Vol.1, No.3, 57-62 (2011)
Open Journal of Ecology
opyright © 2011 SciRes. OPEN ACCESS
Rhizobium alters inducible defenses in broad bean,
Vicia faba
Misty Cree Summers, Edward Brian Mondor*
Department of Biology, Georgia Southern University, Statesboro, USA; *Corresponding Author: emondor@georgiasouthern.edu
Received 18 September 2011; revised 16 October 2011; accepted 28 October 2011.
Conversion of inorganic nitrogen by mutualistic
nitrogen-fixing bacteria is essential for plant
growth and reproduction, as well as the devel-
opment of chemical and mechanical defenses. It
is unclear, however, how these bacteria alter co-
occurring symbioses at higher trophic levels;
e.g., extrafloral nectary (EFN) induction, in re-
sponse to herbivory, to attract defensive mutu-
alist s. We hypothesized that plants colonized by
nitrogen-fixing bacteria would mount a larger
inducible, defensive response than plants lack-
ing symbioses, as defensive traits are costly.
We predicted that bean plants, Vicia faba L.,
harboring Rhizobium leguminosarum bv. viciae
Frank would produce more EFNs upon leaf
damage, than plants lacking the symbionts, as
EFN induction in V. faba is resource-dependent.
Here we report that V. faba colonized by R.
leguminosarum produced similar numbers of
EFNs as did plants without symbionts. Plants
with symbionts, however, produced signifi-
cantly fewer EFNs over 1 week in response to
leaf damage, than those without leaf damage.
As such, nitrogen-fixing bacteria may not al-
ways benefit the host plant, but rather, the utility
of these bacteria may be dependent on the
prevailing ecological conditions.
Keyw ords: Extrafloral Nectary; Inducible Defense;
Mutualism; Phenotypic Plasticity; Rhizobia
Nitrogen-fixing bacteria—host plant mutualisms have
been studied for decades [1,2]. Fixed nitrogen, the con-
version of atmospheric nitrogen to ammonium in plant
root nodules, is used for plant growth and reproduction
as well as chemical defenses [3,4]. Despite their ubiquity,
these symbioses are very host specific; only certain spe-
cies or strains of bacteria can colonize host plants [5,6].
Broad bean, Vicia faba L., roots are one of the most dis-
criminating mutualists, being colonized only by Rhi-
zobium leguminosarum bv. viciae Frank [5,7,8]. This
nitrogen-fixing bacteria increases plant size, vigor, and
seed set [9]. Whether this symbiont alters broad bean
ecological interactions with higher trophic levels, throu-
gh altered defensive capab ilities, however, is unknown.
Defensive mutualisms between plants and predatory
arthropods are common. Over 93 plant families produce
sugar-producing structures, called extrafloral nectaries
(EFNs) [10], which are highly attractive to predatory
ants [11,12]. Plants increasing nectar secretion rates
generally attract more ants, have lower rates of herbivory,
and increased survivorship [12-15]. Increased nectar
production may also be inducible depending upon
environmental conditions [13,16]. In broad bean, EFNs
are located on the stipules at the base of each leaf petiole
[17,18]. Attractive to ants [18,19], broad bean plants
increase EFN numbers when herbivory increases [17,
20]. As broad bean EFNs are visually conspicuous it
may be adaptive to produce a more striking visual dis-
play rather than to augment nectar from existing nec-
taries [17].
The induction of extrafloral nectar [13,16,21] and
nectaries [17,20,22] are believed to be costly as, in se-
veral cases, these responses are resource dependent [14,
20]. Resultantly, many plants have evolved phenoty-
pically plastic responses to augment these defensive
traits only when risk of herbivory increases [23,24]. As
nitrogen-fixing bacteria provide plants with additional
nitrogen, broad bean plants with rhizobia may have the
ability to produce additional defensive structures (e.g.,
EFNs) in response to leaf damage.
As defensive traits have frequently been shown to be
costly, we hypothesized that plants with nutritional sym-
bionts would produce a larger inducible response than
plants lacking nutritional symbionts. More specifically,
we predicted that broad bean plants, V. f a b a , harboring
the nitrogen-fixing bacteria, R. leguminosarum bv. viciae,
M. C. Summers et al. / Open Journal of Ecology 1 (2011) 57-62
Copyright © 2011 SciRes. OPEN ACCESS
would produce more EFNs in response to leaf damage,
than plants lacking these mutualists.
2.1. Experiment
To carry out this study broad bean seeds were spr-
outed in deionized water. Water was changed every 24
hours, for 5 days. After 5 days, sprouted beans (n = 60)
were used for the experiment. Thirty sprouts were each
inoculated with 1g of Royal Peat Inoculant; containing 2
× 108 viable cells of R. leguminosarum bv. viciae per
gram (Becker Underwood, Ames, Iowa 50010). We
chose to use a commercially available inoculant, con-
taining R. leguminosarum bv. viciae, so that others could
easily repeat our experimental protocol. Each seed was
dipped into deionized water and coated with inoculant.
The remainder of the 1g of inoculant was sprinkled on
the soil directly under the sprout. The remaining 30
sprouts were each dipped in water but not inoculated
prior to planting. Sprouted seeds were planted in Sta-
Green All-Purpose Potting Mix (NPK 0.14:0.11:0.08;
Spectrum Brands, A tlanta, GA 3 0328) in 1L pots (ITML
Horticultural Products, Brantford, ON Canada N3T 5M8).
Unsterilized soil was used for this experiment so as not
to kill any beneficial microbes in the soil, which could
alter the experimental results. Plants were grown under
greenhouse conditions (32˚C - 41˚C, 27% - 95% rh,
natural lighting) in a computer-generated random order
(JMP 5.1) [25].
Treatments were randomized using a random number
generator (JMP 5.1) [25]. Pots were then labeled with
the experimental treatments: RD (Rhizobium , leaf dam-
age; n = 15), RND (Rhizobium, no leaf damage; n = 15),
NRD (no Rhizobium, leaf damage; n = 15) and NRND
(no Rhizobium, no leaf damage; n = 15). Plants were wa-
tered every second day. Once plants reached a height of
approximately 10cm, they were staked to help the plant
grow upright. After the fourth week, when plants were
approximately 60 cm tall (mean = 56 ± 9 cm), initial pl-
ant traits were recorded and treatments were conduc-
We recorded the plant traits: plant height, number of
fully expanded leaves, number of partially expanded lea-
ves, and number of EFNs. While each broad bean leaf
pair generally has 0 or 2 EFNs on the associated stipules,
broad beans can alter the total number of EFNs on a pl-
ant by clustering the stipules, and hence the EFNs, on
the apical meristem prior to the leaves unfolding [20].
After recording these traits, leaf damage treatments were
administered to the appropriate plants. For replicates
with leaf damage, the outer one-third of each of the pl-
ants fully and partially ex panded leaf pairs were excised
using floral scissors. To ensure that compounds were not
transferred between plants, the scissors were cleaned
with an alcohol swab after excising tissue from each
After allowing the plants to grow for 1 additional we-
ek, plant traits were again recorded. A few plants, dis-
tributed across treatments, broke before final traits could
be assessed; these plants were not included in any ana-
lyses. We calculated the degree of change in all of the
traits by subtracting the initial trait values from the final
trait values. We also destructively sampled the plants to
assess: total numbers of root nodules, total root nodule
weight, shoot dry weight, and root dry weight (minus
nodules). Once to tal nodule numbers were recorded, no-
dules were excised from the root, placed in aluminum
weighing dishes, and placed in a drying oven (55˚C ±
2˚C) for 2 weeks. Roots and shoots were placed in sepa-
rate brown paper bags and dried similarly. Root nodule,
shoot, and root weights were determined using a micro-
balance (ExplorerPro, Ohaus Corporation, Pine Brook,
NJ 07058).
2.2. Statistical Analyses
For a robust statistical analysis we conducted similar
two-factor ANCOVAs, pre- and post-leaf damage treat-
ments. By conducting similar analyses we could confirm
that significant differences did not exist in the treatment
groups prior to leaf damage. Independent variables in
both analyses were: Rhizobium (no vs. yes), leaf damage
(no vs. yes), and the first-order interaction Rhizobium ×
leaf damage. Covariates in the pre-treatment model were:
number of partially expanded leaves and number of fully
expanded leaves. Covariates in the post-treatment model
were: change in th e number of partially expanded leaves
and change in the number of fully expanded leaves, over
1 week. The dependent variables were: the number of
EFNs per plant (pre-treatment model) or change in num-
ber of EFNs over 1 week (post-treatment model).
To more accurately quantify the effects of rhizobia on
EFN induction we conducted two multiple regression
analyses exclusively on the Rhizobium treatment group
(i.e., there were almost no root nodules on plants in the
“no Rhizobium” group). In the first regression, the in-
dependent variables were: leaf damage (no vs. yes),
change in the number of partially expanded leaves over
1 week, change in the number of fully expanded leaves
over 1 week, and total number of root nodules. In the
second regression, total weight of root nodules was in-
corporated as a variable, instead of root nodule number.
For both multiple regressions the dependent variable was
the change in number of EFNs over 1 week.
To better understand the effects of rhizobia on overall
plant growth, we conducted 2, two-way ANOVAs. Inde-
M. C. Summers et al. / Open Journal of Ecology 1 (2011) 57-62
Copyright © 2011 SciRes. OPEN ACCESS
pendent variables in the analyses were : Rhizobium (no vs.
yes), leaf damage (no vs. yes), and the first-order inter-
action Rhizobiu m × leaf damage. Dependent variables
were: shoot dry weight, and root dry weight (minus no-
All data was analyzed using JMP 5.1 [25]. Post-hoc
treatment effects were determined with Tukey’s Hon-es-
tly Significant Difference (HSD) test.
Prior to leaf damage, there was no difference in the
number of EFNs on inoculated vs. uninoculated plants.
There was also no effect of leaf damage, or any inter-
action between our main variables, showing that we did
not have an apriori differences in our treatment groups,
prior to leaf excision treatments (Table 1). There were
however, positive relatio nships between numbers of par-
tially expanded leaves and fully expanded leaves with
EFN numbers (Table 1). As EFNs are located on the leaf
stipules, a high correlation between leaf pairs and EFN
numbers is not unexpected.
Post-leaf damage, colonization with mutualistic nitro-
gen-fixing bacteria did alter EFN numbers in broad bean
plants. Overall, inoculated plants did not produce more
EFNs over 1 week compared to uninoculated plants (Ta-
ble 1). Plants also did not produce more EFNs in res-
ponse to leaf damage (Ta ble 1). There was, however, a
significant interaction between Rhizobium and leaf da-
mage (Table 1; Figure 1). Plants without nitrogen-
fixing bacteria produced approximately the same number
of EFNs whether or not they suffered leaf damage. Con-
trary to our hypothesis, however, plants with Rhizobi-
um produced fewer EFNs after experiencing leaf da-
mage, compared to inoculated plants without leaf da-
mage (Figure 1). Like in pre-treatment plants, there was
a significant relationship between numbers of partially
Table 1. Effects of Rhizobia and (or) leaf damage on the num-
bers of extrafloral nectaries, in broad bean, before and after
leaf excision.
Pre-treatment Post-treatment
Variable F P F P
Rhizobia (df = 1, 41) 0.010 0.92 0.0130.91
Leaf damage (df = 1, 41) 0.30 0.59 0.201 0.66
Rhizobia × Leaf damage (df = 1, 41) 1.56 0.22 5.60 0.023
*Partially developed leaf pairs
(df = 1, 41) 8.03 0.0071 7.080.011
*Fully developed leaf pairs (df = 1, 41) 7.96 0.0073 2.020.16
*Post-treatment analyses used the “change in partially developed leaf pairs”
over 7 days, following leaf damage treatments (see text for further detai ls ).
Figure 1. Extrafloral nectaries produced by inoculated vs.
uninoculated broad bean, Vicia faba, plants over 7 days when
given mechanical leaf damage. Rhizobia × leaf damage inter-
action; F1,41 = 5.41, P = 0.025. Data are presented as Least
Squared Means. Columns with different letters are significantly
different; Tukey’s HSD (P < 0.05).
expanded leaves and EFN numbers. Unlike the pre-
treatment analysis, however, the relationship dissipated
between numbers of fully expanded leaves and EFN
numbers (Table 1).
Looking more closely at the Rhizobium plants, there
was a trend towards th e total number of root nodu les, bu t
not total weight of nodules, influencing EFN production.
In the first regression analysis, total number of root
nodules was marginally non-sign ificant (F1,21 = 3.31, P =
0.083; Figure 2) in being inversely related to EFN pro-
duction after factoring out the effects of leaf damage
(F1,21 = 6.47 , P = 0.019), change in partially expanded
leaf pairs (F1,21 = 1.99, P = 0.17), and fu lly expanded leaf
pairs (F1,21 = 0.31, P = 0.59). The second regression
analysis showed that the total weight of root nodules was
not significantly related to EFN production (F1,21 = 1.94,
P = 0.18) even when accounting for leaf damage (F1,21 =
Figure 2. The effect of Rhizobium leguminosarum cv. viceae
colonization on EFN production in broad bean plants. There
was a trend towards reduced EFN induction responses as root
colonization rates increased (t1,21 = –1.82, P = 0.083).
M. C. Summers et al. / Open Journal of Ecology 1 (2011) 57-62
Copyright © 2011 SciRes. OPEN ACCESS
4.87, P = 0.039), chang e in par tially expanded leaf pairs
(F1,21 = 2.59 , P = 0.12), and fully expanded leaf pairs
(F1,21 = 0.51, P = 0.48).
In regards to overall plant growth, dry shoot biomass
was not significantly altered by rhizobia colonization
(mean ± 1se; no rhizobia, 1.13 ± 0.04; rhizobia, 1.13 ±
0.04: F1,44 = 0.0060, P = 0.94). There were lower shoot
weights in plants that experienced leaf damage (mean ±
1se; no damage, 1.19g ± 0.04; damage, 1.07g ± 0.04:
F1,44 = 3.98 , P = 0.052), as leaf tissu e had been removed
from these plants. There was no significant interaction
between rhizobia and leaf damage on dry shoot weights
(F1,44 = 0.0113, P = 0.92). Dry root weights were sig-
nificantly lower when colon ized b y rhizob ia (mean ± 1se;
no rhizobia, 0.57 ± 0.04; rhizobia, 0.47 ± 0.03: F1,44 =
4.42, P = 0.041), but there was no effect of leaf damage
on shoot weights (no damage, 0.55g ± 0.04; damage,
0.49g ± 0.04: F1,44 = 1.50 , P = 0.23), and there was no
interaction between these main variab les (F 1,44 = 0.0058 ,
P = 0.94).
Despite their ubiquity, nitrogen-fixing bacteria – host
plant mutualisms are exceedingly specific interactions
[26-29]. Vicia faba, for example, is one of the most
discriminating of the legume hosts towards different
strains of R. leguminosarum [5]. Adding additional
complexity, symbioses are frequently nested within each
other [30], thus changes in one mutualism may indirectly
alter the functioning of other mutualisms. Here we
showed that the presence of nitrogen-fixing bacteria
directly altered the induction of EFNs in broad bean
plants. The direction of this response, however, ran
counter to our hypothesis and prediction; plants with
rhizobia produced fewer EFNs when damaged.
Without rhizobia, plants did not produce more EFNs
when damaged, compared to those that were undamaged.
This might seem contrary to prior experiments, where
damaged plants produce more EFNs compared to unda-
maged plants [17,20,22]. It must be noted, however, that
EFN induction has also been shown to be resource
dependent [20]. It is likely that the plants in our experi-
ments, grown in soil with extremely low fertilizer levels
(NPK 0.14:0.11:0.08), did not have the resources to
produce additional EFNs when herbivory increased. Ex-
trafloral nectary [20] and food body induction [14] are
both influenced by nu trient availability.
Overall, plants with rhizobia did not produce more
EFNs than did plants without nitrogen fixing bacteria.
Plants experiencing leaf damage, however, actually had
decreased EFN numbers compared to undamaged plants.
Furthermore, there were indications that the number of
EFNs decreased as root nodule numbers increased. In
the rhizobia—plant mutualism, symbiotic bacteria con-
sume plant photosynthate in exchange for increased ni-
trogen availability [31,32]. A negative relationship bet-
ween EFN induction and total number of root nodules
indicates a possible tradeoff in resource allocation bet-
ween symbiont nutrition and host plant defense [33]. As
plants have only a finite photosynthetic capability, the
development of rhizobial colonies may come at the ex-
pense of EFN induction. Such tradeoffs may be common
between plants and their mutualistic partners, especially
under relatively nutrient-poor conditions [33].
So why did damaged plants with rhizobia produce
significantly fewer EFNs than undamaged plants with
rhizobia? We advance several, non-mutually exclusive
hypotheses to explain these enigmatic results. First, rhi-
zobia may have contributed to alternate forms of defense,
e.g., increased production of phenolics [3,4]. Upon leaf
damage, it is possible that broad bean plants increase
secondary compound production at the expense of other
forms of defense (e.g., EFN production). Second, rhi-
zobia may enable plants to produce additional EFNs, but
reduced leaf tissue resulting from leaf damage may have
prevented increased expression due to reduced photo-
synthetic capacities [34]. Third, as all plan ts were grown
in a common greenhouse environment, it is possible that
plants received volatile compounds from damaged cons-
pecifics [35,36]. These compounds may have induced
EFN formation in the undamaged rhizobial plants, as
EFN induction is resource dependent [20]. More resear-
ch needs to be conducted to determine the mechanism
underlying this induction, and lack thereof.
Plants may respond very differently when inoculated
with different rhizobia strains [5,6,28]. A more effective
symbiosis might promote EFN induction, even under
adverse circumstances (i.e., when herbivory is intense).
Conversely, ineffective symbioses may lead to decreased
or nearly non-existent nitrogen fixation , leading to a pos-
sible parasitic relationship between the two parties [37,
38]; but see [39] for an alternate viewpoint]. For ex-
ample, bacteria are not needed if nutrient rich soil is al-
ready providing the necessary components for plant
growth. Under this scenario, rhizobia do not carry out
nitrogen fixation, but form small, ineffective nodules [1].
While a very different mutualism, it is important to
note that arbuscular mycorrhizal fungi also significantly
decreased EFN numbers in V. faba, even when plant
growth increased [40]. Unlike nitrogen-fixing bacteria,
mycorrhizal fungi promote phosphorous uptake, in the
absence of other phosphorous sources, leading to aug-
mented plant growth [41,42]. As damaged broad bean
plants increase EFN numbers when sup plemented with a
balanced fertilizer [20], it is uncertain which element
promotes EFN induction.
While many questions remain to be answered about
M. C. Summers et al. / Open Journal of Ecology 1 (2011) 57-62
Copyright © 2011 SciRes. OPEN ACCESS
the nitrogen-fixing bacteria—host plant mutualism, it is
clear that rhizobia alter EFN induction responses in
damaged broad bean, V. faba, plants. Thus, belowground
mutualisms have the potential to alter aboveground
symbioses [43,44]. Corroborating other studies, nitro-
gen-fixing bacteria colonizing the roots of host plants
may span the c ontinu u m from mutuali stic to parasitic [4 5-
48], depending on current ecological conditions, resulting
in altered trophic functi oning.
Authors thank M. Heil and M. Tremblay for insightful discussions
on this topic. Laboratory assistance was provided by T. Brew. Funding
for this project was provided by Georgia Southern University.
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