Allelopatic effects of cyanobacteria extracts containing microcystins on Medicago sativa-Rhizobia symbiosis.

Abstract

The eutrophication of water leads to massive blooms of cyanobacteria potentially producers of highly toxic substances: cyanotoxins, especially microcystins (MC). The contamination of water used for irrigation by these toxins, can cause several adverse effects on plants and microorganisms. In this work, we report the phytotoxic effects of microcystins on the development of symbiosis between the leguminous plant Medicago sativa (Alfalfa) and rhizobia strains. The exposure of rhizobial strains to three different concentrations 0.01, 0.05 and 0.1 μg MC ml(-1) led to decrease on the bacteria growth. The strains of rhizobia Rh L1, Rh L2, Rh L3 and Rh L4 reduced their growth to, respectively, 20.85%, 20.80%, 33.19% and 25.65%. The chronic exposure of alfalfa seeds and seedlings to different MC concentrations affects the whole stages of plant development. The germination process has also been disrupted with an inhibition, which reaches 68.34% for a 22.24 μg MC ml(-1). Further, seedlings growth and photosynthetic process were also disrupted. The toxins reduced significantly the roots length and nodule formation and leads to an oxidative stress. Thus, the MCs contained in lake water and used for irrigation affect the development of symbiosis between M. sativa and Rhizobia.

Full text

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Allelopatic effects of cyanobacteria extracts containing microcystins on
Medicago sativa–Rhizobia symbiosis
Fatima El Khalloufia, Khalid Oufdoua, Majida Lahrounia, Issam El Ghazalia, Sanaa Saqranea,
Vitor Vasconcelosb,c,n, Brahim Oudraa
aLaboratory of Biology and Biotechnology of Microorganisms, Environmental Microbiology and Toxicology Unit, Cadi Ayyad University, Faculty of Sciences Semlalia, P.O. Box 2390,
Marrakech, Morocco
bDepartamento de Biologia, Faculdade de Ciˆ encias, Universidade do Porto, Rua do Campo Alegre, 4169-007 Porto, Portugal
cCIIMAR/CIMAR, Centro Interdisciplinar de Investigac -~ ao Marinha e Ambiental, Rua dos Bragas, 289, 4050-123 Porto, Portugal
a r t i c l e i n f o
Article history:
Received 23 March 2010
Received in revised form
17 September 2010
Accepted 4 October 2010
Available online 27 October 2010
Keywords:
Irrigation water
Cyanotoxins
Legume plants
Medicago sativa
Nodules
Rhizobia
Symbiosis
a b s t r a c t
The eutrophication of water leads to massive blooms of cyanobacteria potentially producers of highly
toxic substances: cyanotoxins, especially microcystins (MC). The contamination of water used for
irrigation by these toxins, can cause several adverse effects on plants and microorganisms. In this work,
we report the phytotoxic effects of microcystins on the development of symbiosis between the
leguminous plant Medicago sativa (Alfalfa) and rhizobia strains. The exposure of rhizobial strains to
three different concentrations 0.01, 0.05 and 0.1 mg MC ml?1led to decrease on the bacteria growth. The
strains of rhizobia Rh L1, Rh L2, Rh L3 and Rh L4 reduced their growth to, respectively, 20.85%, 20.80%,
33.19% and 25.65%. The chronic exposure of alfalfa seeds and seedlings to different MC concentrations
affects the whole stages of plant development. The germination process has also been disrupted with an
inhibition, which reaches 68.34% for a 22.24 mg MC ml?1. Further, seedlings growth and photosynthetic
process were also disrupted. The toxins reduced significantly the roots length and nodule formation and
leads to an oxidative stress. Thus, the MCs contained in lake water and used for irrigation affect the
development of symbiosis between M. sativa and Rhizobia.
& 2010 Elsevier Inc. All rights reserved.
1. Introduction
Eutrophication has become increasingly widespread in aquatic
ecosystems; this phenomenon leads to excessive blooms of cyano-
bacteria(Codd,1995).Severalspeciesofcyanobacteriacausingblooms
are known for their production of various types of toxins namely
hepatotoxins. Of about 30% of the potentially toxic cyanobacteria
species,representing40%ofallgloballyknowntoxicspecieshavebeen
documented in various aquatic ecosystems of Morocco (Oudra et al.,
2008).MicrocystisisthemostcommonbloomforminggenusinAlgeria
and Morocco (Douma et al., 2009). The most common and important
hepatotoxins produced are the microcystins (MC), which are usually
produced by Microcystis aeruginosa (Dittmann and Weigand, 2006).
They include more than 70 structural variants (Zurawell et al., 2005)
being MC-LR the most prevalent one (Dawson, 1998). MC have been
shown to be potent inhibitors of protein phosphatases 1A and 2A for
animals and higher plants (Hastie et al., 2005). Those proteins are
involved in several physiological and molecular processes (Sheen,
1993; Takeda et al., 1994).
Several studies have reported that submerged and emerged
aquatic plants can uptake an MC-LR (Pflugmacher et al., 1998,
2001; Yin et al., 2005). This toxin causes plant growth inhibition
and photosynthetic disorders (Pflugmacher, 2002). The exposure of
Lepidiumsativumto1mgMC-LR l?1inducedasignificantreductionof
root growth (Gehringer et al., 2003). In Lemna gibba, toxic Microcystis
extract cause changes in the peroxidase activity and phenolic
compounds as well as decrease in plant growth and chlorophyll
contents(Saqraneetal.,2007).Asforterrestrialplants,cropirrigation
by water containing cyanobacteria can lead to an exposure of aerial
partsofplantsto cyanobacteriaandtheirtoxins(Abe etal.,1996).The
absorption of cyanobacterial toxins, in sufficient concentrations, by
terrestrial plants may induce morphological (Ko?s et al., 1995;
McElhiney et al., 2001; Pflugmacher et al., 2006) and physiological
disturbances (M-Hamvas et al., 2003; Gehringer et al., 2003;
Chen et al., 2004).
These toxins may influence the germination of seeds as well as
the early stages of plants development (Chen et al., 2004; Saqrane
et al., 2008), the length of primary roots and the photosynthesis
process (Ko?s et al., 1995; Abe et al., 1996; McElhiney et al., 2001;
Pflugmacher et al., 2006). They can also induce leaf necrosis
Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/locate/ecoenv
Ecotoxicology and Environmental Safety
0147-6513/$-see front matter & 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.ecoenv.2010.10.006
nCorresponding author at: Departamento de Biologia, Faculdade de Ciˆ encias,
Universidade do Porto, Rua do Campo Alegre, 4169-007 Porto, Portugal.
Fax: +351 223390608.
E-mail address: vmvascon@fc.up.pt (V. Vasconcelos).
Ecotoxicology and Environmental Safety 74 (2011) 431–438

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(Babicaetal.,2006)andactonthemetabolismofplants(Smithand
Walker,1996).TheexposuretoanMChasinhibitedthegrowthand
development of both rice (Oryza sativa) and rape (Brassica napus)
seedlings. The MC also inhibits the germination rate of seeds and
seedling length in addition to an oxidative stress demonstrated by
the activities of peroxidase and superoxide dismutase enzymes
(Chenetal.,2004).ThetranslocationofMC-LRandLFthroughroots
to shoots for seedlings of 11 agricultural plants was also reported
(Peuthert et al., 2007).
Spray irrigation of lettuce plants (Lactuca sativa) with water
containing Microcystis leads to persistence of colonies and singles
cells of cyanobacterium on the plants leaves after irrigation
(Peuthert et al., 2007) (Codd et al., 1999). Moreover plants could
accumulate an MC in their leaves and roots at concentrations
ranging 0.07–1.2 mg MC g?1fresh weight (Codd et al., 1999)
(Mohamed and Al Shehri, 2009). This study suggested that
ground waters and plants should be continuously monitored in
order to protect the public health against exposure to such potent
hepatotoxins.
The uptake of cyanotoxins in seedlings of several agricultural
plants has a significant negative impact on the various stages of
plants development. This can cause economic losses by the
reduction in yield and productivity, in addition to the impact on
human and animal health, following the consumption of contami-
nated plants. The effects of cyanotoxins, on both aquatic and
terrestrial plants, have been studied. On the other hand, the
investigation of their potential toxic effects on legumes plants is
rare (McElhiney et al., 2001; Pflugmacher et al., 2006).
These plants such as beans, lentils, alfalfa are of great value,
because of their importance for human and animal nutrition and
theirrolewithsymbioticrhizobiainatmosphericnitrogenfixation.
Crop yields are greatly improved in nodulated legume plants;
the rhizobia legumes constitute natural fertilizers. Rhizobia are
bacteria Gram-negative; they have ability to associate with roots
plants and form a specialized organ called the nodule, located on
the roots or rarely on the stems (Zakhia and De Lajudie, 2001).
Symbiotic fixation of atmospheric nitrogen is the main pathway of
atmospheric nitrogen in the nitrogen cycle. The reaction is
catalyzed by nitrogenise enzyme it consists on the reduction of
nitrogen (N2) into ammonia (NH3) available form. Most of the
symbiotic N2in soil occurs in the symbiosis between rhizobia and
plant of the legume family.
McElhiney et al. (2001) have reported that the exposure of
PhaseolusvulgaristoanMC-LRhadnoeffectonseedlingsgrowth.In
the opposite, the development of root system decreased by 30%
comparedtoabeanplantgrowingwithouttoxins(McElhineyetal.,
2001).
Our work investigated the phytotoxic effects of cyanotoxins
(MC) produced by M. aeruginosa from Lalla Takerkoust lake
(Marrakech, Morocco), on the leguminous plant M. sativa and on
symbiotic bacteria (rhizobia). According to our knowledge, the
effects of cyanotoxins on rhizobial growth and their symbiotic
association with alfalfa were not previously reported.
2.Material and methods
2.1.
material
Detection, identification and quantification of an MC in cyanobacteria bloom
Thecyanobacterialbloommaterialwascollected,inSeptember2005,fromLalla
Takerkoust reservoir, with a 27 mm Nitex phytoplankton net. The sample was
freezed and stored at ?25 1C until an MC quantification by the HPLC-PDA analysis.
After de-freezing, the biomass was sonicated (3 Hz?10 min) and centrifuged
(4000 g?12 min). The supernatant was retained and used to expose the seeds
and seedlings.
The aqueous extract of natural cyanobacterial bloom was used, in this report, in
order to take off natural conditions during bloom event.
MC were detected, identified and quantified using a High Performance Liquid
Chromatography (HPLC) system as described in Oudra et al. (2002). In brief, the
detection of an MC was performed with a Merck Hitachi HPLC system with
photodiode array detection system (HPLC-PDA) composed of L-7100 pump, an
L-7200 auto-sampler, a D-7000 interface and an L-7450 photodiode array detector
set at 238 nm. The separation of MCs was achieved using an analytical LiChrospher
R 100, 5 mm ODS column (LiChroCART-250-4 cartridge system, Merck, Germany).
The mobile phase was composed of Milli-Q Plus water (Millipore) and acetonitrile,
both containing 0.05% of tri-fluoro-acetic acid and a flow of 1 mL min?1. A 28–70%
acetonitrile gradient during 30 min was used. The UV-spectrum for each separated
fraction was checked and the MC variants were identified by their characteristic
UV-spectrum(UVmaxabsorbance at238–240 nm). Values ofan MCreported in this
work represent the total amount of the variants found.
2.2. Effects of MC on the growth of rhizobia
2.2.1. Isolation and purification of rhizobial strains
Four strains of rhizobia (Rh L1, Rh L2, Rh L3 and Rh L4) were isolated from root
nodules of alfalfa plants collected from the Marrakech region. Nodules of alfalfa
plant were disinfected with sodium hypochlorite (41) and rinsed several times in
sterile physiological water. The nodule was crushed in a tube containing 1 mL of
sterile physiological water. The suspension was seeded on Petri dishes containing
Yeast Extract Mannitol (YEM) medium agar (Vincent, 1970) with Red Congo. After
incubationduring48 h at28 1C,colonies of rhizobia characterized bya sticky aspect
and off white color (without absorption of Congo red) were isolated and purified on
YEM medium. The strains were stored at ?25 1C in glycerol 25%.
2.2.2. Evaluation of the effect of different MC concentrations on strains of rhizobia
One milliliter of each rhizobial culture in YEM broth medium containing about
109CFU/ml served as inoculums and was added to flasks containing 100 ml of YEM
brothmedium.Thereafter,1 mlofanMCsolutionwasaddedtothemediuminorder
to achieve final concentrations of 0.01, 0.05 and 0.1 mg MC/ml. The control flask was
the YEM broth medium added with 1 ml of sterile distilled water.
The flasks were incubated at 28 1C, in darkness and under continuous agitation
(250 rpm).Therhizobiagrowthwasfollowedbycountingcoloniesformingunitsper
ml (CFU/ml) on YEM medium agar.
2.3. Effects of MC on seeds germination
To evaluate the effect of an MC on the germination, seeds of alfalfa (M. sativa)
weresterilizedwithsodiumhypochlorite121for5 min,followedbythreewashesin
sterile distilled water. Four parallel exposures of seeds were performed and
prepared on three replicates (20 seeds in each Petri dish). The control seeds were
exposed to sterile distilled water. The germination boxes were placed in the
incubator at 25 1C in the dark under sterile conditions. During the germination
process, 2 ml of the aqueous extract were added regularly to prevent dryness. The
rate of germination was determined.
2.4.Effects of MC on the seedlings of M. sativa
Seedlings were grown in sand and watered daily with distilled water. Exposure
of alfalfa began 10 days after sowing and lasted one month. Toxin concentration in
thewaterusedforwateringofseedlingswas0,2.22,11.12or22.24 mgMC ml?1.The
aqueous extract of toxins was supplemented with nutrient solution. At the end of
the experiment, several parameters were evaluated: crop yield, length of the stem
and of the root, number of nodules, chlorophyll fluorescence and the evaluation of
plants defense reaction through the quantification of phenolics content and
peroxidase activity, enzyme involved in the scavenge of the reactive oxygen species
generated in stress conditions.
2.5. Evaluation of M. sativa crop yield
In order to evaluate the effect of an MC on crop production, the plant biomass
was weighed and the results expressed in grams fresh weight per m2per day.
2.6. Effect of MC on M. sativa-rhizobia symbiosis (roots length and number of nodules)
After the exposure period, M. sativa seedlings were used to determine the effect
of MC on the roots nodules formation. Plants were rinsed with distilled water, and
then the length and the number of nodules were determined by visual inspection.
2.7.Chlorophyll fluorescence evaluation
The photosynthetic activity of plants, based on pigment content and fluores-
cence, is an important process, which provides energy for plants development.
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The Chl-fluorescence was determined on dark-adapted leaves. Chlorophyll
fluorescence was measured using a portable fluorometer Handy Plant Efficiency
Analyzer(PEA,HansatechInstrumentsLtd.).Leaffluorescencewasdeterminedafter
illuminationwithalightintensityof3500 mMm?2s?1withdurationflashoflightof
100 s. Thevalue of fluorescence isminimal (F0) when all PSII centers areopen (open
state) and increases with a maximum (Fm), when PSII centers are closed (closed
state).
The ratio (Fm–F0)/Fm, also known as Fv/Fm, was calculated from fluorescence
values F0 and Fm. The Fv/Fm ratio is one of the most common parameters used in
fluorescence. The fluorescence parameters were calculated automatically, using
Handy PEA v 1.3 software.
2.8.Extraction and analysis of phenolic compounds
Phenolic compounds are a family of organic molecules widely present in the
plant kingdom. They are products of secondary metabolism of plants and they
possess high antioxidant capacity.
Determination of phenolic content was performed according to the method
described by El Hadrami et al. (1997) with some modifications. Of about 250 mg
(fresh weight) of M. sativa roots and leaves from each treatment were extracted
three times with 100% methanol at 4 1C under continuous stirring. The homogenate
was centrifuged at 7000 g for 3 min and the supernatant was stored at ?25 1C prior
toanalysis.Thepelletwaskeptfordeterminationoftheperoxidaseactivity.Inorder
to estimate the total phenols concentration (milligram equivalent of catechin per
gramoffreshweight),theFolinCiocalteureagentwasused, andtheabsorbancewas
measured at 760 nm (El Hadrami et al., 1997).
2.9.Extraction and analysis of the peroxidase activity
The extraction was carried out from the pellet previously obtained. It was
conducted with Tris-maleate buffer (0.1 M, pH 6.5) containing Triton X-100
(0.1 g l?1). The peroxidase activity was assayed spectrophotometrically at
470 nm, using guaiacol as a substrate. Plant extract was added to 2 ml of reaction
mixture (solution of 0.1 M Tris-maleate buffer (pH 6.5), 25 mM of guaiacol and
25 mM of H2O2(10%)). The change of absorbance (DDO) at 470 nm was determined
during3 minofincubation,afterthattimethereactionremainedstable(ElHadrami
and Baaziz, 1995). The enzymatic activities were calculated in terms of the protein
content of the sample (Bradford, 1976) and were reported in DDO min?1mg?1of
proteins. Enzymatic assay of each sample was carried out in triplicate.
3. Statistical analysis
Means, standard deviations and standard errors for all experi-
mentalparameterswerecalculatedusingMicrosoftsExcel2007.One-
way analysis of variance (ANOVA) and the Tukey’s test (SPSS v. 17)
were carried out to determine whether treatments were signi-
ficantly different from control group (Po0.05). Pearson’s coefficient
(r2Pearson) was calculated to assess the existence of a correlation
between MC contents of the bloom extracts used in this experiment
and the parameters monitored.
4. Results
4.1. Profile of MC in the M. aeruginosa bloom
The result of HPLC-PDA obtained from the extract of the
M. aeruginosa , revealed a mixture of five variants of microcystins:
DMC-LR (4.20%); MC-(H4)-YR (4.32%); MC-LY (8.32%); MC-FR
(9.45%) and MC-LR (73.71%) (Fig. 1).The total MC content of the
bloom extract was 22.24 mg of MC ml?1.
4.2. Effects of MC on the rhizobial growth
The exposure of rhizobia to cyanobacteria aqueous extract
showed that it had a negative effect on the growth of rhizobial
strains.A significantdecrease inunits forming colonies(UFC ml?1)
compared to the control was registered (Fig. 2). After six days of
exposure to MC extract, there was a decrease of the rhizobia
numbers.ThisreductionwasmorepronouncedforthestrainRhL3.
It reached 31.32%, 32.87% and 33.19% for the respective
concentrations of 0.01, 0.05 and 0.1 mg MC ml?1. The other
strains Rh L1, Rh L2 and Rh L4 showed a comparable reduction
in the order of 20.85%, 20.80% and 25.65%, respectively, mainly for
the highest concentration of microcystins tested (0.1 mg ml?1).
Fig. 1. HPLC-PDA Chromatogram of Microcystis bloom extract; UV-spectrum for confirmation of the presence of all variants according to the standard sample.
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4.3. Evaluation of the germination of M. sativa
Exposure of seeds to MC had an important negative effect on
M. sativa seeds germination. Seeds of alfalfa showed a concentra-
tion-dependent reduction of germination (Fig. 3). The reduction
reached 68.34% for a concentration of 22.24 mg MC ml?1.
4.4. Effects of MC on the seedlings of M. sativa
4.4.1. Effects of cyanotoxins on seedlings growth and development
The exposure to cyanotoxins caused an inhibition of the growth
ofM.sativaseedlings.Thisinhibitionwasobservedfromthe6thday
of exposure and persisted during the assay period (30 days).
A significant difference was recorded between the exposed and
control groups (Fig. 4).
The estimation of crop yield by analyzing the plant productivity
from the cultivated area, showed a reduction of the biomass
depending on the concentrations of MC (Fig. 5). The overall
Fig.2. EvolutionofrhizobiastrainsgrowthRhL1(A),RhL2(B),RhL3(C)andRhL4(D),followingtheexposuretodifferentconcentrationofmicrocystins.Verticalbarsindicate
standard errors (7SE). Significant differences between control and treated strains are indicated by: *at Po0.05.
Fig. 3. Germination rate of M. sativa seeds after four days of exposure to various
concentrations of an MC (Different letters indicate statistically different values at
Po0.05; r2¼ ?0.990 at Po0.01.)
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reduction was 44.9%, 70.31% and 84.24% for batches of M. sativa
exposed, respectively, to 2.22, 11.12 and 22.24 mg MC ml?1.
4.5. Effects of MC on M. sativa-rhizobia symbiosis (roots length and
number of nodules)
The plant exposed to different concentrations of MC revealed a
significant reduction in both the length of roots and the nodule
number. The decrease of roots length is significant for seedlings
exposed to 11.12 and 22.24 mg MC ml?1(Fig. 6A). For the nodules,
the reduction is concentration-dependent (Fig. 6B). This result
shows the negative effect of aqueous extract of cyanobacteria on
roots development.
4.6. Effects of MC on chlorophyll fluorescence
The physiological state of the photosynthetic apparatus was
determined using the Fv/Fm ratio of the fluorescence measure-
ments.ValuesofanFv/Fmlowerthan0.8willrevealtheplantstress
condition, indicating a particular photo-inhibition phenomenon
(Johnson et al., 1993). Chronic exposure of M. sativa seedlings to
different concentrations of an MC led to photosynthetic disorders.
Significant reduction of quantum yield occurred for 11.12 and
22.24 mg MC ml?1(Fig. 7).
4.7. Induction of defense reaction and oxidative stress on seedlings of
M. sativa
Levels of phenolic compounds changed in plants exposed to
differentconcentrationsofanMC.Asignificantincreaseisdetected
in both roots and leaves of M. sativa (Fig. 8A). The increase is
important in roots exposed to 11.12 and 22.24 mg MC ml?1, and in
leavesexposedto22.24 mgMC ml?1.Besides,asignificantincrease
in protein content at both vegetative and underground parts is
recorded for the plants exposed to different concentrations of MCs
Fig. 4. Length of the stem of M. sativa seedlings exposed to different MC
concentrations during 30 days. Vertical bars indicate 7SE. Significant differences
between control and treated plants are indicated by * at Po0.05.
Fig. 5. Evaluation of M. sativa crop productivity after 30 days exposure to three
different concentrations of MC. Vertical bars indicate 7SE. (Significant differences
are indicated by different letters at Po0.05.)
Fig. 6. Roots length (A) and nodules number (B) of M. sativa seedlings exposed to
different concentration of an MC. (Different letters indicate statically different
values at Po0.05.)
Fig. 7. Evolution of ratio (Fv/Fm) in leaves of M. sativa seedlings after 30 days of
exposure to various concentrations of an MC. (Different letters showed significant
differences at Po0.05.)
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compared with controls (Fig. 8B). In addition, specific activities of
peroxidase enzyme have increased significantly in M. sativa leaves
exposed to 11.12 and 22.24 mg ml?1, whereas in roots the activity
increased significantly at 22.24 mg MC ml?1(Fig. 8C).
5. Discussion
The effect of an MC on bacteria has not been much studied
especiallyonthesymbioticbacteria(rhizobia)ofleguminousplants.
Heyduck-S¨ oller and Fischer (2000) showed that the extracellular
substances of a cyanobacterial strain Oscillatoria limnetica FLO1
inhibit Escherichia coli K12 growth. In addition, Islam et al. (2004)
have reported that V. cholerae O139 strain survives better in the
presenceofcyanobacteria:
Anabaena
Hapalosiphon sp. Similarly, Oufdou et al. (1998) reported that
V. cholerae non-O1 is stimulated in the presence of Synechocystis
sp., while this cyanobacterium inhibits E. coli. Valdor and Aboal
(2007)demonstratedthatbothcyanobacterialextractsandpureMC
had negative effects on the growth of microalgae and bacteria, and
the inhibitory effect was more persistent in pure MC than in the
extracts. According to our knowledge, no report was done on the
effect of cyanotoxins on rhizobia.
Thereductionofgerminationrateobtainedcouldbeexplainedby
the effect of cyanobacteria aqueous extract on the metabolic seeds
activities and can explain the lower field production resulting of
irrigation with contaminated water (Pflugmacher et al., 2006;
Crush et al., 2008). Chen et al. (2004) had previously reported
similarresults,usingrice(OryzasativaL.)andrape(BrassicanapusL.)
seeds being the highest inhibition effects measured on rape than on
rice. Significant differences in the germination percentage of rape
seeds were obtained after 10 days of exposure to 0.6 mg MC ml?1.
Exposureofalfalfaseedstocyanobacterialtoxins(MCandAnatoxin-a)
and cyanobacterial cell-free crude extracts (Pflugmacher et al.,
2006) also inhibited the germination process. These investigations
suggest that chronic plant exposure to toxic cyanobacteria
contained in irrigation water could have a net repercussion on
the life cycle of the plants and cause damages to plants crop yield.
Exposure of terrestrial plant seeds such as Lens esculenta,
Zea mays, Triticum durum and Pisum sativum, to cyanobacteria
aqueous extract containing an MC-LR reduced significantly the
germination rate (Saqrane et al., 2008). The effect of an MC on
seed germination rate was concentration-dependent and the
seeds sensitivity was different. L. esculenta seeds were the most
resistant ones, with the maximum germination rate of 74.67%.
P. sativum seems to be the most sensitive species among the four
tested by Saqrane et al. (2008).
Previous studies reported effects on the leaves and roots
development (Ko?s et al., 1995; Kurki-Helasmo and Meriluoto,
1998; McElhiney et al., 2001; Pflugmacher et al., 2006; Crush
et al., 2008). White mustard (Sinapis alba) seedlings exposed to
0.005 mg MC-RR ml?1showed reduction of their length and of the
lateral root formation, as well as an inhibition of protein
phosphatase 1 and 2A (Kurki-Helasmo and Meriluoto, 1998).
Growth of potato (Solanum tuberosum) cultures was completely
inhibited by an MC-LR at 0.5–5 mg kg?1, while growth of common
bean (Phaseolus vulgaris) plants was inhibited at 1.12 mg kg?1
MC-LR (McElhiney et al., 2001). Gehringer et al. (2003) observed a
significant decrease in leaf and root lengths of Lepidium sativum
seedlings caused by MC-extracts and pure MC-LR. Later, it was
reported that different spinach variants were all morphologically
affected after 6-weeks of exposure to cyanobacteria crude extract
containing 0.5 mg MC-LR ml?1(Pflugmacher et al., 2007).
Plants growth inhibition could lead to a decrease in crop yield as
recently confirmed by Saqrane et al. (2009). They showed a net
reduction in plants height, leaf number and root length of Triticum
durum, Z. mays, P. sativum and L. sculenta cultivars exposed to
cyanobacteriaextractcontaining0.5–4.2 mgequivalentMC-LR ml?1.
Effects of cyanotoxins on root growth and development was
recorded by several authors. Kurki-Helasmo and Meriluoto (1998)
have reported that at 0.005 mg MC-RR ml?1inhibited lateral root
formation in white mustard (S. alba). In addition, Gehringer et al.
(2003) observed a significant decrease in leaf and root lengths of
L. sativum seedlings caused by cyanobacteria MC-extract. A net
reduction inroot length was also observed by Saqrane et al. (2009).
According to Saqrane et al. (2008), this reduction of roots
development was showed by the histological modifications of
the primary root tissue of P. sativum seedlings. These cytological
sp.,
Nostoc
sp.and
Fig.8. Phenoliccontent(A),proteinscontent(B)andperoxidaseactivity(C)inroots
and leaves of M. sativa exposed to an MC extract through 30 days. (Different letters
for roots or leaves show significant difference at Po0.05.)
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modifications were evidenced by a delay in the organ root
differentiation and formation of vascular cylinder and inhibition
of lateral root primordial formation.
The obtained results confirm the negative effects of cyanobac-
teria toxins on the photosynthesis of terrestrial plants, reported in
many cases. Cyanobacterial extract treatment produced a concen-
tration and plant species-dependent decrease in the Fv/Fm ratio.
This indicates clearly the effect of MCs on the photosynthetic
activity of all exposed plants, which is, in part; due to a damage of
PSII reaction centers (Saqrane et al., 2009). On the other hand,
broccoli (Brassica oleraceae var. italica) and white mustard (S. alba)
seedlings irrigated with water containing MC showed no effect on
the concentrations and relative ratios of the photosynthetic
pigments chlorophyll a and b (J¨ arvenp¨ a¨ a et al., 2007). The values
of fluorescence in broccoli were all typical for healthy plants
(within 0.82–0.85) (Bj¨ orkman and Demmig, 1987). However,
other studies have reported that the exposure to cyanobacteria
toxinsinducedperturbationsonthechlorophyllcontent.S.tuberosum
showed a reduction of chlorophyll content at 0.005 mg kg?1MC-LR
(McElhineyetal.,2001).MoreoveraqueousextractofanMCcauseda
significant decrease in chlorophyll (a+b) content in Z. mays and
L.esculenta,following30daysexposureto2.1and4.2 mg ml?1ofMCs
(Saqrane et al., 2009).
The results then related the adverse effects of cyanobacterial
crudeextractonphotosynthesisactivity,whichprovidesnecessary
energy for enzyme processes for the reduction of atmospheric
nitrogen into ammonium (Dixon and Wheeler, 1986).
A positive correlation was detected between biochemical
parameters assayed, in the roots and in the leaves, and the
concentrations of an MC, demonstrating the presence of a con-
centration-dependent effect. The increase of the phenolic pool
showed the role of that secondary metabolite in plant protection
against biotic or abiotic stress. Plants exposed to MCs have there-
fore produced phenolic phytoalexins to deal with stressful condi-
tions induced by toxins. On the other hand, reactive forms of
oxygen are commonly produced during normal metabolism of
plants.Severalantioxidantsenzymescontributetotheirremovalin
order to maintain the vital functions of plants. In conditions of
stress, such forms are generated in large quantities and can cause
many cellular disturbances.
The determination of specific peroxidase activity reveals an
increase in plants exposed to an MC. This enzyme plays an
important role in maintaining the redox potential in plant cells
and helps to protect cell membranes from active oxidants
(Pflugmacher et al., 2006). Pflugmacher et al. (2007) observed an
increase in different activities of antioxidant enzymes depending
on the variants of Spinacia oleracea. Pflugmacher et al. (2006) also
detected a high activity of detoxifyingenzymesin M. sativaafter an
exposuretocyanotoxins.Thisexposurehasresultedinasignificant
lipid peroxidation as a consequence of oxidative stress.The
antioxidative system also consists of other enzymes, such as
superoxide dismutase (SOD), catalase (CAT) ascorbate peroxidase
and also non-enzymatic antioxidants, like reduction of glutathione
and vitamins (Asada, 1992; Polle, 2001). The induction of oxidative
stress due to exposure to cyanobacterial toxins was shown in
different plants, such as B. napus, O.sativa and M. sativa (Chen et al.,
2004; Pflugmacher et al., 2006). It has been reported that the
activity of peroxidase (POD) and superoxide dismutase (SOD), two
antioxidant enzymes, was changed in B. napus L. and O. sativa
L. seedlings after exposure to MC (Chen et al., 2004). Also, an
oxidative damage, such as lipid peroxidation, was detected after
theexposureofM.sativaseedlingstothetoxins(Pflugmacheretal.,
2006). The elevation of antioxidative enzymes in spinach, after six
weeks of exposure, clearly indicates that oxidative stress is
promoted in plants by exposure to a cyanobacterial crude
extract containing MC-LR (Pflugmacher et al., 2007).
Theseresultscanthereforeinfertheinvolvementofperoxidases
and phenolic compounds in plant defense process against stress
caused by cyanotoxins introduced via irrigation water, similarly
to that generated by abiotic agents (Pietsch et al., 2001). The
evolution of both parameters can be linked to the process of
detoxification also observed in M. sativa (Pflugmacher et al., 2007).
Themeasuringofthephenoliccompoundslevelsandtheactivityof
peroxidases are two parameters highly associated with an
oxidative stress. They prevent disruptions that result (lipid
peroxidation, DNA) and participate in defense mechanisms of
plants against stress.
The results obtained suppose that the MCs contained on
cyanobacterial crude extract are responsible of the adverse effects
mentioned in this study. However, other substances (lipopolysac-
charides) may also be implicated on those effects when plants are
irrigated with cyanobacterial contaminated water. On the other
hand,theuseofpurifiedMCcouldunderestimatethetoxicityofthe
natural bloom of cyanobacteria (Pereira et al., 2009).
6. Conclusion
An MC causes a decrease in rhizobial growth, depending on the
rhizobial strain and on the concentration of the cyanotoxins. The
contamination of water used for irrigation by an MC affects
physiological and metabolic parameters of the seedlings. The
cyanobacterial toxins induced the inhibition of germination of
alfalfa seeds and the reduction of seedling growth. Furthermore,
thecyanotoxinscausedreductionofrootslength,nodulesnumbers
and oxidative damages.
Acknowledgments
This study is financially supported by the International Foundation
for Sciences (its Project no F/2826-3F). Authors thank F.F. Del Campo
andY.Ouahidforhelpandtechnicalassistance.Thisworkisalsocarried
out within the framework of the cooperation Morocco–Portuguese
collaboration (convention of cooperation CNRST-Morocco/GRICES or
FCT-Portugal; Prof. Brahim Oudra/Prof. V.M. Vasconcelos).
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