Introduction
The atmospheric concentration of methane (CH4) has almost tripled over
the past 150 years, making a substantial contribution to climate change
(Forster et al., 2007). Aerobic soils provide an important habitat for
methanotrophic bacteria and the only significant biological sink
for atmospheric CH4 (20–45 Tg CH4 yr-1) (Forster et al.,
2007). However, CH4 uptake by these soil ecosystems can be impacted by
environmental stress (Kolb, 2009). A common plant physiological response to
ecological stress, such as drought, is the production of ethylene (Morgan
and Drew, 1997). In soils, however, ethylene may be inhibitory to
methanotrophic activity (Jäckel et al., 2004; Pierek et al., 2006; Zhou
et al., 2013) and thereby reduce CH4 oxidation. This potential
interaction needs to be understood, as it may constitute an important
positive feedback loop between climate disruption, soil ecosystem
disturbance, and reduced CH4 removal from the atmosphere (Bousquet et
al., 2006; Zhou et al., 2013).
Conceptual outline of the proposed relationships between soil
CH4 oxidation rates and aboveground plant biomass with regard to the
anticipated effects of the treatments applied in this study. (a) Under
environmental stress, in planta ethylene production is stimulated, resulting in
ethylene exudation into the soil atmosphere and the inhibition of soil
CH4 oxidation by methanotrophs. (b) The application of irrigation (IR)
increases soil moisture while the application of biochar (BC) increases soil
moisture holding capacity, both acting to reduce plant stress and prevent
ethylene exudation into the soil atmosphere. (c) The application of AVG
disrupts ethylene production, thus limiting or preventing the inhibition of
CH4oxidation by the stressed plant.
To test our previous hypothesis that drought-induced in planta ethylene production
reduces soil CH4 oxidation rates (Zhou et al., 2013), we manipulated
plant stress responses by adding the ethylene biosynthesis inhibitor
aminoethoxyvinylglycine ([S]-trans-2-amino-4-(2-aminoethoxy)-3-butenoic acid
hydrochloride; hereafter AVG) (Boller et al., 1979). In addition, the study
tested the hypothesis that addition of biochar (BC) to soils may result in
increased water holding capacity, reducing drought stress and thereby acting
as a potential tool to maintain CH4 oxidation (Karhu et al., 2011).
This is illustrated conceptually in Fig. 1, in which the application of
irrigation (IR) and BC are able to maintain rates of CH4
oxidation by reducing moisture stress and therefore ethylene production,
whereas AVG prevents the production of ethylene after the plant experiences
stress.
Material and methods
Study site
The study site was located in the Bjelke-Petersen Research Station at Kingaroy
(26.53∘ S, 151.83∘ E) in the South Burnett Region of
Queensland, Australia. Precipitation averages 789 mm per annum with erratic
summer droughts frequent in the region. Soil at the field trial site soil is
an acidic Red Ferrosol (pH 5.5) with high cation exchange capacity (Isbell,
1993). The site has a long history of cultivation, supporting peanut and
maize rotations with winter fallows.
Experiment design and management
A full factorial, split-plot design field trial was established as follows:
two IR treatments (IR and no IR) × two BC treatments (BC at 9.2 t ha-1 and no BC) × two
ethylene suppression treatments (AVG and
no AVG). Each treatment had five replicates, producing a total of 40 plots.
Due to practical concerns regarding application and maintenance, the IR
treatments were established in two discrete areas that were spanned by five
blocks. A schematic of the trial site is given in the Supplement (Fig. S1).
The BC treatment was established through application of peanut shell BC to
the surface of the planting zone (∼ 450 mm wide strip each
row) in early 2013. The BC was incorporated into the soil with a rotary hoe
to a depth of 200 mm. The chemical properties of the peanut shell BC are
provided in the Supplement (Table S1).
The site was machine planted with maize cultivar Pioneer 32p55 (Dupont
Pioneer Australia) at a density of approximately 4 plants m-2 in late
January 2014. Compound fertiliser (N : P2O5 : K2O 11.9 : 14.1 : 9.9)
at 180 kg ha-1 and urea at 100 kg ha-1 were applied at sowing.
Trickle tapes, installed into plots receiving IR, were used to distribute
water equivalent to ∼ 50 mm of rainfall whenever there was a
continuous dry spell for 2 weeks throughout the growing season (late
January to late June).
To reduce the in planta production of ethylene, the commercial plant growth regulator
ReTain (containing 15 % AVG; Valent Bioscience Cooperation, Walnut Creek,
CA, USA) was sprayed onto the crop four times from mid-April to mid-June
(the peak maize growth window) at intervals of 3 weeks. During each
event, the treated rows of maize received approximately 750 mL of ReTain
solution (prepared at the label rate of 1 g ReTain l-1 water) directly
to the surface of the plants.
Sample collection and analysis
In late June 2014, six soil cores from 0 to 100 mm depth were collected from
the maize rooting zone of each plot using a 30 mm diameter soil auger. All
samples were collected from the two middle rows of maize in each plot, and
the six soil cores from within each plot bulked to a single plot sample.
After sieving to 2 mm, a 50 g (fresh mass) subsample of each sample was set
aside for CH4 oxidation rate measurements and the remaining material
dried at 105 ∘C for 48 h to determine soil moisture content.
Soil CH4 oxidation rates were determined using the laboratory
incubation. Briefly, about 20 g soil subsamples were incubated in 1 L
glass jars at ambient atmospheric CH4 concentration (assumed to be
1.9 ppm) for 1 week in the dark at 25 ∘C. Headspace gas samples
(approximately 30 mL) were collected through a rubber septum in the jar lid
at the beginning and the end of the incubation, and concentrations of
CH4 were determined using GC-FID (GC-2010 Plus, Shimadzu, Japan). The
CH4 oxidation rates in each jar were calculated from differences in the
headspace CH4 concentration over the incubation time (Zhou et al.,
2008) and adjusted to soil dry weight. Standards were measured once every
10 samples; the coefficient of variation in CH4 oxidation rate was less
than 5 % and control jars had ambient CH4 concentrations.
Statistical analysis
Statistical analysis was carried out in R 3.2.3 (Zhou et al., 2017) using a
multi-factor ANOVA model incorporating an error structure accounting for the
split-plot design associated with the non-random assignment of the IR
treatment. The multi-comparison analysis methods provided in the “easyanova
4.0” R package was used to test for treatment interactions.
Discussion
The increase in CH4 oxidation with the AVG treatment either alone or in
combination with the BC treatment aligns with past studies assessing the
effect of increased ethylene concentrations on soil CH4 oxidation rates
(Jäckel et al., 2004; Xu et al., 2008). This response also supports the
hypothesis that in planta ethylene production in response to stress decreases the
capacity of soil to support methanotrophic activity (Zhou et al., 2013).
The lack of effect of BC on CH4 oxidation is at odds with the results
of previous work (e.g. Karhu et al., 2011; Kim et al., 2017). However, BC
added in this study had no influence on soil moisture content, and this is
proposed to be a key mechanism for BC to support CH4 oxidation in
drought conditions (Karhu et al., 2011). Another reason for this might be
related to the properties of the biochar (C : N ratio of 51.84, 9.2 t ha-1)
used in this study when compared with agricultural soils in
Finland (C : N ratio of 101.07, 9 t ha-1) (e.g. Karhu et al., 2011) and
in East Asia (C : N ratio of 79.65, 2 t ha-1) (e.g. Kim et al., 2017).
The lower C : N ratio of the biochar used in this study can incorporate more N
fertiliser into the soils, which could reduce soil CH4 uptake as N
fertiliser can inhibit methanotrophic activities (see Kolb, 2009). Overall,
the reason why BC addition did not result in increased soil moisture in this
case is unclear. Further studies is needed to investigate the effects of
biochar application on the factors influencing soil CH4 oxidation.
The significant interaction between the AVG and IR treatments is more
difficult to reconcile. The IR treatment was intended to significantly
increase soil moisture content compared to the no IR treatment, reducing
water stress and likely in planta ethylene production. It was noted that increased
soil moisture content can directly influence methanotrophic activity, as
water-driven increases in microbial activity can enhance methanotroph,
whereas water content that exceed field capacity can rapidly decrease
CH4 oxidation rates by reducing gas mobility through soil pores (Le Mer
and Roger, 2001). Given the initial soil water content and scale of the
increase with the IR treatment, direct stimulation of CH4 oxidation was
considered the most likely outcome when considering plant-independent
effects. Consequently, it was anticipated that any effect of AVG on CH4
oxidation (putatively via reductions in ethylene production) would only
manifest without IR, as the IR treatment would make the AVG treatment
redundant. However, CH4 oxidation rates in plots treated with
either IR or IR and AVG in combination were not significantly greater than
untreated control plots. It is possible that the moisture addition
associated with the IR treatment was insufficient to substantially alleviate
plant drought stress, driving an increase in ethylene production, which
could then account for the numerical difference between the AVG and IR
treatments (Fig. 2). The water addition may have also been insufficient to
meaningfully and directly stimulate methanotroph activity. However, it would
be expected that the combination of IR and AVG would support soil CH4
oxidation rates either the same or potentially greater than those observed
for AVG alone. This was not the case, and the explanation for the
significant interaction remains unknown. As discussed above, it is possible
for increased soil moisture content to inhibit CH4 oxidation via
decreased porosity and gas diffusion (see Zhou et al., 2014), but, given the
IR treatment alone did not reduce CH4 oxidation rates relative to the
control, this is not a feasible explanation in this case.
The lack of data explicitly describing ethylene release into soil in
response to the treatments is a limitation to this trial. However, the
quantification of ethylene in soil is not trivial, particularly when
conducted over time (i.e. continuous), and was outside the resources
available for this study. However, given the findings of this study, and
considering treatments were field-based, further investigations of the
interactions between AVG, plant stress, and CH4 oxidation should be
conducted. In these studies, consideration should be given to collection and
integration of ethylene data, particularly given that these data may help
shed light on the nature of any interactions between treatments.
Overall, the findings of this study indicate that application of an ethylene
biosynthesis inhibitor to plant tissue can cause a measurable increase in
the capability of soil to oxidise CH4 under moisture-stressed
conditions. This supports the hypothesis that the stress-induced production
of ethylene by plants can disrupt the activity of methanotrophs and
identify a potential management pathway to help retain, or even enhance,
the methanotrophic capability of soils in productive systems. Given the
global importance of a positive feedback between environmental stress, plant
ethylene production, and lowered microbial CH4 oxidation activity,
further work in this area is needed. In addition, methods to moderate
impacts on the methanotrophic community, such as use of alternative forms or
rates of biochar application, require investigation to enable provision of
important ecosystem services.