Soil Biology and Biochemistry 156 (2021) 108233

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Soil Biology and Biochemistry

journal homepage: http://www.elsevier.com/locate/soilbio

Changes in rhizosphere soil microbial communities across plant

developmental stages of high and low methane emitting rice genotypes
Cristina P. Fernández-Baca a, Adam R. Rivers b, Woojae Kim a, c, Ryo Iwata b, Anna M. McClung a,
Daniel P. Roberts d, Vangimalla R. Reddy e, Jinyoung Y. Barnaby a, *
a
USDA Agricultural Research Service, Dale Bumpers National Rice Research Center, Stuttgart, AR, 72160, USA
b
USDA Agricultural Research Service, Genomics and Bioinformatics Research Unit, Gainesville, FL, 32608, USA
c
Rural Development Administration, National Institute of Crop Science, Wanju, 55365, South Korea
d
USDA Agricultural Research Service, Sustainable Agricultural Systems Laboratory, Beltsville, MD, 20705, USA
e
USDA Agricultural Research Service, Adaptive Cropping Systems Laboratory, Beltsville, MD, 20705, USA

A R T I C L E I N F O A B S T R A C T

Keywords: Rice production is an important source of methane accounting for 11% of global anthropogenic emissions.
Methane Methane emissions can be effectively reduced by management practices and cultivar selection. However,
Methanogens whether cultivars lower methane emissions primarily through mediating whole microbial community shifts or
Methanotrophs
through more targeted interactions with methane-cycling microbes is not understood. Here, we sequenced the
Rice
Rhizosphere
soil metagenomes associated with two rice recombinant inbred lines (RILs) along with their parents, Francis and
Rondo, displaying a range of low to high methane emitting phenotypes. Methane emissions and rhizosphere soil
microbial communities were sampled at booting, heading, grain fill, and maturity growth stages to evaluate how
plant development impacted rhizosphere communities. Methane emissions were low at booting and increased
during heading and grain fill stages where peak methane emissions were observed and returned to basal levels at
maturity. In response to genotype and plant developmental stage, we observed changes in rhizosphere microbial
community structure in several methanogenic archaea as well as bacterial methane oxidizers and sulfur cyclers.
Rice genotype played a larger role in influencing the soil microbial community structure during the reproductive
phases of booting and heading as compared to the ripening phases (grain fill and maturity). Francis showed lower
methane emissions and relative abundance of methanogen populations during the heading stage where methane
emissions were highest for the other genotypes. This indicated that the reduced methane emissions trait was
associated with small changes in the composition of methanogens rather than wholesale community shifts. This
finding suggests future breeding efforts can focus on reducing methane emissions during high methane emitting
phases (i.e., reproductive phases) by selecting genotypes that have lower methanogen populations and higher
methanotroph populations during these developmental stages.

1. Introduction Methane is produced under anoxic conditions typical of flooded rice

Rice has the highest global warming potential (GWP) among the
major cereals due to high methane (CH4) emissions from flooded paddy
soils (Linquist et al., 2012). Methane is a potent greenhouse gas with a
GWP 28 times that of carbon dioxide (CO2) over a 100-year timespan
(Pachauri and Meyer, 2014) and rice production accounts for an esti­
mated 11% of global anthropogenic methane emissions (IPCC, 2013).
Thus, mitigating methane emissions from rice production could signif­
icantly reduce anthropogenic greenhouse gas emissions overall.
fields. These conditions are favorable for methanogens which rely on
reduced conditions as well as soil carbon sources produced by the rice
plant (Liesack et al., 2000). The mcrA gene, which encodes the alpha
subunit of methyl coenzyme M reductase (MCR), has previously been
used to quantify methanogens in rice paddy soils, and other methano­
genic environments, as it is highly conserved and specific to metha­
nogens (Jiang et al., 2019; Lee et al, 2014, 2015; Ma et al., 2012).
Methanogens interact with other members of the microbial community
which ultimately influences the observed methane emissions from rice

* Corresponding author.
E-mail address: jinyoung.barnaby@usda.gov (J.Y. Barnaby).

https://doi.org/10.1016/j.soilbio.2021.108233
Received 28 September 2020; Received in revised form 17 March 2021; Accepted 21 March 2021
Available online 26 March 2021
0038-0717/Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
C.P. Fernández-Baca et al. Soil Biology and Biochemistry 156 (2021) 108233

paddies. Methanotrophic bacteria rely on methane produced by
methanogens and are estimated to oxidize up to 60–70% of methane
produced in paddy soils, significantly reducing methane production
potential from rice agriculture (Neue, 1993; Sigren et al., 1997).
Methanogens can also be outcompeted for substrates, including
plant-derived carbon, by iron and sulfate reducing microbes which, in
turn, decreases the rate of methanogenesis and further reduces methane
emissions from rice paddies (Achtnich et al., 1995; Thauer et al., 2008).
An estimated 80% of the methane produced in paddy soils is trans­
ported through plant aerenchyma to the atmosphere (Yu et al., 1997).
Reported variation in methane emissions by rice cultivars (Gogoi et al.,
2008; Linquist et al., 2018; Simmonds et al., 2015) suggests there is
potential to mitigate methane production from rice cultivation through
the breeding of low-emitting rice varieties. However, the genotypic
differences impacting the rhizosphere microbial communities ultimately
reflected in methane emissions from rice paddy soils are poorly under­
stood. Cultivar differences have been reported to affect the rice rhizo­
sphere composition of methanogens and methanotrophs (Jiang et al.,
2017; Liechty et al., 2020; Ma et al., 2009). However, differences in
plant development may also influence how these communities change
throughout the growing season and, in turn, impact differences observed
in methane emissions. Reports on rice genetic differences impacting
methane emissions have evaluated small sets of diverse rice cultivars
which precludes relating the response to plant genomic differences.
Recombinant inbred line (RIL) mapping populations are developed as a
means of identifying chromosomal regions related to plant phenotypes
derived from the genomes of two intercrossed parental cultivars. Within
Oryza sativa, there are two major sub-species, Indica and Japonica,
which serve as excellent resources for developing genetic mapping
populations because of their compatibility which minimizes segregation
distortion and their allelic diversity. Because of their defined set of ge­
netic alleles, using RILs to compare plant phenotypes is advantageous
over using diverse cultivars and allows for the identification of alleles
that may be driving trait differences.
The purpose of this study was to examine the relationship of plant
growth and development with changes in the methane cycling microbial
community structure in rhizosphere soils and the subsequent effects on
methane emissions. To this end, we selected two cultivars differing in
methane emission profiles, Rondo, an Indica with high methane emis­
sions, and Francis, a Japonica with low methane emissions, and two RIL
offspring that display divergent methane emissions. We sampled at four
developmental stages, booting, heading, grain fill, and maturity/harvest
to evaluate how plant development was related to changes in rhizo­
sphere communities. Ultimately the goal of this research is to better
understand if genotypic differences lead to variations in the methane-
cycling microbial communities thereby affecting methane emissions.
2. Materials and methods
2.1. Plant genetic materials
Two recombinant inbred lines, RIL1 and RIL7, and their parents,
Francis (FRCS) and Rondo (ROND), were selected for this study. Rondo
has been shown to have high methane emissions compared to some
other US rice varieties including Francis (Kim et al., 2018). The two RILs
were selected from a population of 217 progeny to have heading days
within a 10-day window and plant heights within 30 cm to ensure ge­
notypes displayed similar timing for the four developmental stages

Fig. 1. CH4–C efflux (a) and qPCR mcrA gene abundance (b) patterns by genotype and at each plant developmental stage (i.e., booting, heading, grain fill, maturity).
At maturity methane emissions dropped to basal levels and are indicated as “n.d.” for no data. Lower and upper edges of the box correspond to the first and third
quartiles (the 25th and 75th percentiles), the horizontal line indicates the median value and whiskers extend to the smallest or largest values. Statistically significant
differences between genotype means for methane emissions were determined by one-way ANOVA (p < 0.05). Kruskal-Wallis tests (p < 0.05) were used to test
significant differences for mcrA gene abundances. Letters indicate significant differences between genotype by Tukey HSD (p < 0.05) for methane emissions and
Dunn’s test (p < 0.05) for mcrA gene abundance.

2
C.P. Fernández-Baca et al.
being studied (i.e., booting; heading; grain fill; maturity/harvest) and
based on daily and seasonal CH4 emissions (Fig. 1 and Supplemental
Fig. 1).
2.2. Greenhouse experimental design
The study was conducted from April to September 2017 at the USDA-
ARS, Dale Bumpers National Rice Research Center (DBNRRC) in Stutt­
gart, AR. Two independent sets of experiments were conducted simul­
taneously in the greenhouse. One set was used for CH4 measurements
and the second set was used for destructive rhizosphere soil sampling at
the four developmental stages. Each study was conducted with a ran­
domized complete block design with three replications. For each repli­
cate, five seeds were germinated in small pots (7.6 cm length by 7.6 cm
width and 10.2 cm tall) and thinned to one seedling per pot after
emergence. Approximately 4-weeks after sowing, seedlings were trans­
planted to larger PVC pots (30.5 cm diameter by 30.5 cm tall) with one
plant per pot. Pots were filled with soil collected and directly transferred
from DBNRRC fields characterized as a Dewitt silt loam soil (fine,
smectitic, thermic, Typic Albaqualf). Prior to soil collection, basal P and
K fertilizer was applied and incorporated (29 and 84 kg P and K ha− 1,
respectively). Pots were maintained under flooded conditions (water
depth of 5–7 cm above soil surface) from transplanting to maturity.
Nitrogen fertilizer (80 kg N ha− 1) was applied to each pot in a three-way
split in the first two months following transplanting. Control pots were
filled with soil and remained unplanted but underwent the same fertil­
izer and flooding treatments. Greenhouse conditions were monitored
and recorded. The mean air temperature was 24.9 ± 0.14 ◦ C and the
total PAR fluxes were 2398.3 mmol m− 2.
2.3. Methane and soil sampling
Weekly measurements of methane emissions were initiated at
the onset of booting of the first experimental unit and were
continued until maturity following the protocol for greenhouse gas
fluxes in lowland rice (Adviento-Borbe et al, 2013, 2015; Kim et al.,
2018). Gas samples were taken within the 10:00 a.m. and 1:00 p.m.
window over the course of 1 h at 20-min intervals (0, 20, 40, 60
min) after sealing the chamber. Gas samples for all genotypes were
taken in a randomized order within the timeframe. The 25-ml
samples were collected in sealed, pre-evacuated 12 ml glass vials.
Ambient air was collected concurrently with methane samples as a
control. Gas samples were analyzed on a GC-2014 gas chromato­
graph (Shimadzu Scientific, Japan) equipped with a flame ioniza­
tion detector (FID) at 250 ◦ C. A 1 ml headspace sample was
injected into the GC using an autosampler (Bandolero, XYZTEK,
Netherlands). Methane was separated using a column packed with
Hayesep D, 80/100 mesh at 75 ◦ C. Triplicate measurements were
taken once per week per experimental unit and averaged. Methane
emissions measured on the day each experimental unit reached the
designated plant developmental stage (i.e., booting, heading, grain
fill, and maturity) were used to determine genotype methane
emissions for each relevant stage. These CH4 emission measure­
ments were summed across the four plant growth stages to deter­
mine the total or cumulative CH4 flux per genotype.
Rhizosphere soil was sampled as each experimental unit reached
booting, heading, grain fill, and maturity plant developmental
stages. Plants were removed from the pot in their entirety with the
soil-root aggregate intact. The soil-root aggregate was split in two
to allow for sampling from the center of the aggregate at an
approximate depth of 13–18 cm below the crown. The soil covered
roots were shaken to remove loose soil, leaving a thin layer
(roughly 1–2 cm) of soil and then placed in a sterile flask con­
taining 50 ml of phosphate-buffered saline (PBS) solution. The flask
was shaken gently to wash the soil from the roots and sterile for­
ceps were used to remove roots from the soil slurry. This soil slurry
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Soil Biology and Biochemistry 156 (2021) 108233
was used for subsequent DNA extraction (see below). Additionally,
soil samples at an approximate depth of 15 cm below the crown
were taken for total organic carbon analysis at each stage. Percent
of total organic carbon was measured using a total organic carbon
analyzer (Shimadzu Scientific, Japan).
2.4. DNA extraction and quantitative PCR
Soil DNA was extracted from 800 μl of soil slurry using the DNeasy
PowerSoil HTP 96 kit (Qiagen, Germantown, MD) according to manu­
facturer’s instructions. DNA was quantified on a Qubit using the dsDNA
high sensitivity assay (Invitrogen, Waltham, MA). All DNA samples were
normalized to 2.5 ng μl− 1 using deionized, autoclaved water prior to
sequencing preparation.
Gene abundances of mcrA in methanogens was determined via
quantitative PCR (qPCR) on the same diluted DNA used for
sequencing using primers from Pereyra et al. (2010) mcrA_1035F
(5′ -GGTGGTGTMGGATTCACACARTAYGCWACAGC-3′ ) and mcrA_1530R
(5′ -TTCATTGCRTAGTTWGGRTAGTT-3′ ). Reactions were run in triplicate
and each 20 μl reaction was composed of 10 μl Brilliant II SYBR green with
ROX reference dye (Agilent, Santa Clara, CA), 1 μl each of 10 μM forward
and reverse primers, 6 μl of nuclease-free water, and 2 μl of sample DNA.
PCR conditions were as follows: an initial denaturation step of 95 ◦ C for 5
min, followed by 40 cycles of 95 ◦ C for 30 s, 56 ◦ C for 45 s, 72 ◦ C for 60 s and
data acquisition at 80 ◦ C for 10 s (Witarsa et al., 2016). Plasmid standards
were created as described previously (Pereyra et al., 2010; Prasse et al.,
2015). All standard curves were run with a minimum of five dilutions from
103 to 107 copies per μl and had R2 > 0.98 with efficiencies between 98%
and 101%. All reactions were run on the Mx3005P Real-Time PCR in­
strument (Agilent, Santa Clara, CA). Absolute abundances of mcrA genes
were found by adjusting the diluted quantities to their initial
concentrations.
2.5. Shotgun metagenomic sequencing and processing
Shotgun metagenomic library construction was done on the diluted
DNA using the Nextera XT DNA Library Kit (Illumina Inc., www.ill
umina.com, San Diego, CA) following the Illumina protocol. Briefly,
DNA was simultaneously fragmented and tagged with adapter se­
quences. Indexed adapters were added during amplification in limited
cycle PCR. After amplification, AMPure XP beads were used to clean-up
libraries which were then quality checked on the Agilent Bioanalyzer
(Agilent Technologies, Santa Clara, CA). Libraries were normalized and
pooled before sequencing. Samples were sequenced using the Illumina
NextSeq 500 platform at the USDA Agricultural Research Service,
Beltsville Area Research Center, Beltsville, Maryland.
Trimming and removal of contaminant reads was done using BBMap
version 37.66 (https://jgi.doe.gov/data-and-tools/bbtools/bb-tools-
user-guide/bbduk-guide/). BBMap was further used to filter reads
with kmers matching to the reference rice genome. Read searches were
done in DIAMOND (v0.9.17.118) (Buchfink et al., 2015) against the
NCBI NR database retrieved (February 2020) in protein space. Megan 6
(version 6.15.2) command line tool “daa-meganizer” was used to make
taxonomic assignments of the read-based data to the NCBI taxonomy
database (November 2018) while functional gene assignments were
made using SEED DB (May 2015) (Overbeek et al., 2014). Both taxo­
nomic and functional gene assignment were done using the weighted
lowest common ancestor (LCA) algorithm using a top percent setting of
3.0. Read counts from the analysis were exported from Megan and
filtered to exclude those taxa with zero counts for more than 50% of
samples and for taxa with read counts below 100. Filtered read count
data was used for all subsequent analyses including taxonomic analyses
of relative abundance and community composition (principal compo­
nent analysis, PCA), and functional gene analysis of abundance (e.g.,
heatmaps).
C.P. Fernández-Baca et al.
2.6. Sequencing and statistical analyses
All statistical analyses were completed in R (version 3.5.1) (Oksanen
et al., 2017). For normally distributed data (i.e., methane emissions),
one-way ANOVA was conducted at a significance level of p = 0.05 fol­
lowed by TukeyHSD for multiple comparison testing of means using a
significance level of p = 0.05. For non-normally distributed data (i.e.,
mcrA gene abundances), Kruskal-Wallis tests were conducted at a sig­
nificance level of p = 0.05 and was followed by Dunn’s test of multiple
comparisons at a significance level of p = 0.05.
Sequencing produces compositional count data so the data were
transformed using centered log-ratio (CLR) transformation prior to
further analysis according to the methods of Gloor et al. (2017). The CLR
transformation divides each element of the count vector by the geo­
metric mean of all elements then takes the natural log of each ratio,
transforming the data from the simplex to real coordinate space. The
Aitchison distance metric was used for clustering and ordination as it is
more stable to data subsetting and aggregation than more commonly
used metrics including the Bray-Curtis dissimilarity metric (Gloor et al.,
2017).
PCA of the soil microbial community was conducted on the filtered
read counts from shotgun metagenomic sequencing at the species level
using the Aitchison distance metric on the CLR transformed data with
the compositions package in R (Van Den Boogaart et al., 2021). The
variance-based compositional PCA was used in this study as it is more
stable and reproducible than abundance-based principal co-ordinate
analysis (PCoA) and is recommended for use on CLR transformed data
(Gloor et al., 2017). Variable regression calculations were done using
the envfit function in the vegan package of R using methane emissions,
genotype, and developmental stage as factors (Oksanen et al., 2017).
Significant factors were determined from 1000 permutations using a
significance level of p < 0.05 in envfit and were included in the PCA
bi-plots. Ellipses at a confidence level of 0.95 were included alternately
for developmental stage and genotype to examine variability in the
respective groups.
Methane, iron, and sulfur cycling microorganisms as well as func­
tional genes related to these processes were further examined. Manual
searches of read level metagenomic sequencing data were conducted to
identify known methanogens, methanotrophs, and sulfur and iron-
cyclers. Manual searches have the potential to miss taxa that are not
currently known to have methane, iron, and/or sulfur cycling capabil­
ities, leading to underestimation of these populations. For this reason,
we further analyzed the metagenomic data for genes involved in these
pathways. All relevant functional genes, as classified by SEED, were
selected for further analysis. The functional genes were then filtered to
exclude genes with zero counts for more than 50% of samples and for
genes with read counts below 100. These results were presented in a
heatmap to analyze functional gene changes in abundance over time and
by genotype. Significant differences between genotype and develop­
mental stage functional gene abundances were tested by one-way
ANOVA at a significance level of p = 0.05.
3. Results
3.1. Methane emissions and mcrA gene abundances
Methane emissions varied by rice genotype and by developmental
stage (Fig. 1a). Booting stage showed the most variability in methane
fluxes within genotype, however, there were no differences between
genotypes. However, genotypes differed for methane emissions at
heading and grain fill. At heading, Francis was the lowest emitter (mean,
891 mg CH4–C m− 2 d− 1) while RIL1 (mean, 1052 mg CH4–C m− 2 d− 1),
RIL7 (mean, 1240 mg CH4–C m− 2 d− 1), and Rondo (mean, 1410 mg
CH4–C m− 2 d− 1) had higher levels of emissions. At the grain fill stage,
Francis had the lowest emissions (mean, 496 mg CH4–C m− 2 d− 1) fol­
lowed by Rondo (mean, 595 mg CH4–C m− 2 d− 1), whereas RIL1 and
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Soil Biology and Biochemistry 156 (2021) 108233
RIL7 were significantly (p < 0.05) higher (means of 934 mg CH4–C m− 2
d− 1 and 1012 mg CH4–C m− 2 d− 1, respectively). There were no CH4
emissions measured at maturity for any genotype. Results of cumulative
methane emissions (Supplemental Fig. 1) show a consistent pattern for
each genotype, with Francis and RIL1 being lower methane emitters
than Rondo and RIL7.
Gene abundances determined by qPCR of mcrA also varied by ge­
notype and developmental stage, with Rondo having the highest mcrA
gene abundances across all stages however genotypic differences were
significantly different only at the booting stage (Fig. 1b). At heading,
when there were genotypic differences in CH4 emissions, there was a
positive correlation with mcrA gene abundances (R2 = 0.854, p = 0.076)
(Supplemental Fig. 2). In contrast, genotypic differences in CH4 emis­
sions at grain fill, were negatively correlated with mcrA gene abun­
dances (R2 = 0.839, p = 0.084).
3.2. Overall microbial community structure
Shotgun metagenomic analysis revealed the relative abundances of
the 10 most abundant phyla which were represented by bacterial com­
munities that are commonly found in soils (Fig. 2), including Acid­
obacteria, Actinobacteria, Bacteroidetes, Chloroflexi, and Firmicutes.
Principal component analysis (PCA) of microbial community
structure was conducted at the species level on the centered-log ratio
(CLR) transformed taxonomic count data produced by shotgun met­
agenomic sequencing and is shown alternately by developmental
stage (Fig. 3a) and genotype (Fig. 3b). Regression analyses revealed
cumulative methane emissions were a significant factor related to the
overall microbial community structure (p = 0.038, Supplemental
Table 1). Developmental stage and plant genotype were likewise
found to be significant factors (p = 0.0035 and p < 0.0001, respec­
tively, Supplemental Table 1) (Fig. 3a and b). Additionally, methane
emissions were a significant factor associated with the variability in
community structures at both booting and heading stages (p = 0.005
and p = 0.01, respectively) (Supplemental Fig. 3). Differences in
species level microbial community structure were most apparent be­
tween the reproductive phases (i.e. booting and heading) and the
ripening phases (i.e. grain fill and maturity) (Fig. 3a). The microbial
community structure of Francis and RIL1 grouped together whereas
those associated with RIL7 and Rondo grouped closely together
(Fig. 3b). Interestingly, Francis and RIL1 were more similar to each
other and different from RIL7 and Rondo during booting and heading
stages, whereas all genotypes were similar at grain fill and maturity
(Fig. 3b). PCA plots were also created by developmental stage and
genotype (Supplemental Figure 3 and 4, respectively) and results
suggest that microbial communities become more similar over time
for the low methane emitters (Francis and RIL1) but remain relatively
unchanged for the high methane emitters (RIL7 and Rondo). Devel­
opmental stage was a significant factor for Francis (p = 0.0019) and
RIL1 (p = 0.047) but not RIL7 (p = 0.053) and Rondo (p = 0.17)
(Supplemental Fig. 4).
3.3. Methane-cycling microbial community
Methanogen and methanotroph abundances from shotgun meta­
genomic sequencing results were examined at the family level to see if
there were shifts between developmental stages for specific genotypes
(Fig. 4). Francis and RIL1 show a relative increase in Methanobacter­
iaceae from booting to maturity. Rondo likewise shows a slight increase
in Methanobacteriaceae over time but, in general, methanogen abun­
dances remain relatively constant across developmental stages for both
RIL7 and Rondo. A similar increase over time was seen for Meth­
anocellaceae, the most abundant methanogen family, for Francis and
RIL1, but little change was seen in RIL7 or Rondo. In general, metha­
nogen abundances remain relatively constant across developmental
stages for the other families.
C.P. Fernández-Baca et al. Soil Biology and Biochemistry 156 (2021) 108233

Fig. 2. Shotgun metagenomic read-based results of the 10 most abundant phyla across plant genotypes Francis (FRCS), RIL1, RIL7, and Rondo (ROND) shown by rice
developmental stage (i.e., booting, heading, grain fill, maturity).

Fig. 3. Principal component analysis (PCA) of shotgun metagenomic sequencing results of the species level rhizosphere microbial community shown alternately by
plant developmental stage (a) and genotype (b). Genotype was a significant factor contributing to microbial community structure (p < 0.0001) as was developmental
stage (p = 0.0035). Total methane emissions correlated significantly (p = 0.038) with microbial community structure.

Methanotroph families, including Methylobacteriaceae and Metyhlo­
coccaceae, decreased from booting to maturity in Francis and RIL1 but
remained relatively constant in RIL7 and Rondo. Interestingly, meth­
anotroph families known to perform anaerobic methane oxidation
including ‘Candidatus Methanoperedenceae’ (Haroon et al., 2013) and
nitrite-dependent methanotrophs from within the candidate division
NC10 phylum (Ettwig et al., 2009) were also identified at all four
developmental stages. Both of these anaerobic methanotrophs decrease
over time for Francis and RIL1 but remain constant for RIL7 and Rondo.
We further examined if microbial communities known to compete
with methanogens, such as iron- and sulfate-reducing populations,
shifted between developmental stages as compared to the identified
methanogen and methanotroph communities. Individual genotypes
showed community shifts by developmental stage (Supplemental Fig. 5
and 6) with the greatest changes seen from the reproductive to ripening
phases in the low methane emitting genotypes Francis and RIL1.
Abundances of iron and sulfate reducing populations did not vary from
booting to maturity for RIL7 and Rondo, however, there was a small
relative decrease in sulfate reducers for Francis and RIL1 during this
time.
Abundances for genes involved in methane oxidation, methane
production (i.e., acetoclastic methanogenesis, hydrogenotrophic meth­
anogenesis, and methanogenesis pathways common to both), and sulfate
reduction showed changing patterns with developmental stage (Fig. 5,
see Supplemental Table 2 for genes presented in this analysis). In gen­
eral, the abundance of genes involved in methanogenesis was higher in
RIL7 and Rondo during the booting stage as compared to Francis and
RIL1 (Fig. 5a). Interestingly, at booting, RIL7 and Rondo had higher
abundances of genes involved in hydrogenotrophic methanogenesis
(approximately 50% in this group) and general methanogenesis
pathway genes (i.e. MCR) as compared to Francs and RIL1. ANOVA
confirmed there were genotypic differences of methanogenesis path­
ways (p = 0.00254 and p = 0.0011 for hydrogenotrophic methano­
genesis and MCR, respectively, Supplemental Table 3). Generally,
methane oxidation genes appeared highest during booting (Fig. 5a) and
grain fill (Fig. 5c) across all genotypes but were lower during heading
(Fig. 5b). Genes involved in sulfate reduction were present across all
stages and were generally lower in abundance during heading and at
maturity as compared to during booting. Based on ANOVA, both
methane oxidation and sulfate reduction gene abundances were signif­
icantly different (p = 0.0019 and p = 0.0002, respectively, Supplemental
Table 3) by developmental stage but not by genotype.

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C.P. Fernández-Baca et al. Soil Biology and Biochemistry 156 (2021) 108233

Fig. 4. Relative abundance (centered-log ratio, CLR) of methanogen and methanotroph families from shotgun metagenomic sequencing for each genotype Francis
(FRCS), RIL1, RIL7, and Rondo (ROND) shown by rice developmental stage (i.e., booting, heading, grain fill, maturity). Each line represents the mean of three
biological replicates with vertical bars representing the standard deviation for each mean.

Fig. 5. Heatmap of centered-log ratio (CLR) transformed abundances for genes identified from shotgun metagenomic sequencing to be involved in methane cycling
and sulfur reduction for three replicates at each developmental stage; booting (a); heading (b); grain fill (c); and maturity (d) and by genotype; Francis (FRCS), RIL1,
RIL7, and Rondo (ROND).

4. Discussion impact the rhizosphere microbial community (Edwards et al., 2015,

Methane production from rice cultivation is a significant source of
anthropogenic global methane emissions and is driven by the rhizo­
sphere microbial communities found in rice paddies, but how rice ge­
notype modulates the methane cycling community is not well
understood. Previous studies have examined how different rice cultivars
2018; Santos-Medellín et al., 2017). A recent study by Liechty et al.
(2020) has further examined the rice rhizosphere microbiome of known
low vs high methane emitting cultivars, but this was done uncoupled
from direct measurement of cultivar methane emissions. Our study
aimed to explore genotypic influence on the soil microbial community
structure by coupling measured methane emissions to metagenomic

6
C.P. Fernández-Baca et al.
sequencing of the rhizosphere microbial community across four key
developmental stages (i.e. booting, heading, grain fill and maturity). To
this end, we used two rice cultivars differing in methane phenotypes,
Francis having low methane emissions and Rondo having high methane
emissions, and two RIL offspring exhibiting divergent methane profiles,
RIL1 and RIL7.
Our results confirm genotype differences in methane emissions as
previously observed for Francis and Rondo (Kim et al., 2018), however,
these differences were only significant at heading and grain fill. Methane
emissions were variable at booting and were negligible at maturity but
were highest at heading for all genotypes. Previous studies have shown
methane emissions peak late during the reproductive stage (Gogoi et al.,
2008; Simmonds et al., 2015; Adviento-Borbe et al., 2013). The cultivar
with the highest emissions at heading, Rondo, (mean of 1410 mg CH4–C
m− 2 d− 1) was one of the lowest in emissions (p < 0.0001) at grain fill
(mean of 595 mg CH4–C m− 2 d− 1). In contrast, RIL1 and RIL7 had high
emissions during heading and grain fill, before dropping to
non-detectable levels at maturity. Francis consistently had the lowest
methane emissions across all developmental stages (maximum methane
emissions of 890 mg CH4–C m− 2 d− 1 observed at heading).
Copy numbers of mcrA, a gene encoding for the methyl-coenzyme M
reductase (MCR) alpha subunit involved in methanogenesis, were not
found to positively correlate with methane emissions except at heading
stage (Supplemental Fig. 2). In contrast, at grain fill there was an
opposing trend where mcrA gene abundances were negatively correlated
with methane emissions. This may indicate that methanogens are
competing for substrates, such as plant-derived carbon, against iron and
sulfate reducing bacteria during grain fill. Indeed, abundances for genes
involved in sulfate-reduction appear to increase at grain fill as compared
to heading stage (Fig. 5) for RIL7 and Rondo which both show decreases
in methane emissions between these stages (Fig. 1). Somenahally et al.
(2011) found that the abundance of both iron and sulfate reducing
bacteria increased from day 1 to day 60 after sowing and remained
relatively consistent until day 120 after sowing. This trend observed in
our study suggests these sulfate reducing bacteria continue to be
competitive for carbon substrates even while methanogen activity in­
creases. We measured total soil organic carbon over the plant develop­
mental stages and found no significant differences by genotype or
developmental stage (soil carbon mean of 0.8% ± 0.05% across all
samples and stages), indicating the carbon pool was consistent over
time. Alternatively, the negative correlation between mcrA gene abun­
dances and methane emissions during grain fill could indicate that the
overall methanogen presence remains unchanged during this period, but
their activity may decrease or simply be surpassed by the more abundant
methanotrophs. Freitag and Prosser (2009), found that the mcrA tran­
script to gene abundance ratios increase as methane emissions increase,
however, they found emissions can be low even when gene abundances
are high due to the presence of dead and/or inactive methanogens
(Freitag and Prosser, 2009). Thus, although we observed relatively
stable mcrA gene abundances at grain fill, Francis and Rondo showed a
decrease in methane emissions suggesting that methanogen activity for
these genotypes may have decreased. There is also evidence that
methanotrophs are more abundant than methanogens during the grain
fill phase as the gene abundance data shows an increase in genes
involved in methane oxidation from heading to grain fill stage (Fig. 5).
The qPCR-determined mcrA gene abundances were highest during the
reproductive stages (booting and heading) for Rondo and RIL7 but were
lowest for Francis during this time, while RIL1 mcrA gene abundances
increased from booting to heading stage and decreased thereafter.
Although changes in the rhizosphere microbial communities over time
can be measured with qPCR and sequencing at the DNA level (Collins
et al., 2018), it does not give us the capability to examine the activity of
these populations. Thus, further studies examining how the rice geno­
type impacts the methane cycling community and, in turn, methane
emissions from rice paddy soil, would benefit from coupling
qPCR/metagenomic data with RT-qPCR/metatranscriptomic data.
7
Soil Biology and Biochemistry 156 (2021) 108233
Our results confirm previous studies which have found that rice
developmental stage (Breidenbach et al., 2016; Breidenbach and Con­
rad, 2015; Edwards et al., 2018) and genotype (Briones et al., 2002;
Edwards et al., 2015, 2018; Liechty et al., 2020; Santos-Medellín et al.,
2017) can influence the rhizosphere microbial community structure.
PCA revealed that microbial communities of samples from the repro­
ductive phases (i.e., booting and heading) grouped together while those
from the ripening phases (i.e., grain fill and maturity) grouped together
for low methane emitting genotypes (Fig. 3 and Supplemental Fig. 4),
suggesting community structure is influenced more by the phase (i.e.,
reproductive vs ripening) than the specific plant stage for these geno­
types. In contrast, there was no strong developmental stage grouping for
the high methane emitting genotypes RIL7 and Rondo. The microbial
community composition of samples did group by methane emissions
pattern with Francis and RIL1, having low CH4 emissions, clustering
together and apart from Rondo and RIL7, having high CH4 emissions
(Fig. 3b); however, the genotype-associated microbial community
structure becomes more similar between low and high methane emitting
genotypes as the plant ages (Fig. 3a) suggesting this genotype effect is
stronger earlier in plant development.
To further examine the relationship between genotype and plant
development, PCAs for each genotype were made (Supplemental Fig. 4)
which showed that the low methane emitting genotypes (Francis and
RIL1) displayed more distinct grouping by developmental stage than the
high methane emitting genotypes (Rondo and RIL7). Edwards et al.
(2018) observed that root-associated microbial communities changed
throughout the vegetative phase and stabilized during the reproductive
phase similar to what is observed in this study for the low methane
emitters. In contrast, the high methane emitters appear to have a more
stable community throughout their development suggesting these ge­
notypes acquire and stabilize their rhizosphere communities more
quickly than the low methane emitters. It is unclear how much genotype
drives the differences observed in the early-stage plant rhizosphere
community composition; however, plant development, which is geno­
type dependent, does play a role in the process of community stabili­
zation (Edwards et al., 2018). Although genotype and developmental
stage both appear to explain some of this separation in clustering of RIL1
and Francis versus RIL7 and Rondo, there are other factors (e.g., soil
temperature, pH, etc.) not measured in this study that may additionally
explain some of the variation in microbial community structure.
The relative abundance of acetoclastic (acetate-utilizing) and
hydrogenotrophic (hydrogen and carbon dioxide utilizing) metha­
nogens varied by genotype and developmental stage (Fig. 4). Aceto­
clastic methanogens are thought to produce two-thirds of the biogenic
produced methane (Smith and Ingram-Smith, 2007); however, our re­
sults show the relative abundance of hydrogenotrophic methanogens,
Methanocellaceae and Methanobacteriaceae, was greater than that of the
acetoclastic methanogen family, Methanotrichaceae (Fig. 4).
Plant-derived carbon is the most important carbon source for microbial
methane generation and methanogens from the Methanocellaceae family
have been found to incorporate more plant-derived carbon than
methanogens from other families (Lu and Conrad, 2005). The more
versatile Methanosarcinaceae family, which can utilize both acetate and
hydrogen plus carbon dioxide, was abundant across all developmental
stages and genotypes and populations remained relatively stable
through all plant developmental stages likely due to its substrate flexi­
bility (Liu and Whitman, 2008).
Specific rice genotypes revealed changes in methanogen populations
by developmental stage. For example, Francis and RIL1 had an increase
in overall methanogen abundance over time compared to methano­
trophs or iron and sulfate reducing populations, however RIL7 and
Rondo had similar methanogen abundances across all stages (Supple­
mental Fig. 6). For Francis and RIL1, this increase was mostly due to an
increase in Methanobacteriaceae and Methanocellaceae abundance from
booting to maturity (Fig. 4). This is supported by the gene level data
which shows RIL7 and Rondo have higher relative abundances of
C.P. Fernández-Baca et al.
hydrogenotrophic methanogenesis genes particularly at booting and
heading (Fig. 5a and b). In general, the abundance of genes common to
both methanogenesis pathways (i.e. methyl-coenzyme reductase, MCR
genes) are higher in RIL7 and Rondo during booting as compared to
Francis and RIL1 (Fig. 5a) and our qPCR results display a similar trend of
higher mcrA gene abundances for Rondo and RIL7 at booting (means,
1.02 × 106 and 6.25 × 105 copies g− 1 soil, respectively) compared to
Francis and RIL1 (means, 2.92 × 105 and 1.25 × 105 copies g− 1 soil,
respectively) and likewise at heading (means, 6.90 × 105 and 4.09 × 105
copies g− 1 soil, for Rondo and RIL7, respectively, and 5.23 × 104 and
4.27 × 105 copies g− 1 soil, for Francis and RIL1, respectively) (Fig. 1b).
Gene abundances at maturity decrease for RIL7 and Rondo (means, 3.40
× 105 and 4.70 × 105 copies g− 1 soil, respectively) and are more similar
to Francis and RIL1 at this late growth stage (means, 4.18 × 105 and
3.16 × 105 copies g− 1 soil, respectively) (Fig. 1). Overall, the cultivars
having high methane emissions, Rondo and RIL7, had both higher mcrA
gene abundances and a greater proportion of methanogen populations
compared to methanotroph populations at heading stage, which was
likely reflected as higher methane emissions for these genotypes.
However, gene abundances are not reliable predictors of metabolic ac­
tivity, and future work in this realm would benefit from quantifying
transcript abundance in addition to gene abundance.
Within the methanotroph community neither type I (which assimi­
late carbon via the ribulose monophosphate pathway) nor type II
(assimilate carbon via the serine pathway) varied by genotype or
developmental stage (Fig. 4). Liechty et al. (2020) similarly found that
the relative abundances of type I and type II methanotroph 16S rRNA
sequencing reads did not vary between high and low CH4 emitting
cultivars. We found both type II methanotrophs (Methylocystaceae,
Methylobacteriaceae, and unclassified methanotrophs) and type I meth­
anotrophs (Methylococcaceae) across all genotypes and developmental
stages. The cumulative abundance of type II methanotroph families was
greater than that of the single type I methanotroph family found in our
study which supports previous findings (Henckel, Friedrich and Conrad,
1999; Henckel et al., 2000; Lee et al., 2014). Type II methanotrophs are
known to be more resilient under stress (e.g., have the ability to form
resting cells resistant to desiccation and heat stress) (Ho et al., 2013),
more active under high methane concentrations (Henckel et al., 2000),
and more resilient to low oxygen such as those in flooded rice paddies
(Shiau et al., 2018) compared to type I methanotrophs. However, it has
been shown that type I methanotrophs can contribute to methane
oxidation under both low and high methane concentrations (Henckel
et al., 2000). We did not measure gene expression in this study, but it is
likely that both type I and type II methanotrophs were actively oxidizing
methane across genotypes and developmental stages as previous studies
have found activity of both types of methanotrophs in rice paddy soils
(Noll et al., 2008; Qiu et al., 2008). Indeed, type I methanotrophs have
previously been found to be more active than type II methanotrophs in
rice paddy soils despite their overall lower abundances compared to type
II methanotrophs (Noll et al., 2008; Qiu et al., 2008). The relative
abundance of methanogens to methanotrophs (Supplemental Fig. 6),
shows RIL1, RIL7 and Rondo all have relatively larger methanogen
populations as compared to methanotroph populations at heading stage
when methane emissions are highest.
Two anaerobic methanotroph families, ‘Candidatus Methanoper­
edenaceae’ and NC10 phylum bacteria, were identified across all
developmental stages and genotypes. Sequences for both types of
anaerobic methane oxidizers have previously been found in rice paddy
fields (Lee et al., 2015; Wang et al., 2012; Zhou et al., 2014). Candidatus
Methanoperedenaceae’ is an ANME-2d archaeal family that contains the
species ‘Candidatus Methanoperedens nitroreducens’ capable of using a
reverse methanogenesis pathway to oxidize methane coupled to nitrate
reduction (Haroon et al., 2013; Vaksmaa et al., 2017; Welte et al., 2016)
and has also been found to couple methane oxidation to iron and
manganese reduction (Ettwig et al., 2016). Bacteria from the NC10
phylum have been found to couple denitrification to methane oxidation
8
Soil Biology and Biochemistry 156 (2021) 108233
by creating molecular oxygen intercellularly from nitrite reduction to
oxidize methane using the typical methane monooxygenase found in
aerobic methanotrophs (Ettwig et al., 2012; Wu et al., 2011). These two
anerobic methanotrophs can drive denitrification using methane instead
of organic matter and have the potential to mitigate greenhouse gas
emissions from flooded paddy soils (Shen et al., 2012).
Flooded paddy soils are favorable conditions for the degradation of
organic matter leading to competition between microbial communities for
electron donors (Achtnich et al., 1995; Breidenbach et al., 2016).
Generally, electron acceptors with higher redox potentials are reduced
first and for this reason sulfate and iron reducers outcompete metha­
nogens for substrates in high redox potential conditions typical of recently
flooded soils (Achtnich et al., 1995). Our results indicate abundances of
Geobacteraceae, an iron reducer, remain relatively constant from booting
to heading while sulfate reducers, including Desulfovibrionaceae, Desulfo­
bacteraceae, and Desulfobulbaceae, decrease during this time for Francis
and RIL1 but remain unchanged for RIL7 and Rondo. The abundance of
these populations is not necessarily indicative of their activity; thus, these
populations may be present and highly active during booting and remain
present but become less active during heading stage when methane
emissions are highest and presumably when methanogen activity is
dominant. Genes involved in sulfate reduction follow a similar pattern to
sulfate-reducing populations and are most abundant at booting stage
when methane emissions are their lowest. However, at heading stage
there is a decrease in sulfate reduction genes corresponding with an in­
crease in methane emissions. These results suggest sulfate and iron re­
ducers are present and likely active during booting stage, lowering the soil
oxidation-reduction potential thereby creating favorable conditions for
methanogenesis during the later heading stage (Liechty et al., 2020).
Overall, our results show that rice genotypes and plant develop­
mental stage are associated with different soil microbial community
structures. Additionally, rice genotype impacts methane-cycling pop­
ulations and the observed methane emissions to a greater extent during
the reproductive phases of booting and heading as compared to the
ripening phases (i.e., grain fill and maturity). Changes in the iron and
sulfate-reducing communities are less apparent, suggesting their pres­
ence is not necessarily indicative of competition with other microbes.
Our study demonstrates there is potential for developing low methane
emitting rice cultivars through conventional breeding efforts as evi­
denced by the creation of the RILs used in this study which were derived
from high and low emitting cultivars. Future breeding efforts to this end
should focus on mitigating methane emissions during the rice repro­
ductive phase by selecting cultivars that modulate the methane-cycling
community during this critical period. Ultimately, the selection of these
cultivars should be coupled with the identification of plant phenotypic
differences affecting methane-cycling microbial communities in order to
develop trait linked genetic markers that will facilitate future develop­
ment of low-methane emitting rice cultivars.
Accession number(s)
All sequencing data have been deposited as an NCBI BioProject
PRJN663614 under accession numbers SAMN16226752 -
SAMN16226799.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
This research utilized facilities and assistance provided by USDA
Agricultural Research Service Dale Bumpers National Rice Research
Center, Stuttgart, Arkansas and Beltsville Agricultural Research Center,
C.P. Fernández-Baca et al.
Beltsville, Maryland. This study was carried out with the support of the
Cooperative Research Program of the Rural Development Administra­
tion, Republic of Korea and United State Department of Agriculture,
Agricultural Research Service, US (Project title: Development of a
screening methodology for assisting in rice cultivar selection for reduced
methane emissions, Project No.: PJ012431. We would like to thank Mr.
Jonathan Moser for collecting soil and gas samples. We would like to
thank Drs. Sarah Emche and Seonwoo Kim for their technical assistance
in sequencing preparation. We would also like to thank Dr. Stephanie
Yarwood for providing the plasmid standard for qPCR analyses. USDA is
an equal opportunity provider and employer.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.soilbio.2021.108233.
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