Mycorrhizal Inoculation

Sixty pots were inoculated with the AM fungal isolate R. irregularis ‘PH5.’ The isolate is maintained in the AM fungal collection of the Department of Mycorrhizal Symbioses (Institute of Botany, Czech Academy of Sciences, Průhonice, Czech Republic) in sand–zeolite–LT soil (2:2:1, v:v:v) mixture. The AM fungal inoculum cultures, established with Zea mays as the initial and Desmodium sp. as the follow-up, long-term host plant, were 16 months old when used as inoculum source. Inspection under a stereomicroscope had confirmed very abundant intraradical and extraradical sporulation of R. irregularis, as well as an absence of contamination by other AM fungal morphospecies. To prepare the AM fungal inoculum, the host plants’ shoots were removed and the roots were cut into ca 0.5 cm pieces and mixed back into the substrate. The material was subsequently dried at room temperature for 1 week. After thorough homogenization by mixing, the complex AM fungal inoculum (substrate+roots) was weighed into 50 g aliquots, stored temporarily in plastic bags, and then mixed into the upper two thirds of the substrate filled into each mycorrhiza-inoculated pot. This was done simultaneously with the application of cations and P (if applicable) described earlier.

To obtain an appropriate control (non-mycorrhizal, NM) treatment, a “mock” inoculum was produced in exactly the same manner as described above but using NM cultures: the same host plants were grown in the same substrate and under the same conditions as the AM fungal inoculum, but without the AM fungi. Visual inspection of the mock-inoculum cultures under a stereomicroscope confirmed the absence of AM fungal spores and/or mycelium clumps. The mock inoculum was processed and applied into the experimental pots in exactly the same manner as was the AM fungal inoculum (see above).