiocloud上比较并计算菌株W5的全基因组序列与近缘对照菌的全基因组序列的平均核苷酸相似性(ANI值)(Yoon SH,Ha SM,Lim J,Kwon S,Chun J.A  large-scale evaluation of algorithms to calculate average nucleotide identity.Antonie van Leeuwenhoek 2017；110:1281–1286.)。全基因组序列分析显示，菌株W5与Stenotrophomonas rhizophila JCM 13333 T、Stenotrophomonas bentonitica DSM 103927 T、Stenotrophomonas tumulicola JCM 30961 T的平均核苷酸相似性(ANI)分别为84.7％、85.0％、80.7％，这些值均远低于95％—区分原核微生物基因种的ANI阈值(Yoon SH,Ha SM,Lim J,Kwon S,Chun J.A large-scale evaluation of algorithms to calculate average nucleotide identity.Antonie van Leeuwenhoek 2017；110:1281–1286.)。这些结果表明，本发明菌株W5是寡养单胞菌属的一个新发现的基因种。
综上所述，本发明菌株W5与现有寡养单胞菌属菌株有许多显著的差异特征，包括生理生化、细胞化学和基因型等方面。以上数据充分证明了本发明菌株W5代表了寡养单胞菌属的一个新物种，命名为线虫内生寡养单胞菌(Stenotrophomonas nematodicola)。线虫内生寡养单胞菌(Stenotrophomonas nematodicola)W5已经于2020年1月16日保藏于中国普通微生物菌种保藏管理中心，其保藏编号为CGMCC No.19401。
实施例2、线虫内生寡养单胞菌(Stenotrophomonas nematodicola)W5的线虫益生作用研究
1、杀线虫芽孢杆菌B16对菌株W5的抑制作用
实验采用文献(陈志谊,刘邮洲,刘永锋,等.拮抗细菌菌株间的互作关系及其对生物防治效的影响[J].植物病理学报,2005,35(6):539-544.)的测定方法进行。具体如下：
(1)将线虫内生寡养单胞菌(Stenotrophomonas nematodicola)W5和杀线虫芽孢杆菌B16分别接种于NA液体培养基中，在37℃、200rpm的摇床上过夜培养。
(2)在无菌条件下，分别将各个菌液OD 600值调到0.6，将培养后W5涂布于NA固体培养基上，利用无菌滤纸分别浸取B16的培养液，等待完全浸湿，将滤纸片放置于涂布了W5的平板上进行互作培养实验，用浸湿NA培养基的滤纸片做平行对照实验。
(3)封口，放37℃恒温培养箱培养，观察、记录实验结果。
(4)相同方法实验重复三次。
结果：杀线虫芽孢杆菌B16能够抑制线虫内生寡养单胞菌(Stenotrophomonas nematodicola)W5(图3)。
2、菌株W5对杀线虫芽孢杆菌B16在线虫体内定植障碍测试
采用平板转移喂养方法进行实验。具体如下：
(1)按照文献描述的方法(Niu Qiuhong,Huang Xiaowei,Zhang Lin,et al.A Trojan horse mechanism of bacterial pathogenesis against  nematodes[J].Proceedings of the National Academy of Sciences.2010,107(38):16631-16636.Niu Qiuhong,Huang Xiaowei,Hui Fengli,et al.Colonization of Caenorhabditis elegans by Bacillus nematocida B16,a bacterial opportunisticpathogen[J].J Mol Microbiol Biotechnol.2012；22(4):258-67.)用荧光标记杀线虫芽孢杆菌B16的mreB蛋白后对菌株进行平板培养，将秀丽隐杆线虫在NA培养基平板上进行培养，备用。
(2)将线虫内生寡养单胞菌(Stenotrophomonas nematodicola)W5接种在平板上培养24h，备用。然后，将培养24h后的线虫分别转移到接种有W5的新鲜NA平板上继续培养6小时。用线虫转接到无菌NA培养基平板上培养结果作为对照实验。
(3)将用W5喂养后的线虫挑出(100条)，用流动的无菌生理盐水将线虫表面清洗干净，然后转接到生长有杀线虫芽孢杆菌B16的平板上继续培养；对照组中挑取100条线虫直接转移接种到生长有杀线虫芽孢杆菌B16的平板上继续培养，观察。
(4)不同时间段，观察不同组别中线虫体内B16的定植情况。
(5)相同方法试验重复三次。每次每组供试线虫100条。
结果：和不喂养内生寡养单胞菌(Stenotrophomonas nematodicola)W5的线虫相比较，通过平板转移喂养实验后，发现喂养菌株W5的线虫寿命比不喂养W5的线虫寿命延长；使用荧光显微镜对线虫进行观察，喂养菌株W5的线虫体内发现的标记过的芽孢杆菌B16的线虫的数量明显少于未喂养菌株W5的线虫的数量，也就是说，菌株W5对杀线虫芽孢杆菌B16在线虫体内定植形成障碍。具体实验数据：喂养W5的线虫再转接到接种有菌株B16的平板上共培养24h时，线虫的整个虫体都呈现绿色荧光的检出率为0；48h时，有10％的线虫体都呈现绿色荧光；72h时，检测到有10％的线虫虫体都呈现绿色荧光。未喂养过W5的对照组中，24h时，20％的线虫被检测到呈现绿色荧光；48h时，有50％的线虫呈现绿色荧光；72h时，90％的线虫呈现绿色荧光。虫体全部呈现绿色荧光即意味着B16侵染并完全定植于线虫体内(Niu Qiuhong,Huang Xiaowei,Hui Fengli,et al.Colonization of Caenorhabditis elegans by Bacillus nematocida B16,a bacterial opportunisticpathogen[J].J Mol Microbiol Biotechnol.2012；22(4):258-67.)。
3、菌株W5对线虫的益生效果测定
采用平板转移喂养方法进行实验。具体如下：
(1)将线虫内生寡养单胞菌(Stenotrophomonas nematodicola)W5接种在NA平板上培养24h，备用。
(2)将大肠杆菌菌株接种在平板上培养24h，作为对照组备用。
(3)然后，将用NA培养基培养24h后的秀丽隐杆线虫分别转移到接种有W5的平板和大肠杆菌的平板上继续培养，并观察记录线虫存活情况。
(4)相同方法试验重复三次。每次每组供试线虫100条。
结果：和对照组相比较，本发明线虫内生寡养单胞菌(Stenotrophomonas nematodicola)W5喂养线虫后，线虫存活率和寿命都有明显增加。本发明菌株W5喂养的线虫和大肠杆菌喂养的线虫相比较：60h时，将线虫存活率从85％提升至100％；70h时，将线虫存活率从50％提升至80％；80h时，将线虫存活率从30％提升至60％；90h时，将线虫存活率从15％提升至50％；100h时，将线虫存活率从0提升至40％；140h时本发明菌株W5喂养的线虫仍有10％存活率。
工业应用
本发明菌株W5为寡养单胞菌属新物种，命名为线虫内生寡养单胞菌(Stenotrophomonas nematodicola)。同时证明本发明所提供的线虫内生寡养单胞菌(Stenotrophomonas nematodicola)W5具有线虫益生功能，环境中，特别是土壤环境中，适量的线虫对于维持土壤生态系统的稳定性(线虫是土壤生态系统的重要功能组分，在有机质分解、植物营养矿化及养分循环过程中起重要作用)有重要作用，表现为环境友好类生物，人类可以充分利用其线虫有益功能；线虫过度繁殖生长，造成林业灾害时，就表现为林业害虫，在消杀有害的线虫时可以将菌株W5作为靶标制备用于诱杀线虫的制剂。本发明在农林生防方面将具有广泛的应用前景。

Claims (30)

  1. 线虫内生寡养单胞菌(Stenotrophomonas nematodicola)W5，其在中国微生物菌种保藏管理委员会普通微生物中心的保藏号为CGMCC No.19401。
  2. 一种菌剂，其活性成分为权利要求1所述的线虫内生寡养单胞菌(Stenotrophomonas nematodicola)W5。
  3. 根据权利要求2所述的菌剂，其特征在于：所述菌剂包括载体。
  4. 根据权利要求3所述的菌剂，其特征在于：所述载体为固体载体或液体载体。
  5. 根据权利要求4所述的菌剂，其特征在于：所述固体载体为矿物材料、植物材料或高分子化合物；
    所述液体载体为有机溶剂、植物油、矿物油或水。
  6. 根据权利要求5所述的菌剂，其特征在于：所述矿物材料为粘土、滑石、高岭土、蒙脱石、白碳、沸石、硅石和硅藻土中的至少一种；所述植物材料为玉米粉、豆粉和淀粉中的至少一种；所述高分子化合物为聚乙烯醇和/或聚二醇；
    所述有机溶剂为癸烷和/或十二烷。
  7. 根据权利要求2-6中任一所述的菌剂，其特征在于：所述菌剂中，所述活性成分以被培养的活细胞、活细胞的发酵液、细胞培养物的滤液或细胞与滤液的混合物的形式存在。
  8. 根据权利要求2-7中任一所述的菌剂，其特征在于：所述菌剂剂型为液剂、乳剂、悬浮剂、粉剂、颗粒剂、可湿性粉剂或水分散粒剂。
  9. 根据权利要求2-8中任一所述的菌剂，其特征在于：所述菌剂中还添加有表面活性剂、粘合剂、稳定剂和/或pH调节剂。
  10. 权利要求1所述的线虫内生寡养单胞菌(Stenotrophomonas nematodicola)W5作为靶标在制备用于诱杀线虫的制剂中的应用。
  11. 权利要求1所述的线虫内生寡养单胞菌(Stenotrophomonas nematodicola)W5或权利要求2-9中任一所述的菌剂在与杀线虫芽孢杆菌B16互作中的应用。
  12. 权利要求1所述的线虫内生寡养单胞菌(Stenotrophomonas nematodicola)W5或权利要求2-9中任一所述的菌剂在阻碍杀线虫芽孢杆菌B16在线虫体内定植中的应用。
  13. 权利要求1所述的线虫内生寡养单胞菌(Stenotrophomonas nematodicola)W5或权利要求2-9中任一所述的菌剂在提高线虫存活率和/或延长线虫寿命中的应用。
  14. 一种制备用于诱杀线虫的制剂的方法，包括如下步骤：将权利要求1所述的线虫内生寡养单胞菌(Stenotrophomonas nematodicola)W5作为靶标制备用于诱杀线虫的制剂。
  15. 一种阻碍杀线虫芽孢杆菌B16在线虫体内定植的方法，包括如下步骤：利用权利要求1所述的线虫内生寡养单胞菌(Stenotrophomonas nematodicola)W5或权利要求2-9中任一所述的菌剂来阻碍杀线虫芽孢杆菌B16在线虫体内定植。
  16. 一种提高线虫存活率和/或延长线虫寿命的方法，包括如下步骤：利用权利要求1所述的线虫内生寡养单胞菌(Stenotrophomonas nematodicola)W5或权利要求2-9中任一所述的菌剂来提高线虫存活率和/或延长线虫寿命。
  17. 如下任一物质：
    (A1)利用权利要求1所述的线虫内生寡养单胞菌(Stenotrophomonas nematodicola)W5制备的制备用于诱杀线虫的制剂；
    (A2)利用权利要求1所述的线虫内生寡养单胞菌(Stenotrophomonas nematodicola)W5或权利要求2-9中任一所述的菌剂制备的用于阻碍杀线虫芽孢杆菌B16在线虫体内定植的产品；
    (A3)利用权利要求1所述的线虫内生寡养单胞菌(Stenotrophomonas nematodicola)W5或权利要求2-9中任一所述的菌剂制备的用于提高线虫存活率和/或延长线虫寿命的产品。
  18. 菌株，其特征在于：所述菌株为线虫内生寡养单胞菌(Stenotrophomonas nematodicola)菌种中菌株；所述线虫内生寡养单胞菌(Stenotrophomonas nematodicola)菌种中菌株的16S rRNA基因序列与SEQ ID No.1相比至少具有98.7％的相似性，且全基因组序列的平均核苷酸相似性高于96％。
  19. 根据权利要求18所述菌株，其特征在于：所述线虫内生寡养单胞菌(Stenotrophomonas nematodicola)菌种中菌株为革兰氏染色阴性细菌，好氧。
  20. 根据权利要求18或19所述菌株，其特征在于：所述线虫内生寡养单胞菌(Stenotrophomonas nematodicola)菌种中菌株在28℃下，NA培养基上培养48小时后，能够形成湿润，光滑和淡黄色菌落。
  21. 根据权利要求18-20中任一所述菌株，其特征在于：所述线虫内生寡养单胞菌(Stenotrophomonas nematodicola)菌种中菌株的生长耐受范围是22-37℃、0-5％NaCl和pH 6.0-8.0，最适生长条件是28℃、0-1％NaCl、pH 7.0。
  22. 根据权利要求18-21中任一所述菌株，其特征在于：所述线虫内生寡养单胞菌(Stenotrophomonas nematodicola)菌种中菌株的氧化酶和过氧化氢酶测试为阳性，硝酸盐还原阴性，能够使明胶液化；能利用葡聚糖、D-麦芽糖、D-甘露糖、D-果糖，不能利用D-海藻糖、龙胆二糖、蔗糖、水苏糖。
  23. 根据权利要求18-22中任一所述菌株，其特征在于：所述线虫内生寡养单胞菌(Stenotrophomonas nematodicola)菌种中菌株对多粘菌素B与利福平敏感，对妥布霉素、链霉素、奈替米星、四环素、克林霉素、新生霉素、红霉素、万古霉素、庆大霉素、青霉素G、氯霉素、氨苄西林、头孢克罗、卡那霉素具有耐受性。
  24. 根据权利要求18-23中任一所述菌株，其特征在于：所述线虫内生寡养单胞菌(Stenotrophomonas nematodicola)菌种中菌株的主要脂肪酸是C 16:0、Antesio-C 15:0、Iso-C 11:0、Iso-C 15:0。
  25. 根据权利要求18-24中任一所述菌株，其特征在于：所述线虫内生寡养单胞菌(Stenotrophomonas nematodicola)菌种中菌株的极性脂成分包括二磷脂酰甘油、磷脂酰甘油、磷脂酰乙醇胺、未知成分的磷脂。
  26. 权利要求18-25中任一所述菌株在如下任一中的应用：
    (B1)作为靶标制备用于诱杀线虫的制剂；
    (B2)与杀线虫芽孢杆菌B16互作；
    (B3)阻碍杀线虫芽孢杆菌B16在线虫体内定植；
    (B4)提高线虫存活率和/或延长线虫寿命。
  27. 一种制备用于诱杀线虫的制剂的方法，包括如下步骤：将权利要求18-25中任一所述菌株作为靶标制备用于诱杀线虫的制剂。
  28. 一种阻碍杀线虫芽孢杆菌B16在线虫体内定植的方法，包括如下步骤：利用权利要求18-25中任一所述菌株来阻碍杀线虫芽孢杆菌B16在线虫体内定植。
  29. 一种提高线虫存活率和/或延长线虫寿命的方法，包括如下步骤：利用权利要求18-25中任一所述菌株来提高线虫存活率和/或延长线虫寿命。
  30. 权利要求1所述的线虫内生寡养单胞菌(Stenotrophomonas nematodicola)W5或权利要求18-25中任一所述的菌株作为靶标在制备用于诱杀农林害虫的制剂中的应用。
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