Skip to main content
NCBI home page
Saudi Journal of Biological Sciences logoLink to Saudi Journal of Biological Sciences
. 2014 Jan 3;21(4):305–310. doi: 10.1016/j.sjbs.2013.12.005

Impact of bio-fertilizers and different levels of cadmium on the growth, biochemical contents and lipid peroxidation of Plantago ovata Forsk

Irfana Haneef 1,⁎, Shahla Faizan 1, Rubina Perveen 1, Saima Kausar 1
PMCID: PMC4150226  PMID: 25183940

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 application 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 concentration 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.

Keywords: Cadmium stress, Lipid peroxidation, AM fungi, Azotobacter, Plantago ovata

1. Introduction

Naturally, Cd is present in the environment at trace concentrations. 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 production, affects nutrient uptake and homeostasis, and is frequently 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 consequence of the inhibition of chlorophyll synthesis and photosynthesis (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 system (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 without it, solubilize insoluble soil phosphates and produces plant growth substances in the soil. They are in fact being promoted to harvest the naturally available, biological system of nutrient mobilization (Mishra et al., 2013; Venkatashwarlu, 2008). Phosphate absorbers (Mycorrhiza) is a symbiotic association between host plants and certain group of fungi at the root system, in which the fungal partner is benefited by obtaining its carbon requirements from the photosynthates of the host and the host in turn is benefited by obtaining the much needed nutrients especially phosphorus, calcium, copper, zinc etc., which are otherwise inaccessible to it, with the help of the fine absorbing hyphae of the fungus. Azotobacter is aerobic, free living, and heterotrophic in nature. The Azotobacter colonizing the roots not only remains on the root surface but also a sizable proportion of them penetrates into the root tissues and lives in harmony with the plants. They do not, however, produce any visible nodules or out growth on the root tissue.

Plantago ovata is a medicinal-valuable plant, its seeds and shell have a significant role in pharmacy as a laxative compound. In addition, recent researches have shown that P. ovata fiber plays an important role in declining blood cholesterol rate, lipid and sugar. It has valuable features including compatibility to dry and semi-dry climate condition, high production of effective materials and resistance to non-alive tensions especially dry tension (Agarwal and Pandey, 2004).

The aim of the study is to assess the level of cadmium toxicity in terms of various growth and physiological parameters and 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 sterilized with 0.01% mercuric chloride (HgCl2) solution followed by repeated washing with deionized water were sown in circular earthen pots filled with a mixture of farmyard manure in 3:1 ratio making a total of 4.0 kg soil pot−1. Appropriate amount that 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 soil alone and in combination with two biofertilizers and the following treatments were 0Cd (control), 50Cd; 100Cd; 0Cd + AMF; 50Cd + AMF; 100Cd + AMF; 0Cd + A; 50Cd + A; 100Cd + A; 0Cd + AMF + A; 50Cd + AMF + A; 100Cd + AMF + A. AM Fungi (Glomus sps.) and Azotobacter chroococcum obtained from the Division of Mycology, Indian Agricultural Research Institute, New Delhi were used as biofertilizers to study their potential in alleviation of cadmium stress and promotion of growth factors. AM fungi culture was applied as effective soil inoculants in the form of soil layering in previously autoclaved pots at the rate of 2 g per 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. chroococcum cells 5*108 g−1 (1:1 ratio) for 15 min, the seeds were dried for 30 min in shade at ambient temperature (approximately 20 °C) and sown in pots arranged in completely randomized block design. The application rate was same in combination treatments. The plants were collected from each replication for sampling on 45th day after sowing (DAS) for the estimation of growth, photosynthetic pigments, biochemical components and lipid peroxidation. The results were expressed in the average of three replications.

Concentrations of chlorophyll and carotenoid were determined 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. The proline content was quantified by using the acid-ninhydrin procedure of Bates et al. (1973). MDA (malondialdehyde) concentration was determined by a modified version of the method described by Cakmak and Horst (1991).

Data for various growth indices were subjected for analysis of variance using SPSS software version 10.0 Duncan’s multiple range test (DMRT) at the 0.05 level of probability was used to evaluate the difference among treatment means.

3. Results

Plants inoculated with AM fungi and Azotobacter (alone or in combination) showed better growth under cadmium stress. The maximum reduction in growth parameters (root length, shoot length, plant fresh and dry weight) was found at 100 mg Cd kg−1 of soil whereas the addition of AM fungi and Azotobacter showed stimulatory effect on the growth at all levels of treatment (Fig. 1A and B). The total biomass of P. 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 comparison to the control (Fig. 1C and D).

Figure 1.

Figure 1

Open in a new tab

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.

Almost all the biochemical parameters were significantly different among inoculated and non inoculated plants at every level of cadmium treatment than the control. Noteworthy decline in pigment concentration at higher dose of cadmium exposure was noted in total chlorophyll and carotenoid content (Fig. 2A and B). Chlorophyll and carotenoids were found to be highest in plants treated with combination of AM fungi and Azotobacter at 0 mg Cd kg−1. Plant treated with biofertilizers showed marked increase in total chlorophyll and carotenoid content at all levels of cadmium. The effect of AM fungi and Azotobacter treatments was not significant in case of carotenoid content and percent reduction was gradually decreased in inoculated plants.

Figure 2.

Figure 2

Open in a new tab

(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.

Nitrate reductase (NRA) and carbonic anhydrase activity were significantly influenced by Cd concentrations (Fig. 2C and D). Moreover, higher concentration (100 mg kg−1) of Cd showed maximum reduction as compare to the control. However, dual inoculation (AM fungi + Azotobacter) caused maximum 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 significant decrease in proline content as compare to the control. The minimum accumulation (23.09%) of proline content was found in case of combination treatment of AM fungi + Azotobacter at 100 mg kg−1 of Cd.

Malondialdehyde (MDA) content as an index of lipid peroxidation 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 in the level of MDA was found to be maximum in combination treatment of (AM fungi + Azotobacter) and minimum in case of Azotobacter treatment at 100 mg kg−1 of Cd Fig. 2F.

4. Discussion

Cd showed negative effect on the growth whereas addition of bio-fertilizer helps in alleviating the cadmium stress. The dual inoculation of AM fungi and Azotobacter was found to be superior in reduction of cadmium stress and promotion of growth 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 presence of 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 was significantly higher than control. A possible mechanism of this effect is the ability of AMF to bind heavy metals by fungal hyphae outside and inside the roots (Hua et al., 2010). Rhizobacteria (Azotobacter) are able to produce plant hormones or hormone-like substances which can promote plant growth (Abdul-Jaleel et al., 2007).

Biosynthesis of chlorophyll is impeded by Cd. Heavy metals such 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 harvesting and causes impairment of photosynthesis. Mycorrhiza, by improving uptake of Mg can support a higher chlorophyll concentration and subsequently leads to a higher production of photosynthate (Auge 2001; Haneef et al., 2013). The application of Azotobacter showed higher values for chlorophyll and carotenoid was observed during co-inoculation with AM fungi in normal and stress conditions. This may be the result of increased photosynthetic leaf area of plants even when under high levels of salt stress by PGPR (Plant growth-promoting rhizobacteria) inoculation compared to the control where leaf area reduced due to stress (Marcelis and Hooijdonk, 1999; Saghafi et al., 2013). These results corroborate with the findings 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 result corroborates 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 associated with its effect on hormonal level and/or an enhancement of root nitrate reductase (NR). Azospirillum inoculation under abiotic stress enhanced growth, NR and mineral uptake as compared to non-treated plants.

CA activity decreases with increasing cadmium concentration as Cd inhibits enzyme activities and causes alteration of 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 enhancement in CA activity as a result of soil-applied phosphorus in this study could be ascribed to the adequate availability of phosphorus at the site of its metabolism (Naeem et al., 2010). There is a significant correlation between the number of phosphate-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 fungi and Azotobacter helps the host plant during stress condition which results in the synthesis of less amount of proline than non inoculated plants. Proline is an important amino acid in plant under abiotic stress that prevents oxidation of cells from inside. 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. In the present study the application of biofertilizers significantly alleviated the growth inhibiting effects of cadmium stress, as it clearly showed the increased antioxidant enzyme activities with decreased MDA content. Similar results were found in case 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 at 100 mg kg−1 but declined in inoculated plants. The treatment with AM fungi and Azotobacter alone or combination promoted growth factor at all levels of cadmium as compared to the non inoculated control. This clearly showed the beneficial role of biofertilizers in alleviation of cadmium toxicity. Application of biofertilizers is cost efficient and easier to use, it is therefore advisable to enhance the tolerance to cadmium stress in isabgol.

Acknowledgment

The authors are thankful to the Chairman, Department of Botany, Aligarh Muslim University, India for providing necessary facilities.

Footnotes

Peer review under responsibility of King Saud University.

References

  1. Abad J.K.A., Khara J. 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. 2007;10:2413–2417. doi: 10.3923/pjbs.2007.2413.2417. [DOI] [PubMed] [Google Scholar]
  2. Abd El-Samad H.M., El-Komy H.M., Shaddad M.A.K., Hetta A.M. Effect of molybdenum on nitrogenase and nitrate reductase activities of wheat inoculated with Azospirillum brasilense grown under drought stress. Gen. Appl. Plant Physiol. 2005;31:43–54. [Google Scholar]
  3. Abdul-Jaleel C.A., Manivannan P., Sankar B., Kishorekumar A., Gopi R., Somasundaram R., Panneerselvam R. Pseudomonas fluorescens enhances biomass yield and ajmalicine production in Catharanthus roseus under water deficit stress. Colloids Surf. B: Biointer. 2007;60:7–11. doi: 10.1016/j.colsurfb.2007.05.012. [DOI] [PubMed] [Google Scholar]
  4. Agarwal S., Pandey V. Antioxidant enzyme responses to NaCl stress in Cassia angustifolia. Biol. Plant. 2004;48:555–560. [Google Scholar]
  5. Aisha M.H., Al-Rajhi Impact of biofertilizer Trichoderma harzianum Rifai and the biomarker changes in Eruca sativa L. plant grown in metal-polluted soils. World. Appl. Sci. J. 2013;22:171–180. [Google Scholar]
  6. Arumugam R., Rajasekaran S., Nagarajan S.M. 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. 2010;14:113–115. [Google Scholar]
  7. Auge R.M. Water relations, drought and VA mycorrhizal symbiosis. Mycorrhiza. 2001;11:3–42. [Google Scholar]
  8. Bates L.S., Waldren R.P., Teare I.D. Rapid determination of free proline of water stress studies. Plant Soil. 1973;39:205–207. [Google Scholar]
  9. Bhosale K.S., Shinde B.P. Influence of arbuscular mycorrhizal fungi on proline and chlorophyll content in Zingiber Officinale Rosc grown under water stress. Ind. J. Fund. Appl. Life Sci. 2011;1:2231–6345. [Google Scholar]
  10. Brahmaprakash G.P., Sahu P.K. Biofertilizers for sustainability. J. Ind. Inst. Sci. 2012;92:37–62. [Google Scholar]
  11. Cakmak I., Horst J. Effect of aluminium on lipid peroxidation, superoxide dismutase, catalase, and peroxidase activities in root tips of soybean (Glycine max) Physiol. Plant. 1991;83:463–468. [Google Scholar]
  12. Ditoppi L.S., Gabbriell R. Response to cadmium in higher plants. Environ. Exp. Bot. 1999;41:105–130. [Google Scholar]
  13. Dwivedi R.S., Randhawa N.S. Evolution of a rapid test for the hidden hunger of zinc in plants. Plant Soil. 1974;40:445–451. [Google Scholar]
  14. El-Komy M.H., Hamdia M.A., Abd El-Baki G.K. A. Nitrate reductase in wheat plants grown under water stress and inoculated with Azospirillum spp. Biologia Plantarum. 2003;46:281–287. [Google Scholar]
  15. Farahani A., Lebaschi H., Hussein M., Shiranirad A.H., Valadabadi A.R., Daneshian J. 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. 2008;2:125–131. [Google Scholar]
  16. Gianinazzi-Pearson V., Gianinazzi S. The physiology of Arbuscular-mycorrhizal root. Plant Soil. 1983;71:197–209. [Google Scholar]
  17. Giri B., Kapoor R., Mukerji K.G. Influence of Arbuscular mycorrhizal fungi and salinity on growth, biomass and mineral nutrition of Acacia auriculiformis. Biol. Fertil. Soils. 2003;38:170–175. [Google Scholar]
  18. Habibzadeh F., Sorooshzadeh A., Mohammad S.A., Sanavy M., Pirdashti H. Effect of seed inoculation and foliar application of biofertilizers on some biochemical and morphological characteristics of waterlogged-canola. J. Am. Sci. 2012;8:62–68. [Google Scholar]
  19. Haneef I., Faizan S., Perveen R., Kausar S. Role of arbuscular mycorrhizal fungi on growth and photosynthetic pigments in (Coriandrum sativum L.) grown under cadmium stress. World J. Agric. Sci. 2013;9:245–250. [Google Scholar]
  20. Hasan S.A., Barket A., Shamsul H., Aqil A. Cadmium-induced changes in the growth and carbonic anhydrase activity of chickpea. Turk. J. Biol. 2007;31:137–140. [Google Scholar]
  21. Hasan S.A., Fariduddin Q., Ali B., Hayat S., Ahmad A. Cadmium: toxicity and tolerance in plants. J. Environ. Biol. 2009;30:165–174. [PubMed] [Google Scholar]
  22. Hiscox J.D., Israelstem G.F. A method for extraction of chlorophyll from leaf tissue without maceration. Can. J. Bot. 1979;57:1332–1334. [Google Scholar]
  23. Hua J.F., Lin X.G., Bai J.F., Shao Y.F., Yin R., Jiang Q. Effects of arbuscular mycorrhizal fungi and earthworm on nematode communities and arsenic uptake by maize in arsenic-contaminated soils. Pedosphere. 2010;20:163–173. [Google Scholar]
  24. Jan S., Parween T., Mahmooduzzafar, Siddiqi T.O. Comparative effect of Azotobacter, mycorrhizal inoculum and nitrogen fertilizer on biochemical, growth and productivity aspects in Vicia Faba l. Am.-Eur. J. Sustainable Agric. 2009;3:684–693. [Google Scholar]
  25. Jaworski E.G. Nitrate reductase assay in intact plant tissues. Biochem. Biophys. Res. Commun. 1971;43:1274–1279. doi: 10.1016/s0006-291x(71)80010-4. [DOI] [PubMed] [Google Scholar]
  26. Kapoor R., Bhatnagar A.K. Attenuation of cadmium toxicity in mycorrhizal celery (Apium graveolens L.) World J. Microbiol. Biotech. 2007;23:1083–1089. [Google Scholar]
  27. Kucey R.M.N. Phosphate-solubilizing bacteria and fungi in various cultivated and virgin Alberta soils. Can. J. Soil Sci. 1983;63:671–678. [Google Scholar]
  28. Marcelis L.F., Hooijdonk H.V. Effect of salinity on growth, water use and nutrient use in radish (Raphanus sativus L.) Plant Soil. 1999;215:57–64. [Google Scholar]
  29. Mishra D.J., Singh R., Mishra U.K., Shahi S.K. Role of bio-fertilizer in organic agriculture: a review. Res. J. Recent Sci. 2013;2:39–41. [Google Scholar]
  30. Naeem M., Khan M.M.A., Moinuddin, Idrees M., Aftab T. Phosphorus ameliorates crop productivity, photosynthetic efficiency, nitrogen-fixation, activities of the enzymes and content of nutraceuticals of Lablab purpureus L. Scientia Horticulturae. 2010;126:205–214. [Google Scholar]
  31. Padmaja K., Prasad D.D.K., Prasad A.R.K. Inhibition of chlorophyll synthesis in Phaseolus vulgaris seedlings by cadmium acetate. Photosynthetica. 1990;24:399–405. [Google Scholar]
  32. Poschenrieder C., Gunse G., Barcelo J. Influence of cadmium on water relations, stomatal resistance and abscisic acid content in expanding bean leaves. Plant Physiol. 1989;90:1365–1371. doi: 10.1104/pp.90.4.1365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Prasad M.N.V. Cadmium toxicity and tolerance in vascular plants. Environ. Exp. Bot. 1995;35:525–545. [Google Scholar]
  34. Rajeshkumar S., Chandran N.M., Padanilly C.P., Lakew W., Thangavel S. Interaction between Glomus geosporum, Azotobacter chroococcum, and Bacillus coagulans and their influence on growth and nutrition of Melia azedarach L. Turk. J. Biol. 2009;33:109–114. [Google Scholar]
  35. Ramakrishnan K., Selvakumar G. Effect of biofertilizers on enhancement of growth and yield on Tomato (Lycopersicum esculentum Mill.) Int. J. Res. Bot. 2012;2:20–23. [Google Scholar]
  36. Ribaudo C.M., Paccusse A.N., Cura J.A., Frascina A.A. Azospirillum maize association: effects on dry matter yield and nitrate reductase activity. Agr. Trop. Subtrop. 1998;31:61–70. [Google Scholar]
  37. Saghafi K., Ahmadi J., Asgharzadeh A., bakhtiari S. The effect of microbial inoculants on physiological responses of two wheat cultivars under salt stress. Int. J. Adv. Biol. Biom. Res. 2013;1:421–431. [Google Scholar]
  38. Sandalio L.M., Dalurzo H.C., Gomez M., Romero-Puertas M.C., del Rio L.A. Cadmium-induced changes in the growth and oxidative metabolism of pea plants. J. Exp. Bot. 2001;52:2115–2126. doi: 10.1093/jexbot/52.364.2115. [DOI] [PubMed] [Google Scholar]
  39. Sarhan, Taha Z. Effect of biofertilizer and different levels of nitrogen (Urea) on growth, yield and quality of lettuce (Lactuca sativa L.) Ramadi cv. J. Agric. Sci. Tech. B. 2012;2:137–141. [Google Scholar]
  40. Tiwari A., Kumar P., Singh S., Ansari S.A. Carbonic anhydrase in relation to higher plants. Photosynthetica. 2005;43:1–11. [Google Scholar]
  41. Venkatashwarlu, B., 2008. Role of bio-fertilizers in organic farming Organic farming in rain fed agriculture Central institute for dry land agriculture Hyderabad, 85–95.

Articles from Saudi Journal of Biological Sciences are provided here courtesy of Elsevier

RESOURCES