Plants live in tight associations with microbes who colonize their roots and leaves and surrounding soil. Some microbes are harmful and cause disease while others are beneficial and aid in plant nutrient uptake, decomposition of organic materials, protection against pathogens, tolerance against drought and other stressors. Plants can influence microbial communities and vice versa with consequences for the growth of individual plants, the composition of plant communities, and entire ecosystems. In our work, we focus on three main aspects of plant-microbe interactions; 1) how they may aid or hinder plant invasions, 2) how their function changes across environmental gradients, and 3) how they may both cause and prevent disease in plants. On MPG Ranch, three highly invasive weeds of contrasting lie history strategies (cheatgrass, knapweed and leafy spurge) co-occur with remnants of native plant vegetation. Using both observational and experimental approaches, we seek to understand how these weeds alter microbial communities and how this influence invasive success, ecosystem properties and restoration. We also collaborate with researchers from across the world to learn if plant-microbe interactions differ between native and invasive ranges and how this correlates with evolutionary shifts in plant genomes and biogeographical distribution of plant associated microbes. Outcomes of plant-soil microbe interactions depend on the particular plant and fungal species and surrounding environmental conditions. To explore this context-dependency, we use high-throughput sequencing and stable and radioactive isotopes in surveys and experiments to determine if the proportion of fungal guilds (mutualistic mycorrhizal fungi and potential pathogens) change with water and nutrient availability and how these changes relate to plant growth. Because most research occurs in single locations, the generality of findings across locations that differ in environmental conditions is often unknown. To address this, MPG Ranch is part of two global research collaborations, Nutrient Network (https://nutnet.org) and DroughtNet (www.drought-net.org). In these experiments, all researchers apply nutrients, remove herbivores, expose plants to drought, and record responses in both plant and microbial communities using the same protocols, which allow for direct comparisons across sites. The invasive fungal pathogen, Cronartium ribicola, causes the disease commonly known as blister rust in all nine white pine species native to the United States. As one of the only labs in the world to grow C. ribicola in culture, we perform tests of pathogen metabolism when exposed to compounds produced by other fungi found in white pine needles. Greenhouse experiments inoculating trees with these fungi, as well as beneficial ectomycorrhizal fungi, explore how we can improve tree growth and disease resistance. We also use isotopes and controlled field experiments to determine how blue-stain fungi carried by bark beetles can influence wood decomposition in forest ecosystems. As warming climates increase the frequency of bark beetle outbreaks worldwide, this research will help to better estimate future forest carbon storage and release.
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.
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.