1.2.1 Non-dormant chlamydospore
First, Sabourg’s liquid culture medium was prepared by weighing 10.0 g
of peptone, 40 g of glucose, and 1000 mL of distilled water. The
prepared medium was autoclaved at 121 ℃ for 15 min, and the pH was
finally adjusted to 7.0. The sterilized Sabourg’s culture medium was
divided into 500 mL triangular flasks before sterilization, with each
bottle containing 100 mL.
Using a sterilized punch (aperture 5 mm × 5 mm), the PDA agar containing
the aforementioned mycelium was perforated into uniform small circular
blocks, and a sterile technology was used to inoculate the agar blocks
onto a triangular flask containing 100 mL of Sabourg’s liquid medium (10
g of peptone, 40 g of glucose, and 1000 mL of distilled water at pH 7.0)
on an ultra-clean workbench. Eight blocks were inoculated per bottle.
Then, the flask was placed on a shaking table (HZP-250 type, Shanghai
Jinghong Experimental Equipment Co., Ltd.), shaken, and cultivated at 28
℃ and 200 rpm for 72 h, and the cultivation was stopped. The flask was
stored for no more than 3 days at 4 ℃.
Corn and wheat were mixed in a 2:1 ratio, with a material-to-water ratio
of 1.5:1. The mixture was soaked overnight in tap water and divided into
1000 mL triangular flasks. Each triangular flask contains 200 g of grain
(calculated as dry matter) and sterilized under high pressure at 121 ℃
for 20 min. When the culture medium cools to room temperature, the
preserved seed solution is inoculated in a 1:9 ratio (v/w) into a flask
containing grains and then cultured at 28 ℃ for 20 days. When a large
amount of mycelium grows on the grains and when a yellow powdery culture
is obtained, the cultivation can be terminated. 0.05% sterilized
Tweene-80 solution was added to a triangular flask and swirled for 1–2
min to wash the mycelium and spores from the grain. The crude bacterial
suspension was collected by repeated washing three times and filtered by
using a double gauze to remove the mycelium. Then, the suspension was
divided into centrifugal tubes, centrifuged at 1000 rmp for 5 min, and
then removed by enzymolysis. 2% snail enzyme and 2% cellulase were
mixed with the spore suspension to achieve a final concentration of 1%.
The two enzymes were oscillated in a 35 ℃ water bath oscillator for 2 h
and then centrifuged with sterilized distilled water (8000 rpm, 2 min)
three times to remove the remaining enzymes and discard the supernatant.
The concentration of chlamydospore was adjusted by adding distilled
water, and the number of chlamydospore was counted by using a blood cell
counting plate. The average of five times was used to calculate the
concentration of spores per milliliter. The total amount of
chlamydospore and the content of spores in the sample before the test
(spores/mL) were calculated by measuring the volume of spore suspension
and stored at 4 ℃ for use.