Localisation of hydrogen peroxide and peroxidase in gametophytes of Ceratopteris richardii (C-fern) grown in the presence of pathogenic fungi in a gnotobiotic system

The endogenous localisation of peroxidase and hydrogen peroxide (H2O2) was detected when gametophytes of the fern, Ceratopteris richardii, were exposed to the plant pathogenic fungi Sclerotium rolfsii and Sclerotinia sclerotiorum and Phytophthora infestans, an oomycete, in a gnotobiotic system. This was accomplished by light microscopy using 3,3'-diaminobenzidine, guaiacol and H2O2 and starch potassium iodide (KI) staining procedures, which facilitated the observation of the reaction in vivo and in situ, without physically damaging the tissues. All three staining methods promoted staining at the rhizoid regions. Although most of the cells were destroyed when gametophytes were exposed to S. rolfsii and S. sclerotiorum, there was staining where mycelial growth was confluent with cell walls. A qualitative test confirmed that the colour change in starch KI agar medium, as well as in the histochemical test with starch KI, was because of H2O2 secreted by S. rolfsii or S. sclerotiorum and not because of oxalic acid. When gametophytes were exposed to P. infestans, no infection occurred, but localisation of H2O2 and peroxidase was detected irrespective of staining methods tested. Based on the observation on gametophytes grown in presence of P. infestans, it is possible that the peroxidase in plants coupled with H2O2 may prevent the invasion of nonpathogens by functioning as a barrier. This fern-pathogen model system has potential for application as a tool to study the host-parasite interaction in a gnotobiotic system.