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Communications in Soil Science and

Plant Analysis
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Ascophyllum nodosum Extract and Its

Organic Fractions Stimulate Rhizobium
Root Nodulation and Growth of Medicago
sativa (Alfalfa)
a b c
Wajahatullah Khan , Ravishankar Palanisamy , Alan T. Critchley ,
d e b
Donald L. Smith , Yousef Papadopoulos & Balakrishan Prithiviraj
a
Biochemistry Department, Genome Research Chair Unit, College of
Science, King Saud University, Riyadh, Saudi Arabia
b
Department of Environmental Sciences, Faculty of Agriculture,
Dalhousie University, Truro, Nova Scotia, Canada
c
Acadian Seaplants Limited, Dartmouth, Nova Scotia, Canada
d
Department of Plant Science, Macdonald Campus of McGill
University, Ste Anne de Bellevue, Québec, Canada
e
Agriculture and Agri-Food Canada, Atlantic Food and Horticulture
Research Centre, Nova Scotia Agricultural College, Truro, Nova
Scotia, Canada
Accepted author version posted online: 28 Jan 2013.Published
online: 22 Feb 2013.

To cite this article: Wajahatullah Khan , Ravishankar Palanisamy , Alan T. Critchley , Donald
L. Smith , Yousef Papadopoulos & Balakrishan Prithiviraj (2013) Ascophyllum nodosum Extract
and Its Organic Fractions Stimulate Rhizobium Root Nodulation and Growth of Medicago
sativa (Alfalfa), Communications in Soil Science and Plant Analysis, 44:5, 900-908, DOI:
10.1080/00103624.2012.744032

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 Communications in Soil Science and Plant Analysis, 44:900–908, 2013
Copyright © Taylor & Francis Group, LLC
ISSN: 0010-3624 print / 1532-2416 online
DOI: 10.1080/00103624.2012.744032

Ascophyllum nodosum Extract and Its Organic

Fractions Stimulate Rhizobium Root Nodulation and
Growth of Medicago sativa (Alfalfa)

WAJAHATULLAH KHAN,1 RAVISHANKAR PALANISAMY,2

ALAN T. CRITCHLEY,3 DONALD L. SMITH,4
YOUSEF PAPADOPOULOS,5 AND BALAKRISHAN
PRITHIVIRAJ2
Downloaded by [Dalhousie University] at 16:07 29 September 2013

1
Biochemistry Department, Genome Research Chair Unit, College of Science,
King Saud University, Riyadh, Saudi Arabia
2
Department of Environmental Sciences, Faculty of Agriculture, Dalhousie
University, Truro, Nova Scotia, Canada
3
Acadian Seaplants Limited, Dartmouth, Nova Scotia, Canada
4
Department of Plant Science, Macdonald Campus of McGill University, Ste
Anne de Bellevue, Québec, Canada
5
Agriculture and Agri-Food Canada, Atlantic Food and Horticulture Research
Centre, Nova Scotia Agricultural College, Truro, Nova Scotia, Canada

The effects of the seaweed Ascophyllum nodosum extracts (ANE) on nitrogen (N)–fixing
nodules and growth of alfalfa plants were studied under greenhouse conditions. The
treatment of alfalfa roots increased the number of total nodules per plant with ANE
(69%) and organic sub-fractions methanol (20%) and chloroform (35%) at 1 g L−1
concentration. The number of functional nodules was greater per plant in ANE (36%)
and its organic sub-fraction chloroform (105%). Maximum increase in shoot length
was observed in ANE-treated plants (42%) and chloroform-treated plants (42%). Root
length was longer in the chloroform fraction (15%), whereas the shoot dry-weight
accumulation was greater in plants treated with ANE (118%), methanol (85%), and
chloroform (85%) than the control. Root dry-weight accumulation increased in plants
treated with ANE (118%) and chloroform (69%) compared to the control. Further
studies are under way to identify the chemical components in ANE and organic
fractions.

Keywords Alfalfa, Ascophyllum nodosum, nitrogen fixation, plant–microbe

interaction, Sinorhizobium meliloti, symbiosis

Introduction
Alfalfa (Medicago sativa L., Fabaceae) is the most important perennial forage legume
crop and an essential component of many crop rotations. Alfalfa is harvested as hay and
silage, providing a good source of protein for livestock (Wang et al. 2008). It forms a
Received 3 April 2011; accepted 6 September 2011.
Address correspondence to Balakrishan Prithiviraj, Department of Environmental Sciences,
Faculty of Agriculture, Dalhousie University, P.O. Box 550, Truro, NS, B2N 5E3, Canada. E-mail:
bprithiviraj@dal.ca

900
 Seaweed Extract Stimulates Rhizobium Root Nodulation 901

symbiotic association with the bacterium Sinorhizobium meliloti, which is able to fix atmo-
spheric nitrogen (N) (Noel 2009). The successful symbiotic association is the result of
two mutually dependent signalling events: plant-to-bacteria and bacteria-to-plant. Plant-
to-bacteria signalling is mediated by host-specific isoflavonoids that are secreted in root
exudates that function as a chemo-attractant and inducer of nod genes of compatible
rhizobia. For instance, the alfalfa-produced flavone luteolin induces nod genes of S. meliloti
(Subramanian, Stacey, and Yu 2007). Bacteria-to-plant signalling is mediated by bacteria-
secreted lipo-chito-oligosaccharides, commonly referred to as nod factors (D’Haeze and
Holsters 2002). Nod factors are bioactive compounds at picomolar concentrations and may
elicit physiological changes in legume roots, including root hair deformation, induction
of early nodulin genes, calcium spiking, and formation of infection threads (Cullimore,
Ranjeva, and Bono 2001). Environmental, physiological, and chemical factors that alter
signalling events potentially affect the establishment of legume–rhizobia symbioses.
Ascophyllum nodosum (L.) Le Jolis is a brown alga that dominates the rocky intertidal
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zones of the North Atlantic shores of Canada and the estuarine rocky shores of northern
Europe (Ugarte, Craigie, and Critchley 2010). Several beneficial effects of seaweed extracts
on crop plants have been reported (Khan et al. 2009). Seaweeds and seaweed extracts have
been used as a soil conditioner and as foliar sprays to increase crop growth, yield, and
productivity (Khan et al. 2009; Craigie 2010). It has recently been shown that commercial
extracts of A. nodosum improved root and shoot growth in the model plant Arabidopsis
thaliana (Rayorath et al. 2007).
The aim of the present study was to investigate the effects of Ascophyllum nodosum
extracts (ANE) and its organic sub-fractions on root nodulation, growth, and development
of alfalfa plants.

Materials And Methods

Plant Material, Bacterial Strain, Chemicals, and Growth Conditions

Seeds of Medicago sativa L. cv. Caribou were obtained from the Department of Plant
and Animal Sciences, Nova Scotia Agricultural College (Truro, Nova Scotia, Canada).
A culture of Sinorhizobium meliloti strain Rm1021 was a generous gift from Dr. Trevor
Charles, Department of Biology, University of Waterloo (Waterloo, Ontario, Canada). The
culture was maintained on tryptone/yeast extract (TY) media (Vincent 1970) containing
200 µg mL−1 streptomycin. All chemicals were purchased from Sigma-Aldrich (Sigma
Aldrich, Milford, Mass.) unless otherwise indicated.

Plant Growth Conditions

Seeds of alfalfa were sterilized in 5% sodium hypochlorite for 20 min and then rinsed
several times with sterile distilled water. Seeds were planted in plastic pots (two seeds per
10-cm-diameter pot) containing sterile vermiculite. After seedling emergence, the plants
were thinned to one per pot; the retained plants were selected based on overall uniformity of
growth. A total of 10 plants were used per treatment (in a total of five treatments, discussed
later). The pots were placed on a greenhouse bench maintained at a 16:8 h photoperiod with
◦
a light intensity of 200–300 µmol m−2 s−1 , at a temperature of 25 ± 2 C and a relative
humidity of 75%. The plants were watered daily, alternating each day between 50 mL water
and 50 mL nitrogen-free Hoagland solution.
 902 W. Khan et al.

Preparation of A. nodosum Extract and Organic Sub-fraction

An aqueous solution of the commercial formulation (Acadian; Acadian Seaplants Limited,
Dartmouth, Nova Scotia, Canada) of A. nodosum extract (hereafter termed as ANE) was
prepared by dissolving 1 g of the extract powder in 20 mL of sterile distilled water by
steady stirring with a magnetic stirrer for 15 min. The solution was then filter sterilized
using a 0.22-µm SFCA (Surfactant-Free Cellulose Acetate) syringe filter (Corning Inc.,
◦
NY, USA) and stored in sterile glass centrifuge tubes at 4 C until further use. A methanol
organic fraction of ANE was prepared by extracting 10 g in 50 mL methanol for 15 min.
The extract was centrifuged at 4,000g for 10 min, and the supernatant was transferred to a
clean centrifuge tube. The solvent was then evaporated to dryness under a stream of N and
◦
stored in sterile glass centrifuge tubes at −20 C until further use.
The dried methanol organic fraction was resuspended in a minimal quantity of
methanol (100 µL g−1 equivalent) and the treatment volume was made up with sterile
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distilled water for use in experiments. For sub-fractionation, the methanol organic fraction
was prepared as mentioned earlier and the pellet was resuspended in 50 mL of water. This
aqueous solution was further sub-fractionated by sequential extraction with three volumes
(75 mL each) of chloroform and ethyl acetate.
The sub-fractions were dried under a stream of N and stored in sterile glass centrifuge
◦
tubes at −20 C until further use. The dried sub-fractions were resuspended in a minimal
quantity of methanol (100 µL g−1 equivalent) and the treatment volume was made up with
sterile distilled water for use in experiments.

Effects of ANE and Organic Sub-fraction on Root Nodulation of Alfalfa

Roots of 10-day-old alfalfa seedlings were treated with ANE and its organic sub-fractions
(i.e., methanol, chloroform, and ethyl acetate) as follows: A total of 10 pots (10 cm in diam-
eter) containing a single seedling were treated with 100 mL ANE or organic sub-fractions
at a final concentration of 1 g L−1 . The control plants received an equal volume of distilled
water instead of ANE or organic fractions of ANE. Two days after being treated, the plants
were inoculated with 10 mL per plant of S. meliloti 1021 following optical density mea-
surement using spectrophotometer at OD600 0.02 cell suspension (∼10−6 cell mL−1 ). The
pots were irrigated with N-free Hoagland’s solution (Hoagland and Arnon 1950). After
6 weeks, the plants were harvested and the total number of nodules, number of functional
nodules, root length, shoot length, root dry weight, and shoot dry weight were measured.
◦
The plant material was dried at 70 C for 2 days and then weighed to determine accumulated
dry weight. The functional nodules were differentiated based on the visible pink coloration,
associated with the functional leghemoglobin protein (Roponen 1970), as opposed to white
nonfunctional nodules. The number of pinkish color nodules was counted out of the total
number of nodules.

Experimental Design and Statistical Analysis

The experiment was carried out in a completely randomized design with 10 replicates
per treatment. Data on nodulation and growth related variables [root length, shoot length,
shoot dry weight (DW), and root dry weight] were analyzed with CoStat statistical software
(CoHort Software, Monterey, Calif.). When a significant treatment effect (P < 0.05) was
observed by analysis of variance (ANOVA), a least significant difference (LSD) test was
conducted to determine differences among means at P < 0.05. Same letters on the graph
 Seaweed Extract Stimulates Rhizobium Root Nodulation 903

indicate that they are not significantly (P < 0.05) different from each other. Percentage
increase over the control was calculated. Only differences between means at P < 0.05 were
considered to be significant differences.

Results
The treatment of alfalfa roots with ANE and its organic sub-fractions, 48 h preceding
inoculation with S. meliloti, affected the growth and development of the alfalfa plants
(Figures 1a–1e) as well as the number of total nodules and functional nodules per plant
after 6 weeks. The ANE and its organic sub-fractions (methanol and chloroform) at 1 g L−1
concentration significantly (P < 0.05) increased the number of nodules per plant compared
to the control. The percentage increase in number of total nodules and functional nodules
in ANE-treated plants was 20 and 35%, respectively (Figure 2). Similarly the number of
functional nodules was also greater (P < 0.05) per plant in ANE and its chloroform sub-
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fraction (Figure 2). However, the number of total nodules or number of functional nodules
per plant in ethyl acetate sub-fraction treatments did not differ from the control plants.
The greatest percentage increase in number of total nodules (69%) and functional nodules
(105%) per plant was observed in the chloroform sub-fraction (Figure 2).
The ANE and all its organic sub-fractions resulted in greater shoot length (P <
0.05) after 6 weeks of treatment than control plants, except for the plants treated with
ethyl acetate sub-fraction (Figures 1a–1e and 3a). The greatest percentage increase in shoot
length was observed in plants treated with ANE (42%) and chloroform (42%), whereas
methanol and ethyl acetate sub-fractions also caused 26% and 23% increases in shoot
length, respectively, over the control plants (Figure 3a). An increase in root length was also
observed in ANE and its chloroform sub-fraction; however, only the chloroform fraction
was significantly different (P < 0.05) from the control plants (Figure 3b).
The ANE and all its organic sub-fractions also affected the accumulated shoot and
root dry weight (DW) after 6 weeks of the treatment (Figures 4a and 4b). Compared with

Figure 1. Effects of 1 g L−1 ANE and its organic sub-fractions on root nodulation growth and
development of alfalfa plants 6 weeks after the treatment: (a) control, (b) ANE, (c) methanol extract,
(d) chloroform, and (e) ethyl acetate (color figure available online).
 904 W. Khan et al.
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Figure 2. Effects of 1 g L−1 ANE and its organic sub-fractions on number of total nodules per plant
and number of functional nodules, 6 weeks after the treatment. Similar letters at the top of the bars
are not significantly different (P < 0.05) by an ANOVA-protected LSD test. Each value is plotted as
the mean ± SE (n = 10) (color figure available online).

control plants, shoot DW accumulation was greater (P < 0.01) in plants treated with ANE
(118%), methanol (85%), and chloroform sub-fraction (85%). Shoot DW in ethyl acetate–
treated plants did not differ from the control plants (Figure 4a). The root DW was also
significantly increased in ANE- and chloroform-treated plants after 6 weeks. Root DW
accumulation was 118% and 69% greater in ANE- and chloroform-treated plants than the
control plants (Figure 4b). Treating the alfalfa roots with a lower concentration of the
chloroform fraction (0.5 g L−1 ) also resulted in an improved total number of nodules, as
well as the number of functional nodules per plant; this was also concurrent with greater
DW accumulations in shoot and roots (data not shown).

Discussion
Seaweed extracts are known to have positive effects on plant development, including an
enhanced growth rate, prolific root system, and improved biotic and abiotic stress toler-
ance in several crop plants (Somasegaran and Hoben 1994; Nelson and van Staden 1984;
Khan et al. 2009). This study showed that although the ANE and its organic sub-fractions
increased the number of functional nodules over the control plants, statistically only ANE
(36%) and the chloroform sub-fraction (105%) caused significant increases, suggesting
 Seaweed Extract Stimulates Rhizobium Root Nodulation 905
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Figure 3. Effects of 1 g L−1 ANE and its organic sub-fractions 6 weeks after the treatment on
(a) shoot length and (b) root length. Similar letters at the top of the bars are not significantly different
(P < 0.05) by an ANOVA-protected LSD test. Each value is plotted as the mean ± SE (n = 10).

that the two treatments may contain more bioactive compounds that increased the num-
ber of functional nodules. The plants treated with ANE and its chloroform sub-fraction
showed greater root and shoot lengths than the control plants and also caused greater DW
accumulation over the control plants (Figures 3a, 3b, 4a, and 4b).
The possible mechanism behind ANE and its organic sub-fractions enhancing the
number of functional nodules over the control plants could be one of several bioac-
tive compounds that have been suggested to be present in the seaweed extracts. It has
been reported that several cytokinins are found in brown alga extracts, including trans-
zeatin, trans-zeatin riboside, and their dihydro derivatives (Saravanan et al. 2003; Zhang
and Ervin 2008). Cytokinins are known to play a key role in nodule organogenesis,
and addition of external cytokinins to the roots can induce cortical cell division and
may also activate many nodulation-related proteins involved during early nodulation
process (Ferguson and Mathesius 2003; Mathesius et al. 2000; Murray et al. 2007).
Furthermore, it has been shown that the transformed hairy roots of Lotus japonicus with
reduced endogenous cytokinin levels resulted in fewer nodules than wild types, indicat-
ing a positive role of cytokinin in nodulation (Lohar et al. 2004). The cytokinins present
in seaweed formulations include trans-zeatin, trans-zeatin riboside, and dihydro deriva-
tives of these two forms (Stirk and van Staden 1997; Zhang and Ervin 2008). Liquid
 906 W. Khan et al.
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Figure 4. Effect sof 1 g L−1 ANE and its organic sub-fractions 6 weeks after the treatment on (a)
shoot dry weight and (b) root dry weight. The same letters at the top of the bars are not significantly
different (P < 0.05) by an ANOVA-protected LSD test. Each value is plotted as the mean ± SE
(n = 10).

chromatography/mass spectroscopy (LC/MS) analysis of 31 seaweed species representing

various groups revealed that cis-zeatin (cZ) and isopentenyl (iP) conjugates of cytokinins
are the predominant cytokinins (Stirk, Novak, and van Staden 2003). It is a reasonable
assumption that in the present study following treatment with ANE and its organic sub-
fractions, an increase in total nodule number and functional nodule number may be because
of the presence of cytokinin or cytokinin-like compounds or elicitation of endogenous syn-
thesis of cytokinin (Wally et al. 2012). The seaweed concentrates made from the kelps
(Ecklonia maxima, Macrocystis pyrifera) have also shown to exhibit cytokinin-like activity.
Several cytokinins were present in both concentrates, and among them, trans-zeatin-O-
glucoside was the main cytokinin present in these extract (Stirk et al. 2004). Alternatively,
ANE or its organic sub-fractions elicited the endogenous synthesis of cytokinin in planta
that resulted in improved nodulation and growth of alfalfa. In a recent work, Wally et al.
(2012) reported that Ascophyllum nodosum extract induced the synthesis of cytokinins in
the plant. However, further research is needed to confirm this concept.
The seaweed extracts have previously been shown to impart tolerance against several
abiotic stresses (Nelson and van Staden 1984; Zhang and Schmidt 1999, Khan et al. 2009).
Abiotic stress factors can also induce premature nodule senescence (Gogorcena et al.
1995; Gonzalez et al. 1998). Hence, any process that could delay nodule senescence will
 Seaweed Extract Stimulates Rhizobium Root Nodulation 907

eventually result in more nodules. It cannot be established whether the increase observed
in this study was due to the direct result of such effects. However, it has been reported
by Featonby-Smith (1984) that greater root cytokinin reported in seaweed extract–treated
plants. Gersani and Kende (1982) have reported that cytokinins play a role in nutrient mobi-
lization in the vegetative organs of the plant. A recent report by Khan et al. (2010) also
suggested that ANE probably contains compounds that may elicit endogenous cytokinin-
like activity. Hence, it is suggested that in the present study, the improved nodulation and
growth of alfalfa plants following seaweed extracts treatment were probably due to elicitors
of cytokinin synthesis in the extract.

Conclusions
It is concluded that bioactive compound(s) present in ANE extract and its organic sub-
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fractions have affected the legume–rhizobia signaling processes, resulting in more nodules,
more functional nodules, and overall improved growth of alfalfa plants. However, fur-
ther detailed studies are needed to identify the bioactive chemical components in the
organic fractions of ANE extract that were probably responsible for the induction of root
nodulation and growth of alfalfa plants.

Acknowledgments
Research in the BP laboratory is supported by grants from the Atlantic Canada
Opportunities Agency, Acadian Seaplants Limited, and Natural Science and Engineering
Research Council of Canada. The first author thanks the Deanship of Scientific Research,
King Saud University.

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