Patent Application
20110020289
The present invention relates to a novel mycovirus that suppresses a phytopathogenic fungus, and to genes, nucleic acids, and proteins related to the mycovirus, an attenuated phytopathogenic fungal strain incorporating the mycovirus, a plant disease control agent containing the above, a mycovirus production method, a method for attenuating phytopathogenic fungus, a method for controlling plant disease, etc.
As plant diseases, there are those caused by climate, soil, or other environmental cause, those caused by a virus, bacteria, fungus (filamentous fungus), or other infective cause, those caused by a physiological disorder, and those caused by a compounded cause of the above. Even presently, there are numerous plant diseases that are impediments to production of foods, flowers, flowering trees, timbers, etc., and many of these diseases are high in economic impact.
Among plant diseases, fungi represent one of the most important pathogenic factors. It is said that approximately 80% of plant diseases are caused by fungi.
For example, blast is one of the most important plant diseases and occurs in various parts of the world. The pathogen is rice blast fungus (scientific name: “Magnaporthe oryzae”), which is a type of mold (filamentous fungus). For rice blast fungus, a suitable temperature for growth, spore formation, and infection is around 25° C. and the fungus favors humid environments. Outbreak of this fungus thus occurs due to low summer temperatures, heavy rainfall, inadequate sunlight, and other climatic causes to bring about crop failure and quality degradation of rice and apply a serious impact on economy.
There are numerous other plant diseases that are caused by fungi and are high in economic impact, such as sheath blight, rust, powdery mildew, anthracnose, sclerotinia rot, downy mildew, gray mold, etc. Development of novel agricultural chemicals, breed improvement, etc., are thus being carried out even today (in regard to control of blast, see, for example, Patent Documents 1 and 2).
Mycoviruses, which are relevant to the present invention, shall now be described.
A virus that infects fungi is called a mycovirus. Among these, mycoviruses having double-stranded RNA as a genome have been reported. Many of these mycoviruses infect host fungi latently and have hardly any impact on characteristics of the host.
Mycoviruses having a double-stranded RNA as the genome are presently classified into five families, including Partitiviridae, Totiviridae, and Chrysoviridae. Partitiviridae viruses have two straight-chain double-stranded RNAs of substantially the same size inside a virus particle and have a total gene size of 4 to 6 kbp. Totiviridae viruses have a single straight-chain double-stranded RNA of 4 to 7 kbp inside a virus particle. Besides the above, in chestnut blight fungus (scientific name: “Cryphonectria parasitica”), a virus that is present endogenously in the fungus and having a double-stranded RNA of 9 to 13 kbp has been discovered (hypovirus, etc.).
Chrysoviridae viruses have a spherical, virus-like particle and double-stranded RNAs of four components. These viruses, like Partitiviridae and Totiviridae viruses, are known to have a region encoding RdRP (RNA-directed RNA polymerase; RNA-dependent RNA polymerase, the same applies hereinafter). As examples of viruses belonging to Chrysoviridae, Hv145SV (“Helminthosporium victoriae 145S virus,” the same applies hereinafter), PcV (“Pencillium chrysogenum virus,” the same applies hereinafter), AbV1 (“Agaricus bisporus virus 1,” the same applies hereinafter), etc., are known (see Non-Patent Document 1, etc.)
Recently, with respect to specific plant diseases, methods for attenuating the pathogen by a mycovirus, etc., and using the attenuated pathogen to control the disease are being examined and have come to be put to practical use in part. For example, a method of using a full-length cDNA of viral double-stranded RNA that suppresses toxicity of chestnut blight fungus to attenuate the fungus and apply the attenuated fungus to control of chestnut blight (see Non-Patent Document 2, etc.), a method of discovering a double-stranded RNA virus that suppresses violet root rot fungus (scientific name: “Helicobasidium mompa”) and using an attenuated violet root rot fungal strain incorporating the double-stranded RNA to control violet root rot (see Non-Patent Document 3, Patent Document 3, etc.), etc., have been disclosed.
As mentioned above, phytopathogenic fungi have high impacts on quality, yield, etc., of plants and crops. Meanwhile, discoveries of mycoviruses that suppress phytopathogenic fungi have been few and attempts at controlling plant diseases using such mycoviruses have hardly been made. A main object of the present invention is to provide a novel mycovirus that suppresses phytopathogenic fungi and a novel method for controlling plant diseases.
The present inventors independently collected naturally-occurring phytopathogenic fungi and searched for mycoviruses that act suppressingly on these fungi. As a result, we have isolated and identified a novel mycovirus exhibiting a phytopathogenic fungus suppressing action and having four types of double-stranded RNA of 2.8 to 3.6 kb and have determined a full-length sequence of the mycovirus.
The present invention thus provides the novel, naturally-occurring mycovirus, isolated by the present inventors, that exhibits the phytopathogenic fungus suppressing action and has the four types of double-stranded RNA of 2.8 to 3.6 kb, and also provides genes thereof, base sequences thereof, nucleic acids having at least a sequence of a specific portion, within the aforementioned sequences, having a specific function, one or a plurality of proteins encoded by the aforementioned sequences, etc.
The mycovirus has a conserved motif of RdRp (RNA-dependent RNA polymerase) contained in the double-stranded RNA sequence, and the RNA base sequence encoding this region is homologous among known Chrysoviridae viruses. The mycovirus is thus presumed to be a novel species of virus classified under Chrysoviridae.
As mentioned above, the present mycovirus exhibits an action of suppressing growth, etc., of phytopathogenic fungi. Thus, for example, by infecting, etc., a host fungus with the mycovirus and making the mycovirus endogenous in the host fungus, an attenuated strain of the phytopathogenic fungus can be prepared.
Or, for example, by adding (by spraying, coating, etc.) a plant disease control agent, containing at least either or both of the mycovirus according to the present invention and the prepared attenuated strain of the phytopathogenic fungus, to a plant (rice, etc.), there is a possibility of being able to control the corresponding plant disease.
Besides the above, the mycovirus according to the present invention has the following characteristics.
Conventionally, a mycovirus is regarded to be transmitted vertically from cell to cell of a host fungus via hyphal fusion and it is considered that a life cycle of a mycovirus does not have a stage where the mycovirus is present outside a host fungus cell. In contrast, by research of the present inventors, it has become known that the mycovirus according to the present invention can be present extracellularly.
Because a host cell can thus be infected with the mycovirus according to the present invention from outside the cell and without depending on hyphal fusion, the mycovirus can infect widely across fungal hosts and strains that differ in conjugation type and can infect host fungi at high efficiency. That is, there is thus a high possibility of achieving attenuation of phytopathogenic fungi and control of plant diseases easily and highly efficiently.
Also, because the mycovirus according to the present invention can be present extracellularly, for example, by culturing a host fungus in a liquid medium and recovering the mycovirus from a supernatant of the culture, the mycovirus can be produced easily and at a comparatively large amount.
The present invention provides a possibility of controlling plant diseases easily and highly efficiently.
The present invention includes all mycoviruses exhibiting a phytopathogenic fungus suppressing action and having four types of double-stranded RNA of 2.8 to 3.6 kb.
With the present mycovirus, each virus particle has any one of the four types of double-stranded RNA of 2.8 to 3.6 kb, and the mycovirus subsists and repeats generations in a state where virus particles of the four types are present simultaneously. In addition, each of the abovementioned chestnut blight fungus mycovirus and violet root rot fungus mycovirus is made up of a double-stranded RNA of a single component of no less than 12 kb and differs largely in structure from the mycovirus according to the present invention. Therefore, there is a high possibility that the mycovirus according to the present invention is a completely different virus species and there is also a high possibility that a mechanism of attenuation of a host fungus differs greatly as well.
As mentioned above, the mycovirus has the conserved motif of RdRp (RNA-dependent RNA polymerase) contained in the double-stranded RNA sequence, and the base sequence of the RNA encoding this region is homologous among known Chrysoviridae viruses. The mycovirus is thus presumed to be a novel species of virus classified under Chrysoviridae.
Base sequences of the four types of the double-stranded RNA of the mycovirus are indicated by SEQ ID NOS: 1 to 4, and amino acid sequences of proteins encoded by the base sequences are indicated by SEQ ID NOS: 5 to 8.
Although all base sequences shown in the sequence table are indicated as DNA sequences, the sequences according to the present invention includes these sequences and all sequences in the case of RNA (where thymine is replaced by uracil) (the same applies hereinafter).
In regard to the base sequence of the region encoding RdRp (RNA-dependent RNA polymerase), sequence analysis has shown the homology to be 22% and the similarity to be 39% between the mycovirus and Hv145SV, the homology to be 23% and the similarity to be 38% between the mycovirus and PcV, and the homology to be 28% and the similarity to be 45% between the mycovirus and AbV1.
The mycovirus according to the present invention thus includes all viruses for which, with respect to the region encoding RdRP (RNA-dependent RNA polymerase) in the genome of a Chrysoviridae virus (for example, any one of Hv145SV, PcV, and AbV1), the homology is 20% to 100% and the similarity is 40% to 100%. Here “homology” refers to complete matching of base sequences, and “similarity” refers to complete matching of base sequences upon replacement of adenine with guanine or thymine (uracil) with cytosine.
The mycovirus according to the present invention is present endogenously in a predetermined rice blast fungus. The mycovirus can thus be acquired, for example, by separating and recovering the virus from a rice blast fungal strain latently infected by the mycovirus. In addition, the rice blast fungus endogenously containing the mycovirus according to the present invention can be cultured under the same medium and culturing conditions as those of a normal rice blast fungus.
As an example of a mycovirus according to the present invention, for example, MoCV1 (Magnaporthe oryzae chrysovirus 1), identified and named by the present inventors, can be cited. This virus is endogenous at least in an “S-0412-II 1a” strain of rice blast fungus (scientific name: “Magnaporthe oryzae”). Although depositions of this rice blast fungal strain at the International Patent Organism Depositary of the National Institute of Advanced Industrial Science and Technology and at the Patent Microorganisms Depositary of the National Institute of Technology and Evaluation were requested, the depositions were refused on grounds of endogenously containing a virus. The present fungal strain is preserved at the Laboratory of Plant Pathology, Tokyo University of Agriculture and Technology and can be shared with a third party under conditions of adherence to laws and regulations.
The “S-0412-II 1a” strain of rice blast fungus (scientific name: “Magnaporthe oryzae”) has been internationally deposited at the international depository in Germany, DSMZ (Deutsche Sammlung con Mikroorganismen and Zellkulturen GmbH, address: Inhoffenstr. 7B D-38124 Braunschweig GERMANY) (date of deposition: Mar. 25, 2008, deposition number: DSM21334). The same strain has also been internationally deposited at the international depository in the USA, ATCC (American Type Culture Collection, address: University Boulevard Manassas, Va. 20110-2209 USA) (date of deposition: May 15, 2008, deposition number: PTA-9137). The country of origin of this fungal strain is Vietnam.
Known methods may be employed to separate and recover the virus. For example, a fungus body may be frozen by liquid nitrogen, etc., fragmented, and suspended in a predetermined buffer solution and thereafter, the virus can be recovered by separating the virus by ultracentrifugation, etc.
Also, as mentioned above, the present virus can be present extracellularly. Thus, for example, a phytopathogenic fungus (a predetermined rice blast fungus, etc.) containing the mycovirus may be cultured in a liquid medium and the virus may be separated and recovered from a supernatant of the culture. The present virus itself can be prepared at any time at the Laboratory of Plant Pathology, Tokyo University of Agriculture and Technology and be shared with a third party.
The present inventors performed sequence analysis and obtained full-length base sequences of one mycovirus according to the present invention (SEQ ID NOS: 1 to 4). Therefore, the present invention thus includes all of: genes of the mycovirus; nucleic acids having the base sequences or portion thereof; proteins encoded by the base sequences, etc.
The present invention includes mycovirus genes having the four types of sequences of SEQ ID NOS: 1 to 4.
The present invention also includes all nucleic acids having all or a portion of the base sequences. The nucleic acid may be double-stranded or single-stranded, and all of DNAs, cDNAs, RNAs, etc., are included.
The present invention also includes all sequences or any sequence of SEQ ID NOS: 1 to 4, sequences of specific portions in the aforementioned sequences having predetermined functions, cDNAs having base sequences equivalent to the aforementioned sequences, recombinant vectors (plasmids, viruses, etc.) incorporating base sequences equivalent to the aforementioned sequences, etc.
As a result of the sequence analysis, it was found that the conserved motif of RdRp (RNA-dependent RNA polymerase) is present in a sequence portion of SEQ ID NO: 1 and that a sequence portion of SEQ ID NO: 3 is homologous to a double-stranded RNA fragment of a La France disease virus. Thus, in accordance with purpose or application, a nucleic acid, a recombinant vector, etc., having at least these sequence portions as specific portions having predetermined functions may be prepared and used, for example.
In addition, the nucleic acids (or genes) according to the present invention also broadly include nucleic acids homologous to the aforementioned base sequences, for example, nucleic acids that hybridize under stringent conditions with nucleic acids made up of base sequences complementary to the base sequences and exhibits rice blast fungus suppressing action. The stringent conditions here, such as a Tm value of a double-stranded nucleic acid, can be acquired by known arts.
The present invention also includes all proteins encoded by the aforementioned mycovirus genes and nucleic acids. Amino acid sequences of proteins according to the present invention are indicated by SEQ ID NOS: 5 to 8.
Respectively speaking, SEQ ID NO: 5 is an amino acid sequence of an open reading frame in the base sequence indicated by SEQ ID NO: 1, SEQ ID NO: 6 is an amino acid sequence of an open reading frame in the base sequence indicated by SEQ ID NO: 2, SEQ ID NO: 7 is an amino acid sequence of an open reading frame in the base sequence indicated by SEQ ID NO: 3, and SEQ ID NO: 8 is an amino acid sequence of an open reading frame in the base sequence indicated by SEQ ID NO: 4.
All proteins homologous to and maintaining the functions of the proteins having any one of the amino acid sequences of SEQ ID NOS: 5 to 8 are included among the proteins according to the present invention.
For example, by incorporating an abovementioned nucleic acid in a recombinant vector and subjecting the host to forced expression, the corresponding protein can be produced in large quantities. It is considered that coliform bacteria, yeasts, cultured cells, and other known hosts can be used as the host. In consideration that the mycovirus infects fungus and that yeasts have a high proliferation ability and can be used comparatively simply, yeasts are possibly optimal hosts. It is also considered in regard to recombinant vectors that a known vector can be used.
There is a possibility that by using a plurality of vectors incorporating any one of SEQ ID NOS: 1 to 4 and making a plurality of the four types of genes be coexpressed, the mycovirus can be reconstituted. It is considered that a known host and a known recombinant vector can be used in this case as well, and there is a possibility that yeasts are optimal in that a plurality of vectors can be introduced simultaneously.
Attenuated phytopathogenic fungal strains according to the present invention include all attenuated strains endogenously incorporating the mycovirus according to the present invention. That is, for example, both rice blast fungus and other strains already containing the mycovirus endogenously and phytopathogenic fungal strains infected by the mycovirus are included.
As a method for infecting, etc., a phytopathogenic fungus with a mycovirus, for example, there is a method of infecting a host fungus during hyphal fusion as is done conventionally. Also, because as mentioned above, the mycovirus according to the present invention can be present extracellularly, for example, it can be made to infect a host fungus directly from outside the cell.
The abovementioned S-0412-II 1a strain can be cited as an example of the attenuated phytopathogenic fungal strain. This fungal strain is a strain of rice blast fungus infected by the mycovirus according to the present invention. Therefore, its morphological characteristics, culturing characteristics, spore formation, and physiological and chemotaxonomic characteristics are thus basically the same as those of known rice blast fungus. However, in comparison to normal rice blast fungus, the present fungal strain is slower in growth, has hyphae that grow non-concentrically, and is pigmented non-uniformly. Also, abnormal development of aerial hyphae is seen and sector formation and lysis are observed.
Plant disease control agents according to the present invention include all plant disease control agents containing at least one of either the mycovirus according to the present invention or the attenuated phytopathogenic fungal strain according to the present invention. In addition, both the mycovirus and attenuated phytopathogenic fungal strain may be contained and other components may be contained as well.
As the other components, for example, a predetermined carrier, binder, thickener, fixing agent, antiseptic/antifungal agent, solvent, stabilizer, antioxidant, antiultraviolet agent, anticrystallization agent, antifoam agent, property improver, colorant, etc., may be contained. Also, other agricultural chemical components, such as a miticide, nematicide, bactericide, antiviral agent, attractant, herbicide, plant growth regulator, synergist, etc., may be contained.
As a carrier, for example, either or both of a solid carrier and a liquid carrier may be used. Powders of plant and animal origin, such as starch, activated carbon, soy flour, flour, wood flour, fish flour, powder milk, etc., and mineral powders, such as talc, kaolin, bentonite, zeolite, diatomaceous earth, white carbon, clay, alumina, calcium carbonate, potassium chloride, ammonium sulfate, etc., can be cited as examples of a solid carrier. Water, alcohols, such as isopropyl alcohol, ethylene glycol, etc., ketones, such as cyclohexanone, methyl ethyl ketone, etc., ethers, such as propylene glycol monomethyl ether, diethylene glycol mono-n-butyl ether, etc., aliphatic hydrocarbons, such as kerosene, light oil, etc., aromatic hydrocarbons, such as xylene, trimethylbenzene, tetramethylbenzene, methylnaphthalene, solvent naphtha, etc., amides, such as N-methyl-2-pyrrolidone, etc., esters, such as fatty acid glycerin esters, etc., and vegetable oils, such as soy oil, rape oil, etc., can be cited as examples of a liquid carrier.
Starch, dextrin, cellulose, methylcellulose, ethylcellulose, carboxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, carboxymethyl starch, pullulan, sodium alginate, ammonium alginate, alginic acid propylene glycol ester, guar gum, locust bean gum, gum arabic, xanthan gum, gelatin, casein, polyvinyl alcohol, polyethylene oxide, polyethylene glycol, ethylene/propylene block copolymer, sodium polyacrylate, polyvinylpyrrolidone, etc., can be cited examples of a binder, thickener, or fixing agent.
The present control agent is not particularly restricted in dosage form. For example, forms, such as emulsion formulation, suspension formulation, powder formulation, granular formulation, tablet formulation, water-dispersible formulation, water-soluble formulation, liquid formulation, flowable formulation, granular water-dispersible formulation, aerosol formulation, paste formulation, oil formulation, latex formulation, etc., can be applied.
Methods for producing the phytopathogenic fungus suppressing mycovirus according to the present invention include all methods that include at least procedures of culturing a phytopathogenic fungus containing the mycovirus in a liquid medium, etc., and recovering the mycovirus from a supernatant of the culture.
As mentioned above, the mycovirus according to the present invention can be present extracellularly of the host fungus. Thus, for example, by culturing a phytopathogenic fungus, infected by the mycovirus, in a liquid medium, etc., separating the virus from the fungal body by centrifugation, and thereafter separating and recovering the virus from the culture supernatant, the virus can be recovered easily and at a comparatively large amount.
However, the mycovirus according to the present invention is not narrowly restricted to that which is obtained by the above production method. Thus, for example, mycoviruses separated and recovered from rice blast fungi containing mycoviruses endogenously are broadly included in the present invention.
As mentioned above, for example, by making the mycovirus according to the present invention infect, etc., a specific pathogenic fungus, growth, etc., of the host fungus can be suppressed and the fungus can be attenuated. As methods for infecting with the mycovirus, the same methods as those mentioned above may be adopted.
Methods for controlling plant disease according to the present invention include all methods that include at least a step of adding the abovementioned rice blast control agent to a specific plant (such as rice, etc.).
As methods for adding the control agent to a plant, methods of coating top surfaces or rear surfaces of leaves with the control agent, methods of attaching the control agent to top surfaces or rear surfaces of leaves using a predetermined carrier, etc., methods of spraying or supplying the control agent onto leaves, etc., can be cited as examples.
A coating amount or spraying amount of the control agent may be selected as suited according to various conditions, such as concentrations of effective components, form of preparation, type of targeted disease or crop, degree of damage due to disease, location of application, method of application, timing of application, usage amounts and types of chemicals, fertilizers, etc., mixed or used in combination, etc.
For example, there is a possibility that a plant disease can be controlled by spraying or supplying a liquid, containing conidia of an attenuated rice blast fungal strain and adjusted to 1×103 to 1×1010 conidia/mL, at an amount of 1 to 1,000 mL per leaf or by coating or attaching conidia of an attenuated rice blast fungal strain to top surfaces or rear surfaces of leaves at an amount of 1×103 to 1×1010 conidia per 1 mm2 of leaf.
There is a possibility that the present invention can be applied to all plant diseases for which a fungus is a main cause. As plant diseases for which there is a possibility of application, the following can be cited as examples (however, the present invention is not limited to these).
Examples of plant diseases of plants of the family Poaceae include rice blast (causative fungus: “Magnaporthe oryzae”), rice brown spot (causative fungus: “Cochliobolus miyabeanus”), sheath blight (causative fungus: “Thanatephorus cucumeris”), bakanae disease (causative fungus: “Gibberella fujikuroi”), seedling blight (causative fungi: “Fusarium fungi,” “Rhizopus fungi,” “Pythium fungi,” “Trichoderma viride”), false smut (causative fungus: “Claviceps virens”), fusarium blight of wheat (causative fungi: “Gibberella zeae,” “Fusarium avenaceum,” “Fusarium culmorum,” “Monographella nivale”), snow mold (causative fungi: “Pythium fungi,” “Typhula fungi,” “Monographella nivalis,” “Myriosclerotinia borealis”), loose smut (causative fungus: “Ustilago nuda”), stinking smut (causative fungus: “Tilletia controversa”), eye spot (causative fungus: “Pseudocercosporella herpotrichoides”), leaf blotch (causative fungus: “Septoria trifler”), glume blotch (causative fungus: “Phaeosphaeria nodorum”), and powdery mildew (causative fungus: “Blumeria graminis”).
Examples of other plant diseases include melanose (causative fungus: “Diaporthe citri”), melanose-like blemish (causative fungi: “Diaporthe medusa,” “Alternaria citri”), scab (causative fungus: “Elsinoe fawcettii”), brown rot (causative fungus: “Phytophthora citrophthra”), green mold (causative fungus: “Penicillium digitatum”), and blue mold (causative fungus: “Penicillium italicum”) of citruses, blossom blight (causative fungus: “Monilinia mali”), scab (causative fungus: “Venturia inaequalis”), alternaria blotch (causative fungus: “Alternaria mali”), fruit spot (causative fungus: “Mycosphaerella pomi”), sooty blotch (causative fungus: “Gloeodes pomigena”), fly speck (causative fungus: “Zygophiala jamaicensis”), ring rot (causative fungus: “Botryosphaeria berengeriana”), blotch (causative fungus: “Diplocarpon mali”), rust (causative fungus: “Gymnosporangium yamadae”), and valsa canker (causative fungus: “Valsa ceratosperma”) of apple, scab (causative fungus: “Venturia nashicola”), rust (causative fungus: “Gymnosporangium asiaticum”), ring rot (causative fungus: “Botryosphaeria berengeriana”), and phomopsis canker (causative fungus: “Phomopsis fukushii”) of pear, leaf curl (causative fungus: “Taphrina deformans”), brown rot (causative fungi: “Monilinia fructicola,” “Monilinia fructigena”), scab (causative fungus: “Cladosporium carpophilum”), and phomopsis rot (causative fungus: “Phomopsis sp.”) of peach, brown rot (causative fungi: “Monilinia fructicola,” “Monilinia fructigena”) and young fruit rot (causative fungus: “Monilinia kusanoi”) of cherry, scab (causative fungus: “Cladosporium carpophilum”) of Japanese apricot, anthracnose (causative fungus: “Elsinoe ampelina”), ripe rot (causative fungi: “Colletotrichum acutatum,” “Glomerella cingulata”), leaf blight (causative fungus: “Pseudocercospora vitis”), and dead arm (causative fungus: “Phomopsis viticola”) of grape, angular leaf spot (causative fungus: “Cercospora kaki”) and circular leaf spot (causative fungus: “Mycosphaerella nawae”) of persimmon, gray blight (causative fungi: “Pestalotiopsis longiseta,” “Pestalotiopsis theae”), brown round spot (causative fungi: “Pseudocercospora ocellata,” “Cercospora chaae”), blister blight (causative fungus: “Exobasidium vexans”), and net blister blight (causative fungus: “Exobasidium reticulatum”) of tea, gummy stem blight (causative fungus: “Mycosphaerella melonis”), fusarium wilt (causative fungus: “Fusarium oxysporum”), scab (causative fungus: “Cladosporium cucumerinum”), and corynespora leaf spot (causative fungus: “Corynespora cassiicola”) of cucurbitaceous fruits, leaf mold (causative fungus: “Fulvia fulva”), and early blight (causative fungus: “Alternaria solani”) of tomato, brown spot (causative fungus: “Phomopsis vexans”) and leaf mold (causative fungus: “Mycovellosiella nattrassii”) of eggplant, white rust (causative fungus: “Albugo macrospora”), and white spot (causative fungi: “Cercosporella brassicae,” “Pseudocercosporella capsellae”) of brassicaceous vegetables, gray mold (causative fungus: “Botrytis allii”) of onion, leaf spot (causative fungus: “Mycosphaerella fragariae”) of strawberry, early blight (causative fungus: “Alternaria solani”) of potato, stem rot (causative fungus: “Phytophthora sojae”) and purple stain (causative fungus: “Cercospora kikuchii”) of soy, stem rot (causative fungus: “Phytophthora vignae”) of adzuki bean, brown leaf spot (causative fungus: “Mycosphaerella arachidis”) of peanut, cercospora leaf spot (causative fungus: “Cercospora beticola”) and leaf blight (causative fungus: “Thanatephorus cucumeris”) of sugar beet, curvularia leaf blight (causative fungus: “Curvularia fungus”), dollar spot (causative fungus: “Sclerotinia homoeocarpa”), and Helminthosporium leaf blight (causative fungus: “Cochliobolus fungus”) of lawn grass, black spot (causative fungus: “Diplocarpon rosae”) of rose, white rust (causative fungus: “Puccinia horiana”) of chrysanthemum, downy mildew (causative fungi: “Peronospora fungus,” “Pseudoperonospora fungus,” “Plasmopara fungus,” “Bremia fungus”), blight (causative fungus: “Phytophthora fungus”), powdery mildew (causative fungi: “Erysiphe fungus,” “Blumeria fungus,” “Sphaerotheca fungus,” “Podosphaerea fungus,” “Phyllactinia fungus,” “Uncinula fungus,” “Oidiopsis fungus”), rust (causative fungi: “Puccinia fungus,” “Uromyces fungus,” “Physopella fungus”), leaf spot (causative fungus: “Alternaria fungus”), gray mold (causative fungus: “Botrytis cinerea”), sclerotina rot (causative fungus: “Sclerotinia sclerotiorum”), white root rot (causative fungus: “Rosellinia necatrix”), violet root rot (causative fungus: “Helicobasidium mompa”), southern blight (causative fungus: “Sclerotium rolfsii”) of various crops, and various other soil diseases (causative fungus: “Fusarium fungus,” “Rhizoctonia fungus,” “Pythium fungus,” “Aphanomyces fungus,” “Phoma fungus,” “Verticillium fungus,” “Plasmodiophora brassicae,” etc.).
In Example 1, detection of endogenous double-stranded RNAs from 57 rice blast fungal strains was attempted.
First, respective fungal bodies of 57 independently collected strains of rice blast fungus were milled, and after extracting nucleic acids by a phenol-SDS method, DNAs and single-stranded nucleic acids were selectively digested by DNase 1 and S1 nuclease to obtain a solution of double-stranded RNAs endogenous to the fungal bodies. Electrophoresis using a 1% agarose gel was performed at 20V for 18 hours and staining by ethidium bromide was performed.
As a result, double-stranded RNA bands were detected in 11 of the 57 strains. Of these, bands of four components were detected at 2.8 to 3.6 kb in 7 strains, bands of three components were detected at 1.0 to 2.6 kb in 3 strains, and bands of eight components were detected at 1.0 to 3.6 kb in 1 strain.
In the above experimental results, in consideration that a plurality of bands were detected from single strains and in consideration of the lengths of the double-stranded RNAs, there is a high possibility that the double-stranded RNAs detected from the 11 rice blast fungal strains are double-stranded RNAs making up a mycovirus. That is, the results of the present example suggest that a novel mycovirus is endogenous in these rice blast fungal strains.
Based on the results of Example 1, growth rates of strains containing the double-stranded RNAs endogenously and strains not containing any double-stranded RNAs endogenously were compared in Example 2.
Fungal strains with which endogenous double-stranded RNAs were detected and fungal strains with which endogenous double-stranded RNAs were not detected in Example 1 were cultured in a PDA medium and colonies were observed after 6 days and after 10 days from the start of culturing.
As a result, with fungal strains with which endogenous double-stranded RNAs were not detected, hyphae grew in concentric circles and pigmentation was uniform. On the other hand, with fungal strains with which endogenous double-stranded RNAs were detected, growth was slower than fungal strains with which RNAs were not detected, hyphae grew non-concentrically, and pigmentation was non-uniform. Also, abnormal development of aerial hyphae was seen and sector formation and lysis were observed.
That is, with the fungal strains endogenously containing double-stranded RNAs, suppression of growth was observed in comparison to the fungal strains in which double-stranded RNAs were not endogenous. These experimental results suggest that the double-stranded RNAs (mycovirus) obtained in Example 1 are growth suppression factors (attenuation factors) of rice blast fungus.
In Example 3, preparation of mycovirus-cured strains by treatment of fungal strains endogenously containing the double-stranded RNAs with low-concentration cycloheximide (protein synthesis inhibitor) was attempted.
YG plates with 0.25 to 0.50 μg/ml of cycloheximide added were prepared and the strains cultured in Example 2 (strains with which endogenous double-stranded RNAs were detected) were transferred to these plates. Of the formed colonies, portions that recovered to normal growth were transferred to separate YG plates to obtain cycloheximide-treated fungal strains. As a result, the cycloheximide-treated fungal strains exhibited better growth than normal rice blast fungal strains.
Next, from the cycloheximide-treated fungal strains obtained, extraction of double-stranded RNA and detection of double-stranded RNA bands by electrophoresis were attempted using the same methods as those of Example 1. As a result, bands of double-stranded RNA were not detected from the cycloheximide-treated fungal strains.
Mycovirus-cured fungal strains were also prepared by a different method and subject to the same experiment.
After the fungal strains cultured in Example 2 were grown for approximately 1 to 3 weeks on a PDA medium or an oatmeal medium, conidia were isolated, the conidia were transferred one by one to a new PDA medium, and fungal colonies from which the mycovirus was removed were selected as mycovirus-cured fungal strains.
As a result, even with these cured fungal strains, growth became clearly better than that of virus-carrying strains. Also, as a result of attempting detection of double-stranded RNA bands by electrophoresis in the same manner as described above, double-stranded RNA bands were not detected from the cured fungal strains.
These results indicate that the double-stranded RNAs contained endogenously in the rice blast fungus are those of a mycovirus and that this mycovirus is a growth suppression factor of rice blast fungus.
In Example 4, it was examined whether or not the mycovirus according to the present invention is present outside the fungal body of rice blast fungus.
Of the fungal strains with which endogenous double-stranded RNA bands of four components were detected at 2.8 to 3.6 kb in Example 1, three strains were transferred to and cultured in liquid media, and thereafter the culture solutions were centrifuged and culture supernatants were recovered. Next, the culture supernatants obtained were electrophoresed in 1% agarose gel at 20V for 18 hours and stained by ethidium bromide.
As a result, double-stranded RNA bands at the same positions as the fungal endogenous double-stranded RNAs were detected from the culture supernatants of these fungal strains as well.
This result indicates that the mycovirus according to the present invention can be present not only inside the fungal body of rice blast fungus but also outside the fungal body.
In Example 5, it was examined whether or not the mycovirus present outside the fungal body has an infectious ability with respect to normal strains (strains not carrying the mycovirus).
First, the fungal strains with which endogenous double-stranded RNA bands of four or more components were detected at 2.8 to 3.6 kb in Example 1 were transferred to liquid media, and after culturing for 4 weeks, the culture solutions were centrifuged and culture supernatants were recovered. As a result of electrophoresing the culture supernatants in 1% agarose gel in the same manner as in Example 4, the double-stranded RNA bands were observed. The culture supernatants obtained were filtration sterilized using 0.22 μL filters.
Next, 50 ml of YG medium were placed in a 100 ml flask, a normal strain of rice blast fungus (strain not carrying the mycovirus) was inoculated therein, and after culturing for 3 days, 500 μL of the culture supernatant obtained were added and growth of the fungal body was observed.
As a result, on the third day of observation, whereas with a strain to which the culture supernatant was not added, hypha tips grew straightly, indicating normal growth, with a strain to which the culture supernatant was added, hypha tips did not extend much and were in an entangled state.
This result is considered to be due to infection and growth suppression of the normal rice blast fungus by the mycovirus present in the culture supernatant. That is, the present experimental results suggest that the mycovirus present outside the fungal body has an infectious ability with respect to a normal fungal strain.
In Example 6, conidia count measurements were performed on rice blast fungus strains infected with the mycovirus.
The fungal strains with which endogenous double-stranded RNA bands of four or more components were detected at 2.8 to 3.6 kb in Example 1 were transferred to YG plates, and after culturing for 2 weeks, fungal bodies were extracted and collected using a 4 mm cork borer.
5% glycerol, and then, the collected fungal body were placed in a 1.5 ml tube and mixing and suspending were performed for 5 minutes. The number of conidia present in the suspension was then counted using a blood cell counting chamber.
As a result, whereas with the normal strain of rice blast fungus (control, strain not carrying the mycovirus), the conidia count was 33×104 conidia/mL, the conidia count was no more than 1×104 conidia/ml with rice blast fungal strains endogenously containing the mycovirus.
This result indicates that the mycovirus according to the present invention suppresses conidia formation by host fungi. That is, it is suggested that the present invention has a possibility of suppressing proliferation of phytopathogenic fungi and being able to suppress propagation and spreading of infection of the fungi.
In Example 7, the base sequences of the double-stranded RNAs extracted from the rice blast fungus were analyzed.
(1) Extraction and Purification of Double-Stranded RNAs
First, the double-stranded RNAs were extracted and purified by the following procedure.
One of the fungal strains with which double-stranded RNAs were detected in Example 1 was frozen by liquid nitrogen and thereafter milled, and 1 ml of an extraction buffer (2×STE, 1% SDS, 10 mM β-mercaptoethanol) was added per 0.1 g of fungal body. PCI (50% phenol, 48% chloroform, 2% isoamyl alcohol) of an amount equivalent to the extraction buffer was then added to the solution, and the mixture was stirred well at room temperature for 30 minutes and then centrifuged (8,000 rpm, for 20 minutes, room temperature), and a supernatant (aqueous phase) was collected. An equivalent amount of 100% ethanol was added to the collected supernatant (50% ethanol/1×STE solution as the final concentration) to perform ethanol precipitation.
The precipitate (nucleic acid) obtained was dissolved in a 17.5% ethanol/1×STE solution and, after heat treating at 65° C. for 10 minutes, was cooled rapidly in ice, and after centrifuging (8,000 rpm, for 10 minutes, 4° C.), a supernatant was collected. A fibrous cellulose filler (trade name: “CF11,” made by Whatman Inc.) was added to the supernatant, and after stirring for 30 to 60 minutes under a 4° C. condition, the supernatant was filled in a column (diameter: 1.5 cm; height: 12 cm).
Next, the 17.5% ethanol/1×STE solution was supplied to the column and after eliminating a non-adsorbed fraction, a 1×STE solution was added to elute and recover an adsorbed fraction. To eliminate DNA mixed in the eluted fraction, 25 U of DNase 1 (made by Takara Bio Inc.) were added and after letting react for 30 minutes at 37° C., ethanol precipitation was performed and the targeted double-stranded RNAs were obtained.
(2) cDNA Cloning
cDNA was synthesized using the obtained double-stranded RNAs as templates and thereafter, cDNA cloning was performed.
From the obtained double-stranded RNAs, first strand cDNAs were synthesized using Random Primer and SuperScript III reverse transcriptase (made by Invitrogen Corp.; the same applies hereinafter). Then, after treatment by RNase H, second strand cDNAs were synthesized using DNA polymerase I. After terminal blunting the synthesized double-stranded cDNAs by T4 DNA polymerase, purification was performed using a QIAquick PCR Purification Kit (made by Qiagen N. V.; the same applies hereinafter).
Then, after performing terminal phosphorylation, the cDNAs were incorporated in pUC19 plasmid vectors (made by Takara Bio Inc.; the same applies hereinafter), the recombinant vectors were transformed into Escherichia coli, and clones of the respective cDNA were thereby obtained. The respective cDNA clones were then subject to sequence measurement.
As a result of preparing coding sequences based on the respective base sequence information obtained, substantially full-length base sequences were acquired for the four types of double-stranded RNAs present in the fungal body.
(3) Analysis of Both Terminal Sequences by a 5′-RACE Method
In continuation, gene-specific antisense primers were synthesized based on the sequence information, double-stranded RNAs were extracted and purified from the fungal body by the same procedure as described above, cDNAs were synthesized using the SuperScript III reverse transcriptase and the primers and treated with RNase H to obtain single-stranded first strand cDNAs.
Then, using terminal deoxynucleotidyl transferase (made by Takara Bio Inc.) deoxycytosine homopolymer was added as anchor sequences to the single-stranded cDNAs. Adapter primers with deoxyguanine homopolymer attached were then synthesized, gene-specific antisense primers, and portions sandwiched by the two primers were amplified by a PCR method to synthesize cDNAs.
Next, the cDNAs were terminal blunted by T4 DNA polymerase, purified using the QIAquick PCR Purification Kit, and then after performing terminal phosphorylation, the cDNAs were incorporated in pUC19 plasmid vectors, the recombinant vectors were transformed into Escherichia coli, and clones of the respective cDNAs were thereby obtained. The respective cDNA clones were then subject to sequence measurement.
By the above procedure, the full-length sequences of the genetic information of the novel mycovirus were obtained. This mycovirus is made up of four types of double-stranded RNAs. The sequences of the respective double-stranded RNAs are indicated by SEQ ID NOS: 1 to 4. Because the sequence measurements were made upon replacing the double-stranded RNAs by cDNAs, “uracil” is replaced by “thymine” in the sequence table.
As a result of analysis of the base sequences obtained, it was found that a conserved motif of RdRp (RNA-dependent RNA polymerase), present in totiviruses and related viruses, etc., is included in the RNA sequence indicated by SEQ ID NO: 1. It was also found that a region homologous to an L3 double-stranded RNA fragment of a La France disease virus is included in the RNA sequence indicated by SEQ ID NO: 3.
These results indicate that the four types of double-stranded RNAs according to the present invention make up the genetic information of the novel mycovirus.
In Example 8, biochemical characteristics of virus particles of the mycovirus according to the present invention were analyzed.
The S-0412-II 1a rice blast fungal strain was frozen by liquid nitrogen and milled by a mortar. 0.1 M phosphate buffer (pH 7.0) of 4 to 6 times the sample amount was then added to the sample and stirring by a mixer was performed. Then, upon adding butanol/chloroform (volume ratio: 1:1) of 40% the volume of the phosphate buffer, the mixture was stirred for 30 minutes and centrifuged (8,000×g, for 10 minutes), the supernatant was recovered, and this operation was repeated several times.
Next, polyethylene glycol (average molecular weight: 6,000) for achieving a final concentration of 8% and sodium chloride for achieving a final concentration of 1% was then added to and dissolved in the supernatant, and upon leaving for 3 hours at 4° C. to make the viruses aggregate mutually, centrifugation (12,000×g, for 20 minutes) was performed to obtain a virus precipitate aggregated by the polyethylene glycol.
The virus precipitate was then dissolved in an appropriate amount of 0.05 M phosphate buffer (pH 7.0), undissolved impurities were eliminated by centrifugation (6,000×g, for 5 minutes), and the supernatant was recovered.
20% sucrose was then dispensed as a cushion to a height of approximately 1 cm from a bottom of a centrifuge tube, the recovered supernatant was overlaid, ultracentrifugation (100,000×g, for 2 hours) was performed, and the precipitate was dissolved in a small amount of 0.05 M phosphate buffer (pH 7.0) as a partially purified virus preparation.
A portion of the virus preparation was subject to SDS-PAGE (7.5% gel, tris-glycine bugger (pH 8.8), 20 mA, for 90 minutes) and stained by CBB (Coomassie brilliant blue) to perform molecular weight analysis of a principal component of virus protein (coat protein).
As a result, a band of virus particle derived protein was detected at a size of approximately 70 kDa.
Then, by the SDS-phenol method, nucleic acid extraction was performed from the component with which the 70 kDa protein is detected. As a result, double-stranded RNAs of four components of 2.8 to 3.6 kbp were detected.
By the present example, presence of virus particles of the mycovirus according to the present invention was confirmed.
In Example 9, whether or not the present invention is effective for control of a phytopathogenic fungus was examined by a spray inoculation method.
The S-0412-II 1a rice blast fungal strain was inoculated into an oatmeal medium plate and cultured indoors at 25° C. If conidia were not formed adequately on the 15th day after inoculation, approximately 2 mL of sterilized distilled water were poured on the plate, the medium surface was rubbed with a brush to remove aerial hyphae, the plate was placed under a black light for 3 days to induce conidia formation, approximately 1 mL of sterilized distilled water were poured, and the medium surface was rubbed again by a brush to recover conidia along with aerial hyphae to obtain a conidia-containing solution. If conidia were adequately formed on the 15th day after inoculation, approximately 2 mL of sterilized distilled water were poured on the plate, and the medium surface was rubbed with a brush, and recover conidia along with aerial hyphae to obtain a conidia-containing solution. As a control, a completely mycovirus-cured strain of the rice blast fungus was prepared by the same procedure as Example 3, and the conidia-containing solutions was obtained by the same procedure from this fungus.
The conidia-containing solutions were then filtered through Kimwipe or gauze, adjusted to a conidia concentration of 2×106 conidia/mL, and 0.02% (v/v) Tween 20 was added to prepare conidia suspensions.
Next, the conidia suspensions were uniformly sprayed using a nozzle onto rice seedlings (seedlings of 2 to 3 weeks from seeding of a variety selected appropriately in consideration of true rice blast resistance phenotype and fungal race), and after leaving still the plant bodies in an inoculation box at 26° C. and 100% relative humidity for 24 hours, the pots were transferred into a greenhouse with the room temperature being maintained at 23° C. to 30° C. and grown for 7 days after spray inoculation. On the 7th day after spray inoculation, a number per fixed leaf area of disease lesions that have become 3 to 4 mm in size was measured.
The results are shown in
As shown in
In Example 10, whether or not the present invention is effective for control of a phytopathogenic fungus was examined by a punch inoculation method.
Conidia suspensions were prepared by the same procedure as Example 9, the suspensions were deposited on 3% plain agar films, and the agar films were cut to approximately 2 mm square to prepare agar pieces. Fourth leaves of rice grown in a greenhouse at 23° C. to 30° C. were punched by an inoculation punch to form wet wounds, the agar pieces were placed on the wounded portions, and after leaving still the plant bodies in an inoculation box at 26° C. and 100% relative humidity for 24 hours, the pots were transferred into a greenhouse with the room temperature being maintained at 23° C. to 30° C. and grown for 14 days after inoculation. On the 14th day after inoculation, sizes of lesions were measured.
As a result, in comparison to the control, the lesion sizes were significantly smaller in the case of inoculating the conidia suspension prepared from the rice blast fungus infected with the mycovirus according to the present invention. As with Example 9, this result indicates that the present invention is effective for control of the phytopathogenic fungus.
In Example 11, virus particles present outside the fungal body were identified using an electron microscope.
The S-0412-II 1a rice blast fungal strain was cultured in the YG medium (yeast extract: 0.5%; glucose: 2%) and virus particles were isolated from the culture supernatant. The culture supernatant was centrifuged (10,000×g, for 5 minutes) and the resulting supernatant was ultracentrifuged (100,000×g, for 30 minutes) to obtain a precipitate containing the virus particles. The precipitate was dissolved in 0.05 M phosphate buffer (pH 7.0), negative stained using phosphotungstic acid or uranium acetate, and observed by an electron microscope (magnification: ×20,000 to 40,000).
The result is shown in
| Number | Date | Country | Kind |
|---|---|---|---|
| 2008-011012 | Jan 2008 | JP | national |
| 2008-035405 | Feb 2008 | JP | national |
| 2008-104181 | Apr 2008 | JP | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/JP2009/000003 | 1/5/2009 | WO | 00 | 9/23/2010 |
