1.2.1 Preparation of non-dormant chlamydospore
The agar containing mycelium grown on PDA was separated into
6~8 even small rounds with a sterilized hole punch
(aperture 5×5 mm) and inoculated in the above triangular flask
containing Sandburg’s liquid culture medium. The flask was shocked at 28
℃ at 200 r/min for 72 h and the culture was stopped. 10 mL of the
culture containing mycelium was inoculated into a triangle flask
containing 1000 mL of 200 g grains [28]. Then the culture was placed
in an incubator at 28 ℃ for 21 days. When more mycelium grew on the
grain and yellow powder culture was observed by naked eye, it was proved
that more chlamydospore had been produced, and the culture was
terminated. An appropriate amount of 0.05% sterilized Tweene-80
solution was added to the triangular flask, and the solution was swirled
for 1-2 min, then mycelium and spores were washed off the grains, and
the crude spore suspension was collected and placed in ultrasonic cell
pulverization instrument (SCO-2500, Shanghai Shengyan Ultrasonic
Instrument Co., LTD.), and subjected to ultrasonic treatment for 5 min
at 150W and 20 KHZ. At an interval of 30 s, the spore suspension was
filtered through a 300-mesh copper screen, the filtrate was centrifuged
at 1000 r/min for 5 min, and then washed with sterilized distilled water
for 3 times. The sediment (i.e. chlamydospore) was suspended with
sterilized distilled water and counted with a blood cell count plate.
Finally, the concentration of the spore suspension was adjusted with
distilled water to about 1×104 spores /mL for further
use.