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International Journal of Research in Pure and Applied Microbiology

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ISSN 22773843 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 -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, Hodgkins disease, malignant lymphomas, neuroblastoma, rhabdomyosarcoma, Wilms 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 Hodgkins disease [14]. Vincamine and vinpocetine from C. roseus, have vaso-dialating and memory enhancing properties and have been shown to alleviate vascular dementia and Alzheimers 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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Montbriand [18]. Its antibacterial and antidiabetic activities have been reported by Rozas Harnandez [19] and Srinivas et al. [20], respectively. The objective of this study was to investigate interaction between Catharanthus roseus and plant growth promoting four PGPR inoculations (Azospirillum lipoferum, Azotobacter chroococcum, Pseudomonas fluorescens and Bacillus megaterium) by measuring the germination rate, vigour index, Chlorophyll content and NPK content of plant. 2. MATERIALS AND METHODS Pot Culture Experiment A pot culture experiment was conducted during December 2011 to May 2012 at the Department of Microbiology, Faculty of Agriculture, Annamalai University, Annamalai nagar. The annual mean minimum and maximum temperature of experimental area is 25 and 39C, respectively and the mean highest and lowest relative humidity was 96 and 78 per cent respectively. The mean annual rainfall of this area is 1500 mm. The physicochemical properties of the pot soil were analysed. The plant growth promoting rhizobacterial isolates of A. lipoferum CRAS-2, A. chroococcum CRAB-3, P. fluorescens CRPS-5 and B. megaterium CRBA-4 were prepared as pot culture experiment. 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. The seeds of C. roseus were surface sterilized with 80 per cent ethanol and 0.1 per cent mercuric chloride and washed the seeds with sterile distilled water for 3 to 4 times. The seeds were mixed with carrier based plant growth promoting bacteria, either as individual organisms or consortium of organisms separately having a cell load of 1x109 cfu ml-1 and shade dried for 30 min. After shade drying, the seeds were sown at 25 of seeds per pot and finally five seeds were maintained. A control pot without inoculation was also maintained. The experiment was conducted in completely randomized block design with three replications. The treatments are as follows. T0 - Uninoculated control T1 - Azospirillum lipoferum - CRAS-2 T2 - Azotobacter chroococcum - CRAB-3 T3 - Pseudomonas fluorescens - CRPS-5 T4 - Bacillus megaterium CRBA-4 T5 - 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. Uninoculated seeds with distilled water served as control. Seedling height and germination percentage were recorded and vigour index was calculated as per the procedure suggested by Abdul-Baki and Anderson [21]. 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. 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. Root length Plants were pulled carefully without damaging to the roots. 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. Shoot dry weight Plants were pulled carefully without damaging to the shoots. The shoot cut into pieces and dried in an oven at 70C till constant dry weight. The mean was worked out and expressed in g plant-1. Root dry weight The roots were cut into pieces of approximately 5.0 centimeters length and dried in an oven at 70C till constant dry weight. The mean was worked out and expressed in g plant-1. 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]. Estimation of plant nitrogen, phosphorus and potassium content The nitrogen content of the plant samples was analyzed by Microkjeldahl method [23]. The phosphorus content in the plant material was estimated by the method developed by Olsen et al. [24]. The potassium content in the plant sample was estimated following the method of Jackson [25]. 3. RESULTS Seed germination and vigour index of C. roseus To study the effect of single inoculants and consortium of inoculant preparations of PGPR strains viz., A. lipoferum CRAS-2, A. chroococcum CRAB-3, P. fluorescens CRPS-5 and B. megaterium CRBA-4 on the seed germination and vigour index of C. roseus was studied under in vitro condition. The highest percentage of seed germination and vigour index of 87.10 per cent and 1988.10 was recorded for consortium treated C. roseus followed by single inoculant treatment (Table 1). Total chlorophyll content of c. roseus The total chlorophyll content of C. roseus as influenced by inoculant treatments are presented in Table 1. The chlorophyll content of the leaves of C. 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 5.34 mg g-1 of plant-1. Single inoculation treatment of A. lipoferum CRAS-2 recorded 4.60 mg g-1, P. fluorescens (CRPS-5) recorded 4.40 mg g-1, A. chroococcum CRAB-3 recorded 4.12 mg g-1 and B. megaterium CRBA-4 recorded 3.86 mg g-1 respectively.

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Table 1: Effect of PGPR inoculation on the seed germination, vigour index and Total chlorophyll content of C. roseus
Treatments T0 - Uninoculated control T1 - Azospirillum lipoferum T2 - Azotobacter chroococcum T3 - Pseudomonas fluorescens T4 - Bacillus megaterium T5 - Consortium (T 1 + T2 + T3 + T4) Catharanthus roseus Germination (%) 38.00 78.00 67.00 72.80 59.50 87.10 Vigour index 680 1732 1120 1560 740 1988 Total chlorophyll content of Catharanthus roseus (mg g-1 plant-1) 90 DAS 2.60 3.80 3.40 3.60 3.20 4.60 120 DAS 2.90 4.60 4.12 4.40 3.86 5.34 150 DAS 2.85 3.70 3.20 3.42 3.08 4.20 180 DAS 2.38 3.40 2.80 3.90 2.60 4.05

DAS Days after sowing Table 2: Effect of PGPR consortium inoculation on the shoot and root length of Catharanthus roseus
Treatments T0 - Uninoculated control T1 - Azospirillum lipoferum T2 - Azotobacter chroococcum T3 - Pseudomonas fluorescens T4 - Bacillus megaterium T5 Consortium (T 1 + T2 + T3 + T4) Shoot length (cm plant-1)* 90 DAS 120 DAS 150 DAS 180 DAS 26.12 37.40 39.42 48.21 35.25 62.55 64.65 71.33 33.37 48.62 61.63 68.27 31.02 56.21 69.42 75.50 32.15 46.42 55.65 66.32 38.47 56.22 69.47 76.50 Mean 37.79 58.45 52.89 58.04 50.12 60.17 Root length (cm plant1 ) 180 DAS* 18.85 24.13 22.52 23.05 21.08 26.19

*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 - Uninoculated control T1 - Azospirillum lipoferum T2 - Azotobacter chroococcum T3 - Pseudomonas fluorescens T4 - Bacillus megaterium T5 - Consortium (T 1 + T2 + T3 + T4) Shoot dry weight of Catharanthus roseus (g plant-1) 90 DAS 120 DAS 150 DAS 180 DAS 8.72 16.12 21.80 28.12 17.80 26.32 35.12 42.82 13.12 21.65 28.40 36.20 15.70 23.40 31.62 39.25 11.80 19.16 26.18 32.80 19.75 29.70 37.60 47.12 Root dry weight of Catharanthus roseus (g plant-1) 90 DAS 120 DAS 150 DAS 180 DAS 4.80 7.00 8.50 9.68 8.10 12.60 14.80 17.80 6.00 10.80 12.00 13.70 6.90 10.40 13.20 15.20 5.60 9.10 10.20 12.60 9.20 13.80 16.70 19.40

DAS Days after sowing Table 4: Effect of PGPR consortium inoculation on the Nutrient content of Catharanthus roseus
Treatments T0 - Uninoculated control T1 - Azospirillum lipoferum T2 - Azotobacter chroococcum T3 - Pseudomonas fluorescens T4 - 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 1.78 2.02 1.94 1.97 1.90 2.11 1.82 2.14 1.96 2.08 1.92 2.34 1.80 1.96 1.91 1.92 1.88 2.02 1.66 1.92 1.84 1.88 1.82 1.96 0.15 0.23 0.20 0.24 0.26 0.29 0.17 0.26 0.23 0.28 0.30 0.34 0.13 0.22 0.19 0.23 0.24 0.28 0.10 0.21 0.18 0.20 0.20 0.25 1.60 1.93 1.88 1.92 1.86 2.04 1.75 2.01 1.95 1.98 1.92 2.20 1.60 1.89 1.84 1.88 1.80 1.96 1.55 1.86 1.80 1.81 1.78 1.92

Shoot and root length of C. roseus The shoot and root length of the Catharanthus roseus significantly influenced by PGPR. The results are presented in table 2. Shoot and root length varied from 26.12 to 76.50 cm and 18.85 to 26.19 cm respectively. Among the treatments the highest mean value of shoot and root length was recorded in T5 (60.17 and 26.19 cm) followed by T1 (58.45 and 24.13 cm), whereas leased was observed in T0 (37.79 and 18.85 cm). Shoot and root yield of C. roseus The PGPR inoculated treatments increased the shoot and root dry weight and the increase was the most significant in consortium treated. The shoot, root dry weight of 47.12 and 19.40 g plant-1 was recorded respectively by T5 Consortium in C. roseus followed by Seeds inoculated with

A. lipoferum (CRAS-2), P. fluorescens (CRPS-5), A. chroococcum (CRAB-3) and B. megaterium (CRBA-4) respectively (Table 3). Plant nutrient content of c. roseus A nitrogen, phosphorus and potassium content of C. roseus as influenced by the inoculation of PGPR as consortium and as single inoculants was studied. These three major plant nutrient contents significantly varied between the treatments. A positive response due to inoculants was observed at all sampling periods. The NPK content of C. 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. The inoculated treatments increased the leaf nitrogen however, the increase was highly significant in treatment T5 (consortium) (Table 4).

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4. DISCUSSION The plant rhizosphere is a versatile and dynamic ecological environment of intense microbesplant interactions for harnessing essential micro- and macro-nutrients from a limited nutrient pool [26]. In the present investigation, four PGPR isolates were evaluated for their effect on plant growth, yield and nutrient content of Catharanthus roseus in pots conditions for 6 months and to ascertain the involvement of possible PGPR trait(s) in enhancing Catharanthus roseus growth. Catharanthus roseus were tested for its response to biofertilizer inoculation under in vitro condition. Single inoculant preparations A. lipoferum CRAS-2, A. chroococcum CRAB-3, P. fluorescens CRPS-5 and B. megaterium CRBA-4 and the consortium preparation containing all these four plant growth promoting bacterial strains on seed inoculation significantly improved the seed germination, vigour index of C. roseus. The increase is attributed to the potential of inoculant to secrete phytohormone in the spermosphere. Co-culture of microbes performed better than their individual microbes. The combination of bacteria interacts with each other synergistically, provide nutrients, remove inhibitory substances and stimulate each other through physical and biochemical activities. Co-inoculation of PGPR with different beneficial properties may be the future trend for biofertilizer application to enable sustainable production [27]. Pot culture experiment to assess the effectiveness of PGPR consortium versus single inoculant preparations on the growth of C. roseus revealed that the consortium effect was better than the single inoculant effect. Shoot and root dry weight, Shoot and root length, chlorophyll content of Catharanthus roseus significantly increased with seed inoculation of PGPR strains as single inoculant and as consortium preparations. The increase in growth parameters is directly related to increased nutrient content and chlorophyll content. Nitrogen and phosphorus are the major nutrients that determine the growth and yield of any crop plants. The inoculated PGPR strains usually have been found to increase root length and root biomass and this augmented growth of enhanced the mineral uptake in plants [28, 29, 30]. The previous study conducted with P. 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]. Increased growth of plants might have been due to increased efficiency of roots in extracting the nutrients from the soil that are made available by the activity of inoculated bacteria. Many workers have reported the improvement of plant growth by the release of soluble P from inorganic and organic phosphate due to phosphobacteria inoculation [32]. In the present investigation, the consortium used devoid of Rhizobium and hence the possible incompatibility to Rhizobium does not arise. The consortium of Azospirillum, Azotobacter, Pseudomonas and Bacillus showed maximum Shoot and root dry weight, Shoot and root length of C. roseus. The consortium applications increased the root and shoot weight and yield [33, 34].

Attia and Saad [35] reported that combined treatment of Azotobacter + PSB showed significant increases in vegetative growth and chemical composition of the test plant C. roseus. The total chlorophyll of C. roseus increased due to consortium treatment. The single inoculant effect observed in the present study was in conformity with the earlier reports published on several other crops [36, 37, 38]. The research interest in rhizosphere microorganisms has increased because they play significant role in the maintenance of soil tablets. The use of N fixing bacteria that assimilate gaseous nitrogen from the atmosphere includes symbionts of Rhizobium and free living rhizobacteria such as Azospirillum and Azotobacter. The PGPR strains such as Azospirillum, Azotobacter, Pseudomonas and Bacillus when seed treated increased the N, P and K content in different crop plants such as rice, wheat, sorghum, maize, sugarcane, banana, forage crops and medicinal plants. The inoculation of PGPR consortium recorded maximum plant N, P and K contents in both the varieties of C. roseus and in soil which indicated the positive effect of consortium in augmenting the availability of nitrogen and phosphorus to plants. Plants co-inoculated with Azospirillum and phosphobacteria recorded more N and P than the single inoculation of these two biofertilizers [39]. The increase of nitrogen and phosphorus is due to the influence of PGPR in the rhizosphere. PGPR enhanced nutrient uptake from soil solution at a faster rate which increased the accumulation of NPK and thereby dry matter content [40, 41, 42, 30]. 5. CONCLUSION In conclusion, the data presented in this study indicate that the mode of action of PGPR is most probably composed of multiple mechanisms. Further studies are needed to clarify possible interaction in PGPR-host plant association. Therefore, the increased use of various biological processes in soil, of which some examples have been given in the present study, will decisively contribute to make agriculture more productive less harm to environment. 6. REFERENCES 1) Ahmad, F., Ahmad, I., Khan, M.S., 2008. Screening of free living rhizospheric bacteria for their multiple plant growth promoting activities. Microbiological Research 163, 173 181 2) Reed, M.L.E., Glick, B.R., 2004. Applications of free living plant growth-promoting rhizobacteria. Antonie van Leeuwen hoek 86,1 - 25. 3) Reed, M.L.E., Glick, B.R., 2005. Growth of canola (Brassicanapus) in the presence of plant growthpromoting bacteria and either copper or polycyclic aromatic hydrocarbons. Canadian Journal of Microbiology 51, 1061-1069. 4) Glick, B.R., Penrose, D.M., Li, J., 1998. A model for the lowering of plant ethylene concentrations by plant growth promoting bacteria. Journal of Theoretical Biology 190, 63-68.

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Source of support: Nil; Conflict of interest: None declared

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