Abstract:
The present invention is directed to nucleic acids encoding for fusion peptides comprising a fungal targeting agent and a channel-forming domain consisting essentially of amino acids 451-626 of colicin Ia, as well as vectors having the nucleic acids of the invention and host cells having the vectors. The fusion peptides of the peptides of the present invention are particularly useful for the treatment of fungal infections in a wide variety of organisms. The fusion peptides can be prepared from the nucleic acids, such as when a vector having the nucleic acid is included in a host cell.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application is a divisional application of U.S. patent application having U.S. Ser. No. 11/338,117, filed in the U.S. on Jan. 23, 2006 and published as Pre-Grant Publication 2006/0264370, which application claims priority under 35 U.S.C. 119(a) to Chinese Patent Application No. 2005100202199, filed on Jan. 21, 2005, which applications are both incorporated herein by specific reference in their entirety. 
     
    
     BACKGROUND 
       [0002]    Fungus is opportunistic pathogens in humans. Fungus typically does not infect healthy tissues, yet once tissue defense mechanisms have been compromised, they can readily infect the tissue. One typical model of this opportunistic fungal infection is candidiasis, which is caused by  Candida albicans.    
         [0003]      Candida albicans  occurs as normal flora in the oral cavity, genitalia, large intestine, and skin of approximately 20% of humans. The risk of infection increases in children and pregnant women; people who use certain antibiotics or have nutritional and organic disease or immunodeficiency (e.g., AIDS) or trauma; and people with invasive devices, e.g., pacemakers.  Candida albicans  and its close relatives account for nearly 80% of nosocomial fungal infections and 30% of deaths from nosocomial infections in general. 
         [0004]    Historically, opportunistic fungal infections in hospitalized patients were rather unusual. Textbooks from the past described these agents as common contaminants with weak pathogenic potential, and infections were considered extreme deviation from the normal. Older ideas concerning these so-called harmless contaminants are now challenged because in those days immunodeficient and debilitated patients had died from their afflictions long before fungal infection took place. However, currently, with the advent of innovative surgeries, drugs, and other therapies that maintain such patients for expected periods, the survival rates of patients have significantly increased and the number of compromised patients has thus increased. One clinical dilemma that cannot be completely eliminated, even with rigorous disinfections, is the exposure of such patients to potential fungal pathogens from even normal flora. Fungal infections in such high-risk patients progress rapidly and are difficult to diagnose and treat. In one study, fungi caused approximately 40% of the deaths from clinically acquired infections. Up to 5% of all nosocomial opportunistic fungi cause infections. 
         [0005]    Fungi also present special problems in chemotherapy. A majority of chemotherapeutic drugs used in treating bacterial infection are generally ineffective in combating fungal infection. Moreover, the similarity between fungal and human cells often means drug toxic to fungal cells are capable of harming human cells. A few drugs with special antifungal properties have been developed for treatment of systemic and superficial fungal infections. For example, macrolide polyenes represented by amphotericin B, have a structure that mimics some cell membrane lipids. Amphotericin B which is isolated from a species of streptomycin is by far the most versatile and effective of all antifungal drugs. The azoles are broad-spectrum antifungal drugs with a complex ringed structure. As one of the most effective azole drugs, fluconazole, is used in patients with AIDS-related mycoses. 
         [0006]      Magnaporthe grisea  is the pathogen of a devastating fungal disease of rice plants known as rice blast. The fungus can also cause a similar disease in over 50 grasses, including economically important crops such as barley, wheat, and millet.  Fusarium  is another important genus of fungal pathogens, responsible for devastating diseases such as cereal scab. 
       SUMMARY 
       [0007]    The present invention is directed to novel nucleic acid molecules encoding for peptides that include a fungi specific targeting agent, e.g., a pathogenic fungal peptide pheromone, and a channel-forming colicin or a channel-forming fragment thereof (also referred to herein as “domain”). Peptides comprising a pheromone as the fungi specific targeting agent, and a colicin domain, are referred to herein as “pheromonicin peptides”. 
         [0008]    The molecular structure of the formed peptides may have the C-terminus of colicin or a channel-forming domain linked with the N-terminus of a fungi specific targeting agent, e.g., a fungal pheromone, or the N-terminus of colicin may be linked with the C-terminus of a fungi specific targeting agent e.g., a fungal pheromone. The fungal pheromone can be from a pathogenic fungus, e.g.,  Candidas . The molecular weight of the peptide may vary, e.g., from about 26,000 to about 70,000 daltons. 
         [0009]    The nucleic acid molecules of the present invention may be formed by a variety of methods. One method of forming a peptide of the present invention is by inserting a nucleic acid molecule encoding a fungal pheromone into a selected position of a nucleic acid molecule encoding a colicin, or a channel forming domain thereof, then transfecting the mutant plasmid into a host cell, e.g.,  E. coli , to produce the peptide. 
         [0010]    In one embodiment, the peptides encoded by the nucleic acid molecules of the present invention are useful in treating infections of  Candidas  or  Aspergillus  or  Magnaporthes  or  Fusarium . Exemplary infections are those created by  Candida albicans, Candida tropicalis, Candida parapsilokis, Candida krusei, Candida dubliniensis, Cryptococcus neoformans, A. fumigatus, A. flavus, A. niger, Magnaporthe grisea  and  Fusarium moniforme.    
         [0011]    The invention further provides vectors having the nucleic acid molecules that encode the peptides of the invention. The invention also provides cells, e.g., host cells, comprising the vectors of the invention. 
         [0012]    Host cells, including bacterial cells such as  E. coli , insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells), can be used to produce the peptides of the invention. Other suitable host cells are known to those skilled in the art. The invention thus provides methods for producing the peptides of the invention comprising the steps of culturing the host cells of the invention and isolating the peptides of the invention therefrom. 
         [0013]    In another embodiment, the invention provides a method for preparing a peptide which inhibits growth of a fungus comprising: (i) inserting a nucleic acid molecule encoding colicin, or a channel forming domain thereof, into a selected position of a nucleic acid molecule encoding a fungal targeting agent, e.g., a pheromone; (ii) transfecting the mutant plasmid into a host cell, e.g., an  E. coli  cell; and (iii) allowing said host cell to produce said peptide. In further embodiments, the peptide may be purified from the cells. 
         [0014]    In another embodiment, the invention provides a method for preparing a fusion peptide comprising: (i) incorporating a nucleic acid molecule encoding the peptide chain of colicin Ia with a nucleic acid molecule encoding a fungal pheromone such as  Candida albicans  alpha-mating pheromone; and (ii) introducing said nucleic acid molecule encoding the peptide chain of colicin Ia incorporated with said fungal pheromone a following the C-terminus of the colicin Ia to form a nucleic acid molecule that encodes a 639 residue peptide. 
         [0015]    In another embodiment, the invention provides a method for preparing a fusion peptide comprising: (i) incorporating a nucleic acid molecule encoding a peptide chain of colicin Ia with a nucleic acid molecule encoding a fungal pheromone such as  Candida albicans  alpha-mating pheromone; and (ii) introducing said nucleic acid molecule before the N-terminus of said colicin Ia to form a nucleic acid molecule that encodes a 639 residue peptide. 
         [0016]    In one embodiment, the invention provides a method of treating a subject having a fungal infection comprising: administering to a subject a therapeutically effective amount of a fusion peptide of the present invention, e.g., a peptide comprising a colicin Ia with a fungal targeting agent, e.g., a pheromone. Said subject may have a  Candidas  or  Aspergillus  or  Magnaporthe  or  Fusarium  infection. Specifically,  Candidas  or  Aspergillus  or  Magnaporthe  or  Fusarium  may be selected from the group consisting of  Candida albicans, Candida tropicalis, Candida parapsilokis, Candida krusei, Candida dubliniensis, Cryptococcus neoformans, A. fumigatus, A. flavus, A. niger, Magnaporthe grisea  and  Fusarium moniforme . The peptides of the instant invention can also be used to treat clinical fugal infections and other fungal infections in crops. 
         [0017]    The peptides of the invention can be incorporated into pharmaceutical compositions suitable for administration to a subject. In a preferred embodiment, the pharmaceutical composition comprises a peptide of the invention comprising the  C. albicans  alpha-pheromone and a pharmaceutically acceptable carrier. 
         [0018]    The term, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous or parenteral administration (e.g., by injection). Depending on the route of administration, the active compound may be coated in a material to protect the compound from the action of acids and other natural conditions, which may inactivate the compound. 
         [0019]    The foregoing summary is illustrative only and is not intended to be in any way limiting. In addition to the illustrative aspects, embodiments, and features described above, further aspects, embodiments, and features will become apparent by reference to the drawings and the following detailed description. 
     
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         [0020]    The foregoing and following information as well as other features of this disclosure will become more fully apparent from the following description and appended claims, taken in conjunction with the accompanying drawings. Understanding that these drawings depict only several embodiments in accordance with the disclosure and are, therefore, not to be considered limiting of its scope, the disclosure will be described with additional specificity and detail through use of the accompanying drawings, in which: 
           [0021]      FIG. 1  schematically depicts the structure of a recombinant plasmid that contains the gene of colicin Ia with the gene of  Candida albicans  alpha-mating pheromone inserted following the C-terminus of colicin Ia in the plasmid pET-15b to form a plasmid referred to herein as pCHCCA1. 
           [0022]      FIG. 2  schematically depicts the structure of a recombinant plasmid that contains the gene of colicin Ia with the gene of  Candida albicans  alpha-mating pheromone inserted following the N-terminus of colicin Ia in the plasmid pET-15b to form the plasmid referred to herein as pCHCCA2. 
           [0023]      FIG. 3  depicts a growth inhibition assay wherein ATCC 10231  C. albicans  cells were grown in M-H solid medium and exposed to (A) borate stock solution as control, (B) 50 ul amphotericin B (1 ug/ml), (C) 50 ul fluconazole (3 ug/ml), (D) 50 ul pheromonicin-SA (Ph-SA)(50 ug/ml), the fusion peptide against  Staphylococcus aureus  and (E) 50 ul pheromonicin-CA1 (Ph-CA1)(50 u/ml), the peptide produced by the pCHCCA1 plasmid. 
           [0024]      FIG. 4  depicts the inhibition effects of Ph-CA against the growth of ATCC 10231  C. albicans  cells in M-H liquid medium. The amount of sedimentary fungal filaments at the bottom of flasks indicated the inhibition effects of treatment agents. (A) Control, (B) fluconazole, (C) amphotericin B, (D) Ph-SA, (E) Ph-CA2 produced by pCHCCA2 plasmid and (F) Ph-CA1. 
           [0025]      FIG. 5  depicts the inhibition effects of Ph-CA against the growth of  C. albicans  cells in liquid medium. The amount of spores and filaments of ATCC 10231  C. albicans  cells indicated the inhibition effects. (A) Control, (B) fluconazole, (C) amphotericin B, (D) Ph-SA, (E) Ph-CA2 (F) Ph-CA1. X400. 
           [0026]      FIG. 6  depicts the fluorescent imaging of ATCC 10231  C. albicans  cells treated by Ph-CA1 and stained with 50 nM FITC/600 nM propidium iodide. (A) Control, cells were stained by FITC as green, (B) cells became red after 24 hrs Ph-CA1 treatment (10 ug/ml). X400. 
           [0027]      FIG. 7  depicts a growth inhibition assay wherein Huaxi 30168  Cryptococcus neoformans  cells were grown in M-H solid medium and exposed to (A) and (B) 100 ul amphotericin B (2 ug and 0.5 ug/ml respectively), (C) and (D) 100 ul Ph-CA1 (50 ug and 25 ug/ml respectively). 
           [0028]      FIG. 8  depicts a growth inhibition assay wherein Huaxi 30255  Aspergillus flavus  cells were grown in PDA solid medium and exposed to (A), (B) and (C) 100 ul tricyclazole (5 mg, 0.5 mg, and 0.05 mg/ml respectively), (D) 100 ul Ph-CA1 (50 ug/ml). 
           [0029]      FIG. 9  depicts a growth inhibition assay wherein ACCC 30320  Magnaporthe grisea  cells were grown in PDA solid medium and exposed to (A) 100 ul amphotericin B (0.5 ug/ml), (B) 100 ul tricyclazole (0.5 mg/ml), (C) and (D) 100 ul Ph-CA1 (25 ug and 50 ug/ml respectively). 
           [0030]      FIG. 10  depicts a growth inhibition assay wherein ACCC 30133  Fusarium moniforme  cells were grown in PDA solid medium and exposed to (A) control, (B) 100 ul amphotericin B (0.5 ug/ml), (C) Ph-CA1 100 ul Ph-CA1 (50 ug/ml) and (D) 100 ul tricyclazole (0.5 mg/ml). 
           [0031]      FIG. 11  depicts in vivo activity of Ph-CA1 against systemic candidiasis. The  C. albicans  infected mice were untreated or treated by intraperitoneal amphotericin B or Ph-CA1. 
           [0032]      FIG. 12  depicts in vivo activity of Ph-CA1 against systemic candidiasis. The  C. albicans  infected mice were untreated or treated by intravenous amphotericin B or Ph-CA1. 
           [0033]      FIG. 13  depicts the microscopic view of visceral organs of mice treated with Ph-CA1 30 days. (A) liver, (B) kidney and (C) spleen stained with hematoxylin and eosin. 100X. 
       
    
    
       [0034]    All arranged in accordance with at least one of the embodiments described herein, and which arrangement may be modified in accordance with the disclosure provided herein by one of ordinary skill in the art. 
       DETAILED DESCRIPTION 
       [0035]    In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, similar symbols typically identify similar components, unless context dictates otherwise. The illustrative embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects of the present disclosure, as generally described herein, and illustrated in the figures, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are explicitly contemplated herein. 
         [0036]    The antifungal peptides of the present invention comprise a fungi specific targeting agent e.g., a fungal pheromone, and one or more channel-forming colicins or channel-forming domains thereof. The molecular structure is generally either the C-terminus of a colicin or channel-forming domain thereof, linked with the N-terminus of the fungal specific targeting agent, or the N-terminus of the colicin or channel forming domain thereof, linked with the C-terminus of a fungal specific targeting agent. Although full-length colicin may be used in the methods and compositions of the invention, in some embodiments, only a channel-forming domain is used. In a preferred embodiment, the colicin channel-forming domain consists essentially of amino acids 451-626 of colicin Ia. 
         [0037]    Colicins are protein toxins produced by strains of  E. coli . They are generally classified into groups corresponding to the outer membrane receptor on sensitive  E. coli  cells to which they bind, with colicins that bind to the BtuB protein, the high affinity receptor for vitamin B12, being known as the E group. E-type colicins are about 60 kDa proteins that have three functional domains each implicated in one of the three stages of cell killing. The C-terminal domain carries the cytotoxic activity, the central domain carries the receptor-binding activity, and the N-terminal domain mediates translocation of the cytotoxic domain across the outer membrane. Three cytotoxic activities are found amongst E-type colicins: (i) a pore-forming ion channel that depolarizes, the inner membrane (colicin E1); (ii) an H—N—H endonuclease activity that degrades chromosomal DNA (colicins E2, E7, E8 and E9); and (iii) ribonuclease activities (colicin E3, E4, E5 and E6). Colicin-producing bacteria are resistant against the action of their own colicin through possession of a small immunity protein that inactivates the cytotoxic domain. After binding to  E. coli  cell surface receptors, E-type colicins are translocated to their site of action by a tol dependent translocation system. 
         [0038]    The peptides of the present invention maybe prepared by inserting a nucleic acid molecule encoding a fungal pheromone into the selected position of a nucleic acid molecule encoding a colicin, or a channel forming fragment thereof. The resulting transfected mutant plasmid may then inserted into a host cell, e.g.,  E. coli , to produce the peptide. Colicin Ia has the nucleic acid sequence set forth in SEQ ID NO: 1.  Candida albicans  alpha-mating pheromone has the nucleic acid sequence set forth in SEQ ID NO:2 and the amino acid sequence set forth in SEQ ID NO:3. 
         [0039]    The peptides of the invention may be used to treat subjects having a fungal infection, e.g.,  Candidas, Cryptococcus, Aspergillus, Magnaporthes  or  Fusariums . Exemplary fungal infections are oral thrush, oesophageal thrush (Oesophagitis), cutaneous (skin) candidiasis, vaginal yeast infection or candida vaginitis, balanitis, and systemic candidiasis. The peptides of the invention may also be used to treat devastating fungal infections in crops. 
       EXAMPLES 
     Example 1 
       [0040]    A fusion peptide that has been identified as pheromonicin-CA1 (Ph-CA1) was created incorporating a peptide chain of colicin Ia with a  Candida albicans  alpha-mating pheromone, wherein the pheromone was c-terminal to the colicin Ia to produce a polynucleotide having the nucleic acid sequence of SEQ ID NO:4 which encodes a polypeptide having the amino acid sequence of SEQ ID NO:5. 
       Example 2 
       [0041]    A second fusion peptide denominated as pheromonicin-CA2 (Ph-CA2) was created by incorporating a peptide chain of colicin Ia with a  Candida albicans  alpha-mating pheromone, wherein the pheromone is n-terminal to the colicin Ia, to produce a polynucleotide having nucleic acid sequence of SEQ ID NO:6 which encodes a polypeptide having the amino acid sequence of SEQ ID NO:7. 
       Results 
       [0042]    Ph-CA1 had definite antifungal effect on  Candida albicans  (ATCC 10231) in vitro and in vivo. In contrast, Ph-CA2 almost had no effect. One in vitro cell growth inhibition assay was performed with M-H or PDA solid mediums. About 5 ul Cells (10 8  CFU/ml) of  Candida albicans  (ATCC 10231),  Cryptococcus neoformans  (Huaxi 30168 strain, clinical isolated strain by West China Hospital, Sichuan University),  Aspergillus flavus  (Huaxi 30255 strain),  Magnaporthe grisea  (ACCC 30320 strain, Species Conservation Center, Chinese Academy of Agriculture Sciences), or  Fusarium moniforme  (ACCC 30133 strain) were inoculated on the surface of 10 ml M-H or PDA solid mediums contained in disks. Then 50-100 ul amphotericin B (0.5 ug to 2 ug/ml) or fluconazole (3 ug/ml) or tricyclazole (0.05 mg to 5 mg/ml) or Ph-CA1 (25 to 50 ug/ml) either rinsed in a piece of filter paper or contained in a container then being placed on the surface of the medium, and incubated at 35° C. for 2 to 4 days. 
         [0043]    As shown in  FIG. 3 , only an inhibition-zone surrounds Ph-CA1, while no similar zones were observed with other agents.  FIGS. 7 to 10  show that Ph-CA1 had definite antifungal effects against corresponding  Cryptococcus neoformans, Aspergillus flavus, Magnaporthe grisea  and  Fusarium moniforme  cells. On a molar basis, such antifungal effects were one hundred to one thousand times greater than that of known antifungal antibiotics. 
         [0044]    In vitro cell growth inhibition assays were performed in 100 ml Klett flasks containing 10 ml of M-H medium which were monitored turbimetrically with a BioRad 550 microplate reader at OD595 nm every 60 min. The filament (mycelium) precipitation at the bottom of flask was counted with a digital photo-recorder every 6 hrs. Cells were inoculated to an initial cell density of about 2.5×10 5  CFU/ml and shaken at 200 rpm on an orbital shaker at 35° C. Sedimentary fungal filaments appeared in about 36 hrs growing. 
         [0045]    Ph-CA1 and Ph-CA2 were added at the start of the culture. The same amount of borate stock solution (50 mM borate, PH9.0), Ph-SA (pheromonicin constructed by colicin Ia and staphylococcal pheromone AgrD1)(10 ug/ml) and several antibiotics preparations (2 ug/ml amphotericin B, 6 ug/ml fluconazole) were used as controls. All assays were expressed in turbidometric absorbance units measured at 595 nm and pictures of the filament sedimentation at the bottom of the flask were taken. 
         [0046]    Fluconazole and Ph-SA had no effect on the growth of  C. albicans  compared to untreated controls. In contrast, 10 ug/ml Ph-CA1 completely inhibited  C. albicans  growth, as did 2 ug/ml amphotericin B. 10 ug/ml Ph-CA2 had about 30% of the inhibition effect as the Ph-CA1. Considering the difference in molecular weight between Ph-CA1 (70 kDa) and amphotericin B (about 0.9 kDa), the inhibitory effect of Ph-CA 1 against  C. albicans  was approximately ten times greater, on a molar basis, than that of amphotericin B (see  FIG. 4 ). The spores and filaments of 2 ul treated medium were dripped on a slide and observed under microscope. In comparison with control and other treatments, spores were scarcely observed in the amphotericin B and Ph-CA1 (see  FIG. 5 ). 
         [0047]      FIG. 6  shows that after 24 hrs of incubation with Ph-CA1 (10 ug/ml), cell membrane of most  C. albicans  cells (stained by FITC as green in the presence of propidium iodide) was damaged thus the propidium iodide entered into the cell to stain cells red. 
         [0048]    KungMing mice, half male and half female, weighing 18-22 g were injected intraperitoneally with 0.5 ml of  C. albicans  (ATCC 10231), 10 8  CFU/ml. One hour after  C. albicans  injection, mice were injected intraperitoneally with 0.9% saline (A) alone as control (n=10) (C), or with amphotericin B (n=10, 1 ug/gm/day) (B), or with Ph-CA1 (n=10, 5 ug/gm/day) (A) daily for 14 days. The number of surviving animals was determined every 24 hours ( FIG. 11 ). 
         [0049]    KungMing mice, half male and half female, weighing 18-22 g were injected intraperitoneally with 0.7 ml of  C. albicans  (ATCC 10231), 10 8  CFU/ml. One hour after  C. albicans  injection, mice were injected in the tail vein with 0.9% saline alone as control (n=10) (C), or with amphotericin B (n=10, 1 ug/gm) (B), or with Ph-CA1 (n=10, 5 ug/gm) (A). The mice were then injected intraperitoneally with 0.9% saline alone, or with amphotericin B (n=10, 1 ug/gm), or with Ph-CA1 (n=10, 5 ug/gm) each day. The number of surviving animals was determined every 24 hours ( FIG. 12 ). Considering the difference in molecular weight between Ph-CA1 (70 kDa) and amphotericin B (about 0.9 kDa), the in vivo antifungal activity of Ph-CA1 against systemic candidiasis was at least twenty times greater, on a molar basis, than that of amphotericin B. 
         [0050]    KungMing mice (n=10), half male and half female, weighing 18-22 g were injected intraperitoneally with Ph-CA1 (200 ug/mouse/day) for 20 days. The bodyweight of all mice was increased. There was no microscopic evidence of necrosis or inflammation in the livers, kidneys or spleens of mice ( FIG. 13 ). 
         [0051]    A 300 m 2  rice field (seed, gangyou 725) with  Magnaporthe grisea  infection was randomly divided as three zones. The middle 100 m 2  area was treated with water spraying twice as control, the left 100 m 2  area was treated with tricyclazole spraying twice (0.5 mg/ml and 1 mg/ml) and the right 100 m 2  area was treated with Ph-CA1 spraying twice (1 ug/ml and 2 ug/ml) at the tillering stage. The time interval between two sprayings was 7 days. Each 200 leaves were randomly examined in control and treatment areas to determine the protecting efficacy of Ph-CA1. The data are depicted below in Table I. 
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Examining 
                 Grades of impaired leaves 
                 Incident 
                 Infected 
                 Protecting 
               
             
          
           
               
                 date 
                 0 
                 1 
                 3 
                 5 
                 7 
                 9 
                 rate 
                 index 
                 efficacy 
               
               
                   
               
             
          
           
               
                 One day 
                 152 
                 27 
                 14 
                 6 
                 1 
                   
                 24 
                 5.88 
                   
               
               
                 Before 
               
               
                 Treatment 
               
               
                 After 
                 89 
                 57 
                 30 
                 7 
                 7 
                 8 
                 55.5 
                 17.38 
               
               
                 Treatment 
               
               
                 Seven 
                 172 
                 10 
                 12 
                 6 
                   
                   
                 14 
                 4.22 
                 75.83 
               
               
                 days 
               
               
                 Tri- 
               
               
                 cyclazole 
               
               
                 Ph-CA1 
                 67 
                 12 
                 13 
                 8 
                   
                   
                 16.5 
                 5.05 
                 70.94 
               
               
                   
               
             
          
         
       
     
         [0052]    Another 300 m 2  rice field (seed, gangyou 725) with  Magnaporthe grisea  infection was randomly divided as three zones. The middle 100 m 2  area was treated with water spraying once as control, the left 100 m 2  area was treated tricyclazole spraying once (1 mg/ml) and the right 100 m 2  area was treated with Ph-CA 1 spraying once (2 ug/ml) at the head stage. About 200 ears were randomly examined in control and treatment areas to determine the protecting efficacy of Ph-CA1. The data are depicted below in Table II. 
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
             
           
               
                   
                 TABLE II 
               
             
             
               
                   
                   
               
               
                   
                 Grades of impaired ears 
                 Impaired 
                 Infected 
                 Damage 
               
             
          
           
               
                   
                 0 
                 1 
                 3 
                 5 
                 7 
                 9 
                 ears rate 
                 index 
                 rate 
               
               
                   
                   
               
             
          
           
               
                 Control 
                 178 
                 33 
                 22 
                 11 
                 2 
                 2 
                 28.6% 
                 8.33 
                  4.2% 
               
               
                 Tri- 
                 184 
                 16 
                 9 
                 2 
                 2 
                 0 
                 13.62%  
                 3.5 
                 1.63% 
               
               
                 cyclazole 
               
               
                 Ph-CA1 
                 218 
                 19 
                 5 
                 0 
                 0 
                 0 
                 9.92% 
                 1.56 
                 0.53% 
               
               
                   
               
             
          
         
       
     
         [0053]    In both of the above in vivo protecting assays, the concentration of Ph-CA1 used was approximately 500 times smaller than that of tricyclazole. On a molar basis, the protecting effects of Ph-CA1 were three hundred times greater than that of tricyclazole. With these two factors taken together, the total effects of Ph-CA1 against rice blast disease was approximately 10 4  to 10 5  times greater than that of tricyclazole. 
         [0054]    One skilled in the&#39;art will appreciate that, for this and other processes and methods disclosed herein, the functions performed in the processes and methods may be implemented in differing order. Furthermore, the outlined steps and operations are only provided as examples, and some of the steps and operations may be optional, combined into fewer steps and operations, or expanded into additional steps and operations without detracting from the essence of the disclosed embodiments. 
         [0055]    The present disclosure is not to be limited in terms of the particular embodiments described in this application, which are intended as illustrations of various aspects. Many modifications and variations can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and apparatuses within the scope of the disclosure, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the appended claims. 
         [0056]    The present disclosure is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled. It is to be understood that this disclosure is not limited. to particular methods, reagents, compounds compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. 
         [0057]    With respect to the use of substantially any plural and/or singular terms herein, those having skill in the art can translate from the plural to the singular and/or from the singular to the plural as is appropriate to the context and/or application. The various singular/plural permutations may be expressly set forth herein for sake of clarity. 
         [0058]    It will be understood by those within the art that, in general, terms used herein, and especially in the appended claims (e.g., bodies of the appended claims) are generally intended as “open” terms (e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.). It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases “at least one” and “one or more” to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles “a” or “an” limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases “one or more” or “at least one” and indefinite articles such as “a” or an (e.g., “a” and/or “an” should be interpreted to mean “at least one” or “one or more”); the same holds true for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., the bare recitation of “two recitations,” without other modifiers, means at least two recitations, or two or more recitations). Furthermore, in those instances where a convention analogous to “at least one of A, B, and C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “a system having at least one of A, B, and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.). In those instances where a convention analogous to “at least one of A, B, or C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “a system having at least one of A, B, or C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and/or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or “B” or “A and B.” 
         [0059]    In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group. 
         [0060]    As will be understood by one skilled in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” and the like include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 cells refers to groups having 1, 2, or 3 cells. Similarly, a group having 1-5 cells refers to groups having 1, 2, 3, 4, or 5 cells, and so forth. 
         [0061]    From the foregoing, it will be appreciated that various embodiments of the present disclosure have been described herein for purposes of illustration, and that various modifications may be made without departing from the scope and spirit of the present disclosure. Accordingly, the various embodiments disclosed herein are not intended to be limiting, with the true scope and spirit being indicated by the following claims. All references recited herein are incorporated herein by specific reference in their entirety.