Isolation and characterization of mycorrhizae and mycorrhiza-like associations from soil of the Maltese Islands

Abstract

Studies of soil fungal diversity in the Maltese Islands are still considered sporadic and limited. The aim of the present work was to carry out studies to investigate the presence of Trichoderma spp. within the ascomycota, ectomycorrhizae and arbuscular mycorrhizae in the Maltese Islands and eventually to effect their isolation and preservation on different substrates. Soils samples were initially cultured Potato Dextrose Agar medium at a temperature of 27掳C in the light. After microscopic inspection, fast-growing colonies with phialides and conidiophores which are typical of Trichoderma growth, were selected for monoconidial isolation. In total, nine dinstinct isolates were made using a monoconidial isolation technique, and their growth patterns and rate of growth were measured and photographically recorded. To help in identification of isolates, micromorphological studies were carried out using different staining techniques for light microscopy. In addition, DNA barcoding was carried out. Seven strain of the Trichoderma harzianum complex were identified for different soils and media in the Maltese Islands as well as another two new records of Trichoderma species namely T. virens and T. citrinoviride. These Trichoderma isolates were preserved in sterile 99% glycerol, sterile distilled water, Synthetic Nutrient Agar, Potato Dextrose Agar, and spent coffee grounds. Spore and mycelium viability were confirmed after one year for all of them. The search for ectomychorrizae was conducted during the rainy season between 2015 and 2019. Ten records were confirmed, and of which two are new records for the Maltese Islands namely Russula ochrosporea and Hebeloma quercetorum were discovered. For Hebeloma quercetorum it was also possible to measure the rate of growth of the basidiocarp. For the arbuscular mycorrhizae study, plant-traps were set up and their presence was confirmed as vesicles within the roots of Daucus carota, Carya illinoinensis and Quercus ilex. As far as is known, this was the first record of arbuscular mycorrhizae detected in Maltese soils.