Materials and methods for the enhancement of effective root nodulation in legumes

Abstract

The subject invention relates to compounds and compositions which induce transcription of the nolA gene in nitrogen-fixed bacteria, such as Bradyrhizobium japonicum. Novel bacterial strains which are insensitive to NolA, soil inoculants comprising NolA insensitive bacteria and/or nolA inducers, and methods of increasing nitrogen fixation in legumes are also disclosed.

We claim:  
     
         1 . A method of producing a nodulation inoculant containing reduced amounts of cell density factor (CDF) comprising the addition of iron to growth medium for a nodulation inoculant in amounts sufficient to reduce the concentration of CDF.  
     
     
         2 . A method of screening an extract or cell culture supernatant for the presence of an IND- 1 , IND- 2 , CDF, or CDF-like compound comprising: 
 a) obtaining an extract or cell culture supernatant;    b) contacting a host cell transformed with one or more genetic constructs containing a reporter enzyme selected from the group consisting of nolA-lacZ, nodY-lacZ, nodC-lacZ, or nodD-lacZ with said extract or cell culture supernatant; and    c) analyzing the contacted host cell for the modulation or expression of said nolA-lacZ, nodY-lacZ, nodC-lacZ, or nodD-lacZ reporter enzyme.    
     
     
         3 . The method according to  claim 1 , wherein said iron is Fe 3+ .  
     
     
         4 . The method according to  claim 1 , wherein said nodulation inoculant comprises Bradyrhizobium species.  
     
     
         5 . The method according to  claim 1 , wherein said nodulation inoculant comprises  Bradyrhizobium japonicum.    
     
     
         6 . The method according to  claim 1 , wherein medium is liquid.  
     
     
         7 . The method according to  claim 1 , wherein said iron is added prior to the addition of the nodulation inoculant.  
     
     
         8 . The method according to  claim 1 , wherein said iron is added simultaneously with the nodulation inoculant.  
     
     
         9 . The method according to  claim 1 , wherein said iron is added after the nodulation inoculant.  
     
     
         10 . The method according to  claim 1 , wherein said iron is added to the nodulation inoculant and the iron containing inoculant is added to the medium.  
     
     
         11 . The method according to  claim 1 , wherein said iron is separately added to the nodulation inoculant and the medium.  
     
     
         12 . The method according to  claim 1 , wherein the iron has a concentration of at least about 0.5 μM or at least about 0.1M.  
     
     
         13 . The method according to  claim 1 , wherein the iron has a concentration that ranges from 0.5 μM to 1M.  
     
     
         14 . An isolated compound selected from the group consisting of IND- 2  and CDF.  
     
     
         15 . A composition comprising a soil additive or conditioner and a compound selected from the group consisting of IND- 1 , IND- 2 , and CDF  
     
     
         16 . The composition according to claim  22 , wherein the compound is IND- 1  (bis-ethyl-hexyl-ester phthalate).  
     
     
         17 . An isolated bacterial cell defective in recognition of NolA inducer compounds.  
     
     
         18 . The isolated bacterial cell according to claim  21 , wherein said bacterial cell contains a defect in the nwsB gene.  
     
     
         19 . A method of suppressing the nodulation activity of indigenous nodulating bacterial cells comprising the addition of one or more NolA inducers to soil containing said indigenous nodulating bacterial cells.  
     
     
         20 . The method according to  claim 19 , wherein said NolA inducer is bis-ethyl-hexyl-ester.  
     
     
         21 . A composition comprising a carrier and a nodulation inoculant produced according to the process of  claim 1.