Abstract:
The present invention discloses use of borrelidin for preventing and controlling  phytophthora  root rot of soybean. The present invention also discloses formulations of borrelidin seed coating agents and wettable powders and preparation methods thereof. Borrelidin exhibits significant effects on  Phytophthora sojae.  As compared with the conventional fungicide metalaxyl which is used for preventing and controlling  phytophthora  root rot of soybean, the mycelial growth inhibition assay indicates that the IC 50  and IC 95  values of borrelidin are 1/62 and 1/263, respectively, of those of metalaxyl. In the indoor pot-culture experiment, the seed coating agent and wettable powder exhibit significant controlling effects on  phytophthora  root rot of soybean.

Description:
FIELD 
       [0001]    The present invention relates generally to the field of pesticide technology and, more particularly, to a germicide compound for preventing and controlling  phytophthora  root rot of soybean-borrelidin, formulations of its seed coating agents and wettable powders, and methods for preparation thereof. 
       BACKGROUND 
       [0002]      Phytophthora  root rot of soybean is a devastating soybean disease caused by  Phytophthora sajae  Kaufmann &amp; Gerdemann. The disease was first discovered in the northeastern part of the State of Indiana in 1948 and in the northwestern part of the State of Ohio in 1951. In 1955, occurrences of the disease in the United States were first reported by Suhovecky et al, and the disease was thought to be caused by  Phytophthora  pathogens. In 1958, Kaufmann and Gerdemann found out after research of the pathogen that the pathogen is a new  Phytophthora  species and named it  Phytophthora sojae.  The bacterium has a specialized pathogenicity to soybean. The disease is characterized by its wide distribution and is primarily spread through soil and seeds. In the recent years, along with the ceaseless increase in soybean growing area and the increasingly intensification of continuous cropping, the harm of soybean root rot has worsened year after year and resulted in significantly decreased production. In the recent years, soybean production has generally experienced a yearly reduction of 10% to 30%. In seriously affected areas, the disease may cause a production reduction of 60% and even crop failure. It is therefore imperative to prevent and control the disease. 
         [0003]    As there are limited applicable drugs for controlling  phytophthora  root rot of soybean in production, chemical prevention and control are still the primary measures taken for controlling the disease. Metalaxyl is currently one of the most effective drugs for the prevention and control of diseases caused by various  phytophthora  bacteria. However, as metalaxyl is a systemic fungicide with singular site of action, it is extremely easy for pathogenic bacteria to develop resistance to the drug. Numerous researchers have discovered that  phytophthora  is prone to develop resistance to metalaxyl, and this is directly indicated by decline in efficacy or drug failure causing huge production losses. 
         [0004]    Borrelidin is a macrolide with a nitrile functional group, and its structural formula (I) is as shown below. 
         [0005]    Borrelidin was first discovered as a result of its antibacterial activity (Berger et al, 1949), and it was later discovered to exhibit antiviral activity (Dichinson at al, 1965), pest and weed control activities (DE 3607287), angiogenesis inhibition activity (Wakabayashi et al, 1997), antifungal activity (DE19835669; U.S. Pat. No. 6,193,964), antimalarial activity (Otoguro et al, 2003) and other activities. 
         [0000]    
       
                 
         
             
             
         
       
     
         [0006]    Patents DE19835669 and U.S. Pat. No. 6,193,964 disclose the inhibition effects of borrelidin on a number of fungi, including  Alternaria tenius; Botrytis cinerea; Colletotrichum musae; Fusarium oxysporum  f. sp. ( niveum );  Phoma asparagi; Phytophthora capsici; Phytophthora cinnamomi; Pythium aphanidermatum; Pythium splendens; Pythium spinosum; Pythium ultimum; Rhizoctonia solani; Sclerotinia sclerotiorum; Sclerotium rolfsii;  especially on  Pythium  species and  Phytophthora  species. It is also disclosed in the aforementioned patents that the IC 50  values of borrelidin against  Pythium aphanidermatum, Pythium splendens, Pythium ultimum, Phytophthora capsici  are 0.06, 0.03, 0.01, 0.10 and 0.027 respectively. The patents also disclose that borrelidin may be applied to tomato, aubergine, green chilli, cucumber, watermelon, pumpkin, cabbage, pea and other crops, but they have not disclosed the inhibition effects of borrelidin on  Phytophthora sajae  Kaufmann &amp; Gerdemann. 
       SUMMARY OF THE INVENTION 
       [0007]    The present invention aims at providing an application of borrelidin for preventing and controlling  phytophthora  root rot of soybean, which effectively inhibits mycelial growth. 
         [0008]    The present invention overcomes the foregoing problems through the following technical scheme: application of borrelidin for preventing and controlling  phytophthora  root rot of soybean. 
         [0009]    Preferably, the borrelidin is a coating agent or wettable powder. 
         [0010]    Preferably, the seed coating agent comprises the following components in weight percent terms: 
         [0011]    borrelidin: 0.5%˜5%; 
         [0012]    film-forming agent: 1%˜2%; 
         [0013]    thickening agent: 0.5%˜2%; 
         [0014]    dispersing agent: 1%˜3%; 
         [0015]    viscosity stabilizing agent: 1%˜3%; 
         [0016]    defoaming agent: 0.1%˜0.2%; 
         [0017]    warning coloration: a little; and 
         [0018]    the remaining portion is water. 
         [0019]    Preferably, the file forming agent is polyvinyl alcohol (PVA). 
         [0020]    Preferably, the dispersing agent is calcium lignosulphonate. 
         [0021]    Preferably, the viscosity stabilizing agent is n-butyl alcohol. 
         [0022]    Preferably, the thickening agent is bentonite. 
         [0023]    Preferably, the defoaming agent is tributyl phosphate (TBP). 
         [0024]    Preferably, the warning coloration is acid brilliant scarlet. 
         [0025]    Preferably, the method for preparation of the seed coating agent comprises the following steps: 
         [0026]    (1) Mix borrelidin and the warning coloration with some of the water, and add the mixture into a homogenizing machine to undergo grinding and dispersing for 25˜35 minutes; 
         [0027]    (2) Mix the film-forming agent with the remaining amount of water, and add the mixture into a homogenizing machine to undergo grinding and dispersing for 3˜5 minutes; 
         [0028]    (3) Stir the dispersed product obtained from step (2), and add in at the same time the dispersing agent, the viscosity stabilizing agent and the thickening agent; mix thoroughly with the dispersed product obtained from step (1), add in the de-foaming agent, and obtain borrelidin seed coating agent after the completion of defoaming. 
         [0029]    Preferably, the wettable powder comprises the following components in weight percent terms: 
         [0030]    borrelidin: 0. 2%˜5%; 
         [0031]    wetting agent: 2%˜6%; 
         [0032]    dispersing agent: 5%˜10%; and 
         [0033]    filler: 70%˜90%. 
         [0034]    Preferably, the wetting agent is sodium dodecyl benzene sulfonate (SDBS). 
         [0035]    Preferably, the dispersing agent is calcium lignosulphonate. 
         [0036]    Preferably, the filler is inert bentonite. 
         [0037]    Preferably, the method for preparation of the borrelidin wettable powder comprises the following steps: get the various components according to the prescribed weight proportions, mix them evenly; pulverize the mixture and pass through a 40˜45 μm pore size sieve, heat-dry to obtain borrelidin wettable powder. 
         [0038]    The present invention innovatively discloses borrelidin&#39;s characteristic of resistance to  Phytophthora sajae  Kaufmann &amp; Gerdemann, and that the IC 50  and IC 95  values of borrelidin indicated in the mycelial growth inhibition assay are 0.0056 and 0.026 respectively, which are 1/62 and 1/263, respectively, of those of the conventional fungicide metalaxyl which is used for preventing and controlling  phytophthora  root rot of soybean. 
         [0039]    The present invention also innovatively discloses formulations of borrelidin seed coating agents and wettable powders, and methods for preparation thereof. 
     
    
     DETAILED DESCRIPTION 
       [0040]    The present invention is further illustrated in connection with its preferred embodiments as follows. It must however be noted that the scope of the present invention shall not be limited by these embodiments. 
         [0041]    It must be noted that the various raw materials used in the following embodiments are in weight percent terms. 
       Embodiment 1 
     0.5% Borrelidin Seed Coating Agent 
       [0042]    Borrelidin: 0.5%; polyvinyl alcohol (PVA): 2%; calcium lignosulphonate: 3%; bentonite: 1%; n-butyl alcohol: 2%; tributyl phosphate (TBP): 0.15%; acid brilliant scarlet: a little; and the remaining portion is water. 
         [0043]    Processes of preparation of the seed coating agent are as follows:
       1. Mix borrelidin and the acid brilliant scarlet with some of the water, and add the mixture into a high speed homogenizing machine to undergo grinding and dispersing for 30 minutes to produce a standby liquid;   2. Mix the polyvinyl alcohol (PVA) with the remaining amount of water, cause the mixture to undergo high speed homogenizing for 3 minutes, introduce the mixture into a stirred tank for stirring, and add in at the same time the calcium lignosulphonate, the bentonite and the n-butyl alcohol; and mix thoroughly with the standby liquid obtained from step (1) for 2 hours;   3. Finally, add the tributyl phosphate (TBP) into the liquid mixture obtained from step (2), obtain product after the completion of defoaming, and perform analysis to obtain passable seed coating agent of the present invention.       
 
       Embodiment 2  
     1% Borrelidin Seed Coating Agent 
       [0047]    Borrelidin: 1%; polyvinyl alcohol (PVA): 2%; calcium lignosulphonate: 3%; bentonite: 2%; n-butyl alcohol: 2%; tributyl phosphate (TBP): 0.15%; acid brilliant scarlet: a little; and the remaining portion is water. 
         [0048]    Processes of preparation of the seed coating agent are as follows:
       1. Mix borrelidin and the acid brilliant scarlet with some of the water, and add the mixture into a high speed homogenizing machine to undergo grinding and dispersing for 30 minutes to produce a standby liquid;   2. Mix the polyvinyl alcohol (PVA) with the remaining amount of water, cause the mixture to undergo high speed homogenizing for 5 minutes, introduce the mixture into a stirred tank for stirring, and add in at the same time the calcium lignosulphonate, the bentonite and the n-butyl alcohol; and mix thoroughly with the standby liquid obtained from step (1) for 1 hour;   3. Finally, add the tributyl phosphate (TBP) into the liquid mixture obtained from step (2), obtain product after the completion of defoaming, and perform analysis to obtain passable seed coating agent of the present invention.       
 
       Embodiment 3  
     1.5% Borrelidin Seed Coating Agent 
       [0052]    Borrelidin: 1.5%; polyvinyl alcohol (PVA): 1%; calcium lignosulphonate: 2%; bentonite: 2%; n-butyl alcohol: 1%; tributyl phosphate (TBP): 0.15%; acid brilliant scarlet: a little; and the remaining portion is water. 
         [0053]    Processes of preparation of the seed coating agent are as follows:
       1. Mix borrelidin and the acid brilliant scarlet with some of the water, and add the mixture into a high speed homogenizing machine to undergo grinding and dispersing for 30 minutes to produce a standby liquid;   2. Mix the polyvinyl alcohol (PVA) with the remaining amount of water, cause the mixture to undergo high speed homogenizing for 4 minutes, introduce the mixture into a stirred tank for stirring, and add in at the same time the calcium lignosulphonate, the bentonite and the n-butyl alcohol; and mix thoroughly with the standby liquid obtained from step (1) for 1.5 hours;   3. Finally, add the tributyl phosphate (TBP) into the liquid mixture obtained from step (2), obtain product after the completion of defoaming, and perform analysis to obtain passable seed coating agent of the present invention.       
 
       Embodiment 4  
     2% Borrelidin Seed Coating Agent 
       [0057]    Borrelidin: 2%; polyvinyl alcohol (PVA): 2%; calcium lignosulphonate: 2%; bentonite: 2%; n-butyl alcohol: 2%; tributyl phosphate (TBP): 0.15%; acid brilliant scarlet: a little; and the remaining portion is water. 
         [0058]    Processes of preparation of the seed coating agent are as follows:
       1. Mix borrelidin and the acid brilliant scarlet with some of the water, and add the mixture into a high speed homogenizing machine to undergo grinding and dispersing for 30 minutes to produce a standby liquid;   2. Mix the polyvinyl alcohol (PVA) with the remaining amount of water, cause the mixture to undergo high speed homogenizing for 5 minutes, introduce the mixture into a stirred tank for stirring, and add in at the same time the calcium lignosulphonate, the bentonite and the n-butyl alcohol; and mix thoroughly with the standby liquid obtained from step (1) for 1.2 hours;   3. Finally, add the tributyl phosphate (TBP) into the liquid mixture obtained from step (2), obtain product after the completion of defoaming, and perform analysis to obtain passable seed coating agent of the present invention.       
 
       Embodiment 5  
     2% Borrelidin Wettable Powder 
       [0062]    Borrelidin: 2%; sodium dodecyl benzene sulfonate (SDBS): 5%; calcium lignosulphonate: 5%; inert bentonite: 88%. 
         [0063]    Processes of preparation of the wettable powder are as follows:
       1. Compute the dosage of each component in weight percent terms, weigh and mix evenly;   2. Pulverize the ingredients using an airslide disintegrating mill, and pass through a 40-45pm pore size test sieve for grading;   3. Further mix evenly;   4. Heat-dry and perform analysis before storage.       
 
       Embodiment 6  
     5% Borrelidin Wettable Powder 
       [0068]    Borrelidin: 5%; sodium dodecyl benzene sulfonate (SDBS): 5%; calcium lignosulphonate: 5%; inert bentonite: 85%. 
         [0069]    Processes of preparation of the wettable powder are as follows:
       1. Compute the dosage of each component in weight percent terms, weigh and mix evenly;   2. Pulverize the ingredients using an airslide disintegrating mill, and pass through a 40-45 μm pore size test sieve for grading;   3. Further mix evenly;   4. Heat-dry and perform analysis before storage.       
 
       Embodiment 7  
     Mycelial Growth Inhibition Essay 
       [0074]    Indoor assays of active compound borrelidin and active compound metalaxyl for their inhibitory effects against  Phytophthora sajae  Kaufmann &amp; Gerdeman were carried out. 
         [0075]    Growth rate method was used in the assays. The two active compounds were first formulated in a series of concentration gradients.  Phytophthora sajae  Kaufmann &amp; Gerdeman cultures after 7 days of culture were punched using a sterile punch (Ø0.5cm) and put on standby, and the cultures were later transferred onto citrate-acetate (CA) plate culture media and placed at a temperature of 27±1° C., and colony diameters were measured after 7 days of culture. Each treatment process was repeated thrice; the measured results were compared with the inhibition rates of the control group, and their IC 50  and IC 95  values were computed. 
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Inhibitory Effects of Bactericidal Agent on 
               
               
                 Mycelial Growth of  Phytophthora sajae   
               
             
          
           
               
                   
                 Compound 
                 IC 50  (μg/ml) 
                 IC 95  (μg/ml) 
               
               
                   
                   
               
             
          
           
               
                   
                 Borrelidin 
                 0.0056 
                 0.026 
               
               
                   
                 Metalaxyl 
                 0.35 
                 6.85 
               
               
                   
                   
               
             
          
         
       
     
       Embodiment 8 
     Borrelidin Seed Coating Agent Pot-culture Experiment 
       [0076]    For indoor pot-culture experiment, 25 pots were divided into 5 groups of which 4 groups were seed coating agents with different effective contents, seed coating agents were used for soybean seed coating treatment (pesticide/seed mass ratio 1:50), and the other 1 group was the control group with each group having 5 pots. All groups of pots were inoculated with  Phytophthora sajae  Kaufmann &amp; Gerdeman, and each pot was sown with 30 soybean seeds (soybean variety Hefeng No. 25). The disease prevention effects of the seed coating agents are as shown in Table 2. It can be seen from Table 2 that anti-disease soybean seed coating agents have very significant controlling effects on  phytophthora  root rot of soybean. 
         [0000]    
       
         
               
             
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Disease Prevention Effect of Borrelidin Seed Coating Agent 
               
             
          
           
               
                   
                 Disease 
               
               
                   
                 Prevention 
               
               
                 Drug Tested 
                 Effect (%) 
               
               
                   
               
               
                 0.5% Borrelidin Seed Coating Agent (Embodiment 1) 
                 95.4 
               
               
                   1% Borrelidin Seed Coating Agent (Embodiment 2) 
                 96.2 
               
               
                 1.5% Borrelidin Seed Coating Agent (Embodiment 3) 
                 97.4 
               
               
                   2% Borrelidin Seed Coating Agent (Embodiment 4) 
                 97.2 
               
               
                 Control Group 
                 — 
               
               
                   
               
             
          
         
       
     
       Embodiment 9  
     Borrelidin Wettable Powder Pot-Culture Experiment 
       [0077]    All treatment processes were repeated thrice with 10 seedlings being treated each time, and the experiment was performed in a greenhouse with the pots arranged randomly. The drugs to be tested were prepared and delivered separately according to their respective required concentrations in a morning of a sunny day, and inoculation was performed after drug absorption. The hypocotyl inoculation method was used, and the seedlings were cultivated in a humid atmosphere after inoculation. The weather during the experiment was mostly sunny, and the lowest temperature in the greenhouse shed was 18° C., the highest temperature therein was 30° C., the average temperature therein was 24° C., and the relative humidity therein was above 80%. The disease prevention effects of borrelidin wettable powders are as shown in Table 3. 
         [0000]    
       
         
               
             
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Disease Prevention Effect of Borrelidin Wettable Powder 
               
             
          
           
               
                   
                   
                 Disease 
               
               
                   
                   
                 Prevention 
               
               
                 Drug Tested 
                 Preparation Dosage 
                 Effect (%) 
               
               
                   
               
               
                 2% Borrelidin Wettable Powder 
                 1000 times 
                 93.9 
               
               
                 (Embodiment 5) 
               
               
                 5% Borrelidin Wettable Powder 
                 1000 times 
                 97.8 
               
               
                 (Embodiment 6) 
               
               
                   
               
             
          
         
       
     
         [0078]    The foregoing text sets forth only preferred embodiments of the present invention, and it must be noted that various modifications, additions and alterations may be made to the invention by those of ordinary skill in the art without departing from the spirit of the invention, and such modifications, additions and alterations would fall within the scope of the invention.