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International Journal of Research in Pure and Applied Microbiology
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ISSN 2277–3843 Original Article EFFICIENCY OF PLANT GROWTH PROMOTING RHIZOBACTERIA (PGPR) ON ENHANCEMENT OF GROWTH, YIELD AND NUTRIENT CONTENT OF Catharanthus roseus
G.Lenin* and M. Jayanthi Department of Microbiology, Annamalai University, Annamalai Nagar – 608 002, Tamil Nadu, India. E.mail: leninmdu@gmail.com Received 03 October 2012; Accepted 17 October 2012 Abstract Plant growth-promoting rhizobacteria (PGPR) are known in various cropping systems to increase plant growth, vigour and plant nutrient contents. A commercial soil amendment containing a mixture of four PGPR (Azospirillum lipoferum, Azotobacter chroococcum, Pseudomonas fluorescens and Bacillus megaterium) was evaluated for impact on germination, initial growth and nutrient content of Catharanthus roseus. Root inoculation of PGPR strains significantly increased germination rate, vigour index and chlorophyll content (5.34 mg g -1 plant-1 in 120 DAS) compared with the control. In addition, nutrient element contents (P- 2.34, N- 0.34, K-2.20 % in 120 DAS) was investigated and significantly affected by bacterial applications compared with the control. The results of this study suggest that PGPR (Azospirillum lipoferum, Azotobacter chroococcum, Pseudomonas fluorescens and Bacillus megaterium) in combination have the potential to increase the yield, growth and nutrient content of Catharanthus roseus. © 2012 Universal Research Publications. All rights reserved Key words: Catharanthus roseus, PGPR, Germination and Chlorophyll 1. INTRODUCTION Plant growth promoting rhizobacteria (PGPR) are a heterogeneous group of bacteria that can be found in the rhizosphere, at root surfaces and in association with roots [1]. The enhancement of crop plant growth using PGPR is documented [2] and these organisms have been used to reduce plant stress associated with phytoremediation strategies for metal contaminated soils [3]. PGPR enhance plant growth through various forms, such as : (i) reducing ethylene production, allowing plants to develop longer roots and better establish during early stages of growth, due to the synthesis of 1-aminocyclopropane-1-carboxylate (ACC) deaminase which modulates the level of ethylene by hydrolyzing ACC, a precursor of ethylene, in ammonia and α-ketobutyrate [4]; (ii) enhancing asymbiotic nitrogen fixation [5] or indirectly affecting symbiotic N2 fixation, nodulation or nodule occupancy [6]; (iii) producing or changing the Concentration of plant growth regulators like indole acetic acid (IAA) [1]; (iv)raising the solubilisation of nutrients with consequent increase in the supply of bioavailable phosphorous and other trace elements for plant uptake [7]; (v) production of phytohormones such as auxins, cytokinins and Gibberelins [7]; and(vi) synthesis of antibiotic and other pathogen-depressing substances such as siderophores, cyanide and chelating agents that protect plants from diseases [8]. These organisms can also increase plant tolerance to flooding [9], salt stress [10] and water deprivation [11]. Catharanthus roseus (L.) G. Don (Apocynaceae) which is a herbaceous sub-shrub [12], also known as Madagascar periwinkle and Vinca rosea (synonym) worldwide has been extensively studied by several workers and has been identified to be a source of numerous active principles of therapeutic importance. It has more than 400 known alkaloids, some of which are approved as antineoplastic agents to treat leukemia, Hodgkin’s disease, malignant lymphomas, neuroblastoma, rhabdomyosarcoma, Wilm’s tumor and other cancers. Leveque et al. [13] reported anticancerous activity of this plant. C. roseus is one of the most important medicinal plants, being a valuable source of the antitumor agents vinblastine and vincristine, used in chemotherapy of leukemia and treatment of Hodgkin’s disease [14]. Vincamine and vinpocetine from C. roseus, have vaso-dialating and memory enhancing properties and have been shown to alleviate vascular dementia and Alzheimer’s disease [15, 16]. Anna and Bridget in [17] showed anticancerous properties of Vinca alkaloids. C. roseus was also shown to have anticancerous property by

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International Journal of Research in Pure and Applied Microbiology 2012; 2(4): 37-42

chroococcum CRAB-3. Vigour index = Germination per cent x Total length of seedling (mm) Seedling height Seedling from each treatment were taken at random and the length between the collar and tip of primary root and the length of plumule was measured between collar and tip of primary leaves and mean value was expressed in centimeters. Root dry weight The roots were cut into pieces of approximately 5. The seeds of C. lipoferum CRAS-2. A. roseus To study the effect of single inoculants and consortium of inoculant preparations of PGPR strains viz. fluorescens CRPS-5 and B. [20]. roseus as influenced by inoculant treatments are presented in Table 1.Montbriand [18]. T0 . A.10 per cent and 1988. Estimation of total chlorophyll content One gram fresh leaf sample taken from each replication was analyzed for total chlorophyll content by following the method of Talling and Driver [22].Bacillus megaterium – CRBA-4 T5 . vigour index. roseus were surface sterilized with 80 per cent ethanol and 0. After shade drying. The length of the roots from the point of attachment to the stem to the tip of the roots were measured and expressed in centimeter. the seeds were sown at 25 of seeds per pot and finally five seeds were maintained. roseus The total chlorophyll content of C.CRPS-5 T4 . The plant growth promoting rhizobacterial isolates of A. The shoot cut into pieces and dried in an oven at 70ºC till constant dry weight.34 mg g-1 of plant-1. roseus was studied under in vitro condition.T1 + T2 + T3 + T4 (Consortium) Determination of seedling vigour index under in vitro condition Seed germination The number of days taken for 50 per cent of the seeds to show radicle emergence was taken as the day for first count of germination. A. respectively. The mean annual rainfall of this area is 1500 mm.Pseudomonas fluorescens .CRAS-2 T2 .86 mg g-1 respectively. The chlorophyll content of the leaves of C. Determination of plant growth Plant height Height of the plant from ground level of growing point of the stem was measured and the mean was calculated and expressed in centimeter. megaterium CRBA-4 were prepared as pot culture experiment. 2. [24]. RESULTS Seed germination and vigour index of C.Azospirillum lipoferum . roseus followed by single inoculant treatment (Table 1). Single inoculation treatment of A. Preparation of pots and seed inoculation The cement pots of size 1’ x 2’ x2’ filled with land soil and sand in the ratio of 1:1. fluorescens (CRPS-5) recorded 4.Azotobacter chroococcum . lipoferum CRAS-2 recorded 4. roseus increased upto 120 days after inoculation and then gradually decreased with increasing age of the plant. The plant inoculated with consortium treatment (T 5) recorded higher total chlorophyll content of 5. Pseudomonas fluorescens and Bacillus megaterium) by measuring the germination rate. P.Uninoculated control T1 . A. MATERIALS AND METHODS Pot Culture Experiment A pot culture experiment was conducted during December 2011 to May 2012 at the Department of Microbiology.60 mg g-1. Its antibacterial and antidiabetic activities have been reported by Rozas Harnandez [19] and Srinivas et al.12 mg g-1 and B. Root length Plants were pulled carefully without damaging to the roots. 38 International Journal of Research in Pure and Applied Microbiology 2012. Uninoculated seeds with distilled water served as control. P. either as individual organisms or consortium of organisms separately having a cell load of 1x109 cfu ml-1 and shade dried for 30 min. respectively and the mean highest and lowest relative humidity was 96 and 78 per cent respectively. The mean was worked out and expressed in g plant-1. Total chlorophyll content of c. The annual mean minimum and maximum temperature of experimental area is 25° and 39°C. The treatments are as follows. Shoot dry weight Plants were pulled carefully without damaging to the shoots.0 centimeters length and dried in an oven at 70ºC till constant dry weight. The seeds were mixed with carrier based plant growth promoting bacteria. Annamalai University. chroococcum CRAB-3. The physicochemical properties of the pot soil were analysed.10 was recorded for consortium treated C. P. 3. fluorescens CRPS-5 and B. 2(4): 37-42 . The objective of this study was to investigate interaction between Catharanthus roseus and plant growth promoting four PGPR inoculations (Azospirillum lipoferum. phosphorus and potassium content The nitrogen content of the plant samples was analyzed by Microkjeldahl method [23].1 per cent mercuric chloride and washed the seeds with sterile distilled water for 3 to 4 times. Estimation of plant nitrogen. megaterium CRBA-4 on the seed germination and vigour index of C.CRAB-3 T3 . Faculty of Agriculture. chroococcum CRAB-3 recorded 4. The experiment was conducted in completely randomized block design with three replications. The mean was worked out and expressed in g plant-1. Azotobacter chroococcum. Annamalai nagar. Chlorophyll content and NPK content of plant. The highest percentage of seed germination and vigour index of 87. The phosphorus content in the plant material was estimated by the method developed by Olsen et al. The potassium content in the plant sample was estimated following the method of Jackson [25].. Seedling height and germination percentage were recorded and vigour index was calculated as per the procedure suggested by Abdul-Baki and Anderson [21]. megaterium CRBA-4 recorded 3.40 mg g-1. A control pot without inoculation was also maintained. lipoferum CRAS-2.

37 48.84 1. roseus in all the treatments significantly increased upto 120 DAS and thereafter recorded gradual reduction in leaf NPK on 150th and 180th day of sowing.25 11. These three major plant nutrient contents significantly varied between the treatments.90 2.Azotobacter chroococcum T3 .42 48.21 69.22 69.00 67.80 59. The shoot.45 52.Bacillus megaterium T5 . Plant nutrient content of c.04 1.92 2.78 2.00 8. lipoferum (CRAS-2).Uninoculated control T1 .50 Mean 37.95 1.28 0.00 10.97 1.80 3.Uninoculated control T1 .80 6.47 76.60 1.Consortium (T 1 + T2 + T3 + T4) Shoot dry weight of Catharanthus roseus (g plant-1) 90 DAS 120 DAS 150 DAS 180 DAS 8. A positive response due to inoculants was observed at all sampling periods. 39 International Journal of Research in Pure and Applied Microbiology 2012.Uninoculated control T1 . root dry weight of 47.Azospirillum lipoferum T2 .82 1. whereas leased was observed in T0 (37.50 87.20 4.19 cm) followed by T1 (58.13 0.65 66.75 29.24 0.60 9.12 Root dry weight of Catharanthus roseus (g plant-1) 90 DAS 120 DAS 150 DAS 180 DAS 4.60 47.40 3.30 0.26 0. 2(4): 37-42 .80 19.40 3.65 28.70 3.96 1.24 0.16 26.10 10.23 0.86 1.Azospirillum lipoferum T2 .34 1.Azotobacter chroococcum T3 .80 19.90 2.80 1.20 0. the increase was highly significant in treatment T5 (consortium) (Table 4).17 and 26.92 Shoot and root length of C.11 1.40 DAS – Days after sowing Table 4: Effect of PGPR consortium inoculation on the Nutrient content of Catharanthus roseus Treatments T0 . roseus Treatments T0 .80 1.42 75.08 26.85 to 26.55 1.18 32.28 0.12 42.20 5. fluorescens (CRPS-5).88 1.25 62.60 1.15 0.12 and 19.Pseudomonas fluorescens T4 .04 50.66 1.96 1.70 6.88 1.Table 1: Effect of PGPR inoculation on the seed germination.85 3.86 2.84 1.38 3.60 14.32 38.92 1.23 0.10 Vigour index 680 1732 1120 1560 740 1988 Total chlorophyll content of Catharanthus roseus (mg g-1 plant-1) 90 DAS 2.20 3.60 9.19 cm respectively.45 and 24.Uninoculated control T1 .20 15.92 2.20 15.50 9.00 72. roseus as influenced by the inoculation of PGPR as consortium and as single inoculants was studied.93 1.90 10.88 2.90 4. A.60 4.17 Root length (cm plant1 ) 180 DAS* 18.08 4.Azospirillum lipoferum T2 .92 1.12 21.Bacillus megaterium T5 .86 5.34 0.21 35.63 68.65 71.80 7.13 cm).Bacillus megaterium T5 – Consortium (T1 + T2 + T3 + T4) Nitrogen content (%) Phosphorous content (%) Potassium content (%) 90 DAS 120 DAS 150 DAS 180 DAS 90 DAS 120 DAS 150 DAS 180 DAS 90 DAS 120 DAS 150 DAS 180 DAS 1.12 37.33 33. The inoculated treatments increased the leaf nitrogen however.02 56.94 1.70 19.40 31.12 21.Pseudomonas fluorescens T4 .21 0.19 0.27 31.40 39.55 64.17 0.40 g plant-1 was recorded respectively by T5 Consortium in C.88 1.Consortium (T 1 + T2 + T3 + T4) Catharanthus roseus Germination (%) 38.79 and 18. roseus The PGPR inoculated treatments increased the shoot and root dry weight and the increase was the most significant in consortium treated.60 4. chroococcum (CRAB-3) and B.82 13. vigour index and Total chlorophyll content of C.50 32.Azotobacter chroococcum T3 .42 55.20 12.80 17.12 to 76.Azotobacter chroococcum T3 .89 58.02 1.47 56.32 35.75 2. Shoot and root length varied from 26.80 1.85 cm).22 0.62 61.98 1. The NPK content of C. megaterium (CRBA-4) respectively (Table 3).80 16.52 23.25 1.26 0.20 180 DAS 2.60 120 DAS 2.40 13.60 3.96 2.80 3.34 150 DAS 2.15 46.91 1.01 1. P.50 cm and 18.79 58.05 21.80 12.20 13.29 0.12 17.72 16.00 78.70 23.85 24.40 2.78 1.19 *Mean value of three replications DAS – Days after sowing Table 3: Effect of PGPR consortium inoculation on the shoot and Root dry weight of Catharanthus roseus Treatments T0 .92 1.14 1.80 26.81 1. Shoot and root yield of C.00 13.Pseudomonas fluorescens T4 .70 37. phosphorus and potassium content of C.Bacillus megaterium T5 – Consortium (T 1 + T2 + T3 + T4) Shoot length (cm plant-1)* 90 DAS 120 DAS 150 DAS 180 DAS 26.10 0.96 0.89 1. roseus The shoot and root length of the Catharanthus roseus significantly influenced by PGPR.18 0.02 1.12 60. Among the treatments the highest mean value of shoot and root length was recorded in T5 (60.42 3.10 12.12 4.13 22. roseus followed by Seeds inoculated with A.60 3.Azospirillum lipoferum T2 .20 0. roseus A nitrogen.62 39.40 36.05 DAS – Days after sowing Table 2: Effect of PGPR consortium inoculation on the shoot and root length of Catharanthus roseus Treatments T0 . The results are presented in table 2.20 0.08 1.68 8.23 0.20 1.Pseudomonas fluorescens T4 .82 2.80 28.

Li. 37. 2005. will decisively contribute to make agriculture more productive less harm to environment. the consortium used devoid of Rhizobium and hence the possible incompatibility to Rhizobium does not arise. chlorophyll content of Catharanthus roseus significantly increased with seed inoculation of PGPR strains as single inoculant and as consortium preparations. 1998. Catharanthus roseus were tested for its response to biofertilizer inoculation under in vitro condition. four PGPR isolates were evaluated for their effect on plant growth. Further studies are needed to clarify possible interaction in PGPR-host plant association. The single inoculant effect observed in the present study was in conformity with the earlier reports published on several other crops [36. 30]. J. roseus. Shoot and root length of C. Applications of free living plant growth-promoting rhizobacteria.R. Antonie van Leeuwen hoek 86. A. P and K contents in both the varieties of C. remove inhibitory substances and stimulate each other through physical and biochemical activities. The PGPR strains such as Azospirillum. Canadian Journal of Microbiology 51. Single inoculant preparations A. The inoculation of PGPR consortium recorded maximum plant N. CONCLUSION In conclusion. Azotobacter. 38]. megaterium CRBA-4 and the consortium preparation containing all these four plant growth promoting bacterial strains on seed inoculation significantly improved the seed germination. M. roseus increased due to consortium treatment. 30]. The consortium applications increased the root and shoot weight and yield [33. fluorescens showed positive effect on the root initiation and root proliferation due to produced phytohormones (IAA and GA3) in the rhizosphere of black pepper [31]. 6. Journal of Theoretical Biology 190. Azotobacter. 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