Method for down-regulating gene expression in fungi

Abstract

The present invention concerns methods for controlling fungus infestation via dsRNA mediated gene silencing, whereby the intact fungus cell(s) are contacted with a double-stranded RNA from outside the fungal cell(s) and whereby the double-stranded RNA is taken up by the intact fungal cell(s). In one particular embodiment, the methods of the invention are used to alleviate plants from fungus pests. Alternatively, the methods are used for treating and/or preventing fungal infestation on a substrate or a subject in need of such treatment and/or prevention. Suitable fungal target genes and fragments thereof, dsRNA constructs, recombinant constructs and compositions are disclosed.

1 . A method for controlling fungal growth on a cell or an organism or for preventing fungal infestation of a cell or an organism susceptible to fungal infection, comprising contacting fungal cells with a double-stranded RNA from outside the fungal cell(s), wherein the double-stranded RNA comprises annealed complementary strands, one of which has a nucleotide sequence which is complementary to at least part of the nucleotide sequence of a fungal target gene, whereby the double-stranded RNA is taken up into the fungal cells and thereby controls growth or prevents infestation.  
   
   
       2 . A method for down-regulating expression of a target gene in a fungus, comprising contacting fungal cell(s) with a double-stranded RNA outside the fungal cell(s), wherein the double-stranded RNA comprises annealed complementary strands, one of which has a nucleotide sequence which is complementary to at least part of the nucleotide sequence of the fungal target gene to be down-regulated, whereby the double-stranded RNA is taken up into the fungal cells and thereby down-regulates expression of the fungal target gene.  
   
   
       3 . A method according to  claim 1  or  2  wherein said fungal cell(s) is contacted with a composition comprising said double-stranded RNA.  
   
   
       4 . A method according to  claim 1  or  2 , wherein said fungal cell(s) is contacted with double-stranded RNA which is expressed by a prokaryotic (such as a bacterial cell) or eukaryotic (such as a yeast cell) host cell or host organism.  
   
   
       5 . A method according to  claim 1  wherein said double-stranded RNA is expressed by said cell or organism infested with or susceptible to infestation by said fungus.  
   
   
       6 . A method according to  claim 5  wherein said cell is a plant cell or wherein said organism is a plant.  
   
   
       7 . A method according to any of claims  1  or  2 , wherein said double-stranded RNA is expressed from a recombinant construct, which construct comprises at least one regulatory sequence operably linked to said nucleotide sequence which is complementary to at least part of said nucleotide sequence of said fungal target gene to be down-regulated.  
   
   
       8 . A method according to any of claims  1  or  2 , wherein said fungal target gene comprises a sequence which is selected from the group comprising: (i) sequences which are at least 75% identical to a sequence represented by any of SEQ ID NOs 3, 42, 99, 100, 527, 39, 60, 111, 112, 113, 114, 115, 116, 117, 536, 537, 538, 5, 43, 101, 102, 528, 1, 41, 97, 98, 526, 184, 185, 37, 59, 124, 9, 45, 106, 531, 188, 189, 13, 47, 109, 534, 33, 57, 126, 23, 52, 119, 35, 58, 127, 7, 44, 103, 104, 105, 529, 186, 187, 29, 55, 118, 17, 49, 108, 533, 25, 53, 121, 19, 50, 125, 31, 56, 123, 11, 46, 107, 532, 27, 54, 122, 21, 51, 120, 15, 48, 110, 535, 458, 486, 530, 460, 487, 539, 540, 462, 488, 541, 464, 489, 542, 466, 490, 543, 468, 491, 544, 470, 492, 545, 472, 493, 546, 474, 494, 547, 476, 496, 548, 478, 556, 549, 480, 497, 550, 482, 498, 551, 484, 499, 552 and 562 to 859, or the complement thereof, and (ii) sequences comprising at least 17 contiguous nucleotides of any of SEQ ID NOs 3, 42, 99, 100, 527, 39, 60, 111, 112, 113, 114, 115, 116, 117, 536, 537, 538, 5, 43, 101, 102, 528, 1, 41, 97, 98, 526, 184, 185, 37, 59, 124, 9, 45, 106, 531, 188, 189, 13, 47, 109, 534, 33, 57, 126, 23, 52, 119, 35, 58, 127, 7, 44, 103, 104, 105, 529, 186, 187, 29, 55, 118, 17, 49, 108, 533, 25, 53, 121, 19, 50, 125, 31, 56, 123, 11, 46, 107, 532, 27, 54, 122, 21, 51, 120, 15, 48, 110, 535, 458, 486, 530, 460, 487, 539, 540, 462, 488, 541, 464, 489, 542, 466, 490, 543, 468, 491, 544, 470, 492, 545, 472, 493, 546, 474, 494, 547, 476, 496, 548, 478, 556, 549, 480, 497, 550, 482, 498, 551, 484, 499, 552 and 562 to 859, or the complement thereof, or wherein said target gene is an orthologue of a gene comprising at least 17 contiguous nucleotides of any of SEQ ID NOs 3, 42, 99, 100, 527, 39, 60, 111, 112, 113, 114, 115, 116, 117, 536, 537, 538, 5, 43, 101, 102, 528, 1, 41, 97, 98, 526, 184, 185, 37, 59, 124, 9, 45, 106, 531, 188, 189, 13, 47, 109, 534, 33, 57, 126, 23, 52, 119, 35, 58, 127, 7, 44, 103, 104, 105, 529, 186, 187, 29, 55, 118, 17, 49, 108, 533, 25, 53, 121, 19, 50, 125, 31, 56, 123, 11, 46, 107, 532, 27, 54, 122, 21, 51, 120, 15, 48, 110, 535, 458, 486, 530, 460, 487, 539, 540, 462, 488, 541, 464, 489, 542, 466, 490, 543, 468, 491, 544, 470, 492, 545, 472, 493, 546, 474, 494, 547, 476, 496, 548, 478, 556, 549, 480, 497, 550, 482, 498, 551, 484, 499, 552 and 562 to 859, or the complement thereof.  
   
   
       9 . A method for producing a plant resistant against a plant pathogenic fungus, comprising: 
 (a) transforming a plant cell with a recombinant construct comprising at least one regulatory sequence operably linked to a sequence complementary to at least part of a nucleotide sequence of a target fungal gene selected from the group consisting of: 
 (i) a nucleotide sequence of at least 17 nucleotides in length encoding an RNA which is complementary to a fungal target gene comprising a sequence which is at least 75% identical to a sequence selected from the group of sequences represented by any of SEQ ID NOs 3, 42, 99, 100, 527, 39, 60, 111, 112, 113, 114, 115, 116, 117, 536, 537, 538, 5, 43, 101, 102, 528, 1,41, 97,98, 526, 184, 185, 37, 59, 124, 9,45, 106, 531, 188, 189, 13, 47, 109, 534, 33, 57, 126, 23, 52, 119, 35, 58, 127, 7, 44, 103, 104, 105, 529, 186, 187, 29, 55, 118, 17, 49, 108, 533, 25, 53, 121, 19, 50, 125, 31, 56, 123, 11, 46, 107, 532, 27, 54, 122, 21, 51, 120, 15, 48, 110, 535, 458, 486, 530, 460, 487, 539, 540, 462, 488, 541, 464, 489, 542, 466, 490, 543, 468, 491, 544, 470, 492, 545, 472, 493, 546, 474, 494, 547, 476, 496, 548, 478, 556, 549, 480, 497, 550, 482, 498, 551, 484, 499, 552 and 562 to 859, or the complement thereof, or wherein said fungal target gene is a fungal orthologue of a gene comprising any of SEQ ID Nos 3, 42, 99, 100, 527, 39, 60, 111, 112, 113, 114, 115, 116, 117, 536, 537, 538, 5, 43, 101, 102, 528, 1, 41, 97, 98, 526, 184, 185, 37, 59, 124, 9, 45, 106, 531, 188, 189, 13, 47, 109, 534, 33, 57, 126, 23, 52, 119, 35, 58, 127, 7, 44, 103, 104, 105, 529, 186, 187, 29, 55, 118, 17, 49, 108, 533, 25, 53, 121, 19, 50, 125, 31, 56, 123, 11, 46, 107, 532, 27, 54, 122, 21, 51, 120, 15, 48, 110, 535, 458, 486, 530, 460, 487, 539, 540, 462, 488, 541, 464, 489, 542, 466, 490, 543, 468, 491, 544, 470, 492, 545, 472, 493, 546, 474, 494, 547, 476, 496, 548, 478, 556, 549, 480, 497, 550, 482, 498, 551, 484, 499, 552 and 562 to 859, and  
 (ii) a nucleotide sequence comprising a sense strand comprising the nucleotide sequence of (i) and an antisense strand comprising the complement of said nucleotide sequence of (i), wherein the transcript encoded by said nucleotide sequence is capable of forming a double-stranded RNA,  
   (b) regenerating a plant from the transformed plant cell; and    (c) growing the transformed plant under conditions suitable for the expression of the recombinant construct, said grown transformed plant resistant to fungi compared to an untransformed plant, hereby producing a fungus-resistant plant.    
   
   
       10 . A method according to any of claims  1  or  9  wherein said fungal cell(s) is a cell(s) of a plant pathogenic fungus selected from the group consisting of  Acremoniella  spp.,  Alternaria  spp. (e.g.  Alternaria brassicola  or  Alternaria solani ),  Ascochyta  spp. (e.g.  Ascochyta pisi ),  Botrytis  spp. (e.g.  Botrytis cinerea  or  Botryotinia fuckeliana, Cladosporium  spp.,  Cercospora  spp. (e.g.  Cercospora kikuchii  or  Cercospora zaea - maydis ),  Cladosporium  spp. (e.g.  Cladosporium fulvum ),  Colletotrichum  spp. (e.g.  Colletotrichum lindemuthianum ),  Curvularia  spp.,  Diplodia  spp. (e.g.  Diplodia maydis ),  Erysiphe  spp. (e.g.  Erysiphe graminis  f.sp.  graminis, Erysiphe graminis  f.sp.  hordei  or  Erysiphe pisi ),  Erwinia armylovora, Fusarium  spp. (e.g.  Fusarium nivale, Fusarium sporotrichioides, Fusarium oxysporum, Fusarium graminearum, Fusarium germinearum, Fusarium culmorum, Fusarium solani, Fusarium moniliforme  or  Fusarium roseum ),  Gaeumanomyces  spp. (e.g.  Gaeumanomyces graminis  f.sp.  tritici ),  Gibberella  spp. (e.g.  Gibberella zeae ),  Helminthosporium  spp. (e.g.  Helminthosporium turcicum, Helminthosporium carbonum, Helminthosporium mavdis or Helminthosporium sigmoideum ),  Leptosphaeria salvinii, Macrophomina  spp. (e.g.  Macrophomina phaseolina ),  Magnaportha  spp. (e.g  Magnaporthe oryzae ),  Mycosphaerella  spp.,  Nectria  spp. (e.g.  Nectria heamatococca ),  Peronospora  spp. (e.g.  Peronospora manshurica  or  Peronospora tabacina ),  Phoma  spp. (e.g.  Phoma betae ),  Phakopsora  spp. (e.g.  Phakopsora pachyrhizi ),  Phymatotrichum  spp. (e.g.  Phymatotrichum omnivorum ),  Phytophthora  spp. (e.g.  Phytophthora cinnamomi, Phytophthora cactorum, Phytophthora phaseoli, Phytophthora parasitica, Phytophthora citrophthora, Phytophthora megasperma  f.sp.  soiae  or  Phytophthora infestans ),  Plasmopara  spp. (e.g.  Plasmopara viticola ),  Podosphaera  spp. (e.g.  Podosphaera leucotricha ),  Puccinia  spp. (e.g.  Puccinia sorghi, Puccinia striiformis, Puccinia graminis  f.sp.  tritici, Puccinia asparagi, Puccinia recondita  or  Puccinia arachidis ),  Pythium  spp. (e.g.  Pythium aphanidermatum ),  Pyrenophora  spp. (e.g.  Pyrenophora tritici - repentens  or  Pyrenophora teres ),  Pyricularia  spp. (e.g.  Pyricularia oryzae ),  Pythium  spp. (e.g.  Pythium ultimum ),  Rhincosporium secalis, Rhizoctonia  spp. (e.g.  Rhizoctonia solani, Rhizoctonia oryzae  or  Rhizoctonia cerealis ),  Rhizopus  spp. (e.g.  Rhizopus chinensid ),  Scerotium  spp. (e.g.  Scerotium rolfsi ),  Sclerotinia  spp. (e.g.  Sclerotinia sclerotiorum ),  Septoria  spp. (e.g.  Septoria lycopersici, Septoria glycines, Septoria nodorum  or  Septoria tritici ),  Thielaviopsis  spp. (e.g.  Thielaviopsis basicola ),  Tilletia  spp.,  Trichoderma  spp. (e.g.  Trichoderma virde ),  Uncinula  spp. (e.g.  Uncinula necator ),  Ustilago maydis  (e.g.  corn smut ),  Venturia  spp. (e.g.  Venturia inaequalis  or  Venturia pirina ) and  Verticillium  spp. (e.g.  Verticillium dahliae  or  Verticillium albo - atrum ).  
   
   
       11 . (canceled)  
   
   
       12 . A method according to any of claims  6  or  9  wherein said plant is chosen from the group consisting of rice, corn, soybean, cotton, potato, banana, tomato, wheat, sorghum, millet, beans, groundnuts, rapeseed, sunflower and sugarcane; preferably chosen from rice, corn, soybean, cotton, potato, banana and tomato.  
   
   
       13 . A method according to any of claims  6  or  9  wherein said plant is rice and wherein said target gene is a gene from a fungus selected from the group consisting of  Magnaporthe  spp.,  Rhizoctonia  spp.,  Acremoniella  spp.,  Phytium  spp.,  Curvularia  spp.,  Trichoderma  spp.,  Fusarium  spp. and  Rhizopus  spp.  
   
   
       14 . A method according to any of claims  6  or  9  wherein said plant is corn and wherein said target gene is a gene from a fungus selected from the group consisting of  Colletotrichum  spp.,  Gibberella  spp.,  Fusarium  spp.,  Diplodia  spp. and  Puccina  spp.  
   
   
       15 . A method according to any of claims  6  or  9  wherein said plant is soybean and wherein said target gene is a gene selected from the group of  Phakopsora  spp.  
   
   
       16 . A method according to any of claims  6  or  9  wherein said plant is cotton and wherein said target gene is a gene from a fungus selected from the group consisting of  Fusarium  spp. and  Verticillium  spp.  
   
   
       17 . A method according to any of claims  6  or  9  wherein said plant is potato and wherein said target gene is a gene from a fungus selected from the group consisting of  Phytophthora  spp.,  Rhizoctonia  spp. and fungal species that cause wilt, rot or scurf.  
   
   
       18 . A method according to any of claims  6  or  9  wherein said plant is banana and wherein said target gene is a gene from a fungus selected from the group consisting of  Mycosphaerella  spp.,  Cercospora  spp. and  Fusarium  spp.  
   
   
       19 . A method according to any of claims  6  or  9  wherein said plant is tomato and wherein said target gene is a gene from a fungus selected from the group consisting of  Phytophthora  spp. and fungal species that cause foliar disease, wilt or fruit rot.  
   
   
       20 . A method according to any of claims  1  or  9 , wherein said fungal target gene is essential for the viability, growth, development or reproduction of the fungus, preferably said fungal target gene is involved in any of the cellular functions as defined in Table 1; or wherein said fungal target gene is involved in the pathogenicity or infectivity of the fungus, preferably said fungal target gene is involved in the formation of germ tubes, conidia attachment, formation of appressoria, formation of the penetration peg or formation of conidia.  
   
   
       21 . A transgenic plant, or reproductive or propagation material for a transgenic plant or a cultured transgenic plant cell, which expresses or is capable of expressing at least one double-stranded RNA, wherein said double-stranded RNA comprises annealed complementary strands, one of which has a nucleotide sequence which is complementary to at least part of a nucleotide sequence of a fungal target gene, and wherein said double-stranded RNA is taken up by a fungal cell upon plant-fungus interaction.  
   
   
       22 . A transgenic plant, reproductive or propagation material for a transgenic plant, or a cultured transgenic plant cell according to  claim 21 , wherein said fungal target gene is essential for the viability, growth, development or reproduction of the fungus, preferably said fungal target gene is involved in any of the cellular functions as defined in Table 1; or wherein said fungal target gene is involved in the pathogenicity or infectivity of the fungus, preferably said fungal target gene is involved in the formation of germ tubes, conidia attachment, formation of appressoria, formation of the penetration peg or formation of conidia.  
   
   
       23 . A transgenic plant, reproductive or propagation material for a transgenic plant, or a cultured transgenic plant cell according to  claim 21 , wherein said fungal target gene comprises a sequence which is selected from the group comprising: 
 (i) sequences which are at least 75%, preferably at least 80% or 85% identical, preferably at least 90%, 95%, 96%, or more preferably at least 97%, 98% and still more preferably at least 99% identical to a sequence represented by any of SEQ ID NOs 3, 42, 99, 100, 527, 39, 60, 111, 112, 113, 114, 115, 116, 117, 536, 537, 538, 5, 43, 101, 102, 528, 1, 41, 97, 98, 526, 184, 185, 37, 59, 124, 9, 45, 106, 531, 188, 189, 13, 47, 109, 534, 33, 57, 126, 23, 52, 119, 35, 58, 127, 7, 44, 103, 104, 105, 529, 186, 187, 29, 55, 118, 17, 49, 108, 533, 25, 53, 121, 19, 50, 125, 31, 56, 123, 11, 46, 107, 532, 27, 54, 122, 21, 51, 120, 15, 48, 110, 535, 458, 486, 530, 460, 487, 539, 540, 462, 488, 541, 464, 489, 542, 466, 490, 543, 468, 491, 544, 470, 492, 545, 472, 493, 546, 474, 494, 547, 476, 496, 548, 478, 556, 549, 480, 497, 550, 482, 498, 551, 484, 499, 552 and 562 to 859, or the complement thereof, and    (ii) sequences comprising at least 17, preferably at least 18, 19, 20 or 21, more preferably at least 22, 23 or 24 contiguous nucleotides of any of SEQ ID NOs 3, 42, 99, 100, 527, 39, 60, 111, 112, 113, 114, 115, 116, 117, 536, 537, 538, 5, 43, 101, 102, 528, 1, 41, 97, 98, 526, 184, 185, 37, 59, 124, 9, 45, 106, 531, 188, 189, 13, 47, 109, 534, 33, 57, 126, 23, 52, 119, 35, 58, 127, 7, 44, 103, 104, 105, 529, 186, 187, 29, 55, 118, 17, 49, 108, 533, 25, 53, 121, 19, 50, 125, 31, 56, 123, 11, 46, 107, 532, 27, 54, 122, 21, 51, 120, 15, 48, 110, 535, 458, 486, 530, 460, 487, 539, 540, 462, 488, 541, 464, 489, 542, 466, 490, 543, 468, 491, 544, 470, 492, 545, 472, 493, 546, 474, 494, 547, 476, 496, 548, 478, 556, 549, 480, 497, 550, 482, 498, 551, 484, 499, 552 and 562 to 859, or the complement thereof, or wherein said fungal target gene is a fungal orthologue of a gene comprising at least 17 contiguous nucleotides of any of SEQ ID Nos 3, 42, 99, 100, 527, 39, 60, 111, 112, 113, 114, 115, 116, 117, 536, 537, 538, 5, 43, 101, 102, 528, 1, 41, 97, 98, 526, 184, 185, 37, 59, 124, 9, 45, 106, 531, 188, 189, 13, 47, 109, 534, 33, 57, 126, 23, 52, 119, 35, 58, 127, 7, 44, 103, 104, 105, 529, 186, 187, 29, 55, 118, 17, 49, 108, 533, 25, 53, 121, 19, 50, 125, 31, 56, 123, 11, 46, 107, 532, 27, 54, 122, 21, 51, 120, 15, 48, 110, 535, 458, 486, 530, 460, 487, 539, 540, 462, 488, 541, 464, 489, 542, 466, 490, 543, 468, 491, 544, 470, 492, 545, 472, 493, 546, 474, 494, 547, 476, 496, 548, 478, 556, 549, 480, 497, 550, 482, 498, 551, 484, 499, 552 and 562 to 859, or the complement thereof.    
   
   
       24 . A transgenic plant, or reproductive or propagation material for a transgenic plant or a cultured transgenic plant cell according to any of claims  21  or  23  which is a rice plant or reproductive or propagation material for a rice plant or a cultured rice plant cell, wherein the target gene is a gene from a fungus selected from the group consisting of  Magnaporthe  spp.,  Rhizoctonia  spp.,  Acremoniella  spp.,  Phytium  spp.,  Curvularia  spp.,  Trichoderma  spp.,  Fusarium  spp., and  Rhizopus  spp.  
   
   
       25 . A transgenic plant, or reproductive or propagation material for a transgenic plant or a cultured transgenic plant cell according to any of claims  21  or  23  which is a corn plant or reproductive or propagation material for a corn plant or a cultured corn plant cell, wherein the target gene is a gene from a fungus selected from the group consisting of  Colletotrichum  spp.,  Gibberella  spp.,  Fusarium  spp.,  Diplodia  spp. and  Puccina  spp.  
   
   
       26 . A transgenic plant, or reproductive or propagation material for a transgenic plant or a cultured transgenic plant cell according to any of claims  21  or  23  which is a soybean plant or reproductive or propagation material for a soybean plant or a cultured soybean plant cell, wherein the target gene is a gene from the group of  Phakopsora  spp.  
   
   
       27 . A transgenic plant, or reproductive or propagation material for a transgenic plant or a cultured transgenic plant cell according to any of claims  21  or  23  which is a cotton plant or reproductive or propagation material for a cotton plant or a cultured cotton plant cell, wherein the target gene is a gene from a fungus selected from the group consisting of  Fusarium  spp. and  Verticillium  spp.  
   
   
       28 . A transgenic plant, or reproductive or propagation material for a transgenic plant or a cultured transgenic plant cell according to any of claims  21  or  23  which is a potato plant or reproductive or propagation material for a potato plant or a cultured potato plant cell, wherein the target gene is a gene from a fungus selected from the group consisting of  Phytophthora  spp.,  Rhizoctonia  spp. and fungal species that cause wilt, rot or scurf.  
   
   
       29 . A transgenic plant, or reproductive or propagation material for a transgenic plant or a cultured transgenic plant cell according to any of claims  21  or  23  which is a banana plant or reproductive or propagation material for a banana plant or a cultured banana plant cell, wherein the target gene is a gene from a fungus selected from the group consisting of  Mycosphaerella  spp.,  Cercospora  spp. and  Fusarium  spp.  
   
   
       30 . A transgenic plant, or reproductive or propagation material for a transgenic plant or a cultured transgenic plant cell according to any of claims  21  or  23  which is a tomato plant or reproductive or propagation material for a tomato plant or a cultured tomato plant cell, wherein the target gene is a gene from a fungus selected from the group consisting of  Phytophthora  spp. and species that cause foliar disease, wilt or fruit rot.  
   
   
       31 . An isolated double-stranded RNA comprising annealed complementary strands, one of which has a nucleotide sequence which is complementary to at least part of a nucleotide sequence of a fungal target, wherein said fungal target gene is essential for the viability, growth, development or reproduction of the fungus, preferably said fungal target gene is involved in any of the cellular functions as defined in Table 1; or wherein said fungal target gene is involved in the pathogenicity or infectivity of the fungus, preferably said fungal target gene is involved in the formation of germ tubes, conidia attachment, formation of appressoria, formation of the penetration peg or formation of conidia.  
   
   
       32 . An isolated double-stranded RNA according to  claim 31  comprising annealed complementary strands, one of which has a nucleotide sequence which is complementary to at least part of a nucleotide sequence of a fungal target gene, wherein said target gene comprises a sequence which is selected from the group comprising: (i) sequences which are at least 75% identical to a sequence represented by any of SEQ ID NOs 3, 42, 99, 100, 527, 39, 60, 111, 112, 113, 114, 115, 116, 117, 536, 537, 538, 5, 43, 101, 102, 528, 1, 41, 97, 98, 526, 184, 185, 37, 59, 124, 9, 45, 106, 531, 188, 189, 13, 47, 109, 534, 33, 57, 126, 23, 52, 119, 35, 58, 127, 7, 44, 103, 104, 105, 529, 186, 187, 29, 55, 118, 17, 49, 108, 533, 25, 53, 121, 19, 50, 125, 31, 56, 123, 11, 46, 107, 532, 27, 54, 122, 21, 51, 120, 15, 48, 110, 535, 458, 486, 530, 460, 487, 539, 540, 462, 488, 541, 464, 489, 542, 466, 490, 543, 468, 491, 544, 470, 492, 545, 472, 493, 546, 474, 494, 547, 476, 496, 548, 478, 556, 549, 480, 497, 550, 482, 498, 551, 484, 499, 552 and 562 to 859, or the complement thereof, and (ii) sequences comprising at least 17 contiguous nucleotides of any of SEQ ID Nos 3, 42, 99, 100, 527, 39, 60, 111, 112, 113, 114, 115, 116, 117, 536, 537, 538, 5, 43, 101, 102, 528, 1, 41, 97, 98, 526, 184, 185, 37, 59, 124, 9, 45, 106, 531, 188, 189, 13, 47, 109, 534, 33, 57, 126, 23, 52, 119, 35, 58, 127, 7, 44, 103, 104, 105, 529, 186, 187, 29, 55, 118, 17, 49, 108, 533, 25, 53, 121, 19, 50, 125, 31, 56, 123, 11, 46, 107, 532, 27, 54, 122, 21, 51, 120, 15, 48, 110, 535, 458, 486, 530, 460, 487, 539, 540, 462, 488, 541, 464, 489, 542, 466, 490, 543, 468, 491, 544, 470, 492, 545, 472, 493, 546, 474, 494, 547, 476, 496, 548, 478, 556, 549, 480, 497, 550, 482, 498, 551, 484, 499, 552 and 562 to 859, or the complement thereof, or wherein said target gene is an orthologue of a gene comprising at least 17 contiguous nucleotides of any of SEQ ID NOs 3, 42, 99, 100, 527, 39, 60, 111, 112, 113, 114, 115, 116, 117, 536, 537, 538, 5, 43, 101, 102, 528, 1, 41, 97, 98, 526, 184, 185, 37, 59, 124, 9, 45, 106, 531, 188, 189, 13, 47, 109, 534, 33, 57, 126, 23, 52, 119, 35, 58, 127, 7, 44, 103, 104, 105, 529, 186, 187, 29, 55, 118, 17, 49, 108, 533, 25, 53, 121, 19, 50, 125, 31, 56, 123, 11, 46, 107, 532, 27, 54, 122, 21, 51, 120, 15, 48, 110, 535, 458, 486, 530, 460, 487, 539, 540, 462, 488, 541, 464, 489, 542, 466, 490, 543, 468, 491, 544, 470, 492, 545, 472, 493, 546, 474, 494, 547, 476, 496, 548, 478, 556, 549, 480, 497, 550, 482, 498, 551, 484, 499, 552 and 562 to 859, or the complement thereof  
   
   
       33 . An isolated double-stranded RNA according to  claim 32 , wherein at least one of said annealed complementary strands comprises the RNA equivalent of at least one of the nucleotide sequences represented by any of SEQ ID NOs 99, 100, 527, 111, 112, 113, 114, 115, 116, 117, 536, 537, 538, 101, 102, 528, 97, 98, 526, 124, 106, 531, 109, 534, 126, 119, 127, 103, 104, 105, 529, 118, 108, 533, 121, 125, 123, 107, 532, 122, 120, 110, 535, 530, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552 and 562 to 859, or the RNA equivalent of a fragment of at least 17 basepairs in length thereof.  
   
   
       34 . An isolated double-stranded RNA according to  claim 33  wherein the nucleotide sequences comprises at least one of the sequences represented by any of SEQ ID NOs 192, 201, 202, 193, 190, 191, 196, 199, 260, 194, 195, 198 and 197, or a double-stranded fragment of at least 17 basepairs in length thereof.  
   
   
       35 . (canceled)  
   
   
       36 . (canceled)  
   
   
       37 . An isolated double-stranded RNA construct according to  claim 32  comprising at least two copies of said nucleotide sequence complementary to at least part of a nucleotide sequence of a fungal target.  
   
   
       38 . An isolated double-stranded RNA construct according to  claim 37  comprising at least two copies of the RNA equivalent of at least one of the nucleotide sequences represented by any of SEQ ID NOs 99, 100, 527, 111, 112, 113, 114, 115, 116, 117, 536, 537, 538, 101, 102, 528, 97, 98, 526, 124, 106, 531, 109, 534, 126, 119, 127, 103, 104, 105, 529, 118, 108, 533, 121, 125, 123, 107, 532, 122, 120, 110, 535, 530, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552 and 562 to 859, or comprising at least two copies of the RNA equivalent of a fragment of at least 17 basepairs in length thereof.  
   
   
       39 . An isolated double-stranded RNA construct according to  claim 37  comprising at least two copies of the RNA equivalent of the nucleotide sequence as represented in SEQ ID NO 117.  
   
   
       40 . An isolated double-stranded RNA or RNA construct comprising the RNA equivalents of at least two nucleotide sequences independently chosen from the sequences represented by any of SEQ ID NOs 99, 100, 527, 111, 112, 113, 114, 115, 116, 117, 536, 537, 538, 101, 102, 528, 97, 98, 526, 124, 106, 531, 109, 534, 126, 119, 127, 103, 104, 105, 529, 118, 108, 533, 121, 125, 123, 107, 532, 122, 120, 110, 535, 530, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552 and 562 to 859, or fragments thereof of at least 17 basepairs in length.  
   
   
       41 . An isolated double-stranded RNA or RNA construct according to  claim 32 , which comprises at least one additional dsRNA region, at least one strand thereof comprising a nucleotide sequence which is complementary to at least part of a nucleotide sequence of at least one other fungal target gene.  
   
   
       42 . An isolated double-stranded RNA or RNA construct according to  claim 32 , further comprising at least one additional sequence and optionally a linker.  
   
   
       43 . An isolated double-stranded RNA or RNA construct according to  claim 42  wherein said additional sequence is chosen from the group comprising (i) a sequence facilitating large-scale production of the dsRNA construct; (ii) a sequence effecting an increase or decrease in the stability of the dsRNA; (iii) a sequence allowing the binding of proteins or other molecules to facilitate uptake of the RNA construct by a fungal cell(s); (iv) a sequence which is an aptamer that binds to a receptor or to a molecule on the surface or in the cytoplasm of a fungal cell(s) to facilitate uptake, endocytosis and/or transcytosis by the fungal cell(s)* or (v) one or more additional sequences to catalyze processing of dsRNA regions.  
   
   
       44 . (canceled)  
   
   
       45 . An isolated nucleotide sequence encoding a double-stranded RNA according to  claim 32 .  
   
   
       46 . (canceled)  
   
   
       47 . An isolated nucleotide sequence consisting of a sequence represented by any of SEQ ID NOs 3, 99, 100, 527, 192, 39, 111, 112, 113, 114, 115, 116, 117, 536, 537, 538, 201, 202, 5, 101, 102, 528, 193, 1, 97, 98, 526, 184, 185, 190, 191, 37, 124, 9, 106, 531, 188, 189, 196, 13, 109, 534, 199, 200, 33, 126, 23, 119, 35, 127, 7, 103, 104, 105, 529, 186, 187, 194, 195, 29, 118, 17, 108, 533, 198, 25, 121, 19, 125, 31, 123, 11, 107, 532, 197, 27, 122, 21, 120, 15, 110, 535, 458, 530, 460, 539, 540, 462, 541, 464, 542, 466, 543, 468, 544, 470, 545, 472, 546, 474, 547, 476, 548, 478, 549, 480, 550, 482, 551, 484, 552 and 562 to 859, or the complement thereof, or a fragment of at least 17 preferably at least 18,19, 20 or 21, more preferably at least 22, 23 or 24 nucleotides thereof, or the complement thereof.  
   
   
       48 . A recombinant DNA construct comprising the nucleotide sequence of  claim 47  operably linked to at least one regulatory sequence.  
   
   
       49 . A recombinant DNA construct according to  claim 48 , wherein said regulatory sequence is selected from the group comprising constitutive promoters such as any selected from the group comprising the CaMV35S promoter, doubled CaMV35S promoter, ubiquitin promoter, actin promoter, rubisco promoter, GOS2 promoter, Figwort mosaic viruse (FMV) 34S., or tissue specific promoters such as any selected from the group comprising root specific promoters of genes encoding PsMTA Class III Chitinase, photosynthetic tissue-specific promoters such as promoters of cab1 and cab2, rbcS, gapA, gapB and ST-LS1 proteins, JAS promoters, chalcone synthase promoter and promoter of RJ39 from strawberry.  
   
   
       50 . (canceled)  
   
   
       51 . A composition comprising at least one double-stranded RNA, wherein said double-stranded RNA comprises annealed complementary strands, one of which has a nucleotide sequence which is complementary to at least part of a nucleotide sequence of a fungal target gene and further comprising at least one suitable carrier, excipient or diluent.  
   
   
       52 . A composition comprising at least one double-stranded RNA according to  claim 32  and further comprising at least one suitable carrier, excipient or diluent.  
   
   
       53 . A composition comprising at least one nucleotide sequence according to  claim 47;  and further comprising at least one suitable carrier, excipient or diluent.  
   
   
       54 . (canceled)  
   
   
       55 . (canceled)  
   
   
       56 . (canceled)  
   
   
       57 . (canceled)  
   
   
       58 . (canceled)  
   
   
       59 . A cell comprising the nucleotide sequence of  claim 47 .  
   
   
       60 . The cell of  claim 59  wherein said cell is a prokaryotic cell or an eukaryotic cell.  
   
   
       61 . The cell of  claim 60  wherein said cell is a bacterial cell.  
   
   
       62 . The cell of  claim 60  wherein said cell is a plant cell.  
   
   
       63 . A plant comprising at least one double-stranded RNA according to  claim 32 , at least one nucleotide sequence according to  claim 47 , or at least one plant cell of  claim 62 .  
   
   
       64 . The plant of  claim 61  wherein said plant is chosen from the group comprising rice, barley, rye, wheat, miller, lovegrass and crabgrass.  
   
   
       65 . The plant of  claim 63  wherein said plant is rice.  
   
   
       66 . A seed comprising at least one nucleotide sequence according to  claim 47 .  
   
   
       67 . The seed of  claim 66  wherein said seed is of a plant chosen from the group comprising rice, barley, rye, wheat, miller, lovegrass and crabgrass.  
   
   
       68 . The seed of  claim 66  wherein said seed is a rice seed.  
   
   
       69 . A method for treating and/or preventing fungal infestation on a substrate comprising applying an effective amount of a composition according to  claim 52  to said substrate.  
   
   
       70 . A method for treating and/or preventing fungal growth and/or fungal infestation of a plant or propagative or reproductive material of a plant comprising applying an effective amount of a composition according to  claim 52  to a plant or to propagation or reproductive material of a plant.  
   
   
       71 . A method for treating and/or preventing a fungal disease or condition, comprising administering to a subject in need of such treatment and/or prevention, a composition according to  claim 52 , said composition comprising at least one double-stranded RNA or double-stranded RNA construct comprising annealed complementary strands, one of which has a nucleotide sequence which is complementary to at least part of a nucleotide sequence of a fungal target gene that causes the fungal disease or condition.  
   
   
       72 . (canceled)  
   
   
       73 . (canceled)  
   
   
       74 . A kit comprising a double-stranded RNA according to  claim 32 , or a nucleotide sequence according to  claim 47 , or a cell according to  claim 60 , or a composition according to  claim 52  for treating fungal infection of plants.  
   
   
       75 . A method for increasing plant yield comprising introducing in a plant a nucleotide sequence according to  claim 47  in an expressible format.  
   
   
       76 . A method according to  claim 75  wherein said plant is chosen from the group comprising rice, barley, rye, wheat, miller, lovegrass and crabgrass.  
   
   
       77 . A method according to  claim 75  wherein said plant is rice.