|Colonisation; Rumen; Bacteria; Stem; Leaf; Abaxial;
Adaxial; DGGE; QPCR
|With an ever increasing population and increased demands
for ruminant products, the need to ensure future food security is
paramount . This presents a major challenge to find novel strategies
to sustainably increase animal productivity. The main challenge for the
livestock sector is increasing the conversion of plant to microbial protein
within the rumen which is inefficient as only around 30% of ingested
nitrogen is retained by the animal for meat and milk production [2-5].
Thus in pasture-based feeding systems the plant-microbe inter-actome
is central for utilisation of plant nutrients [3,5,6]. Furthering our
understanding of plant-microbe interactions during colonisation and
plant degradation is expected to offer novel opportunities to improve
ruminant nutrient use efficiency in order to sustainably increase meat
and milk availability.
|The ability of the rumen microbiota to attach in a timely manner
to ingested forages in the rumen is central to ruminant nutrient use
efficiency. Indeed, it is known that rumen microorganisms rapidly
attach to recently ingested feed particles [7-13]. We have also
demonstrated that not only does bacterial colonisation of fresh whole
perennial ryegrass in the rumen occur within 15 minutes, but this is not
a stable community with a change in the diversity of attached bacteria
from primary to secondary attached communities occurring within 2
- 4 h of perennial ryegrass colonisation . Rumen microorganisms
colonising the surfaces of forages are confronted with highly variable
physiochemical conditions including nutrient type and availability due
to the heterogeneity of the plant material. This heterogeneity may affect
microbial attachment and subsequent biofilm formation. Indeed, plant
degradation within the rumen is known to be affected by the amount
of cuticle, waxes or lignin and the degree of cross-linkage to other cell wall polymers within the plant [14,15]. McAllister et al. stated that
rumen bacteria do not bind to the waxy cuticle of forage particles due
to the difficulty in degrading these structures in order to access the
plant nutrients . Thus, bacterial attachment to different parts of the
plant material may be different due to the differences in their surface
|We investigated the hypothesis that attachment of the rumen
microbiota to plant material is affected by the physicochemical
properties of forage structures. It has been documented that PRG stem
and leaves differ chemically [16,17], and wax chemistry on abaxial and
adaxial surfaces differ , nonetheless colonisation of PRG by the
rumen microbiota is often investigated without taking into account
that composition of different plant structures vary. These approaches
often miss fundamental information on the interactions of key
microbiota with various plant structures and the subsequent effect on
degradation of various structures within the whole plant. Hence, the
first experiment compared bacterial colonization of fresh perennial
ryegrass stems and leaves and the second experiment assessed attachment of rumen bacteria to each side of the colonised leaf (abaxial
versus adaxial). Knowledge of bacterial colonization of different parts
of forage plants aids our understanding of bacterial niche preference.
Increasing our knowledge of rumen plant-microbe interactions is
paramount in order to develop novel strategies of improving ruminant
nutrient use efficiency and ultimately ensure ruminant food security.
|Materials and Methods
|Growth and preparation of plant material
|Perennial ryegrass, (Lolium perenne cv. Aberdart; PRG) was grown
from seed in plastic seed trays (length 38 cm x width 24 cm x depth 5
cm) filled with compost (Levingtons general purpose). The trays were
maintained in a greenhouse under natural irradiance with additional
illumination provided during the winter months (minimum 8 h
photoperiod) or in a growth cabinet (Sanyo, Osaka) with 16 h of light
(irradiance ~300 μmol m-2s-1) per day. A temperature of 22/19°C day/
night was maintained and plants were watered twice a week.
|In vitro incubations: stem and leaf colonisation
|Following 6 weeks of growth the plant material was cut with
scissors at 3 cm above soil level and divided into stems and leaves.
Sub-samples of stems and leaves were frozen, freeze-dried and stored
at -20°C for chemical analysis and bacterial profiling (0 h samples).
Chemical analyses of stem and leaf samples were carried out as
described in Huws et al. . Subsequently, cut stem or leaves (1 cm;
7.5 g) were added to Duran bottles (250 mL) together with anaerobic
incubation buffer (135 mL pre-warmed to 39ºC  and rumen
fluid inoculum (15 mL, strained through 2 layers of muslin and held
under CO2 at 39°C). Bottles were incubated in a horizontally rotating
rack at 100 rpm and 39°C (Incubator-shaker, LA Engineering, UK).
Incubations were set up in sextuplicate and the bottle contents were
harvested at 0, 0.25, 0.5, 1, 2, 4, 8, 12 and 24 h. At each time interval the
contents of three of the bottles contents were harvested individually by
vacuum filtration through filter paper (11μm2 pore size; ®QL100, Fisher
Scientific, Leicestershire, UK). Retained, colonised plant material was
washed with phosphate buffered saline (PBS), frozen at -20°C, before
freeze-drying and grinding followed by DNA extraction. At each time
interval the contents of the remaining three bottles were oven dried
and plant degradation was measured as % dry matter lost.
|In vitro incubations: adaxial and abaxial colonisation
|Following 6 weeks of growth the plant material was cut with
scissors at 3 cm above soil level and leaves were harvested and cut into
1cm strips. The chopped grass was used as substrate for anaerobic batch
cultures set up in Duran bottles (250 mL) using rumen fluid inoculum
prepared as described above and incubated for 0, 2, 4, and 8 h. Following
incubation, a set of six bottles were harvested at the designated times and
split into two groups of three bottles. The first group of three bottles was
used as positive controls for whole leaf colonisation; the leaf fragments
from those bottles were collected by vacuum filtration through filter
paper (11 μm2 pore size; ®QL100, Fisher Scientific, Leicestershire, UK),
rinsed in PBS then frozen at -20°C before freeze-drying and grinding
followed by DNA extraction. The second group of bottles was used to
assess the quantity and diversity of rumen bacteria on each side of the
colonised leaf. Essentially, at each time interval, abaxial and adaxial
leaf surfaces (from 100 leaf fragments) were placed on 10 microscope
slides (10 per slide) and silicone rubber spread across the leaf surfaces
with a brush. The silicone rubber (Magnacrafty, Midhurst, UK) was
mixed with catalyst (Trylon Ltd, Northants, UK) (7 parts of rubber: 1 part of catalyst) causing it to set in 30 to 60 min. A second microscope
slide was placed on top of the set layer and slight pressure carefully
applied to the upper slide to facilitate the adherence of epidermal and
microbial cells to the rubber film, before leaving overnight at 4ºC. After
setting overnight, the silicone rubber and accompanying epidermal
cells and bacteria were removed from the unwanted side of the leaf
fragments using forceps. The modified leaf blades were then washed
with PBS, frozen at -20°C and then freeze dried and ground. This
method enabled selective removal of the adaxial or abaxial surface, and
thus quantification of the colonisation on the surface left behind on the
leaf fragment. Effective removal was assessed by staining the latex film
after removal from plant fragments using the fluorescent dye DiOC6
(3)-(3,3′-dihexyloxacarbocyanine iodide) (Sigma-Aldrich Company,
Ltd., Dorset, UK) . Removal of bacteria from leaf surfaces was also
visually confirmed by low temperature scanning electron microscopy
of stripped unwanted surfaces as described by Huws et al.  (data
|DNA extraction and denaturing gradient gel electrophoresis
|DNA extraction, PCR-DGGE and subsequent gel fingerprinting,
using the primers 799FGC(5’CGCCCGCCGCGCGCGGCGGGCGG
R1401 (5’CGGTGTGTACAAGACCC3’) was performed as described
by Edwards et al. and Huws et al. [12,13]. Resultant DGGE gels were
scanned using a GS-710 calibrated imaging densitometer (Bio-Rad UK
Ltd, Hemel Hempstead, UK) and the saved image imported into the
software package Fingerprinting (Bio-Rad UK Ltd, Hemel Hempstead,
UK) for analysis. Cluster analysis was performed using Dice, with a
position tolerance of 0.5% and optimisation parameter of 0.5%. The
binary data generated from DGGE based fingerprinting was used to
conduct canonical analysis of principal coordinates (CAP).
|16S rDNA quantitative PCR
|Total bacterial 16S rDNA quantitation using QPCR
was performed as described by Huws et al. using the
primers 520F (5’AGCAGCCGCGGTAAT3’) and 799R2
(5’CAGGGTATCTAATCCTGTT3’) . The quantification was
performed using a 7500 real-time PCR system (Applied Biosystems,
Warrington, UK) using the same standards as described in Huws et
al. . Results were analysed using the 7500 SYSTEM SDS software
(Applied Biosystems, Warrington, UK). QPCRs were performed in
triplicate and assay PCR efficiency was calculated as follows: efficiency
= 10(-1/slope) × 100. QPCR efficiency was always between 90-110%,
and correlations of genomic DNA standards were >0.97.
|Determination of biofilm coverage on abaxial and adaxial
|To estimate the extent of coverage of biofilm communities on
abaxial and adaxial leaf surfaces, four leaves (to allow examination of
two adaxial and two abaxial surfaces) for each of three replicates at
each incubation time were analysed by LTSEM as described by Huws
et al. . The LTSEM analysis was carried out by random scanning of
surfaces at x 2200 magnification and a resolution of 10 μm at 3.0 kV. In
total, 40 frames were analysed per surface and these were recorded as
digital images using SEMAPHORE software. In total 120 digital images
were processed using the digital image analysis software, NIH Image
(Windows version available from Scion Corporation, U.S.A (http://www.scioncorp.com). The digital images were converted to threshold
images, and particle size analysis was subsequently carried out to count and measure the biofilm coverage. The area of particles was computed,
and size and coverage of biofilms quantified in pixels. The analysis was
carried out on particle sizes ranging from 1 to 999999 square pixels.
The area covered by biofilms in the randomly selected frames was
measured as the percentage of pixels detected relative to the area of the
measurement frame (total area of 2015544 square pixels per frame).
|For IVDMD, QPCR and biofilm coverage data, two-way analysis
of variance (ANOVA) was conducted and differences among means
were determined by Duncan’s multiple range tests  using Gen Stat
(Tenth Edition, VSN International Ltd., Hemel Hemstead, UK) .
For plant composition data, one-way analysis of variance (ANOVA)
was conducted and differences among means were determined by
Duncan’s multiple range tests  using Gen Stat. Primer 6 and
PERMANOVA+ (version 6; Primer-E, Ivy bridge, UK) respectively
were used to conduct canonical analysis of principal coordinates
(CAP)  and permutation multivariate analysis of variance
(PERMANOVA)  on DGGE generated binary data.
|Plant dry matter (DM) and chemical composition
|DM did not vary significantly between stem and leaf material
(P>0.05) (Table 1). Nonetheless, chemical compositional differences
were found between stem and leaf in terms total nitrogen, water soluble
carbohydrate (WSC) neutral-detergent fibre (NDF), acid-detergent
lignin (ADL), and alkanes/lipid content (Table 1). Specifically, the
stem contained significantly (P<0.001) more WSC, NDF and ADL but
significantly less total nitrogen than the leaf.
|In vitro dry matter degradation
|IVDMD was significantly different (P <0.001) between leaf and
stem incubations (Table 2). Leaf material was degraded to a greater
extent than stem material with 69.2 and 58.9% being degraded for
leaf and stem material respectively after 24 h incubation (Table 2).
Degradation of leaf was also far more rapid than stem material with
32.2 and 9.1% being degraded for leaf and stem material respectively
after 2 h of incubation (Table 2).
|Attached bacterial diversity
|PCR-DGGE derived UPGMA dendrograms revealed that the
microbiota attached to either stem or leaf were markedly different at
each incubation time (Figure 1A; maximum similarity 40%). As sample
number exceeded gel capacity and comparisons between different
DGGE gels are challenging, multiple gels were run in order to analyse all
samples. Figure 1A shows a representative DGGE gel for one replicate
of the three taken. Some sub-clustering of attached bacteria was also
evident for stem and leaf attached microbiota between 2 and 4 h of
colonisation (Figure 1A; maximum similarity was 40 and 55% between
2 and 4 h leaf and stem attached microbiota respectively). Canonical
analysis of principal coordinates (CAP) ordination confirmed data
for the PCR-DGGE derived UPGMA dendrograms (Figure 1B) by
illustrating that the complexity of microbiota adhering to stem and
leaves differed at each time point, but the 2 to 4 h differences are not
so clear from these graphs. PERMANOVA supported the graphical
data and showed that in the presence of stem or leaf material the
attached microbiota differed significantly from each other (P>0.001,
|PCR-DGGE derived UPGMA dendrograms revealed that the
microbiota attached to either abaxial or adaxial leaf surface did not
differ markedly at any of the time intervals (Figure 2A). Canonical
analysis of principal coordinates (CAP) ordination confirmed data
for the PCR-DGGE derived UPGMA dendrograms (Figure 2B) by
showing similarity in the bacterial 16SrDNA diversity colonising the
abaxial and adaxial leaf surface. Regardless of leaf surface there was
discrimination between populations according to the duration of
incubation (Figure 2B). PERMANOVA also confirmed that bacteria
colonising abaxial and adaxial leaf surfaces did not differ significantly
|Attached bacterial quantity
|The microbiota attached to stem and leaf were not different from
each other in terms of 16S rDNA quantities (Table 3). Nonetheless, time
had a significant effect on quantities of 16S rDNA determined on both
stem and leaf , with 3.0 and 3.1 x times as much attached bacterial 16S
rDNA present following 24 h of incubation on leaf and stem material
respectively, compared to initial 16S rDNA quantities (Table 3).
|Conversely, abaxial and adaxial attached microbiotas were
significantly different from each other in terms of 16S rDNA quantities
(Table 4). After 8 h of incubation bacterial 16S rDNA quantity was
greatest on the adaxial surface compared with the abaxial surface with
2.2 and 3.2 ng g-1 RDM bacterial 16S rDNA present on the abaxial
and adaxial leaf surfaces respectively (Table 4). When added together
abaxial and adaxial 16S rDNA quantities were equal to, or at least
very close, to the data obtained for whole leaves, thus showing that
the stripping methodology was very effective (Table 4). Fluorescent
dye DiOC6 (3)-(3,3′-dihexyloxacarbocyanine iodide) staining of leaf surfaces post stripping as well as LTSEM also confirmed the absence of
bacteria on stripped surfaces (data not shown). No effect of time was
identified in terms of 16S rDNA abundance regardless of leaf surface.
|Biofilm coverage data was in agreement with 16S rDNA QPCR
data in showing that adaxial surfaces had a greater coverage of attached
bacteria than abaxial surfaces (Table 5). Following 1h of incubation
the abaxial surface had 40.6% coverage whilst the adaxial surface had
74.2% coverage (Table 5). By 8h the differential had decreased with the
abaxial surface having 65.9% coverage and the adaxial surface 77.1%
(Table 5). 0h data showed very little colonisation by plant epiphytic
communities (data not shown).
|Increasing the efficiency of feed degradation is a key target for ruminant bioscience. Central to this target is efficient fermentation
of forage, which requires rapid and effective colonisation of newly
ingested feed by rumen bacteria to drive fibre degradation. In this study
we investigated the hypothesis that attachment of the rumen microbiota
to plant material is affected by the physicochemical properties of forage
structures. Within this study we demonstrate that differing plant
parts can have a profound effect on attached bacterial diversity and/
or 16S rDNA quantity. Colonisation of forages by rumen microbiota
is commonly investigated without consideration of the effects of
different plant structures on attachment of the microbiota, and likewise
chemical composition of perennial ryegrass is often assessed using the
whole plant. These approaches often miss fundamental information on
the interactions of key microbiota with various plant structures and
the subsequent effect on degradation of various structures within the
whole plant. The heterogeneity of forage degradation has previously
been shown in Lotus, where tannin containing cells were under graded
by the rumen microbiota . This study aids our understanding of
niche specialisation of the attached rumen microbiota related to plant
chemistry and thereby contributes towards developing novel strategies
of improving ruminant nutrient use efficiency to ensure food security.
|Stem material had more fibre and lignin content than leaves, which
means that this part of the plant is potentially more difficult for the
rumen microbiota to digest. Chemical composition data for stem and
leaves in this study are similar to those reported previously [27,28].
This differential in recalcitrant cell wall structure between stem and
leaves is the most likely explanation for the reduced IVDMD of stem
material compared to leaves. Indeed, Chaves et al. also reported that
DM degradability for leaf is higher than for stem material for perennial
ryegrass, tall fescue, Yorkshire fog, Phalaris and Paspalum grass species
|PCR-DGGE derived UPGMA dendrograms, CAP analysis and
PERMANOVA revealed that microbiota attached to the stem and
leaf was markedly different from each other at all incubation times. QPCR data showed that although diversity of the attached bacteria was
different, bacterial 16S rDNA quantity did not differ in the presence of
stem or leaf material. Shinkai et al. also noted using fluorescence in situ
hybridization (FISH) and QPCR, that more Fibrobacter succinogenes
and Ruminococcus flavefaciens associated with leaves compared to
stems of orchard grass hay . Also noteworthy is the fact that shifts
in attached bacterial diversity occurred between 2 and 4 h incubation
on both stem and leaf material. Indeed, we previously noted this
change from primary to secondary colonisation between these time
intervals . We therefore predict that this differential preference
in colonization behaviour could underlie observed differences in
degradation of leaf and stem material if it is associated with differential
functionalities. This would require more detailed investigation such as
RNA seq to confirm this hypothesis.
|Bacterial communities of abaxial and adaxial leaf surfaces were
similar, but QPCR and quantitative LTSEM data showed that more
bacteria colonised the adaxial surface compared to the abaxial surface.
There are currently few reports regarding differential bacterial diversity
on the adaxial and abaxial surfaces of fresh perennial ryegrass. Akin
found different morphological types of rumen bacteria on different
plant surfaces by electronic microscopy . Results from studies
with mixed bacterial cultures have also shown differences in the
attachment of rumen bacteria to plant cell types e.g. Bacteroides but
not Ruminococcus adhered to intact mesophyll cell wall . It has
been suggested that distinct rumen bacterial species probably colonise
plant surfaces in different ways, due to their distinct growth and
survival requirements . This differential in microbiota attached
to abaxial and adaxial surfaces of perennial ryegrass may be due to
differential chemo-attraction and physical barrier effects described in
early studies [33-35] or differential electrostatic interactions that could
be present on different sides of the leaf . Adaxial surfaces have been
reported to have more waxes containing long-chain alkanes, alcohols,
ketones, and fatty acids as compared to the abaxial surface [36,37].
Therefore, the hydrophobicity of the cell surface may be important in adhesion as hydrophobic interactions tend to increase with the
increasing non-polar nature of one or both surfaces involved i.e.,
the microbial cell surface and the substratum surface [8,38,39]. Most
bacteria are negatively charged but still contain hydrophobic surface
components [40,41], for instance, F. succinogenes cells adhere better
to cationic cellulose ethers than neutral crystalline cellulose, whereas
anionic cellulose-ethers reduced adhesion of this bacterium [8,42]. The
adaxial surface has also been reported to have more stomata present,
which have been reported as easy bacterial plant entry structures
for subsequent plant degradation [7,43]. Irrespective, it appears that
rumen bacteria colonise perennial ryegrass adaxial surfaces to a greater
extent than colonisation of abaxial surfaces.
|In summary we demonstrate that differing plant structures can
have a profound effect on attached bacterial diversity and/or 16S
rDNA quantity likely due to their differing chemistry. This suggests
that the rumen microbiota display niche specialisation in terms of
plant degradation. This data illustrates the importance of investigating
intra-plant colonisation to get a deeper fundamental understanding
of the plant-microbe interactome. Furthering our understanding of
the ruminal plant-microbe interactome, in particular how the plant
cell can be optimised through plant breeding to deliver nutrients in a
manner suitable to maximise microbial efficiency , is fundamental to
the development of novel strategies to increase ruminant production in
order to meet increasing demand for meat and milk.
|We acknowledge funding from COLCIENCIAS, CORPOICA (Colombia) and
the Biotechnology and Biological Sciences Research Council (BBSRC, UK).
We are also grateful to Mark Scott for his technical assistance in setting up the
experiments. The authors have no conflict of interest.
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