Microbial inoculation on plant roots and leaves
We collected microbial communities from plant roots and leaves in the
Gault Nature Reserve in Summer 2020. We sampled 20 individuals of each
of the four species used in the experiments. The roots of each
individual were extracted from the soil and the soil adhering to the
roots was collected. We obtained around 250g rhizosphere soil for each
individual and thus 5 kg for each plant species. The soil from the
different species and individuals were mixed thoroughly and preserved at
-80°C for later use. For leaf-associated microbes, we collected
approximately 150g of plant leaves from each individual into a sterile
roll bag. We added 100ml sterile phosphate-buffered saline to the bag
and agitated the leaves for 5 mins to wash off the microbes on the leaf
surfaces. The resulting leaf wash solution was pooled across species,
and then pelleted for 10 mins at 3000g. We carefully transferred the
pellets into 25% glycerol freezing buffer and stored the extracted
microbes at -80°C for later use.
We inoculated the soil and leaf microbial inocula onto plant communities
in pots in the greenhouse one week after plants established. For soil
microbe inoculation, we thawed the soil-derived inoculum and then
divided it into two equal parts. One part was used for a microbial soil
inoculum and the other part was sterilized in an autoclave for 30
minutes at 121°C used as a control. We added 50g soil of either
microbial inoculum or control into each pot and mixed it with the
background soil substrate. For leaf microbe inoculation, we thawed the
leaf-derived inoculum in glycerol buffer and centrifuged the cells at
3000g for 10 mins. The pellets were collected, resuspended in 300ml 10mM
MgCl2 buffer and diluted to 3L using the same buffer.
This was then equally divided into 3 parts, with one part sterilized in
an autoclave for 30 mins at 121°C as the control, one part diluted by
100 times as a low-concentration microbial inoculum, and the remaining
part used as a high-concentration inoculum. We sprayed approximately 6-8
ml of inoculum onto the aboveground plant surfaces in each pot.
Immediately after inoculation, we increased the humidity of the
greenhouse to 90% for 24 hours to provide a high humidity misting
condition for microbes to establish on leaves . We performed a second
leaf microbe inoculation one week after the first inoculation following
the same procedure as above to ensure a high probability of colonization
of leaves by the microbes in the inoculum.
After microbial inoculation treatments, the pots were arranged randomly
with a 20-cm spacing between pots to avoid contact between leaves and to
reduce the chance of contamination across treatments, and the position
of pots was randomly shifted every two months. We grew plants in the
UQAM greenhouse in Montreal, Canada at 24°C and 50% relative humidity.
After growing for 20 weeks, we harvested and oven-dried the plants at
60°C for 48 hours, and then weighed the plants to determine the biomass
of each plant individual. Meanwhile, we collected 2.5g soil from a
random replicate of each of our treatments (90 samples, i.e. 15 plant
community treatments and 2 soil and 3 leaf treatments) to identity the
soil microbial taxa at the end of our experiments.