CN103114044B - Endophytic fungus strain RR21 and application thereof - Google Patents
Endophytic fungus strain RR21 and application thereof Download PDFInfo
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- CN103114044B CN103114044B CN201310047454.XA CN201310047454A CN103114044B CN 103114044 B CN103114044 B CN 103114044B CN 201310047454 A CN201310047454 A CN 201310047454A CN 103114044 B CN103114044 B CN 103114044B
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Abstract
The invention relates to an endophytic fungus strain mutant RR21 of wild rice and application thereof in regulating plant growth and/or causing diseases to plants, belonging to the fields of microorganisms and microorganism application. Specifically, the invention discloses an endophytic fungus strain RR21, namely Harpophora oryzae R5-6-1-RR21 of which the collection number is CCTCC NO:M2012313. The invention also discloses application of the endophytic fungus strain RR21, which is characterized by causing diseases to plants including rice or barley.
Description
Technical field
The present invention relates to the mutant RR21 of fungal bacterial strain raw in a kind of wild-rice, with and in plant growth regulating and/or make the purposes of plant on causing a disease, belong to microorganism and microbe application field.
Background technology
Endogenetic fungus (endophytic fungi) at least the part of the life history can infect and surely grow in health plant tissue, host is without obvious class fungi (Petrini, 1991 of illness; Wilson, 1995), to be only present in root system of plant different from mycorrhizal fungi, and endogenetic fungus can exist (Faeth & Fagan, 2002) at any histoorgan of the underground and aboveground of plant.Endogenetic fungus conventionally forms reciprocal symbiosis relation with host plant, and endogenetic fungus obtains the required nutrient of growth from host plant on the one hand, and endogenetic fungus can Promoting plant growth on the other hand, the enhancing antibiont of host plant and the ability of abiotic stress.What research was more at present is root nodule symbiote and mycorrhizas homobium, and the symbiote of endogenetic fungus and plant is another manifestation of higher plant and microorganisms symbiosis relation.The mutual of endogenetic fungus and plant studied the concern that is day by day subject to investigator, and becomes the international focus of endogenetic fungus research field.
Endogenetic fungus-plant symbiosis is the coadapted results of both sides, and both are in a kind of running balance.In endogenetic fungus one plant symbiosis is done mutually, the gene expression profile of endogenetic fungus and plant occurs significantly to change, and its express spectra depends on inoculated endogenetic fungus kind; The change of genetic expression shows to exist between endogenetic fungus and plant the interchange (Bailey et al., 2006) of information.
Active oxygen (reactive oxygen species, ROS) is a kind of very important signal transduction mechanism in plant and microbial interaction.Nadph oxidase (NADPH oxidase, Nox) to electron acceptor(EA), causes the formation of active oxygen by the transfer transport of NADPH.Along with Nox family member's discovery, the production process of recognizing active oxygen is the signal pathway of the various atomizations of ubiquitous control, comprising processes such as the breeding of cell, apoptosis, hormone response, programmed cell death, the growth of root hair, spore germinations.In the fragrant post bacterium of ergot mushroom endogenetic fungus fescue grass (Epichlo festucae) of systemic seed vertical transmission and the symbiotic relationship of English ryegrass (Lolium perenne), Endophytic Fungal Hyphae is height limitation growth pattern, surely grow in the intercellular substance of host plant over-ground part, along the direction growth that is parallel to rachis, branch hardly.The fragrant post bacterium NoxA mutant of fescue grass and host plant form hostile interaction, and mutant biomass in plant materials significantly increases, and mycelia form changes, and growth radially; The plant of inoculation mutant is lost apical dominance, the serious passivation of growing, precocious old and feeble, finally dead.Another Nox gene-NoxB does not affect the foundation of determining to grow with symbiotic relationship of the fragrant post bacterium of fescue grass.When the active oxygen that produces when Nox complex body (comprising NoxA, NoxR and RacA) is disorderly, symbiosis interaction is disintegrated, and the relation of plant and endogenetic fungus will be become from reciprocal symbiosis hostile.Therefore, the variation of the active oxygen that endogenetic fungus NoxA the produces growth in plant materials and biomass to it has regulating effect, and maintaining of symbiotic relationship played to vital effect.（Tanaka?et?al.,?2006;?Eaton?et?al.,?2011）。
In addition, many microorganisms can utilize NRPS114 (Nonribosomal peptide synthetase, NRPS) composite structure complexity, miscellaneous biologically active peptides, is used as microbiotic, immunosuppressor, anticancer and antiviral agent, siderophore and bio-surfactant etc.In rye grass endogenetic fungus, be cloned into the gene sidN of a coding NRPS, this gene participates in synthetic siderophore; The mutant of sidN causes normally synthesizing siderophore, the disappearance of endogenetic fungus secretion siderophore ability has changed the running balance of symbiote iron ion, cause intercellular substance free iron ion to increase, cause the increase of reactive oxygen species by Fenton's reaction (Fenton reaction), final symbiotic relationship is transformed to Antagonism by reciprocity, cause the pathology necrosis (Eaton et al., 2011) of plant.
Another important signal transduction pathway is the former activated protein kinase of mitogen (Mitogen-Activated Protein Kinases, map kinase, the MAPK) approach being caused by environment-stress.MAPK approach has important effect to the virulence of many pathomycetes.The fragrant post bacterium of fescue grass sakA genes encoding MAPK, and show requisite effect in plant-endogenetic fungus symbiotic relationship.The disappearance of sakA gene determines that the interaction between fungi and host plant is become pathogenic from symbiosis.SakA deletion mutant is inoculated on plant, plant shows the great variety in the forfeiture of apical dominance, precocious aging and plant evolution, comprise the formation of the similar bulb structure of the base portion of tillering and the forfeiture of anthocyanidin (Eaton et al., 2008).
Endogenetic fungus also produces induction of resistance by induction host plant, thereby improves the ability that Genes For Plant Tolerance biology (pathogenic bacteria, herbivore etc.) and abiotic (salt, arid, high temperature etc.) is coerced.The non-expressor 1(Nonexpressor of pathogenesis related gene of pathogenesis-related genes 1, NPR1) be systemic acquired resistance (systemic acquired resistance, SAR) key controlling gene in approach, between Whitfield's ointment (salicylic acid, the SA) accumulation and SAR genetic expression subsequently of its functional localization in SAR signal transduction.NPR1 gene is cloned and is obtained the earliest from model plant Arabidopis thaliana.Itself does not have bacteriostatic activity NPR1, but its can inducing plant multiple defense response in body, and pathogenic bacteria is not had to strict kind specificity.The system resistance of India's pyriform spore (Piriformospora indica) induction Arabidopis thaliana needs jasmonate acid signal transmission and the NPR1 in tenuigenin.
Plant-endogenetic fungus symbiote is the higher plant found after leguminous plants-root nodule symbiote, mycorrhizas homobium and another manifestation of microorganisms symbiosis relation.But from the depth & wideth of research, compared with leguminous plants-root nodule symbiote, mycorrhizas homobium, the research of plant-endogenetic fungus symbiote is just just in the basis exploratory stage.The mutual of plant-endogenetic fungus studied the concern that is day by day subject to investigator at present, and becomes the international focus of endogenetic fungus research field.
The symbiosis of endogenetic fungus-host plant is the coadapted results of both sides, and both are in a kind of running balance.In India's pyriform spore and Arabidopis thaliana syntaxial system, the beta-glucosidase (PYK10) existing in Arabidopis thaliana root cell endoplasmic reticulum can limit the intrusion of endogenetic fungus India pyriform spore and surely grow, thereby suppressed strong defensive raction, be conducive to both reciprocal symbiosis (Sherameti et al., 2008b); In India's pyriform spore and barley syntaxial system, endogenetic fungus infects the expression that can weaken HvBI-1 gene after root system, and the infection strength that can limit on the contrary mycelia is expressed in crossing of HvBI-1 gene.HvBI-1 gene is very conservative in eukaryote, can suppress programmed cell death, this shows that the Growth and reproduction of Endophytic Fungal Hyphae in pin main body needs plant tissue cell's death (Deshmukh et al., 2006) to a certain degree, and finally both reach equilibrium state.In addition in plant-pathogenic fungi is studied mutually, the programmed death (programmed cell death) or the autophagy (autophagy) that have been found that pathogenic fungi mycelia are essential (Veneault-Fourrey et al. for its successful infected tissue, 2006), but in endogenetic fungus-plant does mutually, in endogenetic fungus, whether also exist similar reaction mechanism and up to the present also do not relate to.
Plant root nodule bacterium (RN) and mycorrhizal fungi (AM) syntaxial system are ubiquity and the most outstanding mode systems in plant-endophyte syntaxial system, and the molecular mechanism of doing mutually for research plant-dark septate endophyte provides basic model.In the white arteries and veins root of leguminous plants and clover symbiote, find the gene that a large amount of symbiosis are necessary.From various symbiosis mutant, clone obtains 26 genes at present, these genes participate in the identification of root nodule bacterium, the early stage reaction of symbiosis signal cascade, root infect with surely grow, formation and the fixed nitrogen of root nodule regulates.These are found to be molecular mechanism and the evolution of understanding better the symbiosis of plant-dark septate endophyte important clue (Johnson et al., 2008) are provided.CSP (the public symbiosis approach of common symbiosis pathway) is prevalent in cogeneration system.Ca ion signal approach is to set up the important channel of endosymbiosis system.CSP participates in the encoding and decoding of Ca ion signal, thereby affects the foundation of AM syntaxial system.At present, there are 7 gene SYMRK, CASTOR, POLLUX, NUP133, NUP85, CCaMK and CYCLOPS to be reported as CSP gene.Confirm that at present paddy rice has three CSP ortholog genes: OsCASTOR, OsPOLLUX and OsCCaMK.Wherein, OsPOLLUX and OsCCaMK are the requisite regulatory factors of paddy rice AM symbiote.In AM and RN cogeneration system, OsCASTOR and OsCCaMK are the molecular basises that maintains CSP function.SYMRK and be rich in the common proteins encoded kinases (Johnson et al., 2008) of leucic repeat region (LRR).In addition, SYMRK can also participate in forming of Ca ion signal by activating by the protein kinase of phosphorylation, regulates the signal conduction in cogeneration system.OsCASTOR and OsPOLLUX are transmembrane channel albumen (Deshmukh et al., 2006), in symbiote, signal transduction are also played a role.At the signal pathway initial stage, the much albumen being positioned on nucleus, such as nucleoporin NUP85, NUP133, participates in transport and the location of Ca ion signal inducible factor.
CCaMK (protein kinase that calcium/calmodulin relies on) may be the demoder of Ca ion signal, affects infecting of mycorrhizal fungi.CYCLOPS/IPD3 is also the downstream modulin of Ca ion signal approach, acts synergistically with CCaMK.In the time that CCaMK causes phosphorylation, CYCLOPS activates Ca ion signal approach downstream gene, thereby makes mycorrhizal fungi and root nodule bacterium successfully infect host.Studies have shown that, paddy rice has all CSP ortholog genes.Wherein, rice Os CASTOR, OsCCaMK, OsCYCLOPS, OsPOLLUX and OsCCaMK also play vital regulating and controlling effect in paddy rice AM symbiote.Make mutually the factor with the screening of SYMRK/DMI2 kinases, result obtains HMGR1 from clover, from white arteries and veins root, obtains SIP.HMGR1 coding mevalonate synthetic enzyme, and this enzyme participates in the synthetic of isoprenoid.Therefore, HMGR1 is considered to participate in infecting and the formation of root nodule of root nodule bacterium.SIP is the DBP with ARID (AT-rich domain) that a class is new, the special promotor in conjunction with NIN.In GRAS region, translation factor NSP1 and NSP2 in leguminous plants RN symbiote, be must and specialization.Except NSP1 and NSP2, translation factor NIN, infects the vital role that is formed with of epiblem and root cortex root nodule to root nodule bacterium.OsNSP1, OsNSP2 with NSP1, NSP2 homology in paddy rice, are also found.In the mutant of white arteries and veins root (pattern leguminous plants) the NSP1-2 disappearance of losing in symbiosis phenotype, rice Os NSP1, OsNSP2 can repair the disappearance of RN symbiosis phenotype completely.CASTOR, POLLUX, CCaMK and CYCLOPS still can find homologue in paddy rice, quite conservative and in symbiotic relationship, have an effect can not be substituted on evolving.The gene of many coding GRAS albumen raises in AM symbiote.At the initial stage nineties, the discovery of root nodule bacterium symbiosis signaling molecule (NFs, Nod facters) has important breakthrough, provides basis for setting forth better subsequently Nod gene function.NF acceptor has identical structure, and this structure is made up of jointly single passage cross-film district and the intracellular kinase region of the outer Methionin induction region (LysM) of born of the same parents.
LysM participates in the binding of peptidoglycan or similar structures molecule, for example chitin oligose.In the mutant of LysM afunction, plant does not produce any reaction (Hiroshi et al., 2010) to the intrusion of root nodule bacterium and NFs.In white arteries and veins root, NFR1 and NFR5 form acceptor complex body, and the NFs of root nodule bacterium secretion is had to specialization identity.Because NFR5 intracellular region territory lacks kinase activity, so NFR1 plays vital effect in intracellular signal being conducted to the process in signal pathway downstream.And the kinase activity of NFR1 is in activation downstream symbiosis signal pathway essential (T. Nakagawa, unpublished result).Complete genome sequencing shows: LysM-RK(LysM receptor-like kinases) gene family can by gene connect and partial replication present diversification.So just increase the possibility that is regulated induction and the intracellular signal conduction of NF by the complex body of multiple LysM-RK genomic constitutions, and be not only confined to NFR1 and NFR5 complex body.So the precise mechanism of host plant identification NFs needs further to be studied.At present, 6 LysM-RK genes in rice genome, have been found.The chitin acceptor CERK1 existing in Arabidopis thaliana, can cause plant congenital immunity power antagonism fungal pathogens.CERK1 also belongs to LysM-RK, and especially, aspect intracellular kinase region, CERK1 and NFR1 have high similarity.Based on structural similarity, NFR1 also has the effect (T.Nakagawa, unpublished results) of of short duration activation resistance related gene.Induction from the symbiosis signaling molecules of root nodule bacterium in white arteries and veins root is regulated by two LysM kinases NFR1 and NFR5.These two kinases root nodule bacterium infect with the forming process of root nodule in essential.In the fragrant post bacterium of interior raw fescue grass and host plant English ryegrass symbiote, mycelia is height limitation growth pattern, surely grows in the intercellular substance of host plant over-ground part, along the direction growth that is parallel to rachis, branch hardly.Between the phenomenon explanation host of this strict control endophyte growth and symbiote, there is signal conduction.
In symbiote, in the process that endogenetic fungus improves at growth and the biomass of promotion plant, hormonal substance plays very important effect.Wherein, novel endogenous plant hormone-solely angle gold plain lactone of sprouting (Strigolactones) that a class is secreted by plant root, can promote the sprouting of metabolism, branch and the spore of fungi, changes physiological function and the mitochondria activity of fungi.Similarly, fungi is releasor molecule also, causes the symbiosis specific reaction of plant root.The β-glucuronidase reporter gene of plant root is activated in the time that hypha,hyphae approaches.Microorganism (such as endogenetic fungus, endogenetic bacteria, root nodule bacterium etc.), by generation or the interference plant hormone route of synthesis of inducing plant hormone, promotes the growth of plant and the increase of biomass.Especially thereby bacterium ACC decarboxylase is interfered the physiological function of host plant by the ethylene levels in regulating plant body.Etc. TAKAKAZU(2010) from cultivated rice stem portion from obtaining endogenetic bacteria, azospirillum Azospirillum sp..By this bacterium the genomic sequence is analyzed, 2 hormone genes involveds are found.AcdS gene, coding ACC decarboxylase.AcdR gene, regulates the expression of acdS.Azospirillum produces ACC decarboxylase, reduces the ethylene content in plant materials, promotes the growth of plant, weakens the signal of environment-stress.In the former research of acdS, never reported.
By the function of research endogenetic fungus mutant, thereby find relevant key gene, the syntaxial system of research endogenetic fungus and plant is significant.Paddy rice is the most important grain plant of China, the syntaxial system of endogenetic fungus and paddy rice, the research of the virulence of endogenetic fungus mutant to paddy rice, can develop and there is corresponding disease resistance defense mechanism biotechnological formulation, to improving rice-cultivating growth, increase biomass, the research and the utilization that improve aspect its viability in adverse circumstance have great importance.
Summary of the invention
The technical problem to be solved in the present invention is to provide mutants which had RR21 of a kind of endogenetic fungus and uses thereof.
In order to solve the problems of the technologies described above, the invention provides a kind of endogenetic fungal bacterial strain RR21, be rice chain Saksenaea vasiformis (Harpophora oryzae) R5-6-1-RR21, its preserving number is: CCTCC NO:M 2012313.
The present invention also provides the purposes of above-mentioned endogenetic fungal bacterial strain RR21 simultaneously: for infecting plant, plant is caused a disease.
Improvement as the purposes RR21 of endogenetic fungal bacterial strain of the present invention: described plant is paddy rice or barley.
Bacterial strain of the present invention is Harpophora oryzae R5-6-1-RR21, depositary institution: Chinese Typical Representative culture collection center, preservation address: Wuhan, China Wuhan University, deposit number: CCTCC NO:M 2012313, August 27 2012 preservation time.
Bacterial strain Harpophora oryzae R5-6-1-RR21 derives from the mutant of the endogenetic fungus rice falciform Saksenaea vasiformis of paddy rice, belongs to mycota, Ascomycota, excrement shell guiding principle, mitosporic fungi Ju Zuo shell section, Saksenaea.This bacterial strain has pathogenic effects on plant.
Brief description of the drawings
Below in conjunction with accompanying drawing, the specific embodiment of the present invention is described in further detail.
Fig. 1: bacterial strain (rice falciform Saksenaea vasiformis mutants which had) Harpophora oryzae R5-6-1-SS1 is 25 DEG C of dark colonial morphologies of cultivating after 5d on CM substratum; Compared with wild-type R5-6-1, R5-6-1-SS1 aerial hyphae is more dense, bacterium colony Dark grey, colony edge irregular and radially extend, melanin deposition also obviously increase.
Fig. 2: the lethal effect of bacterial strain Harpophora oryzae R5-6-1-SS1 to paddy rice: control group 1 is blank, the rice seedlings of not inoculating, control group 2 is the rice seedlings of inoculation R5-6-1, and treatment group is the rice seedlings of inoculation Harpophora oryzae R5-6-1-SS1.The treatment group plant overground part of inoculation R5-6-1-SS1 is organized withered death, and the plant of control group 1 and 2 grows fine;
Fig. 3: fluorescence microscopy Microscopic observation Harpophora oryzae R5-6-1-SS1 determines the situation of growing and the impact on root growth and development root;
A represents root-hair zone shows fluorescent microscopy images,
B represents root-hair zone opticmicroscope figure,
C represents square section, root-hair zone;
Fig. 4: Harpophora oryzae R5-6-1-SS1 infects the pathogenic experiment of barley;
Fig. 5: the barley leaves of micro-Microscopic observation inoculation Harpophora oryzae R5-6-1-SS1 infect the situation of surely growing;
Fig. 6: the cross section situation of surely growing that infects of barley leaves of micro-Microscopic observation inoculation Harpophora oryzae R5-6-1-SS1;
Fig. 7: bacterial strain Harpophora oryzae R5-6-1-S22 is 25 DEG C of dark colonial morphologies of cultivating after 5d on CM substratum; Compared with wild type strain R5-6-1, aerial hyphae is more rare, bacteria colony white, and colony edge is neat also to be extended radially, and melanin deposition also obviously reduces, and does not almost have;
Fig. 8: the growth-promoting functions of rice falciform Saksenaea vasiformis mutants which had Harpophora oryzae R5-6-1-S22 to paddy rice: control group 1 is blank, the rice seedlings of not inoculating, control group 2 is the rice seedlings of inoculation R5-6-1, treatment group is the rice seedlings of inoculation Harpophora oryzae R5-6-1-S22, result display process group plant overground part tissue growth is vigorous, width of blade increases, and chlorophyll content increases, better than the plant growing way of control group 1 and 2.
Fig. 9: fluorescence microscopy Microscopic observation Harpophora oryzae R5-6-1-S22 determines the situation of growing and the impact on root growth and development root;
A represents root-hair zone shows fluorescent microscopy images,
B represents root-hair zone opticmicroscope figure,
C represents square section, root-hair zone;
Figure 10: the colonial morphology of bacterial strain Harpophora oryzae R5-6-1-RR19 on CM substratum.
Figure 11: the lethal effect of rice falciform Saksenaea vasiformis mutants which had Harpophora oryzae R5-6-1-RR19 to paddy rice: control group 1 is blank, the rice seedlings of not inoculating, control group 2 is the rice seedlings of inoculation R5-6-1, and treatment group is the rice seedlings of inoculation Harpophora oryzae R5-6-1-RR19.
Figure 12: fluorescence microscopy Microscopic observation Harpophora oryzae R5-6-1-RR19 determines the situation of growing and the impact on root growth and development root;
A represents root-hair zone shows fluorescent microscopy images,
B represents root-hair zone opticmicroscope figure,
C represents square section, root-hair zone;
Figure 13: Harpophora oryzae R5-6-1-RR19 infects the pathogenic experiment of barley;
Figure 14: micro-Microscopic observation Harpophora oryzae R5-6-1-RR19 infects the situation of surely growing;
A represents the cross section shows fluorescent microscopy images of barley leaves,
The shows fluorescent microscopy images after barley leaves is infected in B representative;
Figure 15; The colonial morphology of bacterial strain Harpophora oryzae R5-6-1-RR21 on CM substratum;
Figure 16: the lethal effect of rice falciform Saksenaea vasiformis mutants which had Harpophora oryzae R5-6-1-RR21 to paddy rice: control group 1 is blank, the rice seedlings of not inoculating, control group 2 is the rice seedlings of inoculation R5-6-1, and treatment group is the rice seedlings of inoculation Harpophora oryzae R5-6-1-RR21.
Figure 17: fluorescence microscopy Microscopic observation Harpophora oryzae R5-6-1-RR21 determines the situation of growing and the impact on root growth and development root;
Figure 18: Harpophora oryzae R5-6-1-RR21 infects the pathogenic experiment of barley;
Figure 19: micro-Microscopic observation Harpophora oryzae R5-6-1-RR21 infects the situation of surely growing.
A represents the cross section shows fluorescent microscopy images of barley leaves,
The shows fluorescent microscopy images after barley leaves is infected in B representative.
Embodiment
With reference to above-mentioned accompanying drawing, the specific embodiment of the present invention is elaborated.
Remarks explanation: following all substratum all need to carry out in a conventional manner the sterilising treatment (121 DEG C, 0.24 MPa, 20 minutes) of High Temperature High Pressure before using.
The acquisition of embodiment 1, endogenetic fungus mutants which had:
Wild-type rice falciform Saksenaea vasiformis strain number is R5-6-1, separates from wild-rice root system in 2007.R5-6-1 bacterial strain be kept at Dutch international fungi preservation center (numbering: CBS125863) and Chinese common micro-organisms preservation center (number: CGMCC 2737).
The conventional things such as Agrobacterium (Agrobacterium tumefaciens) strains A GL1 and plasmid pCAMBIA1300 are this laboratory and preserve.Agrobacterium is generally 28 DEG C of dark culturing, and the minimum medium of growth is LB liquid nutrient medium, in 4% glycerine, at-70 DEG C, preserves.
CM(Complete Medium) substratum: 20 × Nitrate salts 50ml, 1000 × Trace Elements 1ml, D-glucose (glucose) 10g, Peptone (peptone) 2g, Yeast extract (yeast extract) 1g, Casamino acid (casamino acids) 1g, 1000 × Vitamin solution 1ml, with 1mol/L NaOH adjust pH to 6.5, adding distil water is to 1L.
CM solid medium is above-mentioned CM(Complete Medium) add agar powder 12g on the basis of substratum.
20 × Nitrate salts(1000ml) configuration: NaNO
3120g, KCl 10.4g, MgSO
4. 7H2O 10.4g, KH
2pO
430.4g, water is settled to 1L.
1000 × Trace Elements(100ml) configuration: ZnSO
4. 7H
2o 2.2g, H
3bO
31.1g, MnCl
2. 4H
2o 0.5g, FeSO
4. 7H
2o 0.5g, CoCl
2. 6H
2o 0.17g, CuSO
4. 5H
2o 0.16g, Na
2moO
4. 5H
2o 0.15g, Na
4eDTA 5g, water is settled to 100ml.
1000 × Vitamin solution(100ml) configuration: Biotin 0.01g, Pyridoxin 0.01g, Thiamine 0.01g, Riboflavin 0.01g, PABA (p-aminobenzonic acid) 0.01g, Nicotinic acid 0.01g, water is settled to 100ml.
Water agar: distilled water adds 1.5% agar powder (mass ratio that is agar powder and distilled water is 1.5:98.5), and autoclaving, separates for monospore.
DCM(Defined Complex Medium) substratum: for transformant SUR resistance screening, Yeast nitrogen base without amino acids (Difco) 1.7g, Asparagines 2g, NH
4nO
31g, Glucose 10g, is settled to 1L, uses Na
2hPO
4adjust pH to 6.0.
LB liquid nutrient medium (Luria-Bertani): Tryptone (Tryptones) 10g, Yeast extract (yeast extract) 5g, NaCl (sodium-chlor) 10g, add deionized water to 1000ml, is adjusted to pH7.5, autoclaving with NaOH solution.
LB solid medium: Tryptone (Tryptones) 10g, Yeast extract (yeast extract) 5g, NaCl (sodium-chlor) 10g, agar powder 12g, add deionized water to 1000ml, is adjusted to pH7.5, autoclaving with NaOH solution.
Agrobacterium inducing culture AIM formula: 0.8 ml 1.25 K-Phosphate-buffer pH 4.8 (use KH
2pO
4and K
2hPO
4preparation), 20 ml MN-buffer (30 g/l MgSO
47H
2o, 15 g/l NaCl, 1L H
2o
2), 1 ml 1% CaCl
22H
2o (w/v), 10 ml 0.01 % FeSO
4(w/v), 5 ml spore elements (100 mg/l ZnSO
47H
2o, 100 mg/l CuSO
45H
2o, 100 mg/l H
3bO
3, 100 mg/l Na
2moO
42H
2o, 1L H
2o
2) (filtration sterilization), 2.5 ml 20% NH
4nO
3(w/v), 10 ml 50% glycerol (v/v), 40 ml 1 M MES pH 5.5 (with NaOH solution adjust pH), 20% glucose (w/v): add 10 ml in liquid nutrient medium, solid medium adds 5 ml, add water to 1L, solid medium adds 1.5% agar powder, (dimethyl sulfoxide (DMSO) DMSO preparation for 200 mM AS-).
Above-mentioned all substratum all need to carry out the sterilising treatment (121 DEG C, 0.24 MPa, 20 minutes) of High Temperature High Pressure.
Under laboratory condition, can obtain endogenetic fungus mutants which had of the present invention according to the freeze-thaw method method for transformation of Agrobacterium AGL1, i.e. rice falciform Saksenaea vasiformis mutants which had.Specific as follows:
First, Agrobacterium competence preparation: by the fluorescent fusion protein expression vector pKD6-GFP building and pKD5-RED(Li et al. 2012) be transformed into agrobacterium strains AGL1 by freeze-thaw method.
Agrobacterium strains is activated on LB flat board, then forward 28 DEG C of shaken overnight in 5mL LB liquid nutrient medium to and cultivate; This nutrient solution of transferase 12 mL is to 28 DEG C of shaking culture being housed in the triangular flask of 50mL LB liquid nutrient medium to OD600 0.5～1.0; Place and stop growing on ice, 4 DEG C of centrifugal 5min of 3000g; 20 mM CaCl of 1mL precooling for precipitation
2solution suspends, and divides and installs in pre-cooled Eppendorf centrifuge tube, every pipe 0.1mL;
Add carrier (fluorescent fusion protein expression vector pKD6-GFP or the pKD5-RED) plasmid DNA of 1 μ g in above-mentioned centrifuge tube, freezing in liquid nitrogen; 37 DEG C of water-bath 5min thaw; Add 1mLLB liquid nutrient medium, 28 DEG C of jogs are cultivated 2-4h; Cultured bacterium liquid is applied on the LB flat board that contains kantlex (containing 50 μ g/ml), the positive 30min that places, after bacterium liquid is absorbed by substratum completely, is inverted culture dish, cultivates 2-4d for 28 DEG C.
Secondly, the T-DNA of rice falciform Saksenaea vasiformis transforms: select the single colony inoculation of Agrobacterium in 5ml LB liquid nutrient medium (containing 50 μ g/ml kantlex) from the LB flat board (containing 50 μ g/ml kantlex) of above-mentioned cultivation, 200rpm/min, 28 DEG C of incubated overnight; 200-400 μ l nutrient solution was transferred to 5ml containing in the induction liquid nutrient medium (AIM) of 50 μ g/ml kantlex in second day, approximately 0.15,28 DEG C of cultivation of OD value makes OD600 reach 0.5 ~ 0.6 in 5 ~ 6 hours; Wash lower rice falciform Saksenaea vasiformis wild-type conidium from the CM flat board of cultivating about 10 days with 10ml sterile purified water, after three layers of lens wiping paper filter with blood counting chamber counting, and with sterilized water dilution spore concentration be 10
6individual/ml; Get the cultured Agrobacterium AGL-1(of 100 μ l containing carrier) bacterium liquid and the 100 μ l rice falciform Saksenaea vasiformis conidial suspension mixing of having diluted, (200 μ l) are evenly applied to the nitrocellulose filter surface on AIM flat board to mixed solution, AIM is dull and stereotyped containing 200 μ M Syringylethanone (AS, acetosyringone) or contain AS not in contrast, cultivate altogether 48 hours for 22 DEG C; Then nitrocellulose filter is transferred to containing chlorimuronethyl and antibiotic DCM and selected on flat board, in selective medium, containing 300 μ g/ml chlorimuronethyls (SUR), 400 μ g/ml cephamycins (cefotaxime), 60 μ g/ml Streptomycin sulphates (streptomycin), plate is placed at 28 DEG C and cultivates and arrive transformant appearance.
The present invention obtains altogether 87 of transformants, and wherein 4 (being that R5-6-1 is obviously different from wild-type rice falciform Saksenaea vasiformis strain number) carry out preservation, specific as follows:
Strain number is Harpophora oryzae R5-6-1-SS1, depositary institution: Chinese Typical Representative culture collection center, preservation address: Wuhan, China Wuhan University, deposit number: CCTCC NO:M 2012314, August 27 2012 preservation time.
Strain number is Harpophora oryzae R5-6-1-S22, depositary institution: Chinese Typical Representative culture collection center, preservation address: Wuhan, China Wuhan University, deposit number: CCTCC NO:M 2012315, August 27 2012 preservation time.
Strain number is Harpophora oryzae R5-6-1-RR19, depositary institution: Chinese Typical Representative culture collection center, preservation address: Wuhan, China Wuhan University, deposit number: CCTCC NO:M 2012312, August 27 2012 preservation time.
Strain number is Harpophora oryzae R5-6-1-RR21, depositary institution: Chinese Typical Representative culture collection center, preservation address: Wuhan, China Wuhan University, deposit number: CCTCC NO:M 2012313, August 27 2012 preservation time.
Embodiment 2, rice falciform Saksenaea vasiformis mutants which had Harpophora oryzae R5-6-1-SS1(CCTCC NO:M 2012314) morphologic observation:
On CM substratum, observe colonial morphology, color, growth velocity, sporulation quantity and the melanin deposition of this mutants which had.
Observations (Fig. 1) shows: mutants which had Harpophora oryzae R5-6-1-SS1 is 25 DEG C of dark cultivations after 5d on CM substratum, and colony diameter is respectively 5.8cm, 5.5cm.Compared with wild type strain, aerial hyphae is more dense, bacterium colony Dark grey, colony edge irregular and radially extend, the sporulation quantity utmost point significantly improves, and is 3.92 × 10
9/ ware, melanin deposition also obviously increases.
Embodiment 3, rice falciform Saksenaea vasiformis mutants which had Harpophora oryzae R5-6-1-SS1(CCTCC NO:M 2012314) lethal effect to paddy rice:
MS substratum (1L): saltpetre 1900mg/L, ammonium nitrate 1650 mg/L, magnesium sulfate 370 mg/L, potassium primary phosphate 170 mg/L, calcium chloride 440 mg/L, manganous sulfate 22.3 mg/L, zinc sulfate 8.6 mg/L, boric acid 6.2 mg/L, potassiumiodide 0.83 mg/L, Sodium orthomolybdate 0.25 mg/L, copper sulfate 0.025 mg/L, cobalt chloride 0.025 mg/L, ferrous sulfate 27.8 mg/L, Na2EDTA37.3 mg/L, glycine 2.0 mg/L, vitamin 0.1 mg/L, pyridoxine hydrochloride 0.5 mg/L, nicotinic acid 0.5 mg/L, inositol 100 mg/L, 30g sucrose, agar powder 8g, H
2o is settled to 1L, pH value 5.8.
Rice paddy seed (CO39) shells, and with 75% alcohol immersion 5 min, subsequently seed is placed on to the NaClO solution surface sterilization 8-10 min(with 1% in triangular flask and fully shakes up), finally repeatedly rear for subsequent use by sterile water wash.Above-mentioned seed is evenly laid on pre-prepd MS flat board, after seal plate with Parafilm sealed membrane, cultivate (16h illumination/8h dark) at 25 DEG C of constant incubators.After 5 days, axenic germination seed consistent allometry is moved into 1/2 MS(Murashige and Skoog minimum medium) in the flat board of substratum (13cm × 13cm), 5 seedlings of every plate access 3 R5-6-1-SS1 bacterium cakes (diameter: 0.5cm) of cultivating in advance simultaneously.Control group 1 is the CM agar block that does not contain bacterial strain, and control group 2 is the CM agar block containing R5-6-1 bacterial strain.If 5 repetitions.Cultivate altogether after 30 days, observe the growing way of rice seedlings.
The growing way (Fig. 2) of visual inspection rice seedlings, finds: the treatment group plant overground part of inoculation Harpophora oryzae R5-6-1-SS1 is organized withered death, and the plant of control group 1 and 2 grows fine.
Fluorescence microscopy Microscopic observation Harpophora oryzae R5-6-1-SS1 determines the situation of growing and the impact on root growth and development root: observations (Fig. 3) is found: mutant mycelia is concentrated and is distributed in root-hair zone, especially root wool base, mycelia is intensive, hypha biomass is many, and mycelia enters endodermis by cortex, finally infect and surely grow the pillar cell and vascular tissue in root system.Meanwhile, the root Root hair curling of surely being grown is out of shape, and grows limitedly, and length shortens, and occurs dun scab.Compared with wild type strain is only surely grown and surely grown pattern in epidermis with the space constraint of cortex, pathotype bacterial strain can infect the vascular tissue of surely growing root, is nonrestrictive surge type growth.
Embodiment 4, rice falciform Saksenaea vasiformis mutants which had Harpophora oryzaeR5-6-1-SS1(CCTCC NO:M 2012314) pathogenic to infecting of barley leaves:
Barley (ZJ-8) light/6d that secretly grows seedlings, the first leaf of clip surface nondestructive, for inoculation test.25 DEG C, dark rice falciform Saksenaea vasiformis wild-type R5-6-1 and the mutants which had R5-6-1-SS1 of cultivating on CM solid medium, within 10 days, beat and get bacterium cake with the punch tool of 0.8cm bore afterwards, by pure culture biscuits involvng inoculation in vitro barley leaves, 3 bacterium cakes of every leaf, 3 repetitions, 25 DEG C, 12/12h light/dark moisturizing is cultivated, and observes and record incidence after 4d, and test repeats 3 times.
Result (Fig. 4) is found: R5-6-1-SS1 causes serious harm to barley leaves, have very strong pathogenic, inoculation place produces the larger diffustivity black scab of area, and scab can diffuse to healthy area around along vein fast, scab middle section rots gradually, and exists a large amount of mycelia; By contrast, wild type strain does not form scab, pathogenic without infecting.
The barley leaves of inoculating by microscopic examination, the situation of surely growing that infects of observing bacterial strain.Found that: R5-6-1-SS1 can form a large amount of auburn appressoriums (Fig. 5) at blade surface, appressorium forms then infects epidermis and the subcutaneous cell that mycelia is directly infected host, finally surely grow in the vascular tissue of vein, diffuse to periphery position by vein, the blade cell of surely being grown and vascular bundle necrocytosis (Fig. 6).
Embodiment 5, rice falciform Saksenaea vasiformis mutants which had Harpophora oryzaeR5-6-1-S22(CCTCC NO:M 2012315) morphologic observation:
On CM substratum, observe colonial morphology, color, growth velocity, sporulation quantity and the melanin deposition of mutants which had.Observations (Fig. 7) shows: mutants which had is 25 DEG C of dark cultivations after 5d on CM substratum, and colony diameter is respectively 2.8cm, 2.5cm.Compared with wild type strain, aerial hyphae is more rare, bacteria colony white, and colony edge is neat also to be extended radially, and the sporulation quantity utmost point significantly improves, and is 3.34 × 10
9/ ware, melanin deposition also obviously reduces, and does not almost have.
Embodiment 6, rice falciform Saksenaea vasiformis mutants which had Harpophora oryzaeR5-6-1-S22(CCTCC NO:M 2012315) growth-promoting functions to paddy rice:
Method is with reference to embodiment 3.
After 5 days, axenic germination seed consistent allometry is moved into 1/2 MS(Murashige and Skoog minimum medium) in the flat board of substratum (13cm × 13cm), 5 seedlings of every plate, access 3 R5-6-1-S22 bacterium cakes (diameter: 0.5cm) of cultivating in advance simultaneously, control group 1 is the CM agar block that does not contain bacterial strain, and control group 2 is the CM agar block containing wild-type R5-6-1 bacterial strain.If 5 repetitions.Cultivate altogether after 30 days, observe the growing way of rice seedlings.
The growing way (Fig. 8) of visual inspection rice seedlings, find: the treatment group plant overground part tissue growth of inoculation Harpophora oryzaeR5-6-1-S22 is vigorous, with respect to control group 1, width of blade has increased by 39.7%, chlorophyll content has increased by 88.2%, than control group 2, width of blade has increased by 10.5%, and chlorophyll content has increased by 28%.Illustrate: Harpophora oryzaeR5-6-1-S22 is more more remarkable than the growth-promoting functions of wild type strain.
Fluorescence microscopy Microscopic observation Harpophora oryzaeR5-6-1-S22 determines the situation of growing and the impact on root growth and development root: observations (Fig. 9) is found: mycelia mainly distributes and surely grows on root system surface equably, root-hair zone is slightly many, promote the growth of root hair, no pathogenicity.The mycelia of the overwhelming majority is gathered near rhizosphere, only surely grows in epidermal area and exodermis, does not infect endodermis and the middle pillar cell.Harpophora oryzaeR5-6-1-S22 is only limited to rhizosphere around and determines to grow pattern with epidermis and appearance cortex, and the characteristic of promotion host growth, it is more similar to mycorrhizal fungi and fabaceous fixed nitrogen class fungi in soil, be centered around the dietetic alimentation that rhizosphere promotes root system around, increase absorption area, thereby be beneficial to the growth of plant.
Embodiment 7, rice falciform Saksenaea vasiformis mutants which had Harpophora oryzae R5-6-1-RR19(CCTCC NO:M 2012312) morphologic observation:
On CM substratum, observe colonial morphology, color, growth velocity, sporulation quantity and the melanin deposition of mutants which had.
Observations (Figure 10) shows: mutants which had Harpophora oryzae R5-6-1-RR19 is 25 DEG C of dark cultivations after 5d on CM substratum, and colony diameter is respectively 4.5cm, 4.3cm.Compared with wild type strain, aerial hyphae is more dense, bacterium colony Dark grey, colony edge irregular and radially extend, the sporulation quantity utmost point significantly improves, and is 3.94 × 10
9/ ware, melanin deposition also obviously increases.
Embodiment 8, rice falciform Saksenaea vasiformis mutants which had Harpophora oryzaeR5-6-1-RR19(CCTCC NO:M 2012312) lethal effect to paddy rice:
Method is with reference to embodiment 3.
After 5 days, axenic germination seed consistent allometry is moved into 1/2 MS(Murashige and Skoog minimum medium) in the flat board of substratum (13cm × 13cm), 5 seedlings of every plate, access 3 R5-6-1-RR19 bacterium cakes (diameter: 0.5cm) of cultivating in advance simultaneously, control group 1 is the CM agar block that does not contain bacterial strain, and control group 2 is the CM agar block containing R5-6-1 bacterial strain.If 5 repetitions.Cultivate altogether after 30 days, observe the growing way of rice seedlings.
The growing way (Figure 11) of visual inspection rice seedlings, finds: the treatment group plant overground part of inoculation Harpophora oryzae R5-6-1-RR19 is organized withered death, and the plant of control group 1 and 2 grows fine.
Fluorescence microscopy Microscopic observation Harpophora oryzae R5-6-1-RR19 determines the situation of growing and the impact on root growth and development root: observations (Figure 12) is found: mutant mycelia is concentrated and is distributed in root-hair zone, especially root wool base, mycelia is intensive, hypha biomass is many, and mycelia enters endodermis by cortex, finally infect and surely grow the pillar cell and vascular tissue in root system.Meanwhile, the root Root hair curling of surely being grown is out of shape, and grows limitedly, and length shortens, and occurs dun scab.Compared with wild type strain is only surely grown and surely grown pattern in epidermis with the space constraint of cortex, pathotype bacterial strain can infect the vascular tissue of surely growing root, is nonrestrictive surge type growth.
Embodiment 9, rice falciform Saksenaea vasiformis mutants which had Harpophora oryzae R5-6-1-RR19(CCTCC NO:M 2012312) pathogenic to infecting of barley leaves:
Barley (ZJ-8) light/6d that secretly grows seedlings, the first leaf of clip surface abrasion, for inoculation test.25 DEG C, dark rice falciform Saksenaea vasiformis wild-type R5-6-1 and the mutants which had Harpophora oryzae R5-6-1-RR19 of cultivating on CM solid medium, within 10 days, beat and get bacterium cake with the punch tool of 0.8cm bore afterwards, by pure culture biscuits involvng inoculation in vitro barley leaves, 3 bacterium cakes of every leaf, 3 repetitions, 25 DEG C, 12/12h light/dark moisturizing is cultivated, and observes and record incidence after 4d, and test repeats 3 times.
Result (Figure 13) is found: Harpophora oryzae R5-6-1-RR19 causes serious harm to barley leaves, have very strong pathogenic, inoculation place produces the larger diffustivity black scab of area, and scab can diffuse to healthy area around along vein fast, scab middle section rots gradually, and exists a large amount of mycelia; By contrast, wild type strain does not form scab, pathogenic without infecting.
The barley leaves of inoculating by microscopic examination, the situation of surely growing that infects of observing bacterial strain.Found that: Harpophora oryzae R5-6-1-RR19 mycelia is directly infected epidermis and subcutaneous cell from blade wound, finally surely grow in the vascular tissue of vein, diffuse to periphery position by vein, the blade cell of surely being grown and vascular bundle necrocytosis (Figure 14).
Embodiment 10, rice falciform Saksenaea vasiformis mutants which had Harpophora oryzae R5-6-1-RR21(CCTCC NO:M 2012313) morphologic observation:
On CM substratum, observe colonial morphology, color, growth velocity, sporulation quantity and the melanin deposition of mutants which had.
Observations (Figure 15) shows: Harpophora oryzae R5-6-1-RR21 mutants which had is 25 DEG C of dark cultivations after 5d on CM substratum, and colony diameter is respectively 5.4cm, 5.4cm.Compared with wild type strain, aerial hyphae is slightly dense, bacterium colony canescence, and the neat shape of also crawling radially of colony edge extends, and sporulation quantity is 3.25 × 10
9/ ware, melanin deposition also obviously reduces.
Embodiment 11, rice falciform Saksenaea vasiformis mutants which had Harpophora oryzae R5-6-1-RR21(CCTCC NO:M 2012313) lethal effect to paddy rice:
Method is with reference to embodiment 3.
After 5 days, axenic germination seed consistent allometry is moved into 1/2 MS(Murashige and Skoog minimum medium) in the flat board of substratum (13cm × 13cm), 5 seedlings of every plate, access 3 R5-6-1-RR21 bacterium cakes (diameter: 0.5cm) of cultivating in advance simultaneously, control group 1 is the CM agar block that does not contain bacterial strain, and control group 2 is the CM agar block containing R5-6-1 bacterial strain.If 5 repetitions.Cultivate altogether after 30 days, observe the growing way of rice seedlings.
The growing way (Figure 16) of visual inspection rice seedlings, finds: the treatment group plant overground part of inoculation Harpophora oryzaeR5-6-1-RR21 is organized withered death, and the plant of control group 1 and 2 grows fine.
Fluorescence microscopy Microscopic observation Harpophora oryzaeR5-6-1-RR21 determines the situation of growing and the impact on root growth and development root: observations (Figure 17) is found: mutant mycelia enters endodermis by cortex, finally infects and surely grows the pillar cell and vascular tissue in root system.Compared with wild type strain is only surely grown and surely grown pattern in epidermis with the space constraint of cortex, pathotype bacterial strain can infect the vascular tissue of surely growing root, is nonrestrictive surge type growth.
Embodiment 12, rice falciform Saksenaea vasiformis mutants which had Harpophora oryzae R5-6-1-RR21(CCTCC NO:M 2012313) pathogenic to infecting of barley leaves:
Barley (ZJ-8) light/6d that secretly grows seedlings, the first leaf of clip surface abrasion, for inoculation test.25 DEG C, dark rice falciform Saksenaea vasiformis wild-type R5-6-1 and the mutants which had R5-6-1-RR21 of cultivating on CM solid medium, within 10 days, beat and get bacterium cake with the punch tool of 0.8cm bore afterwards, by pure culture biscuits involvng inoculation in vitro barley leaves, 3 bacterium cakes of every leaf, 3 repetitions, 25 DEG C, 12/12h light/dark moisturizing is cultivated, and observes and record incidence after 4d, and test repeats 3 times.
Result (Figure 18) is found: Harpophora oryzae R5-6-1-RR21 causes comparatively serious harm to barley leaves, have stronger pathogenic, inoculation place produces the larger diffustivity black scab of area, and scab can diffuse to healthy area around along vein fast, scab middle section rots gradually, and exists a large amount of mycelia; By contrast, wild type strain does not form scab, pathogenic without infecting.
The barley leaves of inoculating by microscopic examination, the situation of surely growing that infects of observing bacterial strain.Found that: Harpophora oryzae R5-6-1-RR21 mycelia is directly infected host's epidermis and subcutaneous cell, finally surely grow in the vascular tissue of vein, diffuse to periphery position by vein, the blade cell of surely being grown and vascular bundle necrocytosis (Figure 19).
Finally, it is also to be noted that, what more than enumerate is only several specific embodiments of the present invention.Obviously, the invention is not restricted to above embodiment, can also have many distortion.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention, all should think protection scope of the present invention.
Claims (1)
1. endogenetic fungal bacterial strain RR21, is characterized in that: for rice chain Saksenaea vasiformis (
harpophora oryzae) R5-6-1-RR21, its preserving number is: CCTCC NO:M 2012313.
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