Biocontrol formulation containing streptomyces spp., method for preparing the formulation and relevant use

Abstract

The present invention is a method for preparing a biocontrol formulation by the following steps. A Streptomyces bacterium is selected simultaneously with superior sporulation ability, chitinase activity and antagonistic effectiveness against plant fungal pathogens by culturing with fungal pathogens, wherein the selected Streptomyces bacterium is cultured on a chitin-limited plate to assess the ability of sporulation and chitinase activity, and the Streptomyces bacterium with superior sporulation ability is the strain with producing powder-like and maturity form spore chains. Spores from the selected bacterium is collected. The obtained spores are taken as a seed inoculum. Then the seed inoculum is cultured to directly yield a broth medium containing at least 10 9 viable spores/ml which the spores contained in the broth medium are well suspended. The biocontrol formulation is obtained. The present invention further provided a biocontrol formulation containing a high concentration of Streptomyces spp. spores.

1 . A method for preparing a biocontrol formulation comprising: 
 selecting a  Streptomyces  bacterium simultaneously with superior sporulation ability, chitinase activity and antagonistic effectiveness against plant fungal pathogens by culturing with plant fungal pathogens, wherein the selected  Streptomyces  bacterium is cultured on a chitin-limited plate to assess the ability of sporulation and chitinase activity, and the  Streptomyces  bacterium with superior sporulation ability is the strain which producing powder-like and maturity form spore chains,    collecting spores from the selected bacteria,    culturing the spores to obtain a seed inoculum,    amplifying the seed inoculum by a serial fermentor to directly yield a broth medium containing at least 10 9  viable spores/ml which the spores contained in the broth medium are well suspended, and    obtaining the a biocontrol formulation.    
   
   
       2 . The method as claimed in  claim 1 , wherein a final concentration of the seed inoculum is at least 10 8  viable spores/ml.  
   
   
       3 . The method as claimed in  claim 1 , wherein the broth medium is the Czapek's culture medium containing an organic nutrient source.  
   
   
       4 . The method as claimed in  claim 1 , wherein the chitin-limited plate is a sugar-limited plate containing chitin as a major carbon source.  
   
   
       5 . The method as claimed in  claim 1 , wherein the step of collecting spores from the selected bacteria is collected by washing off the selected  Streptomyces  bacteria by sterilized distill water from a colloidal chitin amended potato sucrose agar (PSA) plate to form a spore suspension.  
   
   
       6 . The method as claimed in  claim 1 , wherein the seed inoculum is prepared in a shake-flask.  
   
   
       7 . The method as claimed in  claim 6 , wherein the shake-flask is a 500 to 5000 ml shake-flask.  
   
   
       8 . The method as claimed in  claim 1 , wherein the step of amplifying the seed inoculum by a serial fermentor is stepwisely amplified with the use of 10× series fermentors.  
   
   
       9 . The method as claimed in  claim 1 , wherein the broth medium containing at least 10 9  viable spores/ml is also used as a source inoculum for further amplification.  
   
   
       10 . The method as claimed in  claim 9 , wherein the concentration of the source inoculum is in a range of about 2.0×10 9  to 5.0×10 10  viable spores/ml.  
   
   
       11 . The method as claimed in  claim 10 , wherein the concentration of the source inoculum is in a range of about 5.0×10 9  to 5.0×10 10  viable spores/ml.  
   
   
       12 . The method as claimed in  claim 1 , wherein a condition for the amplifying the seed inoculum are at about 28 to 37° C., 80 to 250 rpm, 0.25 to 0.75 vvm and pH at 5.0 to 8.0.  
   
   
       13 . The method as claimed in  claim 12 , wherein the condition for the amplifying the seed inoculum are at 30° C. under continuous stirring at 200 rpm and aeration at 0.5 vvm for about 4 to 6 days.  
   
   
       14 . A biocontrol formulation prepared according to  claim 1  which comprises at least 10 8  viable spores/ml and barely mycelial remains.  
   
   
       15 . The biocontrol formulation as claimed in  claim 14 , wherein the biocontrol formulation is stored at 6° C. for more than 10 months prior to use.  
   
   
       16 . The biocontrol formulation as claimed in  claim 14 , wherein the biocontrol formulation is a broth medium containing viable spores in a range of about 10 10  spores/ml to 10 11  spores/ml.