Figure: Arabidopsis thaliana roots, hyphae, and chlamydospores of P. indica
(A) Tipical view of Arabidopsis thaliana seedlings grown in glass beads with PNM media and co-cultivated with the beneficial fungus Piriformospora indica for 21 days.
(B) Light microscopy (magnification 100 x). Fuchsin acid/trypan blue method 15 days after co-cultivation with P. indica on PNM medium.
(C) 450-490 nm (magnification 200 x). Fuchsin acid staining method; 15 days after co-cultivation with P. indica on PNM medium.
(D) Live microscopy (magnification 40 x). After 5 days interaction with P. indica on PNM media.
(E) The fluorescent channel at 470 nm (magnification 200 x). Fuchsin acid staining method 15 days after co-cultivation with P. indica on PNM medium.
The vast majority of plants live in symbiotic interaction with mycorrhizal fungi. The fungus delivers soil nutrients to the plant while the plant is responsible for the photoassimilates to the fungus. Mycorrhizal interactions are difficult to investigate at the molecular level because most of the classical model plants do not form mycorrhizal association. As an alternative, endophytic interaction of axenically cultivable Piriformospora indica with the model plant Arabidopsis thaliana might help to understand the basis of the beneficial interactions between two symbionts.
We focus on the identification of Arabidopsis mutants which are impaired in establishing or maintaining the interaction in a beneficial mode. For those studies, reproducible and quantitative co-cultivation parameters are required, therefore, we have developed standardized (co)cultivation conditions for the two symbionts. The mode of interaction and the reproducibility of the data are highly dependent on the quality of the biological material and the co-cultivation conditions. Most important parameters are the age and the density of the fungal mycelium, the age of the seedlings and plants, the ratio between the two symbiotic partners, and the co-cultivation conditions like temperature, humidity, light intensity, spectral distribution, and photoperiod.
We have standardized the growth medium, since even minor changes in the medium have severe consequences for the interaction. Co-cultivation medium is designed such that both organisms can grow in harmony. On plant medium with high levels of nutrients and sucrose, the benefits for plant performance are less pronounced compared to cocultivation conditions on minimal medium. None of the two symbionts should "overgrow and dominate" the partner. Furthermore, shortage in any important nutrients such as N, P, S, or Fe in the medium results initially in a better plant performance in the presence of the fungus, while during later phases of the interaction and after transfer to soil, the plants start to suffer and show growth retardation instead of growth promotion. This can be associated by a shift from mutualism to parasitism in its extreme. Another important parameter for an optimal interaction between the two symbionts is the pH (6.5-7.0) of the medium or the soil. In pot culture and soil experiments, the amount of the inoculum and the growth conditions are crucial. Greenhouse experiments are more variable than experiments in temperature- and light-controlled growth chambers.
The standardized co-cultivation conditions allow us to identify genes, proteins, and other biomolecules which participate in establishing benefits for the plants and/or maintaining the interaction in a mutually beneficial mode (Johnson et al. 2013).