Abstract:
This invention refers to the pure culture of a wild isolate of the fungal species  Fusarium solani  (code name: strain ‘Fs-K’) and to its use for the biological control of plant pathogens. The invention includes the mycelium of the strain ‘Fs-K’ and any genetic modification of it, the asexually produced conidia and chlamydospores of the strain ‘Fs-K’, and the cultures of the strain ‘Fs-K’ (grown on liquid or solid media) which contain the mycelium and/or the conidia and/or the chlamydospores, as well as any extract or ingredient which derives from the growth of the strain ‘Fs-K’ or from the growth of any genetic modification of it. The invention further includes organic substrates for plant growth, as well as any other substrate compositions suitable for the growth of fungi and/or plants which contain forms of the strain ‘Fs-K’ (i.e. any vegetative or reproductive structure of it which is involved in the biological control of diseases). The invention further includes the use of the strain ‘Fs-K’ for the enhancement of plant health and growth, for the protection of plants from pathogenic organisms, and for the regulation of the rhizosphere surroundings.

Description:
DESCRIPTION OF THE INVENTION 
       [0001]    The adverse consequences of the irrational application of chemical compounds (pesticides) for the control of plant diseases are noticeable both on the natural environment and the human health. To avoid the excessive use of synthetic inputs in agriculture, novel biological control agents are being developed for protection against plant pathogens; these antagonistic microorganisms confer to plant disease control by suppressing disease incidence and/or by reducing symptoms severity caused by the pathogens. 
         [0002]    Composting is the biological decomposition of organic wastes and agro-industrial by-products under controlled aerobic conditions to a relatively stable humus-like material called compost. Composts are widely used as soil conditioners and plant growth substrates, and their ability to suppress the growth of plant pests and pathogens (including bacteria, fungi and nematodes) has been repeatedly demonstrated. These suppressive properties are usually attributed to the antagonistic action of the indigenous compost microbiota against the disease-causing agents. 
         [0003]    During the last years a large number of bacteria, yeasts and filamentous fungi were isolated from composts, identified and characterized as regards their suppressive effects against plant pathogens. Various types of biological processes have been proposed for explaining such effects: competition for nutrients and ecological niches, production of cell-wall hydrolytic enzymes and/or of antifungal compounds, parasitism of pathogens, induction of plant defense mechanisms, masking of signals which enhance pathogens growth, etc. 
         [0004]    The present invention describes the isolation of a  Fusarium solani  fungal strain encoded as “Fs-K”, which demonstrates an intense and pronounced antagonistic action (under in vitro conditions) against plant pathogenic fungi and Oomycetes, and with the ability to protect tomato plants from the soil-borne pathogen  Fusarium oxysporum  f.sp.  radicis - lycopersici  and from the leaf pathogen  Septoria lycopersici . Despite the fact that other  Fusarium solani  strains presenting suppressive effects against plant pathogens were also recorded in the past, no strain has ever been reported to provide simultaneous protection from both plant pathogens ( F. oxysporum  f.sp.  radicis - lycopersici  and  S. lycopersici ), which are characterized by different modes of invading the host-plant and by different life habits. In addition, the “Fs-K” strain is an endophyte growing into the plant vascular system, and it is effective at the extremely low ratio of inoculum concentration (conidia) 1:10 3  of antagonist:pathogen. Moreover, the “Fs-K” strain survives for long time periods (minimum one month) in the plant growth substrates, causing thus a satisfactory suppression effect against soil-borne plant pathogens. 
     
    
     
       BRIEF DESCRIPTION OF DRAWINGS (FIGURES) 
         [0005]      FIG. 1 : Mycelium (a) and conidia (b) of the  Fusarium solani  strain Fs-K referred in the invention. 
           [0006]      FIG. 2 : Light micrographs of tomato root tissue at 15 d post-inoculation with  Fusarium solani  strain Fs-K using Chlorazol Black staining. (A) Intracellular growth of mycelium hyphae (arrows) and formation of round bodies (arrowheads), (B) Development of mycelium hyphae into the plant vascular bundle. 
           [0007]      FIG. 3 : Disease incidence in tomato plants caused by inoculation with 10 5  conidia cm −3  of  Fusarium oxysporum  f.sp.  radicis - lycopersici . (A) Disease progress curves in tomato plants grown on peat in the absence (triangles) or presence (diamonds) of conidia (10 4  cm −3 ) of  Fusarium solani  strain Fs-K referred in the invention. Disease incidence was assessed as percentage of dead plants (five pots, each containing five plants per treatment, n=5). Mean values that were statistically different at each time point (t test P≦0.01) are indicated by an asterisk (*). Vertical bars (I) indicate standard errors of means. (B) Disease incidence caused by  Fusarium oxysporum  f.sp.  radicis - lycopersici  in three different tomato cultivars (ACE, VFN8, Pearson) in the absence (−) or presence (+) of 10 4  conidia cm −3  of peat of  Fusarium solani  strain Fs-K. Statistical comparisons within all cultivars were performed by Duncan multiple comparisons tests (a &lt;0.05). Indicator letters in common denote a lack of significant difference. Vertical bars indicate the ½ of the respective standard errors of means. 
           [0008]      FIG. 4 : Effect of  Fusarium solani  strain Fs-K presence on the development of symptoms caused by  Septoria lycopersici  strain NEV on tomato plants grown for 1 month on peat (control) or on peat amended with 10 4  conidia cm −3  of  Fusarium solani  strain Fs-K. Number of lesions was counted per leaf 1 week after inoculation with 10 6  conidia cm −3  of  S. lycopersici  spore suspension. Vertical bars (I) indicate standard deviations of means (n=12). Statistical comparisons were performed by Duncan&#39;s multiple comparisons tests (a &lt;0.005). Letters in common indicate a lack of significant differences. 
           [0009]      FIG. 5 : Linear radial growth of plant pathogens (A)  Fusarium oxysporum  f.sp.  radicis - lycopersici  and (B)  Phytophthora nicotianae  in in vitro tests of the suppressive action of  Fusarium solani  strain Fs-K culture filtrate. The culture filtrate, after the mycelium removal, was sterilized and added into a PDA growth medium in the ratios (v/v) indicated on the graphs. Statistical comparisons were performed by Duncan&#39;s multiple comparisons tests (a &lt;0.005) (n=5). Letters in common indicate a lack of significant difference. Vertical bars (I) indicate the respective standard deviations of means. 
           [0010]      FIG. 6 : Increase of the suppressive effect of a compost derived from spent  Agaricus  mushroom substrate (SMC) against soil-borne plant pathogens through the addition of the  Fusarium solani  strain Fs-K. The graph describes the percentage of disease incidence symptoms in tomato plants after their infection with 10 5  conidia cm −3  of the plant pathogen  Fusarium oxysporum  f.sp.  radicis - lycopersici . Plants were grown on peat or in SMC compost in the absence or presence (10 4            cm −3 ) of the  Fusarium solani  strain Fs-K. Disease incidence was assessed as the percentage of dead plants (five pots, each containing five plants per treatment, n=5). Statistical comparisons were performed by Duncan&#39;s multiple comparisons tests (a &lt;0.005). Letters in common indicate a lack of significant difference. Vertical bars (I) indicate the respective standard deviations of means. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0000]    
       
         
           
             1. Isolation and characterization of  Fusarium solani  strain Fs-K  Fusarium solani  strain Fs-K, an antagonistic fungus, was isolated from the roots of tomato plants (line ACE55) which were cultivated in a peat-compost mixture showing suppressive effects against plant pathogenic fungi. The above compost was produced by agricultural residues derived from the winery and olive-oil extraction processes, in particular grape marc (GM, pressed grape pomace) and extracted olive press cake (EPC), in the facilities of the Institute of Olive and Vegetable Crops (National Agricultural Research Foundation-N.AG.RE.F.) which is located in Kalamata, Greece (Kavroulakis et al., 2005). 
           
         
       
     
         [0012]    Tomato seeds (line ACE 55) were sterilized with 2.5% NaOCl and sown directly in pots containing peat (Sunu Kura, Seda joint-Stock company) and compost (in a ratio of 1:1 dry wt/dry wt). One month after sowing, roots of tomato plants were surface-sterilized in 2.5% NaOCl for 10 minutes and washed with sterilized tap water. The roots were then placed in Petri dishes containing Potato Dextrose Agar (PDA) medium in which 60 μg ml −1  chlorotetracycline, 30 μg ml −1  streptomycin και 30 U penicillin G were added (Zuberer, 1994) and incubated for approximately 4 days at 25° C. Intraradical mycelial hyphae were subsequently post-inoculated in new sterile liquid and solid PDA media. The fungus, described in the present invention, is filamentous with pale-white hyphae and produces asexual conidia ( FIG. 1 ). The fungal strain described in the invention was isolated from a single conidium (spore). This fungus can be cultivated in several growth media, utilizing a broad range of carbon sources. Its optimum pH and temperature for growth are 5.0 and 24° C. respectively. 
         [0013]    This fungal isolate was characterized as  Fusarium solani  strain based on ITS-5.8S rRNA gene sequencing following the experimental procedure described below: DNA from the fungal strain described in the invention was extracted as described by Lee and Taylor (1990). The Internal Transcribed Spacer 1 (ITS1)-5.8S rRNA gene-Internal Transcribed Spacer 2 (ITS2) DNA region was amplified by polymerase chain reaction (PCR) using primers ITS1 (5′-TCC GTA GGT GAA CCT GCG G-3′) and ITS4 (5′-TCC TCC GCT TAT TGA TAT GC-3′) (White et al., 1990). PCR amplification was performed using a denaturation step of 4 min at 94° C., followed by 35 cycles of 30 sec denaturation at 94° C., 30 sec primer annealing at 52° C. and 1 min DNA chain extension at 72° C. Amplification was completed by 10 min DNA chain extension at 72° C. PCR was performed using a PTC-200 thermocycler (MJ Research Inc., USA). PCR products were separated on 1% agarose gels in 1×TAE buffer, extracted using QIAquick gel extraction columns (Qiagen, Hilden, Germany), cloned into the pGEM-T easy vector (Promega, WI, USA) and sequenced. Similarity searches against sequences deposited in nucleotide data bases were carried out using the Blast-N algorithm.
       2. Bioassay for the assessment of the suppressive effects of the fungus described in the present invention against the plant pathogenic fungus  Fusarium oxysporum  f.sp.  radicis - lycopersici          
 
         [0015]    Tomato seeds of different genotypes (ACE 55, Pearson, VFN8, Castlemart) were surface-sterilized in 2.5% NaOCl and sown directly in pots of 15 cm diameter which contained approximately 300 cm 3  peat supplemented with a NPK fertilizer (20-20-20) at a final concentration of 0.8 g l −1 . The pots were placed in a growth chamber which operated at 20°-25° C. with a photoperiod of 16 h and humidity of 65% RH. Plants were irrigated at 48 h intervals and supplemented weekly with a nutrient solution containing micronutrients. 
         [0016]    Plants were routinely inoculated with  Fusarium solani  strain Fs-K at a concentration of 10 4  conidia per cm 3  peat applied one week after sowing. At the stage of the first true leaf emergence, plants were infected with 10 5  conidia of the pathogenic fungus  Fusarium oxysporum  f.sp.  radicis - lycopersici  per cm 3  peat. Control plants were inoculated and infected with a 0.85% NaCl solution only. Five or eight replicates were used for each experimental treatment (replicate pots, each containing five plants). 
         [0017]    Conidia of the antagonistic fungus and the pathogenic fungus were derived from the following procedure: The fungi were cultured in potato dextrose broth (PDB) liquid medium. Removal of mycelial fragments by sieving was subsequently performed and conidia were recovered by centrifugation at 4000 g, washed with 0.85% NaCl, counted using a haemocytometer and re-suspended in the appropriate volume of 0.85% NaCl in order to achieve the desired inoculum concentration. 
         [0018]    Plants were scored either as healthy (lack of disease symptoms or scarce disease symptoms) or as diseased (severe wilting or dead plants). Tomato plants inoculated with the fungus described in the present invention the antagonist) were statistically significantly more tolerant to the disease caused by the phytopathogenic fungus  F. oxysporum  f. sp.  radicis - lycopersici  compared to plants which were cultivated in the absence of the antagonist. All lines and hybrids tested in this bioassay were protected by the fungus described in the invention. 
         [0019]    Plants inoculated with the fungus which is described in the present invention did not develop disease symptoms. Observations were performed following the experimental setup up to the stage of fruit development. The antagonistic fungus can grow endophytically in plants, as it was shown for tomato plants. The fungus was isolated from root and crown tissue but never from the upper parts of the plant. Furthermore, the fungus is able to grow in the vascular system of the plant,
       3. Effects of inoculum concentration (numbers of conidia per cm 3  of growth substrate) of the antagonistic fungus  Fusarium solani  strain Fs-K on the suppressiveness against the plant pathogen  Fusarium oxysporum  f.sp.  radicis - lycopersici          
 
         [0021]    To examine and determine the inoculum concentration which is required to obtain the disease suppression caused by the antagonistic fungus  Fusarium solani  Fs-K, the bioassay procedure described in the section 2 was followed for testing a range of conidia concentrations of the fungus described in the present invention. Specifically, tomato plants (line ACE55) were inoculated with 10 2 , 10 3 , 10 4 , 10 5  and 10 6  conidia per cm 3  peat. The plant pathogen was added after 7 days at a concentration of 10 5  conidia per cm 3  peat. It was found that even in the lowest antagonistic:pathogen ratio tested (1:10 3 ), the fungus described in the invention showed great suppressiveness against the pathogen, resulting in disease reduction by at least 50% (Table 1).
       4. Bioassay for the assessment of suppressive effects of the fungus described in the present invention against the plant pathogenic fungus  Septoria lycopersici          
 
         [0023]    Tomato seeds of the line ACE55 were sown and grown in pots containing peat in the presence or absence of the antagonistic fungus described in present invention, as described in section 2. Three replicates (pots, each containing 5 plants) per treatment were used. Twelve detached leaves (four from each replication) were inoculated by brushing the leaf underside with spore suspensions of  S. lycopersici  NEV [1×10 6  spores cm −3  in 0.01% (v/v) Tween 80] as described by Martin-Hernandez et al. (2000). The underside of control detached leaves was covered with 0.01% (v/v) Tween 80. Five days after inoculation, infection was evaluated by counting the number of necrotic lesions, which are characteristic disease symptoms caused by the pathogen. Leaves from plants inoculated with the antagonistic fungus described in the present invention showed statistically significantly lower numbers of lesions. Thus, the antagonistic fungus described in the present invention also protects plants, preferably tomato plants, from foliar pathogens.
       5. In vitro test of inhibitory effects of broth extract (supernatant) from the liquid culture of fungus  Fusarium solani  Fs-K against the pathogenic micro-organisms  Fusarium oxysporum  f.sp.  radicis - lycopersici  and  Phytophthora nicotianae          
 
         [0025]    Conidia (10 5  ml −1 ) of the fungus  Fusarium solani  Fs-K were inoculated in PDB and incubated for 5 days at 25° C. and 150 rpm. The grown mycelium was removed by filtration and the supernatant, containing compounds secreted by the fungus, was sterilized. The filtered and sterilized growth medium was used in concentrations 5, 10, 25 και 50% per volume in PDA in order to prepare Petri dishes. The plates were then inoculated with the pathogens  Fusarium oxysporum  f.sp.  radicis lycoperisci  and  Phytophthora nicotianae . Radial mycelial growth was determined within a 5 days incubation period and compared to growth in control plates in the absence of the filtered growth medium from the antagonistic fungus. Great inhibition in the radial mycelial growth of the pathogens was observed for media containing 25 and 50% of the filtered growth medium from the cultivation of the fungus  Fusarium solani  Fs-K. In addition, measurements of the mycelial dry weight of the pathogen  Fusarium oxysporum  f.sp.  radicis - lycopersici  showed 30% decrease in dry biomass in the case that 50% culture filtrate was added in the medium compared to the respective control (medium containing 50% PDB and the amount of agar needed) 
         [0000]    This indicates that the fungus of the present invention produces extracellular compounds which can inhibit the radial growth of phytopathogenetic filamentous fungi and oomycetes.
       6. Enhancement of suppressive properties of composts against soil-borne phytopathogenic fungi by inoculation with the antagonistic fungus  Fusarium solani  Fs-K       
 
         [0027]    Composts, produced in the Institute of Kalamata (N.AG.RE.F.-Greece), were used as substrates for the growth of tomato plants (line ACE55). These composts were produced from agricultural residues/by products (e.g. olive tree leaves, olive mill wastewaters, olive press-cake, extracted olive press-cake, spent mushroom substrate and grape marc). Plants were inoculated with the antagonistic fungus described in the present invention at concentration of 10 5  conidia per cm 3  of growth substrate, according to the standard procedure described in section 2. Plants were then infected by the pathogenic fungus  Fusarium oxysporum  f.sp.  radicis - lycopersici  and disease development was determined based on the bioassay described in section 2. 
         [0028]    From the above experimental procedures, it was found that the antagonistic fungus of the present invention can be added in non-suppressive composts, in which it may grow and develop suppressiveness against plant pathogens 
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Effect of inoculum concentration (conidia per cm 3  of growth substrate) of 
               
               
                 antagonistic fungus  Fusarium solani  Fs-K on the control of disease 
               
               
                 incidence caused by  F. oxysporum  f.sp.  radicis-lycopersici  (FORL). 
               
             
          
           
               
                   
                 
                   F. 
                 
                   
               
               
                   
                 
                   oxysporum 
                 
               
               
                   
                 f.sp.  radicis-   
               
               
                   
                 
                   lycopersici 
                 
                 Fs-K:pathogen ratio II   
               
             
          
           
               
                 Fungi 
                 None 
                 Fs-K 
                 (FORL) 
                 1:10 3   
                 1:10 2   
                 1:10 
                 1:1 
               
               
                   
               
               
                 % Disease 
                 0 
                 0 
                 88c 
                 52 b 
                 40 b 
                 22.6 a 
                 18.6 a 
               
               
                 incidence I   
               
               
                   
               
               
                   I Disease incidence was assessed as percentage of dead plants (n = 25 or 30 plants). The antagonist and the pathogen were applied as inocula to the root system. Strain Fs-K was applied one week after sowing; FORL was applied at the stage of first-true-leaf. 
               
               
                   II FORL in the form of 10 5  conidia cm −3  peat; strain Fs-K in the form of 10 2 , 10 3 , 10 4  and 10 5  conidia cm −3  peat. Statistical comparisons between treatments were performed by Duncan&#39;s tests (a &lt;0.05). Letters in common indicate a lack of significant difference.