ORIGINAL ARTICLE Impact of bio-fertilizers and different levels of cadmium on the growth, biochemical contents and lipid peroxidation of Plantago ovata Forsk Irfana Haneef * , Shahla Faizan, Rubina Perveen, Saima Kausar Environmental Physiology Laboratory, Department of Botany, Aligarh Muslim University, Aligarh 202002, UP, India, Received 7 October 2013; revised 11 December 2013; accepted 18 December 2013 KEYWORDS Cadmium stress; Lipid peroxidation; AM fungi; Azotobacter; Plantago ovata Abstract Plantago ovata Forsk. (isabgol) is a valuable medicinal plant; its seeds and shell have a significant role in pharmacy as a laxative compound. Increasing soil contamination with cadmium (Cd) is one of the major concerns and is responsible for toxic effects in plants. This investigation was aimed to analyze the role of biofertilizers in alleviation of cadmium stress, given at the rate of 0, 50, and 100 mg kg 1 of soil. The plants of isabgol, were grown in pots with and without appli- cation of AM fungi and Azotobacter (alone and combination). Cadmium showed negative effect on growth and biochemical component whereas proline and MDA content increase with increasing cadmium concentration. Addition of bio-fertilizer showed better growth and higher pigment con- centration under cadmium stress as compared to the control. The dual inoculation of AM fungi and Azotobacter was found to be the best in reduction of cadmium stress and promotion of growth parameters. ª 2014 Production and hosting by Elsevier B.V. on behalf of King Saud University. 1. Introduction Naturally, Cd is present in the environment at trace concentra- tions. However, human activities, especially some industrial processes and the use of phosphate fertilizers in agriculture, have increased its concentration (Prasad, 1995). Cd is a non- essential element for plant growth, being highly toxic metallic pollutant of soil, inhibits root and shoot growth and yield pro- duction, affects nutrient uptake and homeostasis, and is fre- quently accumulated by agriculturally important crops and then enters the food chain with a significant potential to impair animal and human health (Ditoppi and Gabbriell, 1999). The reduction of biomass by Cd toxicity could be the direct conse- quence of the inhibition of chlorophyll synthesis and photo- synthesis (Padmaja et al., 1990). Excessive amount of Cd may cause decreased uptake of nutrient elements, inhibition of various enzyme activities, induction of oxidative stress including alterations in enzymes of the antioxidant defense sys- tem (Sandalio et al., 2001). Now-a-days, pollution and soil contamination are one of the major concerns and biofertilizers play a very significant role in improving soil fertility by fixing atmospheric nitrogen, both, in association with plant roots and * Corresponding author. Tel.: +91 05712406001. E-mail address: [email protected](I. Haneef). Peer review under responsibility of King Saud University. Production and hosting by Elsevier Saudi Journal of Biological Sciences (2014) xxx, xxx–xxx King Saud University Saudi Journal of Biological Sciences www.ksu.edu.sa www.sciencedirect.com 1319-562X ª 2014 Production and hosting by Elsevier B.V. on behalf of King Saud University. http://dx.doi.org/10.1016/j.sjbs.2013.12.005 Please cite this article in press as: Haneef, I. et al., Impact of bio-fertilizers and different levels of cadmium on the growth, biochemical contents and lipid peroxidation of Plantago ovata Forsk. Saudi Journal of Biological Sciences (2014), http://dx.doi.org/10.1016/j.sjbs.2013.12.005
Transcript
Saudi Journal of Biological Sciences (2014) xxx, xxx–xxx
Peer review under responsibility of King Saud University.
Production and hosting by Elsevier
1319-562X ª 2014 Production and hosting by Elsevier B.V. on behalf of King Saud University.
http://dx.doi.org/10.1016/j.sjbs.2013.12.005
Please cite this article in press as: Haneef, I. et al., Impact of bio-fertilizers and different levels of cadmium on the growth, biochemical cand lipid peroxidation of Plantago ovata Forsk. Saudi Journal of Biological Sciences (2014), http://dx.doi.org/10.1016/j.sjbs.2013.12.005
Environmental Physiology Laboratory, Department of Botany, Aligarh Muslim University, Aligarh 202002, UP, India,
Received 7 October 2013; revised 11 December 2013; accepted 18 December 2013
KEYWORDS
Cadmium stress;
Lipid peroxidation;
AM fungi;
Azotobacter;
Plantago ovata
Abstract Plantago ovata Forsk. (isabgol) is a valuable medicinal plant; its seeds and shell have a
significant role in pharmacy as a laxative compound. Increasing soil contamination with cadmium
(Cd) is one of the major concerns and is responsible for toxic effects in plants. This investigation
was aimed to analyze the role of biofertilizers in alleviation of cadmium stress, given at the rate
of 0, 50, and 100 mg kg�1 of soil. The plants of isabgol, were grown in pots with and without appli-
cation of AM fungi and Azotobacter (alone and combination). Cadmium showed negative effect on
growth and biochemical component whereas proline and MDA content increase with increasing
cadmium concentration. Addition of bio-fertilizer showed better growth and higher pigment con-
centration under cadmium stress as compared to the control. The dual inoculation of AM fungi
and Azotobacter was found to be the best in reduction of cadmium stress and promotion of growth
parameters.ª 2014 Production and hosting by Elsevier B.V. on behalf of King Saud University.
1. Introduction
Naturally, Cd is present in the environment at trace concentra-tions. However, human activities, especially some industrialprocesses and the use of phosphate fertilizers in agriculture,
have increased its concentration (Prasad, 1995). Cd is a non-essential element for plant growth, being highly toxic metallic
pollutant of soil, inhibits root and shoot growth and yield pro-
duction, affects nutrient uptake and homeostasis, and is fre-quently accumulated by agriculturally important crops andthen enters the food chain with a significant potential to impair
animal and human health (Ditoppi and Gabbriell, 1999). Thereduction of biomass by Cd toxicity could be the direct conse-quence of the inhibition of chlorophyll synthesis and photo-synthesis (Padmaja et al., 1990). Excessive amount of Cd
may cause decreased uptake of nutrient elements, inhibitionof various enzyme activities, induction of oxidative stressincluding alterations in enzymes of the antioxidant defense sys-
tem (Sandalio et al., 2001). Now-a-days, pollution and soilcontamination are one of the major concerns and biofertilizersplay a very significant role in improving soil fertility by fixing
atmospheric nitrogen, both, in association with plant roots and
without it, solubilize insoluble soil phosphates and producesplant growth substances in the soil. They are in fact being pro-moted to harvest the naturally available, biological system of
nutrient mobilization (Mishra et al., 2013; Venkatashwarlu,2008). Phosphate absorbers (Mycorrhiza) is a symbiotic asso-ciation between host plants and certain group of fungi at the
root system, in which the fungal partner is benefited by obtain-ing its carbon requirements from the photosynthates of thehost and the host in turn is benefited by obtaining the much
needed nutrients especially phosphorus, calcium, copper, zincetc., which are otherwise inaccessible to it, with the help ofthe fine absorbing hyphae of the fungus. Azotobacter is aero-bic, free living, and heterotrophic in nature. The Azotobacter
colonizing the roots not only remains on the root surface butalso a sizable proportion of them penetrates into the root tis-sues and lives in harmony with the plants. They do not, how-
ever, produce any visible nodules or out growth on the roottissue.
Plantago ovata is a medicinal-valuable plant, its seeds and
shell have a significant role in pharmacy as a laxative com-pound. In addition, recent researches have shown that P. ovatafiber plays an important role in declining blood cholesterol
rate, lipid and sugar. It has valuable features including com-patibility to dry and semi-dry climate condition, high produc-tion of effective materials and resistance to non-alive tensionsespecially dry tension (Agarwal and Pandey, 2004).
The aim of the study is to assess the level of cadmium tox-icity in terms of various growth and physiological parametersand the efficiency of biofertilizers (alone or combination) to
ameliorate cadmium tolerance in inoculated plants.
2. Methods and materials
Healthy and uniform sized P. ovata seeds, surface sterilizedwith 0.01% mercuric chloride (HgCl2) solution followed by re-peated washing with deionized water were sown in circular
earthen pots filled with a mixture of farmyard manure in 3:1ratio making a total of 4.0 kg soil pot�1. Appropriate amountthat is 0, 326.16, and 652.28mg CdCl2 was mixed thoroughly
with 4.0 kg soil to achieve 0, 50, and 100 mg Cd kg�1 of soilalone and in combination with two biofertilizers and the fol-lowing treatments were 0Cd (control), 50Cd; 100Cd;0Cd + AMF; 50Cd + AMF; 100Cd + AMF; 0Cd + A;
50Cd + A; 100Cd + A; 0Cd + AMF +A; 50Cd + AM-F + A; 100Cd + AMF+ A. AM Fungi (Glomus sps.) andAzotobacter chroococcum obtained from the Division of
Mycology, Indian Agricultural Research Institute, New Delhiwere used as biofertilizers to study their potential in alleviationof cadmium stress and promotion of growth factors. AM fungi
culture was applied as effective soil inoculants in the form ofsoil layering in previously autoclaved pots at the rate of 2 gper plant at the depth of 2–3 cm in soil one day before sowing.Azotobacter was used as seed inoculants by dipping the seeds
in sugary solution of bacterial powder containing A. chroococ-cum cells 5*108 g�1 (1:1 ratio) for 15 min, the seeds were driedfor 30 min in shade at ambient temperature (approximately
20 �C) and sown in pots arranged in completely randomizedblock design. The application rate was same in combinationtreatments. The plants were collected from each replication
for sampling on 45th day after sowing (DAS) for the estima-tion of growth, photosynthetic pigments, biochemical compo-
Please cite this article in press as: Haneef, I. et al., Impact of bio-fertilizerand lipid peroxidation of Plantago ovata Forsk. Saudi Journal of Biologic
nents and lipid peroxidation. The results were expressed in theaverage of three replications.
Concentrations of chlorophyll and carotenoid were deter-
mined using the method of Hiscox and Israelstem (1979).The activity of nitrate reductase and Carbonic Anhydrase(CA) was measured following the methods of Jaworski
(1971) and Dwivedi and Randhawa (1974), respectively. Theproline content was quantified by using the acid-ninhydrinprocedure of Bates et al. (1973). MDA (malondialdehyde) con-
centration was determined by a modified version of the meth-od described by Cakmak and Horst (1991).
Data for various growth indices were subjected for analysisof variance using SPSS software version 10.0 Duncan’s multi-
ple range test (DMRT) at the 0.05 level of probability was usedto evaluate the difference among treatment means.
3. Results
Plants inoculated with AM fungi and Azotobacter (alone or incombination) showed better growth under cadmium stress.
The maximum reduction in growth parameters (root length,shoot length, plant fresh and dry weight) was found at100 mg Cd kg�1 of soil whereas the addition of AM fungi
and Azotobacter showed stimulatory effect on the growth atall levels of treatment (Fig. 1A and B). The total biomass ofP. ovata was found to be the highest in case of dual inoculation
of AM fungi and Azotobacter followed by Azotobacter alone,both being significantly different from each other in compari-son to the control (Fig. 1C and D).
Almost all the biochemical parameters were significantly
different among inoculated and non inoculated plants at everylevel of cadmium treatment than the control. Noteworthy de-cline in pigment concentration at higher dose of cadmium
exposure was noted in total chlorophyll and carotenoid con-tent (Fig. 2A and B). Chlorophyll and carotenoids were foundto be highest in plants treated with combination of AM fungi
and Azotobacter at 0 mg Cd kg�1. Plant treated with biofertil-izers showed marked increase in total chlorophyll and caroten-oid content at all levels of cadmium. The effect of AM fungi
and Azotobacter treatments was not significant in case ofcarotenoid content and percent reduction was gradually de-creased in inoculated plants.
Nitrate reductase (NRA) and carbonic anhydrase activity
were significantly influenced by Cd concentrations (Fig. 2Cand D). Moreover, higher concentration (100 mg kg�1) of Cdshowed maximum reduction as compare to the control. How-
ever, dual inoculation (AM fungi + Azotobacter) caused max-imum increment as compared to the control.
The analysis of data Fig. 2E pertaining to proline showed a
significance increase with increasing cadmium concentrations.The increase was maximum (41.19%) at 100 mg kg�1 of Cd.However, AM fungi and Azotobacter treatment showed signif-icant decrease in proline content as compare to the control.
The minimum accumulation (23.09%) of proline content wasfound in case of combination treatment of AM fungi + Azoto-bacter at 100 mg kg�1 of Cd.
Malondialdehyde (MDA) content as an index of lipid per-oxidation in leaves of isabgol plants increased significantly(p < 0.05) with increase in Cd concentration. The rate of this
increase was maximum at 100 mg kg�1 of Cd. The decrease inthe level of MDA was found to be maximum in combination
s and different levels of cadmium on the growth, biochemical contentsal Sciences (2014), http://dx.doi.org/10.1016/j.sjbs.2013.12.005
Figure 1 Growth parameters: (A) root length, (B) shoot length, (C) plant fresh weight and (D) plant dry weight of isabgol plant under
different treatments. Different letters indicate significant difference between means by LSD at 5% level according to DMRT.
Impact of bio-fertilizers and different levels of cadmium 3
treatment of (AM fungi + Azotobacter) and minimum in caseof Azotobacter treatment at 100 mg kg�1 of Cd Fig. 2F.
4. Discussion
Cd showed negative effect on the growth whereas addition ofbio-fertilizer helps in alleviating the cadmium stress. The dual
inoculation of AM fungi and Azotobacter was found to besuperior in reduction of cadmium stress and promotion ofgrowth parameters. These results are in agreement with others
works by Aisha and Al-Rajhi 2013; Arumugam et al., 2010;Sarhan and Taha 2012; Rajeshkumar et al., 2009. The presenceof cadmium in the soil inhibits plant growth by altering the
plant metabolism even at low concentration (Hasan et al.,2009). The effect of inoculation of AM fungi and Azotobacter(alone or in combination) on vegetative growth of isabgol wassignificantly higher than control. A possible mechanism of this
effect is the ability of AMF to bind heavy metals by fungal hy-phae outside and inside the roots (Hua et al., 2010). Rhizobac-teria (Azotobacter) are able to produce plant hormones or
hormone-like substances which can promote plant growth(Abdul-Jaleel et al., 2007).
Please cite this article in press as: Haneef, I. et al., Impact of bio-fertilizersand lipid peroxidation of Plantago ovata Forsk. Saudi Journal of Biologic
Biosynthesis of chlorophyll is impeded by Cd. Heavy metalssuch as Cd2+, Pb2+ and Ni2+ replace the central Mg2+ of
chlorophyll in plants (Gianinazzi-Pearson and Gianinazzi,1983). Such substitution is expected to prevent light harvestingand causes impairment of photosynthesis. Mycorrhiza, by
improving uptake of Mg can support a higher chlorophyll con-centration and subsequently leads to a higher production ofphotosynthate (Auge 2001; Haneef et al., 2013). The applica-tion of Azotobacter showed higher values for chlorophyll and
carotenoid was observed during co-inoculation with AM fungiin normal and stress conditions. This may be the result of in-creased photosynthetic leaf area of plants even when under
high levels of salt stress by PGPR (Plant growth-promotingrhizobacteria) inoculation compared to the control where leafarea reduced due to stress (Marcelis and Hooijdonk, 1999;
Saghafi et al., 2013). These results corroborate with thefindings of Giri et al., 2003; Kapoor and Bhatnagar, 2007;Ramakrishnan and Selvakumar, 2012.
NRA content increased in inoculated plants at all levels of
cadmium as compared to the respective control. This resultcorroborates with the results of Abd El-Samad et al. (2005);El-Komy et al. (2003) and Jan et al. 2009 and Ribaudo et al.
(1998) who indicate that the role of Azospirillum could be
and different levels of cadmium on the growth, biochemical contentsal Sciences (2014), http://dx.doi.org/10.1016/j.sjbs.2013.12.005
Figure 2 (A) Total chlorophyll, (B) carotenoid, (C) nitrate reductase activity, (D) carbonic anhydrase, (E) proline and (F)
malondialdehyde of isabgol plant under different treatments. Different letters indicate significant difference between means by LSD at 5%
level according to DMRT.
4 I. Haneef et al.
associated with its effect on hormonal level and/or an enhance-ment of root nitrate reductase (NR). Azospirillum inoculation
under abiotic stress enhanced growth, NR and mineral uptakeas compared to non-treated plants.
CA activity decreases with increasing cadmium concentra-tion as Cd inhibits enzyme activities and causes alteration of
Please cite this article in press as: Haneef, I. et al., Impact of bio-fertilizerand lipid peroxidation of Plantago ovata Forsk. Saudi Journal of Biologic
their structure. Moreover, Cd causes the stomatal closure,therefore, decreasing CO2 concentration, which is the requisite
factor in the regulation of CA activity (Hasan et al., 2007;Poschenrieder et al., 1989; Tiwari et al., 2005). The enhance-ment in CA activity as a result of soil-applied phosphorus inthis study could be ascribed to the adequate availability of
s and different levels of cadmium on the growth, biochemical contentsal Sciences (2014), http://dx.doi.org/10.1016/j.sjbs.2013.12.005
Impact of bio-fertilizers and different levels of cadmium 5
phosphorus at the site of its metabolism (Naeem et al., 2010).There is a significant correlation between the number of phos-phate-solubilizing bacteria and fungi and the levels of total P
in the soil were observed (Brahmaprakash and Sahu, 2012;Kucey, 1983).
The reduction in proline content with the inoculation of
biofertilizers under cadmium stress was due to that AM fungiand Azotobacter helps the host plant during stress conditionwhich results in the synthesis of less amount of proline than
non inoculated plants. Proline is an important amino acid inplant under abiotic stress that prevents oxidation of cells frominside. Similar results were recorded by Farahani et al. (2008)in Coriandum sativum under drought stress and Bhosale and
Shinde (2011) in Zingiber officinale under water stress.Ditoppi and Gabbriell (1999) reported that the activity of
lipoxygenase increases as a consequent of an increase in lipid
peroxidation due to cadmium toxicity in treated plants. Inthe present study the application of biofertilizers significantlyalleviated the growth inhibiting effects of cadmium stress, as
it clearly showed the increased antioxidant enzyme activitieswith decreased MDA content. Similar results were found incase of canola by Habibzadeh et al. (2012) and in wheat by
Abad and Khara (2007).
5. Conclusion
Toxicity was found at all levels of Cd being maximum at100 mg kg�1 but declined in inoculated plants. The treatmentwith AM fungi and Azotobacter alone or combination pro-moted growth factor at all levels of cadmium as compared to
the non inoculated control. This clearly showed the beneficialrole of biofertilizers in alleviation of cadmium toxicity. Appli-cation of biofertilizers is cost efficient and easier to use, it is
therefore advisable to enhance the tolerance to cadmium stressin isabgol.
Acknowledgment
The authors are thankful to the Chairman, Department of
Botany, Aligarh Muslim University, India for providing neces-sary facilities.
References
Abad, J.K.A., Khara, J., 2007. Effect of cadmium toxicity on the level
of lipid peroxidation and antioxidative enzymes activity in wheat
plants colonized by Arbuscular mycorrhizal fungi. Pak. J. Biol. Sci.
10, 2413–2417.
Abd El-Samad, H.M., El-Komy, H.M., Shaddad, M.A.K., Hetta,
A.M., 2005. Effect of molybdenum on nitrogenase and nitrate
reductase activities of wheat inoculated with Azospirillum brasilense
grown under drought stress. Gen. Appl. Plant Physiol. 31, 43–54.
Abdul-Jaleel, C.A., Manivannan, P., Sankar, B., Kishorekumar, A.,
Gopi, R., Somasundaram, R., Panneerselvam, R., 2007. Pseudo-
monas fluorescens enhances biomass yield and ajmalicine produc-
tion in Catharanthus roseus under water deficit stress. Colloids Surf.
B: Biointer. 60, 7–11.
Agarwal, S., Pandey, V., 2004. Antioxidant enzyme responses to NaCl
stress in Cassia angustifolia. Biol. Plant 48, 555–560.
Aisha, M.H., Al-Rajhi, 2013. Impact of biofertilizer Trichoderma
harzianum Rifai and the biomarker changes in Eruca sativa L. plant
grown in metal-polluted soils. World. Appl. Sci. J. 22, 171–180.
Please cite this article in press as: Haneef, I. et al., Impact of bio-fertilizersand lipid peroxidation of Plantago ovata Forsk. Saudi Journal of Biologic
Arumugam, R., Rajasekaran, S., Nagarajan, S.M., 2010. Response of
Arbuscular mycorrhizal fungi and Rhizobium inoculation on
growth and chlorophyll content of Vigna unguiculata (L.) Walp
Var. Pusa 151. J. Appl. Sci. Environ. 14, 113–115.
Auge, R.M., 2001. Water relations, drought and VA mycorrhizal
symbiosis. Mycorrhiza 11, 3–42.
Bates, L.S., Waldren, R.P., Teare, I.D., 1973. Rapid determination of
free proline of water stress studies. Plant Soil 39, 205–207.
Bhosale, K.S., Shinde, B.P., 2011. Influence of arbuscular mycorrhizal
fungi on proline and chlorophyll content in Zingiber Officinale
Rosc grown under water stress. Ind. J. Fund. Appl. Life Sci. 1,
2231–6345.
Brahmaprakash, G.P., Sahu, P.K., 2012. Biofertilizers for sustainabil-
ity. J. Ind. Inst. Sci. 92, 37–62.
Cakmak, I., Horst, J., 1991. Effect of aluminium on lipid peroxidation,
superoxide dismutase, catalase, and peroxidase activities in root
tips of soybean (Glycine max). Physiol. Plant 83, 463–468.
Ditoppi, L.S., Gabbriell, R., 1999. Response to cadmium in higher
plants. Environ. Exp. Bot. 41, 105–130.
Dwivedi, R.S., Randhawa, N.S., 1974. Evolution of a rapid test for the
hidden hunger of zinc in plants. Plant Soil 40, 445–451.
El-Komy, M.H., Hamdia, M.A., Abd El-Baki, G.K., 2003. A. Nitrate
reductase in wheat plants grown under water stress and inoculated
with Azospirillum spp. Biologia Plantarum 46, 281–287.
Farahani, A., Lebaschi, H., Hussein, M., Shiranirad, A.H., Valada-
badi, A.R., Daneshian, J., 2008. Effects of Arbuscular mycorrhizal
fungi, different levels of phosphorus and drought stress on water
use efficiency, relative water content and proline accumulation rate
of Coriander (Coriandrum sativum L.). J. Med. Plants Res. 2, 125–
131.
Gianinazzi-Pearson, V., Gianinazzi, S., 1983. The physiology of
Prasad, M.N.V., 1995. Cadmium toxicity and tolerance in vascular
plants. Environ. Exp. Bot. 35, 525–545.
Rajeshkumar, S., Chandran, N.M., Padanilly, C.P., Lakew, W.,
Thangavel, S., 2009. Interaction between Glomus geosporum,
Azotobacter chroococcum, and Bacillus coagulans and their influ-
ence on growth and nutrition of Melia azedarach L.. Turk. J. Biol.
33, 109–114.
Please cite this article in press as: Haneef, I. et al., Impact of bio-fertilizerand lipid peroxidation of Plantago ovata Forsk. Saudi Journal of Biologic
Ramakrishnan, K., Selvakumar, G., 2012. Effect of biofertilizers on
enhancement of growth and yield on Tomato (Lycopersicum
