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Pseudomonas fluorescens enhances biomass yield and ajmalicine production in Catharanthus roseus under water deficit stress
Colloids and Surfaces B: Biointerfaces 60 (2007) 7–11
Pseudomonas fluorescens enhances biomass yield and ajmalicine production in Catharanthus roseus under water deficit stress C. Abdul Jaleel, P. Manivannan, B. Sankar, A. Kishorekumar, R. Gopi ∗ , R. Somasundaram ∗ , R. Panneerselvam Stress Physiology Lab, Department of Botany, Annamalai University, Annamalainagar 608 002, Tamilnadu, India Received 22 April 2007; received in revised form 15 May 2007; accepted 20 May 2007 Available online 25 May 2007
Abstract The effect of plant growth promoting rhizobacteria (PGPR) like Pseudomonas fluorescens on growth parameters and the production of ajmalicine were investigated in Catharanthus roseus under drought stress. The plants under pot culture were subjected to 10, 15 and 20 days interval drought (DID) stress and drought stress with Pseudomonas fluorescens at 1 mg l−1 and 1 mg l−1 Pseudomonas fluorescens alone from 30 days after planting (DAP) and regular irrigation was kept as control. The plants were uprooted on 41 DAS (10 DID), 46 DAS (15 DID) and 51 DAS (20 DID). Drought stress decreased the growth parameters and increased the ajmalicine content. But the treatment with Pseudomonas fluorescens enhanced the growth parameters under drought stress and partially ameliorated the drought induced growth inhibition by increasing the fresh and dry weights significantly. The ajmalicine content was again increased due to Pseudomonas fluorescens treatment to the drought stressed plants. From the results of this investigation, it can be concluded that, the seedling treatments of native PGPRs can be used as a good tool in the enhancement of biomass yield and alkaloid contents in medicinal plants, as it provides an eco-friendly approach and can be used as an agent in water deficit stress amelioration. © 2007 Elsevier B.V. All rights reserved. Keywords: Catharanthus roseus; Ajmalicine; Seed priming; Plant growth promoting rhizobacteria (PGPR); Pseudomonas fluorescens; Water deficit stress
1. Introduction Through out the world, the ground water level is inadequate for the cultivation of crops and this water limited condition is threatening problem. A water stress may conceivably arise either from an insufficient or from an excessive water activity in the plant’s environment. In the case of terrestrial plants in nature, the former occurs as a result of a water deficit or drought and therefore is called a water deficit stress (shortened to water stress) or drought stress [1]. The environmental stresses such as drought, Abbreviations: DAP, days after planting; DID, days interval drought; PGPR, plant growth promoting rhizobacteria; MIAs, monoterpenoid indole alkaloids; VCR, vincristine; VLB, vinblastine ∗ Corresponding authors at: Department of Botany, Annamalai University, Annamalainagar 608 002, Tamilnadu, India. Tel.: +91 4144 238248x354; fax: +91 4144 222265. E-mail addresses: [email protected] (C.A. Jaleel), [email protected] (R. Gopi), kalaisomu [email protected] (R. Somasundaram). 0927-7765/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2007.05.012
temperature, salinity, air pollution, heavy metals, pesticides and soil pH are major limiting factors in crop production because, they affects almost all plant functions [2]. Although the general effects of drought on plant growth are fairly well known, the primary effects of water deficit at the biochemical and molecular levels are not well understood [3]. Water stress tolerance is seen in all plant species but its extent varies from species to species. Improving the efficiency of water use in agriculture is associated with increasing the fraction of the available water resources that is transpired, because of the unavoidable association between yield and water use [4]. For the last few decades, several scales of physiological works have been conducted under drought stress in crop plants, but it is not so with respect to medicinal plants [5,3,6]. Numerous microorganisms live in the portion of soil modified or influenced by plant roots so called ‘rhizosphere’ [7]. Among these microorganisms, some have positive effects on plant growth promotion constituting the plant growth promoting rhizobacteria (PGPR) such as Azospirillum, Azotobacter,
8
C.A. Jaleel et al. / Colloids and Surfaces B: Biointerfaces 60 (2007) 7–11
Pseudomonas fluorescens, several gram positive Bacillus sp. [7]. The diazotrophic rhizobiocoenosis is an important biological process that plays a major role in satisfying the nutritional requirements of the commercial medicinal plants. Studies on the diazotrophic population in the rhizosphere region and testing the suitability of the isolated diazotroph as seed inoculant will be highly useful in improving the productivity of commercially important medicinal plants. Diazotrophs secrete plant growth hormones such as auxins, gibberellins and cytokinins [8]. Lot of investigations were already covered the importance of microbial association like arbuscular mycorrhizal symbiosis in drought stressed plants [9,10]. But little attraction is drawn towards PGPR mediated drought stress amelioration in medicinal plants. The strong and rapidly stimulating effect of fungal elicitor on plant secondary metabolism in medicinal plants attracts considerable attentions and research efforts [11]. The reasons responsible for the diverse stimulating effects of fungal elicitors are complicated and could be related to the interactions between fungal elicitors and plant cells, elicitor signal transduction, and plant defense responses [12]. In plants certain secondary metabolite pathways are induced by infection with microorganisms. It was reported that, arbuscular mycorrhizal symbiosis maintained more normal water relations in plants [13]. Catharanthus roseus (L.) G. Don. (Madagascar periwinkle) is a perennial tropical plant belonging to the family Apocynaceae that produces more than 100 monoterpenoid indole alkaloids (MIAs) including two commercially important cytotoxic dimeric alkaloids used in cancer chemotherapy [14]. Periwinkle, native to Madagascar is now found in many tropical and sub-tropical regions of the world. This plant contains anti-cancer alkaloids, vincristine (VCR) and vinblastine (VLB) and antihypertension alkaloid, ajmalicine. In medicinal plants, the content of the economically important metabolite is more important than the yield of the plant part containing the metabolite, as it determines the cost of its extraction [15]. The cell, tissue cultures and biotechnological aspects of this plant are being extensively investigated to increase the yield of the alkaloids [16]. In this context, the cultivation of periwinkle is becoming popular among the farmers. The major problem in the cultivation of periwinkle is environmental stresses such as salinity, water deficits and which created poor yield and establishment at field level. Previous works revealed the influences of triadimefon on the antioxidant metabolism and ajmalicine production [17], paclobutrazol mediated growth regulation [18], salinity problems [19] and salt stress protection by paclobutrazol [20] in C. roseus. Comparatively a little work has been reported on water stress problems and methods to overcome drought stress injuries in this plant [21]. The role of PGPR in this medicinal plant under water deficit stress had attracted little attention. In this experiment, an attempt is made to study the ability of PGPR, Pseudomonas fluorescens in drought stress amelioration, through its effect on growth and ajmalicine production in C. roseus plant under soil water deficits.
2. Materials and methods 2.1. Plant cultivation, drought stress induction and Pseudomonas fluorescens treatments The seeds of Catharanthus roseus (L.) G. Don. (Family: Apocynaceae) were collected from the Department of Horticulture, Faculty of Agriculture, Annamalai University, Tamil Nadu, India. The Pseudomonas fluorescens was obtained from Krishi Care Bioinputs, Chennai, India. The plants under pot culture were subjected to 10, 15 and 20 days interval drought (DID) stress and drought stress with Pseudomonas fluorescens at 1 mg l−1 and 1 mg l−1 Pseudomonas fluorescens alone from 30 days after sowing (DAS) and regular irrigation was kept as control. The pots were covered with a rain out shelter, made up of plastic sheets, whenever rainfall was anticipated and immediately after rain, rain out shelter was pulled back so that, pots received maximum sunlight. Further, the pots were regularly covered with rain out shelter during nighttime. Using this system the pots were protected from rainfall and any external moisture entry. The plants were uprooted on 41 DAS (10 DID), 46 DAS (15 DID) and 51 DAS (20 DID) for analyzing growth parameters and ajmalicine content. 2.2. Analysis of morphological parameters Morphological parameters like root length, plant height and number of leaves per plant were measured in the samples on 40, 45 and 50 DAP. Fresh and dry weights were taken from the samples by using an electronic weighing device (Model-Citizen XK3190-A7M). 2.3. Ajmalicine extraction and quantification Ajmalicine extraction from the roots was carried out by following the standard extraction method [22]. Identification and quantification of ajmalicine was done by preparative Thin Layer Chromatography using silica gel G (Merck) in chloroform:methanol (98:2, v/v) [23] by comparison of Rf values with authentic ajmalicine standard (Himedia, Mumbai). Ajmalicine was spotted with Dragendorff’s reagent [24]. 2.4. Statistics Statistical analysis was performed using one way analysis of variance (ANOVA) followed by Duncan’s Multiple Range Test (DMRT). The values are mean ± S.D. for seven samples in each group. P-values ≤ 0.05 were considered as significant. 3. Results 3.1. Effect of Pseudomonas fluorescens treatments on plant height of C. roseus under drought There was significant variation (p ≤ 0.05) in plant height of C. roseus seedlings treated with Pseudomonas fluorescens
C.A. Jaleel et al. / Colloids and Surfaces B: Biointerfaces 60 (2007) 7–11
Fig. 1. Effects of drought, Pseudomonas fluorescens and their combination on plant hight of Catharanthus roseus plants. Values are given as mean ± S.D. of seven samples in each group. Bar values are not sharing a common superscript (a, b, c, d) differ significantly at p ≤ 0.05 (DMRT).
when compared with drought stressed and well watered controls (Fig. 1). Pseudomonas fluorescens treated plants recorded the highest plant height at all sampling periods in C. roseus (41, 46 and 51 DAP). Individual inoculation of Pseudomonas fluorescens increased plant height of C. roseus. 3.2. Effect of Pseudomonas fluorescens treatments on root length of C. roseus under drought The root length of C. roseus seedlings varied in stress and Pseudomonas fluorescens treatments when compared to wellwatered controls. The maximum root length was recorded in Pseudomonas fluorescens treated plants (Fig. 2).
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Fig. 3. Effects of drought, Pseudomonas fluorescens and their combination on number of leaves of Catharanthus roseus plants. Values are given as mean ± S.D. of seven samples in each group. Bar values are not sharing a common superscript (a, b, c, d) differ significantly at p ≤ 0.05 (DMRT).
3.4. Effect of Pseudomonas fluorescens treatments fresh and dry weights of C. roseus under drought Significant variation (p ≤ 0.05) in whole plant fresh and dry weights of C. roseus seedlings treated with Pseudomonas fluorescens when compared with drought stressed and well-watered controls. Pseudomonas fluorescens treated plants showed the highest fresh and dry weights at all sampling periods in C. roseus (Table 1). 3.5. Effect of Pseudomonas fluorescens treatments on ajmalicine content of C. roseus under drought
The number of leaves was recorded more in the Pseudomonas fluorescens treated C. roseus followed by drought stress in combination with Pseudomonas fluorescens. The low number of leaves was recorded in the water stressed C. roseus (Fig. 3).
During the early stages of plant growth the alkaloid ajmalicine content was less in C. roseus. The lowest content was recorded in roots of control on 40 DAP. But the content increased with drought and treatment with Pseudomonas fluorescens. Drought stressed C. roseus plants showed a significant enhancement in ajmalicine content. The highest content was recorded in the roots of Pseudomonas fluorescens treatments (Fig. 4).
Fig. 2. Effects of drought, Pseudomonas fluorescens and their combination on root length of Catharanthus roseus plants. Values are given as mean ± S.D. of seven samples in each group. Bar values are not sharing a common superscript (a, b, c, d) differ significantly at p ≤ 0.05 (DMRT).
Fig. 4. Effects of drought, Pseudomonas fluorescens and their combination on ajmalicine content of Catharanthus roseus plants. Values are given as mean ± S.D. of seven samples in each group. Bar values are not sharing a common superscript (a, b, c, d) differ significantly at p ≤ 0.05 (DMRT).
3.3. Effect of Pseudomonas fluorescens treatments on number of leaves of C. roseus under drought
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Table 1 Effects of drought, Pseudomonas fluorescens and their combination on fresh and dry weights of Catharanthus roseus plants Days interval drought (DID)
Control
Drought
Drought + P. fluorescens
P. fluorescens
Whole plant fresh weight (g/plant) 10 15 20
10.326 ± 0.333a 14.415 ± 0.529a 17.412 ± 1.131a
8.412 ± 0.300b 11.372 ± 0.496b 14.561 ± 0.696b
11.022 ± 0.355c 14.516 ± 0.568a 18.945 ± 1.229c
12.063 ± 0.292d 16.412 ± 0.531d 19.184 ± 0.747d
1.787 ± 0.068a 1.941 ± 0.076a 2.034 ± 0.123a
1.081 ± 0.035b 1.469 ± 0.064b 1.932 ± 0.095b
1.809 ± 0.058a 1.944 ± 0.073a 2.200 ± 0.131c
1.993 ± 0.037d 2.002 ± 0.064d 2.591 ± 0.130d
Whole plant dry weight (g/plant) 10 15 20
Values are given as mean ± S.D. of seven samples in each group. Values are not sharing a common superscript (a, b, c, d) differ significantly at p ≤ 0.05 (DMRT).
4. Discussion The treatment with PGPR like Pseudomonas fluorescens increased the plant height, root length, number of leaves and ajmalicine content of C. roseus when compared to well-watered control and drought stressed plants. The occurrence of Azospirillum, Pseudomonas in and around the root system of cereals, vegetables and the beneficial effect upon inoculation has been well established. In the present study, the increased growth parameters in C. roseus due to treatment of PGPR might be due to the production of growth hormones (IAA, gibberellins, auxins), by the bacteria [25]. It is worth noting that the increase in growth parameters observed on inoculation of PGPR strains usually has been found to increase the root length and root biomass [26] and this better developed root system may increase the mineral uptake in plants. PGPR plays an important role in plant growth and physiology of the most plants. Pseudomonas fluorescens stimulate plant growth by a variety of mechanisms including production of siderophores, synthesis of antibiotics, production of phytohormones, enhancement of phosphate uptake by the plant, nitrogen fixation and synthesis of enzymes that regulates plant ethylene levels [27]. Fluorescent Pseudomonas sp. has emerged as the largest and potentially plant growth promoting rhizobacteria having the potential as bio-control agent especially to control plant disease [28]. These bacteria are ideally suited as soil inoculants because of the potential for rapid and aggressive colonization in the rhizosphere of crop plants. These bacteria produce siderophores [29], Antibiotics [30] and HCN [31] for plant disease suppression mechanism. In the present study, the PGPR were tested for their effect on growth parameters of C. roseus and the results clearly indicated the PGPR isolates significantly improved plant growth, root length, number of leaves and fresh and dry weights in drought stressed condition. This was in confirmation with early report by Lakshmanan et al. [32] who reported that Azospirillum and Azotobacter isolates to the Senna and Ashwagandha medicinal plants. Siddique [33] reported that tomato crop inoculated with Pseudomonas fluorescens, Azotobacter chroococcum and Azospirillum brasilense either alone or in combination recorded higher plant growth or controlled the nematode, Meloidogyne incognita. Gopal [34] reported that due to PGPR inoculation besides increasing yield also enhanced the alkaloid contents of roots especially withaferin-A in ashwagandha due to the produc-
tion of IAA. In the present study, increase in ajmalicine content in the root may be due to the production of growth promoting substances like IAA by the PGPR. Since the PGPR, which normally induces, gibberellins, auxins and thereby by the rhizobacteria enhanced proliferation of root system, which in turn enhanced mineral uptake and consequently increased dry matter production [35]. Similar trends have been reported by Thosar et al. [36] in Ashwagandha. Continuous availability of growth regulators induced different alkaloids with variable effects among the regulators [37]. 5. Conclusion From the results of this investigation, it can be concluded that bacterial elicitor like Pseudomonas fluorescens treatments had improved number of leaves, plant height, fresh and dry weights and ajmalicine of C. roseus under water deficit. In conclusion, the Pseudomonas fluorescens can protect C. roseus plants from drought stress by partial amelioration of drought induced growth inhibition, apart from their qualities as an efficient PGPR. Further studies are required to confirm whether IAA or gibberellin or both are involved in total alkaloid production and the changes associated with antioxidant defense under treatment with this PGPR in drought stressed C. roseus. References [1] J.K. Zhu, Salt and drought stress signal transduction in plants, Annu. Rev. Plant Biol. 53 (2002) 247–273. [2] H.B. Shao, L.Y. Chuc, G. Wu, J.H. Zhang, Z.H. Lua, Y.C. Hug, Changes of some anti-oxidative physiological indices under soil water deficits among 10 wheat (Triticum aestivum L.) genotypes at tillering stage, Colloids Surf. B: Biointerfaces 54 (2007) 143–149. [3] B. Sankar, C.A. Jaleel, P. Manivannan, A. Kishorekumar, R. Somasundaram, R. Panneerselvam, Drought induced biochemical modifications and proline metabolism in Abelmoschus esculentus (L.) Moench, Acta Bot. Croat. 66 (2007) 43–56. [4] D.W. Lawlor, Limitation to photosynthesis in water stressed leaves: stomata vs. metabolism and role of ATP, Ann. Bot. 89 (2002) 871–885. [5] P. Manivannan, C.A. Jaleel, A. Kishorekumar, B. Sankar, R. Somasundaram, R. Sridharan, R. Panneerselvam, Propiconazole induced changes in antioxidant metabolism and drought stress amelioration in Vigna unguiculata (L.) Walp, Colloids surf. B: Biointerfaces 57 (2007) 69–74. [6] Y. Tan, L. Zongsuo, H.B. Shao, D. Feng, Effect of water deficits on the activity of anti-oxidative enzymes and osmoregulation among three different genotypes of Radix astragali at seeding stage, Colloids Surf. B: Biointerfaces 49 (2006) 60–65.
C.A. Jaleel et al. / Colloids and Surfaces B: Biointerfaces 60 (2007) 7–11 [7] M. Andreselosse, E. Baudoin, P. Vandenkoornhyse, Symbiotic microorganisms, a key for ecological success and protection of plants, C. R. Biol. 327 (2004) 639–648. [8] B.C. Deka, G.C. Bora, A. Shadeque, Effect of Azospirillum on growth and yield of chilli (Capsicum annuum L.) cultivar Pusa Jawala, Haryana, J. Hort. Sci. 38 (1992) 41–46. [9] R.M. Auge, Water relations, drought and vesicular-arbuscular mycorrhial symbiosis, Mycorrhiza 11 (2001) 3–42. [10] Q.S. Wu, R.X. Xia, Y.N. Zou, Reactive oxygen metabolism in mycorrhizal and non-mycorrhizal citrus (Poncirus trifoliata) seedlings subjected to water stress, J. Plant Physiol. 163 (2006) 1101–1110. [11] J. Zhao, T. Lawrence, C. Davis, R. Verpoorte, Elicitor signal transduction leading to production of plant secondary metabolites, Biotechnol. Advances 23 (2005) 283–333. [12] E. Imre, I.E. Somssich, K. Hahlbrok, Pathogen defence in plant-a paradigm of biological complexity, Trends Plant Sci. 3 (1998) 86–90. [13] M.J. Sanchen-Blanco, T. Ferrandes, M.A. Morales, A. Morte, J. Alarcon, Variations in water status, gas exchange, and growth in Rosmarinus officinalis plants with Glomus deserticola under drought conditions, J. Plant Physiol. 161 (2004) 675–682. [14] M. Magnotta, J. Murata, J. Chen, V. De Luca, Identification of a low vindoline accumulating cultivar of Catharanthus roseus (L.) G. Don. by alkaloid and enzymatic profiling, Phytochem. 67 (2006) 1758–1764. [15] A. Levy, Natural and induced genetic variation in the synthesis of alkaloids and other secondary metabolites, in: Improvement of Oilseed and Industrial Crops by Induced Mutations, IAEA, Vienna, 1982, pp. 213–222. [16] D.I. Jacobs, R.V. Heijden, R. Verporte, Proteomics in plant biotechnology and secondary metabolism research, Phytochem. Anal. 11 (2000) 277–287. [17] C.A. Jaleel, R. Gopi, G.M. Alagu Lakshmanan, R. Panneerselvam, Triadimefon induced changes in the antioxidant metabolism and ajmalicine production in Catharanthus roseus (L.) G. Don, Plant Sci. 171 (2006) 271–276. [18] C.A. Jaleel, R. Gopi, P. Manivannan, A. Kishorekumar, B. Sankar, R. Panneerselvam, Paclobutrazol influences on vegetative growth and floral characteristics of Catharanthus roseus (L.) G. Don, Indian J. Appl. Pure Biol. 21 (2006) 369–372. [19] C.A. Jaleel, R. Gopi, B. Sankar, P. Manivannan, A. Kishorekumar, R. Sridharan, R. Panneerselvam, Studies on germination, seedling vigour, lipid peroxidation and proline metabolism in Catharanthus roseus seedlings under salt stress, South Afr. J. Bot. 73 (2007) 190–195. [20] C.A. Jaleel, R. Gopi, P. Manivannan, R. Panneerselvam, Responses of antioxidant defense system of Catharanthus roseus (L.) G. Don. to paclobutrazol treatment under salinity, Acta Physiol. Plant. 29 (2007) 205–209. [21] C.A. Jaleel, P. Manivannan, A. Kishorekumar, B. Sankar, R. Gopi, R. Somasundaram, R. Panneerselvam, Alterations in osmoregulation, antioxidant enzymes and indole alkaloid levels in Catharanthus roseus
[22]
[23]
[24] [25]
[26]
[27] [28] [29] [30]
[31] [32]
[33]
[34]
[35]
[36]
[37]
11
exposed to water deficit, Colloids Surf. B: Biointerfaces 59 (2007) 150– 157. J. Zaho, W. Zhee, Q. Hu, Enhanced ajmalicine production in Catharanthus roseus cell cultures by combined elicitor treatment from Shake-flaskto 20-1 air lift bioreactor, Biotechnol. Lett. 22 (2000) 509–514. J. Renoudin, Reversed phase-high performance liquid chromatographic characteristics of indole alkaloids from cell suspension cultures of Catharanthus roseus, J. Chromatogr. 291 (1984) 165–174. E. Stahl, Thin Layer Chromatography, A Laboratory Handbook, SpringerVerlag, Berlin, 1969. U. Chakraborthy, B.N. Chakraborty, P.R. Chowdhury, C. Tongden, M. Basnet, Investigation on plant growth promoting rhizobacteria of tea rhizosphere, in: 6th International Workshop on PGPR, IISR, Calicut, Kerala, 2003, pp. 78–82. A. Khalid, M. Arshad, Z.A. Zahir, Screening plant growth promoting rhizobacteria for improving growth and yield of wheat, J. Appl. Microbiol. 96 (2004) 473–480. J. Davidson, Plant beneficial bacteria, Biotechnology 6 (1988) 282– 286. J.W. Kloepper, R. Lifshitz, J.A. Kuc, Proposed definitions related to induced disease resistance, Biocontrol Sci. Tech. 2 (1992) 349–351. J.B. Neilands, Microbial iron compounds, Ann. Rev. Biol. 50 (1981) 715–731. L.S. Thomashow, D.N. Weller, R.F. Bonsall, L.S. Perison, Production of the antibiotic phenozine-l-carboxylic acid by fluorescent Pseudomonas spp. in the rhizosphere of wheat, Appl. Environ. Microbiol. 56 (1988) 908–912. C. Blumer, D. Haas, Mechanism, regulation, and ecological role of bacterial cyanide biosynthesis, Arch. Microbiol. 173 (2000) 170–177. A. Lakshmanan, K. Govindarajan, K. Kumar, Effect of seed treatment with native diazotrophs on the seedling parameters of Senna and Ashwagandha, Crop Res. 30 (2005) 119–123. Z.A. Siddiqui, Effects on plant growth promoting bacteria and composed organic fertilizers on the reproduction of Meloidogyne incognita and tomato growth, Bioresour. Technol. 95 (2004) 223–227. H. Gopal, Development of microbial consortium for improvement of growth, yield and alkaloid content of ashwagandha (Withania somnifera, Dunal), J. Herb Spices Med. Plants 8 (2004) 38–43. T.V. Suslow, M.N. Schroth, Rhizobacteria of sugar beets: effects of seed application and root colonization on yield, Phytopathology 72 (1982) 199–206. N.J. Thosar, V.G. Ingle, J.C. Jadhav, Effect of FYM and biofertilizers on dry root and seed yield of Ashwagandha (Withania somnifera), Crop Prot. Prod. 1 (2005) 27–28. M. El-Sayed, R. Verpoorte, Growth, metabolic profiling and enzymes activities of Catharanthus roseus seedlings treated with plant growth regulators, Plant Growth Regul. 44 (2004) 53–58.
Pseudomonas fluorescens enhances biomass yield and ajmalicine production in Catharanthus roseus under water deficit stress C. Abdul Jaleel, P. Manivannan, B. Sankar, A. Kishorekumar, R. Gopi ∗ , R. Somasundaram ∗ , R. Panneerselvam Stress Physiology Lab, Department of Botany, Annamalai University, Annamalainagar 608 002, Tamilnadu, India Received 22 April 2007; received in revised form 15 May 2007; accepted 20 May 2007 Available online 25 May 2007
Abstract The effect of plant growth promoting rhizobacteria (PGPR) like Pseudomonas fluorescens on growth parameters and the production of ajmalicine were investigated in Catharanthus roseus under drought stress. The plants under pot culture were subjected to 10, 15 and 20 days interval drought (DID) stress and drought stress with Pseudomonas fluorescens at 1 mg l−1 and 1 mg l−1 Pseudomonas fluorescens alone from 30 days after planting (DAP) and regular irrigation was kept as control. The plants were uprooted on 41 DAS (10 DID), 46 DAS (15 DID) and 51 DAS (20 DID). Drought stress decreased the growth parameters and increased the ajmalicine content. But the treatment with Pseudomonas fluorescens enhanced the growth parameters under drought stress and partially ameliorated the drought induced growth inhibition by increasing the fresh and dry weights significantly. The ajmalicine content was again increased due to Pseudomonas fluorescens treatment to the drought stressed plants. From the results of this investigation, it can be concluded that, the seedling treatments of native PGPRs can be used as a good tool in the enhancement of biomass yield and alkaloid contents in medicinal plants, as it provides an eco-friendly approach and can be used as an agent in water deficit stress amelioration. © 2007 Elsevier B.V. All rights reserved. Keywords: Catharanthus roseus; Ajmalicine; Seed priming; Plant growth promoting rhizobacteria (PGPR); Pseudomonas fluorescens; Water deficit stress
1. Introduction Through out the world, the ground water level is inadequate for the cultivation of crops and this water limited condition is threatening problem. A water stress may conceivably arise either from an insufficient or from an excessive water activity in the plant’s environment. In the case of terrestrial plants in nature, the former occurs as a result of a water deficit or drought and therefore is called a water deficit stress (shortened to water stress) or drought stress [1]. The environmental stresses such as drought, Abbreviations: DAP, days after planting; DID, days interval drought; PGPR, plant growth promoting rhizobacteria; MIAs, monoterpenoid indole alkaloids; VCR, vincristine; VLB, vinblastine ∗ Corresponding authors at: Department of Botany, Annamalai University, Annamalainagar 608 002, Tamilnadu, India. Tel.: +91 4144 238248x354; fax: +91 4144 222265. E-mail addresses: [email protected] (C.A. Jaleel), [email protected] (R. Gopi), kalaisomu [email protected] (R. Somasundaram). 0927-7765/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2007.05.012
temperature, salinity, air pollution, heavy metals, pesticides and soil pH are major limiting factors in crop production because, they affects almost all plant functions [2]. Although the general effects of drought on plant growth are fairly well known, the primary effects of water deficit at the biochemical and molecular levels are not well understood [3]. Water stress tolerance is seen in all plant species but its extent varies from species to species. Improving the efficiency of water use in agriculture is associated with increasing the fraction of the available water resources that is transpired, because of the unavoidable association between yield and water use [4]. For the last few decades, several scales of physiological works have been conducted under drought stress in crop plants, but it is not so with respect to medicinal plants [5,3,6]. Numerous microorganisms live in the portion of soil modified or influenced by plant roots so called ‘rhizosphere’ [7]. Among these microorganisms, some have positive effects on plant growth promotion constituting the plant growth promoting rhizobacteria (PGPR) such as Azospirillum, Azotobacter,
8
C.A. Jaleel et al. / Colloids and Surfaces B: Biointerfaces 60 (2007) 7–11
Pseudomonas fluorescens, several gram positive Bacillus sp. [7]. The diazotrophic rhizobiocoenosis is an important biological process that plays a major role in satisfying the nutritional requirements of the commercial medicinal plants. Studies on the diazotrophic population in the rhizosphere region and testing the suitability of the isolated diazotroph as seed inoculant will be highly useful in improving the productivity of commercially important medicinal plants. Diazotrophs secrete plant growth hormones such as auxins, gibberellins and cytokinins [8]. Lot of investigations were already covered the importance of microbial association like arbuscular mycorrhizal symbiosis in drought stressed plants [9,10]. But little attraction is drawn towards PGPR mediated drought stress amelioration in medicinal plants. The strong and rapidly stimulating effect of fungal elicitor on plant secondary metabolism in medicinal plants attracts considerable attentions and research efforts [11]. The reasons responsible for the diverse stimulating effects of fungal elicitors are complicated and could be related to the interactions between fungal elicitors and plant cells, elicitor signal transduction, and plant defense responses [12]. In plants certain secondary metabolite pathways are induced by infection with microorganisms. It was reported that, arbuscular mycorrhizal symbiosis maintained more normal water relations in plants [13]. Catharanthus roseus (L.) G. Don. (Madagascar periwinkle) is a perennial tropical plant belonging to the family Apocynaceae that produces more than 100 monoterpenoid indole alkaloids (MIAs) including two commercially important cytotoxic dimeric alkaloids used in cancer chemotherapy [14]. Periwinkle, native to Madagascar is now found in many tropical and sub-tropical regions of the world. This plant contains anti-cancer alkaloids, vincristine (VCR) and vinblastine (VLB) and antihypertension alkaloid, ajmalicine. In medicinal plants, the content of the economically important metabolite is more important than the yield of the plant part containing the metabolite, as it determines the cost of its extraction [15]. The cell, tissue cultures and biotechnological aspects of this plant are being extensively investigated to increase the yield of the alkaloids [16]. In this context, the cultivation of periwinkle is becoming popular among the farmers. The major problem in the cultivation of periwinkle is environmental stresses such as salinity, water deficits and which created poor yield and establishment at field level. Previous works revealed the influences of triadimefon on the antioxidant metabolism and ajmalicine production [17], paclobutrazol mediated growth regulation [18], salinity problems [19] and salt stress protection by paclobutrazol [20] in C. roseus. Comparatively a little work has been reported on water stress problems and methods to overcome drought stress injuries in this plant [21]. The role of PGPR in this medicinal plant under water deficit stress had attracted little attention. In this experiment, an attempt is made to study the ability of PGPR, Pseudomonas fluorescens in drought stress amelioration, through its effect on growth and ajmalicine production in C. roseus plant under soil water deficits.
2. Materials and methods 2.1. Plant cultivation, drought stress induction and Pseudomonas fluorescens treatments The seeds of Catharanthus roseus (L.) G. Don. (Family: Apocynaceae) were collected from the Department of Horticulture, Faculty of Agriculture, Annamalai University, Tamil Nadu, India. The Pseudomonas fluorescens was obtained from Krishi Care Bioinputs, Chennai, India. The plants under pot culture were subjected to 10, 15 and 20 days interval drought (DID) stress and drought stress with Pseudomonas fluorescens at 1 mg l−1 and 1 mg l−1 Pseudomonas fluorescens alone from 30 days after sowing (DAS) and regular irrigation was kept as control. The pots were covered with a rain out shelter, made up of plastic sheets, whenever rainfall was anticipated and immediately after rain, rain out shelter was pulled back so that, pots received maximum sunlight. Further, the pots were regularly covered with rain out shelter during nighttime. Using this system the pots were protected from rainfall and any external moisture entry. The plants were uprooted on 41 DAS (10 DID), 46 DAS (15 DID) and 51 DAS (20 DID) for analyzing growth parameters and ajmalicine content. 2.2. Analysis of morphological parameters Morphological parameters like root length, plant height and number of leaves per plant were measured in the samples on 40, 45 and 50 DAP. Fresh and dry weights were taken from the samples by using an electronic weighing device (Model-Citizen XK3190-A7M). 2.3. Ajmalicine extraction and quantification Ajmalicine extraction from the roots was carried out by following the standard extraction method [22]. Identification and quantification of ajmalicine was done by preparative Thin Layer Chromatography using silica gel G (Merck) in chloroform:methanol (98:2, v/v) [23] by comparison of Rf values with authentic ajmalicine standard (Himedia, Mumbai). Ajmalicine was spotted with Dragendorff’s reagent [24]. 2.4. Statistics Statistical analysis was performed using one way analysis of variance (ANOVA) followed by Duncan’s Multiple Range Test (DMRT). The values are mean ± S.D. for seven samples in each group. P-values ≤ 0.05 were considered as significant. 3. Results 3.1. Effect of Pseudomonas fluorescens treatments on plant height of C. roseus under drought There was significant variation (p ≤ 0.05) in plant height of C. roseus seedlings treated with Pseudomonas fluorescens
C.A. Jaleel et al. / Colloids and Surfaces B: Biointerfaces 60 (2007) 7–11
Fig. 1. Effects of drought, Pseudomonas fluorescens and their combination on plant hight of Catharanthus roseus plants. Values are given as mean ± S.D. of seven samples in each group. Bar values are not sharing a common superscript (a, b, c, d) differ significantly at p ≤ 0.05 (DMRT).
when compared with drought stressed and well watered controls (Fig. 1). Pseudomonas fluorescens treated plants recorded the highest plant height at all sampling periods in C. roseus (41, 46 and 51 DAP). Individual inoculation of Pseudomonas fluorescens increased plant height of C. roseus. 3.2. Effect of Pseudomonas fluorescens treatments on root length of C. roseus under drought The root length of C. roseus seedlings varied in stress and Pseudomonas fluorescens treatments when compared to wellwatered controls. The maximum root length was recorded in Pseudomonas fluorescens treated plants (Fig. 2).
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Fig. 3. Effects of drought, Pseudomonas fluorescens and their combination on number of leaves of Catharanthus roseus plants. Values are given as mean ± S.D. of seven samples in each group. Bar values are not sharing a common superscript (a, b, c, d) differ significantly at p ≤ 0.05 (DMRT).
3.4. Effect of Pseudomonas fluorescens treatments fresh and dry weights of C. roseus under drought Significant variation (p ≤ 0.05) in whole plant fresh and dry weights of C. roseus seedlings treated with Pseudomonas fluorescens when compared with drought stressed and well-watered controls. Pseudomonas fluorescens treated plants showed the highest fresh and dry weights at all sampling periods in C. roseus (Table 1). 3.5. Effect of Pseudomonas fluorescens treatments on ajmalicine content of C. roseus under drought
The number of leaves was recorded more in the Pseudomonas fluorescens treated C. roseus followed by drought stress in combination with Pseudomonas fluorescens. The low number of leaves was recorded in the water stressed C. roseus (Fig. 3).
During the early stages of plant growth the alkaloid ajmalicine content was less in C. roseus. The lowest content was recorded in roots of control on 40 DAP. But the content increased with drought and treatment with Pseudomonas fluorescens. Drought stressed C. roseus plants showed a significant enhancement in ajmalicine content. The highest content was recorded in the roots of Pseudomonas fluorescens treatments (Fig. 4).
Fig. 2. Effects of drought, Pseudomonas fluorescens and their combination on root length of Catharanthus roseus plants. Values are given as mean ± S.D. of seven samples in each group. Bar values are not sharing a common superscript (a, b, c, d) differ significantly at p ≤ 0.05 (DMRT).
Fig. 4. Effects of drought, Pseudomonas fluorescens and their combination on ajmalicine content of Catharanthus roseus plants. Values are given as mean ± S.D. of seven samples in each group. Bar values are not sharing a common superscript (a, b, c, d) differ significantly at p ≤ 0.05 (DMRT).
3.3. Effect of Pseudomonas fluorescens treatments on number of leaves of C. roseus under drought
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Table 1 Effects of drought, Pseudomonas fluorescens and their combination on fresh and dry weights of Catharanthus roseus plants Days interval drought (DID)
Control
Drought
Drought + P. fluorescens
P. fluorescens
Whole plant fresh weight (g/plant) 10 15 20
10.326 ± 0.333a 14.415 ± 0.529a 17.412 ± 1.131a
8.412 ± 0.300b 11.372 ± 0.496b 14.561 ± 0.696b
11.022 ± 0.355c 14.516 ± 0.568a 18.945 ± 1.229c
12.063 ± 0.292d 16.412 ± 0.531d 19.184 ± 0.747d
1.787 ± 0.068a 1.941 ± 0.076a 2.034 ± 0.123a
1.081 ± 0.035b 1.469 ± 0.064b 1.932 ± 0.095b
1.809 ± 0.058a 1.944 ± 0.073a 2.200 ± 0.131c
1.993 ± 0.037d 2.002 ± 0.064d 2.591 ± 0.130d
Whole plant dry weight (g/plant) 10 15 20
Values are given as mean ± S.D. of seven samples in each group. Values are not sharing a common superscript (a, b, c, d) differ significantly at p ≤ 0.05 (DMRT).
4. Discussion The treatment with PGPR like Pseudomonas fluorescens increased the plant height, root length, number of leaves and ajmalicine content of C. roseus when compared to well-watered control and drought stressed plants. The occurrence of Azospirillum, Pseudomonas in and around the root system of cereals, vegetables and the beneficial effect upon inoculation has been well established. In the present study, the increased growth parameters in C. roseus due to treatment of PGPR might be due to the production of growth hormones (IAA, gibberellins, auxins), by the bacteria [25]. It is worth noting that the increase in growth parameters observed on inoculation of PGPR strains usually has been found to increase the root length and root biomass [26] and this better developed root system may increase the mineral uptake in plants. PGPR plays an important role in plant growth and physiology of the most plants. Pseudomonas fluorescens stimulate plant growth by a variety of mechanisms including production of siderophores, synthesis of antibiotics, production of phytohormones, enhancement of phosphate uptake by the plant, nitrogen fixation and synthesis of enzymes that regulates plant ethylene levels [27]. Fluorescent Pseudomonas sp. has emerged as the largest and potentially plant growth promoting rhizobacteria having the potential as bio-control agent especially to control plant disease [28]. These bacteria are ideally suited as soil inoculants because of the potential for rapid and aggressive colonization in the rhizosphere of crop plants. These bacteria produce siderophores [29], Antibiotics [30] and HCN [31] for plant disease suppression mechanism. In the present study, the PGPR were tested for their effect on growth parameters of C. roseus and the results clearly indicated the PGPR isolates significantly improved plant growth, root length, number of leaves and fresh and dry weights in drought stressed condition. This was in confirmation with early report by Lakshmanan et al. [32] who reported that Azospirillum and Azotobacter isolates to the Senna and Ashwagandha medicinal plants. Siddique [33] reported that tomato crop inoculated with Pseudomonas fluorescens, Azotobacter chroococcum and Azospirillum brasilense either alone or in combination recorded higher plant growth or controlled the nematode, Meloidogyne incognita. Gopal [34] reported that due to PGPR inoculation besides increasing yield also enhanced the alkaloid contents of roots especially withaferin-A in ashwagandha due to the produc-
tion of IAA. In the present study, increase in ajmalicine content in the root may be due to the production of growth promoting substances like IAA by the PGPR. Since the PGPR, which normally induces, gibberellins, auxins and thereby by the rhizobacteria enhanced proliferation of root system, which in turn enhanced mineral uptake and consequently increased dry matter production [35]. Similar trends have been reported by Thosar et al. [36] in Ashwagandha. Continuous availability of growth regulators induced different alkaloids with variable effects among the regulators [37]. 5. Conclusion From the results of this investigation, it can be concluded that bacterial elicitor like Pseudomonas fluorescens treatments had improved number of leaves, plant height, fresh and dry weights and ajmalicine of C. roseus under water deficit. In conclusion, the Pseudomonas fluorescens can protect C. roseus plants from drought stress by partial amelioration of drought induced growth inhibition, apart from their qualities as an efficient PGPR. Further studies are required to confirm whether IAA or gibberellin or both are involved in total alkaloid production and the changes associated with antioxidant defense under treatment with this PGPR in drought stressed C. roseus. References [1] J.K. Zhu, Salt and drought stress signal transduction in plants, Annu. Rev. Plant Biol. 53 (2002) 247–273. [2] H.B. Shao, L.Y. Chuc, G. Wu, J.H. Zhang, Z.H. Lua, Y.C. Hug, Changes of some anti-oxidative physiological indices under soil water deficits among 10 wheat (Triticum aestivum L.) genotypes at tillering stage, Colloids Surf. B: Biointerfaces 54 (2007) 143–149. [3] B. Sankar, C.A. Jaleel, P. Manivannan, A. Kishorekumar, R. Somasundaram, R. Panneerselvam, Drought induced biochemical modifications and proline metabolism in Abelmoschus esculentus (L.) Moench, Acta Bot. Croat. 66 (2007) 43–56. [4] D.W. Lawlor, Limitation to photosynthesis in water stressed leaves: stomata vs. metabolism and role of ATP, Ann. Bot. 89 (2002) 871–885. [5] P. Manivannan, C.A. Jaleel, A. Kishorekumar, B. Sankar, R. Somasundaram, R. Sridharan, R. Panneerselvam, Propiconazole induced changes in antioxidant metabolism and drought stress amelioration in Vigna unguiculata (L.) Walp, Colloids surf. B: Biointerfaces 57 (2007) 69–74. [6] Y. Tan, L. Zongsuo, H.B. Shao, D. Feng, Effect of water deficits on the activity of anti-oxidative enzymes and osmoregulation among three different genotypes of Radix astragali at seeding stage, Colloids Surf. B: Biointerfaces 49 (2006) 60–65.
C.A. Jaleel et al. / Colloids and Surfaces B: Biointerfaces 60 (2007) 7–11 [7] M. Andreselosse, E. Baudoin, P. Vandenkoornhyse, Symbiotic microorganisms, a key for ecological success and protection of plants, C. R. Biol. 327 (2004) 639–648. [8] B.C. Deka, G.C. Bora, A. Shadeque, Effect of Azospirillum on growth and yield of chilli (Capsicum annuum L.) cultivar Pusa Jawala, Haryana, J. Hort. Sci. 38 (1992) 41–46. [9] R.M. Auge, Water relations, drought and vesicular-arbuscular mycorrhial symbiosis, Mycorrhiza 11 (2001) 3–42. [10] Q.S. Wu, R.X. Xia, Y.N. Zou, Reactive oxygen metabolism in mycorrhizal and non-mycorrhizal citrus (Poncirus trifoliata) seedlings subjected to water stress, J. Plant Physiol. 163 (2006) 1101–1110. [11] J. Zhao, T. Lawrence, C. Davis, R. Verpoorte, Elicitor signal transduction leading to production of plant secondary metabolites, Biotechnol. Advances 23 (2005) 283–333. [12] E. Imre, I.E. Somssich, K. Hahlbrok, Pathogen defence in plant-a paradigm of biological complexity, Trends Plant Sci. 3 (1998) 86–90. [13] M.J. Sanchen-Blanco, T. Ferrandes, M.A. Morales, A. Morte, J. Alarcon, Variations in water status, gas exchange, and growth in Rosmarinus officinalis plants with Glomus deserticola under drought conditions, J. Plant Physiol. 161 (2004) 675–682. [14] M. Magnotta, J. Murata, J. Chen, V. De Luca, Identification of a low vindoline accumulating cultivar of Catharanthus roseus (L.) G. Don. by alkaloid and enzymatic profiling, Phytochem. 67 (2006) 1758–1764. [15] A. Levy, Natural and induced genetic variation in the synthesis of alkaloids and other secondary metabolites, in: Improvement of Oilseed and Industrial Crops by Induced Mutations, IAEA, Vienna, 1982, pp. 213–222. [16] D.I. Jacobs, R.V. Heijden, R. Verporte, Proteomics in plant biotechnology and secondary metabolism research, Phytochem. Anal. 11 (2000) 277–287. [17] C.A. Jaleel, R. Gopi, G.M. Alagu Lakshmanan, R. Panneerselvam, Triadimefon induced changes in the antioxidant metabolism and ajmalicine production in Catharanthus roseus (L.) G. Don, Plant Sci. 171 (2006) 271–276. [18] C.A. Jaleel, R. Gopi, P. Manivannan, A. Kishorekumar, B. Sankar, R. Panneerselvam, Paclobutrazol influences on vegetative growth and floral characteristics of Catharanthus roseus (L.) G. Don, Indian J. Appl. Pure Biol. 21 (2006) 369–372. [19] C.A. Jaleel, R. Gopi, B. Sankar, P. Manivannan, A. Kishorekumar, R. Sridharan, R. Panneerselvam, Studies on germination, seedling vigour, lipid peroxidation and proline metabolism in Catharanthus roseus seedlings under salt stress, South Afr. J. Bot. 73 (2007) 190–195. [20] C.A. Jaleel, R. Gopi, P. Manivannan, R. Panneerselvam, Responses of antioxidant defense system of Catharanthus roseus (L.) G. Don. to paclobutrazol treatment under salinity, Acta Physiol. Plant. 29 (2007) 205–209. [21] C.A. Jaleel, P. Manivannan, A. Kishorekumar, B. Sankar, R. Gopi, R. Somasundaram, R. Panneerselvam, Alterations in osmoregulation, antioxidant enzymes and indole alkaloid levels in Catharanthus roseus
[22]
[23]
[24] [25]
[26]
[27] [28] [29] [30]
[31] [32]
[33]
[34]
[35]
[36]
[37]
11
exposed to water deficit, Colloids Surf. B: Biointerfaces 59 (2007) 150– 157. J. Zaho, W. Zhee, Q. Hu, Enhanced ajmalicine production in Catharanthus roseus cell cultures by combined elicitor treatment from Shake-flaskto 20-1 air lift bioreactor, Biotechnol. Lett. 22 (2000) 509–514. J. Renoudin, Reversed phase-high performance liquid chromatographic characteristics of indole alkaloids from cell suspension cultures of Catharanthus roseus, J. Chromatogr. 291 (1984) 165–174. E. Stahl, Thin Layer Chromatography, A Laboratory Handbook, SpringerVerlag, Berlin, 1969. U. Chakraborthy, B.N. Chakraborty, P.R. Chowdhury, C. Tongden, M. Basnet, Investigation on plant growth promoting rhizobacteria of tea rhizosphere, in: 6th International Workshop on PGPR, IISR, Calicut, Kerala, 2003, pp. 78–82. A. Khalid, M. Arshad, Z.A. Zahir, Screening plant growth promoting rhizobacteria for improving growth and yield of wheat, J. Appl. Microbiol. 96 (2004) 473–480. J. Davidson, Plant beneficial bacteria, Biotechnology 6 (1988) 282– 286. J.W. Kloepper, R. Lifshitz, J.A. Kuc, Proposed definitions related to induced disease resistance, Biocontrol Sci. Tech. 2 (1992) 349–351. J.B. Neilands, Microbial iron compounds, Ann. Rev. Biol. 50 (1981) 715–731. L.S. Thomashow, D.N. Weller, R.F. Bonsall, L.S. Perison, Production of the antibiotic phenozine-l-carboxylic acid by fluorescent Pseudomonas spp. in the rhizosphere of wheat, Appl. Environ. Microbiol. 56 (1988) 908–912. C. Blumer, D. Haas, Mechanism, regulation, and ecological role of bacterial cyanide biosynthesis, Arch. Microbiol. 173 (2000) 170–177. A. Lakshmanan, K. Govindarajan, K. Kumar, Effect of seed treatment with native diazotrophs on the seedling parameters of Senna and Ashwagandha, Crop Res. 30 (2005) 119–123. Z.A. Siddiqui, Effects on plant growth promoting bacteria and composed organic fertilizers on the reproduction of Meloidogyne incognita and tomato growth, Bioresour. Technol. 95 (2004) 223–227. H. Gopal, Development of microbial consortium for improvement of growth, yield and alkaloid content of ashwagandha (Withania somnifera, Dunal), J. Herb Spices Med. Plants 8 (2004) 38–43. T.V. Suslow, M.N. Schroth, Rhizobacteria of sugar beets: effects of seed application and root colonization on yield, Phytopathology 72 (1982) 199–206. N.J. Thosar, V.G. Ingle, J.C. Jadhav, Effect of FYM and biofertilizers on dry root and seed yield of Ashwagandha (Withania somnifera), Crop Prot. Prod. 1 (2005) 27–28. M. El-Sayed, R. Verpoorte, Growth, metabolic profiling and enzymes activities of Catharanthus roseus seedlings treated with plant growth regulators, Plant Growth Regul. 44 (2004) 53–58.