2.3 Greenhouse experiments
A series of experiments were conducted in a greenhouse with 20-30 °C
night and day temperatures and 65-90% relative humidity. Each
experiment was conducted in a completely randomized design with four
replicates for each treatment and control. Rice seeds were sterilized
with 5% H2O2 then placed in Petri
dishes with moistened filter paper. Weed seeds were sterilized with 75%
ethyl alcohol for 3 min, followed with 3% sodium hypochlorite for 12
min. Sterilized seeds were rinsed with distilled water and then
transferred to Petri dishes with moistened filter paper for
vernalization in a 4 °C refrigerator for 24 h. Dishes of rice and
vernalized weed seeds were pre-germinated in an environmentally
controlled chamber at a temperature of 28 °C in the dark for use in each
experiment.
A preliminary experiment was conducted to investigate the effect of rice
cultivar mixture proportions on four common paddy weeds (barnyardgrass,L. chinensis, C. difformis and E. prostrata ). Eight
pre-germinated seeds were sown in each pot (12 cm diameter ×10 cm deep)
containing 1000 g soil. Four rice seeds in a 3:1, 2:2 and 1:3 (focal
cultivar: related cultivar) ratio
were placed uniformly in the central area while four weed seeds of one
species were sown
surrounding
the rice in the pot (Supplementary Figure 1). Monocultures of eight weed
seeds served as controls. The weed seedlings were harvested after 4
weeks and their dry weights were recorded. This design was replicated
for each focal cultivar and weed species. As a result
(Supplementary Table 2), the 2:2
ratio was used for each subsequent experiment (Experiments 1- 4) as
described below.
Experiment 1- the effect of rice cultivar mixtures on the performance of
four weeds and their mixtures with barnyardgrass. Eight pre-germinated
seeds were sown in each pot as described above. Four rice seeds for each
cultivar pair were placed uniformly in the central area of each pot.
Four weed seeds for each species, or two barnyardgrass with two other
weed seeds, were sown surrounding the rice. Controls were monocultures
of eight weed seeds for each
species and their mixtures with barnyardgrass. Mixtures with
barnyardgrass reflect the dominance of the species as a paddy weed and
its regular co-occurrence with other weeds. Weed seedlings were
harvested at the three-leaf rice stage and their shoots and roots were
separated and dry weight of each was recorded.
Experiment 2- allelochemical production and grain biomass of rice
cultivar mixtures in response to barnyardgrass and its mixtures with
other weeds. Eight pre-germinated seeds were sown in a pot as described
above. Four rice seeds for each cultivar pair were placed uniformly in
the central area. Four barnyardgrass seeds or two barnyardgrass with two
other weed seeds were sown surrounding the rice in each pot.
Monocultures of each rice cultivar and eight weed seeds for each species
and their mixtures with barnyardgrass served as controls. Allelopathic
rice leaves produce allelochemicals at their early growth stages and
then release them into soil through the roots that reach maximal level
at the five-leaf stage and then decrease dramatically (Kong et al.,
2004; Kong, Li, Hu, Xu, & Wang,
2006). This experiment focused on
allelochemical
production but not allelochemical release. Accordingly, rice leaves were
sampled at the three-leaf stage and quantification of rice
allelochemicals conducted as described below. Weed plants and rice
grains were each harvested at the rice mature stage and their dry weight
of each was recorded.
Experiment 3- soil microbial community in response to rice cultivar
mixtures in the presence of barnyardgrass, the dominant weed competitor.
Eight pre-germinated seeds were sown in each pot as described above.
Four rice seeds for each cultivar pair were placed uniformly in the
central area. Four barnyardgrass seeds were sown surrounding the rice in
the pot. Monocultures of barnyardgrass or focal rice cultivar served as
controls. At the three-leaf stage, a soil core with 2.5 cm diameter and
5 cm depth was sampled from the central area of each pot. Visible root
fragments were removed and the sample freeze-dried for the analysis of
phospholipid fatty acids (PLFA) as described below.
Experiment 4- the performance and resource partitioning of barnyardgrass
in response to potential signaling-chemical in rice cultivar mixtures.
Kin recognition in allelopathic rice cultivars involves the
nitrogen-rich compound allantoin (Yang, Li, Xu, & Kong, 2018).
Accordingly, allantoin and its equal quantity of
NH4NO3 were applied into rice cultivar
mixtures in the presence of barnyardgrass. Eight pre-germinated seeds
were sown in each pot (12 cm ×10 cm) containing 1000 g soil. Four rice
seeds for each cultivar pair were placed uniformly in the central area
of each pot. Four barnyardgrass seeds were sown surrounding the rice in
the pot and monocultures of barnyardgrass served as controls. The pots
received either allantoin at a concentration of 0.32 mM (50 μg
g-1 dry soil), NH4NO3at a concentration of 0.63 mM (50 µg g−1 dry soil), or
distilled water, respectively. The
NH4NO3 treatment represents a
non-signaling nitrogen source to contrast with the signaling and high N
content allantoin treatment. Barnyardgrass seedlings were harvested at
three-leaf stage, separating their shoots and roots. The dry weights of
each root and shoot sample were recorded and their carbon and nitrogen
contents were each measured with a CN elemental analyzer (Elementar
vario EL cube, Elementar Analysensysteme GmbH, Langenselbold, Germany),
at a combustion temperature of 960 ℃.
Experiment 5- root placement patterns between focal rice cultivar and
weeds in response to rice cultivars of different relatedness. A series
of window rhizoboxes (30 cm long ×3 cm wide ×30 cm high) were made with
a clear plexiglass sheet containing 2000 g soil. Each rhizobox was
vertically divided into six equal parts, and three rice and three weeds
for each species were sown in the rhizoboxes according to the order of
two
plants of related cultivars, a plant of the focal cultivar, and three
weed plants from the left to the right (Supplementary Figure 2).
Monocultures of six plants barnyardgrass or focal cultivars served as
controls. Window rhizoboxes were placed in racks at an angle of 40° with
the clear plexiglass facing down and away from the light source.
Rhizoboxes were opened after 4 weeks and the root systems were scanned
to a gray scale image and, divided into right and left parts, and the
roots freeze-dried for biomass assessment. Each image was processed with
WINRHIZO (Regent Instruments, Quebec, Canada) to obtain root length, and
horizontal asymmetry in root length. Horizontal asymmetry in root
biomass was also calculated from root dry weight of the left (towards
rice) and right (towards weed) halves of the root system.