Patent Publication Number: US-2018035673-A1

Title: A Mycoherbicide

Description:
FIELD OF THE INVENTION 
     The present invention relates to the use of a consortium of cells of the species  Mycosphaerella polygoni - cuspidati  or the like for the herbicidal control of a plant. The invention also relates to a method for herbicidal control of a plant of the genus  Fallopia . In addition, the invention relates to a method of producing a consortium of cells of the species  Mycosphaerella polygoni - cuspidati . The invention further relates to a composition for the herbicidal control of a plant of the genus  Fallopia.    
     BACKGROUND OF THE INVENTION 
     Introduced in the 19 th  century for ornamental purposes, Japanese knotweed ( Fallopia japonica ) has spread dramatically throughout the UK and continental Europe, as well as many parts of the US and Canada. Today, the species is listed as one of the world&#39;s top 100 invaders. 
     In contrast to its native Japanese range, in its exotic ranges, Japanese knotweed grows aggressively, forming dense stands with severe impacts on native biodiversity. Rapid rhizomatous growth enables Japanese knotweed to force its way through concrete and tarmac with serious consequences to local infrastructures. The estimated annual cost to the British economy is currently £166 million (Williams et al. (2010) The Economic Cost of Invasive Non-Native Species on Great Britain; CABI E-UK report). Mechanical and/or chemical control of Japanese knotweed is both costly and labour-intensive. Thus, classical biological control of Japanese knotweed i.e. the introduction to its exotic ranges of co-evolved, host-specific natural enemies from its native range, which form self-sustaining populations, is an ideal additional tool for the integrated management of Japanese knotweed. 
     In addition, to Japanese knotweed ( Fallopia japonica ), the closely related species giant knotweed ( Fallopia sachalinensis ) and a hybrid between these two species,  Fallopia×bohemica , also pose threats as invasive species. 
     The ascomycete fungus  Mycosphaerella polygoni - cuspidati  is a damaging, co-evolved natural enemy of Japanese knotweed which is found ubiquitously in Japan. Infection with the pathogen causes severe necrotic lesions on Japanese knotweed leaves leading to premature senescence and ultimately leaf drop. It was therefore believed that  M. polygoni - cuspidati  was a highly promising subject for classical biological control of Japanese knotweed. However, the problem with this proposal is that testing against a list of 91 native species revealed that  M. polygoni - cuspidati  causes non-target effects on certain other plant species. More specifically,  M. polygoni - cuspidati  causes non-target effects on the rare British native plant species  Polygonum maritimum  and also, to a lesser degree, the species  Persicaria hydropiper . In particular,  M. polygoni - cuspidati  infection results in necrotic lesions and the formation of spermogonia on  P. maritimum , which constitute the first stage of the ascomycete life cycle. Due to the potential risk posed to  P. maritimum , the introduction of  M. polygoni - cuspidati  in the UK as part of a classical biological control programme against Japanese knotweed would be ill-advised in case there was inadvertent infection of, and thus risk to,  P. maritimum  or other native flora. It is also possible, and perhaps likely, that other countries in the exotic range of Japanese knotweed also have native species that would be at risk from  M. polygoni - cuspidati . As a consequence, the use of  M. polygoni - cuspidati  as a classical control agent agent with a self-sustaining and augmenting field population was seen to be unviable. 
     The present invention arises out of the recognition that  P. maritimum  generally occupies a different habitat (i.e. a coastal habitat) compared with Japanese knotweed and that, therefore, if it were possible to limit the spread of  M. polygoni - cuspidati  then the fungus could be applied to Japanese knotweed as a mycoherbicide, without risking  P. maritimum . The invention further arises from the recognition that  M. polygoni - cuspidati  is heterothallic (that is to say, mating between distinct isolates possessing complementary mating genes, MAT-1 and MAT-2, is required for the development of ascospores and for completion of its life cycle) and it has no asexual conidial stage. This unusual combination of characteristics has resulted in the surprising realization that if  M. polygoni - cuspidati  were applied in the form of only a single mating type then the fungus would not develop ascospores, and, as the fungus does not produce conidia either, this prevents the fungus from reproducing and spreading in the field beyond the site of application. The spread of  M. polygoni - cuspidati  can therefore be very closely controlled. Thus applying  M. polygoni - cuspidati  in the form of only a single mating type enables  M. polygoni - cuspidati  to be used as a herbicide against Japanese knotweed. 
     SUMMARY OF THE INVENTION 
     According to a first aspect of the present invention, there is provided the use of a consortium of cells of the species  Mycosphaerella polygoni - cuspidati or the like for the herbicidal control of a plant of the genus Fallopia , wherein the cells in the consortium are all of the same mating type. 
     Conveniently, the plant is of the species or hybrid:  Fallopia japonica, Fallopia sachalinensis  or  Fallopia×bohemica  preferably  Fallopia japonica  var.  japonica.    
     Preferably, the cells are mycelium cells. 
     Advantageously, the  Mycosphaerella polygoni - cuspidati cells in the consortium are all of mating type  1. 
     Alternatively, the  Mycosphaerella polygoni - cuspidati cells in the consortium are all of mating type  2. 
     According to a second aspect of the present invention, there is provided a method for herbicidal control of a plant of the genus  Fallopia  comprising contacting the plant with a composition comprising a consortium of cells of the species  Mycosphaerella polygoni - cuspidati  or the like, wherein the cells in the consortium are all of the same mating type. 
     Conveniently, the method further comprises the step of maintaining the cells in an aqueous environment on the plant for at least 12 hours, preferably at least 24 hours. 
     Preferably, the method comprises contacting the leaves of the plant with the composition. 
     Advantageously, the plant is of the species or hybrid:  Fallopia japonica, Fallopia sachalinensis  or  Fallopia×bohemica  preferably  Fallopia japonica  var.  japonica.    
     Coveniently, the cells are mycelium cells. 
     Preferably, the  Mycosphaerella polygoni - cuspidati cells in the consortium are all of mating type  1. 
     Alternatively, the  Mycosphaerella polygoni - cuspidati cells in the consortium are all of mating type  2. 
     According to a third aspect of the present invention, there is provided a method of producing a consortium of cells of the species  Mycosphaerella polygoni - cuspidati wherein the cells in the consortium are all of the same mating type comprising the steps of:  
         i. isolating a first individual ascospore from a sample of ascospores of the species  Mycosphaerella polygoni - cuspidati; and      ii. generating at least a first mycelium from said first isolated ascospore.       

     Conveniently, the method further comprises the step of:
         iii. determining the mating type of said first mycelium.       

     Preferably, the method further comprises the steps of:
         iv. isolating a second individual ascospore from a sample of ascospores of the species  Mycosphaerella polygoni - cuspidati;      v. generating at least a second mycelium from said second isolated ascospore;   vi. determining the mating type of said second mycelium;   vii. confirming that the mating type of the first mycelium is the same as the mating type of the second mycelium;   viii. mixing the first and second mycelia.       

     Advantageously, step iii and/or step vi comprise detecting the presence of a biological marker specific for one mating type. 
     Conveniently, the biological marker is a polynucleotide of the sequence of SEQ. ID NO: 1 or 5 or a fragment thereof which is at least 20, preferably at least 50 nucleotides in length. 
     According to a fourth aspect of the present invention, there is provided a composition for the herbicidal control of a plant of the genus  Fallopia  comprising a consortium of cells of the species  Mycosphaerella polygoni - cuspidati or the like, wherein the cells in the consortium are all of the same mating type.    
     Conveniently, the cells are mycelium cells. 
     Preferably, the  Mycosphaerella polygoni - cuspidati cells in the consortium are all of mating type  1. 
     Alternatively, the  Mycosphaerella polygoni - cuspidati cells in the consortium are all of mating type  2. 
     Preferably, the composition further comprises an adhesive agent and/or a wetting agent. 
     According to a fifth aspect of the present invention, there is provided a polynucleotide primer comprising the sequence of any one of SEQ. ID NOS: 9 to 12. 
     According to a sixth aspect of the present invention, there is provided a method for herbicidal control of a plant comprising contacting the plant with a composition comprising a consortium of cells of a fungal species which has the following characteristics:
         i. the fungal species is capable of infecting the plant and causing necrosis in the plant;   ii. the fungal species has no asexual stage in its life cycle;   iii. the fungal species is heterothallic,
 
wherein the cells of the fungal species in the consortium are all of the same mating type.
       

     Definitions 
     As used herein, the term “ Mycosphaerella polygoni - cuspidati” or “M. polygoni - cuspidati ” means a fungus in accordance with the taxonomy set forth in Kurose et al., (2009) Systematics of  Mycosphaerella  species associated with the invasive weed  Fallopia japonica , including the potential biological control agent  M. polygoni - cuspidati . Mycoscience 50, 179-189. 
     As used herein, the term “ Mycosphaerella polygoni - cuspidati  or the like” means the fungus  Mycosphaerella polygoni - cuspidati  and species having at least 98% similarity (or 98% sequence identity) in their ITS (internal transcribed spacer) region to the ITS of  Mycosphaerella polygoni - cuspidati  and meeting the following requirements: i) the fungus infects, and has a herbicidal effect on, a plant of the genus  Fallopia ; ii) the fungus cycles exclusively through its sexual stage (and has no asexual reproduction) and iii) the fungus is heterothallic. 
     As used herein, the term “herbicidal control” means reduction or elimination of plant health or growth and includes (but is not limited to) exfoliation and plant death. 
     As used herein, the term “a plant of the genus  Fallopia ” means any plant of the genus  Fallopia . In some embodiments it is defined as any invasive plant of the genus  Fallopia . It includes the species  Fallopia japonica  (which is also known as Japanese Knotweed) and  Fallopia sachalinensis  and the hybrid thereof,  Fallopia×bohemica . It also includes other hybrid species of the aforementioned species, in particular other hybrids of  Fallopia japonica.    
     As used herein, the term “consortium of cells” means a collection or mixture of cells. In some embodiments the term may be replaced with the term “aggregate of cells” throughout the specification. 
     As used herein, the term “mating type” means the characteristics that define two sexually compatible individuals. 
     The percentage “identity” between two sequences may be determined using the BLASTP algorithm version 2.2.2 (Altschul, Stephen F., Thomas L. Madden, Alejandro A. Schaffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997), “Gapped BLAST and PSI-BLAST: a new generation of protein database search programs”, Nucleic Acids Res. 25:3389-3402) using default parameters. In particular, the BLAST algorithm can be accessed on the internet using the URL http://www.ncbi.nlm.nih.gov/blast/. 
    
    
     
       FIGURES 
         FIG. 1  is a schematic diagram of the life cycle of  M. polygoni - cuspidati.    
         FIG. 2  shows photographs of disease symptoms of  M. polygoni - cuspidati  (ex Mt Hiko, Japan) on Japanese knotweed at four points in the life cycle of  M. polygoni - cuspidati.    
         FIG. 3  is a diagram of the life cycle of  M. polygoni - cuspidati.    
         FIG. 4  is a photograph of disease symptoms of  M. polygoni - cuspidati  (ex Mt Hiko, Japan) on Japanese knotweed 2.5 months after inoculation with ascospores in quarantine greenhouse. 
         FIG. 5  is a photograph of disease symptoms of  M. polygoni - cuspidati  (ex Mt Hiko, Japan) on Japanese knotweed 2.5 months after inoculation with ascospores in quarantine greenhouse, top leaf with white label over it is the control leaf. 
         FIG. 6  is a photograph of Japanese knotweed leaf material infected with  M. polygoni - cuspidati , bearing mature pseudothecia (right hand side) and leaf discs, conditioned in sterile distilled water for ascospore drop, affixed to petri dish lids for suspension over agar to obtain ascospore drop. 
         FIG. 7  is a photograph of PDA (potato dextrose agar) broth inoculated with mycelial plugs of  M. polygoni - cuspidati  after one week incubation on a shaker. 
         FIG. 8  is a photograph of macerated mycelium of  M. polygoni - cuspidati  used for inoculations. 
         FIG. 9  is a photograph of disease symptoms of  M. polygoni - cuspidati  on Japanese knotweed circa 3 weeks after inoculation with mycelium in quarantine greenhouse. 
         FIG. 10  is a photograph of disease symptoms of  M. polygoni - cuspidati  (ex Mt Hiko, Japan) on Japanese knotweed 1 month after inoculation with mycelial broth in quarantine greenhouse. 
     
    
    
     DETAILED DESCRIPTION 
       Mycosphaerella polygoni - Cuspidati  or the Like 
     The ascomycete  Mycosphaerella polygoni - cuspidati  (Mycosphaerellaceae, scomycota), or  Mycosphaerella  leaf-spot, is a fungal pathogen associated with Japanese knotweed ( Fallopia japonica ) in its native range in Japan. It has been shown that the pathogen is widespread in Japan causing extensive damage to its host and to exhibit apparent field host-specificity.  FIG. 1  describes the life cycle of the fungus and  FIG. 2  shows the disease symptoms of the fungus on Japanese knotweed at the end of May, in early June, in early July and in early August. The typical symptoms of the disease caused by the pathogen are chestnut brown circular lesions on the upper leaf surface which are surrounded by a darker, water-soaked halo. With progressing infection these individual lesions coalesce. On the lower leaf surface the appearance of the lesions is pale brown with a prominent grayish-brown halo and whitened veins. Heavy infection results in extensive leaf necrosis, leaf distortion and premature defoliation. Most commonly observed in the necrotic lesions are spermogonia as the first fruiting bodies appearing in the life-cycle. Spermogonia are dark brown to black, immersed to semi-erumpent and appear sometimes in concentric rings within lesions. They occur on both leaf surfaces but are more prominent on the upper leaf surface. Ascomata or pseudothecia as the fruiting bodies appearing subsequently in the life cycle, are less frequent, they are larger and more erumpent than the spermogonia. 
     The ascomycete life cycle generally consists of two stages: a sexual stage during which ascospores are produced as a result of spermatial mating and recombination, as well as an asexual stage characterized by the production of high numbers of conidia for dispersal.  Mycosphaerella polygoni - cuspidati  is known to produce exclusively the sexual ascospore stage, whilst conidia are lacking from the life cycle. Molecular studies using specifically developed markers have further proved the fungus to be heterothallic meaning that mating between two different isolates with complementary spermatial sex gametes (mating type 1 and mating type 2) needs to take place in order for infective ascospores to be produced. Greenhouse inoculation studies with Japanese knotweed using one single isolate (i.e. mating type) of the pathogen have confirmed that while infection will evoke characteristic diseases symptoms including the development of spermogonia producing spermatia, the formation of ascospores is lacking and the life cycle cannot be completed. 
     Referring to  FIG. 3 , the life cycle pattern of  M. polygoni - cuspidati  is shown. Ascospores 1 of the fungus germinate to form mycelium 2 which infects leaves of the target (typically  Fallopia Japonica ) entering via the stomata. As a consequence of infection, circular brown lesions develop on the leaves from necrotic spots. From the lesions, spermogonia 3 grow from which spermatia 4 are produced.  M. polygoni - cuspidati  is heterothallic and the two mating types of  M. polygoni - cuspidati  are mating type 1 and mating type 2. Spermatia of mating type 1 and spermatia of mating type 2 contact with respective, receptive hyphae 7 which are thereby fertilized to produce ascus mother cells 8. The ascus mother cells 8 produce diploid young asci 9 which, through the processes of meiosis and mitosis, form the ascospores 1 at which point the cycle repeats. 
     It is to be noted that  M. polygoni - cuspidati  has no asexual stage to its life cycle. In particular,  M. polygoni - cuspidati  does not form conidia, which are the asexual spores of most ascomycetes. Conidia as the asexual stage of an Ascomycete have never been observed in the field, following greenhouse inoculation or in culture. 
     In embodiments of the present invention, cells of  M. polygoni - cuspidati  are provided. It is to be understood that the cells may be of any strain of  M. polygoni - cuspidati . Such varieties can be characterized as having at least 98%, preferably at least 99% similiarity (identity) in the ITS region to the ITS region of  M. polygoni - cuspidati  as identified in Kurose et al and having the IMI number 401912 and the GenBank accession number AB 434912. Furthermore, the species is heterothallic, has no asexual stage and has the ability to infect  F. japonica.    
     A taxonomic description of  Mycosphaerella polygoni - cuspidati  is provided in Kurose et al., (2009) Systematics of  Mycosphaerella  species associated with the invasive weed  Fallopia japonica , including the potential biological control agent  M. polygoni - cuspidati . Mycoscience 50, 179-189. The fungus was originally described by Hara, Byotyugai Zasshi 5: 617, 1918, but the specimen was lost, hence this pathogen was re-described with a neotype deposited under the IMI number 393529. Hara et al and Kurose et al. and, in particular the taxonomic description of  M. polygoni - cuspidati  therein, are hereby incorporated by reference. 
     As explain above,  M. polygoni - cuspidati  has two mating types and the unique mating type sequences of  Mycosphaerella polygoni - cuspidati  are shown below. 
     
       
         
           
               
            
               
                 MAT1-1-1 sequence (1,296 bp) 
               
               
                 (SEQ. ID NO: 1) 
               
               
                 ATGGCTACCCAAGCCCTGCCAGACACCCTTGACACCCTTCGCGACCTCTT 
               
               
                   
               
               
                 CGCGACCGTCCCCGAGGAGATGGTTACCCAGCTCGTTTCTGAGGTCACGG 
               
               
                   
               
               
                 CTGCGCTGAGCGAAGCAGCAGCTCGACGAGCCACAGCACCGAGACGCACC 
               
               
                   
               
               
                 GCACATGGCGCAATGCCCGGCACGACTGCCACCACCTCGGCTGTAGCGAA 
               
               
                   
               
               
                 GACGCAGCGTGTGGCCGGTGGGCCAAAGAGACCGCTCAACTCCTGGATGG 
               
               
                   
               
               
                 CATTCCGCAGTATGTCCACCTCCCTCCTTGCTCTTGTCTCTTACTGACTT 
               
               
                   
               
               
                 CAACACAGAGTTCTACAACGGCGCGCTCTCGCCGCACACTCAGAAAGTGA 
               
               
                   
               
               
                 TCTCCAAAGCCCTGACGAAGTTTTGGCGTGAGGACCCCTTCGGCGCCAAG 
               
               
                   
               
               
                 TGGTTCGTAACTCCATGTTCTTCACCTGACACTGAAGCATCTGCTAACAT 
               
               
                   
               
               
                 ATCGCAGGGTCATTCTCGCCAAGGCATACAGTGTCGTGCGCGGAAGCCGC 
               
               
                   
               
               
                 GAGAAGCAAGAGGCTCCGCTTGACGAGTTCTTCATCATCTGCGCACCACT 
               
               
                   
               
               
                 CATCGGCGTCATTCCGCCAAACGAGTACATGCAGCAGATGGGATGGCATC 
               
               
                   
               
               
                 TCGGCCCGCCAATGGAGGGCGACAAGGTACCGTGCCCCCCGCAGCCCCTT 
               
               
                   
               
               
                 CGTTGCCTCAGTGCTGACCTCTACCTAGGAGAAGATGCCGCAGATCACTC 
               
               
                   
               
               
                 GCCTCTTCATCCCAAACCTCGACACCTTTCCTGATGCGATCACGATGACC 
               
               
                   
               
               
                 ACACTCAGTGTCGACGATCTGGTCCAGGCCTGCTACAACGCCGGCTACGT 
               
               
                   
               
               
                 CGACGCCAGCAACGACGACTTGCCGAGCGCAAGCACCCAGGGCTCACTCA 
               
               
                   
               
               
                 CCATGGTCGCCCGGCCAACTCCCGTCACCAACGCCGACATGGCTGTGACC 
               
               
                   
               
               
                 ACACATGTGACACCCGTCGCTCAGACTGACATCACCAACCCCGACGTCGA 
               
               
                   
               
               
                 GCCAGATCTTGAGCCAGAGTTCGAGCCAGATTTCGAGCCATTCAACGACC 
               
               
                   
               
               
                 ACCGCCCTGTCCCACTCGACCATGGTCAGTACGCACAACAGACCGTCACG 
               
               
                   
               
               
                 CCCGCTCAGCTGTCCAACAACGAGGCCATCGAGATCGTCCAGTACCACGG 
               
               
                   
               
               
                 CGACGAAGACCTCGGCCTGTTCTTCAACCCTGTCGACGGCATGTTCGGCA 
               
               
                   
               
               
                 CCTTCTTCAACCCAAACGAGAACAACGACGAATTCGCCGCCTTCGACATG 
               
               
                   
               
               
                 GACATCTACAACCACGCCCCGCACACTACCGCAAACGAGGTCCAGCAGTC 
               
               
                   
               
               
                 TGTCCAGAACGCCTCCCTCGACAACACCTTCTACTACGACGGTTGA 
               
            
           
         
       
     
     In a first embodiment, analysis of the cDNA MAT1-1-1 sequence indicates that its structure is as follows. 
     cDNA (exon): 1-259, 309-402, 458-626, 679-1,296 bp
 
intron: 260-308, 403-457, 627-678 bp
 
α-box: 223-259, 309-402, 458-623 bp
 
     Therefore the amino acid sequence of MAT1-1-1 encoded by the nucleotide sequence of SEQ. ID NO: 1 can be predicted as follows. 
     
       
         
           
               
            
               
                 (SEQ. ID NO: 2) 
               
               
                 MATQALPDTLDTLRDLFATVPEEMVTQLVSEVTAALSEAAARRATAPRRT 
               
               
                   
               
               
                 AHGAMPGTTATTSAVAKTQRVAGGPKRPLNSWMAFRKFYNGALSPHTQKV 
               
               
                   
               
               
                 ISKALTKFWREDPFGAKWILAKAYSVVRGSREKQEAPLDEFFIICAPLIG 
               
               
                   
               
               
                 VIPPNEYMQQMGWHLGPPMEGDKEKMPQITRLFIPNLDTFPDAITMTTLS 
               
               
                   
               
               
                 VDDLVQACYNAGYVDASNDDLPSASTQGSLTMVARPTPVTNADMAVTTHV 
               
               
                   
               
               
                 TPVAQTDITNPDVEPDLEPEFEPDFEPFNDHRPVPLDHGQYAQQTVTPAQ 
               
               
                   
               
               
                 LSNNEAIEIVQYHGDEDLGLFFNPVDGMFGTFFNPNENNDEFAAFDMDIY 
               
               
                   
               
               
                 NHAPHTTANEVQQSVQNASLDNTFYYDG 
               
            
           
         
       
     
     Sequence of α-box region in MAT1-1-1 as follows. 
     
       
         
           
               
            
               
                 (SEQ. ID NO: 3) 
               
               
                 CCAAAGAGACCGCTCAACTCCTGGATGGCATTCCGCAAGTTCTACAACGG 
               
               
                   
               
               
                 CGCGCTCTCGCCGCACACTCAGAAAGTGATCTCCAAAGCCCTGACGAAGT 
               
               
                   
               
               
                 TTTGGCGTGAGGACCCCTTCGGCGCCAAGTGGGTCATTCTCGCCAAGGCA 
               
               
                   
               
               
                 TACAGTGTCGTGCGCGGAAGCCGCGAGAAGCAAGAGGCTCCGCTTGACGA 
               
               
                   
               
               
                 GTTCTTCATCATCTGCGCACCACTCATCGGCGTCATTCCGCCAAACGAGT 
               
               
                   
               
               
                 ACATGCAGCAGATGGGATGGCATCTCGGCCCGCCAATGGAGGGCGAC 
               
            
           
         
       
     
     Furthermore, the amino acid sequence of α-box in MAT1-1-1 encoded by the nucleotide sequence of SEQ. ID NO: 3 can be predicted as follows. 
     
       
         
           
               
            
               
                 (SEQ. ID NO: 4) 
               
               
                 PKRPLNSWMAFRKFYNGALSPHTQKVISKALTKFWREDPFGAKWVILAK 
               
               
                 AYSVVRGSREKQEAPLDEFFIICAPLIGVIPPNEYMQQMGWHLGPPMEGD 
               
            
           
         
       
     
     In a second embodiment, analysis of the cDNA MAT1-1-1 sequence indicates that its structure is as follows. 
     cDNA (exon): 1-259, 309-402, 458-626, 679-1,296 bp
 
intron: 260-308, 403-457, 627-678 bp
 
α-box: 225-259, 309-402, 458-625 bp
 
     Sequence of α-box region in MAT1-1-1 as follows. 
     
       
         
           
               
            
               
                 (SEQ. ID NO: 13) 
               
               
                 AAAGAGACCGCTCAACTCCTGGATGGCATTCCGCAAGTTCTACAACGGCG 
               
               
                   
               
               
                 CGCTCTCGCCGCACACTCAGAAAGTGATCTCCAAAGCCCTGACGAAGTTT 
               
               
                   
               
               
                 TGGCGTGAGGACCCCTTCGGCGCCAAGTGGGTCATTCTCGCCAAGGCATA 
               
               
                   
               
               
                 CAGTGTCGTGCGCGGAAGCCGCGAGAAGCAAGAGGCTCCGCTTGACGAGT 
               
               
                   
               
               
                 TCTTCATCATCTGCGCACCACTCATCGGCGTCATTCCGCCAAACGAGTAC 
               
               
                   
               
               
                 ATGCAGCAGATGGGATGGCATCTCGGCCCGCCAATGGAGGGCGACAA 
               
            
           
         
       
     
     In a third embodiment, analysis of the cDNA MAT1-1-1 sequence indicates that its structure is as follows. 
     cDNA (exon): 1-259, 309-402, 458-626, 679-1,296 bp
 
intron: 260-308, 403-457, 627-678 bp
 
α-box: 223-259, 309-402, 458-626, 679-800 bp
 
     Sequence of α-box region in MAT1-1-1 as follows. 
     
       
         
           
               
            
               
                 (SEQ. ID NO: 14) 
               
               
                 CCAAAGAGACCGCTCAACTCCTGGATGGCATTCCGCAAGTTCTACAACGG 
               
               
                   
               
               
                 CGCGCTCTCGCCGCACACTCAGAAAGTGATCTCCAAAGCCCTGACGAAGT 
               
               
                   
               
               
                 TTTGGCGTGAGGACCCCTTCGGCGCCAAGTGGGTCATTCTCGCCAAGGCA 
               
               
                   
               
               
                 TACAGTGTCGTGCGCGGAAGCCGCGAGAAGCAAGAGGCTCCGCTTGACGA 
               
               
                   
               
               
                 GTTCTTCATCATCTGCGCACCACTCATCGGCGTCATTCCGCCAAACGAGT 
               
               
                   
               
               
                 ACATGCAGCAGATGGGATGGCATCTCGGCCCGCCAATGGAGGGCGACAAG 
               
               
                   
               
               
                 GAGAAGATGCCGCAGATCACTCGCCTCTTCATCCCAAACCTCGACACCTT 
               
               
                   
               
               
                 TCCTGATGCGATCACGATGACCACACTCAGTGTCGACGATCTGGTCCAGG 
               
               
                   
               
               
                 CCTGCTACAACGCCGGCTACGT 
               
            
           
         
       
     
     Furthermore, the amino acid sequence of α-box in MAT1-1-1 encoded by the nucleotide sequence of SEQ. ID NO: 14 can be predicted as follows (based on Conserved Domain Database, NCBI which can be accessed on the internet using the URL http://www.ncbi.nlm.nih.gov/cdd/). 
     
       
         
           
               
            
               
                 (SEQ. ID NO: 15) 
               
               
                 PKRPLNSWMAFRKFYNGALSPHTQKVISKALTKFWREDPFGAKWVILAKA 
               
               
                 YSVVRGSREKQEAPLDEFFIICAPLIGVIPPNEYMQQMGWHLGPPMEGDK 
               
               
                 EKMPQITRLFIPNLDTFPDAITMTTLSVDDLVQACYNAGY  
               
               
                   
               
               
                 MAT1-2-1 (1,087 bp) 
               
               
                 (SEQ. ID NO: 5) 
               
               
                 ATGACCACCAGCATCACCACCCACCCCCCGCAGGCGCCGCTGCCAGAGCT 
               
               
                   
               
               
                 CGACCAGTTCTTCCTGAATGAGACTCGCATCACCGACTACATCGTCGAGC 
               
               
                   
               
               
                 AGCGCGTCTGTGGTGCTGACATCATCCGCATCCCCCTAGACGAGGCCCGT 
               
               
                   
               
               
                 GGCTGTGAGAGCGCCCGTATCAAGAGTGCCGTCGAGTAAGTCCATCAGCC 
               
               
                   
               
               
                 GTCTTCACATGTTACGGTTCCCGACTGACCTCTCATAGGCACATGACCAA 
               
               
                   
               
               
                 CACCAGCTTCGAGGTCGTCGTCAGCCTCCCGACGTGGACAGTTGACTTCA 
               
               
                   
               
               
                 TCGCTCAAGGCCCCATGCCGCCCAGCCCGCCCTCGGTCAAGGTGCCTCGC 
               
               
                   
               
               
                 CCAAAGAACGCCTTTGTGATCTACCGCCTTGCCAACCACCAGCGGGTCAA 
               
               
                   
               
               
                 GGCAGCGAATCCTGATCTGCACAACAACGAGATCTGTAAGCATTTCTCTT 
               
               
                   
               
               
                 GCCCCGCCGTGTCAGACTACAACTAACACATCTCTCAGCCAAGATCATCG 
               
               
                   
               
               
                 GCGCCATGTGGGGTCGCGAGAGCCCAGACGTCAAAGACGACTTCAGAGAC 
               
               
                   
               
               
                 CAAGCTGAGGAAGAGAAGCGTCTTCACGCTCTCGCTCACCCAGGCTACCA 
               
               
                   
               
               
                 GTATCAGCCACGCAAGCCTTCTGAGAAGAAGAAGCGCATGACCAAGAACA 
               
               
                   
               
               
                 AGCTGGCCAAGATCGCAGCGAGAGCTCGCAGCGCCGGCGATGACATGGAT 
               
               
                   
               
               
                 GCGACCACCCCACCACCCCAGGTCGCTCACCCGCTCGTCACATTCAACGA 
               
               
                   
               
               
                 CGACGCTCCAGACCGTGTGCCTTTCAACTTCTCTGCTGCCCTCGCCCCAC 
               
               
                   
               
               
                 CGCAGTACGCCGCTCCTCAGGTCGATGCCTACGTCCCAGATGAGTATGCC 
               
               
                   
               
               
                 GAGCAGATTCGCCAGGCCGAGCTCGACAACACCAACTTCGACCGTGCCGA 
               
               
                   
               
               
                 AGCTGCCCTCGACGCTGACTTCAGCGCATTCGATGCTGCCATCCACAACG 
               
               
                   
               
               
                 TCGCTGGCAATGTCGACAACCCAGTCGAATCTTTCGACGCCAACAGCTTC 
               
               
                   
               
               
                 AACGCCGGCGACTTCTACGACCCCTCCTTCAACGTCGACACGTTCTTCGA 
               
               
                   
               
               
                 CTTCACCGCCGCCAGCGGCTTCGTCTACAACGGTTAA 
               
            
           
         
       
     
     In a first embodiment, analysis of the cDNA MAT1-2-1 sequence indicates that its structure is as follows. 
     cDNA (exon): 1-185, 239-435, 489-1,087 bp
 
intron: 186-238, 436-488 bp
 
     HMG box: 339-435, 489-622 bp 
     Therefore the amino acid sequence of MAT1-2-1 encoded by the nucleotide sequence of SEQ. ID NO: 5 can be predicted as follows. 
     
       
         
           
               
            
               
                 (SEQ. ID NO: 6) 
               
               
                 MTTSITTHPPQAPLPELDQFFLNETRITDYIVEQRVCGADIIRIPLDEAR 
               
               
                   
               
               
                 GCESARIKSAVEHMTNTSFEVVVSLPTWTVDFIAQGPMPPSPPSVKVPRP 
               
               
                   
               
               
                 KNAFVIYRLANHQRVKAANPDLHNNEISKIIGAMWGRESPDVKDDFRDQA 
               
               
                   
               
               
                 EEEKRLHALAHPGYQYQPRKPSEKKKRMTKNKLAKIAARARSAGDDMDAT 
               
               
                   
               
               
                 TPPPQVAHPLVTFNDDAPDRVPFNFSAALAPPQYAAPQVDAYVPDEYAEQ 
               
               
                   
               
               
                 IRQAELDNTNFDRAEAALDADFSAFDAAIHNVAGNVDNPVESFDANSFNA 
               
               
                   
               
               
                 GDFYDPSFNVDTFFDFTAASGFVYNG 
               
            
           
         
       
     
     Sequence of HMG box region in MAT1-2-1 as follows. 
     
       
         
           
               
            
               
                 (SEQ. ID NO: 7) 
               
               
                 AAGGTGCCTCGCCCAAAGAACGCCTTTGTGATCTACCGCCTTGCCAACCA 
               
               
                   
               
               
                 CCAGCGGGTCAAGGCAGCGAATCCTGATCTGCACAACAACGAGATCTCCA 
               
               
                   
               
               
                 AGATCATCGGCGCCATGTGGGGTCGCGAGAGCCCAGACGTCAAAGACGAC 
               
               
                   
               
               
                 TTCAGAGACCAAGCTGAGGAAGAGAAGCGTCTTCACGCTCTCGCTCACCC 
               
               
                   
               
               
                 AGGCTACCAGTATCAGCCACGCAAGCCTTCT 
               
            
           
         
       
     
     Furthermore, the amino acid sequence of HMG box in MAT1-2-1 encoded by the nucleotide sequence of SEQ. ID NO: 7 can be predicted as follows. 
     
       
         
           
               
            
               
                 (SEQ. ID NO: 8) 
               
               
                 KVPRPKNAFVIYRLANHQRVKAANPDLHNNEISKIIGAMWGRESPDVKDD 
               
               
                 FRDQAEEEKRLHALAHPGYQYQPRKPS 
               
            
           
         
       
     
     In a second embodiment, analysis of the cDNA MAT1-2-1 sequence indicates that its structure is as follows. 
     cDNA (exon): 1-185, 239-435, 489-1,087 bp
 
intron: 186-238, 436-488 bp
 
     HMG box: 341-435, 489-624 bp 
     Sequence of HMG box region in MAT1-2-1 as follows. 
     
       
         
           
               
            
               
                 (SEQ. ID NO: 16) 
               
               
                 GGTGCCTCGCCCAAAGAACGCCTTTGTGATCTACCGCCTTGCCAACCACC 
               
               
                   
               
               
                 AGCGGGTCAAGGCAGCGAATCCTGATCTGCACAACAACGAGATCTCCAAG 
               
               
                   
               
               
                 ATCATCGGCGCCATGTGGGGTCGCGAGAGCCCAGACGTCAAAGACGACTT 
               
               
                   
               
               
                 CAGAGACCAAGCTGAGGAAGAGAAGCGTCTTCACGCTCTCGCTCACCCAG 
               
               
                   
               
               
                 GCTACCAGTATCAGCCACGCAAGCCTTCTGA 
               
            
           
         
       
     
     In a third embodiment, analysis of the cDNA MAT1-2-1 sequence indicates that its structure is as follows. 
     cDNA (exon): 1-185, 239-435, 489-1,087 bp
 
intron: 186-238, 436-488 bp
 
     HMG box: 348-435, 489-622 bp 
     Sequence of HMG box region in MAT1-2-1 as follows. 
     
       
         
           
               
            
               
                 (SEQ. ID NO: 17) 
               
               
                 CGCCCAAAGAACGCCTTTGTGATCTACCGCCTTGCCAACCACCAGCGGGT 
               
               
                   
               
               
                 CAAGGCAGCGAATCCTGATCTGCACAACAACGAGATCTCCAAGATCATCG 
               
               
                   
               
               
                 GCGCCATGTGGGGTCGCGAGAGCCCAGACGTCAAAGACGACTTCAGAGAC 
               
               
                   
               
               
                 CAAGCTGAGGAAGAGAAGCGTCTTCACGCTCTCGCTCACCCAGGCTACCA 
               
               
                   
               
               
                 GTATCAGCCACGCAAGCCTTCT  
               
            
           
         
       
     
     Furthermore, the amino acid sequence of α-box in MAT1-1-1 encoded by the nucleotide sequence of SEQ. ID NO: 17 can be predicted as follows (based on Conserved Domain Database, NCBI which can be accessed on the internet using the URL http://www.ncbi.nlm.nih.gov/cdd/). 
     
       
         
           
               
            
               
                 (SEQ. ID NO: 18) 
               
               
                 RPKNAFVIYRLANHQRVKAANPDLHNNEISKIIGAMWGRESPDVKDDFR 
               
               
                 DQAEEEKRLHALAHPGYQYQPRKPS 
               
            
           
         
       
     
     Risk of Crossing/Hybridization Between  M. polygoni - Cuspidati  and Other Fungal Pathogens 
     It is considered unlikely that  M. polygoni - cuspidati  could cross or hybridize with other fungal pathogens or  Mycosphaerella  species in the UK. The taxonomic name and position of the pathogen are as follows:  Mycosphaerella polygoni - cuspidati  (Mycosphaerellaceae, Mycosphaerellales, Dothideomycetidae, Dothideomycetes, Ascomycota, Fungi). Morphological characteristics place the leaf-spot pathogen firmly in the genus  Mycosphaerella . Based on morphological data, specifically ascus and ascospore dimensions, as well as molecular data, this leaf spot also falls within the species concept, as described by Hara (1918) from leaf litter from the island of Honshu. But, as no type was designated a neotype description has been published (Kurose et al., 2009) Molecular analysis showed  M. polygoni - cuspidati  sequences (ITS region) to have no close match to those documented in molecular databases (i.e. GenBank).  Mycosphaerella sumatrensis , a recently described species on  Eucalyptus  from Indonesia, showed the nearest match (96-97% homology in ITS region), thus indicating it to be a different species. The low level of homology between  M. polygoni - cuspidati  and  M. sumatrensis  might indicate that the species on  Fallopia japonica  var.  japonica  is taxonomically isolated. A BLAST search gave matches with a number of other  Mycosphaerella  species and their anamorphs but none of these showed more than 90% homology (i.e.  Mycosphaerella  areola 89%,  Mycosphaerella microsora  89%,  Mycosphaerella brassicicola  88%). 
     In the field, fungal species are known to hybridize with closely related species. “The key question, whether a newly formed fungal hybrid will persist in nature, is directly related to the relative fitness of the hybrid” (Schardl &amp; Craven, 2003, Interspecific hybridization in plant-associated fungi and oomycetes: a review.  Molecular Ecology  12: 2861-2873). Hybridization resulting in more virulent hybrids has been shown to occur in the genera  Phytophthora, Ophiostoma  and  Melampsora  (Brasier, 2001, Rapid evolution of introduced plant pathogens via interspecific hybridization.  BioScience  51: 123-133; Schardl &amp; Craven, 2003). On the other hand, based on research undertaken with  Heterobasidion annosum , Garbelotto et al. (2007) Ecological constraints limit the fitness of fungal hybrids in the  Heterobasidion annosum  species complex. Applied  Environmental Microbiology,  73: 6106-6111 propose that “in fungal species complexes characterized by differential host specificity, hybrids may not be generally unfit but may be at an ecological disadvantage with regard to their parents in a habitat specific for one of the parents.” 
     Thus molecular data strongly indicate that there are no closely-related  Mycosphaerella  species with which to hybridise. Furthermore, by using a single mating type isolate for application as a mycoherbicide  M. polygoni - cuspidati  will not be able to produce ascospores in the field, thus will neither reproduce nor spread beyond the area of application. Given that no other  Mycosphaerella  species is known to attack Japanese knotweed outside its native range Japan and  M. polygoni - cuspidati  will only reside in treated Japanese knotweed plants, hybridization will not take place. 
     Genus  Fallopia    
     The genus  Fallopia  comprises around a dozen species of flowering plants in the family Polygonaceae. In embodiments of the present invention, invasive plants of the genus are preferred and plants of the species  Fallopia japonica  (which is commonly known as “Japanese knotweed”) are particularly preferred. Plants of the variety  Fallopia japonica  var.  japonica  are more particularly preferred. However, other embodiments of the invention relate to plants of the species  Fallopia sachalinensis  (which is commonly known as “giant knotweed”). Still further embodiments relate to plants of the hybrid  Fallopia×bohemica  which is a hybrid of the species  Fallopia japonica  and  Fallopia sachalinensis . It is to be understood that the invention also relates to other species within the genus  Fallopia  such as hybrids of  Fallopia japonica  with species other than  Fallopia sachalinensis.    
     Herbicidal Product 
     The principal component of the herbicidal product of the invention is a consortium of cells. In one embodiment of the present invention, the herbicidal products comprises a consortium of cells of  M. polygoni - cuspidati  or the like. In some embodiments, the consortium comprises only cells of a single species but in other embodiments, the consortium comprises cells of a plurality of species such as, for example, i) cells of a first strain of  M. polygoni - cuspidati , ii) cells of a second strain of  M. polygoni - cuspidati  and iii) cells of a different species. It is to be understood that all of the cells of  M. polygoni - cuspidati  and any other cells which are capable of mating with  M. polygoni - cuspidati  are of the same mating type (i.e. either all are mating type 1 or all are mating type 2). However, other cells, which do not mate with  M. polygoni - cuspidati , may, if they have a mating type, be of any mating type. 
     It is preferred that the cells in the consortium are mycelium cells. It is to be understood that these are the haploid mycelial cells that arise from ascospores&#39; germination from and not mycelia from the dikaryophase of the life cycle. In one embodiment, the cells in the consortium are individual, separate cells but in other embodiments, the cells are in the form of part or all of a mycelium or mycelia. In further embodiments, the cells of the consortium are ascospores and in still further embodiments the cells are a mixture of mycelium cells and ascospores. 
     In one embodiment, the consortium of cells is provided as follows. Infected plants are readily available in Japanese knotweed stands at Mount Hiko. Collections of the pathogen are made by sampling infected leaves displaying the above described disease symptoms. Leaves are dried in a plant press for 3-5 days with a daily change of newspaper as the drying paper and then transferred into wax packets. 
     Using a stereomicroscope, the leaf spots are assessed for mature pseudothecia of  M. polygoni - cuspidati . Leaf discs containing these pseudothecia are cut using a hole-punch and immersed in sterile distilled water at 19° C. for 24 hours. Leaf discs (pseudothecia facing down) are affixed to the lid of a Petri dish using double sided sticky tape, suspended over tap water agar (TWA) and incubated at 19° C. for 24 hours. After this incubation period plates are microscopically examined for ascospore discharge.  Mycosphaerella polygoni - cuspidati  ascopores are identified based on morphology and single spore isolates are made by removing individual ascospores together with a block of agar using a sterile hypodermic needle. Each block is placed onto separate TWA agar plates and placed in an incubator at 19° C. Each single spore isolate is then molecularly characterized with respect to its mating type. 
     Multiplex PCR is performed on samples of each spore isolate. Primer sets for differentiating the two mating types are shown below. These primers have been designed originally based on the sequence of each mating type shown above. 
     Primer sets for detecting Mating type 1-1 isolates 
     
       
         
           
               
               
            
               
                   
                 Forward primer: 
               
               
                   
                 (SEQ. ID NO: 9) 
               
               
                   
                 GTTACCCAGCTCGTTTCTGA 
               
               
                   
                   
               
               
                   
                 Reverse primer: 
               
               
                   
                 (SEQ. ID NO: 10) 
               
               
                   
                 AGTGATCTGCGGCATCTTCT 
               
            
           
         
       
     
     Primer sets for detecting Mating type 1-2 isolates 
     
       
         
           
               
               
            
               
                   
                 Forward: 
               
               
                   
                 (SEQ. ID NO: 11) 
               
               
                   
                 CGCCTTTGTGATCTACCGCC 
               
               
                   
                   
               
               
                   
                 Reverse: 
               
               
                   
                 (SEQ. ID NO: 12) 
               
               
                   
                 GCCAGCTTGTTCTTGGTCAT 
               
            
           
         
       
     
     However, it is to be appreciated that mating type characterization may be performed in accordance with alternative methods, for example by detecting the mating type sequences using different sets of primer sequences that are specific for each mating type isolate. 
     After mating type characterization, a mycelial culture is grown up which contains the consortium of cells of the invention. In some embodiments, multiple mycelia cultures of the same mating type are mixed together in order to provide sufficient quantities of cells. 
     In some embodiments, the herbicidal product also comprises such further additives and/or adjuvants, in addition to the consortium of cells, as are appropriate to provide a practical crop protection product. Additives are provided to maintain the long-term viability of the consortium&#39;s propagule and to facilitate ease of handling, storage and application. In preferred embodiments, additives are provided so that the herbicidal product is in the form of a liquid or solution. For example, in one embodiment, the mycelium grown in potato dextrose broth and thus the broth itself may be used in order that the herbicidal product is in the form of a liquid. In another embodiment, the consortium comprises an additive that enhances adherence of the consortium to plant leaves. In another embodiment, the consortium comprises an additive, such as a wetting agent, that maintains the cells in an aqueous environment for an extended period such as at least 12 hours and preferably at least 24 hours. Adjuvants are provided to improve spray delivery of the consortium or enhance the pathological impacts of the propagules. Further details of suitable additives and adjuvants are provided in Fungi As Biocontrol Agents: Progress Problems and Potential edited by T. M. Butt, C. Jackson, N. Magan, which is incorporated herein by reference. 
     In some embodiments the concentration is 2×10 5  mycelial fragments per ml. However, in other embodiments, the concentration is between 1×10 4  and 1×10 6  mycelial fragments per ml, preferably between 1×10 5  and 3×10 5  mycelial fragments per ml 
     Use of the Consortium 
     In use, the consortium is inoculated on the leaves of a plant of the genus  Fallopia  as will now be described. In some embodiments, the herbicidal product comprising the consortium is painted on to the leaves of the plant using a brush. This provides very effective coverage of the leaves but is time consuming and is not appropriate for large scale coverage. Accordingly, in preferred embodiments, the herbicidal product is sprayed on to the leaves of the plant, which permits coverage of multiple plants substantially simultaneously. 
     The cells of the consortium then infect the plant via the leaf stomata and there is fungal growth within the plant. Subsequently, the fungal infection results in defoliation of the plant resulting in death of the plant or a severe setback to its growth which allows other plant species the opportunity to out-compete it during the growing season. Referring to  FIG. 3 , it is to be appreciated that the life cycle of the fungus starts (in the production phase) at the initiation line 5 with the provision of ascospores 1. Furthermore, since only one cell mating type is present in the consortium, the life cycle does not proceed beyond the formation of spermatia 4 and thus stops at the termination line 6. This is very significant since, unlike spores, mycelia cannot be spread by wind and so plant infection is limited to contact only (i.e. the plant initially infected and any other plant where there is leaf-to-leaf contact). As such the effective area of the fungus is controlled and spread is prevented. 
     Since a single application of the herbicidal product may not result in plant death, in some embodiments of the invention, multiple applications of the herbicidal product are inoculated on the plant. Symptoms on the plant leaf are normally visible after 3-4 weeks and it is preferred that such symptoms are assessed before a second application of the product. Therefore, in some embodiments further applications are applied between 1 and 2 months are the preceding application. The number of applications required depends upon various factors such as the age of the plant and the climate. 
     While it is generally desirable to ensure that spraying covers only leaves of plants of the genus  Fallopia , herbicidal product that does fall on other plants is not generally of concern since very few other plants are susceptible to infection by the fungus of the present invention. Furthermore, even if a non-target plant were infected by the fungus of the invention, it would at worst result in infection of plants in physical contact with the originally-infected plant since the presence of only a single mating type in the consortium means that the fungus cannot complete its life cycle to form ascospores. Indeed, spread of the fungus is limited in most circumstances to growth within the plant originally inoculated. 
     EXAMPLES 
     Example 1 
     During survey work in Japan  M. polygoni - cuspidati  was commonly encountered in Japanese knotweed stands at Mount Hiko. Samples of  M. polygoni - cuspidati  were taken and ascospores thereof were inoculated onto leaves of Japanese knotweed in a quarantine greenhouse.  FIG. 4  shows a leaf 2.5 months after inoculation.  FIG. 5  shows a plant 2.5 months after inoculation in which the top leaf with a white label over it is the control leaf. 
     Example 2 
     During survey work in Japan  M. polygoni - cuspidati  was commonly encountered in Japanese knotweed stands at Mount Hiko. Collections of the pathogen were made by sampling infected leaves displaying the above described disease symptoms. Leaves were dried in a plant press for 3-5 days with a daily change of newspaper as the drying paper and then transferred into wax packets. Samples were taken back into quarantine in the UK under licences. 
     Using a stereomicroscope, the leaf spots were assessed for mature pseudothecia of  M. polygoni - cuspidati . Leaf discs containing these pseudothecia were cut using a hole-punch and immersed in sterile distilled water at 19° C. for 24 hours. Leaf discs (pseudothecia facing down) were affixed to the lid of a Petri dish using double sided sticky tape (see  FIG. 6 ), suspended over tap water agar (TWA) and incubated at 19° C. for 24 hours. After this incubation period plates were microscopically examined for ascospore discharge.  Mycosphaerella polygoni - cuspidati  ascopores were identified based on morphology and single spore isolates were made by removing individual ascospores together with a block of agar using a sterile hypodermic needle. Each block was placed onto separate TWA agar plates and placed in an incubator at 19° C. Each single spore isolate can then be molecularly characterized with respect to its mating type. 
     Example 3 
     Four mycelial plugs, 5 mm in diameter, were taken from the actively growing margin of an individual culture of  M. polygoni - cuspidati  using a sterile cork-borer. The mycelial plugs were split into smaller fragments and added to 75 ml of potato dextrose broth (PDB) in a 250 ml conical flask. A flask containing PDB only was used as a control to detect potential contamination during the mass production process. Flasks were sealed with a sterile foam bung and placed on a rotary shaker at 170 rpm at 18° C. for 8 days (see  FIG. 7 ). Subsequently the mycelial broth was transferred to a sterilised blender and macerated for 20 seconds in order to produce a more homogenous inoculum ( FIG. 8 ). 
     The fragmented mycelium broth was applied to plants by using a paintbrush and brushing the inoculum onto the upper and lower leaf surface. Control plants/leaves were inoculated with PDB liquid medium only. Inoculated plants were placed in a customized dew chamber (Mercia Scientific, Birmingham, UK) set at 20° C., without light for 48 h. The viability of the inoculum was assessed as growth of mycelial fragments on TWA. Upon removal from the dew chamber all plants were maintained in a designated quarantine greenhouse chamber under a controlled temperature and light regime of 20° C. and 12-h dark/12-h light and regularly monitored for the development of disease symptoms.  FIG. 9  is a photograph of a leaf c. 3 weeks after inoculation with mycelial broth.  FIG. 10  is a photograph of a leaf 1 month after inoculation with mycelial broth.