Trichoderma spp strains with high capacity of fungus biological control and selection process thereof by molecular markers

Abstract

Trichoderma spp. strains with high capacity for fungus biological control in wide ranges of temperature and pH are described, such strains being compatible with each other. Likewise, a process of selection of such strains through molecular markers is described. The described process reduce the number of necessary experiments to determine if a Trichoderma strain, not previously described, can display a biological activity more acceptable than those well known.

1 .- Trichoderma CIEAV-30 strain with high capacity of biological control in a wide range of phytopathogenic fungus under several different environmental conditions deposited with the ATCC with the number PTA-3950.  
     
     
         2 .- Trichoderma CIEAV-30 strain of  claim 1 , wherein the phytopathogenic fungus are Fusarium sp., Rhizoctonia sp., Phytophtora sp.,  Botrytis cinerea  and Colletotrichum sp.  
     
     
         3 .- Trichoderma CIEAV40 strain with high capacity of biological control in a wide range of phytopathogenic fungus under several different environmental conditions deposited with the ATCC with the number PTA-3951.  
     
     
         4 .- Trichoderma CIEAV-40 strain of  claim 3 , wherein the phytopathogenic fungus are Fusarium sp., Rhizoctonia sp., Phytophtora sp.,  Botrytis cinerea  and Colletotrichum sp.  
     
     
         5 .- Trichoderma CIEAV-62 strain with high capacity of biological control in a wide range of phytopathogenic fungus under several different environmental conditions deposited with the ATCC with the number PTA-3952.  
     
     
         6 .- Trichoderma CIEAV-62 strain of  claim 5 , wherein the phytopathogenic fungus are Fusarium sp., Rhizoctonia sp., and  Botrytis cinerea.    
     
     
         7 .- Trichoderma CIEAV-62 strain of  claim 6 , which produces high amounts of inhibitory substances of phytopathogenic fungus.  
     
     
         8 .- A mixture of Trichoderma strains with high capacity of biological control in a wide range of phytopathogenic fungus under several different environmental conditions comprising at least 2 strains selected from strain CIEAV-30 deposited with the ATCC with the number PTA-3950, strain CIEAV-40 deposited with the ATCC with the number PTA-3951 and strain CIEAV-62 deposited with the ATCC with the number PTA-3952.  
     
     
         9 .- The mixture of Trichoderma strains of  claim 8 , wherein the strains are CIEAV-30 and CIEAV-40.  
     
     
         10 .- The mixture of Trichoderma strains of  claim 8 , wherein the strains are CIEAV-30 and CIEAV-62.  
     
     
         11 .- The mixture of Trichoderma strains of  claim 8 , wherein the strains are CIEAV-40 and CIEAV-62.  
     
     
         12 .- The mixture of Trichoderma strains of  claim 8 , wherein the strains are CIEAV-30, CIEAV-40 and CIEAV-62.  
     
     
         13 .- A process for the selection of Trichoderma strains with high capacity of biological control by molecular markers, comprising the steps of: a step of measurement of in vitro growth rates of each strain of a Trichoderma strains collection under diverse pH and temperature conditions, wherein strains with better growth rates under different testing pH and temperature conditions are identified; a molecular-genetic markers detection step wherein genotipically different groups are identified from strains selected in the growth rate measurement step, remaining located in the same group those strains that display similar patterns of molecular-genetic markers; a step of in vitro confrontation with pathogens, in which strains of diverse groups genetically different identified in the step of molecular genetic markers, are confronted with a great number of pathogens, using for this various strains, patovars and/or races of each pathogen, identifying the strains that allow a higher effective biological than 60% with respect to the total number of strains, patovars and/or races of the corresponding pathogen that were confronted; a step 4 of in vitro measurement of pathogens growth in the presence of secreted substances from strains identified in the molecular genetic markers detection step, wherein strains that display a smaller pathogen growth speed in the presence of substances secreted by such strains are identified; and a step 5 of measurement in planta of the performance of the identified strains in the in vitro measurement step of pathogens' growth wherein strains that display a better biological control are identified on the settlement and/or disease progress in planta through greenhouse assays with plants susceptible to pathogen.  
     
     
         14 .- The process for the selection of Trichoderma strains of  claim 13 , wherein an in vitro measurement step of strains compatibility is accomplished where strains from diverse groups genetically different, identified in the step of molecular genetic markers, are confronted to each other, identifying those strains showing a suitable growth when they are confronted with other strains.  
     
     
         15 .- The process for the selection of Trichoderma strains of  claim 13 , wherein the growth rates measurement step is accomplished by growing the Trichoderma strains in vitro in a suitable culture medium under pH conditions between 5 and 9.  
     
     
         16 .- The process for the selection of Trichoderma strains of  claim 13 , wherein the growth rates measurement step is accomplished by growing the Trichoderma strains in vitro in a suitable culture medium under temperature conditions between 15 and 40° C.  
     
     
         17 .- The process for the selection of Trichoderma strains of  claim 13 , wherein the growth rates measurement step is accomplished by using as culture medium potato-dextrose-agar, measuring the radial growth speed of strains.  
     
     
         18 .- The process for the selection of Trichoderma strains selection of  claim 15 , wherein the culture for the growth rates measurement step is accomplished by adjusting the pH to the value required without a buffer so medium acidification by the microorganism is possible.  
     
     
         19 .- The process for the selection of Trichoderma strains of  claim 13 , wherein the molecular genetic markers detection step is accomplished by the use of selected markers between polymorphism of restriction fragment length “RFLPs”; amplified fragment length polymorphism “AFLPs”; random amplified polymorphic DNA “RAPDs”; microsatellites amplification; or combinations thereof.  
     
     
         20 .- The process for the selection of Trichoderma strains of  claim 13 , wherein the step of in vitro pathogens confrontation is accomplished in the medium potato-dextrose-agar in Petri dishes, determining if the strain provides or not a suitable biological control through the evaluation of strain over-growth against the pathogen's growth, the appearance of a brown to dark brown coloration on the agar, noticed at the bottom of the Petri dish, is indicative of phenolic compounds production by cellular death, as well as the appearance of a light green to dark green coloration that represents the strain capacity of spores production over the pathogen.  
     
     
         21 .- The process for the selection of Trichoderma strains of  claim 20 , wherein an arbitrary range is used in the pathogens confrontation step to evaluate the over-growth capacity, brown coloration and sporulation.  
     
     
         22 .- The process for the selection of Trichoderma strains of  claim 13 , wherein the in vitro pathogens' growth measurement step in the presence of substances secreted by the strains is accomplished by growing the strain to be tested in Petri dishes in a culture medium of potato-dextrose-agar at 27° C., inoculating an agar fragment of the frontal part of the strain radial growth that is tested over a porous membrane in fresh medium, taking off the strain from the Petri dish by removing the porous membrane and inoculating in the same Petri dish with the pathogen of interest, finally determining the radial growth speed of the pathogen, which is a measure of the effect of the secreted or excreted substances from the strain on the phytopathogen.  
     
     
         23 .- The process for the selection of Trichoderma strains of  claim 19 , wherein the molecular genetic markers detection step is accomplished by the use of primers EcoRI 5′AGACTGCGTACCAATC-3′ (SEQ ID NO:1) and Msel 5′GACGATGAGTCCTGAGTAA-3′ (SEQ ID NO:2) with an additional nucleotide (EcoRI+A ans Msel+A) for a pre-amplification.  
     
     
         24 .- The process for the selection of Trichoderma strains of  claim 19 , wherein primers with two additional selective nucleotides (EcoRI+AA, +AC, +AG o +AT/Msel+AA, +AC, +AG, o +AT) are used on a second amplification.