Fungal antagonism of blister rust pathogen, Cronartium ribicola
July 30th, 2019
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Fungal endophytes are microorganisms that live inside plant tissues without causing apparent disease. They often compete with other microorganisms by producing toxic defensive compounds. Western white pines may increase their defenses against forest pathogens, like Cronartium ribicola, the causal agent of white pine blister rust, by hosting diverse fungal endophyte communities. If fungal endophytes produce compounds that can inhibit the growth of C. ribicola, they may also have potential to decrease damage from white pine blister rust disease in infected trees.
To test for inhibition of C. ribicola from fungal endophyte compounds, we used a cell viability indicator dye, resazurin. This highly sensitive method can detect cell viability in as few as 40 living pathogen cells.
Resazurin is used often in medical and agricultural research but is relatively new to the field of ecology. The dye enters living cells and changes from weakly-fluorescent blue to highly-fluorescent pink with active metabolism (Figure 1). Dead cells have no active metabolism and don’t change the dye.
The amount of fluorescence is proportional to the number of metabolically active, viable fungal cells (Figure 2).
We can use resazurin to compare and quantify fluorescence or cell viability between pathogens grown with and without endophytic defensive compounds. So far, we have screened over 70 endophytes for antimicrobial compounds that inhibit C. ribicola metabolism in vitro. The most inhibitory compounds reduced C. ribicola metabolism by >30% after just a few days of exposure (Figure 3).
We selected the endophytes with compounds most inhibitory to C. ribicola and sprayed them onto hundreds of western white pine seedlings in a full factorial greenhouse experiment. We will expose these seedlings to the pathogen in September to determine if endophytes can indeed decrease damage in infected trees.
To test for inhibition of C. ribicola from fungal endophyte compounds, we used a cell viability indicator dye, resazurin. This highly sensitive method can detect cell viability in as few as 40 living pathogen cells.
Resazurin is used often in medical and agricultural research but is relatively new to the field of ecology. The dye enters living cells and changes from weakly-fluorescent blue to highly-fluorescent pink with active metabolism (Figure 1). Dead cells have no active metabolism and don’t change the dye.
The amount of fluorescence is proportional to the number of metabolically active, viable fungal cells (Figure 2).
We can use resazurin to compare and quantify fluorescence or cell viability between pathogens grown with and without endophytic defensive compounds. So far, we have screened over 70 endophytes for antimicrobial compounds that inhibit C. ribicola metabolism in vitro. The most inhibitory compounds reduced C. ribicola metabolism by >30% after just a few days of exposure (Figure 3).
We selected the endophytes with compounds most inhibitory to C. ribicola and sprayed them onto hundreds of western white pine seedlings in a full factorial greenhouse experiment. We will expose these seedlings to the pathogen in September to determine if endophytes can indeed decrease damage in infected trees.
