1.4 NTF classification and identification
Isolation and purification: Using the five-point spreading method, samples were plated on petri-dishes with CMA medium with ~5,000 individuals of Panagrellusredivivus to induce NTF germination. We plated three replicates per sample for a total of 6,750 petri-dishes plated. The petri-dishes were incubated at room temperature (25-28℃). We examined the petri-dishes once after 3 weeks and again after 4-5 weeks using a stereomicroscope to isolate all the species. During each examination, we used single spore isolation to collect and purify any germinated NTF on CMA medium. A sterile toothpick was used during collection. Pure cultures were preserved until we were able to isolate and purify cultures from all specimens.
Morphological identification: All preserved pure cultures were revived in CMA medium and placed on a temporary slide using the inserting and sticking method. Morphological characteristics of NTF, including conidia, conidiophores and chlamydospore, were then photographed using a microscope camera (Olympus BX51, Japan). For each NTF strain, the type of trapping device was observed and confirmed using the observation chamber and nematode induction method. Strains were identified according to Li et al., 2014.
Molecular identification: Each NTF strain was inoculated on PDA medium to gather the mycelium. We then extracted DNA from the mycelium using a CTAB protocol. The fragments of ITS (Internal Transcribed Spacer), TUB (β-tubulin gene), TEF (Translation Elongation Factor 1-alpha) and RPB2 (RNA polymerase II) were amplified by PCR and sequenced by BioSune Biotechnology Co., Ltd. (Shanghai, China). The homologous ITS and TUB sequences were aligned using the BLAST toolkit from NCBI (National Center for Biotechnology Information Search database).
We were able to identify and classify specific NTF species using morphological and molecular identifications, referring to the taxonomic system used by Li et al., 2014.