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.